Gems of Ophthalmology Cornea and Sclera

Gems of Ophthalmology
CORNEA AND SCLERA

Gems of Ophthalmology
CORNEA AND SCLERA
Editors
HV Nema MS
Former Professor and Head
Department of Ophthalmology
Institute of Medical Sciences
Banaras Hindu University
Varanasi, Uttar Pradesh, India
Nitin Nema MS DNB

Professor
Department of Ophthalmology
Sri Aurobindo Institute of Medical Sciences
Indore, Madhya Pradesh, India
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Gems of Ophthalmology—Cornea and Sclera
First Edition: 2018
ISBN: 978-93-5270-248-0
Contributors
Vinay Agarwal MS Frank Joseph Goes MD

Consultant Ophthalmologist Director
Mumbai, Maharashtra, India Oagchirurgie-Oagheekunde
Antwerp, Belgium
Sreedharan Athmanathan MD DNB
Virologist Jaya Gupta MS
LV Prasad Eye Institute Vision Research Foundation
Hyderabad, Telangana, India Sankara Nethralaya
Chennai, Tamil Nadu, India
Mohamed El Bahrawy MD
Consultant, VISSUM
Institute of Ophthalmology Alicante Nidhi Gupta MS
Alicante, Spain Vision Research Foundation
Sankara Nethralaya
Jorge L Alió Del Barrio MD PhD
Consultant
VISSUM Noopur Gupta MD
Institute of Ophthalmology Alicante Dr Rajendra Prasad Centre for
Alicante, Spain Ophthalmic Sciences
All India Institute of Medical Sciences
Jyotirmay Biswas New Delhi, India
MS FMRF FNAMS FIC Path, FAICO
Director Stephen Hilton OD
Department of Uveitis and Ocular Pathology Department of Ophthalmology
Sankara Nethralaya West Virginia University
Chennai, Tamil Nadu, India Morgantown, West Virginia, USA
Charmaine Chai MBBS MMed (Ophth)

Associate Consultant Soosan Jacob MS FRCS DNB
National University Hospital Director and Chief
Singapore Dr Agarwal’s Refractive and Cornea
Foundation
Dr Agarwal’s Group of Eye Hospitals
Rajesh Fogla MS DNB FRCS
Consultant
Sankara Nethralaya
Chennai, Tamil Nadu, India Joveeta Joseph PhD
Microbiologist
Ashok Garg MS PhD FAIMS FIAO Jhaveri Microbiology Centre
Chairman and Medical Director Brien Holden Eye Research Centre
Garg Eye Institute and Research Centre LV Prasad Eye Institute
Hissar, Haryana, India Hyderabad, Telangana, India
vi Gems of Ophthalmology—Cornea and Sclera
G Madhavi DNB N Venkatesh Prajna FRCOphth

Consultant Professor
Srikiran Institute of Ophthalmology Aravind Eye Hospital and
Kakinada, Andhra Pradesh, India Postgraduate Institute
Madurai, Tamil Nadu, India
Parthopratim Dutta Majumder MS
Consultant VK Raju MD FRCS FACS
Department of Uveitis and Ocular Pathology Professor of Clinical Ophthalmology
Sankara Nethralaya West Virginia University
Chennai, Tamil Nadu, India Morgantown, West Virginia, USA
Quresh Maskati MS FCPS DOMS FICS

Srinivas K Rao MD
Consultant
Director
Sankara Nethralaya
Cornea Services, Eye Clinic
Mumbai, Maharashtra, India
Manotosh Ray MD FRCS

Francisco Arnalich Montiel MD
Associate Consultant
VISSUM National University Hospital
Institute of Ophthalmology Alicante Singapore
Alicante, Spain
Ritika Sachdev MD
G Mukherjee MD Dr Rajendra Prasad Centre for
Director Ophthalmic Sciences
Mukherjee Eye Klinik All India Institute of Medical Sciences
New Delhi, India New Delhi, India
Rajib Mukherjee MS Gagan Sahni OD

Director and Senior Consultant Senior Consultant
Cornea and Ocular Surface Contact Lens Designing and Fitting
Mukherjee Eye Klinik Mukherjee Eye Klinik
Prema Padmanabhan DNB FRCOphth

Jorge L Alió y Sanz MD PhD
Director Director, VISSUM
Nethralaya and Cornea Services Institute of Ophthalmology Alicante
Medical Research Foundation Alicante, Spain
Sankara Nethralaya
Chennai, Tamil Nadu, India Savitri Sharma MD
Director of Laboratory Services
LV Prasad Eye Institute Network
Gunisha Pasricha PhD
Head, Jhaveri Microbiology Centre
Assistant Manager Brien Holden Eye Research Centre
Research and Development LV Prasad Eye Institute
Hi Tech Biosciences India Ltd Hyderabad, Telangana, India
Pune, Maharashtra, India
Rajesh Sinha MD FRCS
Shaila Patel MS Professor
Dr Agarwal’s Group of Eye Hospitals and Dr Rajendra Prasad Centre for
Eye Research Centre Ophthalmic Sciences
Chennai, Tamil Nadu, India All India Institute of Medical Sciences
New Delhi, India
Lalitha Prajna MD DNB
Consultant Microbiologist Jerry Tan FRCS FRCOphth FAMS
Aravind Eye Hospital and Consultant
Postgraduate Institute Camden Medical Centre
Madurai, Tamil Nadu, India Orchard Boulevard, Singapore
Contributors vii
Radhika Tandon MD DNB FRCS MRCOphth M Vanathi MD

Professor Professor
Dr Rajendra Prasad Centre for Dr Rajendra Prasad Centre for
Ophthalmic Sciences Ophthalmic Sciences
All India Institute of Medical Sciences All India Institute of Medical Sciences
C Veerajayalakshmi MS
George N Thomas MD
Fellow
National University Hospital Aravind Eye Hospital and
Singapore Postgraduate Institute
Madurai, Tamil Nadu, India
Jeewan S Titiyal MD
Professor Shefali Vyas MD FAAP
Dr Rajendra Prasad Centre for Associate Director
Ophthalmic Sciences Children’s Kidney Center
All India Institute of Medical Sciences RWJ Barnabas Health
New Delhi, India West Orange, New Jersey, USA
Preface
Cornea is one of the important structures of the eyeball. It serves as a window

to convey visual images. Diseases of the cornea impair vision and an individual
loses his independence. In spite of marked reduction in the incidence of
infectious diseases of the anterior segment of the eye, congenital anomalies,
vitamin A-deficiency, traumatic and degenerative diseases of the cornea
cause significant blindness. The corneal blindness can be prevented or cured
by adopting suitable measures. Cornea is the main source of refractive errors
because a major part of refraction occurs at the corneal surface. Refractive
errors contribute to a high percentage of impairment of vision worldwide.
The present book on cornea is designed to provide an up-to-date
information on current important topics. Corneal topography not only helps
in diagnosing refractive errors, but also ectatic conditions like keratoconus
and Pellucid marginal degeneration. It aids in planning the refractive
surgery and fitting of contact lenses. Corneal confocal microscopy is another
diagnostic tool which can distinguish between normal and abnormal corneal
structures at cellular level which is used in the diagnosis of corneal diseases.
Current topics on refractive surgery, such as Laser assisted in situ
keratomileusis (LASIK), LASIK in hyperopia, small incision lenticular
extraction (SMILE) vs LASIK and complications of LASIK and its management
have been described in the book.
Chapter on diagnostic procedures in infectious keratitis presents a detail
account of investigations for the benefit of interested readers. Relatively
uncommon infections like fungal keratitis and Acanthamoeba keratitis are
also included. Corneal dystrophies are described comprehensively with their
latest classification and management.
Keratoconus causes severe visual impairment. Recently, increasing
interest is generated in its management with the introduction of corneal
cross-linking. The book contains chapters on the management of keratoconus
by corneal cross-linking, intrastromal corneal ring segments (INTACS) and
lamellar keratoplasty.
Recent procedures in lamellar keratoplasty are tissue targeted. In
endothelial keratoplasty, normal stroma and the surface are persevered and
Descemet’s membrane and endothelium are targeted resulting in quicker
rehabilitation. While in deep anterior lamellar keratoplasty (DALK), corneal
stroma with Bowman membrane and epithelium are replaced leaving the
healthy Descemet’s membrane and endothelium intact, thus minimizing the
x Gems of Ophthalmology—Cornea and Sclera
chances of rejection. The other procedure is Descemet’s stripping automated

endothelial keratoplasty (DSAEK). It is a much faster keratoplasty and has
a shorter visual recovery time. Introduction of different types of lamellar
keratoplasty may facilitate more corneal transplants because one cornea can
be used for more than one patient. DALK has been described in some detail
while a brief account of various types of lamellar keratoplasty has been given
in the chapter on Advances in keratoplasty.
Chapters on Diagnosis of dry eye, Ocular surface disorder,
Keratoprosthesis, Cystinosis and Corneal changes in contact lens users are
also included in the book.
The editors assure the readers that the major part of the work presented
in this book comes from the Recent Advances in Ophthalmology series edited
by Dr HV Nema and Dr Nitin Nema. In each chapter, author/s have provided
references for the benefit of those who want to read the topic in detail.
The book is multi-authored, therefore, repetition could not be avoided.
Readers can take the advantage of knowing the views of different authors.
However, special effort has been put to avoid ambiguity.
The book is concise and information on corneal diseases is presented
in an easily readable form. The book is profusely illustrated. Postgraduate
students, residents, and general ophthalmologists will find it useful in their
day-to-day clinical practice.
HV Nema MS
Nitin Nema MS DNB
Acknowledgments
We wish to record our grateful thanks to all authors of chapters for their
spontaneity, cooperation and hard work. Some of them have revised their
chapters. Our special thanks go to Drs. Soosan Jacob, Savitri Sharma and
Rajib Mukherjee for contributing their chapters on a short notice.
Credit goes to Mr Jitendar P Vij (Group Chairman), Jaypee Brothers
Medical Publishers (P) Ltd who has agreed to start a new series—Gems of
Ophthalmology. Cornea and Sclera is the first book of this series.
Ms Kritika Dua, Development Editor deserve our appreciation for her
continued interest in refining chapters and eliminating plagiarism.
Contents
1. Corneal Topography 1
Francisco Arnalich Montiel, Jorge L Alió Del Barrio, Jorge L Alió y Sanz
2. Corneal Confocal Microscopy 53

Manotosh Ray, George N Thomas
3. LASIK 78
Frank Joseph Goes
4. SMILE versus LASIK 95

Jorge L Alió y Sanz, Mohamed El Bahrawy
5. LASIK in Hyperopia 107

VK Raju, Stephen Hilton, G Madhavi
6. LASIK—Complications and Management 114

Rajesh Fogla, Srinivas K Rao, Prema Padmanabhan
7. Zyoptix Wavefront-guided Customised Ablation in

Retreated Corneas 127
Jerry Tan
8. Diagnostic Procedures in Infectious Keratitis 139

Savitri Sharma, Sreedharan Athmanathan
9. Fungal Keratitis 169

N Venkatesh Prajna, Lalitha Prajna, C Veerajayalakshmi
10. Herpetic Keratitis 187

Charmaine Chai, Manotosh Ray
11. Acanthamoeba Keratitis—Pathogenesis and Diagnosis 202

Savitri Sharma, Joveeta Joseph, Gunisha Pasricha
12. Corneal Dystrophies 219

Rajesh Sinha, Noopur Gupta, Ritika Sachdev, Radhika Tandon, Jeewan S Titiyal
13. Management of Keratoconus 268

Prema Padmanabhan, Nidhi Gupta
14. Corneal Collagen Cross-linking 276

Ashok Garg
15. Intrastromal Corneal Ring Segments 282

Shaila Patel, Soosan Jacob
16. Deep Anterior Lamellar Keratoplasty 298

Jaya Gupta, Prema Padmanabhan
xiv Gems of Ophthalmology—Cornea and Sclera
17. Dry Eye Disease 311

Vinay Agarwal
18. Ocular Surface Reconstructions 337

Manotosh Ray, Rajesh Sinha, M Vanathi, Noopur Gupta
19. Advances in Keratoplasty 375

Soosan Jacob
20. Keratoprosthesis 392

Quresh Maskati
21. Cystinosis 416

Shefali Vyas
22. Corneal Changes in Contact Lens Users 424

Rajib Mukherjee, Gagan Sahni, G Mukherjee
23. Episcleritis and Scleritis 437

Parthopratim Dutta Majumder, Jyotirmay Biswas
Index 455
1
CHAPTER
Corneal Topography
Francisco Arnalich Montiel, Jorge L Alió Del Barrio, Jorge L Alió y Sanz
BACKGROUND
The cornea is the most important refractive element of the human eye, provid-
ing approximately two-thirds of its optical power, accounting for about 43–44
diopters at the corneal apex.1 Since its surface is irregular and aspherical, it is
not radially symmetric, and simple measurement techniques are inadequate.
The great upsurge in refractive surgery led to a need for improved meth-
ods to analyze corneal shape since refraction and keratometric data alone
were insufficient to predict surgical outcomes. Understanding and quanti-
fying corneal contour or shape has become essential in planning modern
surgical intervention for refractive surgery, as well as in corneal transplan-
tation, and it is also very valuable for assessing optical performance of the
eye. The different methods for evaluating the anterior surface of the cornea,
developed over several centuries, have, in the present era, led to the modern
corneal topography.
FIRST STEPS IN CORNEAL MEASUREMENT

In 1619, Scheiner analyzed corneal curvature by matching the image of a win-
dow frame reflected onto a subject’s cornea with the image produced by one
of his calibrated spheres.
Keratometer
In 1854, Helmhotz described the first true keratometer, which he called

an ophthalmometer.2 With some minor improvements, it is still being used
clinically for calculating refraction, intraocular lens power and contact lens
fitting normal corneas.
2 Gems of Ophthalmology—Cornea and Sclera
This apparatus is based on the tendency of the anterior corneal surface

to behave like a convex mirror and reflect light. The projection of four point,
mire, onto the cornea, creates a reflected image that can be converted into
a corneal radius, ‘r’, using a mathematical equation that considers distance
from the mire to cornea (75 mm in the keratometer), image size and mire
size (64 mm in the keratometer). The corneal radius can be transformed into
dioptric power using the formula:
(Index of refraction of the lens − 1)
DP =
r
The standard keratometric index represents the combined refractive index
of the anterior and posterior surfaces of the cornea, considers the cor-
nea as a single refractive surface, and is 1.3375. Thus, the equation can be
simplified to:
337.5
DP =
r
Although keratometers are still common in ophthalmology clinics, they do

have specific limitations that need to be considered in order to avoid mis-
leading conclusions.
• Most traditional keratometers measure the central 3 mm of the cornea,
which only accounts for 6% of the entire surface.
• It assumes that the cornea is a perfectly spherocylindrical surface, which
it is not. The cornea is aspheric in shape, flattening between the center
and the periphery. Usually the central corneal curvature is fairly uniform,
and this is the reason why it can be used to calculate corneal power in
normal patients. However, this is not true in some pathologies like ectatic
disorders or after refractive surgery.
• The keratometer provides no information as to the shape of the cor-
nea either inside or outside the contour of the mire. Several corneal
shapes can all give the same keratometric value so this apparatus is of
little use should it become necessary to reconstruct the whole corneal
morphology.
Keratoscopy and Photokeratoscopy
Goode presented the first keratoscope in 1847. Placido was the first to photo-
graph the corneal reflections of a series of illuminated concentric rings
(known as Placido’s rings) in 1880 (Fig. 1.1). Finally, in 1896, Gullstrand was
the first to develop a quantitative assessment of photokeratoscopy.3
The keratoscope, like a keratometer, projects an illuminated series of
mires onto the anterior corneal surface, usually consisting of concentric rings.
The distance between the concentric rings or mires gives the observer an idea
of the corneal shape. A steep cornea will crowd of the mires, while a flat cornea
will spread them out. Surface irregularity is seen as mire distortion.
Corneal Topography 3
Fig. 1.1: Placido rings.
When a photographic camera is attached to the keratoscope, we have a

photokeratoscope, which gives semiquantitative and qualitative information
about the paracentral, midperipheral and peripheral cornea.
Based on the mathematical equation, it is possible to calculate corneal
power from object size. Still, photokeratoscopy gives limited information on
the central area, which is not covered by the mires.
Videokeratoscopy
At the beginning of this century, modern corneal topographers were based

on videokeratoscopy.4 A video camera is attached to the keratoscope, and
the information is analyzed by a computer that displays a color-coded map
of power distribution or corneal curvature of the anterior corneal surface
(Fig. 1.2). It overcomes some of the limitations of other methods, since it
measures larger areas of the cornea, with a much larger number of points
thus increasing resolution. Computer technology makes it possible to create
permanent records and do multiple data analyses.
FUNDAMENTALS AND TECHNOLOGICAL APPROACHES

TO CORNEAL TOPOGRAPHY
Shape of the Normal Cornea
The anterior corneal surface is a refractive surface characterized by an almost
spherical shape. The human cornea is not a perfect sphere and is usually
assumed to have a conic section. This model could be represented in a simple
way by means of the equation:
X 2 + Y 2 + (1 + Q) Z 2 − 2 RZ = 0
Fig. 1.2: Videokeratography system.
where the Z-axis is the axis of revolution of the conic, R is the radius at the
corneal apex, and Q is asphericity, a parameter that is used to specify the type
of conicoid.
For a perfect sphere this parameter takes the value of zero (Q = 0), for an
ellipsoid with the major axis in the X-Y plane (oblate surface) the asphericity
is positive (Q > 0), for an ellipsoid with the major axis in the Z-axis (prolate
surface) asphericity is negative (- 1 < Q < 0), while for a paraboloid with its
axis along the Z-axis the value is - 1, and it is less than - 1 for a hyperboloid.
Other parameters have been defined to classify the conicoid form of the
cornea: ‘P,’ the shape factor (P = Q + 1), or the eccentricity value, ‘e,’ defined
as e = − Q .
Several studies have shown that the anterior corneal configuration tends
to be prolate, i.e., the cornea progressively flattens out periphery by 2–4 diop-
ters of flattening.5 The asphericity of the normal cornea depends on the study
ranges from - 0.26 to - 0.11.
This tendency can be detected in the topographic map. Toward the
periphery, dioptric power appears to decline, and the nasal area flattens more
than the temporal area (Fig. 1.3). This could be helpful in distinguishing right
eye topography from the left eye topography. The topographic patterns of the
two corneas of the same individual often show mirror-image symmetry.
Corneal topographic patterns (Figs. 1.4A to D) have been studied in
normal eyes and the following shapes have been found:5 round (23%), oval
(21%), symmetric bow-tie typical for regular astigmatism (18%), asymmetric
bow-tie (32%) and irregular astigmatism (7%). In the round and oval shapes
there is an area of uniform dioptric power close to 43 diopters (D) in the cen-
ter of the cornea. The bow-tie configuration reflects the existence of corneal
Fig. 1.3: Corneal topography in a normal right eye. There is a flattening toward the
periphery, more pronounced at the nasal area.
astigmatism. Depending on the position of the axes, corneal astigmatism is

defined as against-the-rule (the steepest axis is horizontal), with-the-rule
(the steepest axis is vertical) or oblique (the steepest axis is near the meridian
angles of 45° or 135°).
CORNEAL TOPOGRAPY: CURRENT TECHNOLOGIES

Corneal topography is a noninvasive exploratory technique that graphically
describes the geometric characterization of the morphology of the cornea,
that permits us differentiating standard normal patterns form the pathologi-
cal cornea.6 Current topographers are based on either systems based on the
light reflection on the cornea, or systems based on the projection of a slit light
into the cornea with different technology.
Systems Based on the Light Reflection on the Cornea:

Curvature Based Topographers
Placido Disk System

A Placido disk system consists of a series of concentric illuminated
rings or mires that are reflected off of the cornea and recorded by video-
computerized systems.4 The topographer uses an algorithm to translate
these reflected images into radial curvature of the corneal surface. Height
and slope data derived from the radial curvature of the anterior corneal sur-
face are represented as a corneal keratometric map that follows a color scale
developed by the University of Louisiana. Flat curves are represented with
(A) (B)
(C)
(D)
Figs. 1.4A to D: Normal corneal topographic patterns: (A) Oval topographic pattern,
(B) bow-tie pattern that shows an against-the-rule astigmatism, (C) with-the-rule astig-
matism and (D) oblique astigmatism.
cool colors (blue or violet), while warm colors (red or orange) correspond to
high curvature. Mild colors (green or yellow) correspond to medium curva-
ture equal to the reference sphere.
Currently, several companies manufacture instruments called videoker-
atoscopes that picture corneal shape based on the Placido disk method, and,
in fact, this approach has been the most clinically and commercially success-
ful up to the last decade. Two types of Placido targets have been used:
1. Large diameter target (disk-shaped): This is less sensitive to misalign-
ment due to a long working distance, but there can be a loss of data due
to interference by the patient’s brow and nose.
2. Small diameter target (cone-shaped): This is designed for a short working
distance and can be influenced by automatic alignment and focusing or
compensation of misalignment for accuracy. It does not present data loss
due to shadows.
Limitations:
1. Placido-based apparatus creates a 3D system by making geometric
assumptions about the cornea since the apparatus does not measure
corneal surface directly. These assumptions are not accurate for irregular
and aspheric corneas.
2. The reflection technique depends on the integrity and normality of the
tear layer.
Interferometric Method-based Systems
In essence, a reference surface (or its hologram) is compared to the tested

surface, the corneal surface and interference fringes are produced as a result
of differences between the two shapes, which can be interpreted as a contour
map of surface elevations.7
Interference techniques are used in the optical industry to detect lens
and mirror aberrations of subwavelength dimensions. High accuracy is theo-
retically possible, but clinical use has not been very wide-spread as yet.
Moire Deflectometry-based Systems
The deflections of the rays reflected off the corneal surface are analyzed to
build up a surface elevation map.7
Systems Based on the Projection of a Slit Light onto the

Cornea: Elevation-based Topographers
The common denominator of this technology is the projection of a slit light
onto the cornea. Interestingly many of these corneal topographies integrate
a dual technology, and in a first stage they use light reflection on the cornea
by means of a Placido disk to obtain curvature and refractive power data,
followed by capturing the image of the scattered light from the slit light
to measure corneal elevations of the entire corneal segment. From these
two-dimensional (2D) cross-sections, it is possible to create a reliable three-

dimensional model.
Depending on the spatial arrangement of the photographic systems, we
can distinguish the following two different systems.
Systems Based on the Principle of Normal Photography
Its main feature is that the plane of the camera lens is located parallel with the
image.8 When the slit image is on the cornea, it splits into a specular reflection
and a refracted beam that penetrates the corneal surface and is scattered by
the tissue of the cornea. An image of this scattered light within the corneal tis-
sue is captured by an imaging system, which consists of a camera lens located
in parallel with the image. It uses triangulation to measure the elevation of
the anterior and posterior corneal surface with respect to a reference plane.8
The most popular system using this principle is the Orbscan (Bausch &
Lomb Incorporated, USA) (Fig. 1.5), which was the first commercial device
that was able to assess the posterior corneal surface.8 It has dual technology
as it uses Placido disk and slit-based systems to obtain 40 slit images of the
cornea. These images are captured over one second and are then recorded
providing different maps of the anterior and posterior corneal surfaces, and
also pachymetric data.
Systems Based on the Principle of Scheimpflug Photography
Its main feature is that the plane of the camera lens is placed sideways to the
image. Scheimpflug imaging is based on the Scheimpflug principle, which
Fig. 1.5: Orbscan II system.

Fig. 1.6: Scheimpflug-based topographer—Pentacam.
occurs when a planar subject is not parallel to the image plane. In this sce-
nario, an oblique tangent can be drawn from the image, object and lens
planes, and the point of intersection is the Scheimpflug intersection, where
the image is in best focus.9 With a rotating Scheimpflug camera, the devices
can obtain many Scheimpflug images in seconds. The main commercial sys-
tems based on this principle are Pentacam (Oculus, USA), Galilei (Ziemer,
Switzerland) and Sirius (CSO, Italy), which offer repeatable measurements
of the corneal curvature and other anatomical measurements of the anterior
segment (Fig. 1.6).
Although the instruments based on rotating Scheimpflug cameras are
considered the most comprehensive and accurate, they also have some lim-
itations. Lower imaging speed can increase the risk of motion artifacts, even
though there is an inbuilt second camera to control for eye movements. For
example, commercially available Pentacam uses a rotating Scheimpflug cam-
era (180°) to provide a 3D scan of the anterior segment of the eye. It requires
2 seconds to complete 25 radial scans. Moreover, radial scanning may not
provide sufficient scan density of the corneal periphery, needing interpola-
tion. Another limitation is that the instruments using the Scheimpflug prin-
ciple are less accurate in comparison to Placido-based ones in providing
traditional curvature maps of the anterior surface, and only show moderate
agreement in simulated keratometry values. The Sirius system has a dual
technology and combines Scheimpflug camera and a small-angle Placido
disk topographer with 22 rings. The data for the anterior surface are finally
determined by merging the Placido image and the Scheimpflug image using
a proprietary method.
Systems with a single Scheimpflug channel use a mathematical equa-
tion to estimate compensation for an off-center measurement, however, to
properly compensate for an off-center measurement, a dual Scheimpflug

technology is needed.9 Galilei uses a monochromatic slit-light source which
combines dual Scheimpflug cameras and a Placido disk to measure both
anterior and posterior corneal surfaces.
Systems Based on Optical Coherence Tomography
Optical coherence tomography (OCT) of the cornea and anterior segment is

an optical method of cross-sectional scanning based on reflection and scat-
tering of light from the structures within the cornea.10 Measuring different
reflectivity from structures within the cornea by a method of optical inter-
ferometry produces the cross-section image of the cornea and other anterior
segment structures.
In optical interferometry, the light source is split into the reference and
measurement beams. The measurement beam is reflected from ocular struc-
tures and interacts with the reference light reflected from the reference mir-
ror, a phenomenon called interference. The coherent or positive interference
characterized by an increased resulting signal is measured by the interferom-
eter, and, subsequently, the position of the reflecting structure of the eye can
be determined.10
In this way, the structures of the anterior segment can be visualized
with a high degree of resolution (currently 18 microns axial and 60 microns
transverse).
In 2005, a commercial 1310 time-domain OCT system for anterior seg-
ment imaging was launched under the name Visante OCT (Carl Zeiss, Inc.).
Currently although it is widely used OCT device for dedicated in vivo ante-
rior segment imaging and creates pachymetry maps, it cannot perform topo-
graphic analysis of the cornea, mostly because of limitations in acquisition
time. The introduction of Fourier domain OCT in 2002 with the primary
advantage of increased sensitivity or speed and the possibility of 3D imaging
promised to improve the ability of OCT to quantitatively assess the corneal
topography.10 Nowadays, commercial high speed 3D anterior segment OCT
based on swept source OCT provide higher resolution cross-sectional images
that can be used to obtained OCT-based corneal topography. The commer-
cially available OCT-based topographer is SS-1000 CASIA (Tomey Corpora-
tion, Inc., Nagoya, Japan) (Fig. 1.7).
PERFORMING A GOOD TOPOGRAPHY EXAMINATION

Corneal topography is a noninvasive imaging technique for mapping the
surface curvature of the cornea. The specific method varies depending on
the device used, but some aspects are common. The patient is seated facing
a bowl containing an illuminated pattern which is focused on the anterior
surface of his cornea. The reflected pattern is analyzed by a computer that
calculates the shape of the cornea by means of different graphic formulae.11
Although computer programs are created to be very accurate, they cannot
Fig. 1.7: Optical coherence tomography (OCT)-based topographer—CASIA SS-1000.
Fig. 1.8: Distortion of the placido rings because of tear film breakup.
recognize, and account for, every problem. Critical points for precise mea-
surement are accurate alignment, centring and focusing. They depend on the
ability of the examiner to take a good measurement. Another potential source
of error is tear film irregularities, for example focal flattening over a dry patch.
These may be most easily identified on the raw image.
Tear film breakup causes mistracking of the mires and artefacts in the
topography pattern and will apparently look like significant irregularities
(Fig. 1.8). These corneal irregularities could suggest a corneal pathology, such
as keratoconus, and result in wrong diagnosis (Figs. 1.9A and B). To avoid
(A)
(B)
Figs. 1.9A and B: (A) Raw image and (B) topographic irregularities and patches of the
map in the same eye because of a tear film with large instability.
disturbing the tear film, corneal topography should be performed before

administering dilating drops and taking intraocular pressures.
In addition, one must avoid artefacts induced by the nose or the eyelids,
which can lead to a loss of information in certain areas (Figs. 1.10A and B).
These errors are transformed into black areas or areas without data on the
(A)
(B)
Figs. 1.10A and B: Loss of information of certain areas of the cornea due to eyelids not
opened enough. (A) Topographic map (B) Scheimpflug image.
topographic map. Correct positioning of the head, eyes and eyelid opening
should be ensured to avoid these problems.
INTERPRETATION OF CORNEAL TOPOGRAPHY MAPS

Accurate interpretation of corneal shape using color-coded topographic
maps is difficult and confusing for many clinicians, even experienced cornea
specialists. In order to obtain the best performance in the analysis of corneal
maps, several important points must be taken into consideration.
Fig. 1.11: Photokeratoscope raw image.
It is critical to check the raw image first. After that it is necessary to focus
on the scale and step intervals with which the color-coded topographic map
is built up. It is also important to review different topographic displays, espe-
cially when evaluating irregular or postsurgery corneas.
Raw Photoqueratoscope Image
The photokeratoscope image displays the Placido’s rings projected onto the
cornea (Fig. 1.11). When considering a color-coded map, the clinician must
check that the unprocessed data upon which it is based are reliable. If the
videokeratoscope image is irregular, data cannot be processed by the instru-
ment in a meaningful way.
Thus, for Placido disk-based computerized videokeratoscopes, the video-
keratoscope image should not be ignored. In fact, it is recommended to check
this map before referring to any of the other topographic displays, and to go
back to it when there are any doubts regarding the accuracy of the displayed
data. This image provides important information for assessing tear film qual-
ity, mire centring on the cornea, lid opening, or the causes of local irregular-
ities, and other artefacts. If the device used displays computer tracking of the
Placido mires it is important to rule out tracking errors.
Devices that rely only on scanning slit technology to analyze the ante-
rior corneal surface lack of the valuable information provided by the raw-
videokeratoscope image.12 Whether the resulting map is based on reliable
primary data or not is impossible to verify without the raw image. Some
instruments identify regions of uncertainty, showing mire distortions that
cannot be reliable, by leaving blank areas on the color-coded map. Other
instruments merely extrapolate onto the uncertain regions information gath-

ered from adjacent regions with reliable data.
For Scheimpflug technology, its images should also be checked before
looking at the resulting maps, and correct centration and focus should be
assured.
Color-coded Scales
The shape of a cornea can be measured and represented by color-coded

maps in which a given color indicates a different curvature or elevation. The
usual color spectrum for corneal powers shows near-normal power as green,
lower-than-normal power as cool colors (blues) and higher-than-normal
powers as warm colors (reds). Most topographers offer absolute as well as
normalized scales to allow the clinician to customize the information for
maximal clinical value (Figs. 1.12A and B):
• Normalized scale (variable scale) uses a given color for different cur-
vatures or elevations on each cornea analyzed, depending on the range
for that particular cornea, determined by its flattest and steepest values.
These maps are difficult to interpret and can lead to an incorrect diagno-
sis since they may magnify subtle changes in corneal surface if the scale
is too narrow, or minimize large distortions if the scale is too wide. In
addition, color recognition, one of the primary clues used to interpret
on corneal topography, is lost with a variable scale, since it uses different
colors for different eyes.
• Absolute scale (fixed scale) uses the same color for the same curvature
or elevation no matter which eye is examined. However, there are many
different absolute scales since the examiner can choose different vari-
ables such as range or step size (intervals in color changes). For the speci-
fied scale, however, each display will use the same colors, steps and range.
In order to facilitate comparisons over time and between patients, it is
recommended to stick with a given fixed scale for routine examinations
and to change the scale in the particular cases in which this becomes nec-
essary. As an example, the popular Klyce/Wilson scale ranges from 28 D
to 65 D in equal 1.5 D intervals. Currently, there is no consensus as to the
best absolute scale, but in general, dioptric scales with intervals smaller
than 0.5 D are not clinically useful and provide details that are not relevant
and may complicate map interpretation. For corneas with large dioptric
ranges, for instance in advanced keratoconus intervals greater than 0.5 D
are recommended. Regarding scales for elevation maps, elevation steps
of approximately 5 microns appear to be clinically useful.
As mentioned earlier, color pattern recognition makes it possible to iden-
tify common topographic patterns such as the corneal cylinder (Fig. 1.13),
keratoconus (local area of inferonasal steepening) or pellucid marginal
degeneration (butterfly pattern or inferior arcuate steepening), as well as fea-
tures associated with refractive surgery (Fig. 1.14), such as optical zone size,
centration and central islands.
(A)
(B)
Figs. 1.12A and B: Corneal topography map represented using (A) a normalized rela-
tive scale and (B) an absolute scale.
Fig. 1.13: Bow-tie pattern.
Fig. 1.14: Corneal topography after myopic laser in situ keratomileusis (LASIK).
Topographic Displays: Corneal Maps
Maps can be obtained from the anterior and posterior surface except in the
case of pure Placido disk technology.
• Axial map (sagittal map): Although this is the original and most com-
monly used map, its values only provide a good approximation for the
paracentral cornea (Fig. 1.15A). The axial map measures the radius of
(A)
(B)
(Contd.)
(Contd.)
(C)
(D)
Figs. 1.15A to D: Different kinds of topography maps for the same cornea: (A) Sagittal
axial map, (B) instantaneous or tangential map, (C) elevation map and (D) pachymetry
map.
curvature for a comparable sphere (with the same tangent as the point
in question) with a center of rotation on the axis of the videokerato-
scope. Localized changes in curvature and peripheral data are poorly
represented, because of the spherical bias of the reference optical axis.4
However, newer algorithms in some devices (e.g., arc-step method) have
improved the accuracy of curvature measurements in the peripheral
region.
• Local tangential curvature map (instantaneous map): The tangential map
displays the tangential/instantaneous/local radius of curvature or tan-
gential power, which is calculated by referring to the neighboring points
and not to the axis of the videokeratoscope (Fig. 1.15B). Tangential maps
reflect local changes and peripheral data better than axial maps. They are
very useful in detecting local irregularities, corneal ectatic diseases, or
surgically induced changes. For example, in keratoconus corneas with
a displaced apex, tangential maps are less influenced by peripheral dis-
tortion, and can determine the position and extent of the cone more pre-
cisely than axial maps.9
• Refractive map: The refractive map displays the refractive power of the
cornea, which is calculated based on Snell’s law of refraction, assuming
optical infinity. This map correlates corneal shape to vision, and is useful
in understanding the effects of surgery.13
• Elevation map: The elevation map displays corneal height or elevation
relative to a reference plane (Fig. 1.15C), with a presumed assumption
of the shape, which may be the best-fit sphere, best-fit asphere, average
corneal shape, or even based on preoperative data. Points above the ref-
erence surface are positive (hot colors), and points below the reference
surface are negative (cool colors). This map shows the 3D shape of the
cornea and is useful in measuring the amount of tissue to be removed by
a procedure, assessing postoperative visual problems, or planning and/
or monitoring surgical procedure.9
• Difference map: The difference map displays the changes in certain values
between two maps (Fig. 1.16). It is used to monitor any type of change,
such as recovery from contact lens-induced warpage or surgery-induced
changes.
• Relative map: The relative map displays some values by comparing them
to an arbitrary standard (e.g., sphere, asphere or normal cornea) and a
specific mathematical model. This map enhances or magnifies unique
features of the cornea being examined.
• Irregularity map (surface quality maps): The irregularity map uses the
same technique as the elevation map, but takes as a reference surface
the best-fit spherocylindrical toric surface. The difference between the
actual surface and the theoretical surface represents that part of the cor-
nea which cannot be optically corrected. Like refractive power maps, the
irregularity map only has clinical meaning when considering the values
over the pupillary area.
Fig. 1.16: Difference map for evaluating the evolution after implanting a corneal Myor-
ing with central aplannation.
• Corneal thickness maps (Fig. 1.15D): Numerous other displays, includ-

ing 3D maps, astigmatic vector analysis, etc. are available but less used.
QUANTITATIVE DESCRIPTORS OF CORNEAL TOPOGRAPHY:

CORNEAL INDEXES
Color-coded maps provide a rapid visual method for clinical diagnosis, but
do not supply numerical values that can be used for clinical management.
Several corneal indexes describe different features of corneal topography
quantitatively and are of great aid in contact lens fitting, for improved assess-
ment of the optical quality of the corneal surface, and can be used in artificial
intelligence systems to aid in the diagnosis of corneal shape anomalies. Some
of the most useful indexes have been described hereunder.
Basic Topographic Indexes
Simulated Keratometry Reading (SimK Values)
This is a simple descriptor of corneal topography that provides the power

and axes of the steepest and flattest corneal curvatures just as K1 and K2 are
provided by the classic keratometer, to which it correlates well.3 The cylinder
is calculated from the difference between SimK1 and SimK2. Its common
uses are:
• Fitting contact lenses
• Refractive surgery calculations
• Supplying a starting point when assessing an irregular corneal shape,
since it gives the quantity and axis of astigmatism
Minimum Keratometry Reading
Minimum keratometry reading (MinK) is the minimum meridional power

from rings 7, 8 and 9. The average power as well as axis are displayed.
Corneal Eccentricity Index
Corneal eccentricity index (CEI) estimates the eccentricity of the central

cornea, and is calculated by fitting an ellipse to the corneal elevation data.12
A positive value is for a prolate surface, negative value for an oblate surface
(e.g., flattened corneas after myopic refractive surgery), and zero value for a
perfect sphere. Normal central corneas are prolate, meaning they are steeper
in the center than in the periphery, and tend to be around 0.30. This value is
used for:
• Fitting contact lenses
• Approaching to the global shape factor
Average Corneal Power
This is the area-corrected average of corneal power in front of the pupil. It

usually corresponds to the spherical equivalent of the classic keratometer,
except after decentered refractive surgery. It may be helpful in determining
central corneal curvature when calculating the appropriate intraocular lens.
Surface Regularity Index, and Potential Visual Acuity
Surface regularity index (SRI) measures the regularity of the corneal surface
that correlates with the best spectacle-corrected visual acuity assuming the
cornea to be the only limiting factor.14 This index adds up the meridional
mire-to-mire power changes over the apparent pupil entrance. The SRI value
increases with increases in the irregularity of the corneal surface, and its nor-
mal value is less than 1.0. It measures optical quality.
Potential visual acuity (PVA) is a range of the expected visual acuity that
is achievable based on the corneal topography and can be calculated based
on SRI.
Surface Asymmetry Index
Surface asymmetry index (SAI) is a descriptor of the corneal surface that

measures the difference between points located 180° apart in a great number
of equally spaced meridians.15 Therefore, as the cornea becomes less sym-
metric, the index differs more from 0.
Other indexes, some of which will be mentioned below, have been devel-
oped, and might be exclusive to one particular topographer. The clinician
should evaluate the meaning, utility and validity of each index since some
indexes have been tested in peer-reviewed literature while others have not.
Screening Tools and Artificial Intelligence Programs

(Neural Networks) for Classification and Auto Diagnosis
As mentioned earlier, even for experienced personnel, interpretation of
topography can be difficult, particularly when trying to differentiate the early
stages of a disease from a normal cornea (suspected keratoconus), or when
trying to differentiate between two similar conditions (contact lens warpage
versus early keratoconus). Several mathematical algorithms have been devel-
oped to help solve this problem, with high sensitivity and specificity.
Rabinowitz and McDonnell developed the first numerical method for
detecting keratoconus using only topographic data.16 They use the I-S value,
which measures the differences between the superior and inferior paracen-
tral corneal regions, the central corneal power (MaxK), and the power differ-
ence between both eyes. Their study determined the following results:
• Keratoconus suspect: central corneal power > 47.2 D or I-S > 1.4
• Clinical keratoconus: central corneal power > 47.8 D or I-S > 1.9
However, using only these simple measurements for a diagnosis could create
specificity problems.
To solve the specificity problem, the new strategy must be able to detect
and consider the unique characteristics of keratoconus maps, such as local
abnormal elevations. The keratoconus prediction index, developed by
Maeda et al.,17 is calculated from the differential sector index (DSI), the
opposite sector index (OSI), the center/surround index (CSI), the SAI, the
irregular astigmatism index (IAI) and the percent analyzed area (AA). This
method partially overcomes the specificity limitation.
Maeda et al. also developed the neural network model, based on artifi-
cial intelligence.17 It is a much more sophisticated method for classifying cor-
neal topography and detecting different corneal topographic abnormalities;
it employs indexes that were empirically found to capture specific charac-
teristics of the different corneal pathologies, including keratoconus. Further
modifications in neural network approach developed by Smolek and Klyce
supposedly produce 100% accuracy, specificity and sensitivity in diagnosing
keratoconus.
The Pentacam system for instance has developed seven indices of cor-
neal irregularity within the central cornea for the grading and classification
of keratoconus (TKC), as well as the postoperative assessment (Fig. 1.17).
These indices include index of surface variance (ISV), index of vertical asym-
metry (IVA), keratoconus index (KI), central keratoconus index (CKI), index
of height asymmetry (IHA), index of height decentration (IHD) and index of
minimum radius of curvature (Rmin). This machine also provides with two
diagrams that describe the change of corneal thickness in relation to location,
and a progression index of this thickness/location relationship to suggest
the presence or not of an ectatic disease. In addition to this, the Pentacam
tomography includes a new software adaptation called the Belin/Ambrosio
Fig. 1.17: Indices of cornea irregularity—Pentacam.
enhance ectasia display (BAD) that combines both the anterior and poste-
rior elevation data and pachymetric data to orient in the diagnosis of corneal
ectasia.
The Sirius system displays a keratoconus summary to aid in the diagno-
sis and the follow-up of keratoconus combining indices based on curvature,
pachymetry and elevation such as the symmetry index of the front and back
surface, or the Baiocchi Calossi Versaci front and back index (BCV f and BCV b)
to evaluate coma and trefoil aberrations.
The Casia OCT system has a built-in software that estimates ectasia simi-
larity of a scan, and this is calculated as the ectasia similarity score (Fig. 1.18).
This score is presented in percentage of similarity.
CORNEAL ABERROMETRY: FUNDAMENTALS

AND CLINICAL APPLICATIONS
Whenever a point object does not form a point image on the retina, as it
should be in an ideal optical system, one encounters an optical aberration.18
Although one may feel that he is measuring the total refractive error, when
refracting a patient, one is actually only considering two components of a
whole host of refractive components in the optics of the eye. However, these
two components—sphere and cylinder—do constitute the main optical aber-
rations of an eye. Even in a normal eye with no subjective need for refraction,
optical aberrations can be detected.
Since the cornea has the highest refractive power, more than 70% of the
eye’s refraction, it is the principal site of aberrations, although the lens and
the tear film may also contribute to aberrations.19
Fig. 1.18: Ectasia similarity score by Casia S-100.
MEASURING WAVEFRONT ABERRATION

Measuring Total Wavefront Aberration
It is possible to express ideal image formation by means of waves. An ideal

optical system will provide a spherical converging wave centered at the ideal
point image. However, in practice, the resulting wavefront differs from this
ideal wavefront. The deviation from this ideal wavefront is called wavefront
aberration, and the more it differs from zero, the more the real image differs
from the ideal image and the worse the image quality. Ocular wavefront sens-
ing devices use five main technologies to determine the resulting or output
wave:20
1. The Shack-Hartmann method is the most widely used and is inspired by
astronomy technology. It consists of analyzing the wave emerging from
the eye after directing a small low energy laser beam. This reflected wave
is divided by means of a series of small lenses (lenslet array), which gen-
erates focused spots. The position of spots is recorded and compared to
the ideal one. This type of aberrometer provides reproducible measure-
ments in normal eyes but is limited in eyes with significant amounts of
aberrations due to the overlapping of the spots.18
2. The Tscherning technique uses typically a grid that is projected onto the
retina. The distortion of the pattern is analyzed and used to calculate the
wavefront aberration of the eye.21
3. The Ray Tracing system is similar to the Tscherning technique. How-
ever, instead of a grid, a programmable laser serially projects light beams
that form spots on the retina at different locations.21
4. The spatially resolved refractometer evaluates the wavefront profile
using the subjective patient response. This technology is not practical for
clinical use.
5. Piramidal aberrometry is a new wavefront sensor based on the Fou-
cault knife edge.22 The Osiris pyramidal aberrometry system bases his
working principle on a high-resolution four-faced pyramid wavefront
sensor on the focal plane that provides the wavefront gradients in two
orthogonal directions and four pupil images (known as sub-pupil) dis-
tributed by their intensity: each sub-pupil performs a Foucault knife-
edge test to derive slope and shape of the wavefront. Since wavefront is
sampled in the very last stage of the optical path, the resolution of the
device is extremely high compared to commercial sensors. Finally, the
device seems to make possible the analysis of very irregular corneas
probably due to his fuzzy dynamic range and to the absence of problems
related to sample overlapping, so it might allow higher sensitivity than
Hartmann-Shack wavefront sensor.
Measuring Corneal Wavefront Aberration
It is known that 80% of all aberrations of the human eyes occur in the cor-
neal area and only 20% of aberrations originate from the rest of the ocular
Fig. 1.19: Corneal wavefront analysis derived from height topography data.
structures.23 The effect of corneal aberrations is especially important after

corneal surgery such as keratorefractive procedures since the anterior cor-
neal surface is the only one modified.24 The corneal wavefront aberration,
which is the component of the total ocular wavefront aberration attributed
to the cornea, can be derived from the corneal topographic height data. Spe-
cifically, the calculation of wavefront aberrations is performed by expanding
the anterior corneal height data into a set of orthogonal Zernike polynomials
(Fig. 1.19).
Zernike Polynomials
For a quantitative description of the wavefront shape, there is a need for a

more sophisticated analysis than conventional refraction, as the latter only
divides the wavefront in two basic terms: sphere and cylinder. One can obtain
more information by breaking down the wavefront into terms, which are clin-
ically meaningful, besides the sphere and the cylinder. For this purpose, a
standard equation has been universally accepted by refractive surgeons and
vision scientists, known as Zernike polynomials.25
Zernike polynomials are equations which are used to fit the wavefront
data in a 3D way. The wavefront function is decomposed into terms that
describe specific optical aberrations such as spherical aberration, comma,
etc. (Fig. 1.20). Each term in the polynomial has two variables, r (rho) and
q (theta), where r is the normalized distance of a specific point from the cen-
ter of the pupil, and q is the angle formed between the imaginary line joining
the pupillary center with the point of interest and the horizontal. According
to that, we can imagine that aberrations are strongly influenced by pupil size,
Fig. 1.20: Zernike polynomial expansion.
and, therefore, aberrometric measurements should be related to the diameter

of the patient’s pupil.
Zernike terms (Znm ) are defined using a double index notation: (1) a radial
order (n) and (2) an angular frequency (m). When talking about first, second,
third, etc., aberrations we point to indicate the radial order (n). Each radial
order involves n + l terms. There are an infinite number of Zernike terms
that can be used to fit an individual wavefront. However, for clinical practice,
terms up to the fourth radial order are usually considered:25
1. Zernike terms below third order can be measured and corrected by con-
ventional optical means the first order term, the prism, is not relevant
to the wavefront as it represents tilt and is corrected using a prism. The
second order terms represent low order aberrations that include defocus
(spherical component of the wavefront) and astigmatism (cylinder com-
ponent). Wavefront maps that measure only defocus and astigmatism
can be perfectly corrected using spectacles and contact lenses.
2. After the second radial order come the high order aberrations. These are
not measured by conventional refraction or autorefraction. The aber-
rometer is the only method available that can quantify these complex
kinds of distortions.
3. Third order terms describe comma and trefoil defects.
4. Fourth order terms represent tetrafoil, spherical aberration and second-
ary astigmatism components.
Because spherical and comma aberrations refer to symmetrical systems
and the eye is not rotationally symmetrical, the terms spherical-like and
comma-like aberrations are normally used (Fig. 1.21).
Fig. 1.21: Complete corneal wavefront aberration map.
Wavefront Maps
Wavefront map describes the optical path difference between the measured
wavefront and the reference wavefront in microns at the pupil entrance.18
The wavefront error is derived mathematically from the reconstructed wave-
front by one of the techniques described earlier. It is plotted as a 2D or 3D
map for qualitative analysis in a similar fashion to corneal topography maps.
In wavefront error maps, each color represents a specific degree of wavefront
error in microns (Fig. 1.21) and like in corneal topography maps, it is neces-
sary to consider the range and the interval of the scale.
Optical and Image Quality
In order to evaluate the impact of aberrations on visual quality following

quantitative parameters have been defined (Fig. 1.22):
• Peak to valley error (PV error): This is a simple measure of the distance
from the lowest point to the highest point on the wavefront and is not the
best measurement of optical quality since it does not represent the extent
of the defect.25
• Root mean square error (RMS error): This measure is by far the most
widely used. In a simple way, the RMS wavefront error is a statistical
measure of the deviation of the ocular or corneal wavefront from the
ideal25 (Table 1.1). In other words, it describes the overall aberration and
indicates how bad individual aberrations are.
• Strehl ratio: This represents the ratio of the maximum intensity of the
actual image to the maximum intensity of the fully diffracted limited
image, both being normalized to the same integrated flux.25 This ratio
measures optical excellence in terms of theoretical performance results
and it is linked to the RMS by the Maréchal formula.
• Point spread function (PSF): This is the spread function observed on the
retina when the object is a point in infinity.25 PSF measures how well one
object point is imaged on the output plane (retina) through the optical
system. In the eye, small pupils (~1 mm) produce diffraction-limited
PSFs because of the pupil border. In larger pupils, aberrations tend to be
the dominant source of degradation.
• Modulation transfer function, phase transfer function and optical transfer
function: Sinusoidal gratings greatly simplify the study of optical systems,
because irrespective of the amount of eye aberrations, sinusoidal grating
objects always produce sinusoidal grating images.26 Consequently, there
are only two ways that an optical system can affect the image of a grating,
by reducing contrast or by shifting the image at a specific resolution; are
called respectively the modulation transfer function (MTF) and the phase
transfer function (PTF). The eye’s optical transfer function (OTF) is made
up of the MTF and the PTF. A high-quality OTF is, therefore, represented
by high MTF and low PTF.
Fig. 1.22: Visual quality summary obtained with the Sirius CSO topographer. It is possible to visualize the wavefront map (gray scale),
Strehl ratio, point spread function (PSF) and modulation transfer function (MTF) function.
Table 1.1: Reference values for corneal aberrations in the normal population. (RMS: root
mean square; Coma primary coma: terms Z3; Spherical aberration and primary spheri-
cal aberration: term Z4; Spherical-like: terms fourth and sixth order; Coma-like: terms
third and fifth order). Source: Vinciguerra P, Camesasca Fl, Cafossi A. Statistical analysis
of physiological aberrations of the cornea. J Refract Surg. 2003;19(Suppl):S265-9.
Pupil Astigmatism Spherical Coma Spherical- Coma-like

(mm) Total RMS RMS aberration RMS like RMS RMS
3 0.19 ± 0.07 0.14 ± 0.08 0.04 ± 0.03 0.05 ± 0.03 0.07 ± 0.02 0.09 ± 0.03
5 0.53 ± 0.21 0.43 ± 0.24 0.15 ± 0.05 0.14 ± 0.08 0.18 ± 0.05 0.20 ± 0.08
7 1.26 ± 0.43 0.92 ± 0.53 0.52 ± 0.17 0.42 ± 0.23 0.57 ± 0.16 0.52 ± 0.22
Clinical Applications
Aberrometers allow practitioners to gain a better understanding of vision by

measurement of high-order aberrations. These aberrations reflect a refractive
error that is beyond conventional spheres and cylinders. There may be a large
group of patients whose best-corrected visual acuity (BCVA) may improve sig-
nificantly by removing the optical aberrations and this new refractive entity
has been called aberropia. Reduced optical quality of the eye produced by
light scatter and optical aberrations may actually be the root cause of blurred
vision associated with dry eye syndrome and tear film disruption. Measure-
ment of these aberrations are also helpful in keratoconus, post-graft fitting,
irregular astigmatism or when refractive surgery has reduced the patient’s
optical quality.20
Customized ablation patterns, currently in constant evolution, are the
future step in laser technology that should address not only spherical and
cylindrical refractive errors (low-order aberrations), but also high-order
aberrations such as trefoil and comma (Figs. 1.23A to C). Thus, vision can
be optimized to the limits determined by pupil size (diffraction) and retinal
structure and function.
PATHOLOGICAL CORNEA
Corneal topography is a very important tool in the detection of corneal
pathologies, especially ectatic disorders. Screening for these anomalies or
their potential development is a critical point in preoperative evaluation for
refractive surgery. Keratorefractive procedures are contraindicated in these
abnormal corneas.
Keratoconus
Keratoconus is characterized by a localized conical protrusion of the cornea

associated with an area of corneal stromal thinning, especially at the apex
of the cone. The typical associated topographic pattern is the presence of an
inferior area of steepening (Fig. 1.24A to D). In advanced cases, the dioptric
(A)
(B)
(C)
Figs. 1.23A to C: Customized ablation profile designed according to corneal aberra-
tions: (A) Case of early keratoconus with an unaided and corrected distance visual acu-
ity of 0.5, (B) customized transepithelial PRK ablation profile in order to treat only the
coma with the minimal possible ablation depth, (C) topographic outcome 6 months
after simultaneous transPRK and corneal collagen crosslinking with an unaided vision
of 0.8 and corrected distance vision of 1 due to the regularization of the astigmatism.
power at the apex is at or above 55 D.27 In a small group of patients, the topo-
graphic alterations may be centered at the central cornea. In these cases,
there may be an asymmetric bow-tie configuration, and usually the inferior
loop is larger than the superior loop (Figs. 1.23A to C). Keratoconic corneas
have three common characteristics that are not present in normal corneas:
1. An area of increased corneal power surrounded by concentric areas of
decreasing power.
2. An inferior-superior power asymmetry.
3. A skewing of the steepest radial axes above and below the horizontal
meridian.
Keratoconus suspects are problematic. They may signal impending develop-
ment of a clinical keratoconus, but they may also represent a healthy cornea.
(A)
(B)
(Contd.)
(Contd.)
(C)
(D)
Figs. 1.24A to D: Keratoconus topography pattern. It can be observed the inferior
steepening with posterior elevation and corneal thinning.
The lack of ectasia in the fellow cornea does not indicate that the keratoconus
suspect will not progress to true keratoconus. In these cases, the ideal man-
agement is close follow-up of the signs of keratoconus in order to check on
their stability, and a thorough analysis of the videokeratographic indexes.
Pellucid Marginal Degeneration
Pellucid marginal degeneration is characterized by an inferior corneal thin-

ning between 4 and 8 o’clock positions, a narrow band of clear thinned cor-
neal stroma.28 The ectasia is extremely peripheral and it appears just over the
thinned area, presenting a crescent-shaped morphology. This pattern has a

classical ‘butterfly’ appearance that results in a flattening of the vertical merid-
ian and a marked against-the-rule irregular astigmatism (Fig. 1.25A to D).
Keratoglobus
Keratoglobus is a rare bilateral disorder in which the entire cornea is thinned

out, most markedly near the corneal limbus, in contrast to the localized
central or paracentral thinning of keratoconus. It is very difficult to obtain
reliable and reproducible measurements in these cases due to the high
level of irregularity and the poor quality of the associated tear film. Reliable
(A)
(B)
(Contd.)
(Contd.)
(C)
(D)
Figs. 1.25A to D: Pellucid marginal degeneration topography pattern. It can be observed
the crescent-shaped inferior ectasia with posterior elevation and inferior thinning.
topographic examinations show an arc of peripheral increase in corneal

power (steepening) and a very asymmetrical bow-tie configuration.28
Terrien’s Marginal Degeneration
In Terrien’s marginal degeneration, there is a flattening over the areas of

peripheral thinning. When thinning is restricted to the superior and/or
inferior areas of the peripheral cornea, there is a relative steepening of the
corneal surface approximately 90° away from the midpoint of the thinned
area.29 Therefore, high against-the-rule or oblique astigmatism is a common
feature, as this disorder involves more frequently the superior and/or inferior
peripheral cornea. If the area of thinning is small or if the disorder extends
around the entire circumference of the cornea, central cornea may remain
relatively spared with a spherical configuration.
Pterygium
Pterygium is a triangular encroachment of the conjunctiva onto the cornea

usually near the medial canthus. When the lesion continues to grow out onto
the cornea, it could lead to a high degree of astigmatism. When the growth
of pterygium is about 2 mm or more, a flattening of the cornea at the axis of
the lesion occurs. This produces a marked with-the-rule astigmatism, even
of more than 4 D. The evolution of the pathology and the surgical outcome
could be monitored by changes in corneal topography.
Postoperative Cornea in Refractive Surgery
Keratorefractive procedures attempt to alter the curvature of the central and

midperipheral cornea, and usually have a minimal effect on the corneal
periphery. The area in which the curvature is modified is called the optical
zone. This tends to be surrounded by a small zone of altered curvature before
normal cornea is reached at the periphery. The corneal effect of surgery could
be determined by analyzing the difference map between the preoperative
and postoperative measurements.30
Postradial Keratotomy
Radial keratotomy (RK) corrects myopia by placing a series of radial incisions

(nearly full corneal thickness) leaving a central clear zone (optical zone).
These incisions cause a flattening of the central cornea due to retraction of
the most anterior collagen fibers and the outward pressure of the intraocular
force. This area of flattening is surrounded at an approximately 7 mm zone by
a bulging ring of steepening called the paracentral knee or inflection zone.
This increases asphericity and corneal irregularity.
A very typical finding in these corneas is a topographic pattern with a
polygonal shape.18 Depending on the number of incisions made, squares,
hexagons or octagons can be seen. The angles of the polygons correspond
closely to the central ends of the incisions (Fig. 1.26A to D).
Postastigmatic Keratotomy
Astigmatic keratotomy (AK) is a simple modification of the RK that is used

to correct astigmatism. Rather than placing incisions radially on the cornea,
incisions are strategically placed circumferentially on the peripheral cornea at
the steepest meridian. The incisions induce a flattening in that meridian, but
provoke steepening in the perpendicular meridian, in a process called cou-
pling18 (Figs. 1.27A and B). Coupling results from the presence of intact rings
of collagen lamellae that run circumferentially around the base of the cornea.
With the surgery, these rings become oval in the operated meridian and trans-
mit forces to the untouched meridian. The astigmatic change achieved is the
sum of the flattening in one meridian and the steepening on its perpendicular
meridian.
(A)
(B)
(Contd.)
(Contd.)
(C)
(D)
Figs. 1.26A to D: Postradial keratotomy cornea. Observe the anterior and posterior cir-
cumferential elevation but without any alteration in the corneal pachymetry.
Postphotorefractive Keratectomy
Photorefractive keratectomy (PRK) is a procedure which uses a kind of laser

(excimer laser, a cool pulsing beam of ultraviolet light) to reshape the cornea.
To correct myopia, the excimer laser flattens the central cornea by removing
tissue in that area. However, the optical zone needs to be steepened to correct
(A)
(B)
Figs. 1.27A and B: (A) Before and (B) after astigmatic keratotomy. Observe the consider-
able flattening induced by the keratotomy, in this case excessive, generating a second-
ary significant astigmatism in the previously flat axis.
hyperopia. This is achieved by removing an annulus of tissue from the midpe-

riphery of the cornea.
The topographic pattern in myopic corrections shows a flattening of the
central cornea, an oblate profile (Fig. 1.28A to D). Hyperopic corrections have
a pattern of central steepening surrounded by a ring of relative flattening at
the edge of the treatment zone, a prolate profile (Fig. 1.29A to D). In astig-
matic corrections, the treatment zone is oval.18
Inadequate ablations during surgery can be detected postoperatively by

analyzing the resulting corneal topography. Decentrations can only be iden-
tified by a relatively asymmetric location of the treatment area (Fig. 1.30).
Other complicated patterns that may lead to severe visual disturbances are
the presence of focal irregularities or central islands produced by an inhomo-
geneous laser beam or an irregular process of corneal healing.
Postlaser in situ Keratomileusis
Laser in situ keratomileusis (LASIK) is an excimer laser procedure like PRK,

but in this case, tissue is ablated of under a superficial corneal flap in order to
(A)
(B)
(Contd.)
minimize the influence of the epithelium. The topographic patterns for myo-
pic and hyperopic corrections are the same as in PRK (Figs. 1.28 and 1.29).
Although the ablation is covered by a flap of corneal tissue, surface irregu-
larities and central islands may still occur. Decentrations may also occur in
a LASIK ablation, depending on the patient’s ability to maintain eye fixa-
tion during surgery (Fig. 1.30). Epithelial in-growth at the periphery of the
flap-stromal interface produces an area of steepening surrounded by an area
of marked flattening making the corneal surface more irregular.
(Contd.)
(C)
(D)
Figs. 1.28A to D: Topographic pattern after myopic ablation.

Postlaser Thermal Keratoplasty
In laser thermal keratoplasty (LTK), a Holmium laser, is used to heat corneal

stromal collagen in a ring around the outside of the pupil. The heat causes
the tissue to shrink, producing a zone of localized flattening centered on the
spot, and a surrounding zone of steepening. This bulging effect of the central
cornea makes it possible to correct hyperopia. The typical topographic pat-
tern shows the central corneal steepening and a ring of flattening overlying
the spots (Fig. 1.31).
(A)
(B)
(Contd.)
(Contd.)
(C)
(D)
Figs. 1.29A to D: Topographic pattern after a high-hyperopic ablation. In contrast to a
real corneal ectasia, after a hyperopic treatment the posterior corneal surface and the
central corneal thickness are normal.
Postintrastromal Corneal Rings Implantation
Intrastromal rings are small segments or rings, made of a plastic-like sub-

stance, that are inserted into the periphery of the cornea to correct small
degrees of myopia or hyperopia. They act as spacers and by changing the
orientation of the collagen lamellae, depending on their shape and position,
Fig. 1.30: Pattern of a decentered myopic ablation.
Fig. 1.31: Topographic pattern after laser thermal keratoplasty (LTK) for hyperopia.
flatten or steepen the central cornea. Nowadays, intrastromal rings are mainly
used to reduce the corneal steepening and irregular astigmatism associated
with keratoconus (Figs. 1.32A and B).
Postoperative Cornea in Keratoplasty Surgery
Keratoplasty topographies exhibit a wide variety of patterns, depending on

the type of keratoplasty performed, the quality of the surgical procedure,
(A)
(B)
Figs. 1.32A and B: (A) Before and (B) after intracorneal ring segment implantation for
keratoconus.
whether sutures are still in place in the cornea, and the time elapsed after the
procedure. Sutures usually induce a central bulge in the corneal graft and its
removal results in a decrease of the astigmatic component (Figs. 1.33A and
B). The prolate configuration after keratoplasty is the most frequent pattern
with a high degree of irregularity. There can be multiple regions of abnor-
mally high or low power, or both simultaneously in the map. Irregular astig-
matism over the entrance pupil may be detrimental to optimum visual acuity
in the keratoplasty patient.31
(A)
(B)
Figs. 1.33A and B: (A) Before and (B) after graft suture removal on a previous penetrat-
ing keratoplasty. Observe the significant reduction of the topographic cylinder.
Contact Lens-induced Corneal Warpage or Molding
Corneal warpage is characterized by topographic changes in the cornea fol-

lowing contact lens wear (most frequently in wearers of hard or RGP lenses)
as a result of the mechanical pressure exerted by the lens. There are at least
four different forms of noticeable topography changes that usually occur
mixed with one another: (1) peripheral steepening, (2) central flattening,
(3) furrow depression and (4) central molding or central irregularity.18
Inferior corneal steepening (pseudokeratoconus) is caused by a superi-

orly riding contact lens that flattens above the visual axis with an apparent
steepening below. The topographic image could appear similar to keratoco-
nus, but both conditions are easily differentiated (Figs. 1.34A and B). In cor-
neal warpage, the shape indexes do not indicate any keratoconic condition,
and the steep K is not as steep as it is in keratoconus.
Other Uses of Corneal Topography
Corneal topography is a diagnostic tool, but it is also essential before all

refractive procedures, to enable the surgeon to understand the refractive
status of an individual eye, and plan the optimum refractive treatment. The
corneal topography is also used for the following purposes:
(A)
(Contd.)
(Contd.)
(B)
Figs. 1.34A and B: Corneal warpage: (A) soft contact lens removed 1 day before the
measurement; (B) same patient 1 week later without using contact lenses. Observe how
it disappears the inferior asymmetry on the topographic astigmatism.
• To guide removal of tight sutures after corneal surgery (keratoplasty, cat-

aract surgery, etc.) that are causing steepening of the cornea (Figs. 1.33A
and B).
• To help in the AK surgical plan.
• To guide contact lens fitting: election of the probe lens and design of the
lens.
• To calculate the keratometry values for the calculation of the required
intraocular lens power before cataract surgery or refractive lens exchange.
This is an important issue in corneas that have undergone previous
refractive surgery, because it is more difficult to estimate the real kerato-
metric values in order to avoid hyper- or hypocorrections.
• To evaluate the effect and evolution of a keratorefractive procedure.
REFERENCES
1. Kaufman H, Barron B, McDonald M, Kaufman S. Companion handbook to the
cornea. London, Butterworth Heinemann, 1999.
2. Dabezies OH, Holladay JT. Measurement of corneal curvature: keratometer
(ophthalmorneter). In Contact lenses: the CLAO guide to basic science and
clinical practice. Kendall/Hunt Publishing Co. 1995. pp. 253-89.
3. Wilson SE, Klyce SD. Advances in the analysis of corneal topography. Surv Oph-
thalmol. 1991;35:269-77.
4. Corbett M, O’Brart D, Rosen E, Stevenson R. Cornea l topography: principles
and applications. BMJ Publishing Group, 1999.
5. Bogan SJ, Waring GO, Ibrahim O, Drews C, Curtis L. Classification of normal cor-
neal topography based on computerassisted videokeratography. Arch Oph-
thalmol. 1990;108:945-9.
6. Corneal Topography. American Academy of Ophthalmology. Ophthalmol.
1999;106:1628-38.
7. Mejia-Barbosa Y, Malacara-Hernandez, D. A review of methods for measuring
corneal topography. Optom Vis Sd. 2001;78:240-53.
8. Cairns G, McGhee CNJ. Orbscan computerized topography: Attributes,
applications, and limitations. J Cataract Refract Surg. 2005;31:205-20.
9. Cavas-Martinez F, De la Cruz Sanchez E, Nieto Martinez J, Fernandez-
Cañavate FJ, Fernandez-Pacheco D.G. Corneal Topography in Keratoconus:
state of the art.
10. Steinberg J, Casagrande MK, Frings A, Katz T, Druchkiv V, Richard G,
Linke SJ. Screening for Subclinical Keratoconus Using Swept-Source Fouri-
er Domain Anterior Segment Optical Coherence Tomography. Cornea. 2015
Nov;34(11):1413-9.
11. Miller D, Greiner JV: Corneal measurements and tests. In Principles and practice
of ophthalmology. Philadelphia, WB Saunders, 1994.
12. Rao SK, Padmanabhan P. Understanding corneal topography. Curr Opin Oph-
thalmol. 2000;11:248-59.
13. Ambrosio R Jr, Klyce SD, Wilson SE. Corneal topographic and pachymetric
screening of keratorefractive patients. J Refract Surg. 2003;19:24-9.
14. Courville CB, Smolek MK, Klyce SD. Contribution of ocular surface to visual op-
tics. Exp Eye Res. 2004;78:417-25.
15. Klyce SD. Corneal topography and the new wave. Cornea. 2000;19:723-29.
16. Rabinowitz YS, Nesbum AB, McDonnell Pl Videokeratography of the fellow eye
in unilateral keratoconus. Ophthalmol. 1993;100:181-6.
17. Maeda N, Klyce SD, Smolek MK. Neural network classification of corneal
topography. Preliminary demonstration. Invest Ophthalmol Vis Sci. 1995;
36:1327-35.
18. Boyd BF, Agarwal A, Alio JL, Krueger RR, Wilson SE (eds). Wavefront analysis,
aberrometers and corneal topography. Highlights ofophthalmology, 2003.
19. Wilson SE, Ambrosio R. Computerized corneal topography and its importance
to wavefront technology. Cornea. 2001;20:441-54.
20. Rozema JJ, Van Dyck DE, Tassignon MJ. Clinical comparison of 6 aberrom-
eters. Part 1: Technical specifications. J Cataract Refract Surg. 2005;31:
1114-1127.
21. Molebny VV, Panagopoulou SI, Molebny SV, Wakil YS, Pallikaris IG. Principles of
ray tracing aberrometry. J Refract Surg. 2000;16:S572-575.
22. Chamot SR, Dainty C, Esposito S. Adaptive optics for ophthalmic applications
using a pyramid wavefront sensor. Opt Express. 2006;14:518-526.
23. Vincigerra P, Camesasca FI, Calossi A. Statistical Analysis of phisiological aberra-
tions of the cornea. J Refract Surg. 2003;19(suppl):265-9.
24. Joslin CE, Wu SM, McMahon TT, Shahidi M. Higher-order wavefront aberrations
in corneal refractive therapy. Optom Vis Sci. 2003;80:805-11.
25. Thibos LN, Applegate RA, Schwiergerling JT, Webb R. Standards for reporting
the optical aberrations of eyes. J Refract Surg. 2002;18:S652-60.
26. Hamam H. A new measure for optical performance. Optom Vis Sci. 2003;80:
174-84.
27. Rabinowitz YS. Keratoconus. Surv Ophthahnol. 1998;42:297-319.
28. Karabatsas CH, Cook SD. Topographic analysis in pellucid marginal corneal de-
generation and keratoglobus. Eye. 1996;10:451-55.
29. Wilson SE, Lin DT, Klyce SD, Insler MS. Terrien’s marginal degeneration: corneal
topography. Refract Corneal Surg. 1990;6:15-20.
30. Vang L, Koch DD. Corneal Topography and its integration into refractive sur-
gery. Comp Ophthalmol Update. 2005;6:73-81.
31. Krachmer JH, Mannis MJ, Holland EJ, (ed). Cornea. Surgery of cornea and con-
junctiva. St Louis, Elsevier-Mosby, 2005.
2
CHAPTER
Corneal Confocal Microscopy
Manotosh Ray, George N Thomas
Confocal microscopy is an advanced imaging technology that offers several

advantages over conventional wide-field optical microscopy. The operator has
the ability to control the depth of field, eliminate or reduce the background
noise from the focal plane and the capability to obtain precise serial optical
sections from thick specimens. The fundamental of confocal microscopy is its
use of spatial filtering techniques to eliminate out-of-focus light or glare. The
application of this technology permits the acquisition of images of high spatial
resolution and contrast as compared to conventional microscopy. There has
been tremendous interest in confocal microscopy in recent years, due in part
to the relative ease of which extremely high quality images can be obtained
from tissue samples, including the ability to image the cornea in vivo.
The major limiting factor of conventional light microscopy is that when a
particular point of interest is viewed, the reflected light from the surrounding
structures obscures the image produced. The fringing effect produced by this
reflection reduces the image contrast. Therefore, the useful magnification in
slit-lamp biomicroscopes and other similar ophthalmic instruments is lim-
ited to approximately 40 times. Further magnification compromises the image
quality and produces significant image blur. The confocal microscope, on the
other hand, utilizes a principle in which both the illumination and observation
system are focused on a single point. Thus, the spatial resolution is improved
dramatically and the system allows a usable magnification of up to 600 times.
Confocal microscopy employs an oscillating slit aperture in an ophthal-
mic microscope configuration, especially suitable for the analysis of cell lay-
ers of cornea. It can focus through the entire range of a normal cornea from
epithelium to endothelium. A series of scan shows: (a) epithelium, (b) corneal
nerves, (c) keratocytes, (d) endothelium, and (e) a computer-generated slice
of cornea. There are distinct advantages of the confocal microscope over a
Chapter_02.indd 53 06-02-2018 20:14:07

regular light microscope. When a transparent tissue like the cornea is imaged
with a regular microscope, the unfocused layers affect the visibility of the
focused layer. A confocal microscope, on the other hand, can focus on a spe-
cific layer distinctly without being affected by artifact from other layers.
OPTICS
A halogen light source passes through movable slits (Nipkow disk), which is
then passed through a condenser lens (front lens) that projects the light to the
cornea. Only a small area inside the cornea is illuminated to minimize light
scatter. The reflected light passes through the front lens again and is directed
to another slit of same size via a beamsplitter. Finally, the image is projected
onto a highly sensitive camera and displayed on a computer monitor (Fig. 2.1).
The confocal microscope utilizes a transparent viscous sterile gel that is
interposed between the front lens and cornea, to improve the optical inter-
face between the two media. The front lens works on the ‘Distance Immersion
Principle.’ In this principle, the anteroposterior movement of the front lens
enables scanning of the entire cornea starting from anterior chamber and
corneal endothelium to most superficial corneal epithelium. The standard
Fig. 2.1: Optics of confocal microscope.
Chapter_02.indd 54 06-02-2018 20:14:07

Corneal Confocal Microscopy 55
working distance (distance between front lens and the cornea) is 1.92 mm.
Use of standard × 40 immersion lens gives magnified cellular detail and an
image field of 440 × 330 μm. Other lenses (e.g., × 20) can deliver a wide field
image but with less distinct cell morphology. Newer confocal microscopes
(such as the Confoscan 2.0) capture up to 350 images per examination at a
rate of 25 frames per second. The thickness of the imaged layers can be varied
from 3–5 microns depending on the scanning slit characteristics.
Every recorded image is characterized by its position on the Z-axis of
the cornea. Every time a confocal scan is performed, a displayed diagram
shows the depth coordinate on the Z-axis and the level of reflectivity on the
Y-axis. The diagram also displays the distance between two images along
the antreoposterior line. This simultaneous graphic recording is called the
Z-scan graphic. The reflectivity on the Z-scan is entirely dependent on the
tissue being scanned. A transparent tissue displays low reflectivity, whereas a
higher reflectivity is obtained from an opaque layer. Therefore, different cor-
neal layers would display different reflectivities on the Z-scan. The corneal
endothelium displays the maximum reflectivity while that of the stroma is the
lowest. An intermediate reflectivity is obtained from epithelial layers. A typ-
ical Z-scan of entire normal cornea shows high endothelial reflection curves
followed by low stromal reflection and then a late intermediate reflectivity
from superficial corneal epithelium. Thus, confocal miscroscopy allows the
user to perform corneal pachymetry or measure the distance between two
specific corneal layers.
CONFOCAL MICROSCOPY OF THE NORMAL CORNEA

This is a noninvasive technique of imaging of corneal layers that provides
excellent resolution and contrast. A well-executed scan can visualize the cor-
neal endothelium, stroma, subepithelial nerve plexus and epithelial layers
distinctly. The limitations are the inability to image a normal Bowman’s layer
and Descemet’s membrane, since these structures are not visible through
this microscope. However, it is sometimes possible to view these structures
when they are pathological. Eyes with corneal opacity or edema can also be
successfully scanned.1 The quality of an image depends on: (a) centration
of the light beam, (b) stability of the eye, and (c) optimum brightness of the
illumination.
Epithelium
The corneal epithelium has five to six layers. Three different types of cellular
components are recognized in the epithelium:
1. Superficial (2–3 layers): Flat cells
2. Intermediate (2–3 layers): Polygonal cells
3. Basal cells (single layer): Cylindrical cells
Chapter_02.indd 55 06-02-2018 20:14:08

The superficial epithelial cells appear as flat polygonal cells with well-
defined borders, prominent nuclei and a uniform density of cytoplasm. The
main identifying features of superficial epithelial cells are nuclei, which are
brighter than surrounding cytoplasm and usually associated with perinuclear
hypodense rings (Fig. 2.2A and B). The intermediate epithelial cells are sim-
ilar polygonal cells as compared to the superficial layers but their nuclei are
not evident (Fig. 2.3). Basal cell layers are smaller in size and appear denser
than other two layers (Fig. 2.4A and B). The nucleus is also not evident in the
basal layers. The corresponding Z-scans for the superficial and basal layers
have been included, showing the depth of each scan.
Subepithelial Nerve Plexus
Corneal nerves originate from the long ciliary nerve, a branch of the ophthal-
mic division of the trigeminal nerve. Nerve fibers from the long ciliary nerve
form a circular plexus at the limbus. Radial nerve fibers originate from this
circular plexus and run deep into the stroma to form the deep corneal plexus.
Deep vertical fibers that proceed from the deep corneal plexus, run anteriorly
(A)
(B)
Figs. 2.2A and B: Superficial epithelial cells with prominent nuclei with corresponding
Z-scan.
Chapter_02.indd 56 06-02-2018 20:14:09

Fig. 2.3: Intermediate epithelial cells. High cell density with well-demarcated cell
borders.
(A)
(B)
Figs. 2.4A and B: Basal epithelial cells with corresponding Z-scan. A high cell density
seen with well-demarcated cell borders.
Chapter_02.indd 57 06-02-2018 20:14:09

(A)
(B)
Figs. 2.5A and B: Subepithelial nerve fibers with corresponding Z-scan.
to form the subbasal and subepithelial nerve plexuses. Small nerve fibers
from the subbasal plexus terminate at the superficial epithelium.
This complex anatomy was not visualized in vivo until the advent of the
corneal confocal microscope. Generally, the nerve fibers appear bright and
are well-contrasted against a dark background (Fig. 2.5A and B). Confocal
microscopy can visualize the orientation, tortuosity, width, branching pat-
tern and other abnormalities of the corneal nerves.2
Stroma
The corneal stroma represents 90% of the total corneal thickness. It has three
components:
1. Cellular stroma: Composed of keratocytes and constitutes 5% of the
entire stroma.
2. Acellular stroma: Represents the major component (90–95%) of the
stroma, comprising of regular collagen tissue (types I, III and IV) and
interstitial substances.
3. Neurosensory stroma: Represented by stromal nerve plexus and nerve
fibers originating from it.
Chapter_02.indd 58 06-02-2018 20:14:09

The keratocyte concentration is much higher in the anterior stroma and

progressively decreases towards the deep stroma. Generally, the keratocyte
count is approximately 1,000 cells/mm2 in the anterior stroma while the aver-
age value drops to 700 cells/mm2 in the posterior stroma. The confocal image
of stroma shows multiple irregularly oval, round or bean-shaped bright
structures that represent keratocyte nuclei. These nuclei are well-contrasted
against the dark areas which represent acellular matrix (Fig. 2.6). Anterior
stromal keratocyte nuclei assume rounded and bean-shaped morphology
while the same in the rear stroma are more often irregularly oval. A bright
highly reflective keratocyte represents a metabolically activated keratocyte of
a healthy cornea. In a normal healthy cornea, collagen fibers and interstitial
substances appear transparent to the confocal microscope and are impossi-
ble to visualize. It is possible to identify stromal nerve fibers in the anterior
and mid stroma. These nerve fibers belong to the deep corneal plexus and
appear as linear bright thick lines. The stromal nerve fiber thickness is greater
than epithelial nerves. Occasionally, nerve bifurcations are also clearly
(Contd.)
Chapter_02.indd 59 06-02-2018 20:14:10

(Contd.)
Fig. 2.6: Anterior (top image), intermediate (middle image) and deep (bottom image)
stromal keratocytes.
Chapter_02.indd 60 06-02-2018 20:14:10

visible. Representative Z-scans have been included below for the anterior
and intermediate stromal scans.
Endothelium
The endothelium is a non-innervated single layer of cells at the most poste-

rior part of the cornea. The endothelial cell density is maximal at birth and
progressively declines with age. The normal endothelial cell count varies
from 1,600 to 3,000 cells/mm2 (average 2,700 cells/mm2) in a healthy adult.2-4
However, the cornea can still maintain its integrity until the cell count declines
below 300–500 cells/mm2.
Homogeneous hexagonal cells with uniform size and shape represent
healthy endothelial cells. Increasing age and endothelial damage cause
pleomorphism and polymegathism. Confocal microscopy easily identifies
these endothelial cells. These cells appear as bright hexagonal and polygo-
nal cells with no recognizable nucleus. The cell borders are represented by
a thin, nonreflective dark line (Fig. 2.7). A × 20 objective lens provides wide
field with less magnification. It is possible to perform cell count and study
the minute details of cellular morphology. Figure 2.8 shows the correspond-
ing Z-scan of the corneal endothelium. Newer confocal microscopes have
the ability to perform reliable and accurate endothelial cell count measure-
ments (Fig. 2.9A and B).
CONFOCAL MICROSCOPY IN CORNEAL PATHOLOGY

Keratoconus
Keratoconus is a noninflammatory ectatic disorder of the cornea charac-

terized by a localized conical protrusion associated with an area of stromal
thinning. The thinning is most apparent at the apex of the cornea. The steep
conical protrusion of the corneal apex causes high myopia with severe
irregular astigmatism. Other features of keratoconus include an iron ring,
known as Fleischer’s ring, that partially or completely encircles the cone.5
The cone appears as an ‘oil drop’ reflex on distant direct ophthalmoscopy
due to internal reflection of light. Deep vertical folds oriented parallel to
the steeper axis of the cornea at the level of deep stroma and Descemet’s
membrane are known as Vogt’s striae. An acute corneal hydrops appears
when there is a break in the Descemet’s membrane. The corneal edema
usually subsides after few months leaving behind a scar and flattening of
the cornea. The corneal nerves become more readily visible due to thin-
ning of the cornea. High irregular astigmatism precludes adequate spec-
tacle correction. In the early stages, use of contact lenses may improve the
visual acuity. However, contact lens fitting can be extremely difficult and in
advanced cases, it ceases to improve visual acuity optimally necessitating
corneal transplantation.
Chapter_02.indd 61 06-02-2018 20:14:11

Fig. 2.7: Hexagonal endothelial cells in a healthy cornea in high magnification (above)
and low magnification (below).
The most effective way to identify early cases of keratoconus is comput-

erized corneal topography that has become a gold standard for diagnosis
and follow-up of the disease in recent years.6,7 Confocal microscopy is a rel-
atively newer investigative modality to assess the keratoconic cornea. Mor-
phological changes in keratoconus are mostly confined to the corneal apex
and depend on the severity of the disease. The rest of the cornea may appear
Chapter_02.indd 62 06-02-2018 20:14:11

Fig. 2.8: Z-scan of the endothelium.
(A)
(B)
Figs. 2.9A and B: Reliable automated endothelial cell counting by confocal
microscopy.
Chapter_02.indd 63 06-02-2018 20:14:11

Fig. 2.10: Obliquely elongated superficial epithelium in keratoconus.
normal. The typical polygonal shape of superficial epithelial cells is lost. They
appear distorted and elongated in an oblique direction with highly reflective
nuclei (Fig. 2.10). Cell borders are not distinguishable. There may be areas of
basal epithelial loss as evident by a linear dark, nonreflective patch seen on
confocal microscopy. The subepithelial nerve plexus generally appears nor-
mal. However, the subbasal nerve fibers are curved and take the course of
stretched overlying epithelium. The corneal stroma is also affected by kera-
toconus. The confocal images of the stroma are highly specific to the disease.
The characteristic stromal changes are multiple ‘striae’ represented by thin
hyporeflective lines oriented vertically, horizontally or obliquely (Fig. 2.11A
and B). These are confocal microscopic representation of Vogt’s striae.8 In
advanced stages of keratoconus, the keratocyte concentration is reduced in
the anterior stroma. The shape of the keratocytes is also altered. Occasionally,
highly reflective bodies with tapering ends are visible in the anterior stroma
near the apex. The nature of these abnormal bodies is not yet known, how-
ever, it may represent altered keratocytes. The corneal endothelial changes
vary from none to occasional pleomorphism and polymegathism.
Corneal Dystrophies
Corneal dystrophies are inherited abnormalities that affect one or more layers
of cornea. Usually both eyes are affected but not necessarily symmetrically.
They may present at birth but more frequently develop during adolescence
and progress gradually throughout life. The effect on vision is highly variable,
depending on the disorder.
Chapter_02.indd 64 06-02-2018 20:14:12

(A)
(B)
Figs. 2.11A and B: Advanced keratoconus: Striae are seen in the stroma.
Granular Dystrophy
This is an autosomal dominant bilateral noninflammatory condition that

results from deposition of eosinophilic hyaline deposits in the corneal
stroma.9 It specifically affects the central cornea and eventually can cause
decreased vision and eye discomfort. Initially, the lesions are confined to
superficial stroma but with progression of the disease they can involve the
posterior stroma as well.
Chapter_02.indd 65 06-02-2018 20:14:12

Confocal microscopy reveals highly reflective, bright, dense structures

in the anterior and midstroma. Keratocytes are not involved. Depth of stro-
mal involvement may be ascertained by using the Z-scan function. This is an
added advantage over other contemporary investigations that enables the
surgeon to plan for surgical procedures. Confocal microscopy is also useful
in the differential diagnosis and follow-up of the disease.
Posterior Polymorphous Dystrophy
Posterior polymorphous dystrophy (PPMD) is a rare inherited disorder of

the posterior layer of the cornea. It is a bilateral disorder with early onset,
although early stage diagnosis is rare since most of the affected individuals
remain asymptomatic. The characteristic endothelial changes are small vesi-
cles or areas of geographic lesions. In fact, endothelial cells lining of the pos-
terior surface of the cornea have epithelial-like features.10,11 These cells can
also cover the trabecular meshwork, leading to glaucoma in some patients.
Most severe cases may develop corneal edema due to compromised pump
function of the endothelial cells.
Confocal microscopy shows multiple round vesicles at the level of
Descemet’s membrane and endothelium.12 PPMD usually distorts the
normal flat profile of the endothelial cells and present as large dark, cystic
impressions on confocal scan. The endothelial cells surrounding the lesion
appear large and distorted.
Fuchs Endothelial Dystrophy
Fuchs endothelial dystrophy is a chronic bilateral hereditary (variable auto-

somal dominant or sporadic) disorder of the corneal endothelium. It typically
presents after the age of 50 and is more common in females. There is a loss of
endothelial cells that results in deposition of collagen materials in Descem-
et’s membrane. These focal excrescences in Descemet’s membrane are called
corneal guttata, which is the hallmark of this disease. The integrity of the cor-
neal endothelium is essential to maintain the metabolic and osmotic proper-
ties of the entire cornea. Corneal edema in Fuchs dystrophy initially involves
the posterior and midstroma. As the disease advances, the edema progresses
to involve the anterior cornea; resulting in bullous keratopathy.
Confocal microscopy is useful to visualize corneal guttata. This tech-
nique has a distinct advantage over conventional specular microscopy that
fails to visualize the endothelium when there is significant corneal edema.13
The corneal guttata appears dark with a bright central reflex14 (Fig. 2.12).
In advanced stages, the endothelial morphology is altered completely but
distorted cell borders can still be visualized.14 In the early stages of bullous
keratopathy, intraepithelial edema is seen as distorted cellular morphology
with increased reflectivity. Confocal microscopy can also identify the bullae
in the basal epithelial layer.
Chapter_02.indd 66 06-02-2018 20:14:12

Fig. 2.12: Guttata seen in the endothelium in mild Fuchs endothelial dystrophy (above)
and severe disease (below).
Laser in situ Keratomileusis
Traditionally, the cornea is evaluated with slit-lamp biomicroscopy and

computerized corneal topography both pre- and postoperatively. Confocal
microscopy adds a new perspective to the commonly employed investiga-
tions. The functional outcome of laser in situ keratomileusis (LASIK) depends
on many factors including biomechanics, the healing process and the inflam-
matory response of the flap interface that is created between the epithelial
flap and stromal bed. A confocal scan is useful in the following scenarios.
Chapter_02.indd 67 06-02-2018 20:14:12

LASIK is one of the newer techniques of excimer laser refractive surgery

that has a strong record of being successfully used by refractive surgeons for
the correction of various types of refractive errors. LASIK has become the
technique of choice to correct myopia and hyperopia with or without astig-
matism.15 LASIK is a modification of photorefractive keratectomy (PRK)
where an excimer laser is used to ablate superficial corneal stroma after the
epithelium has been removed. LASIK involves the use of a microkeratome or
laser to prepare a hinged corneal flap of uniform thickness. The excimer laser
is subsequently used to ablate the mid-corneal stromal bed and thereafter
the flap is returned to its original position without suturing. After LASIK, the
healing of corneal tissue occurs quickly since there is minimal damage to the
corneal epithelium and the Bowman’s layer.
• Study of corneal flap thickness
• Interface study:
Healing process
Inflammatory response
Abnormal deposits
• Corneal nerve fiber regeneration
• Residual stromal thickness
A well-designed flap is the key to a successful outcome in LASIK. Thinner flaps
are at higher risk for developing flap complications. A few studies employing
confocal microscopy had suggested that the actual flap thickness after LASIK
is consistently lower than the predicted thickness.16 The reasons are not yet
understood. However, corneal edema that may be caused by microkeratome
cut and suction may play an important role. Postoperative scarring and tis-
sue remodeling could be other possible factors. Using a Z-scan, it is possible
to identify the interface that corresponds to a very low level of reflectivity.
The flap thickness is obtained by measuring the distance between the highly
reflective spike from the front surface of the cornea and the low reflective
interface (Fig. 2.13).
The interface usually appears as a hyporeflective space in between the
relatively hyperreflective cellular stroma. This interface can be easily imaged
by a confocal microscope. Typically, the keratocyte concentration is lower
than the normal in the interface. Bright particles and microstriae are consis-
tently visible in the interface. These bright particles most probably originate
from the microkeratome blade and are represented by highly reflective white
bodies (Fig. 2.14). Microstriae are present at the Bowman’s layer. Excessive
interface microstriae and bright particles may lead to astigmatism and even-
tually a poor outcome after LASIK. These microstriae can be imaged with a
confocal microscope, even when the slit-lamp examination is unremarkable.
Diffuse lamellar keratitis (DLK) also known as the ‘Sands of Sahara syn-
drome,’ is a noninfectious inflammation of the interface. The etiology is not
known but it is assumed to be toxic or allergic in nature. In confocal scan,
DLK appears as diffuse and multiple infiltrates in the interface with no ante-
rior or posterior extension (Fig. 2.15).
Chapter_02.indd 68 06-02-2018 20:14:13

Fig. 2.13: Measurement of flap thickness in LASIK via the Z-scan.
Fig. 2.14: Bright high reflective particles at the flap-stroma interface in laser in situ ker-
atomileusis (LASIK).
Subepithelial nerve fibers are also affected by LASIK. Nerves are not usu-
ally visible in immediate postoperative period. However, the regenerating
nerve fibers appear as thin irregularly branching lines when a confocal scan
is performed 5–7 days after surgery. The residual stromal thickness can also
be measured using Z-scan technique as described, while also evaluating the
epithelial flap. A confocal scan image below demonstrates a foreign body
seen under the flap after LASIK (Fig. 2.16).
Chapter_02.indd 69 06-02-2018 20:14:13

Fig. 2.15: Diffuse lamellar keratitis after laser in situ keratomileusis (LASIK). Multiple
infiltrates are seen as bright spots.
Fig. 2.16: A foreign body seen under the flap after laser in situ keratomileusis (LASIK).
Infectious Keratitis
Acanthamoeba Keratitis
Accurate and prompt diagnosis of sight threatening infectious keratitis is

one of the major challenges in ophthalmic practice today. Delay in diagnosis
and inappropriate treatment can adversely affect the visual outcome. Cur-
rent microbiological diagnostic techniques can identify most of the offend-
ing organisms if the tests are carried out meticulously. However, confocal
microscopy can play a significant role in keratitis with a prolonged course,
polymicrobial keratitis and in scenarios when conventional techniques are
Chapter_02.indd 70 06-02-2018 20:14:13

Fig. 2.17: In vivo confocal microscopy demonstrating active Acanthamoeba repre-

sented by bright refractile bodies. A few trophozoites are also visible.
unable to identify any organism. The usefulness of confocal microscopy was

demonstrated in eyes with Acanthamoeba17 and fungal keratitis.18,19
It is frequently difficult to identify Acanthamoeba on the ocular surface
since its presentation can mimic herpetic keratitis and several forms of bac-
terial keratitis. This diagnostic dilemma has often led to delays in making the
correct diagnosis and may affect the visual outcome significantly. Most cases
of Acanthamoeba keratitis is diagnosed on tissue culture, corneal biopsy and
histological analysis.20-22 Confocal microscopy offers a useful noninvasive
technique to diagnose Acanthamoeba keratitis in vivo. Although, confocal
microscopy lacks sufficient resolution to be the only method of diagnosis, it
can be used in screening patients suspected of having Acanthamoeba kera-
titis.23,24 On confocal microscopy, Acanthamoeba are visualized typically as
round or ovoid, highly reflective structures ranging in size from 10 to 25 mm,
which is larger than leucocytes (Fig. 2.17). Sometimes, the internal structure
of Acanthamoeba along with vacuoles are also visible. The double-walled
cystic forms of the parasite are also often well-visualized (Fig. 2.18).
Acanthamoeba are smaller and much more iridescent than corneal paren-
chymal cells such as epithelial cells and keratocyte nuclei.
Mycotic Keratitis
Mycotic keratitis is common in many countries, especially in tropical lati-

tudes. Filamentous fungi are the commonest cause of mycotic keratitis.25 It
is important to establish a specific diagnosis as early as possible to ensure
prompt institution of antifungal therapy. Although confocal microscopic
examination may help in reaching a rapid presumptive diagnosis, the in vivo
confocal microscopic characteristics of fungal keratitis continues to confuse
Chapter_02.indd 71 06-02-2018 20:14:14

Fig. 2.18: In vivo confocal microscopy showing typical double-walled cystic form of
Acanthamoeba.
Fig. 2.19: In vivo confocal microscopy showing fungal hyphae.
ophthalmologists. A clear understanding of the confocal characteristics of

fungal hyphae and spores may play an important role in establishing a rapid
diagnosis. Identification of fungal organisms by confocal microscopy is
important, not only for rapidly diagnosing fungal keratitis, but also for mon-
itoring response to antifungal therapy. On confocal microscopy, the mycotic
organisms appear as thin, extensively branching and beaded filaments.
Sometimes, round to oval spores can also be found (Fig. 2.19). In vivo, confo-
cal microscopy reveals four types of morphologies of mycotic keratitis, such
as: (a) branching hyper-reflective structures, (b) long linear hyper-reflective
structures, (c) short rod hyper-reflective structures, and (d) round to oval
structures (spores). The hyphae must not be confused with corneal nerves
which appear regular, elongated and uniform with sharp margins.
Chapter_02.indd 72 06-02-2018 20:14:14

Corneal Grafts
The confocal microscope is a useful tool to follow-up corneal grafts and to

detect abnormalities that may occur postoperatively. It provides images at
the cellular level to identify pathological changes even before they become
clinically evident. It can also be used to assess the donor cornea.
Corneal graft survival is heavily dependent on the number of healthy
endothelial cells present. Endothelial cell loss occurs rapidly after corneal
transplantation.26 Majority of cell loss takes place during the first two post-
operative years.27 Several studies have suggested that endothelial cell loss is
much higher after corneal grafting when the primary indications are bullous
keratopathy or hereditary stromal dystrophy in comparison to keratoconus
and corneal leukomas.28,29 Another interesting fact is that endothelial cell
loss is greater when corneal transplantation is performed on phakic eyes than
on aphakics.30
Confocal microscopy supersedes conventional specular microscopy
while evaluating endothelial cell characteristics, especially in eyes with stro-
mal edema. During the immediate postoperative period, the endothelium
looks normal and healthy. However, as time progresses, endothelial cell den-
sity decreases as evidenced by pleomorphism and polymegathism. Occasion-
ally, bright pre-endothelial deposits appear, the significance of which is not
yet known (Fig. 2.20).
Reinnervation after grafting is another clinical phenomenon that is
imaged well by confocal microscopy. The first sign of innervation that starts
a few months after keratoplasty is new nerve growth at the periphery of the
graft stroma. However, complete innervation may take many years to develop.
Regenerated nerve fibers look similar to those found in the normal cornea.
Fig. 2.20: Pleomorphism, polymegathism and pre-endothelial deposits in a corneal

graft.
Chapter_02.indd 73 06-02-2018 20:14:14

Fig. 2.21 Coexistence of degenerated and normal endothelial cells in early endothelial
allograft rejection.
Occasionally, they may take a tortuous and convoluted course depending on

age (e.g., older patients) and primary indications of keratoplasty (e.g., bullous
keratopathy, corneal dystrophies).
It is well-known that allograft rejection is one of the most common causes
of graft failure. Graft rejection can be classified as epithelial, subepithelial
and endothelial rejection, of which the endothelial subtype has the worst
prognosis. The confocal microscopic features of epithelial rejection are dis-
torted basal epithelial cells with altered subepithelial reflectivity. Subepi-
thelial rejection is identified by discrete opacities underneath the epithelial
layer.31 Endothelial rejection, on the other hand, is characterized by coexis-
tence of normal looking and degenerated endothelial cells, focal endothelial
cell lesions and bright highly reflective microprecipitates32 (Fig. 2.21).
Intracorneal Deposits
Sources of intracorneal deposits can be exogenous or endogenous. They can

involve various layers of cornea individually or in combination.
• Exogenous sources:
Long-term use of contact lenses
Refractive surgery
Vitreoretinal surgery using silicone oil
Drugs including amiodarone, chloroquine
• Endogenous sources:
Wilson’s disease
Hyperlipidemia
Fabry’s disease
Hemosiderosis
Chapter_02.indd 74 06-02-2018 20:14:15

Fig. 2.22: Intracellular deposits at basal epithelial layer in amiodarone toxicity.
The clinical diagnosis of these conditions is based on slit-lamp biomicros-

copy and systemic features. The knowledge of confocal microscopic features
in these disorders is limited, except in drug-induced keratopathies.
Vortex Keratopathy
Vortex keratopathy, also known as cornea verticillata, is characterized by

whorl-like corneal epithelial deposits. It can be induced by various drugs
[e.g., amiodarone (used for cardiac arrythmias) and antimalarials (chloro-
quine, hydroxychloroquine)]. Clinically, vortex keratopathy is manifested
as golden-brown opacities at the level of the inferior corneal epithelium. On
electron microscopy, they appear as intracytoplasmic lysosome-like lamel-
lar inclusion bodies located at the basal epithelial layer.33 Confocal micros-
copy adds a new perspective to the imaging of this condition. It demonstrates
involvement of the entire cornea, although vortex keratopathy is primarily
a corneal epithelial pathology. The characteristic features are presence of
highly reflective, bright intracellular deposits at the basal epithelial layer
(Fig. 2.22). The overlying epithelium is usually normal. In advanced cases
these microdeposits may extend to the stroma and eventually to the endo-
thelium.34 Stromal keratocyte density is often reduced.
CONCLUSION
Ophthalmic investigations and imaging modalities have advanced tremen-
dously over the past few decades. The confocal microscope is one of these
wonderful innovations that has shed much light on the anatomy and pathol-
ogy of the human cornea. It is becoming increasingly useful in clinical prac-
tice and its indications are continually expanding. Confocal microscopy is
truly an exciting tool that can be useful for the clinical diagnosis, follow-up
and analysis of many corneal lesions.
Chapter_02.indd 75 06-02-2018 20:14:15

ACKNOWLEDGMENT
We would like to acknowledge Nidek Technologies for their contribution of
clinical photographs, as well as Aria Mangunkusumo and Vanathi Ganesh for
their help.
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14. Rosenblum P, Stark WJ, Maumenee IH, et al. Hereditary Fuch’s dystrophy. Am J
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16. Durairaj VD, Balentine J, Kouyoumdjian G, et al. The predictability of corneal
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17. Cavanagh HD, Petrol WM, Alizadeh H, et al. Clinical and diagnostic use of in
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18. Winchester K, Mathers WD, Sutphin JE. Diagnosis of aspergillus keratitis in vivo
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19. Florakis GJ, Moazami G, Schubert H, et al. Scanning slit confocal micros-
copy of fungal keratitis. Arch Ophthalmol. 1997;115:1461-3.
20. Mathers WD, Sutphin JE, Folberg R, et al. Outbreak of keratitis presumed to be
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21. Auran JD, Starr MB, Jakobiec FA. Acanthamoeba keratitis: A review of the litera-
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22. D’Aversa G, Stern GA, Driebe WT Jr. Diagnosis and successful medical treatment
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23. Winchester K, Mathers WD, Sutphin JE, et al. Diagnosis of Acanthamoeba kera-
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24. Chew SJ, Beuerman RW, Assouline M, et al. Early diagnosis of infectious
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25. Wang L, Zhang J, Sun S, et al. In vivo confocal microscopic characteristics of
fungal keratitis. Life Science J. 2008;5(1):51-4.
26. Harper CL, Boulton ML, Marcyniuk B, et al. Endothelial viability of organ cul-
tured corneas following penetrating Keratoplasty. Eye. 1998;12(5):834-8.
27. Vasara K, Setälä K, Ruusuvaara P. Follow-up study of human corneal endothelial
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28. Obata H, Ishida K, Murao M, et al. Corneal endothelial cell damage in penetrat-
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29. Abott RL, Fine M, Guillet E. Long-term changes in corneal endothelium follow-
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30. Ing JJ, Ing HH, Nelson LR, et al. Ten-year postoperative results of penetrating
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31. Cohen RA, Chew SJ, Gebhardt BM, et al. Confocal microscopy of corneal graft
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32. Cho BJ, Gross SJ, Pfister DR, et al. In vivo confocal microscopic analysis of corne-
al allograft rejection in rabbits. Cornea. 1998;17(4):417-22.
33. Ghose M, McCulloch C. Amiodarone-induced ultrastructural changes in human
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368-73.
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3
CHAPTER
LASIK
Frank Joseph Goes
INTRODUCTION
The goal of refractive surgery is to improve the refraction of the eye so that an
ametropic eye becomes emmetropic or approaches emmetropia. The cornea
takes care of two-thirds of the refractive component of the eye and the natural
eye lens of the other one-third. This means that the refractive state of the eye
can be improved by working either on the cornea or the lens. The refractive
power of the cornea can be changed by changing the curvature and/or the
thickness. The same is not applicable on the lens.
However, the natural lens may be removed to decrease the refractive
power of the eye as performed in high myopia (clear lens extraction). Alter-
natively, the natural lens may be replaced by a better adapted lens or by a lens
which restores or improves accommodation using ‘refractive lensectomy’.
If need be a supplementary lens (phakic lens) is implanted to change the
refractive power of the eye.
Theoretically, the natural cornea may be replaced by an artificial cornea to
approach emmetropia but the right material for that purpose is not yet avail-
able. However, an extra material in the cornea (corneal inlay) can be intro-
duced to modify and improve the refractive power of the cornea.
CHANGE IN CORNEAL CURVATURE

Let us focus on modifying the refractive power of the cornea by a technique
called laser in situ keratomileusis (LASIK). ‘Laser-assisted in situ keratomil-
eusis,’ commonly referred to as LASIK, has become the single most common
operation with over 35 million procedures performed worldwide by 2010. It
has evolved into a 10 minutes process that can correct refractive errors with
minimal discomfort and a recovery time of a few hours. It is the result of
Chapter_03.indd 78 06-02-2018 20:37:18

LASIK 79
numerous brilliant ideas and bioengineering accomplishments that have led

to one of the most spectacular medical procedures in the history of medicine.
PERSONAL EXPERIENCE
We were privileged to be the first to introduce an excimer laser (Mel 60 from
Aesculap-Meditec) in the Benelux (Belgium-Netherlands-Luxemburg) in
1992. Later, switched to better adapted models, Mel 70 and 80. We introduced
the VisuMax femtosecond laser from Carl Zeiss Meditec in 2008. A femtola-
ser is used for excision of corneal tissue in our center since 2011. The LASIK
is preferred over photorefractive keratectomy (PRK) from the year 2000. The
differences between both approaches concerning the healing, patient satis-
faction and outcomes are remarkable. With LASIK, patients can enjoy good
vision after a few hours and return to work at the latest after 24 hours. The
postoperative treatment is minimal; a bandage contact lens overnight, arti-
ficial tears during 4 weeks (to be prolonged in some patients) and combined
drops (corticosteroids antibiotics) for 1 week. Patient wears protective gog-
gles for the first 24 hours and has to be instructed not to rub the eye the first
24 hours and to sleep with the protective glasses first night.
In order to make the switch from PRK to LASIK the surgeon has to learn
the handling of the microkeratome. This is now much easier compared to so
many years ago when we started. Training courses, wet labs etc. are available
for the starters and microkeratomes have evolved and became much safer
and more reliable. The introduction of the femtosecond laser eliminating the
need of the microkeratome, made the technique even more reliable, easier
and safer.
BIRTH OF LASIK
Keratomileusis
The concept that a refractive error could be corrected by sculpting corneal

stromal tissue to change its curvature was the idea of José Ignacio Barraquer
Moner in 1948. Barraquer developed a procedure coined as ‘keratomileusis.’
Keratomileusis literally means ‘sculpting of the cornea which involved resect-
ing a disk of anterior corneal tissue which was then frozen in liquid nitrogen’
(Fig. 3.1). The resection was achieved using a manually driven microker-
atome designed specifically for this purpose based on a carpenter’s plane.
Barraquer developed the formulas to derive the volume of tissue removal
required for a particular refractive error correction. His earliest patients were
treated in the early 1960s in Bogotá, Colombia.
Around that time, others were experimenting with Barraquer’s idea.
Krwawicz in Poland, published a paper in 1964 describing a series of three
highly myopic eyes in which he had performed manually a ‘stromectomy.’
Pureskin in Russia described the concept of an incomplete anterior cor-
neal resection to leave a naturally hinged flap in 1967, after which a stromal
Chapter_03.indd 79 06-02-2018 20:37:19

Fig. 3.1: Jose I Barraquer Moner using his first cryolathe to mill the underside of a
resected disk of anterior corneal tissue. This original lathe was a modified Watchmaker’s
lathe.
Courtsey: Carmen Barraquer.
disk was removed by trephination. Early to mid-1980s surgeons from around

the world came to learn this difficult, but miraculous technique.
Barraquer-Krumeich-Swinger Technique
Two of Barraquer’s disciples, Krumeich and Swinger, worked on a refinement

of the technique to perform keratomileusis without freezing referred to as the
Barraquer-Krumeich-Swinger (BKS) technique (Figs. 3.2A and B). The BKS
technique aimed to reduce surgical trauma to the tissues and improve visual
recovery time.
In situ Keratomileusis
At around the same time, another non-freeze technique called, in situ ker-
atomileusis, was developed. The procedure was first performed by Ruiz. He
came up with the idea of passing the microkeratome a second time to resect
the required lenticule directly from the stromal bed.
Automated Lamellar Keratoplasty
Ruiz was then responsible for designing a gear system to automate the pas-
sage of the microkeratome head. This eased the technical challenges of using
a manual microkeratome, therefore avoiding irregular resections and greatly
improving the accuracy.
The procedure became known as ‘automated lamellar keratoplasty (ALK)’.
Chapter_03.indd 80 06-02-2018 20:37:19

LASIK 81
(A)
(B)
Figs. 3.2A and B: (A) Technical diagram of the first manually driven microkeratome
developed by Barraquer for corneal disk resection in keratomileusis (B) photo.
Courtsey: Carmen Barraquer.
In 1988, Ruiz presented a paper demonstrating how a flap could be pro-

duced by stopping the microkeratome before the end of the pass. The flap
would then be tucked under the second microkeratome ring applied for the
stromal resection thus leaving a hinge to simplify the replacement and reduce
cap related complications.
Excimer Laser
In the early 1990s, in situ keratomileusis was combined with the emerg-
ing technology of excimer lasers for corneal tissue ablation to finally
become ‘LASIK,’ with the birth of LASIK as we know it today. In 1970, the
Chapter_03.indd 81 06-02-2018 20:37:19

term excimer laser was introduced to describe a laser built by Basov using
a xenon dimer gas, the name excimer coming from an abbreviation of
‘excite dimer’. The argon-fluoride excimer laser was developed in 1976
(Fig. 3.3). Plume may be seen after an excimer laser pulse (Fig. 3.4).
Fig. 3.3: Basic components of an excimer laser demonstrates the active laser medium
as gas in the storage tank and in the laser cavity, the exciting energy pumping source
initiates the stimulated emission of radiation which is amplified by the mirrors to create
the laser beam.
Fig. 3.4: Plume immediately after an excimer laser pulse.

Courtsey: Alfred Vogel.
Chapter_03.indd 82 06-02-2018 20:37:19

LASIK 83
It was not until 1981 that an argon-fluoride excimer laser (193 nm) was
fired onto organic tissue when Blum, Wynne and Srinivasan, demonstrated
that complex patterns could be made at a micronic level with each pulse
removing a fraction of a micron. This research culminated in excimer lasers
being used for etching microchips. This process of direct splitting of molecular
bonds with minimal adjacent heating using excimer laser-tissue interaction
was coined ‘photoablation.’
Trokel and Marshall later studied the ultrastructural aspects of corneal
photoablation. They compared the quality of the wounds made by an excimer
laser at 193 nm with one at 248 nm as well as made by steel and diamond
blades (Fig. 3.5A to D). The quality of the wounds was best with 193 nm.
The wound quality suggested to Marshall that large area ablation could
be performed in the central cornea, rather than just for peripheral linear inci-
sions (Fig. 3.6). This was described as ‘photorefractive keratectomy (PRK)’.
Then, Marshall demonstrated no changes in corneal transparency 8 months
after PRK in 12 monkey corneas, and McDonald reported stable dioptric
change in a primate cornea with good healing and long-term corneal clarity
up to one year after PRK. In 1985, Seiler performed the first large area abla-
tion in a human eye to remove a corneal scar having previously performed T-
incisions with an excimer laser to correct for astigmatism.
This led to PRK being performed in humans. In the early 1988s McDonald
performed the first PRK on a sighted eye due for enucleation, while at around
the same time L ’Esperance and Seiler began also performing PRK, but in
blind eyes.
In 1991, Dausch and Schroeder presented results in high myopes with the
Aesculap-Meditec excimer laser and later, in 1993, presented the first hyper-
opic ablation profiles.
(A) (B) (C) (D)

Figs. 3.5A to D: Light micrographs of rabbit corneas incised by: (A) an argon-fluoride
excimer laser (193 nm), (B) a krypton fluoride excimer laser (248 nm), (C) a Micra dia-
mond knife and (D) a sharp point steel blade. The wound quality can be seen to be best
with the argon-fluoride excimer laser.
With permission from: Marshall J, Trokel S, Rothery S, et al. A comparative study of cor-
neal incisions inducedby diamond and steel knives and two ultraviolet radiations from
an excimer laser. Br J Ophthalmol. 1986;70(7):482-501.
Chapter_03.indd 83 06-02-2018 20:37:19

Fig. 3.6: Myopic ablation performed using a broad beam laser and a moving iris dia-
phragm. The diameter of the iris diaphragm was gradually reduced which created a
series of small steps. The number of steps was increased to better approximate a curved
surface.
Courtesy: John Marshall.
Laser in situ Keratomileusis
The idea of using an excimer laser to ablate tissue under a flap was spring-
ing up independently in various parts of the world. In 1988, Razhev and
co-workers in Novosibirsk, USSR began using a 5 mm trephine to produce a
central 100 mm deep circular keratotomy and then a scalpel to create a lamel-
lar hinged flap. They then used an excimer laser to ablate the stromal bed
before replacing the lamellar flap in four myopic and five hyperopic eyes and
presented their results with up to 2 years’ follow-up in September 1990 at
Columbia University.
At around the same time, Burrato was performing classical keratomileusis
but from October 1989 he used an excimer laser for ablation on the underside
of the cap and published his first 30 high myopic eyes with few complications
and 1 year follow-up in 1992. In December, 1989 he decided instead to per-
form the excimer laser ablation directly on the stromal bed before replacing
the cap.
Pallikaris also independently conceived of a hinged flap using a microker-
atome, he had specifically designed for rabbit studies and performed the abla-
tion with an excimer laser on the exposed bed followed by replacement of the
flap without sutures. The term LASIK was first used to describe this procedure
in his 1991 paper. Pallikaris treated his first patient in October 1990 and pub-
lished his results on 10 high myopic human eyes with 1 year follow-up in 1994.
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LASIK 85
LASIK COMPLICATIONS
LASIK comes with a number of short- and long-term risks and complica-
tions. Complications typical for PRK are haze, characterised by subepithelial
fibrosis caused by an abnormal wound healing response, leading to epithe-
lial hyperplasia, presence of newly formed and disorganised collagen III.
Besides that, regression is also a much more common complication after PRK
than after LASIK, especially in eyes that have a higher attempted correction
(Cosar).
Topical anesthesia complications can create superficial punctate keratop-
athy or epithelial defect. A bandage contact lens will usually solve the prob-
lem. Complications with eyelashes, drape and speculum may interfere with
the movement of the keratome.
Conjunctiva related complications such as pinguecula, pterygia can make
suction impossible or difficult. Important chemosis causing insufficient suc-
tion, has to be avoided and may lead to postponing the surgery.
Anatomical variations such as a prominent orbital rim, narrow palpebral
fissure, may make placement of the microkeratome difficult. Insertion of the
suction ring without a speculum or using a special speculum can be helpful.
A lateral canthotomy may be tried. If this does not work, one has to convert
towards PRK or laser epithelial keratomileusis (LASEK).
Subconiunctival hemorrhage is a relatively common aesthetic complica-
tion and patient should be informed about it beforehand.
Corneal bleeding may happen when there is limbal hyperemia in con-
tact lens wearers. Application of a dry sponge or eventually soaked with 2.5%
adrenaline (cave the pupil will dilate) may be used.
Flap problems: Faced with a decentered flap or incomplete cut the treatment
can be continued if the exposed stromal bed is large enough to perform the
laser ablation. If this is not the case, a new flap can be planned after 1 week.
Free cap; if the diameter of exposed stroma is large enough, proceed with
treatment and put the flap back with on top a bandage contact lens.
A perforated or buttonholed flap may occur when the cornea is very steep
(more than 46 D) or in case of mechanical defects. In that case, stop the proce-
dure, place a bandage lens and come back in 3-6 months.
Anterior chamber entry may occur in case of keratoconus or irregular cor-
nea. It should be sutured immediately with 10/0 nylon.
Pizza slicing may occur in case of LASIK after previous radial keratotomy.
The pieces have to be put into place (which is easy) and a bandage lens to be
placed for 24 hours.
Photoablation Related Complications
Decentration occurs less frequently with lasers with eye tracking systems.
Central islands (a central zone of minimal 1.5 mm and steepening of at least
3D) may occur with broad beam lasers but is now exceptional. Retreatment
Chapter_03.indd 85 06-02-2018 20:37:20

Fig. 3.7: Customized Topolink ablation with the Mel 80 (F. Goes). Right inferior
before-right superior after ablation; left power difference map showing the ablation
retreatment.
with topoguided ablation is necessary when this does not resolve after
6 months (Fig. 3.7). Interface debris should be cleaned out manually.
Flap wrinkles and folds may induce irregular astigmatism and loss of best
spectacle-corrected visual acuity (BCVA). Wrinkles, occurring during the pro-
cedure should be treated by stretching and repositioning. Wrinkles, occurring
in the early postoperative period may be caused by dry eye syndrome or rub-
bing. Treatment consists of lifting the flap, refloating after stretching, ironing
the flap, hydrating the flap with hypotonic saline, or suturing the flap in recal-
citrant cases.
Postoperative Complications
Diffuse lamellar keratitis or sands of the Sahara syndrome (DLK) may have
several reasons and has four stages. In stages 1 and 2, intensive corticosteroid
treatment may suffice. From stage 3-4 lifting of the flap, cleaning and irriga-
tion of corticosteroids and antibiotics becomes urgent.
Epithelial ingrowth (epithelial cells proliferating in the lamellar interface)
can lead to irregular astigmatism and opacification of the interface resulting
in loss of BCVA and corneal melting. Only when there is a risk of loss of BCVA,
one should intervene. The flap is to be lifted, the bed and the flap are scraped
with a blunt spatula and sponges and a bandage contact lens is applied. Even-
tually the flap has to be sutured.
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LASIK 87
RETREATMENT
In a British study of 360 myopic eyes treated with LASIK about 10% cases
required retreatment (Dick VII). At 1-year follow-up, 56% of the eyes were
within ± 0.50 D spherical equivalent (SE), and 78% were within ± 1.00 D SE.
Seventy-eight percent of the eyes examined at 1-year post-retreatment man-
aged unaided vision of 0.66 or better.1 Among hyperopes, there is a correla-
tion between the preoperative degree of farsightedness and the achievement
of a postoperative refractive error within ± 1 D: in a long-term follow-up study
from Stanford University, the percentage of eyes within ± 1.0 D of emmetro-
pia was 82.4% for low hyperopia (up to + 2.0 D), 75.0% for medium hyperopia
(+ 2.0 to + 4.0 D), and 66.7% for high hyperopia (more than + 4.0 D).
In case of under and or overcorrection, a retreatment has to be considered
in 3 months. In case of severe miscorrection (erroneous treatment planning)
immediate retreatment can be performed. Regression after LASIK may be
caused by epithelial hyperplasia and abnormal corneal wound healing. Mod-
ifying the regression with corticosteroid treatment can be tried.
Iatrogenic ectasia can, most of the time, be avoided by good patient selec-
tion. Candidates with abnormal corneas, the so-called keratoconus suspects,
have to be rejected for treatment. The minimum thickness of residual stroma
to prevent ectasia should be 250 mm. However, some unknown factors can
still be responsible for iatrogenic keratectasias. We are now lucky to have ‘cor-
neal cross-linking’ as a retreatment option. Besides that, intracorneal ring seg-
ments, hard contact lenses and eventually keratoplasty are also available.
The experience of keratomileusis shows that ectasia is a rare phenom-
enon as there were only 45 cases out of 1,606 (2.8%) keratomileusis perfor-
med within 21-year follow-up (Barraquer, 1998 #1298). This population also
included some extremely high myopia. Detecting keratoconus through the
use of front surface topography has been followed by the use of tomography,
enabling evaluation of the posterior surface and corneal thickness progres-
sion. Many keratoconus screening indices have been developed using these
data. Other keratoconus screening techniques have used wave front analy-
sis, and measurements of ocular biomechanics using the Ocular Response
Analyzer. Dan Reinstein introduced the concept of detecting keratoconus
using epithelial thickness profiles. The other factor in reducing the risk of
ectasia has been the introduction of femtosecond lasers, giving surgeons
the ability to make ultra-thin flaps and hence maximize the residual stromal
thickness. Femtosecond lasers also have improved the reproducibility of flap
thickness compared to mechanical microkeratomes, meaning that there are
fewer cases where an unexpectedly thick flap occurs. Mechanical microker-
atomes have also improved dramatically from the early models which had
reproducibility as high as 30 μm, whereas modern models have a reproduc-
ibility of 10 μm.
Night vision problems and glare are present in most of the patients during
the early weeks after surgery. Usually the patient adapts. In case of decentered
Chapter_03.indd 87 06-02-2018 20:37:20

ablation, with large scotopic pupils or induced astigmatism, the complaints

will persist and topography-guided laser has to be considered.
Dry eye is the most frequent complication after LASIK because of the dis-
ruption of the corneal innervation. Patients with preexisting dry eyes should
be preoperatively detected and eventually other treatment modality should
be chosen. Usually, complaints disappear in the first 3 months post LASIK.
Infectious keratitis should be treated aggressively. Culture of the flap, fol-
lowed by cleaning and irrigation with fortified antibiotics may help in control.
Slipped or dislodging flap should be repositioned as soon as possible to avoid
fixed folds.
CUSTOMIZED EXCIMER LASER TREATMENT

The standard ablation pattern treatment using PRK or LASIK consists of treat-
ing only the refractive error of the patient using the nomogram of the particu-
lar platform used. This means that all eyes with a similar refraction receive the
same treatment. When doing customized excimer laser treatment, each eye
is regarded as unique, is measured using wavefront and or topography sys-
tems and a particular unique program is calculated for each eye and applied
accordingly (De Langhe).
Differences exist between ‘wavefront guided’ and ‘topography guided’
customized ablation.
Corneal asphericity is described with a Q value. A cornea which is per-
fectly spherical will have a Q value of 0. If the peripheral cornea is less spherical
(flatter) than the central part, the Q value will be negative; we call this condi-
tion a prolate cornea. In case where the peripheral cornea is more spherical
(steeper) than the central part, the Q value will be positive; we call this condi-
tion an oblate cornea. A prolate cornea will produce better contrast sensitiv-
ity and better night vision than an oblate cornea and therefore all treatment
profiles aim to make the cornea more spherical (prolate) with the peripheral
cornea being flatter than the central cornea.
Wavefront-guided treatment is based upon data acquisition of the total
optical system of the eye; all the media of the eye; cornea, anterior chamber,
lens and vitreous. Measurement is done with a wavefront measuring device
and the differences between a perfect wavefront projected into the eye and
the reflected wavefront from the eye represents the aberrations of the eye.
These aberrations are measured and converted into a specific treatment
program for that particular eye to obtain an ideal cornea (periphery less
spherical than the central part). LASIK has proved to be effective in reducing
refractive errors but it is known to cause or increase high order aberrations
(HOAs) especially spherical aberration and coma. Even with reasonable
good central visual acuity, patients may complain of visual symptoms such
as night vision disturbances and ghosting. Wavefront measurements are
used to measure HOAs. Wavefront-guided ablation can prevent and reduce
Chapter_03.indd 88 06-02-2018 20:37:20

LASIK 89
unwanted side effects, reduce enhancement rates and improve upon the
quality of vision. The goal of wavefront-guided LASIK is to correct all optical
aberrations of the eye which reduce vision.
Topography-guided treatment is based upon data acquisition of the cor-
nea with the help of a topography system. The primary indication is decen-
tered ablation and night vision problems.
Some laser platforms (Carl Zeiss Meditec) use a standard wavefront opti-
mized treatment program. Other laser systems use different devices to mea-
sure and to calculate. The CRS-Master from Carl Zeiss Meditec was used to
design optimal ablation profiles taking into account wavefront data, keratom-
etry, pachymetry, pupillometer and target refraction.
INDICATIONS AND LIMITATIONS OF LASIK

LASIK is probably the most frequently used corneal refractive procedure
worldwide, particularly popular with younger and predominantly myopic
patients. There are limits to its efficiency compelling ophthalmologists to
consider other options (like refractive lens surgery) in patients with severe
myopia or hyperopia. Among ophthalmological societies, there is generally
a consensus that LASIK is best suited for mild and moderate myopia. The
German Ophthalmological Society (DOG) recommends LASIK for up to
- 8.0 D myopia, + 3.0 D hyperopia and 5.0 D astigmatism (Burkhard Dick).
In myopia, Walter Sekundo would be prepared to do femto-LASIK up to
- 6.0 D. In reality he only performs LASIK for the low myopes up to - 2.0. All
the rest is small-incision lenticule extraction (SMILE). He believes that LASIK
for low myopia is a good procedure, but in high myopia it gives poor results
and risks. The results appear to be much better with SMILE. He is convinced
that SMILE is about to replace femto-LASIK. In the last 2 years a snow-ball
effect, in terms of publications, was seen in this field.
LASIK for hyperopia can be safely performed up to + 6.50 D according to
using a third-generation excimer laser with a modern ablation profile, a large
optical zone and a large transition zone (Reinstein). It is possible to measure
epithelial thickness that can be used as a true indicator of how much steepen-
ing can be safely performed after the primary procedure. Therefore, accord-
ing to Reinstein, the best approach is to perform the primary LASIK based
on traditional corneal curvature limits and then assess whether further steep-
ening can be safely performed based on epithelial thickness measurements.
Another critical component for safely treating high hyperopia is for the abla-
tion to be centered on the corneal reflex (or corneal vertex to approximate the
visual axis), particularly given that hyperopic patients tend to have a larger
angle kappa.
According to Dan Reinstein LASIK can be safely performed up to - 14.00 D
in the majority of patients, although there will always be some patients ruled
out by insufficient corneal pachymetry. With the advent of femtosecond
Chapter_03.indd 89 06-02-2018 20:37:20

lasers, it is possible to create ultrathin flaps, for example as thin as 80 mm using

the VisuMax femtosecond laser due to its precision of 4.4 mm. Advances with
aspheric ablation profiles have meant that the spherical aberration is rarely
increased above the tolerable threshold for most patients.
We personally set our limits for LASIK at - 8.0 D for myopia, + 3.5 D for
hyperopia and + 4.0 D for astigmatism, on condition that the patient is a good
candidate.
FEMTOSECOND LASER
Techniques for postoperative management of complications are contin-
ually improving as well. We now have topography-guided custom ablation
solutions that can correct the main causes of irregular astigmatism in small
optical zones and decenterations. Transepithelial PTK can be used to treat
irregular astigmatism by using the epithelium as a natural masking agent for
the stromal surface irregularities very effectively.
The femtosecond laser has become a choicest tool in the hands of many
LASIK surgeons as a result of low incidence of LASIK complications (such
as flap and buttonholes). Additionally, it has good predictability, safety and
a low retreatment rate. In 800 hyperopic eyes of Leccisotti’s series (mean
preoperative SE: + 3.41 D) undergoing femto-LASIK, 9 months postopera-
tively showed that the mean SE was - 0.06 ± 0.26 D, 3 eyes (0.4%) lost two
lines of corrected distance visual acuity (CDVA) and 58 eyes (7.3%) lost one
line. In the long run, an iatrogenic keratectasia becomes less likely but can-
not be ruled out completely. The main risk factor is a preoperative irregular
topography. But thin corneas, deep ablations, thin residual stromal beds and
young patient age at the time of the laser surgery also are other risk factors
(Bragheeth Winkler von Mohrenfels et al.).
The versatility of the femtosecond laser and its effectivity in ophthalmic
surgery (currently a major topic in cataract surgery) has led to the develop-
ment of refractive lenticule extraction (ReLEx)/SMILE (VIII). Both methods
have so far surprised many of the surgeons who have begun to use them.
They are extremely precise and associated with less loss in corneal sensibility
than expected and have proven to be mechanically stable. SMILE seems to
lead to less early corneal nerve damage than LASIK. Presently, only one plat-
form is available in the market to perform the procedures. The indications
for ReLEx/SMILE are myopia between - 3.0 D and - 8.0 D, and astigmatism
up to - 5.0 D.
Future of SMILE ReLEx
In 2010, Carl Zeiss Meditec introduced the femtosecond VisuMax approach,

ReLEx technique, whereby tissue is excised with the help of the femtosecond
laser to adjust the refractive power of the cornea. This approach does, exactly
what Barraquer was doing in the years 1960–1970 but in a more precise and
Chapter_03.indd 90 06-02-2018 20:37:20

LASIK 91
safer way. ReLEx uses the laser to carve out an intrastromal lenticule, which
is then removed in one of two ways to bring about a change in refraction.
The first ReLEx method that was devised, known as femtosecond lamellar
extraction or FLEx, involves using a LASIK-like flap to gain access to the lent-
icule to remove it. In small-incision lenticule extraction (SMILE), the surgeon
induces a refractive change by removing an intrastromal lenticule without
having to create a flap. This method is less invasive, and involves teasing out
the lenticule through a small corneal incision, between 2 and 4 mm wide,
leaving the rest of the cornea intact.
It is expected that ReLEx, SMILE all-femtosecond approach will become
the procedure of future as the cutting precision and visual recovery time con-
tinue to improve. The VisuMax is part of the first-generation of femtosecond
lasers, and there is a scope for improving the technology. The main disadvan-
tage at the moment is the relatively slow visual recovery with fewer patients
experiencing the ‘wow’ effect the day after surgery compared to LASIK. How-
ever, this has been significantly improved by adjusting the energy and femto-
second spot spacing settings to increase the ease of extracting the lenticule
and reducing the total energy being put into the stroma.
The other missing piece of the puzzle is that it cannot be used to treat
hyperopia. However, studies are proceeding in Germany and Nepal where
a hyperopic profile is being developed. It is possible that hyperopic SMILE
could turn out to be more accurate and stable than hyperopic LASIK because
the transition zone may be more reliable.
However, the excimer laser will never be completely replaced. It will be
needed in in the treatment of irregular profiles. The PTK is another procedure
that will always require an excimer laser, while the majority of retreatments
after SMILE will also need to be done as either a LASIK or PRK procedure.
ALTERNATIVES
For higher refractive errors, refractive lens surgery is a viable alternative to
corneal procedures.
Phakic lenses (PIOLs) are one of the available choices. The main advan-
tage of procedure is that the intervention is reversible. However, it comes
at a price that there are number of potential complications associated
with implantation of a phakic IOL. They include inflammations, infection
and toxic reaction may be severe. Both implantable contact lens (ICL) and
iris-fixated PIOL have a tendency to stimulate cataract formation in the
natural lens. More worrisome is the fact that there is usually a consider-
ably higher loss of endothelial cells than would physiologically occur. In a
recently published Japanese study, the mean endothelial cell loss from pre-
operative levels after ICL implantation was 6.2% at 8 years. Patients with a
phakic IOL usually appear to be more comfortable with the quality of their
vision than LASIK patients. Phakic IOLs are a temporary measure according
to some authors (W Sekundo). After having used the Artisan lens for almost
Chapter_03.indd 91 06-02-2018 20:37:20

two decades he switched to cachet, but left it 1 year later and is using now
the ICL. This behavior clearly reflects the experience that sooner or later the
phakic IOLs start to produce problems. Fortunately, we can now repair both
the corneal problems (DMEK) and the lenticular problems (Phako).
The other choice is refractive lens exchange, replacing the natural lens
by a better adapted lens. There are no limits for this technique since refrac-
tive anomalies between - 30 and + 30 can be treated. We know that the most
feared complication infection is extremely rare and that the retinal detach-
ment is nearly nonexistent in hyperopia. Therefore, hyperopic eyes are the
best suited for such a treatment. It will be up to the surgeon if he will take the
risk to operate on an eye with a clear lens.
CONCLUSION
LASIK is an established refractive procedure for correction of mild to moder-
ate myopia, hyperopia and astigmatism. Introduction of femtosecond laser
has made the technique more easy and safe. In the event of complications,
they can be managed using modern repair tools. This is not true for IOLs
since intraocular surgery introduces a number of (albeit rare) catastrophic
complications such as endophthalmitis, suprachoroidal hemorrhage, retinal
detachment and macular edema. The intraocular surgery should be reserved
only for those cases that are outside the limits of LASIK.
Presently or in coming future, LASIK is and will remain the most fre-
quently performed refractive procedure. It should be remembered that in
some patients the success of the surgery may be marred by dry eyes, keratec-
tasia and visual disturbances like glare and halos. However, the LASIK oper-
ated is likely to face a problem in old age especially for calculating the right
IOL power for cataract surgery. But why worry: who knows what kind of tech-
nology may be available by 2030 or 2040?
ACKNOWLEDGMENT
My thanks go to my friends and leaders in refractive surgery: Professor Walter
Sekundo, Germany who introduced the all femtosecond procedure in 2009,
professor Burkhard Dick, Germany and professor Dan Reinstein, UK. They
were very instrumental with their advice in preparing this manuscript.
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13. Krwawicz T. Lamellar corneal stromectomy for the operative treatment of myo-
pia. A preliminary report. Am J Ophthalmol. 1964;57:828-33.
14. Lebedeva LI, Akhmamet’eva EM, Razhev AM, et al. [Cytogenetic effects of
UV laser radiation with wavelengths of 248, 223 and 193 nm on mammalian
cells]. Radiobiologiia.199;30(6):821-6.
15. Leccisotti A. Femtosecond laser-assisted hyperopic laser in situ keratomileu-
sis with tissue-saving ablation: analysis of 800 eyes. J Cataract Refract Surg.
2014;40(7):1122-30.
16. L’Esperance FA Jr, Taylor DM, Del Pero RA, et al. Human excimer laser corneal
surgery: preliminary report. Trans Am Ophthalmol Soc. 1988;86:208-75.
17. McDonald MB, Frantz JM, Klyce SD, et al. One-year refractive results of central
photorefractive keratectomy for myopia in the nonhuman primate cornea.
Arch Ophthalmol. 1990;108(1):40-7.
18. Marshall J, Trokel S, Rothery S, et al. An ultrastructural study of corneal incisions
induced by an excimer laser at 193 nm. Ophthalmology. 1985;92(6):749-58.
19. Marshall J, Trokel S, Rothery S. Photoablative reprofiling of the cornea using an
excimer laser: Photorefractive keratotomy. Lasers Ophthalmol. 1986;1:21-48.
20. Marshall J, Trokel SL, Rothery S, et al. Long-term healing of the central cornea
after photorefractive keratectomy using an excimer laser. Ophthalmology.
1988;95(10):1411-21.
21. Mohamed-Noriega K, Riau AK, Lwin NC, et al. Early corneal nerve damage and
recovery following small-incision lenticule extraction (SMILE) and laser in situ
keratomileusis (LASIK). Invest Ophthalmol Vis Sci. 2014;55(3):1823-34.
Chapter_03.indd 93 06-02-2018 20:37:21

22. Pallikaris IG, Papatzanaki ME, Siganos DS, et al. A corneal flap technique for
laser in situ keratomileusis. Human studies. Arch Ophthalmol.1991; 109(12):
1699-702.
23. Pallikaris IG, Siganos DS. Excimer laser in situ keratomileusis and photore-
fractive keratectomy for correction of high myopia. J Refract Corneal Surg.
1994;10(5):498-510.
24. Pureskin NP. [Weakening ocular refraction by means of partial stromectomy of
cornea under experimental conditions]. Vestn Oftalmol. 1967;80(1):19-24.
25. Razhev A, Lantukh V, Pyatin M. [Ophthalmic devices for corneal microsurgery
on excimer lasers]. Journal de Physique. 1991;1(Suppl III):C7-235.
26. Reinstein D. The birth of LASIK. In: Goes FJ (Ed). The Eye in History; 2013.
Jaypee-Highlights, New Delhi, pp. 431-439.
27. Reinstein D. Personal communication; 2014.
28. Ruiz L. In Situ Keratomileusis. Invest Ophthalmol Vis Sci. 1988;29:392.
29. Sekundo W. Personal communication; 2014.
30. Seiler T, Wollensak J. Myopic photorefractive keratectomy with the excimer la-
ser. One-year follow-up. Ophthalmology. 1991;98(8):1156-63.
31. Winkler von Mohrenfels C, Salgado JP, Khoramnia R, et al. [Keratectasia after
refractive surgery]. Klin Monbl Augenheilkd. 2011;228(8):704-11.
Chapter_03.indd 94 06-02-2018 20:37:21

4
CHAPTER
SMILE versus LASIK
Jorge L Alio, Mohamed El Bahrawy
RECENT EVOLUTION OF LASER REFRACTIVE

SURGERY OF THE CORNEA
The concepts of modern refractive surgery witnessed its breakthrough when
Professor Jose I Barraquer described his coined technique of keratomileusis
in 1949, setting the foundation for all following innovations in this field. The
name ‘excimer laser’ came as an abbreviation of ‘excited dimer’, introduced
by the Russian, Nikolay Basov, in 1970 using a xenon dimer gas. A few years
later, the argon-fluoride excimer laser was developed and was first tried on
an organic tissue by IBM scientists. The introduction of excimer laser to be
used in the human eye was done by Stephen Trokel as a precise and safe tool
of corneal shaping, these concepts later defined the refractive techniques
which are widely used now, when Marguerite McDonald under the super-
vision of Steve Kaufmann, performed the most commonly used epithelium
removal technique photorefractive keratectomy (PRK). Peyman, presented
the first patency using excimer laser as a corneal refractive tool, and it was
accepted in June 1989 (personal correspondence Gholam Peyman). Follow-
ing Ioannis Pallikaris, among others, introduced the most widely used and
commonly accepted technique of laser in situ keratomileusis (LASIK) in
1990.1 Laser refractive surgery has been performed for decades, and there
have been tremendous advancements in terms of technique and technology,
making it increasingly precise and highly predictable.2 LASIK is currently the
most common laser refractive procedure for the treatment of myopia—its
advantages include early postoperative improvement in visual acuity and
minimal postoperative patient discomfort. Although LASIK patients report
95% satisfaction, a spectrum of complicated side effects can negatively
impact results.3
Chapter_04.indd 95 06-02-2018 20:43:28

Femtosecond laser technology was first developed by Dr. Kurtz at the

University of Michigan in the early 1990s4 and was rapidly adopted in the
surgical field of ophthalmology. Femtosecond lasers emit light pulses of
short duration (10−15 seconds) at 1,053 nm wavelength that cause photo-
disruption of the tissue with minimum collateral damage.5 The femtosec-
ond laser has revolutionized corneal and refractive surgery with respect to
its increased safety, precision and predictability over traditional microker-
atomes. Advantages of bladeless femtosecond-assisted LASIK (FS-LASIK)
over conventional microkeratome-assisted LASIK (MK-LASIK) include
reduced dry eye symptomatology, reduced risk of flap button hole or free-
cap formation.6,7
Ever since femtosecond lasers were first introduced into refractive sur-
gery, the ultimate goal has been to create an intrastromal lenticule that can
then be manually removed as a single piece thereby circumventing the need
for incremental photoablation by an excimer laser. A precursor to modern
refractive lenticule extraction (ReLEx) was first described in 1996 using a pico-
second laser to generate an intrastromal lenticule that was removed manually
after lifting the flap;8,9 however, significant manual dissection was required
leading to an irregular surface. The switch to femtosecond improved the pre-
cision10 and studies were performed in rabbit eyes in 199811 and in partially
sighted eyes in 2003,12 but these initial studies were not followed up with fur-
ther clinical trials. Following the introduction of the VisuMaxÒ femtosecond
laser (Carl Zeiss Meditec, Jena, Germany) in 2007,13 the intrastromal lenti-
cule method was reintroduced in a procedure called femtosecond lenticule
extraction (FLEx). The 6-month results of the first 10 fully seeing eyes treated
were published in 200814 and results of a larger population have since been
reported.15,16 The refractive results were similar to those observed in LASIK,
but visual recovery time was longer due to the lack of optimization in energy
parameters and scan modes; further refinements have led to much improved
visual recovery times.17 Following the successful implementation of FLEx, a
new procedure called small-incision lenticule extraction (SMILE) was devel-
oped. This procedure involves passing a dissector through a small 2–3 mm
incision to separate the lenticular interfaces and allow the lenticule to be
removed, thus eliminating the need to create a flap. The SMILE procedure is
now gaining popularity following the results of the first prospective trials.18-29
SMILE OUTCOME
Since the development of the SMILE technique, the exciting new concept of
the flapless nature of the technology, namely the 3rd generation laser refrac-
tive surgery, has driven many authors to approach it and report the results of
SMILE outcomes alone or in comparison with LASIK.
In a study we conducted, we compared the outcomes of a matched cases
of SMILE versus 6th generation excimer laser LASIK patient, where the cases
Chapter_04.indd 96 06-02-2018 20:43:28

SMILE versus LASIK 97
Table 4.1: Refractive outcome of comparative study between SMILE and LASIK.
Comparison SMILE (%) FS-LASIK (%)

20/20 or more 93.75 92.18
20/25 or more 100 96.87
20/40 or more 100 100
No loss of lines 96.87 93.43
Efficacy Lost more than 2 lines 0 0
Gained lines 18.75 (1 line) 18.64 (1–3 lines)
% of cases
84.43 86.25
± 0.5 D
Predictability
% of cases
100 100
± 1.0 D
SMILE: Small-incision lenticule extraction; LASIK: Laser assisted in situ keratomileusis;

FS-LASIK: Femtosecond-assisted LASIK.
were matched by age, gender and spherical equivalent. In the SMILE group;
50% females, 34 years (23:49), - 4.59 diopters (- 2.125:8.37), the LASIK group;
matching SMILE/FLEx cases: of same gender, age (± 1 year), spherical equiv-
alent (± 0.5 D). The study included 16 eyes in each group, and we reported
both SMILE and LASIK had comparable results in terms of safety, efficacy
and predictability, in follow-up of 6 months duration (Table 4.1).
Many other authors reported similar outcomes, still with a disadvantage
of slower refractive recovery in SMILE patients, which is currently witness-
ing significant improvements due to the development of different energy and
spot spacing setting.17,21 Kim et al. reported that age may be a predictor that
influenced visual outcome, as outcomes were better in younger patients of his
study sample but its effect appeared clinically insignificant.22 SMILE surgery
was effective and safe in correcting low-to-moderate astigmatism, and stable
refractive outcomes were observed at the long-term follow-up. The preopera-
tive cylinder ranged from - 2.75 D to - 0.25 D (average of - 0.90 ± 0.68 D), and
the mean postoperative cylinder values were - 0.24 ± 0.29 D, - 0.24 ± 0.29 D
and - 0.20 ± 0.27 D at 1 month, 6 months and 12 months, respectively.23
On the other side, topographic changes and aberrometric changes were
significantly lower in SMILE patients compared with LASIK patients whether
in mild-to-moderate myopia or high myopia as reported by results of our
study (Figs. 4.1A and B and 4.2A and B).
ADVANTAGES OF SMILE IN CASES OF DRY EYE

AND OCULAR SURFACE DISEASE
The flapless nature of SMILE will preserve the important anterior corneal
phase, this will preserve the natural integrity of corneal nerves, which will
significantly influence the ocular surface and tear film stability (Fig. 4.3).
Chapter_04.indd 97 06-02-2018 20:43:28

(A)
(B)
Figs. 4.1A and B: Topographical changes in moderate myopia.
SMILE: Small-incision lenticule extraction; LASIK: Laser assisted in situ keratomileusis.
Central corneal sensitivity exhibited a small decrease and a faster

recovery after the SMILE procedure compared to FS-LASIK during the first
3 postoperative months. Corneal sensitivity after SMILE and FS-LASIK was
similar at 6 months after surgery.24 Qiu et al. in a longitudinal retrospective
study studied 97 consecutive patients (194 eyes) who underwent SMILE
for myopia. Parameters evaluated included: subjective dry eye symptoms
(dryness, foreign body sensation and photophobia), tear film breakup time
(TBUT), Schirmer’s test without anesthesia, tear meniscus height (TMH) and
corneal fluorescein staining. Each parameter was evaluated before, and sub-
sequently at 1 day, 1 week, 1 month and 3 months after surgery. The results
showed that compared with preoperative data, dryness was noted to be sig-
nificantly increased at 1 week and 1 month postoperatively (< 0.01). Symp-
toms of photophobia and foreign body sensation demonstrated significant
differences at 1 day and 1 week as compared with preoperative scores respec-
tively (< 0.01). These values were decreased at 1 month and 3 months post-
surgery (> 0.05). Conversely the corneal staining scores were higher than the
preoperative data at 1 day, 1 week and 1 month (< 0.01), but were close to
Chapter_04.indd 98 06-02-2018 20:43:29

(A)
(B)
Figs. 4.2A and B: Topographical changes in high myopia.
SMILE: Small-incision lenticule extraction; FLEx: Femtosecond lenticule extraction.
Fig. 4.3: Effect of different refractive procedures on the anterior corneal surface.
PRK: Photorefractive keratectomy; LASIK: Laser assisted in situ keratomileusis; SMILE:
Small-incision lenticule extraction.
the preoperative level at 3 months postoperatively. There was a significant

decrease in TMH at 1 week and 1 month (< 0.01), but the value was close to
the preoperative level at 3 months postoperatively (= 0.16). The examination
outcomes of ST were significantly increased at 1 day then reduced at 1 week
Chapter_04.indd 99 06-02-2018 20:43:30

after surgery (< 0.01). Each value subsequently returned to the baseline value
at 1 month and 3 months (> 0.05). TBUT was significantly decreased at all
postoperative time points (< 0.01). It is reported that SMILE resulted in mild
dry eye symptoms, tear film instability and ocular surface damages; however,
these complications can recover in a short period of time.25 This was con-
firmed when compared with FS-LASIK by Li et al. as he reported that SMILE
surgeries resulted in a short-term increase in dry eye symptoms, tear film
instability and loss of corneal sensitivity. Furthermore, SMILE surgeries have
superiority over FS-LASIK in lower risk of postoperative corneal staining and
less reduction of corneal sensation.26
TEAR INFLAMMATORY MEDIATORS IN SMILE

In a study by Gao et al., tears were collected and analyzed for interleukin-6
(IL-6), tumor necrosis factor-alpha (TNF-a), nerve growth factor (NGF) and
intercellular adhesion molecule-1 (ICAM-1) levels using multiplex mag-
netic beads. All measurements were preformed preoperatively and 1 day,
1 week, 1 month and 3 months postoperatively. They reported that in the early
postoperative period, ReLEx SMILE results in milder ocular surface changes
than FS-LASIK. Furthermore, the tear inflammatory mediators IL-6 and NGF
may play a crucial role in the ocular surface healing process following ReLEx
SMILE and FS-LASIK.27 SMILE induces less keratocyte apoptosis, prolifera-
tion and inflammation compared with femtosecond laser LASIK.28
BIOMECHANICAL PROPERTIES OF THE CORNEA IN SMILE

Randleman et al. suggested that the cohesive tensile strength of the stroma is
based on how the stromal lamellae are held together, which decreases from
anterior to posterior within the central corneal region. They used a mathemat-
ical model to predict that the postoperative tensile strength would be higher
after SMILE than both LASIK and PRK, given the fact that the strongest ante-
rior lamellar layer remains intact, enabling it to correct higher levels of myo-
pia with a better safety profile. In our investigation, we studied biomechanical
corneal properties by comparing targeted versus obtained radius of curvature
(Fig. 4.4).
The mean values and standard deviation of the curvature change coeffi-
cient are: [(Paired t-test) SMILE: -1.77 ± 1.72 (%) , FS-LASIK: -1.82 ± 3.76 (%)],
A good correlation for the linear fit: (Pearson Correlation) R = 0.95 for SMILE
group; R = 0.85 for FLEx group. There are not statistically significant differ-
ences (P > 0.1) between two groups. However, the low-standard deviation
of the SMILE group demonstrates a better predictability for this technique
(Figs. 4.5A and B).
Other study used Sheimpflug-based noncontact tonometer, concluded
that no significant modifications in biomechanical properties were observed
Chapter_04.indd 100 06-02-2018 20:43:30

Fig. 4. 4: Mathematical model for calculation of corneal tensile properties.

LASIK: Laser assisted in situ keratomileusis; SMILE: Small-incision lenticule extraction; FLEx: Femto-
second lenticule extraction.
(A)
(B)
Figs. 4.5A and B: Results of biomechanical tensile changes in (A) small-incision lenti-
cule extraction (SMILE) and (B) femtosecond-assisted LASIK (FS-LASIK).
Chapter_04.indd 101 06-02-2018 20:43:31

after SMILE so this procedure could induce only minimal transient alterations
of corneal biomechanics.29 When correlating corneal biomechanical proper-
ties with the induced high-order aberrations. The preoperative chronic renal
failure (CRF) was significantly correlated with the induced 3rd–6th-order
higher-order aberrations (HOAs) and spherical aberration of the anterior
surface and the total cornea after SMILE and FS-LASIK surgeries (P < 0.05),
postoperatively. The CRF was significantly correlated with the induced ver-
tical coma of the anterior and posterior surfaces and the total cornea after
SMILE surgery (P < 0.05). There was a significant correlation between the
CRF and the induced posterior corneal horizontal coma after FS-LASIK sur-
gery (P = 0.013). This indicates that corneal biomechanics affect the surgi-
cally induced corneal HOAs after SMILE and FS-LASIK surgery, which may
be meaningful for screening the patients preoperatively and optimizing
the visual qualities postoperatively.30 On the other hand in high-myopic
patients, FS-LASIK demonstrated a greater increase in posterior corneal ele-
vation than SMILE only at 12 months as well as a greater reduction of CRF
than SMILE, but there were no significant difference between the two groups
over time.31
CONFOCAL MICROSCOPY IN SMILE

In confocal microscopy study, the mean backscattered light intensity (LI) at
all measured depths and the maximum backscattered LI were higher in the
SMILE group than the FS-LASIK group at all postoperative visits. LI differences
at 1-week, 1-month and 3-month visits were statistically significant (P < 0.05).
LI differences at 6 months were not statistically significant. There was no
difference in the number of refractive particles at the flap interface between
the groups at any visit. It may be concluded that SMILE results in increased
backscattered LI in the anterior stroma when compared with FS-LASIK.32 The
decrease in subbasal nerve fiber density was less severe in the SMILE group
than the FS-LASIK group in the first 3 months following the surgery. The sub-
basal nerve density was correlated with central corneal sensitivity.33
CORNEAL CAP PRECISION IN SMILE

There is a significant change in corneal deformation parameters following
SMILE procedure. The changes may be caused predominantly by stromal
lenticule extraction, while lenticule creation with femtosecond laser may
not have an obvious effect on corneal deformation properties.34 A study con-
ducted investigating the morphology of SMILE cap using anterior segment
optical coherence tomography reported that corneal caps of SMILE are pre-
dictable with good reproducibility, regularity and uniformity. Cap morphol-
ogy might have a mild effect on refractive outcomes in the early stage,35 and
the predictability of cap thickness in SMILE surgery does not differ from the
FS-LASIK flaps created using the same femtosecond laser platform.36
Chapter_04.indd 102 06-02-2018 20:43:31

ENHANCEMENTS AFTER SMILE SURGERY

One of the most important challenges facing SMILE technology is the
enhancement methodology in postoperative refractive residuals. In a study
enrolled 28 eyes of which 27 underwent the VisuMaxÒ Circle pattern proce-
dure for refractive enhancement, and 1 for residual lenticule extraction. In all
cases (28 eyes), the lifting of the flap was possible, as planned. In all cases of
refractive enhancement (27 eyes) by LASIK, the exposure of the stromal bed
was sufficient for the necessary excimer laser ablation. No eyes lost two or
more Snellen lines of corrected distance visual acuity (CDVA) and no proce-
dure or flap related complications or serious adverse events occurred. This
initial case series demonstrates that VisuMaxÒ Circle pattern is efficacious
and a suitable method to create a corneal flap for enhancement, following
SMILE.37
INNOVATIVE INDICATIONS OF LASER

LENTICULAR EXTRACTION
• The technique of cryopreservation of corneal lenticules extracted after
small incision ReLEx SMILE and initial results of femtosecond laser intra-
stromal lenticular implantation for hyperopia: The technique seems to be
a safe method of long-term storage of refractive lenticules extracted after
ReLEx SMILE for use in allogeneic human subjects. It may potentially be
a safe and effective alternative to excimer laser ablation for hyperopia
because of the low risks of regression, haze, flap-related complications,
postoperative dry eye and HOAs.38
• ReLEx SMILE Xtra, SMILE with accelerated cross-linking; in patients with
thin corneas and borderline topography: Based on the initial clinical out-
come it appears that SMILE Xtra may be a safe and feasible modality to
prevent corneal ectasia in susceptible individuals.39 Also this has been
investigated in forme fruste keratoconus and irregular corneas, combined
SMILE and intrastromal corneal collagen crosslinking are a promising
treatment option for patients for whom conventional laser refractive sur-
gery is contraindicated.40
• Finally, a feasibility study reported that LASIK can be performed follow-
ing lenticule reimplantation to create presbyopic monovision. The tissue
responses elicited after performing LASIK on corneas that have under-
gone SMILE and subsequent lenticule reimplantation are similar to pri-
mary procedure.41
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35. Zhao J, Yao P, Li M, et al. The morphology of corneal cap and its relation to
refractive outcomes in femtosecond laser small-incision lenticule extraction
(SMILE) with anterior segment optical coherence tomography observation.
PLoS One. 2013;8(8):e70208.
36. Ozgurhan EB, Agca A, Bozkurt E, et al. Accuracy and precision of cap thickness
in small-incision lenticule extraction. Clin Ophthalmol. 2013;7:923-6.
37. Chansue E, Tanehsakdi M, Swasdibutra S, et al. Safety and efficacy of VisuMaxÒ
circle patterns for flap creation and enhancement following small- incision
lenticule extraction. Eye Vis (Lond). 2015;2:21.
38. Ganesh S, Brar S, Rao PA. Cryopreservation of extracted corneal lenticules after
small-incision lenticule extraction for potential use in human subjects. Cornea.
2014;33(12):1355-62.
39. Ganesh S, Brar S. Clinical outcomes of small-incision lenticule extraction with
accelerated cross-linking (ReLEx SMILE Xtra) in patients with thin corneas and
borderline topography. J Ophthalmol. 2015;2015:263-412.
40. Graue-Hernandez EO, Pagano GL, Garcia-De la Rosa G, et al. Combined
small-incision lenticule extraction and intrastromal corneal collagen cross-
linking to treat mild keratoconus: long-term follow-up. J Cataract Refract Surg.
2015;41(11):2524-32.
41. Lim CH, Riau AK, Lwin NC, et al. LASIK following small-incision lenticule ex-
traction (SMILE) lenticule re-implantation: a feasibility study of a novel method
for treatment of presbyopia. PLoS One. 2013;8(12):e830-46.
Chapter_04.indd 106 06-02-2018 20:43:32

5
CHAPTER
LASIK in Hyperopia
VK Raju, Stephen Hilton, G Madhavi
INTRODUCTION
The surgical correction of hyperopia is still in evolution. To correct hyper-
opia surgically, the surgeon has following options: (1) steepening the cen-
tral cornea with excimer laser, (2) steepening the central cornea by collagen
shrinkage, (3) adding a lens of particular power within the cornea or inside
the eye and (4) exchanging the patient’s crystalline lens with one of a dif-
ferent power. In prelaser era thermokeratoplasty, hexagonal keratotomy,
automated lamellar keratoplasty and epikeratophakia were used to correct
hyperopia. However, the procedures are no longer in clinical practice. These
procedures had limited success because they were either difficult to perform,
unstable, unpredictable or caused irregular astigmatism. The surgical correc-
tion of hyperopia has tended to lag behind the advances achieved in treating
myopia even though hyperopia is more common. A 30-year-old patient with
myopia is more likely to seek surgical correction than a 30-year-old patient
with hyperopia, whose accommodative effort achieves good unaided vision
despite the optical error. By the time, hyperopic patients are symptomatic
enough to seek surgical correction, they are older than the corresponding
myopic group. In addition, the hyperopic eye is anatomically different from
the myopic eye. The eye has a shorter axial length, a smaller anterior seg-
ment with narrow angle and smaller corneal diameter and has a tendency for
angle-closure glaucoma due to progressive enlargement of the lens.
SURFACE ABLATIVE PROCEDURES

Steepening the central cornea by surface ablation using photorefractive kera-
tectomy (PRK) or laser epithelial keratomileusis (LASEK) is occasionally per-
formed today.
Chapter_05.indd 107 06-02-2018 20:51:16

Hyperopic LASIK
Early work on hyperopic steepening was with PRK. It involved smaller abla-
tion zones, and preceded the development of tracking. This led to several
problems leading to poor results: midperipheral haze, induced irregular
astigmatism, regression of refractive effect and surgical decenteration.1 Most
of these problems have been overcome with better quality lasers and a better
understanding of the ablation zones required. Hyperopic PRK is still limited
by the large epithelial defect created. This leads to prolonged healing of the
epithelial defect, consequent delayed visual recovery, discomfort and risk of
infection during this period.2 Surface ablation of hyperopia is currently used
on a very limited basis. Epi-LASIK is relatively a new procedure in which an
epithelial flap is created with a keratome.
In correcting hyperopia by LASIK, the cornea needs to be sculpted into
steeper convex lens.3 This is achieved by a peripheral annular ablation with
flattening around the central optical zone which produces central steepen-
ing. It requires larger diameter ablations than myopic treatments (9–9.5 mm).
It is difficult to maintain a homogeneous distribution of energy over a large-
beam cross-section. For this reason, most early lasers were restricted in their
ablation diameters to 6 mm. Various techniques were employed to over-
come this, however, newer developments have the ability to deliver corneal
steepening. Firstly a small beam, flying-spot lasers are able to create an abla-
tion profile out to 9–10 mm. Secondly the tracking of eye movements delivers
the pulse to the correct location. Larger diameter hyperopic ablations take
longer than the equivalent myopic corrections; consequently, eye move-
ments may be more likely to adversely affect the result.4 The LASIK method
for steepening the central cornea is the predominant refractive procedure
for hyperopia up to 4.00 diopters. By applying the midperipheral ablation
under a corneal flap, the surgeon achieves a refractive correction that may be
more stable and predictable than that obtained by PRK. Surface healing and
epithelial hyperplasia are kept to a minimum. A good result is dependent
on the ability to place the entire ablation beneath a corneal cap diameter
larger than the ablation zone. This presents technical challenges because the
hyperopic eye is usually deep set with a smaller corneal diameter.
COMPLICATIONS OF HYPEROPIC LASIK

Flap Complications
Serious complications are rare. Complications arising from the cutting of the
flap are more common in hyperopic LASIK than myopic LASIK. Free flaps,
partial flaps, striae, epithelial defects, epithelial ingrowth diffuse lamellar
keratitis and infection can occur (Fig. 5.1). The microkeratome must predict-
ably produce large diameter cap sizes of 9.5–10.5 mm (Fig. 5.2). A deep-set
eye with small corneal diameter, and very flat or very steep keratometry, if
Chapter_05.indd 108 06-02-2018 20:51:16

LASIK in Hyperopia 109
Fig. 5.1: Epithelial ingrowth after enhancement of hyperopic laser assisted in situ ker-
atomileusis (LASIK).
Fig. 5.2: Large cap in hyperopic laser assisted in situ keratomileusis (LASIK): bleeding
may be encountered. Do not lift the flap until the bleeding stops.
not recognized preoperatively, may lead to serious problems. Large, flat cor-
neas can produce small flaps, which may not be able to encompass a hyper-
opic ablation and small, steep corneas may produce a button hole. Suction
may be more difficult to obtain with any given microkeratome in hyperopic
Chapter_05.indd 109 06-02-2018 20:51:17

LASIK. Very flat and very steep corneas may be contraindication for LASIK.
Hyperopic LASIK patients are generally older, they are more at risk for epi-
thelial defects at the time of surgery, slow recovery and subsequent epithe-
lial ingrowth. Techniques to reduce the epithelial damage in the older age
group include: minimizing the use of topical anesthetic, minimizing the time
between topical anesthesia and keratectomy and keeping the surface moist.
Large flaps in small diameter corneas lead to more intraoperative bleeding.
Neurotrophic epitheliopathy is more likely to produce problems in older
patients who already have marginal dry eyes. Punctal plugs should be used
for managing the dry eye condition.
Ablation-related Complications
LASIK in hyperopics makes central cornea steeper than paracentral cornea.

This is achieved by paracentral flattening of the cornea leaving the central
cornea minimally treated by the laser and the peripheral cornea untreated.
The resulting complex optical shape means that there is more multifocality of
the cornea. This may lead to a little drop in visual acuity, but can give presby-
opic patients better unaided near vision than expected. In hyperopic LASIK,
greater amount of tissue is removed per diopter of correction, compared to
myopic LASIK. A + 3.00 diopter hyperopic correction will take longer abla-
tion than a - 3.00 diopter myopic correction. If active eye tracking is not used,
significant decentration and secondary irregular astigmatism may result.
The volume of tissue removed may also have a role in the predictability of
outcome. The 250 mm rule if residual corneal stroma should be applied to
mid-periphery where the maximum tissue is removed. Hinge protection is
important, as ablation of the undersurface of the hinge may result in irregu-
lar astigmatism. Hyperplasia of the paracentral epithelium tends to fill in the
ablated paracentral zone. This is probably the cause for increased postopera-
tive regression and slower stabilization of refraction. In addition, the steeper
central zone can demonstrate subepithelial haze (Fig. 5.3). This complication
is more common where the central cornea has been left excessively steep and
there is poor quality tear film.
Strategies to obtain accurate intraocular pressure, measurements are an
active area of research. Attention should be paid to the optic disc and visual
fields changes in the postoperative follow-ups. When treatment is based
on refraction, it must be remembered that in hyperopic eyes the values
increase from vertex to the corneal plane, which is opposite of the myopic
eyes.
Management of Complications
The best management of complications is avoidance. Prior proper plan-

ning prevents poor performance. The following points have to be taken into
Chapter_05.indd 110 06-02-2018 20:51:17

Fig. 5.3: Subepithelial haze in hyperopic laser assisted in situ keratomileusis (LASIK).
considerations: (1) patients should be fully informed about all possible com-
plications, (2) eye tracking is essential, (3) spherical corrections of greater
than + 4.00 diopters at corneal plane are not advised, (4) optical zone should
be as large as possible and should dictate the flap size, (5) a large uniform
flap should be created, (6) beware of preoperative keratometry of less than
40.00 D or greater than 47.00 D and (7) center the ablation on the pupil-
lary entrance. Intraoperative problems should be meticulously tackled and
if operative problems occur, consider the following: (1) in the situation of
incomplete flaps, abandon or postpone laser ablation, (2) allow at least 3–6
months to achieve refractive stability before considering surgery again. Doc-
ument the stability, (3) remember the keratometry readings and the stomal
depth. Do not steepen the cornea beyond 50.00 D and always leave 250 mm
of the stromal bed and (4) treat dry eye symptoms aggressively with punctal
plugs and preservative free tear substitutes.
Hyperopic LASIK in special situations:
• Undercorrected hyperopic
• LASIK 2. Overcorrected myopic LASIK
• Overcorrected radial keratotomy
• Correcting hyperopia after previous cataract and implant surgery
Undercorrected Hyperopic LASIK
Stability of refraction needs to be established before proceeding with

enhancement. Hyperopic LASIK takes longer to stabilize than myopic LASIK.
Chapter_05.indd 111 06-02-2018 20:51:17

If the K reading is approaching 49.00–50.00 then it may not be wise to enhance

with corneal surgery. This can lead to discomfort and loss of best corrected
visual acuity. Individual nomograms need to be established. Generally, if an
enhancement for an undercorrected hyperopic LASIK is required, then the
same nomogram is used.
Overcorrected Myopic LASIK
Treating overcorrected myopic, LASIK presents challenge because the cor-

neal cap diameter which may have been adequate for myopic ablation may
not be sufficiently large enough to encompass a hyperopic ablation. Nomo-
grams need to be adjusted. Some series have suggested that standard nomo-
grams are adequate. Others suggest that overcorrected myopic LASIK patients
will get more effect than expected from a secondary hyperopic LASIK proce-
dure. This may be particularly so if the cornea is relatively thin after the initial
procedure. One should approach these eyes with caution.
Overcorrected Radial Keratotomy
Overcorrected radial keratotomy cases are particularly challenging. Ongoing

hyperopic drift can occur for many years or sometimes indefinitely.5 These
eyes are at increased risk of corneal flap complications. Surface laser treat-
ment is not a reasonable option because of the higher incidence of haze and
scarring.5 Topical mitomycin may be effective in minimizing the haze.
Hyperopia after Previous Cataract and Implant Surgery
Generally, this group of patients is in the range of + 1.00 to + 2.00 D. These are
older patients with high incidence of dry eye. They are at high risk of LASIK
induced dry eye symptoms which can be serious. Collagen shrinkage proce-
dures (laser thermal keratoplasty or conductive keratoplasty) may be a better
alternative.
CONCLUSION
For hyperopia, hyperopic LASIK is predominantly the procedure of choice
in most settings. The fact that a range of procedures are available suggests
that clinical decision making is still very important in selecting the best oper-
ation procedure for an individual patient.7,8 As discussed earlier, issues of
tear film, corneal and orbital anatomy, anterior chamber depth, magnitude
of hyperopia and preexisting pathology should be considered before making
a decision. If a conservative approach is adopted, the surgical correction of
hyperopia is worthwhile and rewarding. It will improve with further refine-
ments in laser delivery, ablation patterns and improved corneal contours in
conjunction with wavefront-based analysis and wavefront-guided treatment.9
Chapter_05.indd 112 06-02-2018 20:51:17

The patient–physician relationship is at the core of what we do as doctors.10

Refractive surgery remains one of the most exciting and rapidly developing
fields in ophthalmology. The corrective optics and anatomical characteris-
tics of hyperopia remain a challenge. The market for refractive surgery is con-
sumer driven and potentially very large. While it will likely remain a lucrative
area, it is important to remind ourselves that it is still an elective procedure
with associated benefits and pitfalls.
REFERENCES
1. Lawless MA. Surgical correction of hyperopia. Focal Points: Clinical Modules for
Ophthalmologists. Am Acad Ophthalmol. 2004;22(5):1-14.
2. Varley GA, Huang D, Rapuano CJ, et al. LASIK in hyperopia, hyperopic astigma-
tism, and mixed astigmatism: a report by the American Academy of Ophthal-
mology. Ophthalmology. 2004;111(8):1604-17.
3. Azar DT, Primack JD. Theoretical analysis of ablation depths and profiles in laser
in situ keratomileusis for compound hyperopic and mixed astigmatism. J Cata-
ract Refract Surg. 2000;26(8):1123-36.
4. Cobo-Soriano R, Llovet F, González-López F, et al. Factors that influence out-
comes of hyperopic laser in situ keratomileusis. J Cataract Refract Surg.
2002;28(9):1530-8.
5. Lindstrom RL, Linebarger EJ, Hardten DR, et al. Early results of hyperopic
and astigmatic laser in situ keratomileusis in eyes with secondary hype-
ropia. Ophthalmology. 2000;107:1858-63.
6. Francesconi CM, Nose RA, Nose W. Hyperopic laser assisted in situ kerato-
mileusis for radial keratotomy induced hyperopia. Ophthalmology. 2002;
109(3):602-05.
7. McGhee CN, Ormonde S, Kohnen T, et al. The surgical correction of moder-
ate hypermetropia: the management controversy. Br J Ophthalmol. 2002;
86(7):815-22.
8. Salz JJ, Stevens CA, LADARVision LASIK Hyperopia Study Group. LASIK cor-
rection of spherical hyperopia, hyperopic astigmatism, and mixed astig-
matism with the LADARVision excimer laser system. Ophthalmology. 2002;
109(9):1647-56.
9. Preferred Practice Patterns Committee, Refractive Errors Panel. Refractive Er-
rors. Preferred Practice Pattern. Online. 2002.
10. Raju VK. Refractive Surgery Advertising. J Refract Surg. 2001;17(2):152-3.
Chapter_05.indd 113 06-02-2018 20:51:17

6
CHAPTER
LASIK—Complications
and Management
Rajesh Fogla, Srinivas K Rao, Prema Padmanabhan
INTRODUCTION
Laser in situ keratomileusis (LASIK) is a procedure involving creation of a cor-
neal flap followed by reshaping of the stromal bed with the 193 nm argon flu-
oride excimer laser. It is currently the most commonly performed procedure
for the correction of refractive errors like myopia, astigmatism and hyperopia.
Rapid visual recovery, minimal postoperative discomfort and reduced sub-
epithelial haze are important advantages that LASIK has over surface photo-
refractive keratectomy (PRK).1 However, LASIK exposes the eye to the risks
of creating a flap with the microkeratome, which include creation of button
holes, free caps and thin incomplete or irregular flaps.2 Decentered ablations,
central islands, epithelial ingrowth and infections can often produce irregu-
lar astigmatism with loss of best corrected visual acuity.3 Major studies have
reported that the rate of postoperative complications after LASIK is between
1.8 and 2.6%.4,5 Complications following LASIK can be divided into intraop-
erative complications, early postoperative complications, presenting within
first few days after the procedure and late postoperative complications pre-
senting weeks to months after the surgery.
INTRAOPERATIVE COMPLICATIONS
Partial flap: A partial corneal flap is produced when the microkeratome head
does not complete its full excursion (Fig. 6.1). Farah et al, in their study, have
reported a partial or incomplete flap in up to 3% of cases.6 Various reasons
have been postulated for incomplete pass of microkeratome resulting in a
partial or incomplete flap. These include: (i) power failure, (ii) inadequate
exposure of the globe, (iii) interference by the eyelid, speculum or drape,
(iv) debris along the track and (v) loss of suction during the procedure.4
Chapter_06.indd 114 06-02-2018 20:56:30

LASIK—Complications and Management 115
Fig. 6.1: Incomplete or partial flap.
Management of a partial flap depends on the extent of the uncut flap and
location of the hinge of the reflected flap. If the reflected flap hinge is at the
periphery of the planned treatment zone, the flap hinge can be shielded
with a moist sponge during laser ablation. Besides this one may also con-
sider decreasing the size of the treatment zone. However, this often results in
irregular astigmatism, probably due to the tethering effect of the hinge on the
adjacent cornea. Manual dissection of the flap to its full extent using lamel-
lar dissection should be avoided.5 If the hinge is in the central cornea, the
best option is the reposition the flap and postpone the laser procedure to a
later date. Although, retreatment has been recommended after a duration of
3 months, it can be performed safely as early as 1 month with satisfactory
results comparable to the fellow eye.7 A deeper cut is usually recommended
during retreatment.5 Smaller diameter corneal flap of original thickness can
prevent the formation of sliver of corneal tissue at the flap edge besides a
thicker residual posterior stromal bed.
Partial flap:
• Avoid manual dissection of the flap
• Abort refractive procedure
• Replace flap
• Retreatment 1–3 months later
Flap button holes (Doughnut flap) and thin flap: Thin flap, flap button hole
or doughnut-shaped flap is produced as a result of surfacing of the blade
during creation of the corneal flap (Fig. 6.2). Important risk factors for this
complication include: (i) steep cornea (preoperative keratometry of greater
than 48.0 D), (ii) previous penetrating keratoplasty, (iii) lack of blade sharp-
ness and (iv) sudden reduction of intraocular pressure during passage of
the microkeratome.2 Increased resistance to the oscillatory motion of the
microkeratome blade due to microkeratome malfunction has also been
responsible for this complication.8 The thin irregular flap is repositioned
carefully and protected with a bandage contact lens. Flap button hole is often
Chapter_06.indd 115 06-02-2018 20:56:30

Fig. 6.2: Doughnut flap.
Fig. 6.3: Free flap.
associated with scarring and epithelial ingrowth into the interface, which
may complicate the retreatment at a later date.2 Transepithelial PRK is usu-
ally recommended for the retreatment.2 This is performed when the flap
has healed completely and the refraction is stable for a duration of 3–4 weeks.
This approach usually eliminates subepithelial scarring and epithelial
ingrowth associated with flap button holes. In some cases, a rigid gas perme-
able lens may be necessary to improve vision.
Thin or doughnut flap:
• Replace flap
• Postpone ablation
• Protect with bandage contact lens
• Transepithelial PRK for retreatment
Free cap: Free cap is produced when the corneal flap is cut all 360° without
a hinge (Fig. 6.3). This is often associated with flat corneas, keratometry less
than 38.0 D.2 It can also occur due to reduced IOP and incorrect setting of
the stop in the microkeratome (more common with the Automated Corneal
Chapter_06.indd 116 06-02-2018 20:56:30

Shaper, Chiron Technologies). When this complication occurs, it is extremely

important to locate the free cap as it can sometimes be trapped inside the
microkeratome. The free cap should be placed in a closed antidesiccation
chamber, epithelial side down, on one drop of balanced salt solution (BSS).
If the free cap is of normal thickness, excimer laser ablation can still be per-
formed. The flap should be placed in its original position using the initial ref-
erence marks as guides, with the epithelial side facing up. It can usually be
secured in position using a bandage contact lens. In rare cases, sutures may
be necessary, especially if the flap is easily displaced. These sutures need to
be removed early to minimize scarring.
Corneal perforation: Corneal perforation is a catastrophic complication of
LASIK resulting from a faulty microkeratome assembly. This occurs if the
depth plate is positioned incorrectly, especially with instruments such as the
Automated Corneal Shaper (Chiron Technologies). The damage occurring
with this complication is magnified by the high IOP (> 60 mm Hg) induced
by the suction ring and the sudden drop in IOP following perforation. If it
occurs, it can lead to vitreoretinal complications, besides damage to the iris,
lens and ciliary body.9 If perforation is noted with appearance of aqueous,
suction should be immediately deactivated to relieve pressure on the globe.
Newer designs of microkeratome such as the Hansatome (Bausch & Lomb)
have a fixed depth plate to avoid this complication.
Decentered flap: Decentration of the corneal flap indicates improper align-
ment of the suction ring of the microkeratome (Fig. 6.4).2 Improper align-
ment can occur due to the error made by the surgeon in marking the center of
the pupil. Migration of the suction ring on the corneal surface, as the suction
is applied, can also produce decentered flaps, especially with poor patient
cooperation. Therefore, the surgeon should release the suction as soon as the
misalignment is noted. Delayed release of vacuum results in the formation
of a gutter in the conjunctiva which tends to return the suction ring to the
original position on repeat attempts. In such cases, the procedure should be
Fig. 6.4: Decentered flap.
Chapter_06.indd 117 06-02-2018 20:56:31

Fig. 6.5: Intraoperative bleeding.
postponed to another day. If the corneal flap is decentered slightly, the abla-
tion can still be performed, with a reduction in the size of optic zone. If the
zone of ablation is likely to extend beyond the edge of the cut, the flap should
be replaced and the procedure postponed for 3–4 months.
Intraoperative bleeding: Intraoperative bleeding occurs at the edge of
the corneal flap from the perilimbal vessels, particularly with large diame-
ter flaps (Fig. 6.5). Long-term contact lens wear is usually associated with a
corneal pannus. This should be noted in the preoperative evaluation. Topi-
cal vasoconstrictors like naphazoline can be applied 3–5 minutes before the
procedure. Decentration of the microkeratome can also produce an eccentric
flap with bleeding from the perilimbal vessels. If bleeding occurs, one should
wait for a few minutes before lifting the flap. Persistent bleeding complicates
the procedure with respect to intraoperative visualization, altered stromal
hydration and postoperative interface opacification. The bleeding usually
stops spontaneously and the procedure can be performed as planned. In
cases with persistent bleeding, a Chayet sponge ring can be placed around
the limbus during ablation, which absorbs the blood from the edge.
Epithelial defect: Epithelial defect occurs in a small percentage of patients
undergoing LASIK (< 3%). It is frequently noted at the flap edge, and is possibly
related to the manipulation of the corneal flap or the microkeratome pass.
Excessive use of topical anesthetic agents or preexisting anterior basement
membrane dystrophy are risk factors that predispose to the development of
epithelial defect. Hence, one should consider PRK in patients with history
of recurrent corneal erosion syndrome or anterior basement membrane
dystrophy. Management of epithelial defect includes careful handling of the
corneal flap and repositioning the epithelium to its original position to the extent
possible at the end of the procedure. A bandage contact lens is recommended
to reduce patient discomfort and hasten epithelial healing. There are two
potential complications that may ensue with epithelial defects: (i) infection and
(ii) epithelial ingrowth. Hence, it is important to look out for these
Chapter_06.indd 118 06-02-2018 20:56:31

complications in the postoperative period. Prophylactic antibiotics should be

used after the operations. Topical steroids can be started once the epithelial
defect heals completely.
Epithelial defect:
• Reposit epithelium and place BCL
• Avoid topical steroids
• Watch for infection/epithelial ingrowth
• Consider PRK for fellow eye
EARLY POSTOPERATIVE COMPLICATIONS

Diffuse lamellar keratitis (DLK): DLK or ‘Sands of Sahara’ is a syndrome
characterized by diffuse or multifocal lamellar infiltrates of noninfectious eti-
ology confined to the flap interface after LASIK (Fig. 6.6).10,11 The exact eti-
ology is unknown, although an allergic or toxic reaction is thought to be the
most likely cause.12 The list of inciting agents includes talc from gloves, debris
from the microkeratome blade, toxins liberated by microorganisms and mei-
bomian secretions.11-13 Clinically, the patient presents with pain, photopho-
bia, foreign body sensation and reduced vision. DLK has been described in
four grades:
Grade I: White granular cells in the periphery of the interface
Grade II: White granular cells in the center of the interface
Grade III: Dense aggregates of clumped cells centrally with relative clearing
peripherally and
Grade IV: Scarring and stromal melting14
These diffuse sterile interface infiltrates are usually noticed within 2–6
days of LASIK. Intensive topical steroid therapy leads to resolution of these
infiltrates in most cases of grades I and II severity. In severe cases, it can
be associated with stromal necrosis and melting of the corneal flap. If pro-
gression is noted despite intensive corticosteroid treatment, the flap should
Fig. 6.6: Diffuse lamellar keratitis.
Chapter_06.indd 119 06-02-2018 20:56:31

Fig. 6.7: Flap striae.
be lifted and the bed should be cleaned with Merocel sponges along with
irrigation of the interface. Microbiological investigations should also be per-
formed in these severe cases to rule out any infectious etiology.
Flap wrinkling/striae: Flap wrinkling or striae are not an uncommon com-
plication after LASIK (Fig. 6.7).15 They are most commonly related to the dis-
parity in curvature between the posterior surface of the flap and the stromal
bed after ablation, therefore, seen more often following correction of high
myopic errors.16 Flap striae may also result from improper alignment of the
flap after replacement or movement of the corneal flap due to some manip-
ulation in the postoperative period, such as patient eye-rubbing or pressure
on the eye while sleeping without a protective eye shield. Other predisposing
factors include increased stromal hydration with poor flap adherence and
lagophthalmos.17
Clinically, wrinkles in the stromal tissue of the flap are easily identified
by retroillumination. Peripheral wrinkles may not affect the best corrected
visual acuity (BCVA) and, therefore, can be left alone. However, wrinkles
in the visual axis are likely to affect the BCVA and should be eliminated as
soon as they are identified to prevent permanent irregular astigmatism. It
is essential to lift the flap in the management of flap wrinkles. The flap is
Chapter_06.indd 120 06-02-2018 20:56:32

then hydrated using either isotonic or hypotonic saline solution, followed by

stretching of the posterior surface of the flap with a rounded spatula.2,15-17
Once the flap is repositioned, the surface is further smoothened using a
Merocel sponge. A bandage contact lens is then placed to prevent displace-
ment of the hydrated flap. The wrinkles will still be evident at the end of the
procedure but will resolve by the next day. Some surgeons have advocated to
remove the epithelium overlying the striae to release the traction. Persistent
striae may require suturing of the flap edge to stretch the flap and remove
wrinkles.17
Flap displacements: Flap displacement occurs most commonly in the
first 24 hours after LASIK.2 This may result from dryness due to inadequate
blinking, stromal edema from excessive irrigation, unstable free cap, epithe-
lial trauma or rubbing of the eye.17 Late displacement of the flap has been
reported following trauma.18 Management of flap displacement includes,
flap repositioning, removal of the epithelium from the stromal bed if present
and placement of contact lens or sutures to hold the flap in current position.
Prompt recognition and early surgical intervention are crucial to achieve a
good outcome. It is essential to advise patients to wear safety glasses in any
environment where injury to the eye is possible for several weeks after LASIK.
Interface debris: A variety of exogenous materials including talc, metallic
shavings from the microkeratome blade, lint, fibers of the Merocel sponge,
mucus or oil from the tear film may be introduced into the interface during
LASIK (Fig. 6.8).17 Inert particles that do not produce any inflammation
or surface irregularities can be left alone. However, foreign material in the
pupillary zone needs to be removed. The flap can be lifted followed by
removal of the debris.19-21
Fig. 6.8: Interface debris.
Chapter_06.indd 121 06-02-2018 20:56:32

Fig. 6.9: Interface infiltrates.
Infections: Infection is vision threatening and uncommon complication

after LASIK. Risk factors include trauma and excessive topical corticosteroid
usage. Infections have been reported with microbial agents including virus,
bacteria and fungus.19-21 If an infiltrate is noted in the postoperative period,
it should be treated as infection until proven otherwise (Fig. 6.9). The deeper
location of the microbial infection under the corneal flap makes it difficult to
obtain material for microbiological analysis and reduce the penetration of
the antimicrobial agents. The corneal flap should be lifted to obtain sufficient
material for microbiological analysis. The stromal bed and the undersurface
of the corneal flap can be scraped to debulk the infective load, followed by
irrigation of the interface using appropriate antibiotics. Intensive topical
treatment with fortified antibiotics is mandatory until the infection is con-
trolled. Removal of the corneal flap and penetrating keratoplasty may be
required in advanced cases.
LATE POSTOPERATIVE COMPLICATIONS

Epithelial ingrowth: Epithelial ingrowth is a potential complication of all
lamellar refractive surgical procedures (Fig. 6.10). Ectopic epithelium may
be implanted during flap creation, especially in advanced surgery or may
result from direct ingrowth into the lamellar interface at the edge of the flap.
Incidence of epithelial ingrowth ranges from 1.5–3.5%.9 However, the rates
of epithelial ingrowth have been reported to be as high as 31.4% after hyper-
opic LASIK.15 Potential risk factors for epithelial ingrowth include: (i) poor
flap edge adhesion, (ii) epithelial defects, (iii) flap misalignment, (iv) reuse
of microkeratome blade and (v) flap button holes. Clinical appearance of
epithelial ingrowth varies from peripheral nests of epithelial cells to a cen-
tral peninsula of epithelial pearls extending from the flap edge. Involvement
of the visual axis results in irregular astigmatism and poor visual acuity.
Chapter_06.indd 122 06-02-2018 20:56:32

Fig. 6.10: Epithelial ingrowth (arrow).
Corneal topography shows an irregular pattern corresponding to the involved

area. Keratolysis or melting of the flap is a potential complication of epithelial
ingrowth. The stromal bed, therefore, must be meticulously cleaned of any
epithelial debris before repositioning the flap.3
Management of epithelial ingrowth encroaching on the central cor-
nea involves lifting of the corneal flap followed by debridement of both the
stromal surface and the undersurface of the flap using a spatula or scalpel
blade.22 Manual debridement can be augmented with ablation of both the
stromal bed and the posterior surface of the flap using the excimer laser to
remove the epithelial cells that cannot be seen.5 Small nests of epithelium
in the peripheral cornea can usually be left alone if it is not progressing or
affecting visual acuity.23,24
Keratectasia: Iatrogenic keratectasia is a potential complication after LASIK,
occurring especially in cases where less than 200 microns thick stromal bed
was left after ablation.23 In advanced cases, it resembles a keratoconus like
clinical appearance. It appears that a residual stromal bed of at least 250
microns following ablation is essential to prevent this complication, although
it has been reported to occur in cases with residual bed of greater than 250
microns. Keratectasia occurs with a much more rapid clinical progression
following LASIK in cases of forme fruste keratoconus.24 Altered biomechan-
ical properties of the cornea following LASIK may be responsible for this
complication. Corneal topography reveals progressive steepening at the site
of the pathology. Management includes a rigid gas permeable contact lens
trial to improve visual acuity. Severe cases may require epikeratoplasty or
penetrating keratoplasty. Therefore, it is extremely important to evaluate the
cornea to detect topographic abnormalities before performing refractive sur-
gery. A residual posterior stromal bed of 250–300 microns is recommended to
prevent keratectasia after LASIK.
Chapter_06.indd 123 06-02-2018 20:56:33

Fig. 6.11: Decentered abalation.
Fig. 6.12: Central island.
Decentered Ablations: This complication is seen more commonly in the

early part of the learning curve of the surgeon (Fig. 6.11).5 All refractive
surgical procedures should be centered on the entrance pupil. Decentra-
tion occurs if the laser mires are not aligned properly on the entrance pupil.
Patient’s head position and fixation are also critical in proper centration of
the laser procedure. One should also ensure that the patient is able to visual-
ize the fixation target before the procedure. Use of eye tracking systems can
assist in reducing this complication. With the advent of customized abla-
tions, centration of the laser ablation is absolutely essential for optimal visual
outcome.
Central islands: Central island after LASIK is either due to inhomogene-
ity of the laser ablation or uneven hydration of the stromal bed (Fig. 6.12).10
Obstruction to the laser energy from the plume of the ejected corneal tissue
may also have a role to play in its formation. It is more common with the broad
beam laser systems compared to the scanning or flying spot lasers. Patient
Chapter_06.indd 124 06-02-2018 20:56:33

symptoms include glare, ghostings or undercorrections. Central islands fol-

lowing LASIK have a little tendency to resolve spontaneously as compared
to PRK. Retreatment can be performed using phototherapeutic keratectomy
under the flap, however, there is a tendency for hyperopic shift after the
procedure.
Regression and haze: Regression is uncommon after LASIK, possibly due to
preservation of the Bowman’s layer, unlike PRK.5 Higher refractive errors are
more likely to regress than lower refractive errors. Enhancement procedures
can be attempted once the refraction is stable, provided there is adequate
thickness of residual stromal tissue. Haze occurs rarely following LASIK.
Interface haze can occasionally be seen in the early postoperative period that
responds well to topical steroids.25,26
Retinal complications: Although it has been hypothesized that application
of the suction ring of the microkeratome produces: (i) a rise in intraocular
pressure and (ii) may exert traction on the vitreous base, there is no evidence
that LASIK induces retinal complications. Eyes with high myopia are anyway
at a greater risk of retinal detachment. The incidences of retinal complica-
tions reported in studies of LASIK are consistent with incidences reported in
patients with myopia who have not had LASIK.25,26 Hence, a detailed evalua-
tion of the fundus is essential, both preoperative and at subsequent follow-up
visits, to detect retinal breaks and areas of lattice degeneration and treat them
at an early stage.
REFERENCES
1. Pallikaris IG, Siganos DS. Excimer laser in situ keratomileusis and photo-
refractive keratectomy for correction of high myopia. J Refract Corneal Surg.
1994;10(5):498-510.
2. Ambrósio R Jr, Wilson SE. Complications of laser in situ keratomileusis: etiology,
prevention, and treatment. J Refract Surg. 2001;17(3):350-79.
3. Iskander NG, Peters NT, Penno EA, et al. Postoperative complications in
laser in situ keratomileusis. Curr Opin Ophthalmol. 2000;11(4):273-9.
4. Gimbel HV, Penno EE, van Westenbrugge JA, et al. Incidence and management
of intraoperative and early postoperative complications in 1000 consecutive
LASIK cases. Ophthalmology. 1998;105(10):1839-47.
5. Wilson SE. LASIK: management of common complications. Cornea. 1998;
17(5):459-67.
6. Farah SG, Azar DT, Gurdal C, et al. Laser in situ keratomileusis: literature review
of a developing technique. J Cataract Refract Surg. 1998;24(7):989-1006.
7. Rao SK, Padmanabhan P, Sitalakshmi G, et al. Partial flap during laser in situ
keratomileusis: pathogenesis and timing of retreatment. Ind J Ophthalmol.
2000;48(3):209-12.
8. Leung AT, Rao SK, Cheng AC, et al. Pathogenesis and management of laser
in situ keratomileusis flap buttonhole. J Cataract Refract Surg. 2000;26(3):
W358-62.
Chapter_06.indd 125 06-02-2018 20:56:33

9. Pallikaris IG, Siganos DS. Laser in situ keratomileusis to treat myopia: early ex-
perience. J Cataract Refract Surg. 1997;23(1):39-49.
10. Iskander NG, Peters NT, Penno EA, et al. Postoperative complications in
laser in situ keratomileusis. Curr Opin Ophthalmol. 2000;11(4):273-9.
11. Smith RJ, Maloney RK. Diffuse lamellar keratitis: a new syndrome in lamellar
refractive surgery. Ophthalmology. 1998;105(9):1721-26.
12. Alió JL, Perez-Santonja JJ, Tervo T, et al. Postoperative inflammation, microbial
complications, and wound healing following laser in situ keratomileusis. J Re-
fract Surg. 2000;16(5):523-38.
13. Fogla R, Rao SK, Padmanabhan P. Diffuse lamellar keratitis: are meibomian se-
cretions responsible. J Cataract Refract Surg. 2001;27(4):493-5.
14. Linebarger EJ, Hardten DR, Lindstrom RL. Diffuse lamellar keratitis: diagnosis
and management. J Cataract Refract Surg. 2000;26(7):1072-7.
15. Pannu JS. Incidence and treatment of wrinkled corneal flap following LASIK. J
16. Probst LE, Machat J. Removal of flap striae following laser in situ keratomileusis.
J Cataract Refract Surg. 1998;24(2):153-5.
17. Gimbel HV, Peters NT, Iskander NG, et al. Laser in situ keratomileusis: flap com-
plications and management. J Refract Surg. 2000;16(2 Suppl):S223-5.
18. Lemley HL, Chodosh J, Wolf TC, et al. Partial dislocation of laser in situ ker-
atomileusis flap by air bag injury. J Refract Surg. 2000;16(3):373-4.
19. Davidorf JM. Herpes simplex keratitis after LASIK. J Refract Surg. 1998; 14(6):667.
20. Webber SK, Lawless MA, Sutton GL, et al. Staphylococcal infection under a
LASIK flap. Cornea. 1999;18(3):361-5.
21. Sridhar MS, Garg P, Bansal A, et al. Fungal keratitis after laser in situ keratomile-
usis. J Refract Surg. 2000;26(4):613-5.
22. Helena MC, Meisler D, Wilson SE. Epithelial ingrowth within the lamellar inter-
face after laser in situ keratomileusis (LASIK). Cornea. 1997;16(3):300-5.
23. Seiler T, Koufala K, Richter G. Iatrogenic keratoconus after laser in situ ker-
atomileusis. J Refract Surg. 1998;14(3):312-7.
24. Sieler T, Quurke AW. Iatrogenic keratectasia after LASIK in a case of forme fruste
keratoconus. J Cataract Refract Surg. 1998;24(7):1007-9.
25. Stulting RD, Carr JD, Thompson KP, et al. Complications of laser in situ ker-
atomileusis for the correction of myopia. Ophthalmology. 1999;106(1):
13-20.
26. Curtin BJ. The Myopias: basic science and clinical management. Harper & Row,
Philadelphia, PA: 1985:(334).
Chapter_06.indd 126 06-02-2018 20:56:33

7
CHAPTER
Zyoptix Wavefront-guided
Customised Ablation in
Retreated Corneas
Jerry Tan
INTRODUCTION
With the emergence of wavefront technology and its ability to measure small
and subtle aberrations of the eye, one of the most exciting developments
in the past few years has been the development of wavefront-guided treat-
ments.1-3 With the progress of refractive surgery using customized cornea
ablation, many ophthalmologists have been striving to achieve ‘super’ vision
for their treated patients. The reduction of higher order aberrations has been
shown to improve patient’s vision both in terms of visual acuity and contrast
sensitivity.1-6 Over the past few years, the dominant form of correcting higher
order aberrations in the eye is with the use of a wavefront-guided ablation.
One of the leading systems in this field is the Zyoptix platform developed by
Bausch & Lomb, Inc. Correcting the aberrations is based on an integrated
diagnostic unit composed of an elevation topographer (providing global
pachymetry) and a wavefront analyzer. In this chapter, the results of wavefront-
guided laser in situ keratomileusis (LASIK) enhancements (using the Zyoptix
system) for the correction of residual myopia, astigmatism and higher order
aberrations after previous LASIK surgery have been described. At present,
the Zyoptix wavefront-guided customized ablation is only for myopia and
astigmatism, with the myopia range being from plano to - 12.00 D and astig-
matism 0.00 D to - 6.00 D. The Zyoptix system integrates wavefront analy-
sis, three-dimensional corneal mapping providing corneal pachymetry and
advanced scanning laser technology. The preoperative data required for cus-
tomization is collected by a diagnostic workstation composed of the eleva-
tion topography, Orbscan II and a Hartman Shack based wavefront analyzer,
Chapter_07.indd 127 06-02-2018 21:01:46

Zywave, Bausch & Lomb, Inc. Data collected was incorporated into a software
program called Zylink. This program creates the laser treatment file. The data
is presented to the surgeon in the Zylink software and the surgeon is allowed
to alter the sphere and cylinder but not the cylinder axis. The optical zone
size can also be adjusted depending on the patient’s pupil size as well as the
residual corneal thickness. The treatment file is downloaded into the Bausch
& Lomb 217Z unit, equipped for customized ablation.
MATERIALS AND METHODS

Nineteen eyes were included in this study. All eyes were eligible for Zyop-
tix customized LASIK ablation to correct myopia and astigmatism. In all
cases, they were previously treated with standard LASIK or wavefront-guided
LASIK. The LASIK flap was previously formed either using the Hansatome
microkeratome or the automated corneal shaper (ACS), both by Bausch &
Lomb, USA. The enhancement procedure was performed after a minimum
of follow-up of 3 months. There was also stability of refraction and data mea-
sured which included uncorrected visual acuity, best corrected visual acu-
ity, manifest and cycloplegic refraction, wavefront measurements, corneal
topography, Orbscan and ultrasonic pachymetry and slit-lamp examina-
tion. Examinations were performed preoperatively as well as 1 day, 1 week,
1 month and 3 months after surgery. All eyes fulfilled the full follow-up period
of 3 months. Data collected were incorporated into data collection software,
Data Graph by Ingenieurbüro Pieger GmbH, Germany. Zyoptix enhance-
ment was performed when the total thickness of the cornea was at least 450
microns and the stromal residual bed after ablation was at least 250 microns.
All enhancements were performed by the same surgeon (Jerry Tan). Preop-
eratively, the eyelids were cleaned with iodine and patients were anesthe-
tized with non-preserved amethocaine eye drops. The surgeon and assistants
wore powder-free gloves during instrument set-up and surgery. A drape was
placed over the eye and a lid speculum inserted. The cornea was marked
and the flap was opened at the edge from another mark previously made at
the slit-lamp. A modified cyclodialysis spatula was used to lift the flap. The
amount of ablation was based on the patient’s residual refractive error and
higher order aberrations. The ablation pattern was governed by the Bausch &
Lomb 217Z laser guided by the Zylink software program.
The spot size for the ablation was a mixture of 2 mm and 1 mm spot sizes.
The majority of patients were treated in 4–16 stages. The flap was then repos-
ited to its original position. Irrigation was performed under the flap with
copious amounts of balanced salt solution. The flap was brushed down with
a moist Merocel sponge. The flap was allowed to settle and adhere for a min-
imum period of 3.5 minutes. A striae test was done to confirm adhesion and
the lid speculum was then removed. A soft bandage contact lens was placed
in all eyes, and patient was asked to perform multiple blinks to confirm flap
Chapter_07.indd 128 06-02-2018 21:01:46

Zyoptix Wavefront-guided Customised Ablation in Retreated Corneas 129
adhesion. Postoperative medication was with topical lomefloxacin 0.3% and

tobramycin 0.3% and dexamethasone 0.1% eye drops. The 217Z laser uses fly-
ing spot technology with spot size of 2 mm in the early phases ranging from 4
to 8 stages to perform the major refractive correction. After which, 1 mm spot
laser is used to treat the higher order aberrations, form peripheral blends
and transition zones. Also, the 1 mm spot size is used to smooth over the
2 mm spots resulting in a very smooth ablated surface. Unfortunately, the
1 mm spot size does prolong the treatment time and requires the use of an
eye tracker with high-tracking velocity. The 217Z operates at a frequency of
50 Hz and is supplied with an active eye tracker operating at a frequency of
120 Hz. This eye tracker works within a range of 1.5 mm of eye movement. The
beam profile is a truncated Gaussian beam for maximum smoothing effect
with minimal thermal effect. This truncated Gaussian beam also allows for
small focus changes especially when the cornea is being ablated and when
there are slight height changes while the cornea is thinning during corneal
ablations.
RESULTS
Following Zyoptix enhancement surgery, only one patient lost one line of
visual acuity at 3 months and this patient had evidence of moderately severe
dry eye during the 3 months visit. Her refraction was plano ± 0.25 × 110° and
her visual acuity was 6/7.5. There were 6 eyes that gained one line of vision
and 12 eyes that remained the same (Fig. 7.1).
Predictability
The mean spherical equivalent was - 1.67 and improved to 0.01 ± 0.38 at
3 months postoperatively. This change was statistically significant. At 3 months
Fig. 7.1: Bar diagram showing change in the best corrected visual acuity following
surgery.
Chapter_07.indd 129 06-02-2018 21:01:47

Fig. 7.2: Predictability of spherical equivalent in Zyoptix enhancement (3 months).
follow-up, all 19 eyes had a spherical equivalent within + 1.00 D of emmetro-

pia, and the predictability of this group is represented by the scattergram at
3 months (Fig. 7.2).
Efficacy
At 3 months postoperatively, the mean uncorrected visual acuity improved to

0.94 ± 0.35. Hundred percent of postoperative uncorrected visual acuity was
0.5 or 6/12 better while 12 out of 19 eyes reached 6/6 or better at 3 months
(Fig. 7.3).
Stability
Over a period of 3 months, no eye showed a change of spherical equivalent

greater than or equal to 1.00 D (Fig. 7.4).
Case Study
In addition to the Zyoptix enhancement study, a case of a gentleman,

42-year-old, referred from Hong Kong after treatment with standard LASIK
needs mention. He had a large scotopic pupil of 7 mm and his original refrac-
tion was - 9.50/- 1.00 × 10o. He was treated with standard Planoscan laser
Chapter_07.indd 130 06-02-2018 21:01:47

Fig. 7.3: Bar diagram showing efficiency of procedure in terms of percentage of uncor-
rected visual acuity at 3 months follow-up.
Fig. 7.4: Change in refraction overtime.
treatment, Bausch & Lomb, 217C laser. His right eye had a visual acuity of
6/9 and a residual refractive error of - 1.25/-0.75 × 10o (6/9). His left eye was
also treated at the same time in Hong Kong and he had an original refrac-
tion of - 8.50/- 1.00 × 165o and residual refractive error of plano /- 0.75 × 20o
after LASIK. He was unhappy with his quality of vision and complained of
glare and halos at night. His Orbscan corneal topography is shown with the
anterior float, posterior float, tangential keratometric readings (0.25 D steps)
as well as his corneal pachymetry (residual pachymetry) (Figs. 7.5 and 7.6).
He wanted to apply for a private pilot’s license but unfortunately could not
pass the vision tests. Zywave readings were done to measure his lower and
higher order aberrations and one wavefront reading is shown in (Fig. 7.5).
His pre-enhancement Orbscan is seen in (Fig. 7.6). His higher order aberra-
tions were also analyzed using CT view (Sarver and Associates Inc., FL, USA),
a software program that used graphically chart the lower and higher order
aberrations. These are seen in (Fig. 7.7). A Zyoptix enhancement was done to
Chapter_07.indd 131 06-02-2018 21:01:47

Fig. 7.5: Pre-enhancement zywave.
Fig. 7.6: Pre-enhancement Orbscan showing decentration.
improve his decentration and reduce his lower and higher order aberrations.
Postoperatively his quality of vision improved tremendously and his unaided
visual acuity improved to 6/6. His ablation pattern designed by the Zylink
software is shown in Figure 7.8 and his postoperative corneal topography is
shown in Figure 7.9. A difference map was done and is shown in Figure 7.10.
When comparing his difference map and his ablation pattern, one can
see that the Zyoptix enhancement program gives a consistent and predictable
ablation pattern (Fig. 7.8). With the correction of his decentration, the patient
subsequently had a significant reduction in his preoperative higher order
Chapter_07.indd 132 06-02-2018 21:01:48

Fig. 7.7: Pre-enhancement higher order aberrations (CT view).
Fig. 7.8: Comparison of ablation pattern and difference map.
Fig. 7.9: Postoperative Orbscan 1 month after Zyoptix enhancement.
Chapter_07.indd 133 06-02-2018 21:01:48

Fig. 7.10: Orbscan difference maps derived from pre- and post-Zyoptix enhancement.
Fig. 7.11: Comparison between pre-enhancement and post-enhancement Zernike

coefficients.
aberrations. A comparison between pre- and post-higher order aberrations

is graphically shown in Figure 7.11 using the CT view program (Sarver and
Associates Inc., FL, USA).
DISCUSSION
Even though wavefront-guided LASIK is a procedure that still needs much
research and refinements, the Zyoptix program is able to achieve safe and
excellent results in well-selected cases. With this method, one can improve
both the visual acuity and contrast sensitivity under mesopic and scotopic
conditions. It also provides the ability to improve the quality of vision by
removing and reducing residual refractive errors as well as higher order aber-
rations, not treated and not corrected by the initial standard procedure.7-10
Beyond Zernike second order (defocus and astigmatism), most optical aber-
rations are contained in Zernike third, fourth or fifth terms.11 Beyond the
Chapter_07.indd 134 06-02-2018 21:01:49

fifth order, there is little significant aberration effect in the eye. Wavefront
measurements in an eye with a 3.4-mm diameter pupil requires removal of
wavefront errors up to the fourth Zernike order, in order to achieve diffrac-
tion limited optical performance.11 For a 7.3-mm pupil, aberrations need
to be removed up to the eighth Zernike order.11 From the case study shown,
we can see that Zyoptix enhancement can correct a large amount of higher
order aberrations. However, we are still unable to treat beyond the Zernike
fifth order aberrations. Correcting the higher order aberrations is something
theoretically possible but the laser correction must also take into consider-
ation the effect of wound healing as well as the cornea flap.7-11 In cases where
the flap is already made, as in enhancements, we are correcting aberrations
that were caused by the creation of the LASIK flap. We may be able to achieve
better results in a Zyoptix enhancement procedure than in a primary Zyoptix
procedure. This excludes enhancements that require a second cut. Two-step
LASIK may be a significant advancement in customized ablation. In cases
of mixed astigmatism and residual hyperopia, at present, there is only one
wavefront enhancement system that can be used. This is the Wavelight Alle-
gretto Laser using the wave analyzer based on Tscherning aberrometry. Data
and results have been very scarce, and, at present, there is no large data pre-
sentation using this laser and system for the treatment of residual hyperopia
and mixed astigmatism after primary LASIK. The author has performed a
LASIK ablation in two steps with the LASIK cut being performed first and the
laser treatment being done 3 months later. The fellow eye was done as a single
primary LASIK procedure, both results were excellent. However, when the
patient was interviewed by the author, she felt that it was troublesome having
to come twice for surgery on the same eye. There was the added inconve-
nience of a 3 months delay between the cut and laser ablation. She had to
wear spectacles for 3 months before the laser ablation procedure could be
performed. When asked whether she would do this procedure again in the
same manner, she confessed that she would prefer to do the LASIK proce-
dure all in one step. Even though theoretically, a staged strategy is a solution
for the problem of flap induced aberrations, we must consider that there is a
psychosocial aspect to this treatment. However, in patients who have residual
myopia and astigmatism after primary LASIK, this problem does not arise.
The patient can be encouraged to do a wavefront enhancement as a ‘silver
lining.’ Having to undergo a retreatment, patients should be encouraged to
undergo a wavefront enhancement to remove the aberrations created by the
initial surgery as well as the residual myopia and astigmatism. This is akin
to playing golf where the ball is struck toward the hole, lands on the green
and the second stroke is to put the ball in the hole, hopefully with a short
putt. In our study, enhancing using the Zyoptix system is safe and efficacious.
The Zyoptix system can also correct irregular astigmatism and decentrations
which could never have been effectively treated with standard ablation pat-
terns. This ability to treat irregular contours and decentered ablations will be
probably more important than treating patients for ‘super’ vision. Over the
Chapter_07.indd 135 06-02-2018 21:01:49

past 20 years, refractive surgery has created many visual handicaps. Patients
with small optical zones, irregular contours, interconnecting RK and Tcuts,
wrinkled flaps and button holed flaps have some hope of improving their
vision. One only needs to go to the website ‘www.surgicaleyes.com’ to find
many unhappy patients as a result of our efforts to try and correct their myo-
pia, hyperopia and astigmatism. The use of wavefront-guided enhancements
will give us the ability to correct many of these problems created over the past
20 years. The ability to treat these unfortunate patients is much more import-
ant and satisfying than trying to endow people with ‘super’ vision. Of course,
the ideal situation would be to correct their visual problems and create peo-
ple with ‘super’ vision after wavefront enhancement.
Finally, a word of caution, not all patients can be treated with Zyoptix
enhancement. Patients who require standard LASIK enhancements would
be the ideal situation for a surgeon to start using Zyoptix enhancements.
Potentially, the final result should be better than just doing a standard LASIK
enhancement. The first and most important factor that a surgeon has to
give priority to is the lower order aberrations. The main goal is to achieve a
plano refraction. It is no point to correct higher order aberrations and leave
behind - 0.75 cylinder or sphere. The lower order aberrations always take
precedence over any correction of higher order aberrations. The next group
of patients that I feel would benefit from this Zyoptix enhancement would
be cases of limited amount of induced aberrations or ‘irregular astigmatism.’
These patients typically complain of poor night vision, halos and monocular
diplopia. The main goal is to reduce patient’s symptoms by reducing their
aberrations and achieve plano or maintain plano refraction. In patients with
severe corneal deformation as in the case of a perforated cornea flap with
epithelial in-growth or severely decentered ablation, no treatment is possible
with the wavefront-guided customized ablation as the wavefront patterns are
both bizarre and unreadable (Fig. 7.12). The cornea must also have a resid-
ual stromal bed of 250 microns. At present, customized ablations require
larger amounts of tissue removal and to go beyond the 250 micron limit
Fig. 7.12: Showing (a) good and (b) poor quality centroids.
Chapter_07.indd 136 06-02-2018 21:01:49

would be dangerous. Occasionally, surgeons will venture below 250 microns

but almost every surgeon will not go below 200 microns because of the fear
of postoperative corneal ectasia. There are three golden rules for a patient
inclusion for Zyoptix wavefront customized enhancement: (1) There must be
good centroid quality in the preenhancement wavefronts. (2) The larger the
number of centroids, the better. (3) They must be very distinct, not smudged
and there must be no confusion or misinterpretation by the aberrometer. The
surgeon has to not only look at the color of higher order aberration maps, the
Zernike and RMS values but also at the quality and number of the centroids.
Figure 7.12 is an example of good and poor quality centroid images are shown
in Figure 7.12. There is no major pupil shift. In patients with highly aberrated
eyes, perfect centration is mandatory. Major pupil shifts with undilated versus
dilated pupils is extremely important in the treatment of deformed corneas.
Pupils that decenter greatly with dilation should be cautiously approached
and, at present, wavefront enhancements are best avoided.
The higher order wavefront maps must match the corneal topography
elevation map. This is obvious as the aberrations created in a post-LASIK
eye is most likely caused by the changes in the cornea secondary to surgery,
healing, scarring or a combination of all three. If the higher order aberration
map and corneal topography elevation map do not correspond, this patient
has not only a corneal deformation but other aberrations which can be
induced, for example, by cataractous lens changes. If treatment is still carried
out, the patient and the doctor must be prepared for an unusual response to
the Zyoptix enhancement. Careful examination of the lens should be done
as some of these patients may have a nuclear sclerotic cataract. A Zyoptix
enhancement will only work for a short period of time and further progress
of these cataractous changes will alter both the lower and higher order aber-
rations, resulting in a ‘failed’ enhancement. The Zywave predicted phoropter
refraction (PPR) must be close to or identical to the subjective refraction. In
this manner, one can predict and be confident that the lower order aberra-
tions are not the main cause of the refractive problems and that the higher
order aberrations, once corrected would improve the quality of vision. We
have just started down the path of wavefront customized enhancement and
it is exciting to see the technology progressing so rapidly. The ability to give
improved quality of vision to patients, who have had previous refractive sur-
gery, is a bigger breakthrough than to provide patients with ‘super’ or better
than normal vision. Future improvements in customized ablation will be the
advent of registration (orientating the eye from its iris features to prevent the
effects of cyclotorsion), topography guided ablation, combined topography
and wavefront-guided ablations as well as with high-speed lasers with super
high-speed eye trackers. I think we are still at the ‘base of Mount Everest.’
However, I feel that we have started our ascent. It is going to be a tough climb
but when we get to the top of Mount Everest, what a top of the world feeling it
is going to be. It may or may not be during my lifetime but I am so much glad
to be part of this exciting time.
Chapter_07.indd 137 06-02-2018 21:01:49

REFERENCES
1. Thibos LN. Principles of Hartmann-Shack aberrometry. J Refract Surg.
2000;16(5):S563-5.
2. Mrochen M, Kaemmerer M, Mierdel P, et al. Principles of Tscherning aberrome-
try. J Refract Surg. 2000;16(5):S570-1.
3. Molebny VV, Panagopoulou SI, Molebny SV, et al. Principles of ray tracing aber-
rometry. J Refract Surg. 2000;16(5):572-5.
4. Krueger RR. Technology requirements for Summit-Autonomous CustomCor-
nea. J Refract Surg. 2000;16(5):592-601.
5. Thibos LN. The prospects for perfect vision. J Refract Surg. 2000;16(5):
S540-6.
6. Applegate RA. Limits to vision: can we do better than nature? J Refract Surg.
2000;16(5):547-51.
7. Applegate RA, Hilmantel G, Howland HC, et al. Corneal first surface optical ab-
errations and visual performance. J Refract Surg. 2000;16(5):507-14.
8. Howland HC, Howland B. A subjective method for the measurement of mono-
chromatic aberrations of the eye. J Opt Soc Am. 1977;67(11):1508-18.
9. Applegate RA, Howland HC, Sharp RP, et al. Corneal aberrations and visual per-
formance after radial keratotomy. J Refract Surg. 1998;14(4):397-407.
10. MacRae SM. Supernormal vision, hypervision, and customized corneal
ablation. J Cataract Refract Surg. 2000;26(2):154-7.
11. Alio JL. Results of Zyoptix wavefront-guided customized ablation in virgin
and refracted corneas. Custom LASIK. Surgical Techniques and Complications.
2003. pp. 521-6.
Chapter_07.indd 138 06-02-2018 21:01:49

8
CHAPTER
Diagnostic Procedures
in Infectious Keratitis
Savitri Sharma, Sreedharan Athmanathan
INTRODUCTION
Microbial keratitis may be caused by bacteria, fungi, parasites or viruses
and each of these may produce a spectrum of disease which may or may not
have distinctive clinical appearance. Many a time, it may not be possible to
differentiate between infected or noninfected corneas. Introduction of in
vivo confocal microscopy has helped to confirm clinical diagnosis without
laboratory aid, especially for Acanthamoeba and fungal keratitis, although
the high cost of the equipment remains a limitation.1 Therefore, confirmation
by laboratory methods continues to be the gold standard for the etiological
diagnosis in microbial keratitis. Currently, molecular diagnostic methods
have increased the sensitivity, specificity and speed over the conventional
microbiological techniques of microscopy and culture. To minimize morbidity
that may develop secondary to delay in diagnosis and achieve favorable
outcome within a reasonable cost and time, laboratory investigations are
indicated in patients with suspected microbial keratitis.
Based on the clinical features, there are two entirely different protocols
that are required to be followed while investigating viral and nonviral corneal
ulcers. A combination of the two protocols may be called for when a distinc-
tion of viral versus nonviral is not clinically clear. In the interest of clarity,
this chapter is divided into three parts to describe in vivo confocal micros-
copy and microbiologic procedures required for workup of clinically viral and
nonviral corneal ulcers.
Familiarity of ophthalmologists to functions, limitations and scope of
microbiology laboratory is important for proper and meaningful interpre-
tation of results. A well-equipped ocular microbiology laboratory with well-
trained technical personnel has great advantages over a general microbiology
laboratory in handling and processing minute quantity of ocular samples,
Chapter_08.indd 139 06-02-2018 21:09:28

especially corneal samples. Special orientation towards processing and inter-

pretation of results is of paramount importance.2
CORNEAL CONFOCAL MICROSCOPY

Confocal microscopy is a noninvasive technique that offers high image mag-
nification and contrasts that allow visualization of corneal details in vivo.
Repeated observations can be made which help in diagnosis and follow-up of
cases with microbial keratitis.3 Diagnostic accuracy of confocal microscopy in
microbial keratitis has been studied extensively.1,4 Hau et al. assessed confo-
cal images on two occasions by four observers who were masked to the tissue
diagnosis and calculated the diagnostic accuracy parameters.4 They reported
highest positive likelihood ratio of 2.94 and lowest negative likelihood ratio
of 0.59 with agreement values fair to moderate and did not recommend
stand-alone use of confocal microscopy for the diagnosis of microbial kera-
titis. On the other hand, Vaddavalli et al. compared inter- and intraobserver
variation in analysis and interpretation of confocal microscopy findings with
conventional microbiology findings of the corneal scrapings from patients
with Acanthamoeba or fungal keratitis and found high sensitivity (88.3%) and
specificity (91.1%) of diagnosis by confocal microscopy.1 Figure 8.1A and B
shows the typical branching: (a) fungal filaments with parallel walls and
(b) refractile cysts of Acanthamoeba in the corneal stroma of patients. These
authors recommended the use of confocal microscopy in fungal and Acan-
thamoeba keratitis, particularly when corneal infiltrates were deep seated or
patients were on treatment or microbial keratitis developed after intracorneal
implants like intracorneal ring segments or refractive surgery.
Laboratory Methods for Nonviral Keratitis (Bacterial, Fungal

and Acanthamoeba)
The samples for the microbiologic investigations of a suspected microbial ker-
atitis must be collected before the start of any antibiotic treatment. The treat-
ment can be initiated based on the result of the smears and later if required,
modified in accordance with the culture and sensitivity results. The protocol
essentially consists of four steps: (1) collection, (2) transport, (3) processing of
the clinical samples and (4) interpretation of the results.
Collection of Samples
Before collection of sample from the corneal ulcer itself, it is generally recom-
mended to obtain a culture from the lids and conjunctiva of both the infected
and uninfected eye.5 This procedure is purported to help in two ways; firstly,
the organism(s) grown from the uninvolved eye (indicating normal flora) may
be used for comparative purposes; secondly, in the absence of growth from
the ulcer the organism(s) from the cul-de-sac of the involved eye may well
be the causative organism(s).5 Despite recommendation for this procedure
Chapter_08.indd 140 06-02-2018 21:09:28

Diagnostic Procedures in Infectious Keratitis 141
(A)
(B)
Figs. 8.1A and B: Confocal microscopy image using 40× Zeiss Achroplan lens imaged
on the Confoscan 3 (Nidek) showing (A) interlacing moderate to high reflective
double-walled segmented filaments with a background of inflammatory cells sugges-
tive of filamentous fungi, (B) a cluster of highly reflective Acanthamoeba cysts with low
internal reflectivity.
Courtesy: Dr Pravin K Vaddavalli, LVPEI, Hyderabad.
in several text books, in our experience, samples from lids and conjunctiva
have not yielded useful results in the management of corneal ulcers.6 Simi-
lar observation has been made in the 1994 edition of ‘Laboratory diagnosis of
ocular infections’ published by American Society of Microbiology,7 which is a
deviation from the earlier edition, recommending collection and processing
of samples from the eyelid margins and conjunctiva. Samples collected from
the site of lesion, i.e., the infected corneal tissue are the most valuable for micro-
biological diagnosis of microbial keratitis. If available, any foreign body on the
cornea, contact lens, contact lens case or lens solutions may be collected.
Corneal samples can be collected using the slit lamp or operating
microscope after instillation of topical anesthetic agents (4% Lignocaine
hydrochloride or 0.5% Proparacaine hydrochloride). These anesthetic agents
may have variable effect on the growth of organisms,8 however, allowing
Chapter_08.indd 141 06-02-2018 21:09:29

some time interval between instillation of anesthetic agent and collection of

sample would help reduce their effect, if any.
Cotton swabs are not recommended for collection of corneal samples,
however, calcium alginate swabs, if available, may be used in cases of bac-
terial keratitis.9 Platinum spatula, disposable blade (#15), bent needle, sur-
gical knife, disposable cautery have all been used for collection of corneal
scrapings for microbiological processing. We routinely use blade number 15
on Bard-Parker handle. No difference was found by us in the quantitative
yield of organisms from bacterial and mixed (fungal with bacterial) keratitis
while comparing the use of calcium alginate swab with blade number 15.10
Although, the yield of fungi was more efficient with calcium alginate swab
than with blade in this study we did not recommend replacing blade with
swab. Swabs are likely to get contaminated by normal flora in the tear film
and are less efficient in transferring clinical material onto slides and culture
media. While collecting samples from the corneal ulcer the eyelids must be
held wide apart to reduce inadvertent contamination by the lid margins or
eye lashes. Adherent exudate on the surface of the ulcer may be removed
using a sterile cotton swab prior to collection of scrapings.
The blade or spatula is scraped over the surface of the area of suppuration
by a series of short, moderately firm strokes in one direction to sample both
the central and peripheral margins of the infiltrated area on the cornea. Each
scraping is used to inoculate one medium or to prepare one smear. Viable
organisms may be present throughout the inflamed area or localized to the
advancing margin or the ulcer crater.
In the absence of accessible corneal suppuration, a corneal biopsy can
be done with a disposable skin punch, diamond knife or small corneal tre-
phine.11 Placed in a sterile petri dish, the tissue specimen is minced with
sterile blade and the pieces are used for making a smear for microscopy and
inoculated into various media. Additional corneal scrapings can be obtained
from the base of a partial-thickness corneal biopsy.
Collection of anterior-chamber exudates is advised only under excep-
tional circumstances owing to risk of inoculating organisms into the eye. The
possible circumstances are deep stromal suppuration that cannot be sampled
by an anterior approach and infections that have extended into the anterior
chamber.7
Transport of Corneal Samples to the Microbiology Laboratory
Transportation of corneal scrapings in any transport medium is not recom-

mended. The scrapings are plated directly onto culture media or smeared
onto clean glass slides by the side of the patient in the clinic or operating
room. It would help to maintain a corneal collection kit in the clinic or oper-
ating room containing a set of media, sterile slides (wrapped in foil), spatula/
blades, glass marking pencil, swabs, etc. in case the microbiology laboratory
cannot be reached and requested to provide the materials whenever required
Chapter_08.indd 142 06-02-2018 21:09:29

Fig. 8.2: Corneal scraping collection tray containing culture media, blades, glass slides
marker pen, reagents and coverslips.
(Fig. 8.2). One study has evaluated alternative use of Amies transport medium
without charcoal. Amies medium was found as good as direct patient side
processing of corneal scrapings for culture of bacteria and fungi after storage
for 4 and 24 hours at room temperature.12 However, corneal scrapings were
smeared on slides at patient side, for microscopic examination.
Corneal biopsy tissue can be transported to the microbiology laboratory
in a sterile dry petri-dish or in a sterile bottle. To prevent drying few drops of
sterile saline may be added, however, this runs the risk of bacterial contami-
nation especially with Pseudomonas spp. Aqueous humor is usually collected
and transported in a tuberculin syringe. Exudates from the anterior chamber
may also be directly plated on culture media and smeared on slides and sent
to the laboratory. In case of contact lens associated microbial keratitis, if the
patient presents with contact lenses on the cornea the same may be collected
aseptically with gloved fingers and placed in sterile petri-dish or bottle for
transport. Contact lens cases may be sent to the laboratory in addition to con-
tact lenses.
Processing of Corneal Scrapings
A complete microbiological workup of a nonviral corneal ulcer may require

up to 10 corneal scrapings for a number of smears and culture media
(Table 8.1). In case of small ulcer, with limited material availability, high pri-
ority needs to be given to inoculation of blood agar or chocolate agar and to
prepare only one or two smears. Preferred media may be selectively included
based on clinical impression, for example, non-nutrient agar (NNA) for a
suspected Acanthamoeba keratitis patient. A schematic diagram to guide
nonviral corneal ulcer workup is shown in Figure 8.3.
Chapter_08.indd 143 06-02-2018 21:09:29

Table 8. 1: Sequence of smear preparation and culture media inoculation for the diag-
nosis of nonviral keratitis.
Smears 1. Potassium hydroxide with/without calcofluor white

2. Gram stain
3. Giemsa stain
Media 4. Blood agar—aerobic
5. Chocolate agar
6. Brain heart infusion broth
7. Thioglycollate broth
8. Robertson’s cooked meat broth
9. Nonnutrient agar
10. Sabouraud dextrose agar
Optional smears/media 11. Blood agar—anaerobic
12. Potato dextrose agar
13. Lowenstein—Jensen medium
14. Brain heart infusion broth with antibiotic
15. Extra smear on slide
Fig. 8.3: Schematic diagram for microbiology processing of nonviral keratitis.

KOH: Potassium hydroxide; CFW: Calcofluor white; SDA: Sabouraud dextrose agar; BHI: Brain heart
infusion; NNA: Non-nutrient agar; BA: Blood agar; CA: Chocolate agar; Thio: Thioglycollate broth;
RCM: Robertson’s cooked meat broth.
Direct Microscopic Examination Methods
Material is transferred from the blade/spatula to a glass slide over an area of

a circle approximately 1 cm in diameter (marked on the reverse) to enable
Chapter_08.indd 144 06-02-2018 21:09:29

focused examination under the microscope. While the specimen is thinly

spread for dry smears (Gram, Giemsa, Gomori methenamine silver, etc.),
it can be just placed within the circle for wet smears (KOH, KOH+CFW,
LPCB, etc.) under a coverslip. Table 8.2 outlines the various staining proce-
dures in brief.11 At least two smears should be prepared. For several years, a
combination of KOH + CFW, Gram, and Giemsa stained smears has provided
a high sensitivity and specificity in our laboratory for the detection of bacte-
ria, fungi, microsporidia and Acanthamoeba in corneal scrapings. Common
laboratory light microscope suffices in most instances for the examination of
the smears except when fluorescent stains such as calcofluor white (CFW) or
acridine orange are used which require a fluorescence microscope.
Culture Methods
Inoculation
Sheep blood agar (BA) and sheep blood chocolate agar (CA) plates are inoc-
ulated by lightly streaking both sides of the blade/spatula over a surface in a
row of separate ‘C’ shaped marks without penetrating the agar. This procedure
helps to distinguish valid growth from plate contaminants (Fig. 8.4). Slopes
of sabouraud dextrose agar (SDA) or potato dextrose agar (PDA) in bottles
are similarly inoculated by making a row of streaks from below upwards. Liq-
uid media such as brain heart infusion broth (BHI) is inoculated by agitating
the blade/spatula directly in the broth. To facilitate this procedure without
inviting contamination, the BHI or RCM should be available in screw capped
tubes with the top level of the medium not below 1 cm from the brim of the
tube. The inoculation of thioglycollate broth (thio) requires transfer of the
scraped material onto a cotton or calcium alginate swab and insertion to
the bottom of the tube to facilitate growth of anaerobic bacteria. It is a good
practice to limit the inoculation of NNA with 1–2 strokes in the center of the
plate with minimal disturbance of the surface of the medium. While inoc-
ulating the plates/bottles, care must be taken to minimize exposure of the
medium to the atmosphere.
Corneal biopsy tissue can be cut into small fragments and inoculated
into media or it can be emulsified in sterile saline using tissue homogenizer
and then distributed in culture media, preferably under a biosafety laminar
flow hood.
Aqueous fluid drops can be placed over agar plate surfaces as such with-
out streaking and dropped directly into liquid media, preferably under a bio-
safety laminar flow hood.
Incubation
The inoculated culture media are placed in appropriate incubators. NNA

(after sample inoculation) requires to be overlaid with few drops of heat
Chapter_08.indd 145 06-02-2018 21:09:30

Table 8.2: Common staining procedures for corneal scrapings in the diagnosis of non-
viral keratitis.
Method Steps
A. Gram stain 1. Fix smear in 95% methanol
2. Flood smear with crystal violet for 1 minute
3. Rinse with tap water
4. Flood smear with Gram’s iodine solution for 1 minute
6. Decolorize with acetone-alcohol solution
8. Flood with safranin or dilute carbol fuchsin for 30 seconds
9. Rinse with tap water and allow to dry
B. Giemsa stain 1. Fix smear in fixative for 5 (Diff Quik)TM seconds
2. Dip in reagent A for 5 seconds
3. Dip in reagent B for 5 seconds
4. Rinse in water and allow to dry
C. Potassium hydroxide 1. Add one drop of 10% KOH with 10% glycerol
(KOH) preparation 2. Place a coverslip
3. Apply nail polish around the coverslip edges to prevent
drying (optional)
D. KOH + Calcofluor 1. Add one drop of 10% KOH with 10% glycerol
white 2. Add one drop of 0.1% calcofluor white with 0.1% Evans
blue solution
3. Place a coverslip
4. Examine under UV light (fluorescence microscope)
E. Zeihl Neelsen acid-fast 1. Flood fixed smear with hot (steaming) strong carbol fuchsin
stain and leave for 5 minutes
2. Rinse with water
3. Decolorize with 20% H2SO4 for 1–2 minutes
4. Rinse with water
5. Flood with methylene blue counter stain for 2 minutes
6. Rinse with water and allow to dry
F. Kinyoun’s modification 1. Flood fixed smear with strong carbol fuchsin for 2 minutes
of acid-fast stain 2. Rinse with water
3. Decolorize with 1% H2SO4
4. Rinse with water
5. Flood with methylene blue counter stain for 2 minutes
6. Rinse with water and allow to dry
G. Lactophenol cotton 1. Mix specimen colony in a drop of LPCB
blue 2. Apply coverslip
3. Apply nail polish around edges of coverslip to prevent
drying (optional)
H. Acridine orange 1. Mix specimen in 0.01% of acridine orange
2. Apply coverslip
3. Examine under UV light (fluorescence microscope)
Chapter_08.indd 146 06-02-2018 21:09:30

Fig. 8.4: Blood agar, inoculated with corneal scraping and incubated at 35°C for
48 hours, showing confluent gray, moist colonies (Pseudomonas aeruginosa) on the
inoculum (‘C’ streaks) and a contaminant colony away from the inoculation marks
(arrow).
killed or live E.coli suspension prior to incubation. While BA (aerobic), BHI

broth, RCM broth, thio broth, NNA, SDA and PDA are incubated under nor-
mal atmospheric conditions, CA is incubated in a candle jar which provides
5% CO2, and one BA (anaerobic) is incubated in anaerobic jar or cabinet, if
available. All media are incubated at 35°C (± 1) except SDA and PDA which
are kept at 27°C (± 1) in BOD incubator. Petri dishes are incubated with lids
facing downwards to prevent condensed moisture from dripping onto the
medium. Broth tubes are held upright in racks. Early growth may be detected
on culture plates in most instances within 24–48 hours of incubation, however,
media such as BA (aerobic), CA, thio and BHI that show no growth, should
be incubated until at least 7 days before discarding. In case of no growth, BA
(anaerobic), SDA, PDA and NNA may be incubated until 2 weeks. Incubation
beyond 2 weeks, in our experience, has not resulted in increased positivity.
Instead, incubation longer than 2 weeks may lead to drying of media, and
growth of contaminants due to repeated opening of plates for observation.
We prefer to take reading of plates under laminar flow hood which reduces
contamination due to exposure.
Observation
On solid agar plates growth on inoculation marks (‘C’ streaks) are regarded
important while growth outside the inoculation marks are disregarded as
Chapter_08.indd 147 06-02-2018 21:09:30

contaminants (Fig. 8.4). All culture media, except BA (anaerobic) in anaer-

obic jar/cabinet, must be examined daily for detection of any growth. BA
(anaerobic) may be examined at intervals of 2–3 days for 2 weeks.
Size, color, texture, consistency and number of colonies on the inocula-
tion marks are counted and recorded. Ten or more colonies are considered
confluent. While bacterial and fungal colonies are examined with unaided
eyes, the observation of Acanthamoeba growth requires use of microscope.
NNA plates (with lid on to avoid contamination) are placed under 4 ×
objective lens of the microscope and presence of trophozoites is looked for
in the vicinity of the inoculation mark on the surface of the medium. No col-
onies are formed by Acanthamoeba. Suspected growth will require confir-
mation at higher magnification and examination with open lid.
Growth in liquid media appears as turbidity which requires to be subcul-
tured and Gram stained for identification.
Identification
Microbiological identification details of various organisms that may be iso-

lated from cases of nonviral keratitis are neither the intent nor the scope of
this chapter. Bacterial colonies are usually Gram stained and identified after
consideration of colony characteristics, gram reaction, morphology, and
results of biochemical tests. Conventional procedures may be adopted for bio-
chemical tests or commercial kits from a number of companies (bioMérieux,
France; Lachema, Czech Republic; Organon Technika, USA, etc.)13 may be
obtained.
Identification of fungal species requires observation of growth rate, color,
consistency and texture of the colony and characteristic microscopic fea-
tures. Though most species are identified easily more than 20% of filamentous
fungal isolates may remain unidentified because of the lack of characteristic
spores. Biochemical tests for identification are needed only in case of yeast
or yeast-like fungal growth. Helpful hints for identification are available from
several sources.14
Presence of characteristic cysts and trophozoites on the surface of NNA
helps to identify Acanthamoeba genus (Fig. 8.5). Speciation of this genus is
presently not in the realm of a clinical ocular microbiology laboratory.15 A
recently introduced procedure of zoospore demonstration in our laboratory
helps identify Pythium insidiosum.16
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing is done in vitro to identify the response

of an organism to a panel of selected drugs. Commercially available panels
for Gram-positive and Gram-negative bacteria are used to determine sen-
sitivity by disk-diffusion method. In this method (Kirby-Bauer) the bacte-
ria is cultured on Mueller-Hinton agar (MHA), and antibiotic impregnated
Chapter_08.indd 148 06-02-2018 21:09:30

Fig. 8.5: Acanthamoeba trophozoites (irregular, vacuolated) on the surface of non-

nutrient agar (NNA) with E. coli (original magnification × 500).
disks are applied. Antibiotic susceptibility testing of fastidious organisms

such as Streptococcus pneumoniae requires MHA with 5% sheep blood. After
incubation, the diameter of the zone of inhibition around each disk gives
an approximation of susceptibility or resistance of the organism (Fig. 8.6).
Commercially available kits provide a zone size interpretative chart to facil-
itate interpretation. Slow-growing bacteria and anaerobes cannot be reli-
ably tested with disk-diffusion method. Susceptibility of bacterial isolates
to antibiotics is well standardized by clinical laboratory standards institute
and guidelines are available for disc diffusion assay (CLSI M02-A10, 2009) as
well as for broth dilution and agar dilution methods (CLSI M07-A8, 2009) for
determination of minimum inhibitory concentration (MIC) of antibacterial
agents. Estimating the MIC of antibiotics may provide a more useful informa-
tion than labeling organisms as sensitive or resistant7 especially because the
results of disk-diffusion tests relate to levels of drug achievable in serum and
do not relate directly to concentration of drug produced in the preocular tear
film and ocular tissues by standard routes of administration.
For the dilution methods to determine MIC, the antibiotic is serially
diluted and added to tubes with broth or wells of a microtiter plate or incor-
porated into agar plates. A standard suspension of the organism is then
inoculated. The MIC is recorded as the lowest concentration with no visible
growth. The tubes or wells with inhibited growth can be subcultured and the
lowest concentration with no growth is recorded as minimum bactericidal
concentration. A simple method of MIC determination has become available
in the form of E-test, which is a commercially available quantitative antimi-
crobial susceptibility test based on diffusion. It combines the simplicity and
Chapter_08.indd 149 06-02-2018 21:09:31

Fig. 8.6: Antibiotic susceptibility test on Mueller-Hinton agar for Pseudomonas aerugi-
nosa isolated from corneal ulcer. The diameter of zone of inhibition around antibiotic
disks is measured and reported as sensitive, intermediate or resistant (disk diffusion
test). Note the greenish pigmentation produced by the organism in the medium.
flexibility of disk diffusion test with the ability to determine MICs of up to five
antibiotics at one time. Availability of this technique has made it possible to
test bacterial isolates for MIC of antibiotics on a routine basis. Large number
of publications are available where E-test has been used for determination
of MIC of antibacterial antibiotics against bacterial isolates from microbial
keratitis.17,18
The availability of antifungal and antiamebic susceptibility testing is lim-
ited. In vitro test methods are diverse for fungi19 and Acanthamoeba20 and
clinically predictive value of the results obtained is not known. MIC testing
of antifungal drugs is performed for yeasts and filamentous fungi by broth
or agar dilution methods. Disk diffusion method similar to bacterial sus-
ceptibility testing is available for yeasts (CLSI M44-A, 2009) and some of the
non-dermatophyte filamentous fungi (CLSI M51-A, 2010). E-test of several
antifungals is also available for yeast and filamentous fungi. However, unlike
bacterial isolates, routine testing of fungal isolates for susceptibility to anti-
fungal drugs is yet to become common place in microbiology laboratories.
Molecular Methods
Molecular techniques are extremely sensitive, specific and ideal for detec-
tion of organisms that are difficult to culture such as viruses, microsporidia,
Propionibacterium acnes, Toxoplasma gondii, etc. or that take long time to
grow, such as Mycobacterium tuberculosis. Their application in the diagnosis
of nonviral keratitis is limited as the conventional techniques are easier to
employ, cost effective and have reasonable sensitivity and specificity. Several
Chapter_08.indd 150 06-02-2018 21:09:31

studies21-23 have compared the most commonly used molecular method of

polymerase chain reaction (PCR) with conventional methods of diagnosis of
fungal keratitis. A higher positivity and good correlation has been reported
with PCR compared to conventional methods. However, as of now, all studies
recommend the use of PCR as an adjunct to conventional techniques and
not as a replacement. Comparison of three different pan fungal primers for
the diagnosis of fungal keratitis has shown the sensitivity of PCR using ITS
primers to be significantly higher than 18S rDNA and 28S rDNA primers.24
ITS region is also ideal to determine DNA sequence based identification of
different species of fungi. DNA technology in the form of DNA chip has been
developed by XCyton Diagnostics Ltd, Bangalore, India which provides a plat-
form to apply multiplex PCR and hybridization for simultaneous diagnosis
of bacterial, fungal and viral eye infections.25 Sequencing of DNA fragments
of organisms often helps identify organisms that are difficult to identify by
conventional methods resulting in recognition of novel organisms. After
reporting the first case of microsporidial keratoconjunctivitis from India, we
found that this infection is common in India.26 Since microsporidia are obli-
gate intracellular organisms (phylogenetically related to fungi) that require
tissue culture for growth, PCR based confirmation of the smear findings is
recommended.27 Although, PCR based diagnosis of Acanthamoeba kerati-
tis have been published,28,29 we believe that the sensitivity of PCR is similar
to conventional method while specificity, positive and negative predictive
values are marginally higher.30 Kim et al. compared PCR to microbial cul-
ture for the detection and identification of bacterial and fungal pathogens
in microbial keratitis and found that PCR identifies potentially pathogenic
organisms.31 They reported higher yield and concordance with fungal than
bacterial, however, concluded that the practical utility of PCR for the diag-
nosis of nonviral microbial keratitis is limited by artefactual amplification of
non-pathogenic or commensal organisms likely to be present on ocular sur-
face. In a well conducted study on fungal keratitis, where PCR has been com-
pared with conventional microbiology methods, Vengayil et al. have similarly
recommended application of PCR as an adjunct rather than primary tool for
diagnosis.23 Quantitative PCR (real-time PCR) detects and quantitative num-
ber of organisms in a clinical sample and the test has begun to be applied
to the diagnosis of bacterial and fungal keratitis.32 While clinical validation
of many of these recently available tests are awaited, there is no doubt that
molecular techniques have a great role to play in identification of otherwise
unidentifiable organisms that can cause microbial keratitis.
Interpretation of Microbiology Results
Microscopy
Commonly used stains for microscopic evaluation of smears are listed in

Table 8.2. The results of smear examination form the basis for provisional
diagnosis and initial choice of an antimicrobial agent.
Chapter_08.indd 151 06-02-2018 21:09:31

(A) (B)
(C)
Figs. 8.7A to C: Corneal scrapings stained with KOH + CFW showing (A) septate fungal
filaments, (B) Acanthamoeba cysts, (C) microsporidia spores under fluorescence micro-
scope (original magnification for all, × 500).
KOH: Potassium hydroxide; CFW: Calcofluor white.
Though reported to be useful in the detection of bacteria in corneal scrap-

ings,33 we do not have much experience with acridine orange. However, we
have used CFW for several years and find the stain very useful in the detection
of fungi, Acanthamoeba and microsporidia in corneal scrapings (Fig. 8.7A to C).
The Gram stain is useful in identifying bacteria, fungi, as well as Acanthamoeba
cysts and microsporidia spores (Fig. 8.8). Precipitated stain, carbon, salt crys-
tals and necrotic debris can lead to troublesome artefacts in Gram-stained
smears. It is easier to detect Gram-positive bacteria (especially S. pneumo-
niae) than Gram-negative bacteria. Gram variable bacteria may sometimes be
seen.34 Fungal hyphae and Acanthamoeba cysts stain variably since their cell
walls do not stain well and may often be seen as negative outlines (Fig. 8.8A to E).
Presence of partially stained or unstained bacilli in Gram-stained smear is
suggestive of Mycobacteria and the same smear restained (after decoloriza-
tion) with Ziehl-Neelsen stain (20% H2SO4) would show the acid fast bacilli
(Fig. 8.9A and B).35 Trophozoites of Acanthamoeba are difficult to recognize
owing to their irregular morphology and similarity to macrophages.36 Giem-
sa-stained smear serves as a supportive smear. Cytological details are seen
well and bacteria, fungi as well as Acanthamoeba cysts can be seen.
Arbitrary quantitation of bacteria per high power field may help deter-
mine the significance as bacteria comprising the indigenous microflora of
the conjunctiva and tear film may be detected in small numbers. Smears with
more than 10 organisms are more determinate. However, detection of bacteria
Chapter_08.indd 152 06-02-2018 21:09:32

(A) (B)
(C) (D)
(E)
Figs. 8.8A to E: Corneal scrapings stained with gram stain showing (A) gram-positive
cocci in pairs (× 1000), (B) gram-negative bacilli (× 1000), (C) septate fungal filaments
(× 1000), (D) Acanthamoeba cysts (× 1000), (E) microsporidia spores (× 500).
in smears often needs to be correlated with corresponding bacterial growth in

culture for determining significance. Failure of an organism, seen in smears,
to grow in culture would indicate either nonviable organisms or sample vari-
ation. Sampling error must always be ruled out in case of discrepant results.
Presence of partially stained or unstained bacilli in Gram- or Giemsa-
stained smears has often indicated possibility of atypical mycobacteria
(Fig. 8.9) and successful diagnosis of the same.35 Thin, branching and beaded
Chapter_08.indd 153 06-02-2018 21:09:33

(A)
(B)
Figs. 8.9A and B: Corneal scraping from a case of Mycobacterium chelonae keratitis
(post-LASIK surgery) showing (A) unstained slender long bacilli in gram stain (arrows),
(B) acid-fast bacilli in Ziehl–Neelsen staining (20% H2SO4) (original magnification
× 1000).
filaments in the smears are indicative of Nocardia species. To confirm the

diagnosis, acid fast stains using 20% H2SO4 (Ziehl-Neelsen technique) in the
former and 1% H2SO4 (Kinyoun method) in the latter (Fig. 8.10A and B) are
extremely rewarding. Fungal filaments may either be hyaline (colorless) or
brown (dematiaceous) and septate or aseptate. They are fluorescent in calco-
fluor white stain and may be variably stained (gram positive, gram negative,
unstained, stippled staining) in Gram stain. Although it is generally difficult
to determine the type of fungus in direct microscopy of corneal scrapings,
broad, aseptate fungal filaments with ribbon like folds may be suggestive of
Pythium sp.16 Culture confirmation is required for species determination.
Chapter_08.indd 154 06-02-2018 21:09:34

(A)
(C)
(B)
Figs. 8.10A and B: Corneal scraping from a case of Nocardia keratitis showing (A) gram
positive, thin, long, beaded, branching filaments in gram stained smear, (B) acid-fast,
thin, beaded, branching filaments in the same smear stained by Kinyoun method (1%
H2SO4) after decolorization (original magnification × 1000).
Cultures
While microscopic examination of the smear provides preliminary evi-

dence, culture isolation gives diagnostic confirmation. Culture report should
indicate the day, the growth appeared and its quantitation or significance.
Less than 10 colonies on only one solid medium or growth in only one liquid
medium is usually equivocal. Growth of organisms such as S. epidermidis,
Corynebacterium species and Propionibacterium species in small numbers
or in a single liquid medium is generally of uncertain significance. The same
organisms, however, may be significant in the presence of a strong predis-
posing factor in the patient. All isolates must be considered in the light of
Chapter_08.indd 155 06-02-2018 21:09:34

clinical relevance and laboratory significance. Laboratory criteria for defini-

tive infection include: (i) confluent growth at the inoculation site on at least
one solid medium, (ii) growth on two or more media, (iii) growth on at least
one medium of the same organism seen in smears and/or (iv) repeat isola-
tion from the same patient. These criteria are more applicable to bacteria and
fungus than Acanthamoeba as it is neither a normal commensal nor a labo-
ratory contaminant.
Antibiotic Susceptibility
Interpretation of agar disk diffusion test (for bacterial susceptibility) that
relate to levels of drug in serum is often controversial. However, since higher
antibiotic concentrations can be achieved in the cornea by topical admin-
istration of antibiotics, an organism labeled as resistant or intermediate in
sensitivity by this test may respond to the drug in vivo. The reverse is unlikely
to be the case.
The quantitative MIC can be compared to the antibiotic concentration
expected at the site of infection. Although resistance breakpoints for ocular
isolates have not been determined and there are no generally accepted cut off
points, results of disk diffusion test have been reported to correlate with MIC
at least in Pseudomonas keratitis.37 For antifungal susceptibility testing MIC,
cut off points available in CLSI guidelines for most drugs is similarly based on
serum levels. Cut off point for most commonly used antifungal agent in fun-
gal keratitis, natamycin, is not available in CLSI guidelines, however; recent
studies have considered MIC greater than 16 mg/mL as sensitive.38,39
MOLECULAR METHODS
The results of PCR on corneal scrapings are usually as good as the choice of
primers (oligonucleotide sequence for a particular gene of a particular organ-
ism) and the stringent performance of the test. Being a highly sensitive test,
instances of false positives can be high if PCR test is not handled carefully.
Any laboratory that undertakes molecular diagnostics must comply with all
requirements to contain amplicon contamination, use appropriate controls
and provide reliable results. The PCR results are best viewed in conjunction
with the clinical impression and, if possible, with another supporting labora-
tory evidence towards the diagnosis.
PROTOCOL FOR VIRAL KERATITIS

The advancement made in the field of laboratory techniques for the diagno-
sis of viral infections in the past decade has been enormous with the intro-
duction of newer techniques and improvization of earlier techniques. These
techniques have been extensively employed for the diagnosis of viral keratitis,
especially in advanced countries. However, owing to the cost constraints, the
techniques are yet to become a routine in most laboratories in India, including
Chapter_08.indd 156 06-02-2018 21:09:35

large university laboratories. Several factors may need to be considered

before a laboratory chooses to adopt advanced techniques for the diagnosis
of viral keratitis or in fact any viral disease. These factors include information
regarding the prevalence of the particular viral infection, need of screening
for the same in a given population, cost of the technique, availability of the
infrastructure, and whether a rapid diagnosis can be provided. An ideal tech-
nique would be cost effective, provide a rapid diagnosis in a reasonable time
frame, easy to perform and interpret and adaptable in routine microbiology
laboratories.
As pertinent in nonviral keratitis, the collection, transport and processing
of corneal samples for the diagnosis of viral keratitis have a distinct protocol
which may be combined with the former in case of clinical uncertainty.
Collection of Samples
A variety of samples including corneal scrapings, corneal swabs, corneal

impression smear and corneal button may be submitted for viral diagnosis.
In addition or instead of corneal samples, conjunctival scrapings/swabs or
aqueous humor may also be helpful in some situations. As is true for most
diseases, collection of clinical sample early in the disease prior to administra-
tion of antimicrobial agents, is most useful for laboratory diagnosis.
Transport of Samples
Unlike the banishment for transport of corneal scrapings (in a transport

medium to the laboratory) in the protocol for nonviral corneal ulcer diagnosis,
the sample for viral diagnosis always needs to be collected in an appropriate
transport medium (except the smears) and sent to the laboratory. Methods
of transport would vary according to the type of sample collected. Table 8.3
outlines the methods of transportation of samples to the virology laboratory.
Processing of Samples
Samples received in a virology laboratory may be processed using a variety of

techniques. The choice of technique would depend on the type of sample and
the specific virus that is being looked for. Most of the procedures can be per-
formed in a moderately equipped laboratory. The procedures standardized
and adopted by us for the diagnosis of herpes simplex virus (HSV) keratitis
are outlined in Table 8.4. Of all available laboratory techniques for diagnosis
of viral infections, only a few can be adopted in a particular laboratory. The
choice is made based on the advantages, disadvantages and cost effective-
ness of the techniques and their overall utilities.
Direct Microscopic Examination (Cytology)
A rapid diagnosis of viral keratitis can be established by observing stained

smears of corneal scrapings, conjunctival scrapings/swabs or centrifuged
Chapter_08.indd 157 06-02-2018 21:09:35

Table 8.3: Methods of transportation of specimens to the virology laboratory for inves-
tigation of viral keratitis.
A. Corneal scrapings
1. Smear on glass slide, air dry and send for staining/IF/IP
2. Transfer in a vial (0.5 to 1 mL) of viral transport medium (VTM) and send for culture. It
can be stored at 4°C without freezing
3. Transfer on a cellulose acetate membrane, air dry, fix in acetone/methanol and send
for staining/IF/IP
4. Transfer in 1 mL of PBS/MEM/HBSS and send for PCR
B. Corneal impression smear on glass slide or cellulose acetate membrane
Air dry, fix in acetone/methanol/15 minutes and send for staining/IF/IP
C. Corneal/conjunctival swab
1. Use cotton swab to collect material and transfer in VTM and send for culture. It can
be stored at 4°C without freezing
2. Dry swab and calcium alginate swabs are unacceptable
D. Corneal button
1. Place in VTM and send for culture
2. Place in 10% buffered formalin and send for histopathology
3. Place in PBS/MEM/HBSS and send for PCR
E. Aqueous humor
1. Place few drops in VTM and send for culture
2. Place in sterile tube/eppendorf and send for PCR or staining/IF/IP
PBS: Phosphate buffered saline; MEM: Minimum essential medium; HBSS: Hank’s balanced
salt solution; PCR: Polymerase chain reaction.
Table 8.4: Methods for laboratory diagnosis of viral

keratitis at LV prasad eye institute.
1. Non-specific smear examination (cytology) methods

Papanicolaou stain
Giemsa stain
2. Cell-associated antigen detection methods
Direct/Indirect immunofluorescence assay (IF)
Indirect immunopeoxidase assay (IP)
3. Virus isolation (tissue culture) methods
Conventional tissue culture
Shell-vial technique
4. Molecular virology method
Polymerase chain reaction
deposits of aqueous humor (cytospin).40 This may be accomplished using

nonspecific staining techniques such as Giemsa, Papanicolaou, and Hema-
toxylin-Eosin stain. These techniques help visualize multinucleated giant cells
Chapter_08.indd 158 06-02-2018 21:09:35

Fig. 8.11: Corneal scrapings from a case of herpes simplex virus (HSV) keratitis showing
multinucleated giant cell (Giemsa stain, original magnification × 1000)
(Fig. 8.11) which is a nonspecific finding in cytology smears, usually associated

with viral infections,41 koilocytic changes and intranuclear/intracytoplasmic
inclusions, and various inflammatory cells which are predominantly lympho-
cytes. Koilocytic changes are characteristic perinuclear clearing (halo) with
increase in density of surrounding rim of cytoplasm, classically described
in Human papilloma virus infected squamous epithelial cells of the cervix.
Intranuclear inclusions are more efficiently seen in Papanicolaou stain than
Giemsa stained smears, however, Giemsa stain is good for enumerating cell
types. Though these staining techniques have the advantage of being rapid
and inexpensive they are often nonspecific and offer low sensitivity in the
diagnosis of viral infection. For example, these stains cannot differentiate the
intranuclear inclusions of HSV from that of Varicella zoster virus (VZV).
Specific cytology techniques used for viral diagnosis are techniques that
indirectly suggest the presence of viral antigen in the clinical sample. Detec-
tion of cell associated viral antigen in a corneal scraping, impression cytology
or conjunctival scraping is very useful in the diagnosis of viral keratitis.42,43
We have been routinely using direct and indirect immunofluorescence (IF)
(Fig. 8.12) and indirect immunoperoxidase (IP) (Fig. 8.13) assays in the
diagnosis of HSV, VZV keratitis and adenoviral keratoconjunctivitis. Both
these tests are rapid, specific and sensitive when suitable monoclonal or
purified polyclonal antibodies are used in the test system. Relatively higher
sensitivity and lower specificity is achieved with purified polyclonal antibod-
ies while monoclonal antibodies show high specificity but lose out on sensi-
tivity, IP assay has distinct advantages over IF assay. The former provides a
permanent preparation for records and utilizes an ordinary light microscope
while the latter has the inherent problem of quenching (fading) of fluores-
cence and requires a sophisticated and expensive fluorescence microscope.
Chapter_08.indd 159 06-02-2018 21:09:35

Fig. 8.12: Corneal scraping from a case of herpes simplex virus (HSV) keratitis showing
presence of HSV-1 antigen in the epithelial cells (indirect immunofluorescence assay,
original magnification × 250).
Fig. 8.13: Corneal scraping from the same patient of herpes simplex virus (HSV) keratitis
(Fig. 23.12) showing presence of HSV-1 antigen (stained brown) in epithelial cells (indi-
rect immunoperoxidase assay, original magnification × 500).
In addition, the IP technique can be used on paraffin embedded tissue while

the IF technique provides better results with frozen tissue sections.
Detection of soluble viral antigens in corneal scrapings collected in buffer
and body fluids including tears, aqueous and vitreous have been described
using enzyme-linked immunosorbent assay (ELISA),44 latex agglutination,45
and radio immunoassay (RIA). Results obtained by ELISA and RIA are more
objective compared to IF and IP assays (which tend to be subjective), how-
Chapter_08.indd 160 06-02-2018 21:09:36

Table 8.5: Rapid diagnostic tests for viral keratitis.
Type of test Time Viruses detected

I. Less than 1 hour
1. Giemsa stain (Diff Quik)TM 5 minutes HSV 1/2, VZV, Adenovirus
2. Papanicolaou stain 45 minutes HSV 1/2, VZV, Adenovirus
3. HSV test kit (Sure Cell Herpes)â 20 minutes HSV 1/2
4. Latex agglutination (Virogen)â 35 minutes HSV 1/2
II. Between 1 and 6 hours
1. Immunofluorescence 2–3 hours HSV 1/2, VZV, Adenovirus
2. Immunoperoxidase 4–5 hours HSV 1/2, VZV, Adenovirus
3. HSV antigen detection (Herp check)â 5 hours HSV-1
4. ELISA 3–4 hours HSV ½
HSV: Herpes simplex virus; ELISA: Enzyme-linked immunosorbent assay; VZV: Varicella zoster
virus.
ever, we do not have experience using these techniques for the diagnosis of
viral keratitis. Some of the rapid methods of antigen detection in viral keratitis
are described in Table 8.5.
Tissue Culture Methods
Classically described techniques of virus isolation have been embryonated

eggs and animal inoculation, which are not favored by most virology laborato-
ries for routine diagnostic purposes. Much favored technique is that of tissue
culture, especially cell cultures. Established cell lines such as HeLa, Vero, HEp 2,
MRC-5, etc. have been used for isolation of HSV from corneal scrapings.46
We have recently succeeded in using immortalized human corneal epithelial
cell line47 for isolation of HSV.
Growth of virus in the cell lines can be determined either by character-
istic cellular changes or cytopathic effect (CPE) as shown in Figure 8.14 or
by IF or IP techniques, which detect viral antigens in the infected cell lines
(described above). Appearance of CPE may take several days but antigens
can be detected even before CPE occurs, thereby rendering the latter a more
rapid method. Viruses may be cultured in cell lines maintained in tubes (tube
culture) or on cover slips in vials (shell vial).48 Shell vial technique is a mod-
ification of conventional tissue culture technique wherein entry of virus into
the monolayer of susceptible cells (on a coverslip in a vial) is facilitated by
centrifugation of the vial containing cells and the clinical sample (spin ampli-
fication).49 The virus growth occurs in a shorter period (18–72 hours) by this
method and additionally, both IF and IP techniques can be performed easily
on the cover slips retrieved from the vials for antigen detection. Both these
factors are responsible for increased sensitivity of shell vial technique in iso-
lation of viruses.
Chapter_08.indd 161 06-02-2018 21:09:36

Fig. 8.14: Monolayer of Vero cell line showing cytopathic effect caused by HSV-1 indi-
cating growth of the virus in the cells (tube culture, phase contrast, original magnifica-
tion × 200).
Molecular Methods
As mentioned earlier, more than any other infection, molecular methods

are most suited for the diagnosis of viral infections. PCR technique has been
employed extensively for the detection of viral DNA in clinical samples which
is one of the best indications for diagnostic use of this technique.50 By virtue
of being extremely sensitive and specific and at the same time simple and
rapid. PCR is presently the most sought after technique for viral diagnosis. In
our experience, combination of cytology coupled with antigen detection by
IF or IP techniques and viral DNA detection by PCR may obviate the role of
cumbersome procedures of viral isolation by tissue culture, in the diagnosis
of viral keratitis.51 By cost considerations, setting up and running a molecu-
lar virology laboratory may be less expensive than tissue culture laboratory.
Diagnosis of atypical herpetic epithelial keratitis using primers for 142 base
pair segments of the DNA polymerase gene of the HSV genome by PCR have
been reported.52,53 A variety of primers for PCR diagnosis of HSV type 1/2
and VZV infections of the eye (other than keratitis) have been described and
they can be adapted for the diagnosis of keratitis.54,55 A nested PCR for stro-
mal herpetic keratitis diagnosis has also been described.56 We have designed
a multiplex PCR for the detection of Herpes simplex virus-1, Varicella zos-
ter virus and cytomegalovirus in ocular specimens.57 Real time PCR is being
increasingly used for viral load estimation in various tissues including corneal
tissue. Rapid diagnosis of adenoviral keratoconjunctivitis has been reported
using fully automated real-time PCR.58 Apart from diagnosis, a wide range of
Chapter_08.indd 162 06-02-2018 21:09:37

Fig. 8.15: Detection of HSV-1 by polymerase chain reaction (PCR) in a corneal scraping
from a case of herpes simplex virus (HSV) keratitis. Agarose gel electrophoresis (Ethid-
ium Bromide stained) showing negative control (lane 1), positive control (lane 2), test
samples (lanes 3, 4) and molecular weight marker (lane 5). Note the band of 221 bp size
(HSV-1, Glycoprotein D gene specific) in lanes 2 and 3. Sample V 459/01 is positive and
V460/01 is negative for HSV-1 DNA.
utility of real-time PCR has been reported such as assessment of prevalence

and clinical consequence of HSV type-1,59 and quantitation in tear fluid and
aqueous humor.60 In case of typical herpetic epithelial keratitis usually a large
number of HSV DNA copies are detected compared to stromal keratitis.61
As has been mentioned in the protocol for nonviral keratitis guarding
against false positives in PCR based test is a great challenge to a molecular
virology laboratory. Top of the line quality control, appropriate controls and
good laboratory practices are mandatory to obtain reliable laboratory reports.
Interpretation of Virology Results
As indicated in Table 8.4, our laboratory routinely performs a variety of tech-

niques for the laboratory diagnosis of viral keratitis which includes cytology
by Giemsa stain, antigen detection by IF/IP techniques, and PCR (Fig. 8.15).
In an analysis of 170 clinically suspected cases of HSV keratitis whose cor-
neal scrapings were tested by PCR, antigen detection by IF, and cytology by
Giemsa stain, the sensitivity of the tests was 100%, 85.7% and 57.1% respec-
tively.51 A laboratory diagnosis of HSV keratitis was offered when HSV-1 anti-
gen was detected and/or HSV DNA was detected by PCR with or without
cellular changes in Giemsa stained smear. PCR results were interpreted with
caution when this test alone was positive. It was always correlated with clin-
ical findings and with the results of other tests. False positives were avoided
by using a different primer set and adopting a reduced sensitivity PCR.62
Chapter_08.indd 163 06-02-2018 21:09:37

It is evident from our observations that adopting a single technique alone

may result in under diagnosis. Giemsa stain, though less sensitive than oth-
ers, is a valuable test. Presence of multinucleated giant cells, intranuclear
inclusions, and koilocytic changes are indicative of HSV/VZV infection. Initi-
ation of antiviral therapy is indicated based on these smear findings coupled
with positive antigen detection by IF or IP assays. Both IF and IP assays detect
viral proteins in the absence of viable virions when cultures would be nega-
tive. We have often been more successful in detecting the viral antigen than
isolating the virus. We recommend that antigen detection assays and Giemsa
or Papanicolaou staining be done in the laboratory diagnosis of viral keratitis
where facilities for culture and PCR are unavailable.
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45. Kowalski RP, Gordon YJ. Evaluation of immunologic tests for the detection of
ocular herpes simplex virus. Ophthalmology. 1989;96(11):1583-6.
46. Simon MW, Miller D, Pflugfelder SC, et al. Comparison of immunocytology to
tissue culture for diagnosis of presumed herpesvirus dendritic epithelial kerati-
tis. Ophthalmology. 1992; 99(9):1408-13.
47. Athmanathan S, Reddy SB, Nutheti R, et al. Comparison of an immortalized hu-
man corneal epithelial cell line with Vero cells in the isolation of herpes simplex
virus-1 for the laboratory diagnosis of Herpes simplex keratitis. BMC Ophthal-
mol. 2002;2:3.
48. Athmanathan S, Bandlapally S, Rao GN. Comparison of the sensitivity of
a 24 h-shell vial assay, and conventional tube culture, in the isolation of
Herpes simplex virus-1 from corneal scrapings. BMC Clin Pathol. 2002;2(1):1.
49. Johnson FB, Luker G, Chow C. Comparison of shell vial culture and the
suspension-infection method for the rapid detection of herpes simplex
viruses. Diagn Microbiol Infect Dis. 1993;16(1):61-6.
50. Podzorski RP, Persing DH. Molecular detection and identification of micro-
organisms, Chapter 13. In: Murray PR, Baron EJ, Pfaller MA, Tenover FC,
Yolken RH (Eds). Manual of Clinical Microbiology (6th edition). Washington DC:
American Society of Microbiology; 1995.
51. Subhan S, Jose RJ, Duggirala A, et al. Diagnosis of herpes simplex virus-1 kera-
titis: comparison of Giemsa stain, immunofluorescence assay and polymerase
chain reaction. Curr Eye Res. 2004;29(2-3):209-13.
52. Tei M, Nishida K, Kinoshita S. Polymerase chain reaction detection of Herpes
simplex virus in tear fluid from atypical herpetic epithelial keratitis after pene-
trating keratoplasty. Am J Ophthalmol. 1996;122(5):732-5.
53. Koizumi N, Nishida K, Adachi W, et al. Detection of herpes simplex virus DNA in
atypical epithelial keratitis using polymerase chain reaction. Br J Ophthalmol.
1999;83(8):957-60.
54. Yamamoto S, Langston DP, Kinoshita S, et al. Detecting herpesvirus DNA in uve-
itis using the polymerase chain reaction. Br J Ophthalmol. 1996;80(5): 465-8.
55. Cunningham ET, Short GA, Irvine AR, et al. Acquired immunodeficiency syn-
drome—associated herpes simplex virus retinitis. Clinical description and use
of a polymerase chain reaction—based assay as a diagnostic tool. Arch Oph-
thalmol. 1996;114(7):834-40.
56. Kudo E, Shiota H, Kinouchi Y, et al. Detection of herpes simplex virus DNA in
tear fluid of stromal herpetic keratitis patients by nested polymerase chain re-
action. Jpn J Ophthalmol. 1996;40:390-6.
57. Chichili GR, Athmanathan S, Farhatullah S, et al. Multiplex polymerase chain
reaction for the detection of herpes simplex virus, varicella-zoster virus and
cytomegalovirus in ocular specimens. Curr Eye Res. 2003;27(2):85-90.
58. Koidl C, Bozic M, Mossbock G, et al. Rapid diagnosis of adenoviral keratocon-
junctivitis by a fully automated molecular assay. Ophthalmology. 2005;112(9):
1521-8.
59. Remeijer L, Duan R, van Dun JM, et al. Prevalence and clinical consequ-
ences of herpes simplex virus type 1 DNA in human cornea tissues. J Infect Dis.
2009;200(1):11-9.
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60. Fukuda M, Deai T, Higaki S, et al. Presence of a large amount of herpes simplex
virus genome in tear fluid of herpetic stromal keratitis and persistent epithelial
defect patients. Semin Ophthalmol. 2008;23(4):217-20.
61. Kakimaru-Hasegawa A, Kuo CH, Komatsu N, et al. Clinical application of
real-time polymerase chain reaction for diagnosis of herpetic diseases of the
anterior segment of the eye. Jpn J Ophthalmol. 2008;52(1):24-31.
62. Yamamoto S, Shimomura Y, Kinoshita S, et al. Detection of Herpes simplex virus
DNA in human tear film by the polymerase chain reaction. Am J Ophthalmol.
1994;117(2):160-3.
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9
CHAPTER
Fungal Keratitis
N Venkatesh Prajna, Lalitha Prajna, C Veerajayalakshmi
INTRODUCTION
The incidence of fungal keratitis has shown a dramatic increase in the recent
years. In fact, in country like India, fungi have nearly replaced bacteria as
the most common cause of infectious suppurative keratitis. This increase
is thought to be due to a combination of various factors namely increased
clinical suspicion, advances in diagnostic techniques and paradoxically the
advancement in the field of antibacterial therapy which has proportionately
reduced the incidence of bacterial keratitis. Morbidity in fungal infections
tends to be greater than that in bacterial keratitis because the diagnosis is
often delayed and the available drugs are not very effective.
EPIDEMIOLOGY
Fungal keratitis is most often encountered in rural populations, where agri-
cultural workers are exposed to corneal injury contaminated with organic
matter much more frequently than population at large.1 It is relatively
uncommon in the western world. Even, when it happens, it is usually caused
by Candida. Filamentary fungi, especially, Aspergillus and Fusarium are the
common cause of mycotic keratitis in most of the developing world.
CLASSIFICATION OF FUNGI
Fungi are eukaryotic and heterotrophic organisms. Although numerous fungi
have been implicated, the pathogenic fungi, which cause significant keratitis,
can be divided into filamentous, yeast and dimorphic forms.
Chapter_09.indd 169 06-02-2018 21:17:32

Filamentous Fungi
Filamentous fungi are also known as moulds, they occur as long filaments,
called hyphe, which grow by apical extension and produce feathery aerial
colonies above the culture media. They can be further divided into septate
and nonseptate organisms. The septate filamentary fungi are the most com-
mon cause of keratomycosis. They are divided into nonpigmented monilial
(which include Fusarium sp., Aspergillus sp. and Acremonium sp.) and pig-
mented dematiaceous (Curvularia sp. and Lasiodiplodia sp.) varieties. The
nonseptate filamentary fungi (Mucor, Absidia and Rhizopus sp.) are import-
ant causes of orbital disease and endogenous endophthalmitis, but do not
commonly produce corneal disease.
Yeasts
Yeasts are fungi with usual and dominant growth as unicellular organisms
and form creamy, pasty colonies, which may be mistaken for staphylococcal
colonies. They divide by asexual budding, forming pseudohyphae and do not
form mycelium in culture. The most common fungi in this category are the
Candida sp. and Cryptococcus sp., which are part of the normal flora of skin,
respiratory tract and conjunctiva and act as opportunistic pathogens.
Dimorphic Fungi
Dimorphic fungi have two distinct morphologic forms: the yeast phase that
occurs in tissues and a mycelial phase, which occurs in media and natural
surfaces. These fungi, which include in this category are Blastomyces, Coccid-
ioides, Histoplasma and Sporothrix, exhibit properties of molds when, culti-
vated at 25°C and of yeasts when grown at 37°C.
RISK FACTORS
Fungi are ubiquitous organisms present almost everywhere in the environ-
ment. Although the eye is continuously exposed to these pathogens, the
normal defense mechanisms, such as the eyelids, tear components and the
corneal epithelium, provide adequate protection. An epithelial defect is a
prerequisite for these organisms to set up an infection.
The importance of trauma that is often too trivial and frequently associ-
ated with plant material has been well documented in the initiation of fungal
infection caused by filamentous fungi.
Contact lens wear is an uncommon risk factor in fungal keratitis. These
organisms have been shown to grow within the matrix of soft contact lenses.2
Filamentous fungi were more commonly associated with cosmetic lens wear
and yeasts from therapeutic lens use.
Corticosteroids appear to activate and increase the virulence of
fungi. The other factors uncommonly reported include vernal or allergic
Chapter_09.indd 170 06-02-2018 21:17:33

Fungal Keratitis 171
keratoconjunctivitis, neurotrophic ulcers and penetrating keratoplasty. The

predisposing factors for the development of fungal keratitis in patients after
penetrating keratoplasty include suture problems, topical steroid therapy,
chronic antibiotic use, contact lens wear, graft failure and persistent epithelial
defects. Fungal corneal ulcers have been reported following refractive
surgical procedures like radial keratotomy, photorefractive keratotomy and
more recently following Laser in situ keratomileusis (LASIK) procedures,
either in the immediate postoperative period (direct surgical contamination)
or later (following trauma).
The principal risk factors for yeast or Candida keratitis are prolonged
epithelial ulceration, penetrating keratoplasty and therapeutic contact lens
wear. In addition systemic diseases, like Sjogrens syndrome, erythema mul-
tiforme, immunodeficiency, endocrinopathy, diabetes and hypovitaminosis
A, have been shown to predispose to candidal infection.
Certain investigators have highlighted the association between the pre-
vailing climatic conditions and the incidence of fungal keratitis. In India, the
incidence is highest in the harvesting seasons September-October. There is
less seasonal variation with regard to yeast infections.
PATHOGENESIS
The filamentous fungi affect normal eyes of immunocompetent hosts after
corneal abrasion or trauma, from some kind of vegetable matter. The yeasts
usually cause keratitis in immunocompromised individuals. Their pathoge-
nicity is related to a decrease in the systemic or local defense mechanisms
either by a direct effect on the immune system (topical steroids) or by an
alteration in the normal epithelial barriers (i.e., persistent epithelial defects,
bandage contact lenses, neurotrophic keratitis, topical anesthetics, etc.) with
predisposing systemic and/or eye disease. The dimorphic fungi are a rare
cause of keratitis.
The exact mechanisms underlying the pathogenesis of fungal infections
are unclear. As compared to bacteria, the fungi are relatively nonimmuno-
genic, partly because of their large size, which prevents them from being
engulfed by the neutrophils, and partly because they do not secrete chemo-
tactic factors which attract inflammatory cells. After entering through a cor-
neal epithelial defect, the fungi elaborate toxic substances and enzymes such
as proteases, hemolysins and exotoxins. Fusarium sp. is especially known to
possess specific cellular and molecular attributes, which aid to cause virulent
reaction. They can adhere to biopolymers and have the ability to produce tox-
ins and elaborate enzymes.3
A few strains of Aspergillus produce aflatoxins and ochratoxins.4 The
conidia of Aspergillus fumigatus have been shown to bind to and degrade
basement membrane laminin, an extracellular matrix glycoprotein found
in basement membranes. They have the capacity to penetrate an intact
Descemet’s membrane. The resultant host inflammatory response subsequently
Chapter_09.indd 171 06-02-2018 21:17:33

contributes to a part of the tissue damage. Activation of the complement

system leads to concentration of polymorphonuclear inflammatory cells in
the corneal cells that liberate proteolytic enzymes. Encapsulated Cryptococcus
neoformans, Candida albicans and the conidia of A. fumigatus all activate the
complement system by either classic or alternative pathways.
Fungi can penetrate deep into stroma and through an intact Descemet’s
membrane. It is thought that once fungus gains access into anterior chamber
or to the iris and lens, eradication of the organism becomes extremely diffi-
cult. Likewise, organisms that extend from cornea into sclera become diffi-
cult to control.
C. albicans strains have also been known to produce a variety of pro-
teolytic enzymes. The most important of these is an aspartyl acid protease
that can act on a wide variety of tissue proteins and is thought to contribute
to the invasiveness of the organism. In addition, various virulence factors,
such as phospholipase A and lysophospholipase, have been identified with
Candida sp.
CLINICAL FEATURES
Filamentary Fungi
Unlike bacterial infections, there is less pain, conjunctival congestion, dis-

charge and chemosis early in the course of fungal infection and the symp-
toms are far less than what is expected of the size of the ulcer.
The earliest finding may be a small nonspecific stromal infiltrate with
surrounding unhealthy looking epithelium, in which case it is indistinguish-
able from a bacterial infection. Commonly the patient presents with a central
or a paracentral ulcer with feathery stromal margins (Fig. 9.1). The common
misdiagnosis, which might be made at this stage, may be a dendritic keratitis
Fig. 9.1: Typical fungal ulcer with feathery margins.
Chapter_09.indd 172 06-02-2018 21:17:33

caused by herpes simplex. These fungal pseudodendritic lesions are shorter,

stockier and are associated with surrounding stromal infiltration. In addition,
a mirror image of the pseudodendritic lesion in the deeper stromal layers can
also be seen. The adjacent Descemet’s membrane may be thrown into folds.
With time, the ulcer starts to become larger and elevated above the level
of the corneal surface. The edges of the ulcer look serrated, somewhat looking
like a can opener capsulotomy. The surface looks dirty white and dry and has
a rough texture. The serrated edges and the dry elevated rough surface can be
considered pathognomonic of a fungal lesion (Fig. 9.2). The stromal lesions
are present beyond the size of the epithelial defect and have a feathery pat-
tern. In rare instances, the lesion may be entirely in the posterior aspect of the
corneal stroma without an accompanying epithelial defect. In these cases,
the posterior stromal lesions also have a feathery edge like paint sprayed over
a wall.
Foci of infiltration can be seen several millimeters away from the main
area of involvement. These are called satellite lesions and they may remain
isolated from the main lesion or may be connected with the main ulcer by a
thin line of stromal infiltration (Fig. 9.3). The epithelium can be intact over
the infiltrate. An endothelial plaque may also be present. Like in many other
keratitis, a ring infiltrate may surround the primary lesion, most likely repre-
senting an antibody response to fungal antigen (Fig. 9.4).
Hypopyon can be present in varying proportions and the amount is not
directly proportional to the size of the ulcer.
As the lesions progress, the pain gets intense and the ulcer involves
almost the whole of the cornea and starts to lose its characteristic pattern.
The lesions look more suppurative and soupy. The edges of the ulcer may start
becoming rounded like a bacterial ulcer, but the edges of the deeper stro-
mal lesions have the characteristic feathery pattern until the final course of
Fig. 9.2: Ulcer with raised surface.
Chapter_09.indd 173 06-02-2018 21:17:33

Fig. 9.3: Multiple satellite lesions.
Fig. 9.4: Corneal ulcer with serrated and immune ring.
the disease. As the size of the ulcer becomes larger, it becomes more flushed
with the surface and assumes a smooth surface. A significant majority of the
deeper stromal ulcers perforate over time.
In the ulcers caused by the Dematiaceous fungi, there may be macro-
scopic pigment deposition over the surface. The color may vary from a thick
blackish brown pigmentary lesion to a less mottled greenish-gray pigment
dispersed over a whitish base (Fig. 9.5). The denser lesion can be mistaken for
a uveal prolapse. The lesion caused by Dematiaceous fungi are more elevated
than those caused by nonpigmentary filamentous fungi and has a character-
istic mushroom head appearance. These lesions can be dissected from the
surface using a Bard Parker knife and has a tough, leathery attachment to the
ulcer surface.
Chapter_09.indd 174 06-02-2018 21:17:34

Fig. 9.5: Pigmented fungal ulcer with macroscopic pigments over the surface of the
ulcer.
In contrast to the filamentary fungal keratitis, the stromal keratitis caused

by Candida may be more localized and have a collar-button configuration,
often with a small ulcer and an expanding infiltrate. They are usually super-
imposed on a chronic debilitated ocular condition. The lesions tend to be
oval, plaque like and elevated. They are well-outlined and surrounded by
stromal edema and more closely resemble the bacterial keratitis. If untreated,
the keratitis evolves to produce dense suppuration and necrosis of the deep
stroma.
It should be emphasized that the differentiation between an advanced
keratitis caused by bacteria and fungi is difficult to distinguish by clinical
features alone. Future developments in the field of confocal microscopy may
become a useful clinical tool for the diagnosis of fungal keratitis. The lack of
experience and the cost of the instrument may be the limiting constraints for
its widespread use.
HISTOPATHOLOGY
Nonreplicating fungi, and their mycotoxins, proteolytic enzymes and solu-
ble fungal antigens are capable of inducing severe inflammatory reactions.
These agents can result in necrosis of the corneal lamellae, and incite an
antigenic response with immune ring formation and hypopyon. The inflam-
matory response tends to be less marked than seen in bacterial keratitis
though the epithelium often remains intact over the infective lesion. The
classical histopathologic findings in fungal keratitis include fungal hyphal
elements oriented perpendicular to the normal corneal lamellae, and the
tendency for the hyphal elements apparently to penetrate Descemet’s mem-
brane and spread into the anterior chamber (these two features are consid-
ered to be suggestive of progressive pathogenicity). When this occurs, it is
Chapter_09.indd 175 06-02-2018 21:17:34

seen as retrocorneal or anterior chamber inflammatory mass adjacent to an

area of deep keratitis.
LABORATORY DIAGNOSIS
A standard microbiologic baseline workup should be performed in every
case of suspected infectious keratitis. This is even more crucial in the regions
of the world, where fungi and bacteria cause keratitis, almost in equal pro-
portions. In addition, combined infections with bacteria and fungi, or even
with multiple fungi, can be detected through this routine examination.
Scrapings are made from the base and the edges of the ulcer under top-
ical anesthesia using a Bard-Parker knife or a Kimura spatula. The lesions
caused by fungi often feel gritty and have a leathery attachment to the base of
the ulcer. Calcium alginate swabs have been reported to increase the recov-
ery rate of the organisms. In deeper lesions, a corneal biopsy may be required
for obtaining adequate specimen.
Direct microscopic evaluation is the most valuable and rapid diagnos-
tic tool for the detection of fungal elements in corneal scrapings. The ini-
tial smears can be examined using a potassium hydroxide (KOH) mount
preparation and a Gram stain. The KOH preparation is a simple, reliable and
reproducible screening method for identifying filamentary fungi. KOH has
been used as a 10–20% suspension, either plain or with ink or with lactophe-
nol cotton blue. Proteinaceous components such as host cells are partially
digested by the alkali, leaving intact the polysaccharide containing fungal
cell walls (Fig. 9.6). However, the sensitivity of this method is highly variable
ranging between 33% and 94%. A recent study done by Sharma et al. aimed
to look at the sensitivity, specificity and predictive values of KOH prepa-
ration and to compare its efficacy with other methods of corneal scraping
Fig. 9.6: Fungal filaments in potassium hydroxide (KOH) wet mount under 40× magni-
fication.
Chapter_09.indd 176 06-02-2018 21:17:34

Fig. 9.7: Fungal filaments seen in Gram staining under 100× magnification.
examinations used in the diagnosis of mycotic keratitis.5 KOH is an excellent

and simple tool and has a definite place in the armamentarium of diagnostic
techniques.
Giemsa and Gram stain techniques are equally sensitive in detecting fun-
gal elements. The Gram stain does not stain the cell wall or septa of hyphal
fragments but is absorbed by the protoplasm (Fig. 9.7). Yeasts typically stain
dark blue and can be distinguished from bacteria, foreign material, precip-
itate and other artifacts. Hyphal fragments and yeasts appear dark blue or
purple in Giemsa stain. Gomori methenamine silver and Periodic acid–Schiff
stains are other methods used when conventional staining techniques yield
no results. However, these methods are time consuming, tedious and difficult
to interpret.
Though the use of calcofluor white, acridine orange and fluorescein con-
jugated lectins yield rapid results, special infrastructural requirements make
them inapplicable in most situations. However, these are the stains of choice
for the detection of yeasts.
The findings of direct examination can be confirmed by culture. Sab-
ouraud dextrose agar and Brain-heart infusion broth are the most commonly
used primary isolation media for fungi. Cycloheximide should never be incor-
porated in Sabouraud, because it inhibits the saprophytic fungi predominantly
responsible for keratitis. The media are kept at room temperature (25°C) for
the isolation of fungi. Since bacterial pathogens are always a consideration in
the differential diagnosis of suppurative keratitis, additional material is inocu-
lated in a row of C streaks on fresh sheep blood agar or chocolate agar media.
Most fungi show mycelial growth on blood agar within 24–72 hours. Yeasts
grow on these media at 30°C and are visible in 18–24 hours.
In our laboratory, we prefer to use potato dextrose agar as the princi-
pal culture media to isolate fungi, since this medium is thought to have a
Chapter_09.indd 177 06-02-2018 21:17:35

profound effect on the conidiogenesis and the surface character of Fusarium

colonies. Colonies of Fusarium are usually white in the early stages of devel-
opment, but often show reverse pigmentation. The classification is based
on microscopic features of conidia that form on specialized hyphae called
conidiophores.
Colonies of A. fumigatus are white at first, but as spores are produced,
they become velvet green owing to the pigmentation of the conidia. Asper-
gillus niger colonies are also white during the initial growth phase but turn
completely black on sporulation. The typical colony of Candida sp. on Sab-
ouraud dextrose agar has a dirty white color. It is opaque with a smooth, flat,
round contour and a pasty soft consistency. When grown on blood agar 35°C,
they are slow-growing and nonhemolytic. Candida colonies have a distinc-
tive fruity and yeasty odor.
Identification of C. albicans is usually based on the formation of germ
tubes in vitro and further speciation is based on sugar fermentation. Classi-
fication of noncandidal yeasts is based on biochemical testing. Since most of
the ocular fungal isolates grow quicker, it may not be absolutely necessary for
the incubation period to be prolonged than a week.
Scraping also provides for debridement of organisms and the epithelium,
which may provide a barrier to antifungal drug penetration. When scrapings
are negative, a diagnostic superficial keratectomy or corneal biopsy may be
performed and sent for smear, culture and histopathological examination. In
some cases of deep keratitis, a 27 G hypodermic needle or a 6-0 silk suture
can be introduced into the infiltrate to obtain a specimen for culture.
A polymerase chain reaction (PCR) has been used in an animal model
to develop a sensitive, specific and rapid test to diagnose fusarium keratitis.
Even as the availability of in vitro susceptibility testing for fungi have been
reported, there is yet no agreement on the standardization and applicability
of these tests and currently do not enjoy widespread usage.
MEDICAL TREATMENT
All the available antifungal agents are fungistatic and not fungicidal. The pen-
etration of the drugs is poor and has to be aided by repeated debridement,
which acts by debulking of the pathogenic organism. The treatment sched-
ules are usually prolonged and often leading to a poor compliance with med-
ical therapy.
Most of the antifungal drugs exhibit their effect through their actions on
the fungal cell membrane. In addition to its barrier function, the fungal cell
membrane controls the movement of electrolytes and thereby controls the
internal homeostasis of the cell. Ergosterol is the predominant sterol unique to
the fungal cell membrane, while mammalian cell membranes are composed
of cholesterol. Most antifungals capitalize on this important difference in
plasma membrane constituents to damage the fungal cells, while minimizing
Chapter_09.indd 178 06-02-2018 21:17:35

damage to the host cells. The following three major classes of antifungal
drugs are available:
1. Polyenes
2. Imidazoles
3. Fluorinated pyrimidines
Polyenes
Polyenes constitute the first line of the antifungal agents. They bind prefer-
entially to ergosterol in the fungal plasma membrane, thereby altering mem-
brane permeability and disrupting the fungal cell. Larger polyenes (such as
nystatin and amphotericin B) create channels that span the cell membranes
and allow electrolyte movement. Small polyenes such as natamycin are too
small to bridge the width of the cell membrane and causes localized mem-
brane disruptions thus altering permeability.6
Natamycin
Natamycin is a tetraene antibiotic. Discovered in 1958, it has proved itself to

be the most valuable ocular antifungal agent available till date. The drug is
available as a 5% suspension and must be shaken well before use. Natamy-
cin has a broad spectrum of activity against filamentous fungi. When applied
topically, it adheres to the ulcer site like a rope, which may act as a reservoir
of drug. In general, topical natamycin is well tolerated and its corneal toxicity
is rare. It may cause conjunctival necrosis when given subconjunctivally and
therefore, this route is not preferred.
Amphotericin B
Amphotericin B is a heptene polyene. It is still considered the drug of choice

for treatment of life threatening infections. It is also the drug of choice for
candidal keratitis and Cryptococcus. The drug is variably active against fila-
mentary fungi and is not recommended as the first line treatment for these
organisms. Rifampin reportedly increases the effectiveness of amphotericin
B against Aspergillus sp. but not against Candida sp. The drug is insoluble in
water, unstable at 37°C and degrades rapidly if exposed to light. However, sol-
ubility can be improved by addition of deoxycholate. It is relatively toxic and
cannot penetrate intact epithelium. The adverse effects of the topical applica-
tion include a stinging sensation, chemosis and punctate epithelial keratitis.
They are mostly thought to be due to the presence of bile salt used to stabilize
the drug in solution.
Amphotericin B is dispensed in 20-mL vials for intravenous use contain-
ing 50 mg powder. The powder is initially reconstituted to a concentration
of 5 mg/mL in 10 mL of sterile water for injection. It should be prepared in
Chapter_09.indd 179 06-02-2018 21:17:35

distilled water because it precipitates in sodium chloride solution. An initial

dose of 0.15% is applied for every 5 minutes for half an hour as a loading dose
and then hourly. Subconjunctival injections can cause conjunctival necrosis
and should be avoided. Systemic administration of amphotericin B is neph-
rotoxic and is ineffective in the treatment of keratitis.
Occasionally, intracameral injection of amphotericin B (5–10 mg in 0.1 mL)
can be a useful alternative treatment of severe fungal keratitis with intraocu-
lar extension that is resistant to conventional therapy.7
Nystatin
Nystatin is another polyene antifungal agent that is used as an ointment or
formulated eye drops (50,000 units/mL) and can be used for the treatment of
superficial candidal keratitis. The ointment preparation causes severe sting-
ing sensation and patient compliance is poor for prolonged usage. It is too
toxic for parenteral administration.
Imidazoles
This group constitutes the imidazoles and the structurally related N-

substituted triazole. The imidazoles include clotrimazole, miconazole,
econazole and ketoconazole. The triazoles include fluconazole and itracon-
azole. These drugs exhibit their antifungal activity by having two mechanisms
of action. In lower concentration, they are fungistatic by inhibiting sterol 14
alpha-demethylase; a microsomal P-450 related enzyme, which is required
in the demethylation of lanosterol in the synthesis of ergosterol.8 At higher
concentrations, they are fungicidal which is due to direct membrane damage
to the phospholipids present in the fungal cell wall. However, they are never
able to achieve fungicidal concentration in the human cornea.
Clotrimazole
Clotrimazole is used extensively topically for the treatment of skin and genital
candidal infection. A topical ophthalmic preparation of clotrimazole can be
made with 1% clotrimazole in arachis oil. A dermatological cream containing
clotrimazole 1% is well tolerated when applied to the eye. The drug is too
toxic for systemic use. Though not preferred, it can be used in the treatment
of aspergillus keratitis.
Miconazole
Miconazole can be used through various routes—topically, subconjuncti-

vally or intracamerally. It can be prepared for topical application as a 1% drop
in arachis oil or a 2% cream. The commercially available intravenous prepa-
ration can also be administered topically (10 mg/mL) or by subconjunctival
injection (5–10 mg). The drug is relatively unstable in solution and has to be
Chapter_09.indd 180 06-02-2018 21:17:35

reformulated weekly. It is active against mainly yeasts and is variably active

against filamentary fungi.
Econazole
Econazole is a dichloroimidazole that exhibits a wide spectrum of activity

against filamentous fungi. A topical preparation of econazole 1% can be pre-
pared and is well tolerated.
Ketoconazole
Ketoconazole is more water-soluble and is better absorbed after systemic
administration than other imidazoles. It is available as 200 mg tablets. The
usual dose for adults is 200–400 mg/day, which can be increased to 800 mg or
more. It can also be administered as a topical preparation in concentrations
ranging from 1% to 5%. Clinically, the agent has been shown to be effective
against Candida, Aspergillus, Fusarium and Curvularia sps in particular.9
The most common side effects are dose-dependent nausea, anorexia
and vomiting. Ketoconazole inhibits steroid biosynthesis in fungi. Several
endocrinological abnormalities like decreased libido and testosterone lev-
els, menstrual irregularities and suppression of the ACTH-stimulated plasma
cortisol response have been reported. It blocks the hepatic microsomal sys-
tem, interfering with the metabolism of several drugs such as cyclosporine.
Fluconazole
Fluconazole, a water-soluble triazole is available in an oral dose of 100 mg

capsules and intravenous solution (sterile solution in 0.9% sodium chloride,
2 mg/mL: 100 mL unit). Although the MICs of fluconazole are higher than
other azoles in most susceptibility test systems, the in vitro activity of fluco-
nazole does not parallel the efficacy in infections in animals and in clinical
trials in humans.10 The recommended dose of the drug is 200–400 mg/day
and is the same for oral and intravenous route. It is useful in candida keratits.
Itraconazole
The newer oral triazole antifungal agent itraconazole may also be a help-
ful adjunctive agent in the treatment of fungal keratitis, based on in vitro
activity, pharmacokinetic properties and limited clinical trials in nonocular
infections. However, its drawback is that it is quite hydrophobic, and, being
90% protein bound in the serum, it does not permeate the tissues as well as
fluconazole.
Voriconazole
Voriconazole is one of the recent drugs which is useful in the management
of fungal keratitis.11,12 It is a synthetic derivative of fluconazole in which
Chapter_09.indd 181 06-02-2018 21:17:35

the triazole group is substituted with a fluoropyrimidine moiety and has

an increased potency and in vivo efficacy. Addition of a methyl group to
the propyl backbone increases the affinity of the drug for the target enzyme
(14-a-sterol demethylase). It shows a broader spectrum of activity against
Fusarium, Aspergillus, Scedosporium, Paecilomyces, and Candida. As with
other triazole antifungal agents, voriconazole exerts its affect primarily
through inhibition of cytochrome P-450 dependent 14-sterol demethylase,
an enzyme responsible for conversion of lanosterol to 14-demethyl lanosterol
in ergosterol biosynthetic pathway.
Voriconazole is available in vials containing 30 mg lyophilized powder.
It has excellent corneal penetration because of its low molecular weight
(349 g/mol) and its lipophilic properties. It can be used topically in the form
of 1% eye drops and can be titrated based on epithelial healing. In deeper
lesions, intrastromal voriconazole may be given in the dose of 50 mg/0.1 mL.
Oral voriconazole may be supplemented in the dose of 5 mg/kg body weight.
A baseline liver function test should always be performed before starting oral
voriconazole therapy.
The adverse effects with systemic voriconazole therapy include tran-
sient visual disturbances (photopsias, photophobia or color vision defects),
formed visual hallucinations and elevated liver enzymes.
Fluorinated Pyrimidines
Flucytosine
Flucytosine is a fluorinated pyrimidine and is selectively taken by suscepti-

ble fungi and deaminated to fluorouracil which blocks thymidine synthesis.
Since mammalian cells do not normally metabolize flucytosine, it does not
inhibit metabolic processes in the mammalian cells. It is effective against
Candida and Cryptococcus and certain strains of Aspergillus, but is not effec-
tive against Fusarium and Cephalosporium. However, some fungal strains
lack the specific permease to transport the drug into the cells and they are
resistant to flucytosine. Topical flucytosine is used as drops in a 1% solution
every hour and is well tolerated. Therapeutic levels can also be achieved in
adults with the administration of a dose of 50–150 mg/kg/day in divided
doses. Flucytosine should not be given alone in the treatment of keratomyco-
sis for fear of inducing resistance. Its topical form is relatively nontoxic to the
eye. When administered systemically, it may cause a transient depression of
the bone marrow and diarrhea.
PRINCIPLES OF THERAPY
As mentioned earlier, the treatment for fungal keratitis is usually prolonged.
Improvement may not be visible for several days. Lack of progression of the
stromal infiltrate is the first sign that the antifungal drug is effective. This is
followed by a rounding of the feathery margins, blunting of the perimeters
Chapter_09.indd 182 06-02-2018 21:17:35

and reduction in cellular infiltrate and edema in surrounding stroma. Pro-

longed conjunctival injection, protracted epithelial ulceration, punctate
corneal epithelial erosions and diffuse stromal haze imply drug toxicity. The
appearance of a transient hypopyon in the face of an improving corneal pic-
ture has been attributed to hypersensitivity or a toxic reaction.
The initiation of treatment can be performed as soon as the results of
the KOH mount and the Gram stain are obtained. These tests can readily be
performed at the office of the physician itself. Studies have questioned the
reliability of the positivity of culture as the gold standard and have recom-
mended that all smear positive cases of mycotic keratitis be treated accord-
ingly despite negative culture reports.13 If the smears and the culture results
are negative, but the clinical features are strongly in favor of fungal keratitis,
we recommend initiation of topical antifungal therapy, especially in coun-
tries where there is a high prevalence. Culture results are useful in certain
situations and can play an effective role in identification of the species, which
is useful for epidemiological purposes.
For suspected filamentous fungal keratitis, natamycin 5% drops should
be administered hourly for the initial 24–48 hours and at 2-hour intervals.
The dosage can then be gradually reduced depending upon the response.
If natamycin is not available, then 1% econazole or 1% miconazole can be
used.
For yeast keratitis, topical amphotericin B 0.1–0.25% drops are initially
applied at 15-minute intervals for the first 24–48 hours and then frequency
of application is gradually reduced for the next 4–6 days. If initial therapy was
topical natamycin or amphotericin B drops alone and the stromal suppuration
is progressing, oral ketoconazole or fluconazole should be added for either
filamentous fungal or yeast keratitis. Subconjunctival miconazole (5 mg)
or fluconazole (1 mg) can be given.
Prolonged treatments with systemic antifungals are reserved for deep
keratitis associated with scleritis and endophthalmitis. The most frequently
used oral antifungal has been ketoconazole. Recent evidence suggests that
fluconazole may penetrate better into the cornea after systemic adminis-
tration than some other azoles and associated with fewer side effects.14 The
other drugs, which have been tried for the treatment of fungal keratitis in past
include silver sulfadiazine and sulfacetamide.
The issue of synergy or antagonism when the antifungal drugs are com-
bined has given varying results. A combination of flucytosine and miconazole
or natamycin was found to be synergistic in vitro against aspergillus kerati-
tis.15 Certain investigators routinely use flucytosine topical (1% solution) and
systemic (oral 150 mg/kg) in combination with amphotericin B in the man-
agement of candida keratitis.16 Antagonism has been reported when ampho-
tericin B and the imidazoles have been used systemically.17
Supplementary therapy of keratomycosis includes a cycloplegic-mydriatic
agent, such as atropine 1% twice a day, and topical beta blocker or oral
carbonic anhydrase inhibitor to control secondary ocular hypertension.
Chapter_09.indd 183 06-02-2018 21:17:35

Corticosteroids do not have any role in the treatment regimen of fungal

keratitis and is not recommended in any stage of the disease.
SURGICAL TREATMENT
Regular debridement of the ulcer using a scalpel or a Kimura spatula is an
invaluable step to ensure adequate therapeutic levels of the antifungals into
the deeper stromal layers. Some authors feel that surgery is seldom needed
during the acute spread of the infection.18 Studies conducted at the Bascum
Palmer Institute indicate that therapeutic keratoplasty was required seven
times more frequently in fungal keratitis than in bacterial keratitis. We also
feel that the surgical modality of excisional keratoplasty has an important role
to play in the treatment of fungal keratitis and perhaps in some parts of the
world, it may well be the primary choice of treatment.
Therapeutic keratoplasty has to be contemplated when the ulcer pro-
gresses despite specific antifungal therapy. The progression is characterized
by increased area and depth of stromal suppuration, stromal ulceration to
the point of extreme thinning of the cornea, dense fibrinous exudate in the
anterior chamber and frank perforation. However, a small perforation devel-
oping during the course of the healing can be managed by the use of a tissue
adhesive.
The goals of the therapeutic keratoplasty are to remove the infected part
of the cornea, and restore the integrity of the globe. A recurrence of infection
in a graft is more difficult to treat than a graft rejection. Even if one of these
transplants is rejected, a second keratoplasty for optical rehabilitation can
be performed later. A large-sized graft should be used without any fear of
rejection.
Local anesthesia may suffice in a majority of the cases. In these cases,
O Brien’s facial nerve block is given first prior to the ciliary block to prevent
squeezing of the eyelid and resultant inadvertent perforation. The initial
incision is performed using a hollow trephine needle. The anterior cham-
ber is entered using a sharp razor blade. If the area of suppuration is large
and asymmetric, decenteration of the trephination or a free-hand dissection
to encompass the infected areas should be done. The size of the trephina-
tion should preferably leave 1–1.5 mm clear zone of the uninvolved cornea
to reduce the possibility of removal of infective organisms peripheral to the
trephination. Thicker fibrous membranes covering the surface of the iris or
the lens can be peeled off using forceps or in some cases excised using scis-
sors. A peripheral iridectomy should be performed in all cases to prevent
secondary glaucoma. If involvement of the posterior segment or endophthal-
mitis is suspected, an antifungal agent such as amphotericin B (5 microor-
ganism/0.1 mL) is injected. The intact lens should not be removed as much as
possible and localized cataracts should warrant an extraction at a later date in
a separate sitting. The graft is then secured with 10 nylon interrupted sutures.
The advantage of these interrupted sutures is that they can be removed
Chapter_09.indd 184 06-02-2018 21:17:36

(A) (B) (C)

Figs. 9.8A to C: Cear graft without any infiltrate after surgery.
selectively if future infiltrations develop. Results of therapeutic keratoplasty

are often encouraging (Figs. 9.8A to C). The recurrence of the infection in the
graft starts from the periphery.
Postoperatively, topical natamycin 5% is continued for a period of
1–2 months. During this time, the usage of topical steroids is not recom-
mended and supplementary topical nonsteroidal anti-inflammatory drugs
may be used. Steroids can be used cautiously after the first postoperative
month follow-up.
CONCLUSION
In conclusion, fungal keratitis is fast becoming a silent epidemic, especially
in developing countries. A lot of research is clearly needed in the field of
antifungal pharmacology to limit the morbidity caused by these infections.
Until then, early surgical intervention may be the preferred mode to eliminate
the infection.
REFERENCES
1. Forster RK. Fungal keratitis and conjunctivitis. Clinical disease. In: Smolin G,
Thoft RA (Eds). The Cornea: Scientific Foundation and Clinical Practice. Boston:
Little Brown; 1994. pp. 229-52.
2. Churner R, Cunningham RD. Fungal-contaminated soft contact lenses. Ann
Ophthalmol. 1983;15(8):724-7.
3. Nelson PE, Dignani MC, Anaissie EJ. Taxonomy, biology, and clinical aspects of
Fusarium species. Clin Microbiol Rev. 1994;7(4):479-504.
4. Bennett JE. Mycoses: Aspergillus. In: Mandell GL, Douglas RG Jr, Bennett JE
(Eds). Principles and Practice of Infectious Disease. New York: Churchill Living-
stone; 1990. pp. 1958-62.
5. Sharma S, Silverberg M, Mehta P, et al. Early diagnosis of mycotic kerati-
tis: predictive value of potassium hydroxide preparation. Ind J Ophthalmol.
1998;46(1):31-5.
6. Medoff G, Kobayashi GS. Strategies in the treatment of systemic fungal infec-
tions. N Engl J Med. 1980;302(3):145-55.
7. Yilmaz S, Ture M, Maden A. Efficacy of intracameral amphotericin B in the
management of refractory keratomycosis and endophthalmitis. Cornea.
2007;26(4):398-402.
Chapter_09.indd 185 06-02-2018 21:17:36

8. Sud IJ, Feingold DS. Mechanisms of action of the antimycotic imidazoles.

J Invest Dermatol. 1981;76(6):438-41.
9. Thienpont D, Van Cutsem J, Van Gerven F, et al. Ketoconazole—a new broad
spectrum orally active antimycotic. Experientia. 1979;35(5):606-7.
10. Saag MS, Dismukes WE. Azole antifungal agents: emphasis on new triazoles.
Antimicrob Agents Chemother. 1988;32(1):1-8.
11. Hariprasad SM, Mieler WF, Lin TK, et al. Voriconazole in the treatment of fungal
eye infections: a review of current literature. Br J Ophthalmol. 2008;92(7):871-8.
12. Sponsel W, Chen N, Dang D, et al. Topical voriconazole as a novel treatment for
fungal keratitis. Antimicrob Agents Chemother. 2006;50(1):262-8.
13. Jones DB. Initial therapy of suspected microbial corneal ulcers. II. Specific anti-
biotic therapy based on corneal smears. Surv Ophthalmol. 1979;24(2): 97-116.
14. O’Day DM. Orally administered antifungal therapy for experimental keratomy-
cosis. Trans Am Ophthalmol Soc. 1990;88:685-725.
15. Johns KJ, O’Day DM. Pharmacologic management of keratomycoses. Surv
Ophthalmol. 1998;33(3):178-88.
16. Abad JC, Foster SC. Fungal keratitis. International Ophthalmol Clini. 1998;
36(3):1-15.
17. Brajtburg J, Kobayashi D, Medoff G, et al. Antifungal action of amphotericin
B in combination with other polyene or imidazole antibiotics. J Infect Dis.
1982;146(2):138-46.
18. O’Day DM. Fungal keratitis. In: Pepose JS, Holland GN, Wilhelmus KR (Eds). Oc-
ular Infection and Immunity. St. Louis: Mosby; 1996. pp. 1048-61.
Chapter_09.indd 186 06-02-2018 21:17:36

10
CHAPTER
Herpetic Keratitis
Charmaine Chai, Manotosh Ray
INTRODUCTION
Herpetic keratitis is an infection of cornea caused by the herpes simplex virus
(HSV) type 1 or 2. Like many viruses, the HSVs are present in most adults. The
viruses in the herpes family usually live around the nerve fibers in humans
without ever causing any problem. Occasionally, the viruses will start to mul-
tiply, or they will move from one area of the body to another, and that is when
herpetic disease breaks out. This often happens when some other disease
process significantly weakens the immune system of the body. It can mani-
fest in various forms depending on the level of the corneal involvement. The
pathogenesis and management are different in an epithelial keratitis, stromal
keratitis or endothelitis along with iridocyclitis. An early diagnosis is neces-
sary in order to initiate the appropriate treatment.
This chapter describes the common manifestations of herpetic keratitis
and its management based on our experiences.
EPIDEMIOLOGY
HSV keratitis can affect all ages. HSV is an endemic throughout the world and
humans are the only known natural reservoir. The studies suggest that nearly
90% of world population is infected by latent HSV-1 infection by the age of 60.
HSV-1 is primarily transmitted through direct contacts. The rate of infection is
affected by the amount of exposure to potential sources of the infection. The
socio-economic status of the patient plays a significant role. Children with
lower socio-economic status have higher rate of seroconversion than with
middle class and above. The reported incidence of new cases of ocular HSV
is about 8.4–13.2 cases per 100,000 per year.1-3 HSV-1 has been found to be
the main causative virus in herpetic eye disease, accounting for about 95%
Chapter_10.indd 187 06-02-2018 21:30:06

of ocular HSV. HSV-2 is less commonly isolated, and more commonly associ-
ated with genital herpes.
HSV is a common cause of corneal infection and one of the leading infec-
tious causes of corneal blindness worldwide. Ocular herpes is typically uni-
lateral. However, bilateral herpetic manifestations have been observed in
children and atopic individuals.4-6 The herpetic eye disease study (HEDS),
a multi-arm placebo controlled trial was designed to determine best treat-
ments and prophylaxis for HSV keratitis and to investigate the risk factors
of the disease. HSV epithelial and stromal keratitis were accounted for 47%
and 16% of ocular HSV infection respectively in the HEDS trial.7 The study
focussed on use of oral acyclovir to prevent the epithelial and stromal kerati-
tis and thus provided valuable epidemiological data, especially in regards to
the recurrence rates of the disease. HEDS found an ocular HSV recurrence
rate of 32% over 1 year.7 A history of previous episode of HSV stromal keratitis
and a high number of previous episodes were associated with a higher risk of
subsequent attacks. The reported risk of recurrence after the first episode is
about 9.6% in the first year, and 22.9% at 2 years. The cumulative risk of recur-
rence was about 50% at 10 years.1 The number of recurrences was strongly
associated with past episodes. Therefore, a history of HSV stromal keratitis
and a high number of previous episodes increase the risk of future recur-
rence. The study also suggested that short intervals between attacks tend to
be associated with shorter intervals between future attacks.7
Ocular HSV infection is responsible for visual disability in approxi-
mately 1,000,000 people each year worldwide. It is also estimated that there
are 1,000,000 new cases and 900,000 recurrent infections each year globally.
The average time from onset of symptoms to resolution of an active herpetic
eye infection varies between 17 and 28 days. Therefore, the number of global
man-hour loss from HSV eye infection is alarmingly significant.
ETIOLOGY
HSV keratitis is caused by the HSV-1 and HSV-2, that are parts of the human
herpes virus family. These are double-stranded DNA viruses made up of a
core DNA surrounded by icosahedral-shaped capsid. HSV-1 and HSV-2 are
typically differentiated by virus specific antigens. HSV-1 primarily affects the
oropharynx region while HSV-2 typically involves the genital area, although
there are exceptions of the rule. Transmission of the disease occurs by direct
contact with infected secretions or lesions.
After primary infection, the HSV virus can remain dormant within the
trigeminal nerve or manifest with frequent reactivation. Primary infection
can be asymptomatic or present as a benign form of blepharoconjunctivitis.
Ocular manifestations are the result of ocular inflammation and viral activ-
ity. This is more often secondary to viral reactivation as compared to primary
infection. Active viral replication has been demonstrated in tear samples
collected from patients with active epithelial and stromal disease.8 The viral
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Herpetic Keratitis 189
load detected in the tears was found to be higher in epithelial compared to

stromal keratitis.9 Periodic shedding of HSV into the tears can occur even in
asymptomatic individuals. Hence, virus transmission can also occur through
asymptomatic individuals.
CLINICAL PRESENTATION
HSV keratitis has multiple manifestations. The nature of these manifestations
is distinctive and can readily be distinguished by careful examination of indi-
vidual layers of the cornea. HSV keratitis involving different layers of cornea
has functionally distinct pathogenesis. HSV epithelial keratitis is believed to
be a result of direct infection of epithelial cells, while immune mechanisms
are involved in HSV stromal keratitis. Therefore, epithelial keratitis typically
requires antiviral therapy, while stromal keratitis would require topical ste-
roids in addition to antiviral therapy.
Primary ocular herpes may manifest as blepharitis, conjunctivitis or HSV
keratitis. Typically, these patients present with unilateral red eye, vesicles on
the lids and occasionally with punctate epithelial keratitis. Recurrent dis-
ease can manifest as corneal or adnexal infections. HSV keratitis is classified
according to the level of involvement of the corneal layers (Table 10.1). The
pathogenesis differs depending on the types of manifestation.
HSV epithelial keratitis manifests as a dendritic or geographic ulcer. Den-
dritic ulcers are the commonest presentations of HSV keratitis. Classical fea-
tures of the ulcer are linear branching pattern with terminal bulbs and swollen
epithelial borders. Untreated, dendritic ulcers progress to form geographic
ulcers characterized by swollen epithelium and scalloped or geographic bor-
ders. This occurs as a result of direct virus infection of the corneal epithelial
cells. Hence, antiviral therapy is the mainstay of treatment. Neurotrophic
keratopathy may present with punctate epithelial erosion or irregular cor-
neal surface. Eventually, these lesions may progress to a persistent epithelial
Table 10.1: Classification of herpes simplex keratitis.
Type of HSV keratitis Clinical manifestation

Epithelial Dendritic ulcer with terminal bulb that stains
l Dendritic with fluorescein
l Geographic
Stromal keratitis Stromal infiltrates with or without ulceration

l Necrotising Deep stromal vascularization
l Non-necrotising, immune stromal Immune ring
keratitis
Endothelial keratiits Corneal edema with keratic precipitates
l Disciform keratitis
l Diffuse keratitis
l Linear keratitis
Chapter_10.indd 189 06-02-2018 21:30:06

defect. Typically, these are interpalpebral oval lesions with smooth borders.
Stromal ulceration is not uncommon in advanced stage.
HSV stromal keratitis may be the primary manifestation of herpetic ker-
atitis or it may be secondary to other form of keratitis, e.g., epithelial, neu-
rotrophic or even endothelial form of HSV keratitis. Nearly one quarter of
epithelial keratitis patients develop stromal keratitis. HSV stromal keratitis
may manifest as necrotizing or non-necrotizing forms. The manifestation is
primarily attributed to immune related mechanisms. Hence, topical steroids
are necessary on top of topical antiviral therapy. Necrotizing stromal kera-
titis is believed to result from active viral replication in stromal keratocytes
and thereby producing a severe host inflammatory response. Clinical lesion
is characterized by dense stromal infiltrate, ulceration and necrosis and
often difficult to differentiate from other forms of microbial keratitis. Non-
necrotizing stromal keratitis is less severe focal or multifocal stromal infil-
trates associated with immune ring and deep corneal neovascularization.
Multiple recurrences are common.
Though disciform keratitis can present with stromal involvement, it is
more accurately a form of endothelial keratitis presenting with a focal area
of corneal edema, associated keratic precipitates, anterior chamber reaction
associated with accompanying corneal hypoesthesia.
HSV endothelitis can manifest either as disciform, diffuse or linear
endothelitis based on the area of corneal involvement. Herpetic endotheli-
tis is characterized by keratic precipitates, stromal and epithelial edema in
absence of any stromal vascularization. Corneal edema is due to endothelial
decompensation.
RISK FACTORS
Various factors may increase the virus reactivation. Compromised cell medi-
ated immunity has been suggested to increase the risk of HSV disease and
recurrences (Table 10.2). Both systemic and local factors that alter the host
immunity can increase the risk of HSV keratitis.
Higher incidence of reactivation has also been reported in HIV,10-12
atopic and diabetic patients. However, the incidence of HSV keratitis was
found to be similar in HIV positive patients compared to those who were
tested negative for HIV.11 Atopic individuals were found to have a higher risk
of HSV keratitis, and this risk was greater in patients with severe atopy.13 Var-
ious factors have been postulated to contribute to this. These patients have
altered cell mediated immunity with relative imbalance between type 1 and
type 2 response, with a reduction in type 1 response allowing virus reactiva-
tion.14 In addition, many of these patients remain on chronic immunosup-
pression treatment for their atopic condition. A patient with severe eczema
was treated with a course of oral prednisolone and long-term topical steroids.
She presented with conjunctival injection and blurring of vision shortly after
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Table 10.2: Risk factors for herpes simplex virus (HSV) keratitis.
Systemic risk factors Local risk factors

Pre-existing conditions that reduce host immunity Chronic use of topical medications
l HIV infection
11,12 l Corticosteroids
l Organ transplant recipients l Prostaglandin analogs

60
l Diabetes mellitus
Surgical procedures
l Measles infection
l Laser procedures
Altered immunity l LASIK
l Children l Collagen cross-linking
l Atopy l Cataract surgery
l Prolonged use of systemic immunosuppression l Corneal transplant
l Immune stressors
LASIK: Laser in situ keratomileusis.
(A) (B)
Figs. 10.1A and B: Anterior segment photographs of a patient with severe eczema who
presents with epithelial keratitis after starting oral steroids.
starting the oral prednisolone. On examination, a geographic ulcer was seen

(Figs. 10.1A and B). The patient was given topical acyclovir and the oral
steroid was stopped. The lesion responded well to treatment and healed
within 2 weeks.
HSV keratitis can present more aggressively in children, with more exag-
gerated inflammatory response and frequent recurrences leading to increased
corneal scarring. This is especially detrimental since children are susceptible
to amblyopia secondary to corneal opacity and the induced astigmatism.15
Bilateral diseases are also seen more frequently as compared to adults.
HSV reactivation has also been reported to occur as a result of local
trauma. Several case reports have been published reporting incidences of HSV
keratitis after yttrium aluminium garnet (YAG) laser,16,17 photodynamic ther-
apy,18 argon laser trabeculoplasty19 and laser in situ keratomileusis (LASIK).20
Recurrences have also been reported after collagen cross-linking.21-23
Surgical trauma is a risk factor for recurrences, which is further aug-
mented by the use of postoperative topical steroids. Antiviral prophylaxis can
be considered in a select group of patients with a history of HSV keratitis.
Chapter_10.indd 191 06-02-2018 21:30:07

(A) (B)
Figs. 10.2A and B: Anterior segment photographs of a patient who presents with a
herpetic dendritic ulcer 1 week after cataract surgery.
Few case reports of HSV keratitis during the first few postoperative weeks
after cataract surgery have been reported.24,25 A patient who presented with
left eye irritation and redness 1 week after an uneventful phacoemulsifi-
cation was on tobramycin and dexamethasone eye drops (Figs. 10.2A and
B). He responded well to acyclovir therapy and his vision remained good
(6/7.5).
The reported incidence of newly acquired herpetic keratitis after pene-
trating keratoplasty is 1.2 per 1,000 person-years, and it usually occurs in the
first 2 postoperative years.26 Herpetic keratitis can occur as a new onset after
transplantation or as a reactivation.27,28 Recurrence of herpetic keratitis is the
main reason for graft failure in patients who undergo keratoplasty for HSV
keratitis.29 Around 33% of donor corneas with primary graft failure were found
to be positive for HSV-1 DNA.30 Recurrence may be related to regeneration of
corneal innervation or due to virus shedding into the tear film. Oral antiviral
prophylaxis is recommended in patients with a history of herpetic keratitis
and may be continued while the patient is maintained on topical steroids for
the first 1 to 2 years after transplantation.31,32 This has been found to reduce
the recurrence rate and graft failure in this group of patients.33,34 Although,
there is no clear regime or guideline, studies suggest a maintenance dose of
400mg oral acyclovir twice a day. Topical acyclovir prophylaxis, on the other
hand, is not routinely used as it may lead to epithelial toxicity and is possibly
not so effective in preventing recurrences.35
A patient who underwent an endothelial keratoplasty about 2.5 years
back and was maintained on long-term topical prednisolone acetate eye
drops, as part of his postoperative regime, presented with a dendritic ulcer in
the right eye (Figs. 10.3A and B). His ulcer resolved upon treatment with top-
ical acyclovir but he subsequently developed a metaherpetic ulcer with an
overlying epithelial defect and corneal thinning. The epithelial defect healed
after 3 weeks with treatment but the residual stromal thinning remained. His
vision was ‘counting fingers’ which was contributed by his advanced glau-
coma which was being treated with topical antiglaucoma therapy.
Chapter_10.indd 192 06-02-2018 21:30:07

(A) (B)
Figs. 10.3A and B: Anterior segment photographs at presentation and upon resolution
of a patient with metaherpetic ulcer after endothelial keratoplasty.
COMPLICATIONS
Corneal Scar
Recurrent keratitis can lead to visual loss from scarring and induced astigma-
tism. A visible corneal scar was seen in 18% of primary presentation and 28%
of recurrent disease. About 73–90% still maintained a visual acuity of 6/12 or
better for at least 5 years. Significant scarring and astigmatism may require
subsequent keratoplasty.
Neurotrophic Keratopathy
Herpetic keratitis can result in corneal neurotrophy and persistent corneal

defect. Metaherpetic ulcers can occur following dendritic or geographic
ulcers due to inability of the epithelium to heal. It occurs in the absence of
any live virus. The edges of the ulcer may be rolled up and do not stain with
Rose Bengal, while the base of the ulcer will stain, exhibiting a ‘reverse stain-
ing pattern’. Without adequate treatment, this can progress to corneal thin-
ning and perforation.
Bullous Keratopathy
Endothelial decompensation can result from chronic endothelitis. The

reported incidence of endothelial dysfunction is about 0.01% from recurrent
ocular HSV.1
Corneal Perforation
Destructive intra-stromal inflammation in necrotizing stromal keratitis

may lead to devastating complications including severe corneal thinning and
corneal perforation. The complication may occur within a short span of time
in spite of adequate treatment.
Chapter_10.indd 193 06-02-2018 21:30:07

Fig. 10.4: Anterior segment photograph demonstrating a dendritic ulcer, which stains
with fluorescein.
MANAGEMENT
Epithelial Keratitis
Epithelial keratitis is the most common ocular presentation of HSV, account-

ing for 50–80% of ocular herpes. It can occur due to actively replicating virus.
It classically presents as a dendritic ulcer (Fig. 10.4) with branching pattern or
a geographic ulcer. This pattern stains with fluorescein or Rose Bengal, high-
lighting the branching lesions with terminal bulbs. Management of epithelial
keratitis is primarily with antivirals. The use of steroids is contraindicated in
the presence of epithelial involvement.
Antiviral agents stop viral replication by interfering with viral DNA
synthesis during transcription of the viral genome. Various topical antiviral
therapies have been investigated. A Cochrane systemic review compared the
relative effectiveness of antivirals agents, interferon and corneal debride-
ment in the treatment of HSV epithelial keratitis.36 The review found that
trifluridine and acyclovir are more effective than idoxuridine or vidarabine.
Gancyclovir was also found to be as effective as acyclovir. Oral acyclovir alone
or a combination of oral acyclovir and a topical antiviral appeared as effective
as a topical antiviral monotherapy. The review found that there is no strong
evidence supporting the efficacy of combining antiviral treatment with either
Interferon or corneal surface debridement. Corneal debridement was con-
sidered to remove or destroy the virus infected cells physically. However,
debridement alone is not an effective form of treatment.
Oral acyclovir may be an alternative to a topical antiviral agent. Oral acy-
clovir 400 mg 5 times a day is as efficacious as topical acyclovir ointment.37,38
Despite the widespread use of acyclovir, HSV resistance still appears low.39
The efficacy of valacyclovir and famacyclovir in the treatment of herpetic epi-
thelial keratitis is not known.
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Stromal Keratitis
There are two main forms of stromal keratitis. Non-necrotizing keratitis

accounts for about 88% and necrotizing stromal keratitis accounts for about 7%.
The remaining 5% is a mix of both. Disciform keratitis has frequently been
considered as a form of stromal keratitis, though it is primarily an endothe-
litis with secondary stromal edema.40 Around 20–60% of recurrent herpetic
keratitis presents as stromal keratitis. This can lead to a significant visual
impairment as a result of stromal inflammation and scarring.
Necrotizing keratitis is often characterized by necrosis and ulceration and
can lead to corneal perforation without treatment. This is due to combination
of immunological and viral-related mechanisms. Non-necrotizing stromal
keratitis is marked by stromal inflammation. This is likely due to recruitment
of T lymphocytes causing an immunopathologic response.41,42 The host
response contributes to scarring and corneal vascularization. The role of viral
antigen in the pathogenesis of non-necrotizing stromal keratitis is not clear.
Management strategies of stromal keratitis have been largely drawn from
evidences of HEDS.43 HEDS comprises of two randomized, double-masked,
placebo-controlled multicentre studies that were performed to look at the
role of topical steroids in stromal keratitis, and the role of oral acyclovir when
used together with topical antiviral (1% trifluridine) and topical steroids (1%
prednisolone phosphate). The study established the efficacy of using topical
steroids in controlling the host immune response in stromal keratitis, but did
not find a benefit of adding oral acyclovir for patients already on treatment
with both topical antiviral and topical steroid. The study methodology treated
these patients with 10 weeks of tapered topical steroids and antivirals. How-
ever, the optimal duration of taper of topical steroids remains unanswered
and is often based on clinical response. Hence, topical steroids and antivirals
remain the mainstay of treatment in stromal keratitis to reduce inflammation
and halt viral replication.
Topical acyclovir penetrates the corneal wall in the presence of an intact
epithelium,44 and is preferred over topical trifluridine or gancyclovir. Topical
trifluridine and topical gancyclovir do not penetrate the cornea well in the
presence of an intact epithelium. Also, long-term use of topical trifluridine
is associated with epitheliopathy. Where topical acyclovir is unavailable, oral
acyclovir can be used instead together with topical steroids. In patients with
stromal keratitis and epithelial disease, topical steroids should be withheld or
used judiciously until the epithelial disease has resolved.
Other forms of treatment that have been reported include:
• Cyclosporin A (CsA): Several small studies suggest a benefit of CsA in the
treatment of stromal keratitis, even after failing treatment with topical
steroids.45-47 These studies found a resolution of inflammation in about
83% of non-necrotizing disease, and about 50% in necrotizing disease.48
However, these studies lack a control group. CsA serves as a steroid-
sparing agent and is particularly useful in patients with exaggerated
Chapter_10.indd 195 06-02-2018 21:30:08

(A) (B)
Figs. 10.5A and B: (A) Anterior segment photographs of a patient presenting as first
onset disciform keratitis and (B) a patient with recurrent disciform keratitis.
adverse response to steroids or in those that require prolong treatment

with topical steroids.
• Amniotic membrane transplant (AMT): AMT has been investigated in the
treatment of necrotizing stromal keratitis.49,50 Amniotic membrane may
help reduce the stromal inflammation and stabilize the ocular surface.
It is a possible treatment option in patients with severe and persistent
corneal inflammation. However, these have been limited to small retro-
spective series and further randomized controlled trials are necessary to
establish their roles.
Herpetic Endothelitis
Herpetic disciform keratitis is a form of endothelitis. It presents with a focal,

discrete area of corneal edema with associated keratic prescipitates and
anterior chamber activity. The treatment is similar to stromal keratitis, with
the use of topical antivirals together with topical steroids.51,52 Oral acyclovir
appeared to be as effective as topical acyclovir ointment.53 Oral acyclovir may
be preferred in the absence of topical acyclovir, since topical trifluridine and
gancyclovir do not achieve adequate corneal penetration.
A woman presented with a first episode of eye redness and blurring of
vision for 1 month before seeking consultation. After examination, there was
a focal area of cornea edema seen inferiorly with associated keratic precipi-
tates, posterior synechiae and mild cellular activity (Fig. 10.5A). Her best cor-
rected visual acuity was 6/12 and her intraocular pressure was elevated at
28 mm Hg. Antiviral treatment together with topical prednisolone eye drops
was started and slowly tapered over few months with good response. Other
patient with disciform keratitis presented with recurrent episodes of keratou-
veitis (Fig. 10.5B). She was previously treated elsewhere and had about 7–8
episodes of recurrence over 13 years. She responded well to oral acyclovir
with topical steroids and was subsequently started on long-term prophylac-
tic acyclovir at 400 mg twice a day. Treatment guideline for HSV keratitis is
shown in Table 10.3.
Chapter_10.indd 196 06-02-2018 21:30:08

Table 10.3: Suggested treatment guidelines.
Type of keratitis Suggested regime Choice of medications

Epithelial keratitis Antiviral agents alone Topical acyclovir or
for 1–3 weeks Gancyclovir gel 0.15% or topical
Avoid topical steroids trifluridine 1%
OR
Oral acyclovir 400 mg 5 ×/day

(Alternatives: valacyclovir, famciclovir)
Stromal keratitis Antiviral PLUS topical Topical acyclovir or
steroids over at least Oral acyclovir
10 weeks (Alternatives: valacycovir, famciclovir)
AND
Topical prednisolone 1%
Endothelial keratitis Antiviral for 1–2 weeks Topical acyclovir or
PLUS topical steroids Oral acyclovir
(Alternatives: valacycovir, famciclovir)
AND
Topical prednisolone 1%
Patients with high risk Low dose oral antiviral Oral Acyclovir 400 mg twice/day or
of recurrence: treatment for at least Oral valacyclovir 500 mg once/day
Multiple previous
l 1 year (Alternative: famciclovir)
episodes of ocular
HSV
Post-keratoplasty
l
patients for HSV

related scarring
Post cataract or
l
laser in patient with

known history of
ocular HSV
PREVENTION
Prevention of Recurrent HSV Keratitis
Recurrent herpetic keratitis is seen in about 32% of patients over 1 year.7 It

causes irregular astigmatism, corneal scarring and loss of vision. The HEDS2
was conducted to look at the efficacy of low dose oral acyclovir in preventing
recurrent herpetic keratitis.7,54 The study found that a low dose of 400 mg oral
acyclovir twice a day over 1 year reduced the risk of recurrent stromal keratitis
by 50% in patients with a history of stromal keratitis. However, there was no
Chapter_10.indd 197 06-02-2018 21:30:08

significant difference in recurrence when compared to placebo at 6 months

after stopping of treatment. The addition of high dosed (400 mg 5 times a day)
oral acyclovir in the treatment of epithelial keratitis to patients already on
treatment with topical antiviral agent did not prevent subsequent episodes
of stromal keratitis.
Valacyclovir 500 mg once a day may be a more convenient alternative and
has been shown to be as effective as oral acyclovir 400 mg twice a day in pre-
venting recurrences of ocular HSV.55
Acyclovir resistance is seen in about 0.32% of immunocompetent indi-
viduals. This is higher among the immunocompromised, up to an estimate
of 10.9%.56,57 It is associated with thymidine kinase gene mutation. Cross-
resistance is seen in other medications such as valacyclovir, ganciclovir and
famciclovir, which also rely on thymidine kinase phosphorylation.58 Alterna-
tive medications would include foscarnet, cidofovir or trifluridine that work
via a different mechanism.
HSV Vaccination
A randomized controlled trial conducted in 10 patients over 1 year investigated

the use of a heat-shock inactivated HSV-1 vaccine. The vaccine was found to
reduce the number and duration of HSV-1 related ocular recurrences.59,60
Larger studies are necessary to establish the role of such vaccinations.
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8. Fukuda M, Deai T, Higaki S, et al. Presence of a large amount of herpes simplex
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9. Kakimaru-Hasegawa A, Kuo CH, Komatsu N, et al. Clinical application of

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10. Burcea M, Gheorghe A, Pop M. Incidence of Herpes Simplex Virus Kerati-
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with acquired immune deficiency syndrome. Ophthalmology. 1989;96(10):
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14. Carr DJ, Härle P, Gebhardt BM. The immune response to ocular herpes simplex
virus type 1 infection. Exp Biol Med (Maywood). 2001;226(5):353-66.
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segment: characteristics, treatment, and outcomes. Ophthalmology. 2012;
119(10):2003-8.
16. Hou YC, Chen CC, Wang IJ, et al. Recurrent herpetic keratouveitis following YAG
laser peripheral iridotomy. Cornea. 2004;23(6):641-2.
17. Huang SC, Wu WC, Tsai RJ. Recurrent herpetic keratitis induced by laser iridec-
tomy: case report. Changgeng Yi Xue Za Zhi. 1999;22(3):515-9.
18. Yoon KC, Im SK, Park HY. Recurrent herpes simplex keratitis after verteporfin
photodynamic therapy for corneal neovascularization. Cornea. 2010; 29(4):
465-7.
19. Reed SY, Shin DH, Birt CM, et al. Herpes simplex keratitis following argon laser
trabeculoplasty. Ophthalmic Surg. 1994;25(9):640.
20. Jain V, Pineda R. Reactivated herpetic keratitis following laser in situ keratomil-
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21. Kymionis GD, Portaliou DM, Bouzoukis DI, et al. Herpetic keratitis with iritis af-
ter corneal crosslinking with riboflavin and ultraviolet A for keratoconus. J Cat-
22. Al-Qarni A, AlHarbi M. Herpetic keratitis after corneal collagen cross-linking
with riboflavin and ultraviolet-A for keratoconus. Middle East Afr J Ophthalmol.
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23. Yuksel N, Bilgihan K, Hondur AM. Herpetic keratitis after corneal collagen
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Ophthalmol. 2011;31(6):513-5.
24. Barequet IS, Wasserzug Y. Herpes simplex keratitis after cataract surgery. Cor-
nea. 2007;26(5):615-7.
25. Patel NN, Teng CC, Sperber LT, et al. New-onset herpes simplex virus keratitis
after cataract surgery. Cornea. 2009;28(1):108-10.
26. Remeijer L, Doornenbal P, Geerards AJ, et al. Newly acquired herpes simplex
virus keratitis after penetrating keratoplasty. Ophthalmology. 1997;104(4):
648-52.
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27. Rezende RA, Uchoa UB, Raber IM, et al. New onset of herpes simplex virus
epithelial keratitis after penetrating keratoplasty. Am J Ophthalmol. 2004;
137(3):415-9.
28. Mannis MJ, Plotnik RD, Schwab IR, et al. Herpes simplex dendritic keratitis after
keratoplasty. Am J Ophthalmol. 1991;111(4):480-4.
29. Sterk CC, Jager MJ, Swart-vd Berg M. Recurrent herpetic keratitis in penetrating
keratoplasty. Doc Ophthalmol. 1995;90(1):29-33.
30. Cockerham GC, Bijwaard K, Sheng Z-M, et al. Primary graft failure: a clinicopath-
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31. Foster CS, Barney NP. Systemic acyclovir and penetrating keratoplasty for her-
pes simplex keratitis. Doc Ophthalmol. 1992;80(4):363-9.
32. van Rooij J, Rijneveld WJ, Remeijer L, et al. Effect of oral acyclovir after penetrat-
ing keratoplasty for herpetic keratitis: a placebo-controlled multicenter trial.
Ophthalmology. 2003;110(10):1916-9.
33. Goodfellow JF, Nabili S, Jones MN, et al. Antiviral treatment following penetrat-
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34. Barney NP, Foster CS. A prospective randomized trial of oral acyclovir after pen-
etrating keratoplasty for herpes simplex keratitis. Cornea. 1994;13(3): 232-6.
35. Ghosh S, Jhanji V, Lamoureux E, et al. Acyclovir therapy in prevention of re-
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2008;145(2):198-202.
36. Wilhelmus KR. Antiviral treatment and other therapeutic interventions for her-
pes simplex virus epithelial keratitis. Cochrane Database Syst Rev. 2015;1.
37. Collum LM, McGettrick P, Akhtar J, et al. Oral acyclovir (Zovirax) in herpes sim-
plex dendritic corneal ulceration. Br J Opthalmol. 1986;70(6):435-8.
38. Collum LM, Akhtar J, McGettrick P. Oral acyclovir in herpetic keratitis. Trans
Opthalmol Soc UK. 1985;104(pt 6):629-32.
39. Christophers J, Clayton J, Craske J, et al. Survey of resistance of herpes sim-
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40. Holland EJ, Schwartz GS. Classification of herpes simplex virus keratitis. Cornea.
1999;18(2):144-54.
41. Russell RG, Nasisse MP, Larsen HS, et al. Role of T-lymphocytes in the pathogen-
esis of herpetic stromal keratitis. Invest Ophthalmol Vis Sci. 1984;25(8): 938-44.
42. Metcalf JF, Kaufman HE. Herpetic stromal keratitis-evidence for cell-
mediated immunopathogenesis. Am J Opthalmol. 1976;82(6):827-34.
43. Wilhelmus KR, Gee L, Hauck WW, et al. Herpetic eye disease study. A controlled
trial of topical corticosteroids for herpes simplex stromal keratitis. Ophthalmol-
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44. Poirier RH, Kingham JD, de Miranda P, et al. Intraocular antiviral penetration.
Arch Ophthalmol. 1982;100(12):1964-7.
45. Rao SN. Treatment of herpes simplex virus stromal keratitis unresponsive to
topical prednisolone 1% with topical cyclosporine 0.05%. Am J Opthalmol.
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46. Heiligenhaus A, Steuhl KP. Treatment of HSV-1 stromal keratitis with topical
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435-8.
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47. Gündüz K, Ozdemir O. Topical cyclosporin as an adjunct to topical acyclovir

treatment in herpetic stromal keratitis. Ophthalmic Res. 1997;29(6):405-8.
48. Knickelbein JE, Hendricks RL, Charukamnoetkanok P. Management of herpes
simplex virus stromal keratitis: an evidence-based review. Surv Ophthalmol.
2009;54(2):226-34.
49. Heiligenhaus A, Li H, Hernandez Galindo EE, et al. Management of acute ulcer-
ative and necrotising herpes simplex and zoster keratitis with amniotic mem-
brane transplantation. Br J Opthalmol. 2003;87(10):1215-9.
50. Shi W, Chen M, Xie L. Amniotic membrane transplantation combined with anti-
viral and steroid therapy for herpes necrotizing stromal keratitis. Ophthalmol-
ogy. 2007;114(8):1476-81.
51. Collum LM, Logan P, Ravenscroft T. Acyclovir (Zovirax) in herpetic disciform ker-
atitis. Br J Opthalmol. 1983;67(2):115-8.
52. Power WJ, Hillery MP, Benedict-Smith A, et al. Acyclovir ointment plus topical
betamethasone or placebo in first episode disciform keratitis. Br J Opthalmol.
1992;76(12):711-3.
53. Porter SM, Patterson A, Kho P. A comparison of local and systemic acyclo-
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54. Acyclovir for the prevention of recurrent herpes simplex virus eye disease. Her-
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55. Miserocchi E, Modorati G, Galli L, et al. Efficacy of valacyclovir vs acyclovir for
the prevention of recurrent herpes simplex virus eye disease: a pilot study. Am
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56. Danve-Szatanek C, Aymard M, Thouvenot D, et al. Surveillance network for her-
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57. Stránská R, Schuurman R, Nienhuis E, et al. Survey of acyclovir-resistant
herpes simplex virus in the Netherlands: prevalence and characterization.
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58. Morfin F, Thouvenot D. Herpes simplex virus resistance to antiviral drugs.
J Clin Virol. 2003;26(1):29-37.
59. Pivetti-Pezzi P, Accorinti M, Colabelli-Gisoldi RA, et al. Herpes simplex virus vac-
cine in recurrent herpetic ocular infection. Cornea. 1999;18(1):47-51.
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log therapy. Surv Ophthalmol. 2008;53(Suppl 1):S93-105.
Chapter_10.indd 201 06-02-2018 21:30:08

11
CHAPTER
Acanthamoeba Keratitis—
Pathogenesis and Diagnosis
Savitri Sharma, Joveeta Joseph, Gunisha Pasricha
INTRODUCTION
Bacterial and fungal keratitis are well-documented in the literature.1-3 In
contrast, Acanthamoeba keratitis (AK) is a relatively recent development. The
number of these cases has been increasing especially in developed countries
due to high incidence of contact lens wearers.4,5 In India, however, corneal
trauma remains the major risk factor.6,7 While our institute reported the clin-
ical and laboratory findings of AK in 38 patients, which is among the largest
series from India,6 the largest series on clinical outcome was published by
Robaei et al.4 from Moorfields Eye Hospital. Well-documented studies have
shown that about 1.5–4% of laboratory-proven infective keratitis in India is
caused by Acanthamoeba and that the majority of the cases the affected per-
son do not wear contact lens.6,7
Diagnostic tests for identification of Acanthamoeba in scrapings or
biopsies of the cornea are simple and can be adapted by any microbiology
laboratory with facilities for smear examination and culture. All medium
to large size ophthalmology setups can easily incorporate procedures for
the diagnosis of Acanthamoeba in the laboratory. However, basic research
related to Acanthamoeba is confined to large tertiary eye care centers with
research facilities. Efforts have been made to achieve molecular typing of
Acanthamoeba isolated from Indian patients and also to develop molecular
diagnostic methods, which are highly sensitive as well as specific for the diag-
nosis of AK.8 A working diagnosis of AK can be made from the demographic
and clinical examination which may be aided by in vivo confocal microscopy.
Some studies have focused on the tissue reaction and pathogenesis of Acan-
thamoeba in the cornea.9
At present, much is known about the epidemiology, risk factors, patho-
genesis, genetics, clinical features and treatment of Acanthamoeba and the
Chapter_11.indd 202 06-02-2018 21:52:21

Acanthamoeba Keratitis—Pathogenesis and Diagnosis 203
keratitis caused by it. It is beyond the scope of this chapter to discuss recent
advances in all aspects. Therefore, this chapter is confined to the advance-
ments made in recent times with respect to classification, molecular typing,
pathogenesis and diagnosis of AK.
Classification and Molecular Typing
The term ‘acanth’ came from the Greek word ‘acanth’ meaning ‘spikes’ and
‘amoeba’ was added to indicate the projections of spike-like structures
(now known as acanthopodia) on its surface.5 Although, the genus Acan-
thamoeba was first identified in 1931, there was considerable confusion
about its taxonomic classification in the literature. Volkonsky divided the
existing genus Hartmanella into three genera, i.e., Hartmanella, Glaeseria
and Acanthamoeba.10 In 1975, Visvesvara and Balamuth identified definable
and demonstrable differences in the trophozoite and cyst stages of Acan-
thamoeba and Hartmanella.
Many approaches have been used for the subgenus classification of Acan-
thamoeba, which mainly include classification based on: (i) morphology of
the cysts, (ii) isoenzyme electrophoretic patterns, (iii) mitochondria restric-
tion fragment length polymorphism (mtRFLP), (iv) sequencing of nuclear
and mitochondria genes and (v) riboprinting.
In 1977, Pussard and Pon proposed the classification based on the mor-
phology of cysts. They established 18 different species in three distinct groups
(Table 11.1).10
The systematic classification of Acanthamoeba based on cyst morphol-
ogy has been deemed ambiguous and vague. It can define an isolate up to the
genus level, however, variations occur in cyst forms within the species and
clonal population. This fact makes classification using morphology as a sub-
jective process. Also, this system does not show genetic relationship between
the strains.11 Constituents of the growth medium seems to alter the morphol-
ogy of the cyst reducing its reliability as a taxonomic characteristic.12
Table 11.1: Morphological classification of Acanthamoeba.
Group I Group II Group III

A. astronyxis A. castellanii A. culbertsoni
A. comanodoni A. rhysodes A. royreba
A. echinulata A. mauritaniensis A. palestinensis
A. tubiashi A. divionensis A. lenticulata
A. griffini A. pustulosa
A. polyphaga
A. lugdunensis
A. quina
A. triangularis
A. hatchetti
Chapter_11.indd 203 06-02-2018 21:52:21

Isogenic electrophoretic patterns have been shown to demonstrate intra-

generic relationship and to test morphological classification.13 Zymograms
of acid phosphatase, leucine aminopeptidase, malate dehydrogenase, pro-
pionyl esterase, glucose phosphate isomerase, phosphoglucomutase and
alcohol dehydrogenase suggested changes in the taxonomy within the mor-
phology group 2 of Pussard and Pon.13 However, the drawback of this method
of classification was that different zymoderms might exist within a species,
which suggested that neither isoenzyme pattern nor morphological analysis
be used alone for subgenus classification. Different groups of Acanthamoeba
described by several studies were often found inconsistent with species and/
or morphological group characteristics.14
Species classification was difficult and the taxonomy of a number of
strains was unreliable mandating new approaches for classification. Byers
et al. used electrophoretic patterns following digestion of mitochondrial
DNA (mtRFLP) with restriction enzymes.15 They demonstrated high degree
of molecular diversity among strains classified as a single species. Similar
results were obtained by others.16,17
Eukaryotes have both cytoplasmic and mitochondrial ribosomes. Both
types of ribosomes consist of large subunit (LSU) and small subunit (SSU)
ribonucleoproteins. Rns are the nuclear genes coding for 18S rRNA found in
the cytoplasmic SSU while rns are mitochondrial genes coding for 16S rRNA
found in mitochondrial SSU.14 Based on analysis of complete sequences of
nuclear ribosomal subunit RNA genes (Rns), Gast et al. proposed four distinct
sequence types. They were designated as sequence types T1-T4.14 T1 included
A. castellanii V006, T2 included A. palestinensis Reich, T3 included A. griffini
S7, while the fourth sequence type T4 included 15 isolates classified as A.
castellanii, A. polyphaga, A. rhysodes and 10 other isolates of Acanthamoeba,
all obtained from keratitis patients. They found that T4 has a worldwide
distribution. Data also showed that T4 included three different species A.
castellanii, A. polyphaga and A. rhysodes. This classification confirmed the
inconsistencies of the morphological classification. Nevertheless, even this
classification was inadequate for full phylogenetic differentiation of branch-
ing orders within the T4 sequence type. Ledee et al. analyzed rns sequences
(16S rDNA) of 68 strains of Acanthamoeba.18 The phylogeny, based on mito-
chondrial rns sequences, was mostly consistent with that observed with
nuclear Rns DNA. Both Rns and rns sequences are suitable for Acanthamoeba
genotyping.
Since it is known that nuclear rRNA sequences are useful for identifica-
tion and differentiation of Acanthamoeba isolates, Chung et al. subjected 23
reference strains of Acanthamoeba, for classification at the subgenus level
by riboprinting, i.e., PCR/RFLP analysis.19 The dendrograms based on ribo-
prints coincided well with grouping based on morphology of cysts and that
with dendrogram constructed by Stothard et al.12 which is based on rRNA
gene sequences. Currently, there are 17 rRNA gene sequence-based geno-
types (T1-T17), each genotype having more than 5% sequence divergence
Chapter_11.indd 204 06-02-2018 21:52:22

between different genotypes. T4 genotype have been associated with the

majority of human infections due to Acanthamoeba.20-22
Pathogenesis
The low incidence of AK, despite its widespread prevalence in nature, has at
least two mutually compatible explanations: First, Acanthamoeba is a weak
pathogen; and second, there is high degree of innate host resistance against
it. The mechanisms involved in corneal tissue damage and invasion by the
ameba are poorly understood, especially those related with early events of
amebae-cornea interaction. There are very few studies on the host immune
response to Acanthamoeba infection. They mostly describe the late stage of
the disease, since corneal transplantation specimens were mainly available
from patients treated previously for study.23-25 Most of the understanding
of corneal invasion by Acanthamoeba comes from animal models. Number
of animal models26-30 and corneal cell primary cultures31 have been used
to study AK. Inability to check the repeatability of these experiments and
the need to sacrifice animals regularly are the major disadvantages of these
methods.32 Cell lines other than corneal cells have also been used as infec-
tion models for Acanthamoeba,33 but these studies yield data which are not
specific to interaction of Acanthamoeba with the cornea.16 Recently, primary
cultures34 and immortalized human corneal epithelial cell lines35 have been
developed and they are more characteristic of the in vivo situation.
Some authors believe that initial insult to the cornea in form of trauma,
chemicals, organic matter, insect or microtrauma because of contact lens
wear is required for the infection to occur.36,37 On the other hand, Omana-
Molina et al., in their study on Chinese hamsters have described that Acan-
thamoeba species is capable of producing damage to intact hamster cornea
without producing a previous artificial lesion.38 Once the ameba is present on
the cornea, an important first step in the infectious cascade of AK is its attach-
ment to the intact corneal epithelium. Thus, AK occurs in a sequential man-
ner and is initiated by the pathogens’ adherence to the host cells, followed by
invasion of the corneal layers necessary for Acanthamoeba to establish cor-
neal infection.39 In the initial stages of adhesion, cytoplasmic projections or
acanthopodia of the trophozoites come in contact with the superficial cells of
the cornea. Soon after trophozoites adhere completely and separate the cell
junction of the corneal epithelial cells, and, eventually desquamate them.38
Trophozoites can adhere more intensely with the epithelial surface, thus tro-
phozoites are probably more important than the cysts in initiating human
corneal disease.40,41
The study of human corneal constituent which acts as a substrate for
acanthamoebic growth will definitely lead to a better understanding of
the pathogenesis of AK.42 Yang et al. have demonstrated that corneal epi-
thelium expresses Acanthamoeba reactive mannose glycoprotein and the
parasites express a mannose-binding protein.43 The authors propose that the
Chapter_11.indd 205 06-02-2018 21:52:23

interaction between the mannose-binding protein of the amebae and man-

nose glycoprotein of the corneal epithelium is one of the mechanisms of
Acanthamoeba adhesion to the corneal surface.43 This finding was vali-
dated by Leher et al. who found that involevement of the mannose recep-
tors induced the release of serine protease thereby mediating cytolysis of the
epithelial cells of the cornea.39 Their study demonstrated that while adhe-
sion of trophozoites to epithelial cells was essential for initiating cytolysis, it
was unnecessary once the mannose receptor was engaged. Using mannose
receptors, the authors proposed that Acanthamoeba trophozoites were capa-
ble of mediating both contact dependent and contact independent cyto-
pathic effect.39 Studies on rabbit corneal epithelial cell (SIRC) lines suggested
that interstrain differences of Acanthamoeba in adherence correlates with
observed variation in the rate of progression and virulence in vivo.44
After adhering to corneal epithelial cell, amebae require cellular elements
for their sustenance. The cell surface of the A. castellanii is a highly special-
ized region that is not active in the active transport of solutes, but is involved
directly in the uptake of nutrients by endocytosis, membrane fusion events and
cell motility.41 It feeds on complex macromolecules found most commonly
in living cells for its nutrition. Acanthamoeba feeds directly on the dense cel-
lular epithelial layer causing disruption and eventually there is access to the
corneal stroma, which provides further nutritional support through its kerato-
cytes. Vemuganti et al. have shown that one of the modes of keratocyte loss
in AK is apoptosis which explains disproportionate loss of keratocytes.9 The
plentiful food supply allows the organism to subsist in the stroma for a long
period of time.42 Moore et al. suggested that the trophozoites of A. castellanii
use two methods of penetration in entering human corneas in vitro. The first
method involves the secretion of material, which mainly includes enzymes
that interferes with the junction of the surface squamous epithelium. Acan-
thamoebae are known to have several enzymes that include ribonucleases,
phosphatase, proteinase, a-glucosidase, (3-Nacetylglucosaminidase and
13-glucuronidase).40 Plasma membrane of Acanthamoeba has enzymes like
phospholipase A, lysophospholipase, acetyl Co-A hydrolase, palmitoyl Co-A
synthetase,41 alkaline phosphatase and 5¢-nucleotide activities and Mg ++
adenosine.41 Acyl Co-A: lysolecithin acyltransferase, CDP choline: 1, 2-diacyl-
glycerolcholine phosphotransferase are present in the microsomal fraction.45
Thompson and Shultz reported substantial levels of two phospholipases, glu-
cose-6-phosphatase and 5¢-nucleotidase in both rough and smooth endoplas-
mic reticula but found that NADPH cytochrome C reductase and rotenone
insensitive NAPH cytochrome C reductase are present only in smooth surface
membrane.46 Moore et al. suggested that rough ER plays an important role
in elaborating substances that break the desmosomes of the squamous epi-
thelium.40 Because of the enzymatic action, trophozoites separate adjacent
surface cells, extend pseudopodia into the separated area and move under
the surface of the epithelium without causing damage to overlying cells. This
finding may explain the clinical signs of stromal infiltrates without associated
Chapter_11.indd 206 06-02-2018 21:52:23

epithelial signs.40 Apart from many enzymes, collagenase was also attributed
to pathogenicity of Acanthamoeba, since collagenase from the axenic cul-
tures of A. castellanii digested collagen shields and purified type I collagen in
vitro. This finding further suggests that the stromal degradation in AK may be
caused by parasite derived collagenase.47
Antibodies to free living amebae have been reported to prevent their
adhesion and spread. Antibodies also inhibit phagocytic property of amebae
and promote neutrophil-mediated killing of ameba.48
Based on results of a histopathological study of 30 cases of AK, four-stage
pathogenetic sequence of events after initial breaching of the epithelium by
Acanthamoeba have been described.23 They are:
Stage 1: Initial infection: Initial infection involves breaching of the surface
epithelium. At this stage, there is no inflammatory response, because
intact amebae do not induce inflammatory response. Hence at this
stage opsonization of the parasite by antibody and complement must
be occurring.
Stage 2: Keratocyte depletion: Keratocyte depletion occurs in the second stage
of the infection, which is seen in anterior part of stroma. Larkin and
colleagues proposed that this kertocyte loss is not dependent on the
inflammatory cell infiltration and is a consequence of their being
consumed by the trophozoites. However, keratocyte loss in deeper
stroma (independent of inflammatory response) was also reported to
be due to apoptosis.9
Stage 3: Inflammatory response: Neutrophils with some macrophages were
shown to be the main composition of the inflammatory response.
Garner found dearth of lymphocytes and plasma cells as a result of
stromal vascularization which acts as a barrier to invasion by rela-
tively immobile cells.
Stage 4: Stromal necrosis: Garner observed reduced thickness of stromal col-
lagen, which was accompanied by acute inflammatory cell infiltra-
tion. He attributed lysis of stromal collagen to enzymes released by
neutrophil and other collagenolytic activity. There was minimal or no
neutrophil infiltration in this stage.23
Vemuganti et al. studied corneal tissues in detail from five patients with
AK. Florid granulomatous reaction with multinucleated giant cells in the pos-
terior stroma and around Descemet’s membrane were seen in tissues from
all cases (Fig. 11.1). Immunostaining characterised the inflammatory cells in
the corneal stroma to be T-cell population. In the granulomatous region the
cells were positive for T-cells, macrophages (Fig. 11.2) and negative for B-cell
marker.49
Immune-biology of Acanthamoeba Keratitis
The interactions of Acanthamoeba to its cell receptor(s) in the host is not

clearly understood. Though both innate and adaptive immunity are said to
Chapter_11.indd 207 06-02-2018 21:52:24

Fig. 11.1: Corneal button section from a case of Acanthamoeba keratitis showing periph-
eral vascularization and corneal inflammation involving full thickness of stroma. Granu-
lomatous inflammation and multinucleated giant cells are seen in the deep stroma with
a detached Descemet’s membrane (hematoxylin and eosin, 200 ´).
Fig. 11.2: Corneal button section from a case of Acanthamoeba keratitis showing immu-
nopositive staining of macrophages with CD 68 antobody (Immunoperoxidase stain,
DAB chromogen and counterstained with hematoxylin, 400 ´).
Chapter_11.indd 208 06-02-2018 21:52:26

be involved in resistance to AK, some studies have suggested that it is the

innate immune response that causes release of host factors from infiltrating
cells during rapidly progressing stromal necrosis. Stewart et al. in their in
vitro study have shown that macrophages can directly kill trophozoites by a
strong chemotactic response to Acanthamoeba infection.50 Van Klink et al. in
their experiment on Chinese hamster selectively depleted out macrophages
with liposomes containing dichloromethylene diphosphonate (C12MDP-
L1P) and found that macrophage depletion affected the incidence, severity
and chronicity of keratitis.51
The absence of macrophages in corneal biopsy specimens and corneal
button from penetrating keratoplasy patients was explained by these authors.
They pointed out that all the previous histopathologic studies on human cor-
neal specimens and in experimental animals had been done on later stages of
the disease and not during the acute phase. They believed that macrophages
serve as an important barrier to corneal infection and exert their effect by
preventing the initiation of infection and appearance of clinical signs. Thus,
they suggested that macrophages act as a first line of defense and eliminate
significant number of Acanthamoeba trophozoites.51
Neutrophils kill ameba only when they are activated by lymphokines. In
addition, these altered neutrophils cannot act in the absence of complement
or antibody. Therefore, combined action of lymphokines, complement and
antibody is needed for killing mechanism of neutrophils. Neutrophils kill-
ing mechanism involves both the oxidative respiratory system and enzymes
which are found in its azurophilic granules such as myeloperoxidase. Respi-
ratory burst and release of lysosomal enzymes from neutrophils is augmented
by T-cells and macrophage cytokine. TNFa is responsible for NADPH oxidase
activation resulting in the release of azurophilic granules.51 The part played
by macrophages and neutrophils in pathogenesis of AK indicates the import-
ant role of innate immune system in controlling infection.52
Inflammatory cytokines play a major role in keratitis. They regulate the
inflammatory response to pathogen invasion and induce pathogen clearance
and host cell death. Toll-like receptors (TLRs) additionally help in the detec-
tion of pathogens by recognizing pathogen-associated molecular patterns.
They also initiate the signal for the synthesis of cytokines and innate defense
molecules in host cells.52,53 Ren et al.53 analyzed in vitro the TLR signal path-
way involved in Acanthamoeba infection and found up-regulation of TLRs 2
and 4 expression along with IL-8, TNF-a and IFN-b. Additionally, there was
a down regulation of MyD88, NF-kB, p-IkB and p-ERK1/2 in HCE cells. They
also found that TNF-a and IL-8 levels peaked at 6 hours and decreased there-
after, suggesting that they were early response cytokines in the inflammatory
cascade leading to activation of lymphocytes in response to Acanthamoeba
infection.53
Further, the TLRs initiate intracellular signals and gene expression of
downstream molecules through various signaling pathways.54-58 Through an
Chapter_11.indd 209 06-02-2018 21:52:26

Fig. 11.3: In vivo confocal microscopy of the cornea in a patient with Acanthamoeba
keratitis showing double walled cysts of Acanthamoeba.
MyD88-dependent pathway, TLR4 signals activate the mRNA expression of

NF-kB, leading to the production of IL-6, -8 and TNF-a.59 Simultaneously,
signals are also transduced through the MyD88-independent pathway to acti-
vate transcription factors, such as interferon regulatory factor -3 and -7 and
ERK1/2, leading to the synthesis of IFN-b. The latter pathway and the produc-
tion of IFN-b.53
Diagnosis
Early diagnosis is essential to the optimum clinical outcome of AK. The clin-
ical features, although occasionally pathognomonic, may be misleading. A
number of reports have dealt with the clinical diagnosis of AK,60-62 however,
misdiagnosis due to resemblance with viral and fungal infection is common.6
Corneal or conjunctival swabs are not useful for the diagnosis of AK.63 Labo-
ratory diagnosis is highly rewarding with corneal scraping or corneal biopsy
specimen.
With the availability of confocal microscope, Acanthamoeba cysts may
be visualized in the cornea of the patient. Confocal microscopy use in AK
diagnosis is attractive because it can provide a noninvasive method to image
corneal Acanthamoeba cysts, and allows examination of corneal structures at
a cellular level in real time. High contrast images of coronal corneal sections
containing trophozoites or cysts are visualized on a video monitor (Fig. 11.3).
While trophozoites may be mistaken for inflammatory cells, the charac-
teristic morphology of cysts can be well appreciated.64-66 A prospective,
nonrandomized, observational clinical trial was conducted at our institute
to investigate the role of confocal microscopy as a diagnostic modality in
Chapter_11.indd 210 06-02-2018 21:52:27

microbial keratitis. A sensitivity of 88.3% and a specificity of 91.1% was

found and we concluded that the confocal microscope was accurate and
reliable for the etiological diagnosis of AK.67
Conventional Laboratory Procedures
Diagnosis of Acanthamoeba corneal infections is not very difficult since

the procedures are within routine laboratory techniques for bacteria and
fungi with minor modifications. Two protocols have been described for the
investigation of infective keratitis—clinically viral and non-viral keratitis.68
The protocol for non-viral keratitis includes a combination of smears and cul-
tures of corneal scrapings such that the diagnosis of bacterial, fungal and AK
can be made. The procedure is described in detail in our earlier publication.68
While smears stained with calcofluor white or Gram stain provide early diag-
nosis, culture confirmation on nonutrient agar with Escherichia coli may take
1-3 days. The medium, however, should be incubated for up to two weeks
before concluding a negative culture.69
A flat-bottomed tissue culture flask containing a suspension of Esche-
richia coli in one-fourth Ringer’s solution (3 ´ 108/mL determined by optical
density) has been described as an alternative to non-nutrient agar. After inoc-
ulation with corneal scrapings the flask is incubated at 37°C and examined
daily under an inverted microscope for trophozoites.70 Samples may be trans-
ported in phosphate buffered saline to a distant laboratory without adversely
affecting the survival of cysts in the corneal scraping. Acanthamoeba may
grow on media such as blood agar and chocolate agar. However, these media
are not recommended for the diagnosis of Acanthamoeba although they are
included for exclusion of bacterial or fungal etiology of keratitis.
Corneal biopsy material may be processed similar to corneal scrapings
if the corneal stromal infiltrates are deep. Most staining procedures such as
calcofluor white, Gram, Giemsa, fluorescein conjugated lectin, hematoxylin
and eosin delineate the cyst of Acanthamoeba very well showing the char-
acteristic morphology of polygonal, double walled structure with central
nucleus.71-73 Trophozoites, on the other hand, may be difficult to distinguish
from inflammatory cells.74 Immunostaining with either indirect fluoros-
cent antibody75-77 or immunoperoxidase technique74 have been described.
However, these antibodies are not available commercially and are confined
to laboratories working on Acanthamoeba. These stains can be used on cor-
neal scrapings as well as corneal tissue sections. Apart from hematoxylin and
eosin stain, histopathology sections can also be stained with Masson’s tri-
chrome stain, periodic acid-Schiff, and Gomori methenamine silver stain for
the demonstration of Acanthamoeba cysts and trophozoites, although it may
be difficult to differentiate trophozoites.
Molecular Methods of Diagnosis

Diagnosis of AK using molecular methods have been described by sev-
eral authors and some of these methods have been concurrently used for
Chapter_11.indd 211 06-02-2018 21:52:27

subgenus classification of Acanthamoeba. Vodkin et al.78 and Lehmann et

al.79 were among the first to use polymerase chain reaction (PCR) for the
detection of Acanthamoeba using primers which amplified 272 bp of 18S
rRNA gene (18S rDNA). These primers were found to be more sensitive than
culture of corneal scrapings.79 They succeeded in identifying Acanthamoeba
DNA in tear samples of patients with AK and concluded that PCR of tear sam-
ples may complement the results of PCR with corneal epithelial samples,78
especially in culture negative cases.
Schroeder et al. have reported a detailed analysis of 18S rDNA sequ-
ences of 80 isolates of Acanthamoeba and described another primer pair
(JDP1-JDP2) that was shown to be highly genus specific.80 They demonstrated
a cross-reaction of the primers used by Lehmann et al. to that of related ame-
bae such as Balamuthia and Hartmanella species. JDP1-JDP2 primer pair has
been recently evaluated in a clinical study for its sensitivity in the diagnosis
of AK. This study compared the PCR assay with conventional microbiological
tests for the diagnosis of AK. The results of the study confirmed the efficacy
of PCR assay, although the sensitivity was equal to that of smear examination
of corneal scrapings.8 Specific fluoroscent oligonucleotide probes (genus and
subgenus) for the detection of Acanthamoeba in clinical specimens and cul-
tures have also been described.81
In a prospective study, the multiplex Acanthamoeba beta-globin PCR
(MAB-PCR) was tested in patients with suspected infectious keratitis82 and
was found to be useful in the simultaneous detection of pathogenic free-
living amebae in the same sample. In another study, use of real-time PCR
for the simultaneous detection of 10 different genotypes of Acanthamoeba
was found to be highly sensitive.83 Loop-mediated isothermal amplification
(LAMP) is a newly developed highly specific, efficient, and less time con-
suming DNA amplification technique which rapidly amplifies target DNA
sequences under isothermal conditions was evaluated, using 18S rDNA gene
for specific detection of Acanthamoeba from corneal scraping samples and
was found particularly suitable for a rapid and accurate diagnosis of AK.84 It
takes 2–3 hours lesser than PCR, and thus offers a rapid, highly sensitive and
specific, simple and affordable diagnostic modality for patients suspected
of AK, especially in resource limited settings. Currently, in the armamen-
tarium of rapid detection of Acanthamoeba in clinical samples are included
matrix-assisted laser desorption-ionization time-of-flight (MALDI-ToF mass
spectrometry and 1H NMR spectroscopy.85 The value of these tests in the
diagnosis of AK is yet to be determined.
CONCLUSION
AK remains a difficult condition to diagnose and treat. A better understand-
ing of the disease process at the molecular level is essential if we are to pro-
vide a faster and reliable diagnosis and a more effective treatment.
Chapter_11.indd 212 06-02-2018 21:52:27

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1369-73.
62. Lindquist TD. Treatment of Acanthamoeba keratitis. Cornea. 1998;17(1):
11-6.
63. Wright P, Warhurst D, Jones BJ. Acanthamoeba keratitis successfully treated
medically. Br J Ophthalmol. 1985;69:778-82.
64. Chew SJ, Beuerman RW, Assouline M, et al. Early diagnosis of infectious
keratitis with in vivo realtime confocal microscopy. CLAO J. 1992;18(3):
197-201.
65. Mathers WD, Sutphin JE, Folberg R, et al. Outbreak of keratitis presumed to be
caused by Acanthamoeba. Am J Ophthalmol. 1996;121(2):129-42.
66. Pfister DR, Cameron JD, Krachmer JH, et al. Confocal microscopy findings of
Acanthamoeba keratitis. Am J Ophthalmol. 1996;121:119-28.
67. Vaddavalli PK, Garg P, Sharma S, et al. Role of confocal microscopy in the di-
agnosis of fungal and acanthamoeba keratitis. Ophthalmology. 2011; 118(1):
29-35.
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68. Sharma S, Athmanthan S. Diagnostic Procedures in Infectious Keratitis. In: Di-

agnostic Procedures in Ophthalmology. In: Nema HV, Nema N (Ed.). New Delhi:
Jaypee Brothers Medical Publishers (P) Ltd. 2002. pp. 232-53.
69. Illingworth CD, Cook SD. Acanthamoeba keratitis. Sury Ophthalmol. 1998;
42(6):493-508.
70. Wilhelmus KR, Osato MS, Font RL, et al. Rapid diagnosis of Acantha-
moeba keratitis using calcofluor white. Arch Ophthalmol. 1986;104(9):
1309-12.
71. Robin JB, Chan R, Andersen BR. Rapid visualization of Acanthamoe-
ba using fluorescein-conjugated lectins. Arch Ophthalmol. 1988;106(9):
1273-6.
72. Thomas PA, Kuriakose Th. Rapid detection of Acanthamoeba cysts in corneal
scrapings by lactophemol cotton blue staining. Arch Ophthalmol. 1990;
108(2):168.
73. Sharma S, Srinivasan M, George C. Acanthamoeba keratitis in non-contact lens
wearers. Arch Ophthalmol. 1990;108(5):676-8.
74. Sharma S, Athmanathan S, Ata-Ur-Rasheed, et al. Evaluation of Immuno-
peroxidase staining technique in the diagnosis of Acanthamoeba keratitis. Indi-
an J Ophthalmol. 2001;49(3):181-6.
75. Epstein RJ, Wilson LA, Visvesvara GS, et al. Rapid diagnosis of Acanthamoeba
keratitis from corneal scrapings using indirect fluoroscent antibody staining.
Arch Ophthalmol. 1986;104(9):1318-21.
76. Visvesvara GS, Mirra SS, Brandt FH, et al. Isolation of two strains of Acanthamoe-
ba castellanii from human tissue and their pathogenicity and isoenzyme pro-
files. J Clin Microbiol. 1983;18(6):1405-12.
77. Blackman HJ, Rao NA, Lemp MA, et al. Acanthamoeba keratitis successfully
treated with penetrating keratoplasty: Suggested immunogenic mechanisms
of action. Cornea. 1984;3:125-30.
78. Vodkin MH, Howe DK, Visveswara GS, et al. Identification of Acanthamoeba at
the generic and specific levels using the polymerase chain reaction. J Protozo-
ol. 1992;39(3):378-85.
79. Lehmann OJ, Green SM, Morlet N, et al. Polymerase chain reaction analysis of
corneal epithelial and tear samples in the diagnosis of Acanthamoeba keratitis.
Invest Ophthalmol Vis Sci. 1998;39:1261-5.
80. Schroeder JM, Booton GC, Hay J, et al. Use of subgenic 18S ribosomal
DNA PCR and sequencing for genus and genotype identification of Acan-
thamoeba from humans with keratitis and from sewage sludge. J Clin
Micrbiol. 2001;39(5):1903-11.
81. Stothard DR, Hay J, Schroeder-Diedrich Jill M, et al. Fluoroscent oligonu-
cleotide probes for clinical and environmental detection of Acanthamoeba
and the T4 18S rRNA gene sequence type. J Clin Microbiol. 1999;37(8):
2687-93.
82. Qvarnstrom Y, Visvesvara GS, Sriram R, et al. Multiplex real-time PCR assay
for simultaneous detection of Acanthamoeba spp., Balamuthia mandril-
laris, and Naegleria fowleri. J Clin Microbiol. 2006;44(10):3589-95.
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83. Goldschmidt P, Degorge S, Benallaoua D, et al. New tool for the simultaneous
detection of 10 different genotypes of Acanthamoeba available from the Amer-
ican Type Culture Collection. Br J Ophthalmol. 2009;93:1096-100.
84. Mewara A, Khurana S, Yoonus S, et al. Evaluation of loop-mediated iso-
thermal amplification assay for rapid diagnosis of Acanthamoeba keratitis.
Indian J Med Microbiol. 2017;35(1):90-4.
85. Del Chierico F, Di Cave D, Accardi C, et al. Identification and typing of free-living
Acanthamoeba spp. by MALDI-TOF MS Biotyper. Exp Parasitol. 2016;170:82-9.
Chapter_11.indd 218 06-02-2018 21:52:29

12
CHAPTER
Corneal Dystrophies
Rajesh Sinha, Noopur Gupta, Ritika Sachdev, Radhika Tandon, Jeewan S Titiyal
INTRODUCTION
Corneal dystrophies are a heterogenous group of rare and inherited corneal
diseases that are typically bilateral, symmetric, noninflammatory, slowly
progressive and usually bear no relationship to environmental or systemic
factors. The word dystrophy is derived from Greek literature (dys = wrong, dif-
ficult; trophe = nourishment). Clinically, the corneal dystrophies are divided
into three groups based on the principal anatomic location of abnormali-
ties. These may affect the corneal epithelium and its basement membrane
or Bowman layer and the superficial corneal stroma (anterior corneal dys-
trophies), the corneal stroma (stromal corneal dystrophies) or Descemet
membrane and the corneal endothelium (posterior corneal dystrophies).
Most corneal dystrophies have no systemic manifestations and present with
variable shaped corneal opacities in a clear or cloudy cornea and they affect
visual acuity to different degrees.
PREVALENCE
Most of the published reports derive their prevalence data from the number
of cases of corneal dystrophy in patients undergoing keratoplasty. Some of
the recent reports are summarized in Table 12.1. While this may be an indi-
cator of the prevalence of cases severe enough to warrant a corneal graft,
it is not a true estimate of the prevalence of corneal dystrophy in the entire
population.
CLASSIFICATION OF CORNEAL DYSTROPHIES

The increasing availability of genetic analyses highlighted the shortcomings of
the phenotypic method of classification of corneal dystrophy. Abnormalities
Chapter_12.indd 219 06-02-2018 21:56:22

Table 12.1: Prevalence of corneal dystrophy in patients undergoing keratoplasty.
Iran3 Canada4 India5 Singapore6 USA7 USA8

1994– 1996– 1997– 1982– 2001–
2004 2004 2003 1991–2003 1996 2005
Total cases 19,668 794 2,022 901 4,217 1,162
No. of cases with 1,272 101 78 64 978 126
corneal dystrophy (6.47%) (13%) (3.85%) (7.1%) (23.2%) (10.8%)
in different genes may produce a single phenotype, whereas various defects

in a single gene can manifest as varying phenotypes.1 The International
Committee for Classification of Corneal Dystrophies (IC3D) was developed
to incorporate the traditional classification of corneal dystrophies with new
genetic, clinical and pathologic information.2 The anatomic classification
continues to group dystrophies according to the structures predominantly
involved. Each dystrophy carries a template summarizing genetic, clinical
and pathologic information. A category number from 1 through 4 is assigned
depicting the level of evidence supporting the existence of the particular dys-
trophy. The most defined dystrophies belong to category 1 (a well-defined
corneal dystrophy with a gene that has been mapped, identified and specific
mutations are known) and the least defined belong to category 4 (a suspected
dystrophy without substantial genetic evidence).2
Category 1: A well-defined corneal dystrophy in which the gene has been
mapped and identified and specific mutations are known.
Category 2: A well-defined corneal dystrophy that has been mapped to one
or more specific chromosomal loci, but the gene(s) remains to be
identified.
Category 3: A well-defined corneal dystrophy in which the disorder has not
yet been mapped to a chromosomal locus.
Category 4: This category is reserved for a suspected new, or previously doc-
umented, corneal dystrophy, although the evidence for it, being a
distinct entity, is not yet convincing.
The category assigned to a specific corneal dystrophy can be expected to
change over time as knowledge progressively advances. Eventually, all valid
corneal dystrophies should attain the classification of category 1.
THE IC3D CLASSIFICATION2 (C = CATEGORY)

Epithelial and Subepithelial Dystrophies
• Epithelial basement membrane dystrophy (EBMD)—majority degener-

ative, some C1
• Epithelial recurrent erosion dystrophy (ERED) C4, (Smolandiensis vari-
ant) C3
• Subepithelial mucinous corneal dystrophy (SMCD) C4
Chapter_12.indd 220 06-02-2018 21:56:23

Corneal Dystrophies 221
• Mutation in keratin genes: Meesmann corneal dystrophy (MECD) C1

• Lisch epithelial corneal dystrophy (LECD) C2
• Gelatinous drop-like corneal dystrophy (GDLD) C1
Bowman Layer Dystrophies
• Reis–Bucklers corneal dystrophy (RBCD)—granular corneal dystrophy

type 3 C1
• Thiel–Behnke corneal dystrophy (TBCD) C1, potential variant C2
• Grayson–Wilbrandt corneal dystrophy (GWCD) C4
Stromal Dystrophies
1. TGFBI corneal dystrophies

A. Lattice corneal dystrophy
a. Lattice corneal dystrophy, TGFBI type (LCD): Classic lattice
corneal dystrophy (LCD1) C1, variants (III, IIIA, I/IIIA, and IV)
are C1
b. Lattice corneal dystrophy, gelsolin type (LCD2) C1 (this is not a
true corneal dystrophy but is included here for ease of differen-
tial diagnosis)
B. Granular corneal dystrophy (GCD) C1
a. Granular corneal dystrophy, type 1 (classic) (GCD1) C1
b. Granular corneal dystrophy, type 2 (granular-lattice) (GCD2) C1
c. Granular corneal dystrophy, type 3 (RBCD) = Reis–Bucklers C1
2. Macular corneal dystrophy (MCD) C1
3. Schnyder corneal dystrophy (SCD) C1
4. Congenital stromal corneal dystrophy (CSCD) C1
5. Fleck corneal dystrophy (FCD) C1
6. Posterior amorphous corneal dystrophy (PACD) C3
7. Central cloudy dystrophy of Francois (CCDF) C4
8. Pre-descemet corneal dystrophy (PDCD) C4
Descemet Membrane and Endothelial Dystrophies
• Fuchs’ endothelial corneal dystrophy (FECD) C1, C2, or C3

• Posterior polymorphous corneal dystrophy (PPCD) C1 or C2
• Congenital hereditary endothelial dystrophy 1 (CHED1) C2
• Congenital hereditary endothelial dystrophy 2 (CHED2) C1
• X-linked endothelial corneal dystrophy (XECD) C2
As clinical manifestations widely vary with the different entities, corneal
dystrophies should be suspected when corneal transparency is lost or corneal
opacities occur spontaneously, particularly in both corneas, and especially in
the presence of a positive family history or in the offspring of consanguineous
parents.
Chapter_12.indd 221 06-02-2018 21:56:23

The management of the corneal dystrophies varies with the specific dis-
ease. Some are treated medically or with methods that excise or ablate the
abnormal corneal tissue like phototherapeutic keratectomy (PTK). Corneal
transplantation—penetrating or lamellar—may be required for visual reha-
bilitation in advanced cases. Other less debilitating or asymptomatic dystro-
phies do not warrant treatment. The prognosis varies from minimal effect on
the vision to corneal blindness, with marked phenotypic variability.
EPITHELIAL AND SUBEPITHELIAL DYSTROPHIES

Epithelial Basement Membrane Dystrophy
Alternative names: Map-dot-fingerprint dystrophy, Cogan microcystic epi-

thelial dystrophy, anterior basement membrane dystrophy, dystrophic recur-
rent erosion.
EBMD is characterized by recurrent corneal erosions (Fig. 12.1) as a
result of abnormal epithelial-basement membrane adhesion complexes.9
This dystrophy is the most commonly encountered anterior corneal dystro-
phy in clinical practice.
Inheritance
Most cases have no definite hereditary patterns. Autosomal dominant inher-

itance has been documented in a few cases.10
Genetic Locus
The genetic locus has been mapped to chromosome 5 (5q31); gene TGFBI
has been isolated in a minority of cases.11,12
Fig. 12.1: Recurrent corneal erosions in basement membrane dystrophy.
Chapter_12.indd 222 06-02-2018 21:56:25

Fig. 12.2: Map like areas with dots in a case of epithelial basement membrane dystro-
phy (EBMD).
Symptoms
The disease manifests in adult life around 30 years of age. Familial cases may
manifest earlier in childhood.
Patients may remain asymptomatic or develop recurrent erosions with
pain, lacrimation and blurred vision. Visual acuity is usually not affected.
Irregular astigmatism and increase in higher-order aberration may cause
blurred vision.
Signs
This disease is characterized by the appearance of maps, dots and fingerprint

lines.5
• Maps appear as gray geographical patches, best observed on broad tan-
gential illumination (Fig. 12.2).
• Dots (Cogan) are irregular round, oval or comma-shaped gray-white
intraepithelial opacities; clustered like an archipelago in the central cor-
nea. These occur in combination with other signs, especially with maps.
• Dots (Blebs of Bron and Brown) are small clear round dots clustered
together, visible only on retroillumination.
• Fingerprint lines are parallel, curvilinear branching lines with club-
shaped terminations. Refractile lines are seen on retro illumination.
Combination of maps and dots are noted most frequently, followed by
maps alone (Fig. 12.3).
Chapter_12.indd 223 06-02-2018 21:56:26

Fig. 12.3: Map like areas in Cogan microcystic epithelial dystrophy.
Histopathology
The major pathology lies in the abnormal synthesis of the epithelial basement
membrane. Recurrent erosions occur due to lack of hemidesmosomal con-
nections between the epithelial cells and the abnormal basement membrane.
Maps are areas of projections of the abnormal multilamellar basement
membrane into the epithelium; fingerprint lines represent rib-like intraep-
ithelial extensions of basal laminar material; dots represent intraepithelial
pseudocyst containing cytoplasmic debris.13
In vivo confocal microscopy, images document the abnormal epithelial
basement membrane protruding into the corneal epithelium, epithelial cell
abnormalities and microcysts.14 No abnormalities are observed in superfi-
cial epithelial cells or the stroma. Confocal microscopy has been reported
to assist in the diagnosis of EBMD in patients suffering from recurrent ero-
sion syndrome, particularly in patients with no corneal changes visible
biomicroscopically.
Management
Corneal scrapping may be performed in cases of recurrent corneal erosions.

Following the procedure, a soft contact lens is placed for 24–48 hours and
topical antibiotics instilled. A 5-year cumulative probability of recurrence of
up to 44.7% has been reported following epithelial debridement for anterior
basement membrane dystrophy.7
Conservative therapy with hypertonic sodium chloride (to dehydrate
the epithelium allowing it to adhere better) along with lubricating eye drops
Chapter_12.indd 224 06-02-2018 21:56:26

may be useful in reducing the frequency and severity of attacks. Torres Perez
et al.15 suggested that treatment of recurrent corneal erosions with erosion
debridement may be better than stromal punctures with a 23- to 25-gauge
needle since it implies less potential risks. Anterior stromal puncture by
Nd:YAG laser has been reported to be an effective and simple procedure to
treat recurrent corneal erosion with minimal complications.16
PTK using an excimer laser with low pulse energy and low number of
pulses has been reported as an effective and minimally invasive treatment
modality to achieve a fast and durable epithelial closure, to prevent recurrent
corneal erosions and to increase visual acuity in most patients. A success rate
of 84.6 to 100% has been reported by various authors.17-20 Shallow ablations
(mean ablation depth 4.6 microns) have been recommended by Zaidmman
et al. in view of decreased complications.18,19
Epithelial Recurrent Erosion Dystrophy
Alternative name: Franceschetti recurrent epithelial dystrophy.21

Variant: Dystrophia Smolandiensis.
Inheritance
Inheritance pattern is autosomal dominant. The genetic locus remains

unknown.22
Symptoms
Most patients experience attacks of redness, photophobia, epiphora and ocu-

lar pain due to corneal erosions (Fig. 12.4). Some may complain of sensitive
Fig. 12.4: Epithelial recurrent erosion dystrophy (ERED).
Chapter_12.indd 225 06-02-2018 21:56:27

eyes for years. Exposure to sunlight, dust and smoke and lack of sleep can
precipitate attacks. Attacks generally decline in frequency and intensity and
cease by the age of 50 years.
Signs
Recurrent corneal erosions appear typically at 4–6 years of age but occasion-
ally as early as 8 months of age. These may be precipitated by minimal trauma
or may be spontaneous. The cornea may develop subepithelial haze or blebs
between attacks. The Smolandiensis variant is characterized by recurrent
corneal erosions, followed by the formation of central corneal keloid like
opacities.23
Histopathology
Light microscopic examination reveals epithelial hyperplasia, absence of

Bowman’s layer and subepithelial fibrosis in cases with Dystrophia Smolan-
diensis; the specimen being positive for Congo red, suggesting an amyloid
deposit. The general morphological pattern of pathology (true keloid forma-
tion, absence of Bowman’s layer, subepithelial fibrosis and abnormal sub-
basal nerves) probably reflects a novel phenotypic expression of the healing
response to recurrent erosion of the corneal epithelium.23
Management
Recurrent erosions are managed similar to cases with EBMD.

In the Smolandiensis variant, a quarter of patients eventually require cor-
neal grafts at mean age of 44 years. The opacities recur within 15 months in
the graft periphery, but the central graft can remain clear for many years.
Subepithelial Mucinous Corneal Dystrophy
Inheritance
This dystrophy has an autosomal dominant pattern of inheritance. Genetic

locus and gene remain unknown.24
Symptoms
The onset is characterized by frequent and recurrent corneal erosions in the

first decade. These subside during adolescence with the formation of subep-
ithelial opacities, causing progressive decreased vision.24
Signs
Bilateral and homogenous subepithelial haze is noted. The haze is most

dense centrally, and fades towards the periphery.
Chapter_12.indd 226 06-02-2018 21:56:27

Histopathology
Light microscopy reveals a subepithelial band of eosinophilic, periodic

acid Schiff-positive, alcian blue-positive, Masson trichrome-positive
hyaluronidase-sensitive material anterior to the Bowman layer. The overly-
ing epithelium is thinned out. Immunohistochemistry staining is positive for
combination of chondroitin-4-sulfate and dermatan sulfate.24
Management
Initial treatment includes management of recurrent corneal erosions. The

superficial location of pathology makes PTK a potential treatment modality.
Meesmann Corneal Dystrophy
Alternate name: Juvenile hereditary epithelial dystrophy.

This bilateral, diffuse corneal dystrophy involves the accumulation of
intracytoplasmic debris in the corneal epithelium, manifesting clinically with
the formation of epithelial cysts.
Historical Perspective
This dystrophy was first described clinically by Pameijer.25 The histopatho-

logical description was given by Meesmann.26
Inheritance
Autosomal dominant with incomplete penetrance and variable expressibility

is seen in a majority of cases. Autosomal recessive form has been reported by
Stocker and Holt.27
Genetic locus has been mapped to chromosome 12q13 (KRT3); and the
gene Keratin K3 (KRT3) has been implicated.28 Locus 17q12 (KRT12) and
gene Keratin K12 (KRT12) has been isolated in cases with the Stocker–Holt
variant.29 These genes are known to encode cytoskeletal proteins.
Onset and Course
Clinical signs may be visible as early as 12 months of age and increase

throughout life.
The dystrophy follows a slowly progressive course and majority of the
patients may remain asymptomatic till the fourth or fifth decade of life.30,31
Patients with the Stocker–Holt variant demonstrate more severe signs and
symptoms with earlier onset compared with classic MECD.27
Symptoms
Patients are usually asymptomatic till the fourth or fifth decade of life. Pho-
tophobia, redness and pain may occur due to recurrent corneal erosions
Chapter_12.indd 227 06-02-2018 21:56:27

with the rupture of the epithelial cysts. Most patients retain good functional
vision, few may complain of blurred vision secondary to corneal irregularity
and scarring.
Signs
Corneal involvement is usually bilateral. Multiple, tiny epithelial vesicles

extend to the limbus and are most numerous in the interpalpebral area with
clear surrounding epithelium. These appear as white spots on focal illumina-
tion and are seen as refractile cysts of retroillumination. Cysts may coalesce
to form refractile linear opacities with intervening areas of clear cornea.
Stocker–Holt variant encompasses the entire cornea. Fine, grayish punc-
tate epithelial opacities that take up fluorescein and fine linear opacities in
whorl-like pattern are visible.27
Histopathology
Light microscopy demonstrates intraepithelial cysts filled with periodic acid

Schiff-positive cellular debris. The epithelium may be thickened and disorga-
nized. Bowman’s layer and anterior stroma are unaffected.
Transmission Electron Microscopy reveals intracytoplasmic ‘peculiar
substance’ representing a focal collection of fibrogranular material sur-
rounded by tangles of cytoplasmic filaments.
Tuft et al.32 reported hyporeflective areas in the basal epithelium ranging
from 40-150 mm in diameter, with potential reflective spots inside visible on
confocal microscopy.
Associations
Cremona et al.33 reported a rare case of bilateral and symmetric MECD con-
current with bilateral EBMD and bilateral but asymmetric PPCD in a patient
of Armenian origin.
Management
Most patients remain asymptomatic and may not require any treatment.
Palliative treatment includes ocular lubricants, cycloplegia and therapeutic
contact lenses. In severe cases, management with epithelial debridement,
PTK and lamellar keratoplasty has been advocated.34 Yeung et al.35 have sug-
gested keratectomy with mitomycin C application in recurrent cases of Mees-
man’s dystrophy.
Lisch Epithelial Corneal Dystrophy
Alternative names: Band-shaped and whorled microcystic dystrophy of the

corneal epithelium.36
Chapter_12.indd 228 06-02-2018 21:56:27

Inheritance
X-chromosomal dominant inheritance with genetic locus at Xp22.3 has been

documented. The gene remains unknown.37
Symptoms
The disease onset is in childhood with a slowly progressive course. Most

patients are usually asymptomatic. Patients may report blurred vision if the
pupillary zone is involved.
Signs
Direct illumination reveals localized gray opacities of varying patterns: whorl-

like, radial, band-shaped, flame or feathery-shaped or club-shaped.38-40
Indirect illumination reveals intraepithelial multiple, densely crowded micro-
cysts. The surrounding epithelium appears clinically normal. Similar degrees
of opacities are noted in both men and women.
Histopathology
Light microscopy documents diffuse cytoplasmic vacuolization of all cells in

the affected area.
Management
The corneal abnormalities have been reported to recur after corneal scrap-
ping.41 Lisch et al.42 reported that wearing contact lenses for a longer dura-
tion causes a significant regression of corneal opacities in LECD (two cases
reported). The etiology of this phenomenon was interpreted as a contact lens
induced thinning of corneal epithelium and reduction of epithelial layers.
Gelatinous Drop-like Corneal Dystrophy
Alternative names: Subepithelial amyloidosis; primary familial amyloidosis.43
Inheritance
Inheritance pattern is autosomal recessive. The genetic locus has been iso-
lated to 1p32; and tumor-associated calcium signal transducer 2 (TACSTD2,
previously M1S1) gene has been implicated.44,45
Symptoms
Patients present with significant decrease in vision, photophobia, irritation,

redness and lacrimation.
Chapter_12.indd 229 06-02-2018 21:56:27

Fig. 12.5: A case of Gelatinous droplet corneal dystrophy with sub-epithelial, multiple,
nodular lesions.
Signs
Onset of the disease is by the first to the second decade of life. Initial subep-
ithelial lesions appear similar to band-shaped keratopathy. As they progress
to form groups of small multiple nodules (Fig. 12.5), they acquire a mulberry
configuration. These lesions show late staining with fluorescein, implying
hyperpermeability of the corneal epithelium. Superficial vascularization may
be noted. As the disease progresses, patients may develop stromal opacifi-
cation (Fig. 12.6) or develop larger nodular kumquat-like lesions. This dys-
trophy is usually found in Japanese people, but has been reported in other
regions of the world as well.46
Histopathology
Light microscopy demonstrates subepithelial and stromal amyloid deposits.

Disruption of epithelial tight junctions in the superficial epithelium and the
presence of amyloid in the basal epithelial layer is visible on transmission
electron microscopy.
Management
Corneal transplantation is required for visual rehabilitation.47,48 Deep lamel-

lar keratoplasty (DLKP) has been reported to successfully treat GDLD.49
Recurrence is common after keratoplasty, the disease may recur in nearly
half the grafts. Lasram et al.50 reported that the five cases of GDLD treated by
Chapter_12.indd 230 06-02-2018 21:56:27

Fig. 12.6: Stromal opacification and larger nodular kumquat-like lesions in a case of
gelatinous droplet corneal dystrophy.
them required multiple keratoplasties at a mean interval of five years because

of recurrence of the disease on the corneal graft.
Ito et al.51 reported that PTK may be a safe and useful modality to remove
corneal opacities that recur after lamellar grafts.
BOWMAN LAYER DYSTROPHIES

Reis–Bucklers Corneal Dystrophy
This dystrophy primarily involves the Bowman’s layer with secondary alter-
ations in the epithelium and the stroma.
Alternative names: Corneal dystrophy of Bowman layer, type 1; geo-
graphic corneal dystrophy (Weidle); superficial GCD; atypical GCD; GCD,
type 3; anterior limiting membrane dystrophy, type 1.
Historical Perspective
This dystrophy was first reported by Reis in 1917.52 Detailed description was
given by Buckler in 1949.53
Inheritance
Autosomal dominant inheritance with variable expressibility has been noted.

Genetic locus lies at 5q31; gene TGB1 has been implicated.54-56
Chapter_12.indd 231 06-02-2018 21:56:27

Fig. 12.7: Reis Buckler dystrophy showing irregular and coarse geographic opacities in
the Bowman’s layer and superficial stroma.
Symptoms
Recurrent corneal erosions manifest as pain, redness and tearing in the first
decade of life. These attacks become less severe after the second decade with
progressive deterioration of vision. The visual loss is attributable to the dif-
fuse opaque irregular surface.
Signs
Irregular and coarse geographic-like opacities are seen in the Bowman’s layer
and superficial stroma (Fig. 12.7), secondary to generalized replacement of
the Bowman’s layer by irregular collagen fibers.57 Opacities may be linear,
geographical, honeycomb or ring like and are best seen with broad oblique
illumination. Peripheral cornea is usually spared, although a diffuse haze
extending up to the limbus may be seen in advanced cases. Corneal sensa-
tions are decreased and prominent corneal nerves may be noted.6
Histopathology
The Bowman’s layer is replaced by a mass of irregularly placed collagen fibers,

which in advanced cases can extend to the subepithelial stroma. Epithelial
cells and anterior stromal keratocytes show degenerative changes such as
swelling of the endoplasmic reticulum and vacuole formation. The posterior
epithelial layer shows a saw-tooth configuration.
Subepithelial electron-dense, rod-shaped bodies are noted on electron
microscopy. These rod-shaped bodies are immunopositive for transforming
Chapter_12.indd 232 06-02-2018 21:56:27

growth factor beta–induced protein (keratoepithelin). Electron microscopy

is necessary to distinguish RBCD from the TBCD where curly fibers are pres-
ent.58 Laser confocal scanning may also enable differentiation of Theil–Benke
and Reis–Bückler dystrophy in vivo.59 In Thiel–Behnke corneal dystrophy,
the deposits in the epithelial basal cell layer show homogeneous reflectivity
with round edges accompanying dark shadows. In contrast, deposits in Reis–
Bücklers corneal dystrophy in the same cell layer show extremely high reflec-
tivity from small granular materials without any shadow. In each dystrophy,
Bowman’s layer is replaced totally with pathological materials; the reflectivity
of those materials is reported to be much higher in Reis–Bückler corneal dys-
trophy than in Thiel–Behnke corneal dystrophy.
Management
Recurrent corneal erosions are treated in initial stages. PTK has been reported
to be an effective modality for the treatment of this dystrophy. Recurrence
is common after this procedure. Dinh et al.60 reported that 47% of the eyes
with Reis–Bucklers dystrophy developed clinically significant recurrence
after an average of 21.6 months after PTK. Adjunctive application of topical
Mitomycin-C 0.02% may be helpful in reducing the recurrence of the disease
after PTK.61 Corneal electrolysis has been reported to effectively treat subep-
ithelial opacities in RBCD.62 Keratoplasty may be required in severe cases.63
Thiel–Behnke Corneal Dystrophy
Alternative names: Corneal dystrophy of Bowman layer, type 2 (CDB2);

honeycomb-shaped corneal dystrophy; anterior limiting membrane dystro-
phy, type 2; curly fibers corneal dystrophy; Waardenburg–Jonkers corneal
dystrophy.
Inheritance
Autosomal dominant inheritance with genetic locus at 10q24 has been

demonstrated.64 The gene remains to be isolated.
Symptoms
Recurrent erosions begin in childhood. Slowly, progressive deterioration of

vision occurs with increasing corneal opacification. The erosions are less fre-
quent, and the onset of visual impairment is later than in RBCD.
Signs
Symmetrical subepithelial reticular honeycomb-like opacities are noted,

sparing the peripheral cornea.65 Corneal sensations are normal. In advanced
Chapter_12.indd 233 06-02-2018 21:56:28

cases, opacities can progress to deep stromal layers and corneal periphery.
It may be impossible to distinguish it clinically from Reis–Buckler corneal
dystrophy.
Histopathology
A fibrillogranular material is deposited under the epithelium and projects

into the overlying cells in a ‘saw tooth’ configuration. The epithelial basement
membrane is thickened.
Electron microscopy demonstrates pathognomonic curly collagen fibers
with a diameter of 9–15 nm and distinguishes this dystrophy from RBCD.
These curly fibers are immunopositive for transforming growth factor beta-
induced protein (keratoepithelin).58 The confocal images may also enable dif-
ferentiation from RBCD.8
Grayson–Wilbrandt Corneal Dystrophy
Inheritance
Autosomal dominant inheritance pattern is seen. The genetic locus remains

unknown.
Symptoms
The onset of the disease occurs at 10–12 years, later than RBCD. Corneal ero-
sions are less severe and less frequent than in RBCD and TBCD. Visual acuity
is usually preserved.
Signs
Bowman layer demonstrates diffuse gray-white mound-like opacities extend-
ing anteriorly into the epithelium. The intervening cornea is clear, and the
peripheral cornea is spared. The corneal sensations are preserved, unlike in
RBCD.66,67
Histopathology
Accumulation of abnormal material (PAS positive) in the basement mem-

brane with disruptions in the Bowman’s membrane is noted.
STROMAL DYSTROPHIES
Corneal stromal dystrophies are a group of inherited disorders of the cornea
(Table 2) that are caused by progressive accumulation of deposits within the
stroma. These deposits are not caused by inflammation, infection or trauma,
but by genetic mutations that lead to abnormal proteins resulting in the
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Table 12.2: Stromal corneal dystrophies and their characteristics.
Stromal corneal
dystrophy Inheritance Onset Clinical features Histology
Lattice type 1 AD First decade Subepithelial Amyloid; Congo
5q31 dots-coalesce into red
TGFBI typical, branching
lattice lines-
corneal haze
Lattice type 2 AD Middle Systemic Amyloid
9q34 age with features- deposits (corneal
GSN progressive progressive facial stroma and renal
facial palsy palsy; renal and glomeruli)
cardiac failure
Granular type 1 AD First decade Superficial and Amorphous
5q31 central white, hyaline deposits-
TGFBI crumb like stain with mason
opacities—become trichrome
confluent and
progress deeper
and peripherally-
spare limbus
Granular type 2 AD 4th-5th Superficial discrete, Amorphous
5q31 decade white, crumb like hyaline deposits-
TGFBI opacities stain with mason
trichrome
Granular type 3 AD Late in life Few, superficial dis- Amorphous
(Avellino) crete, ring shaped, hyaline deposits
white, crumb like + Amyloid
opacities deposits
Macular type 1 AR 2nd decade Progressive, gen- Abnormal
16q22 eralized, corneal closely packed
CHST6 haze with focal, collagen; lack of
poorly delineated proteoglycans;
opacities; reduced aggregations
corneal thickness of dermatan
and chondroitin
sulfate
Macular type 2 AR 2nd decade Progressive, gen- Abnormal
16q22 eralized, corneal closely packed
CHST6 haze with focal, collagen; lack of
poorly delineated proteoglycans;
opacities; reduced aggregations
corneal thickness of dermatan
and chondroitin
sulfate
(Contd.)
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(Contd.)
Stromal corneal
dystrophy Inheritance Onset Clinical features Histology
Congenital AD Before birth Moderate to severe
stromal dystrophy 12q13.2 visual loss; diffuse
DCN corneal clouding;
flake-like opacities
throughout stroma
Fleck dystrophy AD At birth Normal small –
2q35 discrete dandruff or
PIP5K3 ring-shaped, fleck
like opacities
Posterior AD Infancy or Mildly affected –
amorphous Unknown childhood diffuse sheet-like
corneal dystrophy opacities
(PACD) especially in poste-
rior corneal stroma
accumulation of insoluble material within the stroma. These disorders may

or may not affect vision and may or may not be symmetrical. They usually
present in the second to third decade of life. The major corneal dystrophies
include lattice, granular and macular dystrophy.
Lattice Dystrophy
Lattice dystrophy gets its name from an accumulation of amyloid deposits or

abnormal protein fibers throughout the middle and anterior corneal stroma.
Inheritance
This dystrophy usually begins before the age of 20 years, and is inherited as an
autosomal dominant disorder.
Symptoms and Signs
Early symptoms tend to be a ‘foreign body’ sensation and a slight deteriora-

tion in vision due to clear, comma-shaped overlapping dots and branching
filaments in the corneal stroma, creating a lattice effect. As the dystrophy pro-
gresses, these lines become thicker and opaque that imparts a ground glass
haze to the cornea and cause diminution of vision.
Types of Lattice Corneal Dystrophy
A network of delicate criss-cross branching filamentous opacities are seen

within the cornea in two genetically distinct inherited disorders, one caused
by specific mutations in the TGFBI (transforming growth factor-beta) gene
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with no systemic manifestations (LCD1) and the other resulting from a muta-
tion in the gelsolin gene (LCD2). LCD 2 has systemic manifestations.
Lattice corneal dystrophy, type 1: (LCD1; Biber-Haab-Dimmer dystrophy)
is a rare form of human corneal dystrophy that becomes apparent in both
eyes towards the end of the first decade of life, but occasionally it begins in
middle life and is rarely seen in infancy. LCD1 was first described by Swiss
ophthalmologist Hugo Biber in 1890.68 It has no systemic manifestations. The
disease is bilateral, usually noted before the end of the first decade of life.
Inheritance
LCD1 is caused by mutations in TGFBI gene encoding keratoepithelin. Trans-

forming growth factor, beta-induced, 68kDa, also known as TGFBI (initially
called BIGH3, BIG-H3), is a protein which is encoded by the TGFBI gene in
humans.69 This gene encodes a protein that binds to types 1, 2 and 4 colla-
gens. It is found in many extracellular matrix proteins modulating cell adhe-
sion and serves as a ligand recognition sequence for several integrins. The
protein is induced by transforming growth factor-beta and acts to inhibit cell
adhesion.
Symptoms
Recurrent corneal erosions manifest as pain, redness and tearing in the first
decade of life. These attacks become less severe after the second decade with
progressive deterioration of vision.
Signs
Filamentous opacities appear in the cornea with intertwining delicate

branching processes, particularly within the central corneal stroma, while the
peripheral cornea remains relatively transparent. Corneal sensation is often
diminished and the interwoven linear opaque filaments have some resem-
blance to nerves. Recurrent corneal erosions may precede the corneal opac-
ities and even appear in individuals lacking recognizable stromal disease
(Fig. 12.8). Both corneas are usually symmetrically involved, but sometimes
one cornea remains clear or has discrete rather than the linear opacities.
Histopathology
On light microscopy, epithelial atrophy and disruption with degeneration

of basal epithelial cells; focal thinning or absence of Bowman layer that
progressively increases with age; eosinophilic layer between epithelial base-
ment membrane and Bowman layer; and stromal deposition of amyloid
substance distorts the architecture of corneal lamellae. Amyloid deposits
have characteristic staining. Deposits stain positive with Congo red. Green
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Fig. 12.8: A case of lattice dystrophy showing lattice lines in the corneal stroma.
birefringence is visible with a polarizing filter and red-green dichroism when

a green filter is added with this stain. Metachromasia is noted with crystal
violet and fluorescence is noted with use of thioflavin T staining. Descemet
membrane and endothelium are normal.
Like nerves, the linear deposits of LCD1 are argyrophilic in silver impreg-
nated preparations, but nerves have not been identified in relation to the
eosinophilic amyloid deposits. Amyloid deposits occur throughout the cor-
neal stroma70 and coincide with the lattice pattern of lines and other opaci-
ties.71 The amyloid seems to react mainly with antibodies to the N-terminal
sequence of TGFBIp and not with those to the C-terminal portion.72 The
majority of cases of LCD1 throughout the world have been associated with a
C ® T transition at nucleotide 417 (417 C ® T) in exon 4 of the TGFBI gene.
This causes a p.Arg124Cys mutation in the affected codon.73
Lattice corneal dystrophy, type 2: (LCD2, systemic amyloidosis, familial
amyloid polyneuropathy, type 4, Finnish or Meretoja type) LCD2 is most
common in Finland, where the disease was first discovered and most exten-
sively studied.74 In this disorder, both corneas contain randomly scattered
short fine glassy lines which are less numerous, more delicate and more radi-
ally oriented than those in LCD1 (Fig. 12.9). The peripheral cornea is chiefly
affected and the central cornea is almost spared. The cornea has fewer amor-
phous deposits than LCD1.
Inheritance
Finnish type amyloidosis is a form of amyloidosis associated with gelsolin

gene.75 In persons, homozygous for the relevant mutation in the GSN gene,
the disorder begins earlier.
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Fig. 12.9: Branching filaments in the corneal stroma, creating a lattice effect. A network of
delicate, criss-cross filamentous opacities are seen within the cornea in lattice dystrophy.
Symptoms and Signs
The abnormal protein fibers in LCD1 accumulate under the outer corneal
layer—the epithelium. This can cause erosion of the epithelium, manifesting
as recurrent corneal erosion. These erosions alter the normal corneal curva-
ture, resulting in temporary vision problems; and expose the nerves that line
the cornea, causing severe pain. Even the involuntary act of blinking can be
painful. Recurrent epithelial erosions are common particularly from the first
decade of life.
In LCD2, corneal sensitivity is reduced or absent. Visual acuity is usu-
ally normal until the sixth decade because the dystrophy progresses from the
peripheral to central cornea. Dry eye symptoms are frequent, and corneal
erosions may occur late in life. It has a slowly progressive course; the majority
of affected individuals are in good health till the seventh decade. At around
40 years of age, some people with lattice dystrophy will have scarring under
the epithelium, resulting in a haze on the cornea that can greatly obscure
vision. In LCD2, the corneal abnormalities are accompanied by a progres-
sive and bilateral cranial and peripheral neuropathy, dysarthria, a dry and
extremely lax itchy skin with amyloid deposits. Associated conditions include
cutis laxa76 and ataxia,77 a characteristic ‘mask-like’ facial expression, pro-
truding lips with impaired movement, pendulous ears and blepharochalasis
are associated systemic features.
Histopathology
The amyloid in LCD2 is composed of a mutated 71 amino acid long fragment

of gelsolin and this protein accumulates in the corneal stroma. In LCD2,
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amyloid deposits are found in the cornea, scleral, choroidal and adnexal
blood vessels as well as in the lacrimal gland and perineurium of ciliary
nerves. The amyloid is also found in the heart, kidney, skin, nerves, wall of
arteries and other tissues.78 Amyloid is deposited in the cornea in lattice
lines, as a discontinuous band under Bowman layer and within the sclera.
Streak-like deposits are seen between corneal lamellae, especially in the
limbal cornea. Immunohistochemistry demonstrates deposition of mutated
gelsolin in the conjunctiva, in the sclera, in the stroma of the ciliary body,
along the choriocapillaris, in the perineurium of ciliary nerves, in the walls
of ciliary vessels and in the optic nerve. Extraocularly, amyloid is found in
arterial walls, peripheral nerves and glomeruli. The amyloid within the cor-
nea in LCD2 reacts with the antigelsolin antibody.79 Two single base substi-
tutions in the GSN gene, located on human chromosome 9 (9q34), which
encodes the actin modulating protein gelsolin are known to cause LCD2
(p.Asp187Asn, p.Asp187Tyr).
Management
Recurrent epithelial erosions are treated with preservative free tear substi-
tutes, ointments, eye patching, bandage contact lens or amniotic membrane
transplantation for persistent defects. With effective care, these erosions usu-
ally heal within three days, although occasional sensations of pain may occur
for the next 6 to 8 weeks. PTK may be tried in all patients with superficially
accentuated opacities in lattice dystrophy before undergoing a more invasive
procedure, such as lamellar or penetrating keratoplasty (PK).80
Corneal transplant is indicated in cases with epithelial scarring and cor-
neal haze. Although, people with lattice dystrophy have an excellent chance
for a successful transplant, the disease may also arise in the donor cornea
in as little as three years. A corneal graft may be necessary in LCD1 by 20
years of age, but is usually not indicated until after the fourth decade. LCD1 is
slowly progressive and usually substantial discomfort and visual impairment
occurs before the sixth decade. The outcome of penetrating graft is excellent,
but amyloid may deposit in the grafted donor tissue some 2–14 years later.
Incomplete removal of the recipient stroma by DLKP can lead to the
recurrence of amyloidosis in the residual stroma in patients with LCD.81 The
corneal lesions in LCD2 rarely warrant a PK, but when performed a neu-
rotrophic persistent epithelial defect may develop. The results of the graft
are generally good, but it is possible that the dystrophy may recur in the
donor cornea within five to ten years. In one study, about half of the trans-
plant patients with lattice dystrophy had a recurrence of the disease from
between two to 26 years after the operation. Of these, 15% required a second
corneal transplant.82 Early lattice and recurrent lattice arising in the donor
cornea respond well to treatment with the excimer laser or anterior lamellar
keratoplasty.
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Granular Corneal Dystrophy
Subtle differences in the clinical appearance of the discrete corneal opaci-

ties permit two types of GCD to be recognized: GCD type 1 (GCD1) and GCD
type 2 (GCD2). GCD2 tends to have fewer corneal deposits than GCD1 and
the corneal deposits in GCD2 sometimes resemble a combination of GCD
and LCD. GCD has been extensively studied in Denmark by Moller.83
Inheritance
GCD shows an autosomal dominant mode of inheritance. It is caused by

mutations in TGFBI gene encoding keratoepithelin.
Granular corneal dystrophy, type 1: (GCD1, corneal dystrophy Groenouw,
type 1) is a rare form of human corneal dystrophy. It was first described by
German ophthalmologist Arthur Groenouw in 1890.84
Symptoms
Light sensitivity and ‘foreign body’ sensation may be a problem. Vision is not
usually severely affected under the age of 50 years.
Signs
It usually occurs before the age of 20 years. Early on, the vision is not affected
but grayish dots can be seen through a microscope. Slowly, the dots become
larger and more apparent and become visible to the naked eye. The clinical
picture is characterized by multiple, small, white discrete irregular-shaped
sharply demarcated spots that resemble bread crumbs or snowflakes and
become apparent beneath Bowman zone in the superficial central corneal
stroma (Fig. 12.10). They initially appear within the first decade of life and
may be evident by 3 years of age. The opaque spots are often arranged in
lines and with time they gradually enlarge and become more numerous. In
children, the external corneal surface is smooth, but in adults it may become
uneven. While some patients have only a few corneal granules, others even-
tually have multiple opacities and subsequently, the cornea becomes mark-
edly opaque. Visual acuity is more or less normal. By the end of the second
decade, opacities are present in the central and superficial cornea but rarely
in the deep stroma. Intervening tissue between the opacities and the periph-
eral 2–3 mm of the cornea usually remains crystal clear (Fig. 12.11). The
opaque spots eventually extend throughout the central two-thirds of the cor-
nea. On confocal microscopy, multiple, hyper reflective opacities are evident.
Confocal microscopy with anterior segment optical coherence tomography
may provide sufficient diagnostic information to diagnose corneal granular
dystrophies in a clinical setting.85
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Fig. 12.10: Granular corneal dystrophy (GCD) with granules composed of extremely
small, translucent dots and opacities that do not extend to the corneal limbus.
Fig. 12.11: Intervening tissue between the opacities and the peripheral 2–3 mm of the
cornea usually remains crystal clear in a case of granular corneal dystrophy (GCD).
Histopathology
The light microscopic and transmission electron microscopic (TEM) appear-

ance and staining attributes of the corneal deposits in GCD are diagnostic.
Eosinophilic deposits in GCD consist predominantly of an extracellular
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deposition of mutant TGFB1 protein, which stains a brilliant red with the
Masson trichrome stain. With the reticulin stain, the accumulations appear
to contain tangles of argyrophilic fibers. The deposits react with histochem-
ical methods for protein as well as with antibodies to TGFB1 protein. The
granules stain positively with luxol fast blue and are reported to stain posi-
tively with antibodies to microfibrillar protein. By TEM, characteristic elec-
tron dense, discrete, rod-shaped or trapezoid bodies are evident.86 Some
rod-shaped structures appear homogeneous without a discernible inner
structure; others, however, are composed of an orderly array of closely
packed filaments (70–100 nm in width) orientated parallel to their long axis,
while others appear moth-eaten with variable-shaped cavities containing
fine filaments. Descemet membrane and the corneal endothelium are unre-
markable, and so is the cornea between the deposits.
Granular corneal dystrophy, type 2: (GCD2, Avellino corneal dystrophy,
combined granular–lattice corneal dystrophy) was first described by Folberg
et al. in 1988.87 The ancestry of some families with GCD2 have been traced to
the Avellino district of Italy (hence the synonym Avellino corneal dystrophy).
Symptoms
Glare and photophobia are early symptoms. Visual acuity decreases as opaci-
fication progresses with age. Recurrent erosions are seen frequently. Homo-
zygote has more severe symptoms and show rapid disease progression.
Signs
Slit-lamp examination reveals well-defined granules that appear white on

direct illumination. On retroillumination, these granules are composed of
extremely small, translucent dots with the appearance of vacuoles, glassy
splinters or crushed bread crumbs. Opacities do not extend to the corneal
limbus. In children, there may be a vortex pattern of brownish granules
superficial to the Bowman layer. In later life, granules may extend into the
deeper stroma down to Descemet membrane.
As the condition progresses, the opacities become more confluent in
the superficial cornea, resulting in a significant reduction of visual acuity. In
most cases of GCD, visual acuity remains good until late in the course of the
disease. Confocal microscopy demonstrates features of both GCD1 and LCD.
Reflective, breadcrumb-like round deposits with well-delineated borders or
highly reflective, irregular trapezoidal deposits are present in the anterior
stroma (similar to GCD1). Linear and branching deposits with changing
reflectivity are observed (similar to LCD).
Histopathology
On light microscopy, corneal opacities extend from the basal epithelium

to the deep stroma. Although there is deposition of both typical GCD1
Chapter_12.indd 243 06-02-2018 21:56:29

deposits and amyloid; individual opacities stain with either Masson tri-
chrome or Congo red. Homozygotes demonstrate more severe findings. On
TEM, anterior stromal rod-shaped, electron-dense deposits composed of
extracellular masses of fine, electron-dense, highly aligned fibrils are noted.
An extremely common ultrastructural finding is the presence of randomly
aligned fibrils of amyloid.
Management
Many individuals with GCD never require corneal grafting because vision is
usually not sufficiently impaired. Until relatively recently, a PK had been the
traditional method for treating GCD, but postoperative recurrent disease can
be detected in the donor tissue and even along the suture tracts within sev-
eral years, particularly in GCD2.88 After a PK, the graft usually remains free
of recurrence for at least 30 months, but the opacities may recur in the grafts
within a year, usually superficial to the donor tissue, even with lamellar grafts
or at the host-graft interface.
PTK has been advocated as an initial therapy for GCD, but recurrent
disease is still a common complication. Similarly, deep anterior lamellar
keratoplasty (DALK) using the Melles technique89 and automated lamellar
keratoplasty90 can restore and preserve useful visual function for a signifi-
cant period in these patients in spite of recurrence. Intralase femtosecond
assisted lamellar keratoplasty has also been described recently to treat Avel-
lino dystrophy.91 Injury to the central cornea may result in exacerbation of
the corneal dystrophy. GCD2 may also be exacerbated by laser epithelial
keratomileusis (LASEK) and radial keratotomy (RK).92 LASIK,93 LASEK and
other forms of refractive surgery are hence contraindicated in individuals
with GCD2.
Macular Corneal Dystrophy Fehr Corneal Dystrophy, Corneal

Dystrophy Groenouw, Type 2
It is the least common but the most severe of the three major stromal corneal
dystrophies.
Inheritance
This dystrophy is inherited in autosomal recessive fashion and is thought to

be caused by the lack or abnormal configuration of keratan sulfate (KS). KS
is one of the major glycosaminoglycans of the corneal stroma and plays an
important role in corneal transparency. Most cases of MCD are caused by
mutations in CHST6 (carbohydrate sulfotransferase 6) gene.94 The most fre-
quent abnormalities are missense and nonsense single nucleotide polymor-
phisms in CHST6 that alter a conserved amino acid.
Chapter_12.indd 244 06-02-2018 21:56:29

Symptoms
Eye pain from recurrent corneal erosions can occur but is much less com-
mon than in patients with lattice or granular dystrophies. Over time, the non-
transparent areas progressively merge as the entire corneal stroma gradually
becomes cloudy, causing severe visual impairment.
Signs
MCD involves the entire thickness of the cornea and is more superficial
centrally and deeper peripherally. The first signs are usually noticed in the
first decade of life, and progress afterwards, with opacities developing in the
cornea.
It is characterized by multiple, gray-white opacities in the corneal stroma
that extend out into the peripheral cornea. These stromal opacities are dis-
tributed throughout the cornea without clear spaces. Initially, diffuse stromal
haze develops extending to the limbus; later, superficial, central, elevated,
irregular whitish opacities (macules) develop and give the condition its name
(Fig. 12.12). Unlike granular dystrophy, there are no clear areas between cor-
neal opacities. There are also more posterior peripheral white lesions. The
cornea is thinner than normal in early disease. In the advanced stage, the
corneal endothelium is affected and Descemet membrane develops guttate
excrescenses. In addition, the stroma thickens from the imbibition of water
from endothelial decompensation.
Fig. 12.12: Macular corneal dystrophy with stromal opacities distributed throughout
the cornea without clear spaces.
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Histopathology
Macular dystrophy is characterized by an intracellular storage of glycosami-

noglycans within keratocytes and the corneal endothelium combined with
an extracellular deposition of similar material in the corneal stroma and
Descemet membrane. The deposits stain with alcian blue. Three immuno-
phenotypes of MCD are recognized,95 one has no detectable KS in the serum
or cornea (MCD type 1), another has normal amounts of KS in the serum and
cornea (MCD, type 2) and a third lacks detectable antigenic KS in the serum,
but has stainable KS in the keratocytes (MCD, type 1A). Because KS in the
serum appears to be predominantly derived from the normal turnover of car-
tilage, these studies strongly suggest that the defect in KS synthesis in MCD is
not restricted to corneal cells and that this condition is one manifestation of
a systemic disorder of KS.96
On electron microscopy, delicate fibrillogranular material can be dis-
cerned within the vesicles. Some corneal endothelial cells contain simi-
lar material. Numerous electron-lucent lacunae are randomly distributed
throughout the cornea and some lacunae are filled with clusters of abnormal
sulfated proteoglycan filaments. The collagen fibrils have a normal diameter,
but the interfibrillar spacing of collagen fibrils in affected corneas is less than
that in the normal cornea. This close packing of collagen fibrils seems to be
responsible for the reduced corneal thickness in MCD. The posterior non-
banded portion of Descemet membrane contains numerous corneal guttae.
Management
In MCD, vision can be restored by corneal grafting, but the disease may recur
in the graft after many years. Conventionally, the condition affects the entire
corneal stroma, Descemet membrane and the corneal endothelium; a lamel-
lar keratoplasty will not excise all of the pathologic tissue. However, DALK
with the big-bubble technique can be considered for visual rehabilitation in
cases with no significant Descemet membrane and endothelial involvement
(Fig. 12.13).97
Congenital stromal corneal dystrophy: (CSCD; Witschel dystrophy) is an
extremely rare, nonprogressive form of corneal dystrophy.
Inheritance
Its inheritance is autosomal dominant and is linked to mutations in DCN

gene encoding decorin. This protein is a component of connective tissue,
binds to type 1 collagen fibrils, and plays a role in matrix assembly protein.
Symptoms
Corneal erosions, photophobia and corneal vascularization are absent. Stra-

bismus or primary open angle glaucoma was noted in some of the affected
patients.
Chapter_12.indd 246 06-02-2018 21:56:29

Fig. 12.13: Deep anterior lamellar keratoplasty in a case of macular dystrophy.
Signs
The main features of the disease are numerous opaque flaky or feathery areas
of clouding in the stroma that multiply with age and eventually preclude visi-
bility of the endothelium. Thickness of the cornea stays the same, Descemet’s
membrane and endothelium are relatively unaffected, but the fibrills of col-
lagen that constitute stromal lamellae are reduced in diameter.
Histopathology
In CSCD, the morphologic abnormalities include a peculiar arrangement of

tightly packed lamellae having highly aligned collagen fibrils of an unusually
small diameter.98 The abnormally small stromal collagen fibrils and disor-
dered lamellae suggest a disturbance in collagen fibrogenesis.
Management
PK or lamellar keratoplasty may be eventually required when vision becomes

significantly impaired. Associated manifestations may be managed with
spectacles or contact lenses for correction of refractive errors; patching and/
or surgical correction for strabismus may be required in the initial stages of
the disease.
Fleck Corneal Dystrophy
Inheritance
It is caused by mutations in PIP5K3 gene.
Chapter_12.indd 247 06-02-2018 21:56:29

Symptoms
The disease is nonprogressive and in most cases asymptomatic, with mild

photophobia reported by some patients.
Signs
FCD affects males and females equally and has been observed throughout
life and even in children as young as 2 years. Multiple nonprogressive, sym-
metric minute opacities, some of which resemble ‘flecks’, are scattered in
the stroma of affected patients. Other opacities look more like snowflakes or
clouds with distinct borders in the central and peripheral cornea with inter-
vening portions of the cornea being normal. The corneal epithelium, Bow-
man layer, and Descemet membrane are unremarkable. Corneal sensation is
usually normal.
Histopathology
Corneal tissue with FCD has rarely been examined, but some keratocytes
contain fibrillogranular material within intracytoplasmic vacuoles or pleo-
morphic electron-dense and membranous intracytoplasmic inclusions.99
The stored material reacts positively with alcian blue, colloidal iron, Sudan
black B and oil red O stains and is partially sensitive to hyaluronidase and
b-galactosidase.
Management
As it does not affect vision and is usually asymptomatic and does not require
treatment. LASIK does not stimulate visually significant exacerbation of
FCD.100
Posterior Amorphous Corneal Dystrophy
Inheritance
The chromosomal locus of the gene responsible for the autosomal dominant
PACD has not been determined.
Symptoms
Visual acuity is usually minimally impaired.
Signs
It is characterized clinically by irregular, amorphous sheet-like opacities in

posterior corneal stroma and Descemet membrane. The abnormalities are
Chapter_12.indd 248 06-02-2018 21:56:29

observed in infancy and childhood and, in contrast to the traditional cor-

neal dystrophies, non-corneal manifestations have been reported including
abnormalities of the iris (irido-corneal adhesions, corectopia, and pseudo-
polycoria). Transparent corneal stroma may intervene between opacities,
while the Descemet membrane and the corneal endothelium may show focal
abnormalities.
Histopathology
Disorganized posterior stromal collagen lamellae, and an attenuated corneal

endothelium have been observed.101 A zone of collagen fibers may interrupt
the Descemet membrane beneath the anterior banded layer.
Management
Treatment is usually not required for this dystrophy as it does not affect vision.
DESCEMET MEMBRANE AND ENDOTHELIAL DYSTROPHIES

Fuchs’ Endothelial Corneal Dystrophy
FECD is a bilateral, progressive disease involving the corneal endothe-

lium manifesting as corneal edema and progressive deterioration of vision
(Fig. 12.14). The condition was first described by Austrian Ernst Fuchs (1851–
1930), after whom it is named.102
Fig. 12.14: Fuchs’ dystrophy with increased corneal thickness due to endothelial
decompensation.
Chapter_12.indd 249 06-02-2018 21:56:29

Inheritance
The inheritance of FED is autosomal dominant with genetic and environ-

mental modifiers such as increased prevalence in the elderly and in females.
Cases without known inheritance may constitute the majority.
Genetic Locus
Genetic loci involved in FECD have been isolated at 13pTel –13q12.13, 15q
and 18q21.2 –q21.32.
Early-onset variant FECD may involve locus 1p34.3 –p32 and the gene
affecting collagen type 8, Alpha 2–COL8A2. Some authors have disputed the
role of these genes in the pathogenesis of FCD and the genetic basis of this
disease remains to be fully elucidated.103-106
Associations
Fuchs’ dystrophy has been reported to be associated with cardiovascular dis-

ease, keratoconus, age-related macular degeneration, short axial length, nar-
row anterior chamber angles and open angle glaucoma.107,108
Signs and Symptoms
Most cases begin in the fourth decade or later but the early variant starts in
the first decade.107 Most studies suggest that FED more commonly affects
females, with as high as 4:1 female preponderance. The increased incidence
in females has led to speculation about the role of hormones in the patho-
genesis of FED.
Stage 1: This is the stage of cornea guttata. It occurs in the fourth or fifth
decade of life when slit lamp examination by specular reflection
reveals cornea guttata in the central part of the corneal endothe-
lium. The excrescences of corneal guttata increase in number and
may become confluent, resulting in a beaten metal appearance of
the endothelial surface. Pigment dusting of the endothelium may
be noted. The condition spreads from the center toward the periph-
ery. The corneal thickness and visual acuity remain unaffected. The
patient is asymptomatic.
Stage 2: This stage is characterized by deterioration in vision caused by incip-
ient edema of the corneal stroma. As the stroma becomes edema-
tous, the cornea acquires a ground glass appearance. The patients
complain halos around lights, blurred vision and glare. Vision may
improve as the day progresses owing to evaporation and subsequent
corneal deturgescence.
Stage 3: The edema progresses to involve the epithelium at this stage. Epi-
thelial microcysts manifest as bedewing on retroillumination. As
these cysts enlarge and coalesce, they form larger intraepithelial or
Chapter_12.indd 250 06-02-2018 21:56:30

subepithelial bullae. When these bullae rupture, they cause pain and
discomfort.
is stage is characterized by corneal scarring following rupture
Stage 4: Th
of bullae cause diminution of vision, while ameliorating the pain.
Peripheral vascularization may be noted.
Histopathology
Light microscopy reveals diffuse thickening and lamination of Descemet

membrane with hyaline excrescences on thickened Descemet’s membrane
(guttae). Degeneration, thinning, and reduction of endothelial cells is noted.
Transmission Electron Microscopy demonstrates multiple layers of basement
membrane-like material on the posterior part of Descemet’s membrane with
degeneration of endothelial cells.108 Stromal thickening secondary to edema
causes disorganization and disruption of the lamellar pattern of collagen
fibers. Confocal Microscopy reveals polymegathism and pleomorphism of
the endothelial cells. Subepithelial bullae formation may be seen on the ante-
rior corneal surface. Subepithelial fibrosis may be seen subsequent to rup-
ture of bullae. The Bowman membrane is normal, unless it has been involved
in ulcer formation and keratitis, after the rupture of a bulla.
The following 5 stages on specular microscopic evaluation may be seen,
as described by Laing et al.109
Stage 1: The guttate excrescences are seen as dark structures with sharply
defined single bright spots at their center. They are smaller in size
than a single endothelial cell and do not lie near the boundary wall
of the cell.
Stage 2: The excrescence is almost the size of the endothelial cell. The sur-
rounding cells have a stretched appearance.
Stage 3: The excrescence is considerably larger, and many cells are involved in
one lesion. The dark structure is 5–10 times the size of an endothelial
cell. The adjacent cells are abnormal and have missing boundaries.
Many lesions are seen close to each other, but they do not coalesce.
The excrescences are of 2 types, a smooth round shape or a rough
excrescence.
Stage 4: The individual excrescences have coalesced. The net result is multi-
lobed, rather than a round outline. The dark areas have many bright
spots. The multilobulated structures cover considerable area. The
cells between the excrescence masses tend to become abnormal.
Coalesced areas contain both the smooth and the rough variety of
excrescences.
Stage 5: An organized mosaic of endothelial cells is difficult to see. Many
stages may be observed in the different areas of the same eye.
Management
Conservative treatment: Patients of FCD with clear corneas (Stage 1) do not
require treatment. When corneal decompensation sets in medical therapy
Chapter_12.indd 251 06-02-2018 21:56:30

may be initiated and can be continued till good functional vision is main-
tained beyond which keratoplasty is necessary.
• Dehydrating agents
Sodium chloride 5% eye drops especially in the early hours of the
day; sodium chloride ointment is used at bedtime.
Glycerine can be used for diagnostic purposes such as fundus evalu-
ation as it causes rapid dehydration and clearing of the cornea.
• A hair dryer, placed at arm’s distance, can be used to enhance evapo-
ration and cause corneal deturgescence for temporary improvement of
symptoms.
• Antiglaucoma agents should be administered in cases with concomitant
glaucoma. Topical carbonic anhydrase inhibitors should be avoided they
hinder the activity of endothelial pump.
• Supportive treatment for ruptured bullae
Anterior stromal punctures may be indicated
Soft therapeutic contact lenses
Cycloplegics, local antibiotics with bandaging of the affected eye
Excimer laser PTK, amniotic membrane graft, or a conjunctival flap
can also be considered for patients not willing for keratoplasty or in
those with a poor visual prognosis.110-113
Keratoplasty: Changing Trends
Corneal transplantation remains the definitive treatment for advanced cases

of FCD. PK has been performed for many years to restore vision in patients
with FCD. Chung et al. suggested in patients with insufficient endothelial cell
density like in FCD, a large trephine size could reduce chronic endothelial
cell loss.114
Endothelial keratoplasty (EK) which selectively replaces the posterior
layers of the cornea has recently emerged as an alternative to PK for patients
with endothelial diseases, including Fuchs’ endothelial dystrophy.115 EK pro-
vides distinct advantages over PK, in that it is a less invasive procedure and
leads to more rapid recovery of vision. Additionally, this procedure does not
require long-term corneal sutures, eliminating problems with suture break-
age, suture abscesses, astigmatism, and wound dehiscence. Disadvantages
of EK include the need for specially prepared donor tissue and additional
surgeon training or experience. Patients with significant corneal scarring and
vascularization (Stage 4) may also not be amenable to DSAEK and require a
full thickness graft.
Refractive ametropia and astigmatism are reported to be significantly
less in Descemet’s stripping automated endothelial keratoplasty (DSAEK)-
treated eyes than in PK-treated eyes, even after suture removal and arcuate
keratotomy.116 DSAEK seems to be superior to PK in treating Fuchs’ endothe-
lial keratoplasty in terms of faster visual recovery and decreased dependence
on contact lenses and glasses. DSAEK, however, has a significant learning
Chapter_12.indd 252 06-02-2018 21:56:30

curve and may have initial endothelial cell loss greater than the conventional
full thickness graft.117
Cataract extraction may be required in many of the patients of FCD. Antic-
ipating the correct intraocular lens power for a patient undergoing cataract
surgery alone followed by DSAEK or combined cataract surgery with DSAEK
requires understanding the hyperopic shift that can occur with DSAEK and
incorporating this correction preoperatively in the intraocular lens power
selection.118
Descemet’s membrane endothelial keratoplasty (DMEK) has been
reported to effectively restore physiologic pachymetry and clarity, but donor
preparation and attachment currently are more challenging than with
DSAEK.119 This limits the widespread acceptance of the DMEK procedure
over the currently popular DSAEK.
Posterior Polymorphous Corneal Dystrophy
Alternative name: Schlichting dystrophy.120

In PPCD, dystrophic endothelial cells acquire epithelial characteristics,
leading to secondary abnormalities in the Descemet’s membrane.121
Inheritance
Autosomal dominant inheritance with low penetrance and variable expres-

sion is seen.122-126 Isolated unilateral cases have been reported, with similar
phenotype but no heredity.
PPCD 1: Genetic locus isolated to 20p11.2–q11.2; gene remains unknown.
PPCD 2: G enetic locus mapped to 1p34.3–p32.3; gene collagen type 8 alpha 2,
COL8A2 is involved.
PPCD 3: Genetic locus localized to 10p11.2; gene two-handed zinc-finger
homeodomain transcription factor 8 is implicated.
Associations
Associations with Alport syndrome, keratoconus and keratoglobus have been

reported.127-129
Symptoms
Endothelial alterations often asymptomatic and are noted on slit lamp exam-
ination. Endothelial changes often unchanged over years. Rarely exten-
sive and progressive visual impairment may occur due to stromal clouding
(Fig. 12.15). Vision loss may also be secondary to iridiocorneal adhesions and
glaucoma.
Signs
Disease onset is in childhood and involvement is often asymmetric. Deep

corneal lesions of various shapes including nodular, vesicular (isolated,
Chapter_12.indd 253 06-02-2018 21:56:30

Fig. 12.15: Posterior polymorphous membranous dystrophy.
Fig. 12.16: Stromal edema in a case of Posterior polymorphous membranous dystrophy.
in clusters, or confluent) and blister-like lesions are observed.130 Stromal

and epithelial edema due to endothelial decompensation may be seen
(Fig. 12.16). Peripheral iridocorneal adhesions have been reported in about
25% of cases, and elevated intraocular pressure in 15% of cases.130 Lipid
deposition or band keratopathy have been reported in advanced cases. A
congenital variant may manifest with corneal edema at birth.
Chapter_12.indd 254 06-02-2018 21:56:30

Histopathology
‘Epithelialization’ of endothelial cells occurs and they are seen to acquire

characteristics like cytoplasmic keratin, desmosomal junctions and micro-
villi. Metaplastic fibroblast-like endothelial cells have also been reported.131
The altered endothelial cells are capable of migrating over the trabecular
meshwork, thereby producing glaucoma. The posterior nonbanded layer of
the Descemet’s membrane is abnormal in cases of PPMD, thickened with
deposition of abnormal collagen. Rare cases of the congenital variant may
involve the anterior banded layer. Stromal and epithelial changes may occur
secondary to edema due to endothelial dysfunction.
Confocal microscopy demonstrates vescicular and linear abnormalities
along with bright nucleus like structures in the endothelial cells.132,133 Vesic-
ular lesions appear as rounded dark areas with some cell detail apparent in
the middle giving a doughnut-like appearance. Railroad track appearance of
band-like dark area with irregular edges enclosing some smaller lighter cells
resembling epithelium-like cells may be noted.
Management
Most cases are asymptomatic and may not require any treatment. Corneal
transplantation may be required in severe cases. In a series of 120 cases of
PPMD, 10% were reported to require a corneal graft. Coexisting glaucoma
and iridiocorneal adhesions may compromise graft survival in these cases.
Congenital Hereditary Endothelial Dystrophy 1
CHED type 1 is characterized by bilateral, diffuse, noninflammatory corneal

edema with markedly increased corneal thickness. It has not been consis-
tently associated with any systemic disease.
Inheritance
Autosomal dominant inheritance with genetic locus at 20p 11.2–q11.2 (peri-

centromeric region) is seen. The gene involved remains to be isolated.134
Signs
Children affected with CHED1 have clear corneas at birth. Corneal clouding
with a diffuse haze begins from the first to second year of life and progresses
to a ground-glass appearance over 5–10 years. Thickening of the cornea can
be upto 2–3 times normal thickness. The corneal opacification extends upto
the limbus and there are no interevening clear areas (Fig. 12.17). Bullae are
unusual despite the extensive stromal edema, unlike in cases with FCD.135
Coexisting congenital glaucoma and band keratopathy have also been
reported.136
Chapter_12.indd 255 06-02-2018 21:56:30

Fig. 12.17: Milder form of congenital hereditary endothelial dystrophy (CHED). The
hazy cornea does not preclude iris details completely.
Symptoms
Early symptoms include photophobia and tearing. Progressive corneal cloud-

ing leads to blurred vision. Visual acuity in cases of CHED1 tends to be better
than in CHED2 and nystagmus is absent.137
Histopathology
Diffuse thickening and lamination of Descemet membrane and atrophic

endothelial cells are seen on light microscopy. Stromal thickening is associ-
ated with disorganized arranged of the collagen fibers with disruption of the
lamellar pattern.135 Subepithelial fibrosis has been noted in some cases of
CHED1.
Congenital Hereditary Endothelial Dystrophy 2
Alternative Name: Maumenee corneal dystrophy.138
Inheritance
Autosomal recessive inheritance pattern is seen. Genetic locus has been

localized to 20p13 (telomeric portion); the Solute carrier family 4, sodium
borate transporter, member 11 gene has been implicated.139,140
Signs and Symptoms
This form is more severe than CHED1 with onset at birth or in the neonatal
period. Corneal clouding is dense at the time of presentation and does not
Chapter_12.indd 256 06-02-2018 21:56:30

Fig. 12.18: Diffuse corneal clouding (ground glass appearance) in a case of congenital
hereditary endothelial dystrophy (CHED).
tend to progress (Fig. 12.18). Nystagmus is usually present, possibly second-

ary to the severe visual impairment.137 Congenital glaucoma may be coex-
isting but falsely elevated intraocular pressure measurement due to stromal
edema may lead to misdiagnosis.136,141 Band keratopathy and subepithelial
amyloidosis has been reported in a few cases.142-144
Histopathology
Diffuse thickening and lamination of Descemet membrane with deposition

of multiple layers of basement membrane-like material on the posterior part
of Descemet membrane has been noted. Endothelial cells appear atrophic
and may be absent.
Management of CHED
Corneal transplantation remains the definitive treatment for CHED. Pediat-

ric keratoplasty, however, is a surgically challenging procedure and demands
intensive postoperative monitoring and therapy. The timing of the surgery
remains controversial in view of the possible challenges and complications.
While some recommend early surgery, others suggest delaying the proce-
dure as long as the patient demonstrates good fixation and normal align-
ment.145,146
Schaumberg et al. reported 2 year survival rate of first grafts performed
for CHED at a median age of 40 months to be 71%.147 However, just four of
the 10 eyes attained a visual acuity of 20/200 or better. They concluded that
PK for CHED in children has a reasonable chance of surgical success when
Chapter_12.indd 257 06-02-2018 21:56:30

performed at a young age; however, the prognosis for improved visual acuity
in children appears to be more guarded. Javadi et al. reported the absence of
a relationship between postoperative visual outcome and age at keratoplasty,
and advocated a conservative approach and careful risk-benefit ratio evalua-
tion in patients with CHED.148
DSAEK remains a theoretical option in the treatment of CHED but tech-
nical difficulties during Descemet’s membrane scoring and stripping and
poor visualization have been reported to prevent successful completion of
the procedure.149
X-linked Endothelial Corneal Dystrophy
Inheritance
X-chromosomal dominant inheritance with genetic locus at Xq25 has been

reported, the involved gene remains unknown.150
Symptoms
Males may complain of progressive blurred vision while females are asymp-
tomatic.
Signs
Congenital clouding ranging from a diffuse haze to a ground-glass, milky

appearance have been reported in male patients with advanced involve-
ment.150 Moon crater-like endothelial changes have been observed in both
males and females.
Histopathology
Light microscopy reveals irregular thickening of Descemet membrane with
small excavations and pits (moon crater endothelial changes).150
Management
Corneal transplantation may be required in advanced cases.
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study of 64 families. Arch Ophthalmol. 1978;96(11):2036-9.
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107. Hogan MJ, Wood J, Fine M. Fuchs’ endothelial dystrophy of the cornea. Am J
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110. Sharma N, Prakash G, Sinha R, et al. Indications and outcomes of photo-

therapeutic keratectomy in the developing world. Cornea. 2008;27(1):
44-9.
111. Pires RT, Tseng SC, Prabhasawat P, et al. Amniotic membrane transplanta-
tion for symptomatic bullous keratopathy. Arch Ophthalmol. 1999; 117(10):
1291-7.
112. Alino AM, Perry HD, Kanellopoulos AJ, et al. Conjunctival flaps. Ophthalmolo-
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113. Zemba M. Palliative treatment in bullous keratopathy. Oftalmologia. 2006;
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114. Chung SH, Kim HK, Kim MS. Corneal Endothelial Cell Loss after Penetrating
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115. Rose L, Kelliher C, Jun AS. Endothelial keratoplasty: historical perspec-
tives, current techniques, future directions. Can J Ophthalmol. 2009;44(4):
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117. Price MO, Gorovoy M, Benetz BA, et al. Descemet’s Stripping Automated Endo-
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118. Eghrari AO, Daoud YJ, Gottsch JD. Cataract surgery in Fuchs corneal dystro-
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13
CHAPTER
Management of Keratoconus
Prema Padmanabhan, Nidhi Gupta
INTRODUCTION
The therapeutic options in the management of keratconus have increased in
the last few decades. The problems in keratoconus which need to be addressed
stem from two important characteristics of the disease: first, the progressive
nature of keratoconus and second, the poor corneal optics associated with it.
Until as recently as the beginning of the present century, it was only the opti-
cal effects of keratoconus that were treated and the disease itself was allowed
to worsen until it needed a corneal transplant. With a better understanding of
the biomechanics of the cornea and etiopathogenesis of keratoconus, a more
systematic and targeted approach to treat it is now possible.
The treatment options for keratoconus are:
Contact Lenses
Contact lenses have been the mainstay of treatment for keratoconus, provid-
ing a uniform refractive surface and improving the vision quality. Although,
there have been improvements in lens materials and designs, contact lens
fitting in patients with keratconus is still a challenge. Commonly used contact
lenses include soft toric lens, and rigid gas permeable lens. Some lens sys-
tems are specially designed for keratconus like the Rose K lens, the piggy back
lens system, the soft perm hybrid lens. Scleral lenses like the Boston scleral
contact lens has also proved useful in eyes with irregular astigmatism, when
other lenses have failed.
The choice of contact lens often depends on the severity of kerataconus.
Soft toric lenses are usually adequate for milder forms of the disease. As the
disease progresses, more complex, rigid gas permeable lenses like the multi-
curve and biaspheric lenses may be tried. A hybrid lens, with a rigid central
Chapter_13.indd 268 06-02-2018 22:16:51

Management of Keratoconus 269
optic and a soft hydrophilic peripheral skirt is also a popular design (e.g.,
SynergEyes, Ca, USA). This combination combines the quality of vision pro-
vided by a rigid lens with the comfort of a soft contact lens
There are, broadly speaking, three approaches to rigid contact lens fitting
for keratoconus:
1. ‘The 3-point touch’ is the classical design used to fit contact lenses for
keratoconus, especially when the cone is centrally located. The 3-point
touch refers to the support provided for the lens by an area of central
bearing and two other areas at the corneal mid periphery. The apical
bearing should not exceed 2–3 mm because of the risk of corneal ero-
sions and scarring.
2. The apical clearance designs, on the other hand, vault over the corneal
vertex and are suited for small central or slightly paracentral cones.
3. Large and flat lens designs are required for eyes with displaced apexes. In
general, lower the apex, larger the lens diameter required for centration.
Multiple trial lens fittings may be required before the right choice of con-
tact lens with the optimal fit is obtained.
While contact lenses are useful in trying to improve vision, they also carry
the potential risk of damage to the cornea—whether by inducing corneal
hypoxia and oxidative stress or by entrapment of biodebris and tears. The
abrasive potential of contact lenses could lead to epitheliopathy, especially if
the lubricating effect of the tears is inadequate. Apical contact could be asso-
ciated with epithelial trauma and apical scarring.
Newer modalities of custom wavefront guided soft contact lenses have
been tried in an attempt to decrease the higher order aberrations associated
with a distorted cornea.1
The Boston scleral lens prosthetic devices are fluid ventilated rigid gas
permeable lenses with diameters ranging from 14.5–24 mm which cover the
limbus and create an expanded precorneal tear film over the ocular surface.
These have also proved useful in eyes with irregular astigmatism.2
Intracorneal Ring Segments
These are C-shaped PMMA implants meant to be inserted into pockets cre-
ated in the deep stroma of the mid peripheral cornea. The creation of the
scleral pocket can be done by specially designed dissectors or more accurately
and predictably by the femtosecond laser.3 Originally designed to correct low
grades of myopia, they are used in ectatic corneal disorders to produce cor-
neal flattening. The degree of corneal flattening is a function of ring thickness
and diameter. However, no accurate mathematical relationship has been
worked out so far to be able to predict the effect of these ring segments on the
curvature or refraction of the cornea. Several nomograms have been proposed
by different authors, some based on refraction, others on topographic profile.
The mean keratometric change has been reported to vary from 2 D to
9 D4,5 with most studies reporting more than 50% of patients experiencing
Chapter_13.indd 269 06-02-2018 22:16:51

an improvement in their best corrected visual acuity (BCVA).6 The intracor-

neal ring segments (ICRS) implantation is also useful in improving contact
lens tolerance in patients whose cornea was so steep that contact lenses were
too unstable for their comfort.7
Postoperative complications include ring segment extrusion or migra-
tion, corneal vascularization or infiltrates along the channel.
ICRS implantation has been used successfully in conjunction with col-
lagen cross linking.8 The main advantages of the ICRS include the fact that
there is no removal of corneal tissue (unlike laser techniques) and that the
changes they induce are reversible when removed.
Corneal Collagen Cross-linking
Recent studies have begun to unravel the biochemical basis for some of the
morphological changes seen in keratoconus. There is a distinct thinning of
collagen fibrils and an alteration of the normal configuration of the lamellae.
An increased expression of proteolytic enzymes, including matrix metallopro-
teinases (MMP), 1, 2 and 13, which are known to cleave the collagen molecule
have been noted. There is also an impairment of MMP 8 which participates
in collagen remodeling and a decrease in the protease inhibitors. All these
factors explain why the stiffness of keratoconic cornea is only about 60% of
normal. Therefore, collagen becomes the target of therapeutic attention. Col-
lagen is an insoluble extracellular glycoprotein that accounts for 71% of the
dry weight of the cornea and is the major stress-bearing component of the
cornea. It is made of 3 polypeptides twisted into a left-handed triple heptix,
called tropocollagen, which is the precursor of collagen. Non-disulfide cross-
links among strips of tropocollagen form collagen fibers. Wollensak and co-
workers9 reported using ultraviolet-A (UV-A) radiation combined with ribofla-
vin to increase the covalent bonds between collagen fibers.
Riboflavin acts a photosensitizer, it absorbs energy and converts from its
ground state to its excited singlet or triplet states. The triplet state binds to the
substrate to produce substrate free radicals, which react further with molec-
ular oxygen to form superoxide anions and singlet oxygen. These singlet oxy-
gen radicals produce covalent bonds between the aldehyde and amino ends
of collagen fibers.
After extensive laboratory and animal studies, a standard protocol for the
clinical application of collagen cross-linking (CXL) has been advocated.10,11
After de-epithelialization of the central 7 mm of the cornea, the stroma is sat-
urated with 0.1% riboflavin in a 20% dextran solution (which typically takes
30 mts) and the cornea then exposed to UV-A radiation with a wavelength
of 370 nm for 30 minutes. The surface irradiance of 3 mw/cm2 limits the
keratocyte death to a depth of approximately 300 m. Experimental evidence
and theoretical constructs infer that this energy will not damage the corneal
endothelium or crystalline lens, if the cornea is more than 400 m thick.10,11
The rigidity of corneal tissue, as expressed by the Young’s modulus, has
Chapter_13.indd 270 06-02-2018 22:16:51

been shown to increase by a factor of 4.512 and to be confined to the anterior

200 m of stoma. In addition, CXL has been shown to increase stromal shrink-
ing temperatures13 and to increase resistance to enzymatic digestion.14
Clinical studies also support these observations. In the largest published
series of 241 eyes, followed up for up to 6 years after CXL, the study found
statistically significant improvements in astigmatism, BCVA and maximum
simulated keratometry values at 12 months. A mean flattening of 1.91 D was
noted among 54% of eyes.15 The only randomized controlled trial published
so far also shows statistically significant differences between the treatment
and control groups in terms of steepest K and BCVA in up to a12 months fol-
low up period.16
Improvement in visual acuity could be attributed to a flattening of the
cornea, a reduction in the magnitude of astigmatism improved symmetry
and homogeneity of the corneal surface and a reduction in higher order aber-
rations.
Although, the procedure has proved its efficacy and safety, the potential
for adverse outcome must not be overlooked. It would be prudent to ensure
that the minimum corneal thickness is at least 400 m. This is best assessed
by elevation mapping using the computerized videokeratograph rather than
an ultrasonic pachymeter which may miss the steepest location. In patients
with a corneal pachymetry slightly less than 400 m, the use of hypoosmolar
solutions of riboflavin has been tried to swell the corneal stroma to more than
400 m before proceeding with the UV-A radiation.17 It is difficult to be certain,
however, that the over-hydrated corneal stroma will respond similarly to the
CXL procedure and that this modification to the technique affords adequate
endothelial protection.18 There is also the potential risk of all the complica-
tions associated with the creation of an epithelial defect—delayed healing,
sterile or infections infiltrates. Although most clinical studies report stabiliza-
tion if not an actual improvement in the severity of the keratoconus. Koller et
al.19 documented a progression of the disease in 7.6% and a loss of 2 or more
lines of BCVA in 2.9% of eyes, 1 year after CXL.
Surgical Management of Keratoconus
Contact lens failure or intolerance and visual impairment because of central

corneal scarring are indications for surgical intervention to restore corneal
anatomy and improve vision quality. At this time, keratoconus remains a
leading indication for corneal grafting in developed countries.20,21
Traditionally, full thickness corneal transplantation procedure has been
commonly performed for advanced keratoconus. Penetrating keratoplasty
(PK) is a safe and effective treatment for this condition with excellent graft
survival rates of over 90% even after 5–12 years.22-24 Recovery of visual acuity
was good,25 reporting 91.5% of patients achieving a BCVA of 20/40 or better.
Although, PK has successful outcomes, it breaches the structural and immu-
nological integrity of the eye and sacrifices the healthy endothelium of the
Chapter_13.indd 271 06-02-2018 22:16:51

host cornea. The transplanted allograft is at risk for rejection, which leads
to concomitant endothelial cell loss with subsequent risk of graft failure. A
rejection rate of 4–30% has been reported between 3 and 10 years after PK
for keratoconus with most of them occurring in the first 2 years.26,27 In addi-
tion, continuous attrition of corneal endothelium following PK is reported to
occur with a rate several times higher than in the normal population.28
Lamellar keratoplasty (LK) aims to selectively replace the abnormal lay-
ers of corneal stroma while preserving the host’s healthy endothelium. This
eliminates the risk of endothelial graft rejection and has been reported to
have minimal detrimental effect on endothelial cell count. Deep anterior
lamellar keratoplasty (DALK) is an almost full thickness LK where the host
bed is dissected up to the Descemet’s membrane. However, allograft rejection
in the form of epithelial and stromal rejection may occur in LK, occurring in
3%–8% of patients with keratoconus. DALK requires a shorter postoperative
regimen of steroid than PK, which leads to lower rates of glaucoma and cat-
aract. DALK also avoids most of the complications associated with intraoc-
ular surgery especially with an open-sky approach, such as anterior uveitis,
peripheral synechiae, secondary glaucoma, endophthalmitis, and expulsive
hemorrhage.
Preference for PK in the last century was mainly because of the technical
difficulties and poor visual outcomes resulting from lack of quality and reg-
ularity of interface with different LK techniques. LK for keratoconus was first
suggested by Enrique Malbran in 1964. Several variations in the technique
have been described29,30 but the Big Bubble technique of Anwar31 is consid-
ered the most elegant one, with the most consistent results. Although, some
studies indicated that the visual outcomes after DALK are comparable with
those after PK, several other studies have documented that it is inferior to PK
in terms of BCVA. This difference is most often technique dependent and can
invariably be attributed to the irregularity at the host donor interface limit-
ing the vision after LK. Should the interface between the host and donor be
smooth and the corneal stroma be removed down to the DM, as with the big
bubble technique, the optical quality of the interface is excellent.
The correction of irregular astigmatism has been one of the challenges in
the management of keratoconus. Whether due to the primary condition or
the result of keratoplasty, the restoration of good functional vision in these
patients has been elusive. While gas permeable contact lenses can be used in
mild or moderate cases of keratoconus and in some cases following kerato-
plasty, they have their limitations.
The application of customized topography-guided laser ablations has
been an interesting application of the excimer laser in recent times. Ker-
atoconus has always headed the list of absolute contraindications for laser
refractive surgery, because the tissue removal accompanying the ablative
procedure would further destabilize the already weak biomechanics of the
cornea. However, the combination of the ablative procedure with corneal
collagen cross-linking is now looked upon as a possible approach to improve
Chapter_13.indd 272 06-02-2018 22:16:52

the corneal contour and, therefore, the quality of vision and to concurrently
strengthen the residual stroma with a cross-linking procedure.
A major consideration in planning combined approach is the estimation
of the postablative, precross-linking pachymetry which needs to be more
than the stipulated limit of 400 m.
Patients treated with simultaneous topography-guided PRK with CXL
have been shown to have a significant improvement in uncorrected and
BCVA.32,33 More importantly, their topography reflected the improvement in
regularity, symmetry and contour of the cornea. It is, of course, very import-
ant to restrict this treatment to carefully selected eyes and limit the abla-
tive procedure to permissible levels. The objective of this approach is not a
refractive correction but a topographic improvement with a biomechanical
stabilization.
CONCLUSION
In conclusion, the methods to diagnose and techniques to treat keratoconus
have vastly improved in recent times. Not only can patients of keratoconus
hope for a better quality of vision, there is also strong evidence that the pro-
gression of the condition can now be effectively arrested.
REFERENCES
1. Marsack JD, Parker KE, Niu Y, et al. On-eye performance of custom wave-
front-guided soft contact lenses in a habitual soft lens-wearing kerato-
conic patient. J Refract Surg. 2007;23(9):960-4.
2. Jacobs DS. Update on scleral lenses. Curr Opin Ophthalmol. 2008;19(4):
298-301.
3. Ertan A, Kamburoğlu G. Intacs implantation using a femtosecond laser for
management of keratoconus: comparison of 306 cases in different stages. J
4. Coskunseven E, Kymionis GD, Tsiklis NS, et al. One-year results of intrastromal
conreal ring segment implantation (keraring) using femtosecond laser in pa-
tients with keratoconus. Am J Ophthalmol. 2008;145(5):775-9.
5. Zare MA, Hashemi H, Salari MR. Intracorneal ring segment implantation for
the management of keratoconus: safety and efficacy. J Cataract Refract Surg.
2007;33(11):1886-91.
6. Piñero DP, Alio JL. Intracorneal ring segments in ecstatic corneal disease—a
review. Clin Exp Opthalmol. 2010;38(2):154-67.
7. Alió JL, Shabayek MH, Artola A. Intracorneal ring segments for keratoco-
nus correction: long-term follow-up. J Cataract Refract Surg. 2006;32(6):
978-85.
8. Chan CC, Sharma M, Wachler BS. Effect of inferior-segment intacs with and
without C3-R on keratoconus. J Cataract Refract Surg. 2007;33(1):75-80.
9. Wollensak G. Cross-linking treatment of progressive keratoconus: new hope.
Curr Opin Ophthalmol. 2006;17(4):356-60.
Chapter_13.indd 273 06-02-2018 22:16:52

10. Wollensak G, Spörl E, Reber F, et al. Corneal endothelial cytotoxicity of ribofla-

vin/UVA treatment in vitro. Ophthalmic Res. 2003;35(6):324-8.
11. Spoerl E, Mrochen M, Sliney D, et al. Safety of UVA-riboflavin cross-linking of
the cornea. Cornea. 2007;26(4):385-9.
12. Wollensak G, Spoerl E, Seiler T. Stress-strain measurements of human and por-
cine corneas after riboflavin-ultraviolet-A-induced cross-linking. J Cataract Re-
fract Surg. 2003;29(9):1780-5.
13. Spoerl E, Wollensak G, Dittert DD, et al. Thermomechanical behavior of
collagen-cross-linked porcine cornea. Ophthalmologica. 2004;218(2):
136-40.
14. Spoerl E, Wollensak G, Seiler T. Increased resistance of crosslinked cornea
against enzymatic digestion. Curr Eye Res. 2004;29(1):35-40.
15. Raiskup-Wolf F, Hoyer A, Spoerl E, et al. Collagen crosslinking with riboflavin
and ultraviolet-A light in keratoconus: long-term results. J Cataract Refract
Surg. 2008;34(5):796-801.
16. Wittig-Silva C, Whiting M, Lamaoureux E, et al. A randomized controlled
trial of corneal collagen cross-linking in progressive keratoconus: preliminary
results. J Refract Surg. 2008;24(7):S720-5.
17. Hafezi F, Morchen M, Iseli HP, et al. Collagen crosslinking with ultravi-
olet-A and hypoosmolar riboflavin solution in thin corneas. J Cataract
Refract Surg. 2009;35(4):621-4.
18. Snibson GR. Collagen cross-linking: a new treatment paradigm in corneal
disease—a review. Clin Exp Opthalmol. 2010;38(2):141-53.
19. Koller T, Mrochen M, Seiler T. Complication and failure rates after corneal cross-
linking. J Cataract Refract Surg. 2009;35(8):1358-62.
20. Paglen PG, Fine M, Abbott RL, et al. The prognosis for keratoplasty in keratoco-
nus. Ophthalmology. 1982;89(6):651-4.
21. Pramanik S, Musch DC, Sutphin JE, et al. Extended long-term outcomes
of penetrating keratoplasty for keratoconus. Ophthalmology. 2006;113(9):
1633-8.
22. Ing JJ, Ing HH, Nelson LR, et al. Ten-year postoperative results of penetrating
keratoplasty. Ophthalmology. 1998:105(10):1855-65.
23. Sit M, Weisbrod DJ, Naor J, et al. Corneal graft outcome study. Cornea.
2001;20(2):129-33.
24. Thompson RW Jr, Price MO, Bowers PJ, et al. Long-term graft survival after pen-
etrating keratoplasty. Ophthalmology. 2003;110(7):1396-1402.
25. Fukuoka S, Honda N, Ono K, et al. Extended long-term results of penetrating
keratoplasty for keratoconus. Cornea. 201;29(5):528-30.
26. Sharif KW, Casey TA. Penetrating keratoplasty for keratconus: complications
and long-term success. Br J Ophthalmol. 1991;75(3):142-6.
27. Olson RJ, Pingree M, Ridges R, et al. Penetrating keratoplasty for keratoco-
nus: a long-term review of results and complications. J Cataract Refract Surg.
2000;26(7):987-91.
28. Patel SV, Hodge DO, Bourne WM. Corneal endothelium and postopera-
tive outcomes 15 years after penetrating keratoplasty. Am J Ophthalmol.
2005;139(2):311-9.
Chapter_13.indd 274 06-02-2018 22:16:52

29. Archila EA. Deep lamellar keratoplasty for dissection of host tissue with
intrastromal air injection. Cornea. 1984-1985;3(3):217-8.
30. Amayem AF, Anwar M. Fluid lamellar keratoplasty in keratoconus. Ophthalmol-
ogy. 2000;107(1):76-9.
31. Anwar M, Teichmann KD. Big-bubble technique to bare Descemet’s membrane
in anterior lamellar keratoplasty. J Cataract Refract Surg. 2002;28(3): 398-403.
32. Kymionis GD, Kontadakis GA, Kounis GA, et al. Simultaneous topography-
guided PRK followed by corneal collagen cross-linking for keratoconus. J Re-
fract Surg. 2009;25(9):S807-11.
33. Kanellopoulos AJ. Comparison of sequential vs same-day simultaneous colla-
gen cross-linking and topography-guided PRK for treatment of keratoconus. J
Refract Surg. 2009;25(9):S812-8.
Chapter_13.indd 275 06-02-2018 22:16:52

14
CHAPTER
Corneal Collagen Cross-linking
Ashok Garg
INTRODUCTION
In last one decade, corneal refractive surgery has advanced rapidly with
excellent visual results worldwide. Refractive surgeons have come across
the problem of post-refractive keratectasia or corneal ectasia. Due to effect
of excimer laser photoablation on the corneal biomechanical properties, a
significant decrease in the biomechanical assets was found after surgery. This
implies that due to creation of flap and subsequent corneal thinning by abla-
tion weakens the cornea and decreases its elastic properties. This leads later
to corneal ectasia. This is the indicator for the clinical significance of evalu-
ating corneal biomechanical properties, specifically in the cornea hysteresis
and becomes the resistance factor in screening of refractive surgery patients.
Similarly, in keratoconus (a progressive noninflammatory cone like ectasia),
the corneal hysteresis (CH) and corneal resistance factor (CRF) are signifi-
cantly lower than in the normal eyes and post-LASIK surgery, corneas low
values of CH means that the cornea is less capable of absorbing the energy of
the air pulse whereas low values of CRF indicates that corneal rigidity is lower
than normal. The corneal biomechanical properties are primarily deter-
mined by the collagen fibers and the degree of interfibrillar linkage. Corneal
ectatic conditions, whether inflammatory or noninflammatory, have weak
interfibrillar linkage strength.
WHAT IS CROSS-LINKING?
Cross-linking of human collagen is a physiologic process. Corneal collagen
cross-linking, also known as C3-R/CCL/CxL treatment, is a new approach
to increase the mechanical and chemical stability of corneal tissue. The pri-
mary aim of this treatment is to create additional chemical bonds inside the
Chapter_14.indd 276 06-02-2018 22:25:20

Corneal Collagen Cross-linking 277
corneal stroma by means of a highly localized photopolymerization while

minimizing exposure to the surrounding structure of the eye. This procedure
was first developed by Theo Seile Wollensak and Eberhard Spoerl in 1998 at
the University of Dresdan, Germany. They did this procedure in cases of pro-
gressive keratoconus and post-refractive corneal ectasia. Followed this, other
studies were undertaken by Caporossi, Roberto Pinelli and their colleagues
in Italy and Brian Boxer in the USA.
There are several techniques of cross-linking. The most promising tech-
nique in cornea is use of ultraviolet (UV) light and riboflavin (Vitamin B2
solution) for inducing cross-linking to increase biomedical rigidity of the cor-
nea. This slows down or even stops the progressive thinning of the cornea.
The photopolymerization is performed by means of a nontoxic and soluble
photomediator (riboflavin) and a wavelength, which is absorbed strongly
enough to protect deep layers of the eye (Riboflavin-UVA technique).
PHYSIOLOGY OF CORNEAL COLLAGEN CROSS-LINKING

In this procedure, custom-made riboflavin eye drops are applied to the cor-
nea, which is then activated by UV light. Using UVA at 370 nm, the photosen-
sitizer riboflavin is excited into its triplet state and thus generating reactive
oxygen species (ROS), which is mainly singlet oxygen, and to a much less
degree of superoxide anion radicals. The ROS can react further with various
molecules including chemical covalent bonds bridging amino groups col-
lagen fibrils/type 2 photochemical reaction (Figs. 14.1 and 14.2). The wave-
length of 370 mm of UVA is chosen because of an absorption peak of riboflavin
at this wavelength. Biomechanical studies have shown an increase in the
Fig. 14.1: Bonding tissues and cross-linking.
Chapter_14.indd 277 06-02-2018 22:25:20

Fig. 14.2: Strengthening of corneal fibers by C3-R treatment.
Fig. 14.3: UV-Xtm illumination system.
corneal rigidity of 328.9% in human cornea after cross-linking (Fig. 14.3).

The increase on biomechanical rigidity after C3-R is probably caused by an
increase in the collagen fiber diameter due to interfibrillar and intrafibrillar
covalent bonds by photosensitized oxidative cross-linking. The cross-linking
results in more compact stronger corneas are more resistant to biomechani-
cal deformation or ectasia.
Indications for Corneal Cross-linking Treatment
• Progressive keratoconus
• Iatrogenic post-refractive keratectasia (post-LASIK ectasia)
• Pellucid marginal degeneration
Exclusion Criteria
• Corneal thinness less than 400 mm at thinnest position

• Keratometric readings above 60 D
Chapter_14.indd 278 06-02-2018 22:25:20

• Active ocular disease

• Herpes keratitis
• Diabetes
• Pregnancy
• Previous ocular surgery other than laser refractive surgery
• Immunocompromised patients
• Patients with known sensitivity
Parameters for Cross-linking Treatment

• Disorder should be progressive in nature
• Thinnest corneal pachymetry higher than 400 mm
• No central corneal scarring
• Maximum corneal curvature should not exceed 60 D
Preoperative Work-up
• Visual acuity assessment (UCVA, BCVA, contrast sensitivity)

• Intraocular pressure recording
• Detailed slit-lamp examination specially for Vogt’s striae, Fleischer’s ring
and corneal scarring
• Slit-lamp photographs of corneal changes
• Pentacam evaluation for central corneal thickness and thinnest pachy-
metry
• Corneal topography
• Optical coherence tomography (OCT) examination
Steps of Corneal Cross-linking Technique
The procedure takes place ambulatory and takes about 1 hour (Fig. 14.4).
• First eye is anesthetized with topical proparacaine 0.5% eye drops. Then
manually debride the corneal epithelium (thin surface layer). It is
abraded in the central 7 mm of cornea in order to allow penetration of
stroma. Riboflavin solution, containing 0.1% riboflavin and 20% dextran
T-500 in isotonic sodium chloride solution (pH 7.0), is applied every 3
minutes for the first 30 minutes. This is followed by irradiation of cornea
with 365 nm UVA using UV-Xtm for 30 minutes. Riboflavin drops are then
continued for another 30 minutes at an interval of every 5 minutes as
the eye is exposed to a UVA light positioned above the cornea to deliver
predetermined dose of UVA light. The distance between the UV delivery
system and cornea should be 5 cm (50 mm) so as to deliver a dose of
3 mw/cm2 (total of 5.4 J/cm2 in 30 minutes). As the UVA light interacts
with the riboflavin chemical bonds (cross-links) between the corneal
collagen molecules and makes the cornea stiffer. As a result, the corneal
collagen tissue is stronger and can more uniformly retain its natural
Chapter_14.indd 279 06-02-2018 22:25:20

Fig. 14.4: Corneal collagen cross-linking (controlled UVA radiation is applied to corneal
stroma to stiffen the cornea).
curved shape rather than bowing forward into the cone-like shape,
which is the hallmark of keratoconus and corneal ectasia. At the end of
treatment, the cornea is flushed with BSS and a bandage contact lens is
placed over the cornea.
Postoperative Follow-up
Patient is prescribed topical antibiotic drop, nonsteroidal anti-inflammatory

drop and lubricating eye drop in the postoperative period. Eye may be lit-
tle painful after the treatment and pain may disappear after 48 hours. Until
the closure of epithelial defect, the patient is followed-up every day. Bandage
contact lens is taken off when epithelial defect heals completely. Subsequent
follow-up should be taken at 1 week, 4 weeks, 12 weeks, 24 weeks and 1 year
respectively. On each follow-up refraction, keratometry, slit-lamp and penta-
cam examinations are mandatory. OCT is done during 1 month and subse-
quent visits on usual basis.
FUTURE PROSPECTS
Corneal collagen cross-linking with riboflavin and UVA for the treatment of
progressive keratoconus and post-refractive keratectasia is relatively safer
and effective. The ability to permanently strengthen the inherently weak
cornea is a major advancement and achievement of this technique. C3-R
treatment alone or combined with intacs implantation in keratoconus are
allowing improved vision and comfort to the patients. C3-R is a simple, safe
and effective procedure in the management of progressive ectatic disorders
of the cornea. It will be a standard treatment in near future.
Chapter_14.indd 280 06-02-2018 22:25:20

Several clinical research works are going on for the further improvement
and wider applications of this treatment. New clinical research works have
started for possible combining of C3-R treatment with topography-guided,
advanced surface ablation, intacs implantation, orthokeratology and con-
ductive keratoplasty. Possible C3-R treatment applications in treating corneal
edema, bullous keratopathy, corneal decompensation and corneal melting
are also being investigated with a lot of hope and promise.
BIBLIOGRAPHY
1. Hadley JC, Meek KM, Malik NS. The effect of glycation on charge distribution
and swelling behavior of corneal and scleral collagen. Invest Opthalmol Vis Sci.
1996;37:1010.
2. Hafezi F, Kanellopoulos J, Wiltfang R, Seiler T. Corneal collagen crosslinking
with riboflavin and ultraviolet A to treat induced keratectasia after laser in situ
keratomileusis. J Cataract Refract Surg. 2007;33:2035-40.
3. Kohlhaas M, Spoerl E, Speck A, et al. Eine neue behandlung der keratektasie
nach LASIK durch kollagenvernetzung mit riboflavin/UVAlicht. Klin Monatsbl
Augenheilkd. 2005;22:430-6.
4. Kymionis DG, Bouzoukis DI, Diakonis VF, et al. Diffuse lamellar keratitis after
corneal cross-linking in a patient with post-laser in situ keratomileusis corneal
ectasia Cataract Refract Surg. 2007;33:2135-7.
5. Kymionis DG, Portaliou DM, Bouzoukis DI, et al. Herpetic keratitis with iritis af-
ter corneal crosslinking with riboflavin and ultraviolet A for keratoconus J Cat-
aract Refract Surg. 2007;33:1982-4.
6. Sady C, Hosrof K, Nagaraj RH. Advanced Maillard reaction and crosslinking
of corneal collagen in diabetes. Biochem Biophys Res Commun. 1995;214:
793-7.
7. Spoerl E, Huhle M, Seiler Th. Induction of cross links in corneal tissue. Exp Eye
Res. 1998;66:97-103.
8. Spoerl E, Mrochen M, Sliney D, et al. Safety of UVA–Riboflavin Cross—inking of
the Cornea. Cornea. 2007;26:385-9.
9. Wollensak G, Spoerl E, Seiler T. Riboflavin/ultraviolet-a-induced collagen
crosslinking for the treatment of keratoconus. Am J Ophthalmol. 2003;135:
620-7.
10. Wollensak G, Spoerl E, Wilsh M, et al. Keratocyte apoptosis after corneal colla-
gen cross-linking using riboflavin/UVA treatment. Cornea. 2004;23:43-9.
11. Wollensak G. Crosslinking treatment of progressive keratoconus: new hope.
Curr Opin Ophthalmol. 2006;17:356-60.
12. Zhao HR, Nagaraj RH, Abraham EC. The role of D- and e amino groups in
the glycation-mediated cross-linking of ãB-cristallin. J Biol Chem. 1997;272:
14465-9.
Chapter_14.indd 281 06-02-2018 22:25:20

15
CHAPTER
Intrastromal Corneal
Ring Segments
Shaila Patel, Soosan Jacob
INTRODUCTION
Intrastromal corneal ring segments (ICRS) are small pieces made of synthetic
material which are implanted in the deep corneal stroma to alter the corneal
curvature.1-7 Implantation of ICRS is a safe, reversible procedure that leaves
the central visual axis of the cornea unaltered and has been used to treat kera-
toconus,8,9 pellucid marginal degeneration,10 post-LASIK ectasia,11 post-PRK
ectasia12 and myopia.13-16
In general, different types of ICRS differ in diameter, design, optical zone
and arc length. These induce different effects: smaller the optical zone and
the greater the ring segment diameter, stronger the effect obtained and vice
versa.
HISTORICAL BACKGROUND
The concept of inserting polymethyl methacrylate (PMMA) segments as cor-
neal inserts was first introduced by Fleming in 1987.17,18 ICRS were initially
designed to correct low degree myopia by flattening the central corneal cur-
vature.14 Intacs was approved by the FDA for the correction of low myopia,
between 1 and 3 D in 1996.19-21 The first Intacs implantation for improving
visual acuity (VA) in a keratoconic patient was performed by Joseph Colin in
1997.8
TYPES OF ICRS
• Intacs (Addition Technology Inc.) and Intacs SK (Addition Technology
Inc.) (Fig. 15.1)
• Ferrara rings (Ferrara ophthalmics)
Chapter_15.indd 282 06-02-2018 22:32:17

Intrastromal Corneal Ring Segments 283
Fig. 15.1: Intacs.
• Kerarings (Mediphacos)
• Corneal allogenic intrastromal ring segments [(CAIRS)—Soosan Jacob]
The middle two types of ICRS comes in same design and diameter, but
are produced by two different companies.
STRUCTURE OF ICRS
IntacsÒ and Ferrara Ring segments are made of semicircular PMMA pieces.
Intacs are available in two broad categories: regular rings and severe kerato-
conus or steep keratometry (K)—SK rings, which are used for steeper corneas
based on the K readings on topography. Comparative structural characteris-
tics of different types of ICRS are described in Table 15.1.
MECHANISM OF ACTION
The corneal ring complies with Barraquer and Blavatskaya postulates, accord-
ing to which an addition in the corneal periphery results in its flattening, and
the ring diameter determines how much the cornea will be flattened. Thus,
more the tissue added (increasing ring thickness), and smaller the diameter,
greater will be the myopia correction obtained.22,23
INDICATIONS FORICRS
• Keratoconus
• High irregular astigmatism after penetrating or lamellar keratoplasty
• Myopia of 1 to 3 D
Chapter_15.indd 283 06-02-2018 22:32:17

Table 15.1: Comparative structural characteristics of different types of intracorneal ring

segments (ICRS).
Intacs regular Intacs SK

Characteristics segment segment Ferrar A rings Cairs
Shape Hexagonal Oval cross- Triangular Rectangular
transverse shape, section shape section
and a conical
longitudinal
section
Diameter External Internal Internal
diameter: diameter: diameter:
8.10 mm 6.0 mm; 4.40 mm
Internal diameter: Outer diameter: External

6.77 mm 7.0 mm diameter:
5.60 mm
Size 0.25–0.45 mm 0.21, 0.40, 0.45, 0.20–0.35 mm
thickness in 0.05 and 0.50 mm
mm increments thickness
Arc length 150 150 160
(degrees)
• Corneal ectasia after excimer laser

• Irregular astigmatism after radial keratotomy
CONTRAINDICATIONS FOR ICRS

• Highly advanced keratoconus with significant apical opacity and
scarring
• Corneal hydrops
• Thin corneas with thickness below 300 microns in the ring track
• Patients with intense atopy (should be treated before the implant)
• Any ongoing infectious process, i.e., local or systemic
ICRS AND MYOPIA

The Intacs were approved by FDA in 1996,19 for correction of low myopia
between 1 and 3 D.
Advantages of ICRS over Laser Correction for Myopia
• It is tissue saving as the cornea is flattened without removing tissue from

its center (as in myopia correction by excimer laser).
• Central cornea remains untouched as tissue is added to at the periphery
of the cornea.
• It is a reversible procedure. The cornea regains its preoperative shape
after removal of the Intacs. Its VA essentially returns to former levels
Chapter_15.indd 284 06-02-2018 22:32:17

within 1–7 weeks.26 Asbell et al. reported that eyes returned to preopera-
tive refractive status within 3 months after Intacs removal.16
• Intacs being adjustable, can be replaced by thinner or thicker rings as
needed. These exchange procedures were successful in improving
uncorrected visual acuity (UCVA).24
Disadvantages of ICRS over Laser Correction for Myopia
• Do not correct the astigmatism that accompanies myopia (unlike laser

procedure)
• Manual Intacs implantation (i.e., the original method of implantation)
has a steep learning curve
• Femtosecond laser-assisted Intacs increases the cost of the procedure by
limiting its availability
• A pair of Intacs incurs a high cost for the surgeon comparable to the cost
of the patient’s laser surgery
For all the above-mentioned reasons, laser treatment has become more
popular for low myopia correction.
ICRS AND KERATOCONUS

Keratoconus is a bilateral, non-inflammatory disease characterized by pro-
gressive corneal steepening. The irregular cornea created by keratoconus
often decreases best spectacle corrected visual acuity (BSCVA) and prevents
patients from wearing contact lenses. Intacs regularize the corneal contour
enough so that patients can again wear contact lenses or spectacles. Kerato-
conus also aims at stabilizing the cone through its flattening effects. Several
studies have demonstrated that in keratoconic eyes, post-Intacs implanta-
tion, topography has regularized and UCVA has improved.6,24-32
Indications for Corneal Rings in Keratoconus
• Unsatisfactory VA with glasses33

• Contact lens intolerance
• Mild to moderate keratoconus
• Keratometry reading less than 58 D
• Clear cornea and optical zone with no corneal scarring
• Patients who desire modest improvements in their VA
• Corneal thickness greater than 450 mm in the area of the proposed tun-
nels, where Intacs are expected to be implanted, as marked by the marker
and measured by pachymetry.25,34
SURGICAL TECHNIQUES
Intacs are implanted in a circumferential corneal channel created in the
corneal stroma. A small incision made at 70% of the corneal thickness, as
Chapter_15.indd 285 06-02-2018 22:32:18

measured by ultrasound pachymetry, is an entry point through which the

stromal rings are inserted into the channel. The incision and tunnels are cre-
ated either mechanically by surgical dissection or with the use of femtosec-
ond laser.
The Manual Technique Steps
• The center of the cornea is marked as a reference point.

• A special marker is then used to mark the incision points on the steep
axis of the cornea and the Intacs tunnels.
• Corneal thickness is measured at the incision site and the marked Intacs
tunnels position by ultrasound pachymetry.
• The calibrated diamond knife is set to a depth between 70 and 80%.
• A 1.2–1.8 mm radial incision is created in the marked position. Pocketing
hooks create corneal pockets on each side at the bottom of the incision,
and then a special glide is inserted on both sides in order to check the
pocket depth.
• A semi-automated suction ring is placed around the limbus and the vac-
uum system is started. The vacuum system has two grades of suction: low
suction (400–500 mbar) and high suction (600–667 mbar). At the begin-
ning low suction is applied, and once contraction of the suction ring is
ascertained, a higher level of suction is applied.
• Two semi-circular dissectors are placed (one clockwise and the other
counter-clockwise) into the pocket after inserting the special glide. The
dissectors pass under the glide in order to ascertain the proper depth,
and are advanced by rotational movement creating two semi-circular
tunnels with specific diameters.
• ICRS are then inserted either clockwise or counter-clockwise (in Intacs
with 2 holes) and placed at least 1 mm away from the incision.
The Femtosecond Laser Technique
Femtosecond laser is an infrared laser with a wavelength of 1052 nm. It

induces an optical breakdown in corneal tissue without thermal or shock-
wave damage to the surrounding tissues.35
Steps of the Procedure
• The center of the cornea is marked, and the corneal thickness is mea-
sured by pachymetry in the area of the ring insertion.35
• The suction ring is centered, and the eye is fixated by applanating the
cornea with a disposable glass lens. This maintains a precise distance
between the laser head and the focal point.
• The entry point is created, the corneal tunnel is created at 70–80% of the
corneal thickness, and the ring segments are inserted in the tunnel.
Chapter_15.indd 286 06-02-2018 22:32:18

TURNAROUND AND DOUBLE-PASS TURNAROUND

TECHNIQUES IN EYES WITH FALSE CHANNEL DISSECTION
These were described by one of the authors, Soosan Jacob.36 After creating
a femtosecond channel for Intacs, if resistance to insertion is noted, blind
forceful forward movements should be avoided to prevent creation of false
channel. In cases of false channel, turnaround technique is used in which the
segment is removed, turned around, and inserted in the opposite direction
through the femtosecond channel. With the help of forceps it is advanced for-
ward as far as possible until it is completely within the channel and cannot
be further moved with the forceps. The second segment introduced in the
same direction, now acts as an instrument to advance the leading edge of
the first segment forward into the correct plane by opening the femtosecond-
dissected channel in the area of false dissection. After this segment has
been placed in the intended site, the second segment is manipulated into its
intended site using a reverse Sinskey hook to engage the positioning hole.
With symmetric segments, the same technique is applicable even if one seg-
ment has been implanted and the second segment causes false channel dis-
section. In this case, the second segment is removed and used to advance the
first segment so it lies in the area of obstruction.
In asymmetric implantation, turn around technique can be followed if
the first segment has entered a false channel. If the second segment is not
passing easily after successful placement of the first segment, it is removed
and a double-pass turnaround technique is performed.36 In this case, the
obstructed segment is removed and used to manipulate the asymmetric first
segment (already positioned in its intended place) forward and out through
the incision site. This externalized segment is reintroduced and used as a tool
to reposition the second segment so both segments eventually come to rest
in their respective planned sites segment insertion. Alternately, the first seg-
ment can be removed and used to position the second segment into desired
position using the turnaround technique.
INCISION, RING POSITION AND NOMOGRAMS

Different combinations of Intacs can be implanted:
• Two symmetrical segments37
• Two asymmetrical segments38,39
• One segment
In 2006, Colin5 used a nomogram that was based on the spherical equiv-
alent (SE), location of the cone, and asymmetric astigmatism induced by
keratoconus. Rabinowitz recommends making the incision on the steepest
keratometric meridian, so that the Intacs will bisect the thinnest part of the
cornea, which will make the cornea physiologically normal by thickening the
thin area. In almost all of the cases of oval and central cones, this placement
entails a temporal incision.7 Shetty et al. have also proposed a nomogram40
Chapter_15.indd 287 06-02-2018 22:32:18

in their study where patients underwent Femtosecond laser-assisted Intacs

with collagen cross-linking (CXL) simultaneously and showed significant
improvement in keratoconus patients.
COMPLICATIONS
Intraoperative Complications
The traditional mechanical technique of tunnel creation can lead to com-

plications including anterior and posterior perforations during channel cre-
ation, segment decentration, asymmetry of the implants, inadequate depth
of channel, etc. Other complications include mechanical epithelial defects at
the keratotomy site, extension of the incision towards the central visual axis
or towards the limbus and shallow placement and/or uneven placement of
the Intacs segments.35
Postoperative Complications
Kanellopoulos et al.38 reported a 35% postoperative complication rate. Com-

plications included segment movement and exposure and corneal melting
after mechanical tunnel dissection. This higher incidence was not reported
in other studies.
Other postoperative complications reported include corneal neovas-
cularization, mild channel deposits around Intacs ring segment,41 segment
migration, corneal haze around segments or at the incision site, corneal melt-
ing, night halos, chronic pain,42 epithelial plug at the incision site,43 focal
edema around segments,35 infectious keratitis44 and segment extrusion. Shal-
low implantation is one of the causes of extrusion. It has been associated with
ring migration superficially, stromal thinning and epithelial breakdown.44-47
The femtosecond laser offers several advantages that could reduce these
complications due to more precise localization of the channel by manipula-
tion of its dimensions, depth, diameter and width. Creation of the side-cut
incision using the femtosecond laser rarely requires suture placement for
closure. Although there are many advantages of channel creation using the
femtosecond laser, Ertan and Kamburog˘lu48 observed decentration after
Intacs placement.
RECENT ADVANCES: CORNEAL ALLOGENIC

INTRASTROMAL RING SEGMENTS
One of the authors, Soosan Jacob, has invented a novel technique known as
CAIRS which utilizes allogenic corneal tissue as a spacer graft and behave
similar to Intacs (Fig. 15.2). In this procedure, two ring segments of appro-
priate sizes are made from donor corneal tissue after removing epithelium.
Chapter_15.indd 288 06-02-2018 22:32:18

Fig. 15.2: Corneal allogenic intrastromal ring segment (CAIRS).
Similar to Intacs, these segments are then inserted into a femtosecond laser-
dissected channel via two opposite placed incisions on the flat axis. The
results obtained on our initial subset of patients are very good. This is a cost-
effective procedure as compared to Intacs with similar benefits of improve-
ment in topography and UCVA as well as BSCVA (Figs. 15.3 and 15.4). It also
does away with the complications that can be associated with the implan-
tation of synthetic material into the cornea including migration, intrusion,
extrusion, vascularization, infection, necrosis and melt. A study on a larger
group of patients is being conducted.
COMBINING CROSS-LINKING WITH INTACS IN

KERATOCONUS AND POST-LASIK ECTASIA
Intacs alone may not stop progressive keratoconus.30 This is to be expected,
especially in cases of rapidly progressing keratoconus, because Intacs
insertion does not treat the underlying structural problem of weakened col-
lagen. Therefore, it is logical to combine cross-linking with Intacs insertion in
patients with progressive keratoconus to ensure stability.
Chan et al.49 reported that combining CXL with Intacs augmented the
flattening effects of Intacs. There was statistically greater reduction in cylin-
der and K-values in the Intacs with CXL group than in the Intacs-only group.
Ertan et al.50 concluded that CXL had an additive effect on Intacs implanta-
tion in these eyes and may be considered as an enhancement and stabilizing
procedure.
Chapter_15.indd 289 06-02-2018 22:32:18

Fig. 15.3: Pre- and post-intacs implantation showing improvement in topography.
Fig. 15.4: Pre- and post-CAIRS implantation showing improvement in topography.

CAIRS: Corneal allogenic intrastromal ring segments.
Study by Kamburoglu and Ertan51 and Barbara et al.52 demonstrated the

additive effect of CXL after Intacs in post-LASIK ectasia.
ICRS AND CACXL FOR THIN CORNEAS

This technique was described by Soosan Jacob in 201353 and utilizes a ultra-
violet (UV) barrier-free soft contact lens saturated in riboflavin to increase
the functional corneal thickness. A SofLens daily disposable soft contact lens
(Bausch & Lomb) made of hilafilcon B without UV filter and of negligible
Chapter_15.indd 290 06-02-2018 22:32:19

power is immersed in isotonic riboflavin for 30 minutes during the same time
that the deepithelialized cornea is also saturated with riboflavin. At the end of
30 minutes, riboflavin penetration through the cornea is confirmed by visual-
ization of a green flare in the anterior chamber. The soft contact lens is placed
on the corneal surface, and pachymetry is remeasured. The SofLens soft con-
tact lens has an absolute thickness of 90 mm and together with the precor-
neal riboflavin film gives an additional thickness of approximately 100 mm.
Once the combined pachymetry is confirmed to be 400 mm or above, cross-
linking is proceeded by using either standard cross-linking at 3 mW/cm2 for
30 minutes or accelerated cross-linking. The author’s personal preference is
to use an accelerated protocol at 10 mW/cm2 for 9 minutes, as the shortened
treatment time decreases intraoperative dehydration caused by dextran-
containing solutions while at the same time producing effective cross-linking
(unpublished data).
Contact lens-assisted corneal collagen cross-linking (CACXL) has the
advantages of being independent on the swelling properties of the cornea.
The functional corneal thickness is artificially increased by adding a ribofla-
vin-saturated layer (thin precorneal riboflavin film, riboflavin-soaked bar-
rier-free contact lens and a thin precontact lens riboflavin film). The mean
additional thickness is increased by 107.9 ± 9.4 mm (minimum of 90 mm, which
is the absolute thickness of the contact lens measured after riboflavin satura-
tion).14 This increased functional thickness decreases UV irradiance at the
endothelial level to within safe limits. It also decreases stromal levels of UV
transmittance to approximately 60 to 70%. In our studies, these levels were,
however, sufficient to get clinical effects in the form of demarcation line, with
no patients showing signs of progression. The contact lens used should not
have any built-in UV barrier, and this may be confirmed by going through its
product literature and by checking UV transmittance with a digital UV meter.
UV transmittance will be decreased with contact lenses that have UV barrier.
Studies have shown that contact lenses made of narafilcon A and senofilcon
A have the highest blockage and the lowest transmittance, whereas hilafil-
con B, filcon IV, nelfilcon A, enfilcon A, lotrafilcon A and lotrafilcon B have
the highest UVA transmittance.54 There is increased biomechanical efficacy
of collagen cross-linking in thin corneas as compared to thick corneas due to
increased oxygen availibilty.55
In corneas with preoperative pachymetry less than 350 mm, the combined
functional pachymetry after application of the contact lens may not reach
400 mm. If this occurs, a combination of hypoosmolar and CACXL techniques
may be used. A few drops of distilled water is applied on the deepithelialized
corneal surface to minimally increase the corneal thickness by hydration.
This combination of techniques combines the principles of hypo-osmolar
CXL with CACXL. The degree of hydration of the cornea and the increase in
thickness that is required is much less as compared to conventional hypoos-
molar CXL. Excessively increased spacing between collagen fibrils may
Chapter_15.indd 291 06-02-2018 22:32:19

disallow cross-linking from occurring if a large degree of hydration is needed.

However, when combined with CACXL, the amount of hydration required is
generally negligible and easily attained. ICRS can be performed sequential
or simultaneous with cross-linking.
ICRS AND POST-LASIK ECTASIA

Post-LASIK ectasia is equivalent to iatrogenic keratoconus with all of the
visual disturbances of keratoconus. It consists of a progressive corneal steep-
ening, usually inferiorly, with an increase in myopia and astigmatism, loss of
UCVA and BSCVA and a significant increase in corneal aberrations. Ectasia
has also been reported in few cases after PRK.56-59
ICRS improves visual function by decreasing irregular astigmatism and
reducing corneal steepening.60 Improvement in VA and reduction in sphero-
cylindrical error and keratometry have been reported after ICRS implanta-
tion in post-LASIK ectasia60-65 in severe cases61 with long-term refractive
stability even after 5 years.11 Several studies have shown the beneficial effect
in post-LASIK ectasia with implantation of a single inferior Intacs segment66
and Intacs SK.67 The postoperative complications are the same as in kerato-
conus patients.68 Two cases of flap slippage, months after Intacs implantation
in post-LASIK ectasia patient have been reported.7
ICRS AND PELLUCID MARGINAL CORNEAL

DEGENERATION
Pellucid marginal corneal degeneration is a progressive non-inflammatory
ectatic disorder that frequently involves the inferior cornea in a crescentic
fashion. The involved area, which is located 1–2 mm from the corneoscle-
ral limbus, is 1–2 mm in width, and usually extends from the 4 o’clock to the
8 o’clock meridians.69 Patients in their 40s and 50s, usually present reduced
VA due to high irregular astigmatism.70
A number of surgical procedures have been suggested to improve VA in
PMD, including thermokeratoplasty, epikeratophakia, total lamellar kerato-
plasty, crescentic lamellar or full thickness resection, large eccentric pene-
trating keratoplasty, central penetrating keratoplasty, crescentic lamellar
keratoplasty and a combination of the last two procedures.71-76 All of these
procedures require extensive surgery with unpredictable visual results.
Intacs improve UCVA and BSCVA in patients suffering from PMD.77-79 Pin-
ero et al. reported significant reduction in the manifest astigmatism, astigma-
tism, higher order residual and coma-like aberrations. The sphere, inferior-
superior symmetry and manifest astigmatism were reduced along with rel-
ative centration of the peripheral corneal protrusion.80 Thus, Intacs have
promising role in treating PMD and maybe used as an alternative to the other
available surgical options.
Chapter_15.indd 292 06-02-2018 22:32:19

CONCLUSION
ICRS is a safe and reversible procedure that was first introduced to treat low
myopia from 1 to 3 D, but has eventually evolved to treat various other con-
ditions, like keratoconus, pellucid marginal degeneration, post-LASIK ecta-
sia, post-PRK ectasia. Its most favourable characteristics are reversibility to
preoperative refractive status within few months of explantation and sparing
of visual axis. It has reduced the need forother challenging surgical options,
like deep anterior lamellar keratoplasty and penetrating keratoplasties for
patients with corneal ectasia. ICRS have been instrumental in creating a pos-
itive impact on the quality of life in keratoconic patients.81
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Chapter_15.indd 297 06-02-2018 22:32:19

16
CHAPTER
Deep Anterior Lamellar
Keratoplasty
Jaya Gupta, Prema Padmanabhan
INTRODUCTION
Historically, lamellar keratoplasty (LK) was the first technique used in corneal
transplantation. With the instruments and microscopes available then, con-
ducting lamellar dissection was a technically difficult and time-consuming
procedure. The irregularities in the stromal bed often resulted in an interface
haze, which reduced the optical clarity of the cornea, and hence affecting the
visual recovery of the patient. LK was soon replaced by penetrating kerato-
plasty (PK), which was relatively simpler and faster procedure resulted in bet-
ter visual outcomes. For over a century, then, PK has been the mainstay of
surgical treatment for corneal disease, irrespective of how superficial or deep
the pathology lay.
A better understanding of the surgical anatomy of the cornea and a sound
logic of a targeted tissue replacement has spearheaded the return to LK in
the last few decades. Innovative surgical techniques and the design of appro-
priate instrumentation have refined the procedure to such an extent that the
visual outcome of LK today is as good as PK. Sometimes it is better than PK.1
LK is broadly categorized as either anterior or posterior (endothelial)
keratoplasty. Anterior keratoplasty is indicated in eyes where the pathology
spares the endothelium, whereas endothelial keratoplasty is indicated where
the pathology is confined to the endothelium.
The arrangement of the corneal lamellae and the distribution of the ker-
atocytes allow a smoother and clearer interface when the lamellar dissection
is done in the deeper layers of the stroma as compared to the anterior stroma.
While the actual depth of deep lamellar keratoplasty (DLK) is not specified
anywhere, the term, deep anterior lamellar keratoplasty (DALK), is generally
used to describe a technique where a plane of cleavage is created between the
Descemet’s membrane (DM) and the corneal stroma. A donor corneal button,
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Deep Anterior Lamellar Keratoplasty 299
denuded of its DM, is then sutured to the recipient. Since the donor endothe-
lium is not transplanted, the criterion for tissue selection is less stringent.
This near-full-thickness graft has an optical clarity nearly as good as that
of the penetrating graft and yet avoids the complications of an intraocular
surgery such as intraocular inflammation, anterior synechiae, endophthal-
mitis or suprachoroidal hemorrhage. It also preserves the structural integrity
of the globe better than a penetrating graft would. Above all, the retention of
the host endothelium eliminates the risk of endothelial graft rejection which
is one of the main causes of graft failure.
Sometimes a thin layer of stroma is left on the recipient bed. This is some-
times referred to as ‘Near-Descemet’s deep lamellar keratoplasty’. The thick-
ness and texture of the residual stroma determines, to a large extent, the final
visual outcome. It is believed that a residual stroma of more than 20 m may
cause the visual acuity to deteriorate.2
INDICATIONS OF DEEP ANTERIOR LAMELLAR

KERATOPLASTY
Optical
• Keratoconus
• Corneal stromal dystrophies
• Corneal stromal degenerations
• Deep corneal scars (traumatic, postinfection and other stromal scars)
Tectonic

• Peripheral corneal thinning (Mooren’s ulcer, Terrien’s marginal degener-
ation, collagen disease and other autoimmune diseases)
• Post-LASIK keratectasia
• Corneal melt (autoimmune or neurotrophic)
• Infectious keratitis
Ocular Surface Disease
• Stevens–Johnson syndrome (SJS)

• Chemical or thermal burns
• Ocular surface squamous neoplasia
Miscellaneous
• Post-excision of corneal lesions (pterygium, dermoid and tumors)

The most common indication for DALK is keratoconus. A best corrected
visual acuity (BCVA) of 20/40 (6/12) or better has been reported in 77.8–92.3%
of keratoconus patients after DALK.3-5 Acute hydrops or the presence of deep
scars involving the DM in the visual axis are relative contraindications.
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Most types of hereditary corneal stromal dystrophies are also well suited
for DALK,6 with the exception of macular corneal dystrophy.7 The involve-
ment of the deeper layer of the stroma, and sometimes the endothelium is
possibly responsible for the higher recurrence rate and poorer outcomes of
LK in eyes with macular corneal dystrophy.3
DALK or near-Descemet’s lamellar keratoplasty can also be done as a
tectonic procedure to restore the integrity of the globe in cases of corneal
thinning or even a descemetocele.
DALK may actually be superior to PK in the surgical management of
infectious corneal ulcers, if it is possible to completely remove the infected
stroma and still retain the DM. On the other hand, PK involves higher risk
of intraocular extension of the infection leading to endophthalmitis. Post-
operative inflammation, anterior synechiae, secondary glaucoma and graft
rejection are also more likely to occur after a therapeutic PK as compared to
DALK. Anshu et al.8 showed significantly higher graft survival rate (90% after
DALK compared to 78.4% after PK) and less endophthalmitis following DALK
for infectious keratitis not responding to the medical treatment.
Large diameter lamellar keratoplasties may be required to restore a sta-
ble ocular surface in cases of chemical injuries, SJS and ocular cicatricial
pemphigoid (OCP) associated with corneal thinning. This may be combined
with a keratolimbal allograft when associated with a significant limbal stem
cell deficiency. This procedure would tectonically support a future central
optical lamellar or PK.
SURGICAL TECHNIQUES
The surgical procedure has evolved over the years, with different techniques
to achieve the plane of cleavage between the corneal stroma and DM, for
obtaining the smooth interface that offers the best optical clarity.
DALK can be performed under either general or local anesthesia. How-
ever, general anesthesia is preferable as this minimizes intraoperative up
thrust and hence prevents an inadvertent DM tear which may occur due to
suboptimal akinesia.
Layer-by-layer Manual Dissection
Essentially, the procedure involves layer-by-layer removal of host tissues

until the deep stroma or the bare DM is exposed. This technique is time con-
suming and leaves a relatively rough surface.9
Techniques, like ‘intrastromal air injection’ or ‘segmental removal of host
stroma’ have been described to improve visibility of DM.
Air-guided Deep Stromal Dissection
Melles10 injected air into the anterior chamber (AC) that creates a mirror
reflex to guide surgical instruments directly into the space between DM and
Chapter_16.indd 300 06-02-2018 22:34:11

the posterior stroma. The difference in refractive index between air and cor-
neal tissue creates a reflex of the surgical knife. The distance between the
instrument and reflex can be used to judge the amount of remaining stromal
tissue.
Big Bubble Technique of Anwar
The more popular ‘big bubble’ technique described by Anwar and Teichman
takes advantage of the loose adhesion between Descemet’s and the poste-
rior stroma. In this technique, a partial thickness trephination up to 60–80%
depth is performed (Fig. 16.1). A 30-gauge needle bent 60° (bevel facing
downward) at 5 mm from the tip is attached to a 5 mL air-filled syringe. The
needle tip is introduced at the edge of the partial thickness trephination,
deep into corneal stroma, and gradually advanced to the midperipheral cor-
nea stroma, while keeping the tip under direct visualization. The plunger of
the air-filled syringe is pressed forcibly until entry of air into the stroma is
noted (Fig. 16.2). A sudden easing of the resistance is accompanied by the
appearance of a whitish circular semi-opaque disk (big bubble) (Fig. 16.3).
Air is further injected gradually to enlarge this disk, reaching up to the edge
of trephination mark. Peripheral paracentesis performed at this stage to
lower the intraocular pressure. The superficial stromal layer, approximately
40–50% is removed by lamellar dissection (Fig. 16.4). A sharp 15° slit knife
is used to enter the air space within the bubble. The anterior stromal layers
are then dissected over a blunt iris spatula introduced into the space created
by the big bubble. Blunt-tipped Vanna scissors are used to remove anterior
Fig. 16.1: Partial thickness trephination of host.
Chapter_16.indd 301 06-02-2018 22:34:11

Fig. 16.2: A sharp 30 gauge needle in position for air injection in ‘big bubble’ technique.
Fig. 16.3: Formation of ‘big bubble’ circular disk with dense white edge.
stromal tissue along the edge of partial thickness trephination (Figs. 16.5 to
16.7). Donor tissue oversized by 0.5 mm is taken (in cases of keratoconus,
same sized donor may be used to decrease myopia) and the DM, along with
the endothelium, is peeled off with a fine-tipped tying forceps. Identification
of the edge of the DM is assisted either by a dry Weck-Cel sponge or by try-
pan blue dye to stain the DM (Fig. 16.8). The donor lenticule is placed on the
Chapter_16.indd 302 06-02-2018 22:34:11

Fig. 16.4: Anterior debulking after air injection.
Fig. 16.5: Excision of stroma safely separated from Descemet’s membrane by visco-
elastics.
recipient bed (Fig. 16.9). Four cardinal sutures are passed first. Rest of the 14
interrupted sutures are then passed using 10-0 monofilament nylon suture.
Alternatively, following the application of cardinal sutures, a single continu-
ous suture may be applied, taking care to include 90% of donor and recipient
thickness (Fig. 16.10).
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Fig. 16.6: Remaining stroma being lifted off Descemet’s membrane and excised with
curved corneal scissors.
Fig. 16.7: Clear Descemet’s membrane after removal of anterior stromal tissue.
‘Small Bubble Technique’ to Aid in Anwar’s ‘Big Bubble

Technique’ of DALK
To confirm that a ‘big bubble’ has been achieved a ‘small air bubble’ can
be injected into the AC via a limbal paracentesis. If the small bubble is then
Chapter_16.indd 304 06-02-2018 22:34:12

Fig. 16.8: Descemet’s membrane edge is identified using trypan blue dye and then
peeled off the donor button.
Fig. 16.9: Suturing the donor cornea to the host.
seen at the corneal periphery, it confirms that the big bubble separation of
DM has been successfully accomplished, as the convexity of the bubble is
not seen in the corneal periphery, this means that it is present centrally,
beneath the opaque corneal stroma and, therefore, the big bubble has not
been achieved.
Chapter_16.indd 305 06-02-2018 22:34:12

Fig. 16.10: Final postoperative picture of donor disc in position with interrupted and
continuous suture.
The big bubble technique may not be possible or even advisable in the
following situations:
• Keratoconus with acute hydrops or a post-hydrops scar
• Macular dystrophy is believed to be associated with fragility of DM
• Failure of achieving a big bubble in spite of several attempts
• Needle-perforation during air injection in the big bubble technique
Familiarity with different techniques is useful to allow the surgeon to
choose the appropriate one for each eye and to allow him to switch from one
technique to another in case the situation demands it. For example, the big
bubble technique may not work in an eye with dense and deep stromal scars,
in which case a layer by layer dissection may be required.
COMPLICATIONS
Descemet’s Membrane Perforation
Inadvertent microperforations of DM often occur during the process of dis-

section. It is best to reform the AC with air and continue to dissect the cornea
at a site remote from the area of perforation.
The management of DM perforation depends on the size and location of
the perforation and the step of surgery at which it occurred.
• If the perforation occurs with the needle during air injection, it is best
to avoid baring the DM. A thin layer of stroma is retained to cover the
perforation site.
Chapter_16.indd 306 06-02-2018 22:34:12

• If the perforation occurs during stromal dissection, then the air or C3F8
or SF6 is injected into the AC to tamponade the break. Stromal dissection
could then be resumed from another site, leaving a cushion of stroma
around the area of perforation.
• If the perforation is inferiorly located, it may be difficult to tamponade
with air or gas. Cyanoacrylate glue can be used as a very thin layer to seal
the perforation.
• If the perforation is large, for example while trephining the edges, then it
may be more prudent to convert to PK.
Pseudoanterior Chamber
A pseudoanterior chamber or double anterior chamber (Fig. 16.11) usually

forms secondary to breaks in the DM. Occasionally, retained viscoelastic
material in the interface leads to a detached DM.11 A shallow pseudocham-
ber may be self-limited and may resolve after a few days. Large pesudocham-
bers may require surgical intervention in the form of injection of expandable
gases like sulfur hexafluoride.1
Pupillary Block Glaucoma
Sometimes an air bubble left in the AC can block the pupil, leading to poste-
rior accumulation of aqueous and angle-closure. It can be avoided by dilating
the pupil or limiting the size of air bubble and also a periodic check of eye in
immediate postoperative period.
Folds and Wrinkles
In keratoconus, there is a disparity between the surface area of the donor

cornea and the posterior lamellar bed of the recipient which manifests as
Fig. 16.11: Visante ocular coherence tomography (OCT) image of an eye with a pseudo-
anterior chamber.
Chapter_16.indd 307 06-02-2018 22:34:12

Descemet’s folds and wrinkles secondary to compression at the interface.

They are usually concentric, outside the visual axis and thus, of no visual con-
sequence. If they occur in the visual axis, as is more likely in patients with
central cones, they may affect the quality of vision.
Graft Rejection
Although, DALK eliminates the risk of endothelial rejection, other types of

graft rejection i.e., epithelial, subepithelial or stromal may still occur.1 The
clinical course of subpeithelial and stromal graft rejection after DALK is very
similar to that of PK.
POSTOPERATIVE EVALUATION AND FOLLOW-UP

Patients receive topical antibiotic drops (e.g., 0.3% ciprofloxacin hydrochlo-
ride 4 times a day), topical corticosteroid drops (e.g., 1% prednisolone acetate
eye drops 4 times day) and preservative-free artificial tears every 2 hours for
the first month, which are subsequently tapered over the next six months.
Where DALK is performed for scars following herpes simplex keratitis, long-
term prophylactic systemic acyclovir is advised.
Follow-up visits are scheduled as for a PK. At each visit, uncorrected
visual acuity (UCVA), BCVA, detailed slit lamp evaluation and assessment of
intraocular pressure must be conducted. Serial corneal pachymetry, at suit-
able intervals, helps monitor graft outcome. Videokeratography is performed
to assess the amount of postoperative astigmatism and to plan selective
suture removal at an appropriate time. Visual rehabilitation can take a year
or longer; keratoconus patients do notice a dramatic improvement earlier
than others, on account of the immediate corneal flattening and reduction
of myopia alone.
CLINICAL OUTCOME
Visual Acuity
The recovery of vision to some extent depends on the technique and smooth-
ness of interface (Fig. 16.12). The big-bubble technique ensures baring of the
DM and is associated with the best visual outcomes. Postoperative BCVA
of 20/40 or better has been reported in 92.3% of eyes following DALK for
keratoconus.
Refractive Outcome
As with PK, unpredictable refractive errors and irregular astigmatism are the
limiting factors for a satisfactory visual outcome. A wide range of refractive
errors from -13 D to +7 D has been reported12,13 while astigmatism more
than4 D has been reported in 34.4% of patients undergoing DALK.3
Chapter_16.indd 308 06-02-2018 22:34:12

Fig. 16.12: Mild interface haze following deep anterior lamellar keratoplasty (DALK).
Endothelial Cell Loss
The loss of endothelial cells after DALK usually occurs in the early postop-
erative period (approximately, 11% in the first 6 months)14 due to surgical
trauma, but soon stabilizes to the physiological rate of 1–2% per year. In
comparison, the endothelial cell loss is about 33% over a 2-year-period fol-
lowing PK.15
CONCLUSION
DALK is the preferred surgical treatment option for corneal opacification or
disease in which the endothelium is healthy. It is preferred over PK, primar-
ily because it minimizes the risks of intraocular complications and immune
graft rejection. It is, therefore, ideally suited in ‘high risk’ keratoplasties like
in young patients and in vascularized corneas where the corneal pathology
spares the endothelium or where large grafts are indicated.
REFERENCES
1. Karimian F, Feizi S. Deep anterior lamellar keratoplasty: indications, surgi-
cal techniques and complications. Middle East Afr J Ophthalmol. 2010;17(1):
28-37.
2. Ardjomand N, Hau S, McAlister JC, et al. Quality of vision and graft thickness in
deep anterior lamellar and penetrating corneal allografts. Am J Ophthalmol.
2007;143(2):228-35.
Chapter_16.indd 309 06-02-2018 22:34:13

3. Feizi S, Javadi MA, Jamali H, et al. Deep anterior lamellar keratoplasty in pa-
tients with keratoconus: big-bubble technique. Cornea. 2010;29(2):177-82.
4. Anwar M, Teichmann KD. Deep lamellar keratoplasty: surgical techniques for
anterior lamellar keratoplasty with and without baring of Descemet’s mem-
brane. Cornea. 2002;21(4):372-82.
5. Fogla R, Padmanabhan P. Results of deep lamellar keratoplasty using the
big-bubble technique in patients with keratoconus. Am J Ophthalmol.
2006;141(2):254-9.
6. Kawashima M, Kawakita T, Den S, et al. Comparison of deep lamellar kerato-
plasty and penetrating keratoplasty for lattice and macular corneal dystro-
phies. Am J Ophthalmol. 2006;142(2):304-9.
7. Marcon AS, Cohen EJ, Rapuano CJ, et al. Recurrence of corneal stromal dystro-
phies after penetrating keratoplasty. Cornea. 2003;22(1):19-21.
8. Anshu A, Parthasarathy A, Mehta JS, et al. Outcomes of therapeutic deep lamel-
lar keratoplasty and penetrating keratoplasty for advanced infectious keratitis:
a comparative study. Ophthalmology. 2009;116(4):615-23.
9. Tsubota K, Kaido M, Moden Y, et al. A new surgical technique for deep lamel-
lar keratoplasty with single running suture adjustment. Am J Ophthalmol.
1998;126(1):1-8.
10. Melles GR, Rietveld FJ, Beekhuis WH, et al. A technique to visualize corneal inci-
sion and lamellar dissection depth during surgery. Cornea. 1999;18(1): 80-6.
11. Hirano K, Sugita J, Kobayashi M. Separation of corneal stroma and Des-
cemet’s membrane during deep lamellar keratoplasty. Cornea. 2002;21(2):
196-9.
12. Watson SL, Ramsay A, Dart JK, et al. Comparison of deep lamellar keratoplasty
and penetrating keratoplasty in patients with keratoconus. Ophthalmology.
2004;111(9):1672-82.
13. Fontana L, Parente G, Tassinari G. Clinical outcomes after deep anterior lamellar
keratoplasty using the big-bubble technique in patients with keratoconus. Am
J Ophthalmol. 2007;143(1):117-24.
14. van Dooren BT, Mulder PG, Nieuwendaal CP, et al. Endothelial cell density af-
ter deep anterior lamellar keratoplasty (Melles technique). Am J Ophthalmol.
2004;137(3):397-400.
15. Saw VP, Ng T, Crouch R, et al. Deep anterior lamellar keratoplasty using the
manual dissection technique of Melles: a histopathological correlation. Cor-
nea. 2006;25(8):882-5.
Chapter_16.indd 310 06-02-2018 22:34:13

17
CHAPTER
Dry Eye Disease
Vinay Agarwal
INTRODUCTION
Keratoconjunctivitis sicca (KCS) is one of the most common diseases seen by
ophthalmologists. In the current scenario of growing population and increas-
ing environmental factors, it is becoming even more prevalent. Dry eye is not
a trivial complaint. The symptoms cause significant discomfort and substan-
tially reduce the sufferer’s quality of life. This chapter is intended to give clini-
cians an update on dry eye disease.
The content is divided into segments to allow readers to access specific
facts with ease. The first section summarizes the symptoms of the condition
and highlights the scale of the problem. Further, the current concepts linking
the ocular surface and the tear film as a single functional unit are described.
The dysfunction of any one of the components can lead to a problem of ‘dry
eye’. It is basically the result of a localized immune-mediated inflammatory
response involving both the lacrimal glands and the ocular surface.
The theoretical aspects of the immunological research elucidating the
underlying pathophysiology of dry eye is omitted. Instead, there is a dis-
cussion on a not so often discussed aspect of dry eye, the inflammation, in
various disease entities like allergies, conjunctivitis, blepharitis and even
eyedrops.
The discussion of the methods of diagnosing the dry eye status is included
to elucidate the details of the techniques to enable general ophthalmologists
to adopt them in their practices. The materials needed for conducting most of
the clinical tests are now available in the country. The last segment includes
the treatment options available to treat dry eyes depending on the various
needs of patients and the underlying causative factors.
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THE SCALE OF PROBLEM OF DRY EYE DISEASE

Normal sight depends on a moist ocular surface. This moisture is maintained
by a complex interplay of various factors: sufficient quantity of tears, a normal
composition of tear film, normal lid closure and regular blinking of the lids.
The tear film and the ocular surface form a stable system which can lose its
equilibrium through numerous disturbing factors.
The general understanding of the causes and pathology of the dry eye
have advanced significantly in recent years. We now know that the dry eye is
a multifactorial disease that exhibits primary and secondary changes in the
ocular surface. We now understand that the tear film and the ocular surface
are interdependent.
DEFINITION OF DRY EYE DISEASE

The modern definition of dry eye disease is based on the concept of the three
layers of the tear film devised by Holly and Lemp.1 Also, secondary factors
such as pathological changes to the eyelids, cornea or conjunctiva can them-
selves disturb the normal function of the tear film. Neurotransmitters, hor-
mones and immunological processes play an important role in the regulation
of tear production by the lacrimal gland. Various environmental factors like
contact lenses, pollution and working at video display terminals can affect the
tear film.
The multiplicity of causes and effects make a global definition of dry eye
difficult. However, the following definition has been proposed: Dry eye is
a disease of the ocular surface attributable to different disturbances of the
natural function and protective mechanism of the external eye, leading to an
unstable tear film during the open eye state. Recent studies have shown that
immunologic changes play a role in the pathogenesis of the dry eye even in
post infectious and age-related conditions.2 In addition to the term ‘dry eye,’
which is established worldwide, the term ‘ocular surface and tear disorder’
has been suggested.3
PREVALENCE
How the dry eye disease is widespread in the community? Is it a disease seen
in the general population that needs the attention of the general ophthal-
mologists? Data on the epidemiology of dry eye is sparse even in western lit-
eratures. The differences in the definition and inclusion criteria in different
studies create more confusions. Available data suggest that it is a significant
problem in the older age group. In a community study in Sweden, the preva-
lence rate of 15% was found in the general population aged 55–72 years. This
was done on the basis of symptoms of dry eye disease and positive findings on
Schirmer’s test, tear-film breakup time or Rose Bengal staining.4 A recent Jap-
anese study revealed a 17% rate of positive symptoms of dry eye.5 Most other
studies reveal a prevalence rate of between 11% and 17%. These studies found
Chapter_17.indd 312 06-02-2018 22:53:18

Dry Eye Disease 313
that symptoms of dry eye disease are more frequent in individuals above
50 years of age, however, they found no association of symptoms with sex.
The prevalence of dry eye disease in the community may increase in the
future as the proportion of individuals over the age of 60 years is growing. It
would thus be fair to state that an ophthalmologist has to acquire the knowl-
edge to both manage and educate the patients about the condition in the best
possible way.
OCULAR SURFACE AND TEAR FILM

With improving understanding of the interaction at the ocular surface, we
now realize that the tear film, corneal epithelium, conjunctiva, the lacrimal
glands and the eyelids act as a functional unit (Table 17.1).
All the components of the functional unit are in anatomic connectivity
and share feedback mechanisms. This connectivity causes them to react to
a stimulus as a single unit. This is exemplified by the simultaneous reactions
elicited amongst the various components by a single insult 6 (Fig. 17.1).
Table 17.1: Ocular surface and tear film functional unit.
1. Corneal epithelium
2. Limbal epithelium
3. Conjunctival epithelial cells
4. Conjunctival goblet cells
5. Mucoepidermal junction of lid
6. Meibomian glands
7. Lacrimal glands
All above structures are interconnected and regulated by various hormones, tear film, nerves,
blood, cytokines and lid movements.
Fig. 17.1: The relationship of various units of the ocular surface and tear film.
Chapter_17.indd 313 06-02-2018 22:53:18

The ocular surface is the interface between the eye and the outer world. It
must function optimally to provide a refractive surface to enable the sharpest
vision, and also react quickly to resist injury and protect the ocular structures.
It is also important to realize that the ocular surface has to maintain the integ-
rity in the face of continuous challenges by the shearing forces of blinking, air
currents, humidity variations, foreign bodies and attacks by microorganisms.
This causes the ocular surface and each of the components to be in a highly
dynamic state to meet the changing needs created by changing environmen-
tal conditions.
Among all the components of the ocular surface, the tear film is the most
dynamic. It can be considered as an extracellular matrix playing a complex
and active role with surrounding tissues. It can be compared to the extracel-
lular matrix because it too functions to provide nutrients and communication
pathways, distributes regulatory factors and provides a pathway for cells to
reach the epithelium.
Tears cleanse, lubricate and nourish the surface of the eye, and also pro-
vide physical and immune protection against infection. The tear film-air
interface is the most powerful refractive surface of the eye. A small change
in the tear film stability and/or volume will result in a significant change in
the quality of the image at the retina; thus maintenance of a stable tear film is
essential for clear vision.7
TEAR FILM REGULATION

Normally, when the eye is open, tear film stability is affected by the exposed
(interpalpebral) area of the ocular surface and the time spent between blinks
(inter-blink interval). Even with normal components in the tear fluid, eyelid
blinking is essential to achieve adequate tear spread onto the entire ocular
surface and to form a stable preocular tear film.8
Thus, with respect to tear function and regulation, the compositional
aspect, which involves the ocular surface epithelia (corneal and conjunctival)
and external adnexae, and the hydrodynamic aspect, which involves eyelid
blinking and closure, constitute key elements in regulation and maintenance
of a stable tear film (Fig. 17.2).
The ocular surface is regulated through a reflex loop. The two major
components of the protective mechanism, namely the compositional and
the hydrodynamic aspects, are controlled by two major reflex arcs. Both the
arcs use the first (ophthalmic) branch of the trigeminal nerve (V) to transmit
the sensory stimulus. The aqueous secretion of the compositional aspect is
mediated by the parasympathetic branch of the facial nerve (VII). The eyelid
blinking or the hydrodynamic aspect is mediated by the motor branch of the
facial nerve.
The spreading of the tear fluid on the ocular surface to form the complexly
structured preocular tear film is the consequence of blinking. The sequential
Chapter_17.indd 314 06-02-2018 22:53:18

Dry Eye Disease 315
Fig. 17.2: Elements of protective mechanism of the ocular surface.
operation of the orbicular and levator muscles of the lids spreads the tear
fluid and reconstructs the tear film architecture disturbed by the evaporation
of water and by environmental contamination during the inter-blink period.
The movement of the lids exerts a significant pressure on the bulbar surface at
each blink, with a retropulsion of the eye by 0.7–1 mm (up to 2 mm on forced
blinking). If not protected by an efficient viscoelastic tear film, the ocular sur-
face epithelia can be damaged by the applied shear forces.
COMPOSITION OF TEAR FILM

Tears are a complex solution composed of water, enzymes, proteins, immuno-
globulins, lipids, various metabolites and exfoliated epithelial and polymor-
phonuclear cells. Due to the highly dynamic nature of the tear film, defining
its exact composition at a particular point of time is impossible. Its specific
content will vary depending upon the challenges with which the ocular sur-
face has to deal.
The tear film is classically described as having three layers as shown in
Figure 17.3. This architecture of the precorneal tear film being three layered
structure is controversial. There is now growing evidence that it is two layered
structure where under the lipid layer lies an aqueous-mucin gel, in which the
mucins have a decreasing gradient of concentration from the epithelium to
the surface.9 The source and function of the various layers are mentioned in
Table 17.2.
An analogy which may help simplify the importance of the various com-
ponents of the tear film is that of a goldfish. The epithelial cells on the surface
of the eye are similar to a fish. And the tear film surrounding the cell is like
a fish bowl, containing a proper balance of hydration, pH, osmolarity, tem-
perature, oxygenation, nutrients, amino acids, glucose and waste removal.
When there is dirty water in the fish bowl, which equates to an imbalance
within the tear film, the goldfish begins to die just as the epithelial cell would
(Fig. 17.4).
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Table 17.2: Functions of various layers of precorneal tear film.
Mucin layer Aqueous layer Lipid layer

Source Conjunctival goblet cells, Main and accessory Meibomian
conjunctival and corneal lacrimal glands glands
epithelial cells
Function Formation of glycocalyx Creating the proper Prevents the
renders the ocular surface environment for the evaporation of
hydrophilic epithelial cells tears. Enhances
Allows the viscosity of Provides essential stability of the
tears to change as per the nutrients and oxygen to tear film
shear rate of blinking the cornea
Maintains the dioptric Allows cell movement
integrity of the tear film in over the ocular surface
the inter-blink interval
Prevents adhesion of Provides many of the
foreign bodies to the growth factors necessary
ocular surface for ocular surface health
Fig. 17.3: Normal tear film.
DYSFUNCTION OF THE OCULAR SURFACE AND TEAR FILM

Deficiencies of any of the three layers of the tear film, defective spreading of
the tear film, systemic diseases and some systemic and topical medications
can disturb the ocular surface or tear film and cause dry eye disease.
Chapter_17.indd 316 06-02-2018 22:53:19

Dry Eye Disease 317
Fig. 17.4: The gold fish analogy for functions of tear layers (concept first used by Dr
Mark Abelson).
The National Eye Institute’s classification of dry eye has two major divi-
sions: aqueous deficient and evaporative dry eye.10 It helps separate patients
according to the main causative factor of the disease. However, the clinical
picture is often a mix of the two pathogenic pathways (a reduced tear produc-
tion often results in defective oily layer spreading and in excessive evapora-
tion, and meibomian gland disease is often associated with a hyposecretive
dry eye).
The Vicious Cycle of Increased Evaporation and Ocular

Surface Damage
From clinical point of view, it is well-recognized that, in early cases, dry eye
patients characteristically have hyperemia confined to the interpalpebral con-
junctiva with vital dye staining localized to the same area. Furthermore, the
initial areas of the bulbar conjunctiva to be stained are the nasal quadrants. If
we consider the tear turnover in front of the eye, there is the longest period of
contact between the altered hyperosmotic tears and the conjunctival epithe-
lium. This suggests that the hyperosmotic tears act as toxic agents towards the
conjunctival epithelia, both by direct osmotic mechanism and by inflamma-
tory activity.
Tear evaporation has a definite role in ocular surface disease (Fig. 17.5).
It triggers the vicious cycle of evaporative dry eye, and also has a role in the
build up and maintenance of the vicious cycle of damage in tear deficient
dry eye.
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Fig. 17.5: Role of tear evaporation in ocular surface disease.
Finally, it has been proposed that the prolonged stimulation of enhanced

secretion by the lacrimal gland, caused by the chronic increased evaporation
of water from the tear film, could result in neurogenically induced and immu-
nologically mediated inflammation of the lacrimal gland itself, slowly trans-
forming an evaporative dry eye into a tear deficient dry eye.11
CHRONIC INFLAMMATION AND DRY EYE DISEASE

Whatever the initial cause of dry eye disease is, chronic dryness of the ocular
surface results in excessive nervous stimulation aimed at triggering the mech-
anisms of regulation and repair. It has been hypothesized that these nervous
stimuli lead to neurogenic inflammation, activation of T-cells. This leads to
the subsequent release of inflammatory cytokines in the lacrimal glands,
tear film and conjunctiva. Various other mechanisms activate the cytotoxic
phenomenon.
The inflammatory reactions of the ocular surface result in gradual dys-
function and destruction of the lacrimal gland and impairment of the con-
junctival epithelium. In addition, inflammatory mediators may inhibit neural
signals to the lacrimal gland, thus depriving the gland of the trophic stim-
ulation needed for its maintenance resulting in its progressive destruction
(Fig. 17.6).
Reduced levels of immunosuppressive androgens after menopause may
enhance both dryness and inflammatory stimulation.
Thus, despite the initial cause of lacrimal gland atrophy, a local inflam-
matory reaction is present at the ocular surface. It is hypothesized that sub-
clinical hormonal or autoimmune factors aggravated by exogenous factors
such as pollution, local infection, allergies result in a cascade of cellular reac-
tion with which apoptotic cell death and inflammation may be closely linked.
The inflammation may be preexisting and induce dryness, it may be concom-
itant, or it may appear after the administration of anti-allergic treatments,
especially those containing preservatives (Fig. 17.6). Once dry eye disease
has developed, inflammation becomes the key mechanism of ocular surface
injury as both the cause and consequence of cell damage.
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Dry Eye Disease 319
Fig. 17.6: The vicious cycle of dry eye disease.
Inflammation and Dry Eye
The dry eye is classified into two groups: tear deficient dry eye and evapora-
tive dry eye. The tear deficient dry eye can be further classified into Sjögren’s
syndrome dry eye, an autoimmune disorder, and non-Sjögren’s syndrome dry
eye, which encompasses the range of other causes of tear deficiency.
Sjögren’s Syndrome
It is an autoimmune disorder characterized by chronic inflammatory infil-

tration of exocrine glands and systemic immune reactivity, either isolated
(primary Sjögren’s syndrome) or associated with other connective tissue dis-
eases (secondary Sjögren’s syndrome) like rheumatoid arthritis, and may be
associated with sarcoidosis or lymphoproliferative syndromes. In the eye, the
autoimmune process involves not only the lacrimal gland but also the entire
conjunctival surface.
Dry Eye Disease and Allergies
Induction of dry eye disease is commonly associated with chronic aller-

gic conjunctivitis. Chronic allergy results in chronic or regularly reactivated
stimulation of the local immune system resulting in damage to the ocular sur-
face, especially the loss of goblet cells.
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In practice, within a few weeks or months after the acute allergic epi-
sode, dry-eye like symptoms follow due to lack of normal homeostasis in the
ocular surface. In such cases, the anti-allergic medications are not of much
use and may in fact aggravate the symptoms. The patients improve by with-
drawal of aggressive treatments and prescription of tear substitutes (usually
preservative-free). These tear substitutes break the cycle of the rubbing the
irritated eyes, causing mast cell degranulation and more irritations as resul-
tant. Artificial tears also wash away inflammatory mediators and restore a
more stable tear film.
Dry Eye Disease and Conjunctivitis
Dry eye disease commonly occurs after an episode of viral keratoconjuncti-

vitis or severe acute or subacute conjunctivitis. These diseases (or the treat-
ments) may lead to the loss of goblet cells from the conjunctival epithelium
and release of inflammatory cytokines.
Patients usually complain of persistent symptoms and are continued on
the same treatment for the original condition. This treatment is not only inap-
propriate but it may also be toxic, whereas these patients are actually suffer-
ing from the vicious cycle of secondary tear film alterations. Discontinuation
of antibiotics, steroids and all preservative-containing eyedrops are manda-
tory for the relief of symptoms and progressive improvement of the tear film
and ocular surface.
Dry Eye Disease and Blepharitis
Blepharitis, an extremely frequent cause of dry eye disease, has infectious

and inflammatory components (Figs. 17.7A to C). It results in impairment
of the lipid layer of the tear film and an increased rate of tear evaporation.
In addition, dysfunction of the meibomian glands and poor elimination of
abnormally thick and viscous lipid secretions provide favorable conditions for
secondary bacterial infection at the base of the eyelashes. The toxins released
by the bacteria aggravate the condition and produce lesions on the cornea
adjacent to the lid margins.
Meibomian gland dysfunction (posterior blepharitis) is itself a major
cause of dry eye and ocular discomfort. These patients present with com-
plaints of a chronic gritty irritation in their eyes that is worse upon awaken-
ing. This is caused by an accumulation of inflammatory mediators beneath
the eyelids during sleeping. As the condition progresses the resulting tear
film disturbances cause worsening of symptoms later in the day.
Dry Eye Disease and Eyedrops
Due to the various causes mentioned and discussed above, the patients may
be using tear substitutes instilled many times per day for the relief of dry eye
symptoms. Sometimes they are unable to get relief, indeed the condition may
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Dry Eye Disease 321
(A) (B)
(C)
Figs. 17.7A to C: (A) Blepharitis showing enlarged dilated blood vessels on the lid mar-
gin, (B) matted eyelashes and (C) with obstructed meibomian glands.
be worse. In these cases, aggressive treatments should be discontinued and

the use of additional toxic drugs avoided. This is in order to prevent the devel-
opment of a vicious cycle of ocular surface and tear film impairment. On stop-
ping the treatment, many patients experience less discomfort.
Superficial punctate keratitis extending beneath the palpebral fissure
and conjunctival staining in the lower nasal part, where irritant eyedrops
tend to accumulate, are indicative of the toxic effect of treatment. Some
toxic effects of the eyedrops are not clinically evident. They can be assessed
by subtle signs like reduced tear-film breakup time or, sometimes, only on
impression cytology. Many reports have suggested the involvement of pre-
servatives, especially benzalkonium chloride (BAK), a member of the quater-
nary ammonium family, in these toxic effects.
Most eyedrop formulations contain preservatives which are cytotoxic
to different degrees. In animals, chronic instillation of preservatives leads to
the onset of inflammatory conjunctival infiltration and damage to the muco-
sal cells.12 These results are similar to those observed in humans after the
chronic use of antiglaucoma eyedrops.13 This points to the problem of ren-
dering topical drugs less toxic. Despite a higher cost, the use of single-dose
units or preservative-free multimode devices should, therefore, be recom-
mended for chronic and repeated use, especially in dry eye syndrome.
DIAGNOSIS OF DRY EYE

Dry eye disease can be classified as tear-deficient, in which there is a defi-
ciency of aqueous tear secretion, or evaporative in which the cause is exces-
sive evaporation.
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Each form of dry eye has certain global features in common.

• A set of characteristic symptoms
• Ocular surface damage
• Reduced tear film stability, and
• Tear hyperosmolarity
Increasingly, an inflammatory component has become apparent, which
contributes not only to symptoms, but also to the disease process itself.
A diagnostic trap, in patients who are highly symptomatic but who do not
appear to have signs of dry eye, is the failure to recognize the presence of
superior limbic keratoconjunctivitis (SLK) which can be overlooked if not
searched for routinely. Also SLK and dry eye can occur together.
The global features of dry eye disease can be identified by the following
types of diagnostic tests.
• Symptom questionnaires
• Staining to identify the ocular surface damage
• Tear breakup time to assess tear instability, and
• Osmometry for tear hyperosmolarity
System Questionnaires
Various questionnaires have been designed, most of them consist of questions

relating to six symptoms.
1. Do your eyes ever feel dry?
2. Do you ever have gritty or sandy sensation in your eyes?
3. Do your eyes ever have a burning sensation?
4. Are your eyes ever red?
5. Do you notice much crusting on your lashes?
6. Does your eye ever get stuck shut in the morning?
Even these questionnaires fail to find a correlation between symptoms,
tear deficiency and ocular surface damage.
Ocular Surface Staining
Epithelial staining to the exposed surface of the eye can be demonstrated

with vital and supravital stains. Staining of cornea occurs preferentially over
its lower part, often more nasally than temporally, and frequently in conti-
nuity with the bulbar conjunctival stain. Staining of the bulbar conjunctiva
occurs over a wedge-shaped zone nasally and temporally, which in advanced
dry eye may become confluent. Staining on the conjunctiva may be present in
the absence of corneal stain in milder forms of dry eye. The basic guidelines
for using the vital dyes for staining the ocular surface are given in Table 17.3.
The following dyes are in routine use.
Fluorescein
Fluorescein staining is the standard method to demonstrate ocular surface
damage. This orange dye, which fluoresces green when excited by blue light,
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Dry Eye Disease 323
Table 17.3: Tips on using vital dyes for staining the ocular surface.
1. The time to use one of these dyes is after completing the external eye examination
and refraction
2. In the case of Rose Bengal, use an anesthetic drop first
3. If the patient has mild dry eye, the dye will permeate the nasal bulbar conjunctiva and
possibly the superior edge of the lower lid as well
4. In more severe cases, the staining may be more intense and involve the cornea as well
as the temporal bulbar conjunctiva
i. Test all refractive surgery candidates in this way, since dry eye can affect healing
and outcome of corneal surgery
ii. Test if a patient wants refractive surgery because he or she finds contact lenses
uncomfortable
iii. In the presence of staining, consider infectious conjunctivitis and blepharitis in
addition to the dry eye
Fig. 17.8: Corneal fluorescein staining in keratoconjunctivitis sicca (KCS) showing stain-
ing is in the lower area of the cornea.
is applied to the eyes with a fluorescein-impregnated strip wetted with a ster-

ile drop of saline. Optimal results are obtained by viewing through a yellow
barrier filter, such as Kodak Wratten 12 absorption filter, used in combination
with the standard blue exciter filter of the slit-lamp biomicroscope. This tech-
nique reveals damage on both the corneal and the conjunctival surface.
Usually staining has a characteristic distribution and is confined to the
exposed interpalperbral area of the ocular surface (Fig. 17.8), but in severe
dry eye, staining may extend to the unexposed surface of the globe, particu-
larly the upper bulbar conjunctiva. In the absence of the yellow filter, staining
is poorly seen on the conjunctiva.
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Fig. 17.9: Rose Bengal staining of the conjunctiva in keratoconjunctivitis sicca (KCS).
Rose Bengal
In India, Rose Bengal is available as impregnated strips similar to the fluo-

rescein dye strips. Instillation is best preceded by topical anesthesia to limit
stinging, and care should be taken to remove the excess dye from the lid mar-
gins on eye closure with a tissue to avoid unsightly staining of the lid skin. The
amount of staining seen is dose-dependent.
Rose Bengal stain is supposed to demonstrate the ocular surface damage
by being taken up by dead and degenerated cells (Fig. 17.9). However, it is
now believed that the Rose Bengal staining of ocular surface is due to the loss
of the normal mucin layer in dry eye disease, allowing the dye to stain live
epithelial cells that would normally be protected by mucin in healthy eyes.
Recent research has suggested that staining in dry eye is associated with an
altered glycosylation of the surface mucin of apical conjunctival cells. Thus, it
appears that although stained cells are not necessarily degenerated, they may
suffer from impaired expression of membrane mucin.
Other Dyes
Lissamine green is another dye now available in India. This dye, when viewed
in white light, produces a staining pattern similar to Rose Bengal. The staining
is best seen over the white of the sclera and least on the cornea, over a dark iris.
Like fluorescein it is well tolerated. This dye has an advantage over the stinging
and pain commonly experienced with Rose Bengal.
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Dry Eye Disease 325
ASSESSMENT OF TEAR FILM STABILITY

Noninvasive Tear Breakup Time
A correlation between ocular discomfort and tear-film breakup time exists. In

hundreds of dry-eye patients, it has been observed that within one second of
tear-film breakup, 73% of the patients experience ocular awareness followed
by discomfort. This manifestation of ocular discomfort may stimulate the eye
to blink, replenishing the tear film and providing protection to the ocular
surface. If the patient has a short tear-film breakup time, due to disease or
other factors such as systemic medications known to cause ocular drying, or
an altered blink rate, as a result of staring at a computer screen, symptoms
and signs can be exacerbated. Therefore, the relationship between tear-film
breakup time and blink rate is critical. How does one assess the noninvasive
tear breakup time (NIBUT)? If you have your patient stare straight ahead and
monitor the time from his last complete blink and the moment he reports
ocular awareness, this time will be within approximately one second of his
tear-film breakup time (Table 17.4).
Interblink Interval and Ocular Protection Index
Interblink interval (IBI) can be calculated by observing the patient as he reads

the vision chart without his being aware that the blink rate is being measured.
The number of blinks per minute divided by 60 will give the IBI.
The understanding of the ocular protection index (OPI) brings us to the
concept of interaction of IBI and BUT (Table 17.5). It also explains the con-
Table 17.4: Noninvasive breakup test.
Improved understanding of tear-film breakup time and its relationship to ocular aware-
ness allows for a simple test.
l Obtain a stop watch or clock
l Blink twice, then stare straight ahead
l Record the time between the last complete blink and the first sensation of ocular
awareness
This time in seconds is the noninvasive breakup time.
Table 17.5: Calculating ocular protection index.
l Visual count of blinks/min while patient reads vision chart

l IBI is determined by dividing blinks/min by 60
l Measure tear-film breakup time using the slit-lamp or the noninvasive test
l If BUT > IBI, patient is protected
l If BUT < IBI, patient is at risk
l Severity may be determined by the degree of discrepancy between IBI and BUT
IBI: Interblink interval; BUT: Breakup test.
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Fig. 17.10: Interaction between the blink rate and ocular discomfort.
cept of discomfort in dry eye patients (Fig. 17.10). When the IBI is longer than
the ability of the tear film to protect the intact ocular surface, the surface will
begin to show signs of drying. The severity will depend on the degree of mis-
match between the spread of the tear film at the completion of the blink and
the onset of the next blink.
Tear Meniscus Height
Evaluation of the tear meniscus height is a rough estimate of tear quantity,

and assessing the amount of debris in the tear film also demonstrates the
tear quality. Several authors have suggested that a decreased tear meniscus
height indicates decreased aqueous; however, there has been minimal work
to systematically classify clinical ranges in normals and dry eye patients. A
tear meniscus height for dry eye is approximately 0.25 mm in height, while
normals demonstrate a height of around 0.50 mm. Therefore, clinically a cut-
point value of 0.3 mm is considered indicative of dry eye.
Fluorescein Tear Breakup Test
Fluorescein tear breakup test (FTBUT) is a provocative test in the sense that
the intillation of fluorescein dye shortens the normal breakup time. Breakup
is best observed with the use of blue exciter filter and yellow barrier filter
while the patient refrains from blinking. The yellow filter is, however, not
essential. The breakup time is the time that elapses from the last blink to the
first appearance of a dark spot in the fluorescein stained film. In general, a
breakup time of less than 10 seconds suggests an unstable tear film. Tear
breakup time is reduced in all forms of dry eye.
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Dry Eye Disease 327
Assessment of Aqueous Tear Production
Schirmer Test
The Schirmer I test without anesthetic is a test of reflex secretion in response

to conjunctival stimulation. It is a useful test for the evaluation of dry eye,
although the diagnosis or exclusion of dye eye cannot be made on the basis of
this test alone. Less than 6 mm of wetting after 5 minutes indicates a diagnosis
of tear deficiency. The reliability of the test may be affected by environmental
conditions such as temperature or humidity.
The Schirmer I test can be performed after instillation of a topical anes-
thetic, and it measures the basal secretion rate in the absence of a reflex com-
ponent. Nasal anesthesia reduces the Schirmer value obtained from the test.
The Schirmer II test assesses basal and reflex secretion of tears in
response to nasal stimulation in addition to the conjunctival stimulation.
This test is very uncomfortable for the patient, as it involves vigorous stim-
ulation of the nasal mucosa. Wetting of the strip in response to this test has
been shown to be reduced in the more severe forms of dry eye, such as in
Sjögren’s syndrome.
Assessment of Oil Glands
Clinical assessment of the extent of meibomian gland dysfunction is a method

used to assess extent of evaporative dry eye. This simplest method of assess-
ing meibomian glands involves quantification of occluded gland orifices and
grading the quality of expressed oil secretion.
There are several other tests which are not mentioned in the above dis-
cussion because of their lack of standardization or availability in our country.
Some of these are the phenol red thread test, tear meniscometry, meibome-
try and lysozyme assays. However, a sequence of tests has been suggested for
the diagnosis of dry eye disease in an outpatient set-up (Table 17.6).
TREATMENT OF DRY EYE

The most important factor in the success of dry eye therapy is that the patient
must be fully informed about the nature of his or her eye disease and the goals
of therapy. This is because the conventional management of dry eye is only
palliative.
Currently, the choice of therapy for dry eye disease may be determined
by the severity of the condition. Mild cases may be successfully managed
with artificial tears applied up to four times daily. In moderate disease, where
damage to the ocular surface is limited to certain zones, use of unpreserved
artificial tears up to 12 times per day and an unpreserved lubricating oint-
ment at bedtime may be needed. Severe dry eyes will need additional therapy
in the form of some tear conserving therapies.
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Table 17.6: Sequence of tests for the diagnosis of dry eye disease.
1. Noninvasive tests:
a. Noninvasive tear breakup test (to assess tear stability)
b. Tear meniscus height and quality (to assess tear volume)
2. Minimally invasive tests:
a. Fluorescein tear breakup time (to assess tear stability)
b. Staining of bulbar conjunctiva and cornea (to assess ocular surface damage)
3. Tests of tear volume or secretion:
a. Schirmer I test (to assess basal tear flow)
b. Schirmer II test in response to nasal stimulation (to assess basal and enhanced
reflex tear flow)
4. Additional dye tests:
a. Rose Bengal staining (to assess ocular surface damage)
b. Lissamine green staining (to assess ocular surface damage)
5. Oil glands assessment
A 5 minutes gap is recommended between invasive and noninvasive tests.
Tear Substitution
Tear replacement by topical artificial tears and lubricants is currently the most
widely used therapy for dry eye, and a variety of components and preserva-
tives are used to formulate a considerable number of preparations. The goal
of tear substitutes is to increase humidity at the ocular surface and to improve
lubrication, with subsequent secondary benefits.
The use of artificial tears has obvious limitations too. Natural tears are a
complex composition of water, salts, hydrocarbons, proteins and lipids which
artificial tears cannot completely substitute. In addition, the integrity of the
three-layered emulsion of the aqueous, the mucin and the lipid, which is so
vital for the effective functioning of the tear film, cannot be reproduced by
artificial components.
Furthermore, artificial tears are delivered intermittently rather than con-
tinuously as are natural tears. To overcome this problem somewhat, newer
formulations contain ingredients to increase their contact time with the ocu-
lar surface (Table 17.7). For example, carboxymethylcellulose has twice the
mucoadhesive strength compared to hydroxypropylcellulose. The downside
of this would be in terms of an increased viscosity leading to irritation, blur-
ring of vision, sticky eyelids and a sensation of heavy eyelids.
What’s Inside the Bottle
An ocular medication is much more than the active drug and its other com-
ponents may present difficulties for some patients. This is especially true
for patients who overuse their artificial tear products, use multiple ocular
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Dry Eye Disease 329
Table 17.7: Properties of some components used in artificial tear substitutes.
Component Properties Advantages Disadvantages

Cellulose esters Viscoelastic, increase the Good retention Only of benefit
(hypromellose, viscosity of tears, large time on the in aqueous
hydroxyethyl- increase in viscosity when ocular surface, tear deficiency,
cellulose, concentration is moder- mix well with hypromellose can
carboxymethyl- ately increased, sometimes other ophthalmic cause crusting of
cellulose) co-formulated with elec- products, viscosity eyelids, mimick-
trolytes, as hypotonic is not affected by ing blepharitis
blinking
Polyvinyl alcohol Synthetic polymer, high Good retention Tends to blur,
viscosity when eye is static, time on ocular vision often
shear thin during blinking surface uncomfortable to
or eye movement, max- patients
imizing thickness of the
tear film while minimizing
drag
Povidone Synthetic polymer, co- Beneficial in mucin Little clinical
(polyvinyl formulated with electro- layer deficiency experience
pyrrolidone) lytes, superior wetting
ability when co-formulated
with polyvinyl alcohol
medications, suffer from chronic eye diseases like dry eye or glaucoma or
require post-surgery dosing of medication.
Ocular medications are composed of unique mixtures of the active drug,
a preservative, the drug delivery system, viscosity-increasing agents, buffers
and stabilizers and a vehicle by which all the above ingredients are ‘carried’.
Of these, it’s the preservative that is most often been considered the culprit
in damaging the corneal epithelium leading to the disruption of glycocalyx,
when the eyedrops are used beyond the recommended dosing.14 The cyto-
toxic damage to epithelium makes the tear film unstable and leads to keratitis
medicamentosa (Fig. 17.11).
Preservatives in Artificial Tears
Previous generations of artificial tears contained more toxic preservatives,

such as thimerosal, which was found to cause sensitization and lead to
drug-induced allergic conjunctivitis in some patients. Current products are
preserved with less toxic agents like BAK and disodium EDTA. Even newer
agents are now available with a form of preservative that either dissipates or
changes to water upon contact with the tear film.
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Fig. 17.11: Diffuse superficial punctate keratitis due to keratitis medicamentosa.
Ocular preservatives are divided into two types: (1) chemical and (2) oxi-
dative. Chemical preservatives alter the cell membrane permeability and lyse
the cytoplasmic contents. Oxidative preservatives penetrate the cell mem-
branes and interfere with cell’s functions.
Common chemical preservatives found in ophthalmic preparations are
BAK, chlorobutanol and sorbate. Sodium perborate, stabilized oxychloro
complex (SOC), and polyquad are newer proprietary preservatives that may
be safer to the corneal epithelium. Of these, BAK is still the most prevalent
and its cytotoxicity is well-documented.
Benzalkonium chloride: BAK is a quaternary ammonium compound that is
often used in conjunction with disodium EDTA. It is a chemical detergent pre-
servative that is chemically stable, does not degrade easily, even at high tem-
peratures, and is very effective and fast acting against many microorganisms.
BAK acts upon microorganisms by altering the cell membrane permeability
and lysing the cytoplasmic contents. It has also been shown to increase the
corneal penetration of some drugs by causing a separation of the epithelium.15
BAK has been the gold standard of preservatives for years, and is the
most common antimicrobial preservative currently used in topical multi-
use ophthalmic solutions. Reports have shown that BAK can accumulate in
ocular tissues and cause different types of cell death with frequent dosing. At
concentrations and dosing used clinically, however, BAK does not appear to
have significant adverse effects unless its frequency of use exceeds four to six
times daily. This becomes a concern when patients use other drops on top of
chronic medications, such as tear substitute drops.16 It is important to recall
that preservatives themselves are bactericidal.
Chlorobutanol: This is an alcohol-based preservative used in artificial tears
and as an active ingredient in certain sedatives and anesthetics. It has a wide
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Dry Eye Disease 331
range of antimicrobial action. It works by disorganizing the lipid structure

of the cell membrane thereby increasing the permeability of the cell. In one
double-blind crossover study, however, chlorobutanol 0.5% in artificial tears
was shown to cause irritation in over half of the subjects.
Sodium perborate: This is one of the first oxidative preservatives developed
and it works by oxidizing the cell walls or membranes and disrupting the cel-
lular function. When combined with water, sodium perborate is converted to
hydrogen peroxide, allowing low levels of this preservative to be effective at
destroying the microbes. Also, it is unique in the fact that, when it makes con-
tact with the tear film and/or ocular tissue, it changes into simple oxygen and
water.
Stabilized oxychloro complex (SOC Purite): This is an oxidative preserva-
tive introduced several years ago. The sodium chlorite found in SOC has been
used since the mid-1940s in water purification. It is also used in the tooth-
paste, mouthwash and some antacids.
SOC has a wide spectrum of antimicrobial activity and has been shown
to destroy the fungus, Aspergillus niger, one of the most difficult organisms
to kill. SOC was found to be safe and well-tolerated when dosed frequently,
according to a 62-patient study in which patients dosed an SOC-based pre-
servative four to eight times daily for a four-week period.
Nonpreserved Drugs
Preservative-free drugs may eliminate the risk of toxic side effects which
can make them attractive treatment options. Studies support the belief that
preservative-free preparations are safe to use in patients, especially with fre-
quent dosing.
Nonpreserved artificial tears have an extra advantage over preserved
ones. They may be the best choice for patients immediately following eye sur-
gery due to the increased viscosity and pH buffering.
Though preservative-free drugs may avoid some toxic side effects, they
have certain inherent disadvantages. While it is true that a preservative may
be the cause of an allergic reaction, it is difficult to determine whether the
problem is caused by the preservative, the drug, the drug delivery system or
the buffers and stabilizers. Nonpreserved preparations may still hold some
risk therefore.
Nonpreserved drugs are only available in unit-dose vials which may be
more difficult for a patient to use correctly affecting compliance. Poor com-
pliance may hinder a nonpreserved drug’s effectiveness even if it is more
comfortable to use. This can be especially important when it is used con-
comitantly with multidose glaucoma medications for which compliance is
vital. Unit-dose vials are also more expensive than multidose containers.17 In
addition, patients with advanced rheumatoid arthritis may find it difficult to
Chapter_17.indd 331 06-02-2018 22:53:21

squeeze the drops from the single-use vials. They may be tempted, then, to
use the vial for more than one application.
Tear Preservation
Occlusion of the Tear Drainage System
Occlusion of the lacrimal puncta or cannaliculi prevents the drainage of natu-

ral and artificial tears and is currently the most common non-pharmacological
therapy for dry eye disease.
Punctal occlusion improves the quantity and quality of the aqueous
component of the tear film, relieving symptoms and signs of dry eye, making
patients more comfortable and reducing the need for artificial tears.
Controversy about many of these claims remains. It has been suggested
that punctal occlusion can decrease tear production, clearance and ocular
surface sensation.18 Many of the problems caused by occlusion of tear film
drainage are derived from delayed tear clearance and turnover, both of which
are already present in dry eye state. Delayed tear clearance can increase accu-
mulation of pro-inflammatory cytokines in the tear film causing desensitiza-
tion of the corneal surface and promoting inflammation. Delayed clearance of
tears can also cause increased toxicity of preservatives in the tear substitutes.
To minimize these risks as well as that of epiphora after occlusion, most
authorities recommend assessing the result of temporary occlusion with
absorbable or removable plugs or inserts prior to considering permanent
occlusion.
Usually occlusion of only one punctum, the inferior punctum, provides
sufficient relief from the symptoms of dry eye, sometimes both puncta may
need to be occluded. Most authors reserve these for moderate to severe dry
eyes, and only after frequent use of unpreserved artificial tears and lubricants
remains insufficient.
Methods of Occlusion
Various surgical, thermal, and tamponade methods have been reported.19

Surgical methods are rarely used as they are extremely difficult to reverse. The
punctum patch, in which an autologous conjunctival graft covers the punc-
tum, seems easy to perform, and can be removed if occlusion needs to be
reversed (Figs. 17.12A to C).
Thermal methods (cautery, diathermy or laser) produce cannalicular
occlusion by destroying and shrinking cannalicular walls. Recanalization is,
however, more common with these methods of occlusion.
Tamponade methods occlude the drainage system with a foreign body.
They are by far the most popular and commonly performed techniques, as
they require no surgery and can be easily performed as an outpatient pro-
cedure. Absorbable inserts made of collagen are available locally. They are
Chapter_17.indd 332 06-02-2018 22:53:21

Dry Eye Disease 333
(A) (B)
(C)
Figs. 17.12A to C: Punctum patch (A) the conjunctival piece overlying the punctum
is separated and removed, (B) the autologous conjunctival graft from nearby bulbar
conjunctiva is raised and (C) patched over the punctal opening (adapted after Murube).
inserted into the vertical or the horizontal canaliculus after topical anesthe-
sia and punctal dilatation. The collagen inserts dissolve slowly over 2 weeks.
Non-absorbable tamponade can be achieved by intracanalicular occlu-
sion or by punctal occlusion. Punctal plugs made of silicone, HEMA or,
more recently, acrylic are inserted into the vertical portion of the canalicu-
lus with the head of the plug left protruding from the punctum (Fig. 17.13).
The intracanalicular plug is a silicone or acrylic plug that is inserted past
the punctum into the horizontal portion of the canaliculus until it becomes
lodged just in front of the common canaliculus.
Treatment of Underlying Causes of Dry Eye Disease
The therapies described in the previous sections only improve the signs
and symptoms of dry eye, but do not treat the underlying condition. New
Chapter_17.indd 333 06-02-2018 22:53:22

Fig. 17.13: Punctal plug in place for punctal occlusion.
findings are demonstrating that a chronic immune-mediated inflamma-

tory process plays an essential role in the pathogenesis of dry eye disease.
Increased inflammatory cytokines have also been demonstrated, as has been
lack of hormonal support or altered innervations.
The following are the main anti-inflammatory, immunomodulatory, or
hormonal therapeutic agents tested or under investigation for the treatment
of dry eye.
Cyclosporin A
Cyclosporin is an established immunomodulator drug used to prevent rejec-

tion after organ or tissue transplantation and as a treatment for a variety of
autoimmune diseases. Early treatment with cyclosporin have shown good
results in patients with KCS. This product is likely to be of significant benefit
in patients with dry eyes that do not have extensive lacrimal gland damage
and where reversal of inflammation in the conjunctiva is likely to be useful.
Trials have shown that cyclosporin A ophthalmic emulsion results in a sig-
nificant improvement in the signs and symptoms of dry eye disease.20 It has
recently been approved by FDA.
Topical Corticosteroids
Topical corticosteroid therapy is useful for short-term treatment to reduce

the initial inflammation. Long-term use would be hampered with the signif-
icant risk of side effects like raised intraocular pressure, cataract formation,
and secondary infections. Topical steroids (preferably nonpreserved) are,
therefore, more suitable for acute management of dry eye exacerbations.
Systemic Tetracyclines
In severe blepharitis, especially in rosacea-associated meibomitis (especially

if associated with KCS), oral tetracycline and its derivatives have become the
treatment of choice.
Chapter_17.indd 334 06-02-2018 22:53:22

Dry Eye Disease 335
Sexual Hormones
The relationship between hormone levels and tear production is complex,

but it is still unclear how different sexual hormones work to regulate the func-
tional activity of the lacrimal tissue. Human trials are lacking though animal
studies suggest that androgen therapy suppresses the inflammation and stim-
ulates the function of lacrimal glands
Topical Autologous Serum
Topical autologous serum has been proposed to deliver components such as

vitamin A, EGF, and TGF beta to the ocular surface of patients with dry eye
disease. Patients using autologous serum diluted 20% in saline for four weeks
show improvement in Rose Bengal and fluorescein staining. Practical prob-
lems with the use of such a product are obvious, the patient’s blood needs to
be extracted and processed and the serum can only be kept refrigerated for
a short period of time. However, it is of significant benefit in a selected group
of patients, especially those with secondary persistent epithelial defects.
REFERENCES
1. Holly FJ, Lemp MA. Tear physiology and dry eyes. Surv Ophthalmol. 1977;
22(2):69-87.
2. Stern ME, Buerman RW, Fox RE, et al. The pathology of dry eye: the interaction
between the ocular surface and lacrimal glands. Cornea. 1998;17(6): 584-9.
3. Tseng SC, Tsubota K. Important concepts for treating ocular surface and tear
disorders. Am J Ophthalmol. 1997;124(6):825-35.
4. Jacobsson Lt, Axell TE, Hansen BU, et al. Dry eyes and mouth—an epidemi-
ological study in Swedish adults, with special reference to primary Sjögren’s
syndrome. J Autoimmun. 1989;2(4):521-7.
5. Hikichi T, Yoshida A, Fukui Y, et al. Prevalence of dry eye in Japanese eye centers.
Graefes Arch Clin Exp Ophthalmol. 1995;233(9):555-8.
6. Wilson SE, Liang Q, Kim WJ. Lacrimal gland HGF, KGF, and mRNA levels increase
after corneal epithelial wounding. Invest Ophthalmol Vis Sci. 1999; 40(10):
2185-90.
7. Reiger G. The importance of the precorneal tear film for the quality of optical
imaging. Br J Ophthalmol. 1992;76(3):157-8.
8. Doane MG. Interaction of eyelids and tears in corneal wetting and the dy-
namics of the normal human eyeblink. Am J Ophthalmol. 1980;89(4):
507-16.
9. Dilly PN. Structure and function of the tear film. Adv Exp Med Biol. 1994;
350:239-47.
10. Lemp MA. Report of the national eye institute/industry workshop on clinical
trials in dry eyes. CLAO J. 1995;21(4):221-32.
11. Stern ME, Beuerman RW, Fox RI, et al. The pathology of dry eye: the interaction
between the ocular surface and lacrimal glands. Cornea. 1998;17(6): 584-9.
Chapter_17.indd 335 06-02-2018 22:53:22

12. Becquet F, Goldschild M, Moldovan MS, et al. Histopathological effects of top-

ical ophthalmic preservatives on rat corneoconjunctival surface. Curr Eye Res.
1998;17(4):419-25.
13. Broadway DC, Grierson I, O’brien C, et al. Adverse effects of topical anti-
glaucoma medication. I. The conjunctival cell profile. Arch Ophthalmol.
1994;112(11):1437-45.
14. Berdy GJ, Abelson MB, Smith MA, et al. Preservative-free artificial tear pre-
parations. Assessment of corneal epithelial toxic effects. Arch Ophthalmol.
1992;110(4):528-32.
15. Green K, Tonjum A. Influence of various agents on corneal permeability. Am J
Ophthalmol. 1971;72(5):897-905.
16. De Saint Jean M, Brignole F, Bringuier AF, et al. Effects of benzalkonium chloride
on growth and survival of Chang conjunctival cells. Invest Ophthalmol Vis Sci.
1999;40(3):619-30.
17. Wilson LA. To preserve or not to preserve, is that the question? Br J Ophthalmol.
1996;80(7):583-4.
18. Yen MT, Monroy D, Pflugfelder SC. Punctal occlusion decreases tear produc-
tion, clearance, and ocular surface sensation. Invest Ophthalmol Vis Sci.
1999;40:980-1.
19. Murube J, Murube E. Treatment of dry eye by blocking the lacrimal canaliculi.
Surv Ophthalmol. 1996;40(6):463-80.
20. Sall K, Stevenson OD, MundoWrf TK, et al. Two multicenter, randomized stud-
ies of the efficacy and safety of cyclosporine ophthalmic emulsion in mod-
erate to severe dry eye disease. CsA Phase 3 Study Group. Ophthalmology.
2000;107(4):631-9.
Chapter_17.indd 336 06-02-2018 22:53:22

18
CHAPTER
Ocular Surface Reconstructions
Manotosh Ray, Rajesh Sinha, M Vanathi, Noopur Gupta
INTRODUCTION
Anatomically, the ocular surface comprises cornea, conjunctiva and the
limbus. However, functionally ocular surface is a much bigger unit compris-
ing both main and accessory lacrimal glands, ocular adnexa and tear film
in addition to cornea, conjunctiva and limbus. The anatomical ocular sur-
face is dependent on adjacent structures, such as the anterior lamellae of
the lids, the lashes, and lacrimal system for normal function. The role of ocu-
lar surface is to maintain optical clarity of the cornea by regulating hydra-
tion and to protect the globe from toxic, infectious and mechanical trauma.
Ocular surface is highly complex in its functionality that is maintained by an
intricate homeostatic system. Severe ocular surface disorders (OSD) cause
significant ocular morbidities and eventually, corneal blindness in a large
number of patients. The management of OSD has greatly evolved in the past
3 decades with tremendous improvements in clinical knowledge and scien-
tific advancements.
In the past, patients with severe OSD had a foregone conclusion of grim
prognosis with only available temporary symptomatic measures including
tarsorrhaphy, Gunderson’s flap or artificial tears. Recent scientific advance-
ments have provided clearer understanding of the ocular surface. Newer
therapeutic and surgical methods provide advanced options for OSD man-
agement in an attempt to maintain reasonable eyesight. These days, we have
a number of potent tools in our armamentarium to deal with severe OSD,
namely, stem cell transplantation, amniotic membrane graft (AMG), tissue
engineering products, range of prosthetic devices and bioartificial microsys-
tem. Ocular surface reconstruction (OSR) has emerged as the treatment
modality of choice for OSD refractory to conventional medical therapies.
Chapter_18.indd 337 06-02-2018 23:03:21

OCULAR SURFACE
Cornea, conjunctiva and limbus are lined by nonkeratinized squamous epi-
thelium. The conjunctival epithelium contains mucin producing goblets
cells. Corneal epithelium, which is distinctly different from conjunctiva pro-
vides intraocular homeostasis, assists refractive function and maintain avas-
cularity of cornea. It has a regenerative property and aptly supported by tear
film, lacrimal glands and eyelids. The primary function of ocular surface is to
provide clear vision. The cornea contributes approximately two-thirds of total
refractive power of the eye. The intricate relationships between ocular sur-
face and preocular tear film ensure the health of the ocular surface. A normal
tear film is fundamental to maintain the integrity and the function of human
cornea and conjunctiva. It’s a two-way contribution to develop a sustained
relationship. On one hand, a stable tear film protects ocular surface epithe-
lium, and on the other hand, the epithelia also actively participate in forming
a stable tear film. The tear film plays a significant role through its antimicro-
bial, mechanical, optical and nutritional properties.
The function of the eyelids includes ocular protection, maintenance and
dispersion of tear meniscus and to minimize the tear film disintegration.
Controlled eyelid blinking mechanically spreads the tear film to lubricate
the ocular surface. The blinking movement protects the ocular surface from
unwanted stimuli and allows the eyelids to spread the tear meniscus over the
ocular surface. Impairment of this protective reflex mechanism may make
the ocular surface susceptible to pathologic insults. Blinking also activates
the lacrimal pump when tear is removed from the ocular surface and drained
into the nasolacrimal system via puncta. Eyelid disorders, like trichiasis,
ectropion or entropion have deleterious effects on tear film stability and can
adversely affect the ocular surface integrity. Advances in microsurgical tech-
niques and understanding the role of the limbal stem cells have led to great
improvements in both visual acuity and quality of life.
EVOLUTION OF OCULAR SURFACE TRANSPLANTATION

The management of severe ocular surface diseases has hugely benefited
from recent major breakthroughs in this field. Previously, the available OSR
techniques, e.g., penetrating and lamellar keratoplasties, superficial kerato-
plasties, tarsorrhaphy, etc. had resulted in recurrent conjunctivalization of
corneal surface. Frustrating results from the earlier techniques were observed
mainly due to non-regeneration of corneal epithelium after sloughing of
donor epithelium. This resulted in eventual conjunctivalization leading to
permanent corneal opacification. However, the modern treatment of severe
OSD is quite different.
The concept of limbal stem cells and the effect of their deficiency are para-
mount in understanding of modern OSR. Limbal stem cell deficiency (LSCD)
has many deleterious complications. Stem cells in the palisades of Vogt
participate in regeneration and preservation of corneal transparency as well
Chapter_18.indd 338 06-02-2018 23:03:21

Ocular Surface Reconstructions 339
Table 18.1: Indications for ocular surface reconstruction.
Conjunctival autograft Limbal autograft/allograft Amniotic graft

Pterygium Chemical injury Fornix reconstruction
Bullous keratopathy Thermal burns Chemical injury
Reconstruction of canthal angle Contact lens keratopathy Immune melts
Fornix reconstruction Persistent epithelial defect Pterygium
Post-excision of conjunctival Post-multiple surgery limbal Conjunctivochalasis
tumor depletion OSSN
Symblepharon repair Chronic medication toxicity Limbal dermoid
Stevens-Johnson syndrome Symblepharon
Ocular cicatricial pemphigoid Leaking blebs
Aniridia
Atopy
OSSN: Ocular surface squamous neoplasia.
as avascularity.1 The diminished or lost regenerative capacity observed in

LSCD is characterized by persistent epithelial defects, corneal erosion, con-
junctivalization, chronic inflammation and extensive neovascularization.2-4
Other complications of severe OSD include keratinization, symblepharon
formation, eyelid scarring, trichiasis and severe dry eye. Patients with severe
LSCD experience acute photophobia, pain and loss of vision. In fact, LSCD is
one of the major causes of corneal blindness.2 Total limbal stem cell depletion
requires limbal stem cell transplantation along with keratoplasty, as the later
procedure alone cannot restore the useful vision. Thus, the general objective
of OSR is to restore the anatomical and physiological ocular surface and also
to prevent recurrence of pre-existing ocular surface disease. Table 18.1 rep-
resents broad indications and current approach to the management of OSD.
CONJUNCTIVAL TRANSPLANTATION
In 1977, Thoft described the procedure of conjunctival transplantation. It
is now recognized as the forerunner of modern ocular surface transplanta-
tion surgery. He performed conjunctival autograft in a patient with chemical
injury.5 Conjunctival autograft procedure is based on the theory of conjunc-
tival transdifferentiation. Autologous conjunctival transplantation has lim-
ited value in repopulating the corneal surface unless associated limbal cells
are also harvested for grafting. However, conjunctival grafting can be useful
in suppressing inflammation and scarring in the traumatized cornea and,
thereby, promoting corneal cells proliferation indirectly. This procedure is
considered by many to set the standard in the surgical technique of pteryg-
ium today, since the procedure can provide excellent cosmesis, is safe and
has a low recurrence rate. With the advancement of newer microsurgical
techniques, the role of conjunctival grafting is primarily limited to pterygium
surgery and occasional fornix reconstruction (Figs. 18.1 and 18.2).
Chapter_18.indd 339 06-02-2018 23:03:21

Fig. 18.1: Fleshy pterygium.
Fig. 18.2: Same patient in Figure 18.1 after pterygium excision and conjunctival auto-
grafting.
Efficacy of conjunctival autograft: Plenty of studies available to prove

beyond doubt that conjunctival autografts reduce the recurrence rate signifi-
cantly after pterygium surgery. One such randomized study demonstrated
that conjunctival autografting could achieve a low recurrence rate (2%) in
a population in which bare sclera recurrence rate was 61% for primary
Chapter_18.indd 340 06-02-2018 23:03:22

pterygium, and 82% for recurrent pterygium.6 Other published studies on

conjunctival autografting report varying recurrence rates from 2–39%.7-10 It
is likely that differing surgical technique may account for the wide range of
recurrences reported in pterygium surgery, while inconsistency in defining
recurrence and varying periods of follow up remain additional factors to
consider when comparing these studies.
Traditionally, during pterygium surgery, the conjunctival autografts are
secured in place with absorbable sutures. Cohen et al. first described the
use of fibrin glue during pterygium surgery in 1993.10 Since then, there have
been several reports on the safety and efficacy of fibrin glue in pterygium sur-
gery.11,12 Fibrin glue is a two-component tissue adhesive that mimics natural
fibrin formation. A recent retrospective study on a large cohort suggested that
pterygium surgery with tissue glue leads to significantly lower recurrence rate
when compared with sutures.13
The basic components of fibrin glue are fibrinogen and thrombin. The
commercial product is a two-component system from human plasma that
contains more than fibrinogen and thrombin. The first component contains
highly concentrated fibrinogen, factor VIII, fibronectin and traces of other
plasma protein. The second component contains thrombin, calcium chloride
and antifibrinolytic agent, such as aprotinin. Mixing of two-components pro-
motes clotting and formation of fibrin cross-linking.
Surgical Technique
Conjunctival autografting for pterygium involves three principal steps. These

are:
1. Excision of pterygium from corneo-scleral surface
2. Harvesting the conjunctival graft usually from superior bulbar
conjunctiva
3. Securing the free conjunctival graft on the bare sclera either with suture
or tissue adhesives
The aim is to ensure complete pterygium tissue removal without exces-
sive adjacent tissue damage or scarring. The principles of pterygium excision
are complete removal of all pterygium tissue at Bowman’s layer of cornea and
the sclera and minimizing scarring and astigmatism at the cornea.
Surgical Steps
1. Anesthesia: Subconjunctival or regional anesthesia is applied. For

conjunctival graft harvesting, peribulbar or retobulbar anesthesia is
preferred.
2. Stabilization and exposure of the globe: A corneal traction suture is
applied at 12 o’clock position. This provides excellent horizontal mobili-
zation while excising the pterygium, and vertical mobilization while har-
vesting the graft.
Chapter_18.indd 341 06-02-2018 23:03:22

3. Pterygium excision: The author prefers to excise pterygium from body

and proceed to the head. This approach curtails the vascular supply to
the head and significantly reduces the bulk and facilitates subsequent
excision. Care should be taken not to initiate pterygium body dissection
too far from the limbus, since there is significant horizontal tissue retrac-
tion. The body of the pterygium along with underlying tenon’s is dis-
sected using a Westcott scissors. The limbal attachment of the lesion is
exposed by reflecting the excised body toward the cornea. A no 64 Beaver
blade is used to peel the pterygium tissue from the limbus and Bowman’s
layer of the cornea. The blade is oriented vertically and scraped horizon-
tally to scrape the pterygium tissue from corneal attachment in a lamel-
lar fashion. Hemostasis of the scleral bed is achieved with a wet field
cautery.
4. Harvesting the conjunctival autograft: Since this grafting is integral in
preventing recurrence of pterygium, a meticulous surgical technique for
harvesting the autograft is needed to ensure consistently improved sur-
gical results. It is of paramount importance to have a thin, Tenon’s free
conjunctival graft of adequate size.
Once pterygium is excised, the globe is rotated inferiorly to expose
the superior bulbar conjunctiva. This is the ideal site for the graft har-
vesting as it is covered by the upper lid and adequate tissue available to
fashion a graft. Harvesting a thin, Tenon’s free conjunctival graft requires
practice since no distinct tissue plane exists between conjunctival epi-
thelium and the Tenon’s layer. A fine initial nick at the epithelial layer
allows epithelium to be lifted up without inclusion of the Tenon’s cap-
sule. Blunt tipped conjunctival forceps and scissors prevent inadvertent
buttonholing of the graft. The superficial dissection is carried forward to
the limbus.
The size of an autograft is determined by the size of the recipient bed.
Careful measurement with a caliper and marking of the recipient and
donor bed is useful in preparing the accurate size of the autograft. It is
always better to have a slightly oversized graft in view of tissue retraction.
It is essential that the conjunctival graft be positioned correctly with the
epithelium facing up. Inadvertent inversion of the graft results in slough-
ing within weeks after surgery. To prevent this, one should slide the graft
from the harvest site into recipient bed without lifting away from cornea.
Graft inversion can be detected by fluorescein staining when examined
under cobalt blue light.
5. Securing the conjunctival autograft: Conjunctival autograft is secured in
place with interrupted 10-0 vicryl sutures. Initial sutures are placed at the
limbal limits of the graft followed by superior and inferior edges. The graft
is secured with underlying episclera to prevent displacement. Finally, a
central limbal suture is also necessary to prevent forward migration of
the graft.
Chapter_18.indd 342 06-02-2018 23:03:22

Fig. 18.3: Conjuctival autografting after pterygium excision; outline of the graft is high-
lighted with fluorescin.
When securing the conjunctival autograft with fibrin glue, the graft can
be placed on the cornea with the stromal side facing upward (Fig. 18.3). The
two-components of tissue glue are mounted on two separate syringes on a
Duploject injection system. Two to three drops of the adhesive are placed on
the scleral bed and the conjunctival graft is quickly flipped over the area of
conjunctival defect. The graft is smoothed out swiftly with a non-toothed for-
ceps while thrombin is breaking down the fibrinopeptides to form the fibrin
clots. The graft-fibrin glue is left alone for one or 2 minutes to form firm adhe-
sions (Fig. 18.4).
Postoperative Treatment
Postoperatively, for about a month, the eyes are treated with topical steroid
antibiotic combinations to reduce the ocular surface inflammation. Before
discontinuing, the dosage can be tailed off slowly. Premature suture breakage
with localized graft retraction requires early re-suturing to prevent recurrence.
Complications
Pterygium surgery is generally a safe procedure. Being an extra-ocular sur-

gery, the chances of sight-threatening complications are almost unknown.
However, complications may still occur both at intraoperative as well as post-
operative stages. The list of complications associated with conjunctival auto-
grafting are depicted in Table 18.2.
Chapter_18.indd 343 06-02-2018 23:03:22

Fig. 18.4: Conjunctival autograft in situ using fibrin glue.
Table 18.2: Complications of pterygium surgery

with conjunctival autograft.
Intraoperative
Rectus muscle disinsertion
Corneo-scleral perforation
Graft inversion
Early postoperative
Graft hemorrhage
Graft oedema
Graft retaction
Graft necrosis in inverted graft
Dellen formation
Late postoperative
Conjunctival granulomas
Epithelial inclusion cyst
Steroid induced glaucoma
Corneal astigmatism and scarring
Recurrence
AMNIOTIC MEMBRANE TRANSPLANTATION

The observations of Brown,14 who used the rabbit peritoneum to promote
healing and prevent spread of necrosis of ocular surface in acute burns,
prompted Sorsby et al.15,16 to use chemically processed, dry amniotic
Chapter_18.indd 344 06-02-2018 23:03:23

Fig. 18.5: Histology of amniotic membrane (AM).
membrane as a patch for treating acute ocular burns. Kim and Tseng in
1995,17 advocated the use of preserved human amniotic membrane for the
management of various OSD.18,19 Amniotic membrane enhances growth and
differentiation of conjunctival epithelial cells20 and is reported to inhibit sub-
conjunctival scar tissue formation.21 Amniotic membrane is considered to be
a favorable substrate for OSR.22-30
Characteristics of Amniotic Membrane
Amniotic membrane (AM) is the inner layer of the fetal membranes. It con-
sists of an epithelium, thick continuous basement membrane and an avas-
cular stromal matrix that contains a high concentration of basic fibroblast
growth factor,31 basement membrane components32-34 and several trophic
factors (Fig. 18.5). Recent evidence indicates that AM has anti-inflammatory
antiproteocytic35,36 and antimicrobial activities.24,25 The stromal matrix of the
AM contains protease inhibitors37 and anti-inflammatory proteins.38 It sup-
presses TGF-b signaling, proliferation and myofibroblast differentiation of
human corneal and limbal fibroblasts.39,40 It is avascular and antiangiogenic.
It does not express histocompatability antigens and has antibacterial proper-
ties. AM facilitates epithelial cell migration, reinforces adhesion of basal epi-
thelial cells, diminishes their apoptosis and promotes their differentiation.
Wound Healing Features of Amniotic Membrane
The rapid healing and reduction of ocular inflammation following amni-

otic membrane transplantation (AMT) has been explained by the following
mechanisms:
Chapter_18.indd 345 06-02-2018 23:03:23

• The AM provides a new basement membrane, which forms a substrate

for enhancing adhesion and growth of epithelial progenitor cells, includ-
ing stem cells.
• AM also exerts an anti-inflammatory effect. The expression of 1L-1a and
1L-1b is markedly suppressed when human epithelial cells are cultured
on the AM stromal matrix.41
• AM stromal matrix has a direct anti-scarring function secondary to its
suppression of TFG-b signaling and myofibroblast differentiation.
• All these acts in combination to restore the micro-environmental condi-
tions conducive to the growth of the epithelial progenitor cells.
• AM is also thought to promote nerve regeneration by maintaining nerve
growth factors signaling.42
Thus, effective restoration of stromal thickness to normal or near-
normal levels in deep corneal ulcerations using AMT has made it an alter-
native option in management of corneal ulcerations, especially in the devel-
oping countries with preservation facilities of corneal tissues. If the need be,
penetrating keratoplasty (PK) can be performed after AMT in the setting of
anuninflammed ocular surface.
Indications for AMT
Preserved AM has been used in the management of persistent epithelial de-

fects with and without stromal thinning and perforation (i.e., neurotrophic
keratopathy), herpetic infection, autoimmune diseases, shield ulcer, infecti-
ous keratitis and in extensive infectious scleral and corneoscleral ulcers.
AMT combined with glue may also be used for treating small corneal
perforations.
The common indications of AMT include:43
• AMT in the presence of stem cell deficiency
Ocular chemical injury
• AMT in the absence of stem cell deficiency
Corneal epithelial defects
Corneal/Corneoscleral ulcers
Bullous keratopathy
• AMT for conjunctival reconstructions
Pterygium
Conjunctivochalasis
Ocular surface squamous neoplasia (OSSN)
Limbal dermoid
Symblepharon
Conjunctival lesions
Leaking blebs
• AMT in ocular cicatricial diseases
Toxic epidermal necrolysis
Chapter_18.indd 346 06-02-2018 23:03:23

Ocular cicatricial pemphigoid

Oculopalpebral and reconstructive surgery
• Other indications of AM use
Stem cell cultures
Preparation of AM
AM is the innermost semi-transparent layer of the foetal membranes and

is separated from the placenta. The human placenta is obtained following
an elective caesarean delivery from a consenting donor tested negative for
human immunodeficiency virus (HIV), hepatitis B, C and syphilis. Under a
lamellar flow hood, the placenta is cleaned of blood clots with sterile saline
solution containing 50 mg/mL of amphotericin B. The AM is separated from
the chorion by blunt dissection and flattened on to nitrocellulose membrane
of appropriate size with the epithelium-basement membrane surface up. This
is stored at -70–80°C in a sterile vial containing Dulbecco’s Modified Eagle
Medium (DMEM) and sterile glycerol (sterilised by autoclaving) in a ratio of
1:1 (vol/vol) along with 3.3% L-glutamine, 25 mg/mL gentamicin, 50 units/mL
penicillin 100 mg/mL ciprofloxacin and 0.5 mg/mL amphotericin B. The vial
is thawed at room temperature for 10 minutes or at 4°C for 30 minutes. The
membranes can be used for up to 6 months after preparation, but the cellular
viability is found to be reduced by more than 50% in 2 months. Damage of
AM cells due to cryopreservation results in decreased levels of AM associated
of growth factors. Upon thawing, before transplantation, the surgeon should
ensure the epithelial side of the AM identified by its stickiness to the tip of the
cotton swab.
AMT Surgical Techniques
Amniotic Membrane Overlay

A single layer or multilayered of AM with stromal side down is placed over the
epithelial defect trimmed at the edges and secured to the cornea with 10.0
nylon suture with buried knots. This is performed in cases with nonhealing
epithelial ulcerations with minimal or no stromal thinning. It is also useful in
the effective management of persistent epithelial defects of the cornea and in
cases of acute chemical injuries.
Amniotic Membrane Inlay
In cases of epithelial defects/ulcerations associated with stromal thinning,

AM are placed stromal side down, layer-by-layer, filling up the stromal defect.
The layers are trimmed to fit the size of the ulcer defect. The topmost layer is
fashioned to cover the area of defect and is sutured to the cornea with 10.0
nylon suture with buried knots (Fig. 18.6). The underlying layers, compactly
Chapter_18.indd 347 06-02-2018 23:03:23

Fig. 18.6: Diagrammatic representation of ‘filling-in’ or layered inlay.
Fig. 18.7: Healing metaherpetic ulceration after amniotic membrane inlay.
packed under the larger top layer, are left unsutured. A compact packing of
the stromal ulceration aids in good healing (Fig. 18.7).
Amniotic Membrane Inlay with Overlay
After compact packing of the stromal ulcer defect with multiple layers of AM
and sealing it with a sutured top layer of membrane, an overlay of multilay-
ered AM patch covering the whole cornea may be placed and sutured to the
underlying conjunctiva (Fig. 18.8). Multilayered inlay with onlay grafting of
AM helps in achieving early ocular surface stabilization in severe grades of
ocular chemical injury (Figs. 18.9 and 18.10).
Chapter_18.indd 348 06-02-2018 23:03:23

Fig. 18.8: Multilayered inlay + onlay amniotic membrane grafts (AMG) in neurotrophic
ulceration.
Fig. 18.9: Multilayered amniotic membrane transplantation (AMT) with suture fixation
with symblepharon shell in situ.
Fig. 18.10: Multilayered inlay with onlay amniotic membrane grafts (AMG) in acute
chemical injury.
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Fig. 18.11: Diagrammatic representation of the patch technique.
Amniotic Membrane Patch Technique
AM can be effectively used for patching the cornea by placing the basement
membrane side downward and the stromal side up and suturing it to the sur-
rounding perilimbal conjunctiva (Fig. 18.11). This effectively serves acting as
a patch for the cornea.
Amniotic Membrane for Corneal Perforation
Perforations measuring less than 2 mm in size can be treated with this tech-
nique. The bottom of a perforation is closed with an appropriately sized AM
and multilayer AM graft is then placed packing the defect. A larger piece of
AM is placed on the uppermost to cover the whole ulcer area, trimmed to fit
the ulcer and sutured with an interrupted 10-0 nylon suture.
Amniotic Membrane and Glue in the Management

of Corneal Perforation
The technique consists using of a high-viscosity sodium hyaluronate vis-
coelastic material to restore the anterior chamber depth followed by a
debridement of the ulcer. The perforation site is filled with the human fibrin
glue (HFG) on the corneal surface. The glue plug is then secured with an
AMT to avoid its extrusion and an extended-wear bandage contact lens is
applied.
An alternative technique is the placement of an AM of optimal size in the
anterior chamber directly under the corneal perforation lesion. The cyanoac-
rylate tissue adhesive can then be applied over the perforation site and sealed
successfully.
AMG with glue fixation or suture fixation achieves effective OSR in cases
of symblepharon after chemical injury (Fig. 18.11).
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AMT in Infectious Corneal Ulcers
Pathogenic microorganisms produce disease in the cornea by direct inva-

sion, secretion of toxic products or by both. Most of the proteolytic enzyme
is endogenously released by epithelial and stromal cells and polymorphonu-
clear cells. A series of matrix-metalloproteinases (MMPs) influence corneal
wound healing. Infectious ulcerative keratitis can be modified by controlling
MMP activity in tissue. MMP-2 and MNIP-9 can degrade basement mem-
brane collagens (types 4 and 7).
AM is reported to contain tissue inhibitors of MMP-1 and MMP-2
which can inhibit collagen destruction by MMP-2 and MMP-9. It contains
proteinase inhibitors, like a1 anti-chymotrypsin, a2 macroglobulin, a1 anti-
trypsin, a2 antiplasmin and inter a1 trypsin inhibitor. It also has inhibitory
effects on various proteases in polymorphic nuclear cells.
These suggest the possibility of modulating therapeutic effect of AMT on
infectious keratitis. It also provides an effective barrier against PMN in the
tear film and prevents stromal infiltration of inflammatory cells to the injured
cornea, besides its influences on wound healing. Additional mechanism
involved may be the effect of AMT mimicking collagen shield when the AM
is presoaked in anti-infective agents, thus providing long-term drug delivery
effect. Within 48–72 hours of effective and appropriate antimicrobial therapy,
progression of the keratitis is halted in bacteria induced corneal ulceration.
When the pathogen activity is suppressed and clinical signs indicate improve-
ment of the lesion AMG graft can be applied safely and effectively. AM graft
has also been found to be effective in promoting conjunctival reepitheliali-
zation and reducing scleral melts and inflammation in extensive infectious
scleral and corneoscleral ulcers. AMT has been used as an adjunctive therapy
to promote wound healing and prevent perforation in bacterial keratitis.
AMT in HSV-1 Necrotizing Keratitis
Stromal herpes simplex keratitis (HSK) is an immune-mediated disease.

After reactivation from latency in the trigeminal ganglion, herpes simplex
Fig. 18.12: Symblepharon release and amniotic membrane transplantation (AMT) with
fibrin glue.
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Fig. 18.13: Multilayered amniotic membrane grafts (AMG) after ocular surface squa-
mous neoplasia (OSSN) resection.
virus (HSV) antigens are expressed and corneal antigens exposed. CD4+
T-cells are the principal mediators of the inflammatory response. The cor-
neal damage is caused by proinflammatory molecules and reactive radicals.
Neutrophils, T-cells and macrophages contribute to tissue destruction in the
cornea. The healing process in necrotizing herpes keratitis may be promoted
by AMT which has been found promoting epithelialization, reduce stromal
inflammation and ulceration in HSV-1 keratitis in the animal model study.
AMT in Other Conditions

AM grafting helps ocular surface stabilization in various conditions including
pterygium excision, dermoid excision and excision of OSSN (Fig. 18.13).
Advantages of AM Grafting
The thick basement membrane of the AM facilitates epithelial migration

and reinforces the adhesion of basal epithelial cells causing rapid epithe-
lization. It also promotes epithelial differentiation and prevents epithelial
apoptosis.
Preserved AM expresses mRNAs for several growth factors and it con-
tains various growth factor proteins that might benefit epithelialization. In a
deep stromal ulcer abundant release of inflammatory cytokines and proteo-
lytic enzymes by stromal keratocytes and inflammatory cells induces more
rapid dissolution of the AM necessitating multiple grafting.
Fixation of AM in AMT can be facilitated with either HFG or sutures. Histo-
pathological evaluation has revealed the integration of the transplanted AM44
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Fig. 18.14: Healed neurotrophic ulcer after amniotic membrane transplantation (AMT).
into the host cornea which can be either superficial, intrastromal, intraepi-
thelial or subepithelial. AM graft causes less vascularization on healing and
is relatively easy to perform (Fig. 18.14). It provides more acceptable cosme-
sis, does not disturb healthy conjunctiva and acquires progressive transpar-
ency with time leading to improvement in visual acuity. Hence, AMT is more
advantageous than conjunctival grafting. An AMT procedure produce sless
tissue inflammation compared to tissue adhesives. Performing multilayer
AMT successfully manages small corneal perforations and help averting an
emergency tectonic keratoplasty. An optical PKP can be performed later in a
more favorable setting with a better outcome of vision.
Nonsurgical Innovations
AMX or amniotic membrane extract: This is a lyophilized human AM

currently under evaluation and potentially can be applied topically to the
ocular surface. The AMX preparation is in the form of a lyophilized powder
which requires the addition of saline for reconstitution as an eye drop. As it
is non-preserved, it must be retained in normal refrigeration for no longer
than 72 hours. When applied topically, it promotes wound healing and works
same way as transplanted AM. Preliminary report reveals that the therapeu-
tic efficacy of AMX is comparable to AMG, but further studies are necessary
before it is made commercially available.
Prokera: This is another innovation in the field of AM. Prokera is a cryopre-
served AM clipped into a polycarbonate ring set. The polycarbonate rings are
available as 16 mm diameter and are slightly thicker than ordinary soft con-
tact lens. When applied to the eye with OSD, the AM gets absorbed and only
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Fig. 18.15: Location of limbal stem cells.
the ring is left behind which can be removed conveniently. Prokera remains
in the eye up to 30 days. However, it facilitates healing of most defects within
7–10 days.
LIMBAL STEM CELL TRANSPLANTATION

Corneal limbal stem cell transplantation is a process whereby corneal stem
cells at the limbus (corneoscleral junction) are replaced surgically in order to
regenerate the ocular surface and helps in restoration of vision. The host stem
cells normally reside in the limbal area and are responsible for the homeosta-
sis, maintenance and regeneration of ocular surface epithelium (Fig. 18.15).
Transplantation is required when the host stem cells have been severely
damaged due to disease or injury.
Relevant Anatomy
The ocular surface is a complex biological layer consisting of the mucosal

epithelial lining limited by the mucocutaneous junction of the superior and
inferior eyelid margins. Mainly, it is composed of the corneal and conjunc-
tival epithelium separated by the limbus. The precorneal tear film, neuronal
innervation and the protective blink reflex help sustain a favorable milieu for
ocular surface stability and regeneration.
The corneal epithelium plays an essential role in maintaining corneal
clarity and regular refractive surface of the cornea. The corneal epithelium is
composed of non-keratinised, stratified squamous epithelial cells. Its thick-
ness is approximately 50 mm (five to six layers of cells). The most superficial
Chapter_18.indd 354 06-02-2018 23:03:24

layer of the epithelium consists of flattened cells with microvilli. The tear film
protects the cornea from dryness and aids in maintenance of a regular and
smooth epithelial surface. The conjunctival epithelium is 1–2 cell layers thick
with goblet cells intermixed with epithelial cells. The desquamated cells of
the corneal epithelium are replenished by a small population of stem cells
located in the basal layer of the limbal epithelium.
Limbal Stem Cells
The adult eye harbors stem cells near the corneal limbus, in the conjunc-
tivalfornices, the pars plana and pars plicata of ciliary body and the adult
human retina. The corneal limbus harbors the corneal epithelial stem cells
and contributes to the unique microenvironment of the stem cell niche sur-
rounded by tissue, cells, and substrates, which control the self-renewal and
differentiation potential of stem cells. Stem cells are defined by their capac-
ity of unrestrained or prolonged self-renewal that can produce differentiated
progenitor cells. They maintain homeostasis in normal conditions as well as
following injury. Stem cell niche is a special milieu consisting of several cellu-
lar and extracellular components in the vicinity. The niche is responsible for
the biologic regulation of stem cells.44
At the corneoscleral limbus, there is a gradual shift from the stratified,
nonkeratinized squamous epithelium of the cornea to the stratified, nonke-
ratinized columnar epithelium with mucin-secreting goblet cells of the con-
junctiva. It has 7–10 layers of cells that are arranged as palisades of Vogt and
are home to the stem cells and transient amplifying cells, an intermediate
population of progenitor cells (Fig. 18.16). Limbal stem cells are shown to be
abundant in the superior and inferior limbus as compared with the temporal
Fig. 18.16: SCs (white)—basal limbal epithelium; Transient amplifying cells—basal epi-
thelia of limbus and peripheral cornea; Postmitotic and terminally differentiated cells—
suprabasal and superficial layers.
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Table 18.3: Putative markers for corneal limbal epithelial stem cells.
Corneal epithelium Limbal epithelium

Markers Basal Suprabasal Suprabasal Basal
Cytoplasmic marker
Keratin K3/K12 ++ ++ + -
Keratin K19 - - - ++
Nuclear marker
p63 (+) - (+) ++
Cell surface markers
Connexin 43 ++ + + -
Transporter
ABCG2 - - - ++
and nasal limbus. The pigmented perilimbal area contains the undifferen-
tiated limbal epithelial stem cells (LESC) that reside in the limbal epithelial
crypts.45 Limbal stem cells act as a physiological barrier to the ingress of con-
junctival cells across the cornea.
Stem cells at the limbus are characterized by slow cycling and possess
potential for rapid proliferation. The differential expression of putative limbal
stem cell markers, like keratins, vimentin and integrins provides direct evi-
dence that basal limbal epithelium contains the unique and least differenti-
ated cells of corneal epithelium (Table 18.3). Keratin 19 (CK 19) is expressed
in the basal limbal epithelium in adults, suggesting the persistence of embry-
onic young (stem) cells at the limbus in adults. Keratin 3 (CK 3), which is the
differentiation keratin, is demonstrable in the entire corneal epithelium and
suprabasal limbal epithelium, but not in the basal epithelium of the limbus.
The ATP binding cassette transporter protein, ABCG2, has been proposed as
a possible LESC marker.46
Limbal Stem Cell Deficiency
LSCD may be primary or secondary (Table 18.4). Primary LSCD is charac-

terized by the absence of identifiable external factors and results due to an
insufficient microenvironment that is unable to support the limbal stem cells.
Secondary LSCD occurs due to the destruction of limbal stem cells by extrin-
sic factors (Fig. 18.17). It may be diffuse (total) or sectoral (partial) depending
on the degree of involvement.
The clinical features of LSCD include photophobia, blurred or decreased
vision, ocular discomfort, tearing or recurrent episode of pain from epithelial
breakdown and history of chronic inflammation and redness. In partial stem
cell deficiency, a clear line of demarcation may be visible between corneal
and conjunctival phenotype of cells.
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Table 18.4: Causes of limbal stem cell deficiency.
Primary/Hereditary
Aniridia
Congenital erythrokeratodermia
Keratitis associated with multiple endocrine deficiencies
Secondary/Acquired (more common)
Chemical or thermal injuries
Stevens-Johnson syndrome
Toxic epidermal necrolysis
Ocular cicatricial pemphigoid
Multiple ocular surgeries or cryotherapies involving limbus
Immunological disorders
Ultraviolet and ionizing radiation
Contact lens wear
Extensive microbial infection
Chronic limbitis (vernal, atopy, phlyctenular)
Limbal tumors
Antimetabolite use (5- FU, mitomycin C)
Neurotrophic (neural and ischemic) keratopathy
Pterygium
Peripheral ulcerative keratitis (Mooren’s ulcer)
Chronic bullous keratopathy
Trauma
Fig. 18.17: Total and partial limbal stem cell deficiency (LSCD).
The clinical signs of LSCD may vary depending on its severity, and may
include:
• Loss of limbal architecture and palisades
• Irregular, thinned out epithelium
• Stippled fluorescein staining of the area covered by abnormal epithelium
• Filaments and erosions
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• Superficial and deep vascularization of cornea (conjunctivalization)

• Persistent epithelial defects leading to ulceration, melting and
perforation
• Chronic inflammation
• Fibrovascular pannus
The clinical diagnosis of LSCD can be confirmed histologically by the
use of impression cytology which confirms the presence of conjunctival
goblet cells on the corneal surface. Immunohistochemically, the absence of
a cornea-type differentiation, such as the absence of keratin CK 3 (marker
of corneal phenotype), presence of cytokeratin 19 to confirm a conjunctival
phenotype, and the presence of mucin in goblet cells, can be demonstrated
by monoclonal antibodies.
Management
The goal of treatment for severe LSCD is to restore the anatomic and phys-
iologic environment of the ocular surface by reconstruction of the corneal
and conjunctival epithelium. The conservative options available for man-
aging patients with LSCD are intensive non-preserved lubricants, bandage
contact lenses and autologous serum eye drops. Management strategies for
LSCD have greatly evolved in the recent years with improved understanding
of the role of limbal stem cells, advanced microsurgical techniques and better
immunosuppressive regimen.
In patients with total LSCD, limbal auto- or allo-transplantation are
indicated for OSR. Pellegrini et al.47 first demonstrated that LESCs can be
expanded ex-vivo in humans to generate cohesive sheets of corneal epithe-
lium which can effectively restore the corneal surface of patients with LSCD.
Limbal stem cell transplantation is a surgical treatment to address LSCD and
restore a corneal epithelial phenotype. A holistic approach toward manage-
ment of LSCD includes treatment of underlying systemic disease, ocular
adnexal pathology and dry eye.
Partial LSCD
Asymptomatic patients with partial and peripheral conjunctivalization of

the corneal surface may not need any intervention. It has been shown that
repeated debridement of migrating conjunctival epithelium in the acute
phase following injury, known as sequential sector conjunctival epitheliec-
tomy can reduce or prevent conjunctival ingrowth in cases with partial stem
cell deficiency.48
The transplantation of AM as an inlay to promote corneal epithelial
migration into the area of LSCD may be successful in partial LSCD and
reconstruction of the ocular surface (Fig. 18.18). AM is thought to provide
growth factors that encourage corneal regeneration from the patient’s own
limbal stem cells. Human AM has been shown to facilitate wound healing by
promoting epithelial cell migration and adhesion by containing growth
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Fig. 18.18: Amniotic membrane transplantation (AMT) for limbal stem cell deficiency
(LSCD).
factors that encourage healing and retarding corneal neovascularization

through its anti-inflammatory properties.49
Surgical Techniques for Limbal Cell Transplantation
Numeroustechniques have been described to replace limbal stem cells with

the aim of achieving a stable ocular surface. Although, all the techniques are
similar in principle, the variation lies in the source of donor stem cells and
the carrier tissue used for transplantation of stem cells to the recipient site
(Fig. 18.19). Currently, the main clinical procedures that are in vogue, include
a conjunctival limbal autograft (CLAU) using tissue from the fellow eye; a
living related conjunctival limbal allograft (lr-CLAL), where a living relative
donates conjunctiva and limbal tissue; and keratolimbal allograft (KLAL),
utilizing a cadaveric donor where the peripheral cornea is used to transfer
the limbal stem cells. Ex vivo expanded limbal stem cells or oral mucosa cells
are also being used successfully to recreate a healthy ocular surface.
Preoperative Assessment
Survival of limbal stem cells depends on the limbal stem cell niche that is,
in turn, maintained by the tear film, corneal vascularity and innervation. To
achieve higher level of sucess rates in limbal transplantation, several issues
are addressed:
• Preoperative correction of eyelid, eyelashes and fornix abnormalities
prior to transplantation for optimizing tear film status
• Adequately control inflammation using topical and systemic medica-
tions for at least 3–6 months before surgery
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Fig. 18.19: Preoperative and postoperative photographs after stem cell transplantation.
• Correct assessment of the extent of LSCD

• Management of glaucoma
• Choosing the best method to restore limbal stem cells and individualiz-
ing therapy according to patients’ condition, including:
CLAU for unilateral disease
lr-CLAL for bilateral limbal deficiency (preferably partial limbal
involvement) with moderate to severe conjunctival disease
KLAL for bilateral limbal deficiency with minimal to moderate con-
junctival disease
Combined lr-CLAL and KLAL, for bilateral limbal deficiency and
severe conjunctival disease
Conjunctival Limbal Autograft
Limbal autograft transplantation is limited to unilateral cases where the

healthy fellow eye is a suitable source of limbal stem cells. In these cases, the
Chapter_18.indd 360 06-02-2018 23:03:25

(A)
(B)
Figs 18.20A and B: Conjunctival limbal autograft in a patient with chemical injury.
(A) Preoperative, (B) Postoperative.
procedure involves lamellar removal of two segments of conjunctiva, includ-

ing 4–6 mm of conjunctival tissue (nearly 2–3 clock hours), moving anteriorly
to remove a partial-thickness limbal epithelium of about one-third thickness.
Preparation of the recipient eye begins with 360° peritomy and sharp and
blunt dissection of the fibrovascular pannus over the cornea. Subsequently,
the donor segments are secured at the 6 and 12 o’clock positions with sutures
or bioadhesive fibrin glue (Fig. 18.20A and B).
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The main concern with this procedure is, inducing stem cell deficiency
in the fellow eye. The risk to the donor eye is extremely low if the donor eye
is truly healthy with no long-term contact lens usage or subclinical exposure
to original trauma and less than 6 clock hours of limbal tissue is removed.50
Although CLAU is an autograft and there is no risk of immunologic rejec-
tion, like all forms of stem cell transplantations, it must be considered only
after adequate control of ocular inflammation to provide a better environ-
ment for transplanting cells.
Living-related Conjunctival Limbal Allograft
The surgical technique of lr-CLAL is similar to CLAU. However, the source of

stem cell is a living relative instead of the fellow eye. HLA typing on all poten-
tial donors is helpful in finding more compatible tissue for transplantation.
Potential donors with long-term contact lens usage and glaucoma, who may
eventually require trabeculectomy, should be excluded. Serologic testing of
potential donors for syphilis, hepatitis B, C and HIV infection should be per-
formed to avoid risk of transmission to the recipient.
This procedure provides conjunctival and limbal stem cells to the host
with some degree of histocompatiblity. The use of allogenic grafts is associ-
ated with the risk of graft rejection and can be suspected with the development
of inflammation along with acute or chronic severe surface abnormalities.
The clinical features of limbal allograft rejection vary according to the presen-
tation, whether acute or chronic, of the rejection episode. Acute rejection is
associated with intense sector injection at the limbus, edema and infiltration
of the lenticule, punctate keratopathy and epithelial defects. In low-grade
rejection, there is mild diffuse or perilimbal injection, elevated perilimbal
area, punctate epithelial keratopathy and epithelial irregularity.51 These
patients require long-term systemic immunosuppression therapy with cyc-
losporine, FK 506 or mycophenolate mofetil.
In addition to stem cells, live related CLAL provides healthy conjuncti-
val tissue, but it does not cover 360° of the limbus that may allow conjunc-
tivalization of the surface in total stem cell deficiency. Hence, lr-CLAL may
result in better outcome for patients with partial stem cell deficiency. In severe
cases wherein the patient has extensive conjunctival disease and total LSCD,
combined KLAL and lr-CLAL, called the ‘Cincinnati procedure,’ maximizes
the advantages inherent in both the procedures. Introduced by Holland et al.,
the Cincinnati procedure begins with recipient eye preparation as for stan-
dard KLAL.52 Conjunctival tissues are placed superiorly and inferiorly and
keratolimbal tissue is used to fill in the gaps nasally and temporally. Systemic
immunosuppression is required and these patients may be at higher risk for
immunologic rejection because two different types of antigenic tissues are
used.
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Fig. 18.21: Kerato-limbal graft.
Keratolimbal Allograft
KLAL uses peripheral cornea as the carrier for allogenic cadaveric stem cells.
Tissue from the youngest possible donor with an upper limit of 50 years is
recommended.53 In this procedure, the central cornea is removed with a 7.50-
mm trephine. The rim is bisected and excess peripheral tissue is removed
along with the posterior two-thirds of the stroma, Descemet’s membrane
and endothelium (Fig. 18.21). The host bed is prepared by performing 360°
conjunctival peritomy and releasing areas of symblepharon. Superficial ker-
atectomy is performed to peel off pannus and conjunctivalized tissue, thus
creating a regular and smooth surface (Fig. 18.22A and B).
The prepared limbal grafts are secured to the eye using 10-0 nylon sutures
or fibrin glue, trying to match the donor’s and recipient’s limbus. KLAL does
not provide conjunctival tissue and, therefore, it is the procedure of choice
for patients with primary limbal involvement with minimal conjunctival
involvement, including aniridia. Immunosuppressive therapy is required
postoperatively.
Bioengineered Ocular Surface Equivalents/ex vivo Stem

Cell Expansion
Transplantation of cultivated limbal stem cells for the treatment of partial
and total LSCD results in successful corneal epithelialization and stability
of regenerated epithelium giving relief in patient’s discomfort and visual
Chapter_18.indd 363 06-02-2018 23:03:26

(A)
(B)
Figs. 18.22A and B: Preoperative (A) and postoperative (B) outcome of keratolimbal
allograft.
acuity. It is a major breakthrough in the field of OSR. Ex vivo cultivated stem

cell transplantation provides a useful method to restore the stem cell popu-
lation in LSCD,54 with the expanded LESC retaining their in vivo properties
of being slow cycling, label retaining and undifferentiated.55 This procedure
eliminates the need for immunosuppression as autologous tissue is utilized.
Moreover, removing a small limbal biopsy of about 1–2 mm from the healthy
eye does not pose a considerable risk. In particular, the limbal niche may
need to be restored for the procedure to become effective over time.
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Fig. 18.23: Culture of corneal limbal epithelial stem cells (CLESC) over human amniotic
membrane (HAM).
This method uses completely autologous xenobiotic-free bioengineered

ocular equivalent for clinical transplantation. For this procedure, a small
limbal biopsy (2 mm2) is required. This is then cultivated on various sub-
strates, such as AM or fibrin-based substrates with cell culture media, to
obtain sheets of cell layers with confluence (Fig. 18.23). This is then trans-
planted on to the diseased eye with the aid of sutures or fibrin glue after
dissecting the pannus tissue. The composite AM ex vivo cultured LEC grafts
acts as a biological bandage providing sufficient time for the patients’ own
endogenous limbal epithelial stem cell population to regenerate.
Oral mucosal cells have been used as an alternative source of stem cells
for treating LSCD. A biopsy of mucosal epithelium and a small amount of sub-
mucosal tissue is harvested under local anesthesia. Prior to biopsy, a compre-
hensive oral hygiene program is followed, including tooth decay treatment,
regular brushing and iodine gargle. The biopsy size varies between 2–9 mm.
The use of autologous oral mucosal epithelium as a source of cells for treating
bilateral LSCD has potential advantages. As the cells are autologous, there
is no risk of immune-mediated rejection. The oral mucosa is thought to be
at a lower stage of differentiation than epidermal keratinocytes, they divide
rapidly, and they can be maintained in culture for prolonged periods without
keratinization.56 Moreover, keratin 3 is expressed by both corneal epithelium
and oral mucosa, but not by epidermis suggesting that gene expression in
oral and corneal epithelium may be similar.
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Postoperative Management and Immunosuppression
Postoperative management of patients, who undergo limbal stem cell trans-

plantation, is one of the most important factors that determine the success
rate and outcome. Postoperatively, topical antibiotic is used until the surface
is completely epithelialized and topical steroids are used to reduce inflam-
mation. The health of the ocular surface should be optimized with the use of
non-preserved artificial tears, punctal occlusion, bandage lens, tarsorrhaphy
and trichiasis removal. Any factor that destabilizes the ocular surface needs
to be addressed aggressively.
Transplantation of an allograft poses the risk of rejection even in HLA-
matched recipients. Therefore, all allografts, such as KLAL and lr-CLAL need
prolonged systemic immunosuppression. The goal of immunosuppression is
to eliminate eye inflammation and prevent allograft rejection. Topical immu-
nosuppressants are usually insufficient in controlling allograft rejection after
KLAL. In general, it is recommended to co-manage the patient with an organ
transplant immunologist to minimize the risk of adverse effects.57
Prognosis and Outcome
The outcome of stem cell transplantation can be adversely affected by several

risk factors threatening the ocular surface health. The ocular surface health
depends on a stable tear film, which occurs due to the adnexal glands, eye-
lids, neuroanatomic integration of two neural reflexes controlling the secre-
tion of different tear components and eyelid closure. Preoperative clinical
characteristics and risk factors that lead to ocular surface deficits, include
infrequent blinking, blink-related microtrauma, conjunctival inflamma-
tion, increased intraocular pressure, aqueous-deficient dry eye and previ-
ous failed corneal or stem cell graft. Younger patient’s age and performing
PK simultaneously with KLAL may affect the outcome adversely.58 Current
consensus is that autologous limbal grafts (CLAU) have a better prognosis
than allogenic grafts (KLAL). The most significant advantage of CLAU is the
absence of immunologic rejection.
Intraoperative complications of the recipient include damage caused to
the muscle during symblepharon release, bleeding during superficial kera-
tectomy, and corneal perforation. Thick donor lenticules result in improper
surfacing of tear fluid with subsequent dellen formation. Friction due to lid
movements may dislodge the lenticules. Postoperative complications include
epithelial breakdown, limbal allograft rejection, infection and secondary
glaucoma. Early recognition and treatment of limbal allograft rejection is
important to avoid stem cell death and ocular surface breakdown. Infective
keratitis may occur following limbal stem cell transplant.
Conventional PK has been shown to have uniformly poor results in view
of the hostile microenvironment of diseased eyes in LSCD. The judicious
evaluation and management of OSD is important for proper outcome in
Chapter_18.indd 366 06-02-2018 23:03:27

these cases. Currently available therapeutic options are able to rehabilitate

the ocular surface in a reasonably large proportion of eyes. Ongoing research
into the regulatory mechanism involved in the differentiation of limbal stem
cells, the structural and biochemical components of the stem cell niche may
open up exciting frontiers leading to an enhancement of our therapeutic
armamentarium in successfully managing these disorders.
NON-AMNIOTIC SUBSTRATE
Primary limitation of stem cell transplantation is rejection. There have been
tremendous interest and effort in the recent past for developing non-AM sub-
strates that can support confluent growth of stem cells. Although cost and
technical difficulties limit the availability, many scientists have demonstrated
the feasibility of this procedure.
Fibrin Substrate
Fibrin substrates are being investigated for cultivating autologous limbal

stem cells. Rama et al. had successfully transplanted autologous limbal stem
cells cultivated on a fibrin substrate.59 Following transplantation, 78% of the
subjects demonstrated complete epithelization within a week. Other import-
ant observations were significant regression of inflammation and vascular-
ization and formation of a normal looking epithelium within a month. Higa
et al. developed carrier-free corneal epithelial sheets grown on a biodegrad-
able fibrin sealant.60 In comparison to AMT, these carrier-free sheets demon-
strated more differentiation.
Ex vivo Fabrication and Transplantation of Autologous

Oral Mucosal Tissue
This technique is based on a temperature-responsive cell culture surface that
enables harvesting intact transplantable epithelial cells. Nishida et al. had
performed this technique on a group of patients with bilateral total corneal
stem cell deficiencies.61 The authors had demonstrated reasonable success
after this procedure.
Bioengineered Corneal Endothelial Monolayer
Lai et al. had cultivated bioengineered human corneal endothelial cell mono-
layer sheet graft on a thermoresponsive surface, and then transplanted into
rabbit corneas with denuded endothelium.62 Corneal function and clarity
were restored after 6-month observations.
Human Anterior Lens Capsular Scaffold
Human anterior lens capsule can be used as scaffold for ex vivo expansion
of limbal stem cells. There are studies demonstrating excellent growth of
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limbal stem cells on both autologous and allogenic capsules. In one such
study, Galal et al. reported 95% or better cell viability when stem cells were
grown on anterior lens capsule.63 Thus, anterior lens capsule, which is easily
obtained from cataract surgery, can be a potential substrate for future OSR.
Contact Lens as Substrate
Artificial substrates, like contact lenses, are recently been postulated as pos-
sible scaffold to cultivate the cells. It is assumed that the close proximity of
the contact lenses and ocular surface helps to propagate and migrate cells
from the former to replenish the damaged ocular surface. It is also postulated
that secretory factors from contact lenses not only promotes healing, but also
activates limbal stem cells, and thereby prevent neovascularization.64
Implantable and Prosthetic Devices
Corneal blindness is the second most common cause of global blindness

ranking only after cataract. An estimated 10–15 million people are blind,
worldwide, due to cornea-related diseases. Corneal transplant remains the
only option for most of these patients. PK is the frequently performed and
most successful organ transplantation with a success rate nearly 90% in low-
risk cases. However, success rate of PK is dramatically reduced in patients
with OSD, like ocular cicatricial pemphigoid, Stevens–Johnson syndrome,
keratoconjunctivitis sicca and chemical burns. Moreover, shortage of the cor-
neal graft also remains a significant roadblock.
Transplantation of an artificial or synthetic cornea using biocompatiable
materials, such as polymethylmethacrylate (PMMA), is a potential alterna-
tive technique to restore vision in these patients with poor prognostic cor-
neal diseases. However, these extremely invasive procedures are not without
problems. Numerous complications including intractable glaucoma, retro-
prosthetic membrane, implant extrusion and endophthalmitis pose enor-
mous challenge to the success of the surgery.
Keratoprosthesis would be discussed in appropriate place and beyond
the scope of this chapter.
BIOENGINEERED CORNEA
Tissue engineering is an evolving area of research in OSR that is keenly being
monitored by the ophthalmologists worldwide. The concept of bioengineered
cornea came from accomplishment of successful in vitro reconstruction of
skin cells and blood vessels in recent years.64 An ideal bioengineered cor-
nea must be optically transparent and compatible with host ocular surface
into which it is being transplanted. Moreover, it should have enough tensile
strength to bear intraoperative manipulations.
Human cornea has five layers: epithelium, Bowman’s membrane, stroma,
Descemet’s membrane and endothelium. A successful bioengineered cornea
Chapter_18.indd 368 06-02-2018 23:03:27

must have at least three of these five layers, namely, epithelium, stroma and
endothelium. Now, each of these individual components has been success-
fully regenerated separately. As we know, corneal epithelium can be cultured
using limbal stem cells on different substrates that act as scaffold, such as
AM and cross-linked human fibrin gel.65 The stromal component has been
reconstructed by mixing corneal keratocytes with human collagen types 1
and 3 or even with bovine collagen type 1.66 Corneal endothelial cells have
been regenerated successfully on human type 4 collagen.67 Now the chal-
lenge remains to grow all these components simultaneously.
Many research groups are currently working to bioengineer complete
cornea equivalents. Some of them used a collagen sponge or gel to culture all
three layers of cornea.68 When cultured on a collagen sponge, the epithelial
and endothelial cells formed a monolayer on its surface, while keratocytes
displayed migration and proliferation and eventually repopulated the colla-
gen sponge matrix. Though the collagen sponge had initially demonstrated
excellent transparency, extensive contraction of the matrix had limited its
ability of wound healing and maintaining the clarity. Attempts have been
made to bioengineer corneal tissue using human or bovine collagen thermo-
gel as substrate.68 In this unique technique, collagen sheets were prepared
by inducing human fibroblasts, and then it was implanted with human cor-
neal epithelial and stromal cells. Advanced research even attempted seeding
endothelial cells in addition to epithelial and stromal cells.69 The resultant
tissue though demonstrated excellent wound healing properties; it did not
have enough structural integrity to withstand transplantation. Recently, Li F
et al. had experimented a scaffold using type 1 collagen cross-linked with a
copolymer based on N-isopropylacrylamide, acrylic acid and acryloxysuc-
cinimide.70,71 The final product was a gel that had an excellent transparency,
tensile strength and could withstand the intraoperative manipulations. This
substrate had demonstrated in vivo regeneration of neural ingrowths in addi-
tion to usual epithelium and stroma. The neural growth is an important ele-
ment that improves epithelial growth and protects them from external injury.
Bioengineering is an exciting upcoming field and continues to be a chal-
lenge to the researchers. For many, this is the future to the OSR. The principal
requirements of a bioengineered cornea, i.e., optical clarity, tensile strength
and neural regeneration have already been achieved. The challenge remains
to fine-tune these techniques. It is probably no more a distant reality when
these bioengineered corneas are replacing diseased corneas during OSR.
Tremendous progress has been made toward regenerative medicine,
implantable devices and prosthesis with the development of biodegradable
scaffold in recent years. These scaffolds support growth and differentiation of
stem cells. Scientists are in the process of developing bioengineered lacrimal
glands. This can potentially restore the tear film to ensure surgical success.
Bioengineered cornea, hopefully, will replace the tissue when other forms
of transplantation are not feasible. Thus, future for OSR looks great and it is
going to be extremely exciting for the corneal surgeons.
Chapter_18.indd 369 06-02-2018 23:03:27

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19
CHAPTER
Advances in Keratoplasty
Soosan Jacob
INTRODUCTION
The oft-performed keratoplasty is the most successful of all organ transplants,
even without routine human leukocyte antigen (HLA) typing and matching
or systemic immunosuppression. This is because of the immune privilege
that the corneal allograft has. However, this advantage is lost with corneal
neovascularization, inflammation, infection, atopy, etc. In general, the goal
of keratoplasty is to have a clear graft with regular surface and minimal refrac-
tive error as all of these add up to good functional vision.
CLASSIFICATION
Corneal transplantation can be classified as follows:
A. Purpose:
1. Optical: To restore vision
2. T ectonic: To restore corneal structure and enhance strength of the
cornea
3. Therapeutic: To eliminate infectious load
4. Cosmetic: To restore a normal appearance to the eye
B. Depth:
1. Penetrating keratoplasty (PK): Transplantation of all the layers of the
cornea
2. Lamellar keratoplasty (LK): Transplantation of selective layers of the
cornea
C. Methods:
1. Manual keratoplasty: This includes conventional keratoplasty where
the host and graft are manually fashioned, using trephines or rarely
with manual dissection
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Femtosecond-assisted keratoplasty: Femtosecond laser is used in cut-

2.
ting the host and the graft tissue
PENETRATING KERATOPLASTY
Penetrating keratoplasty involves transplantation of full thickness of the cor-
nea. The main indication for PK is in those cases where the corneal pathology
involves both the anterior and posterior cornea. It is a commonly performed
procedure, when the facility/expertise for lamellar grafts is unavailable. A PK
procedure is technically easy to perform and has good visual outcome. How-
ever, it takes longer time forvisual recovery and poor visual quality due to
induction of astigmatism and high spherical refractive errors. It also carries
the risk of suture-related problems and traumatic wound rupture. Full thick-
ness grafts have issues with long-term prognosis in terms of allograft rejec-
tion and long-term graft failure because of continuing endothelial cell loss.
PK can be performed manually, using trephines or with the femtosecond
laser (Fig. 19.1A and B).
LAMELLAR KERATOPLASTY
Lamellar keratoplasty is classified as:
• Anterior lamellar keratoplasty (ALK)
• Posterior lamellar keratoplasty (PLK)
Anterior Lamellar Keratoplasty
ALK involves replacement of anterior layers of the host cornea. Indications

include corneal scars and dystrophies, where the endothelium is normal;
ectasias, degenerations, ocular surface disorders and corneal ulcers without
anterior chamber penetration.
Depending on the level of pathology, ALK can be classified as:
Superficial Anterior Lamellar Keratoplasty
In superficial anterior lamellar keratoplasty (SALK) procedure, the dissection

depth is less than one-third or 160 mm thickness of the cornea.
Automated Lamellar Therapeutic Keratoplasty
In automated lamellar therapeutic keratoplasty (ALTK) technique, both

host and donor cuts are made with a microkeratome. This allows smoother
lamellar dissection as well as better apposition than a manual dissection per-
formed at a superficial plane. ALTK can be used for anterior to mid-stromal
corneal opacities.
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Advances in Keratoplasty 377
(A)
(B)
Figs. 19.1A and B: A: Primary graft failure; B: Clear graft after a repeat penetrating ker-
atoplasty.
Femtosecond-assisted Lamellar Keratoplasty
In femtosecond-assisted lamellar keratoplasty (FALK) technique, the dissec-

tion is performed with femtosecond laser instead of microkeratome (Moria
ALTK microkeratome). It can be performed as a conventional graft using a
combination of anterior side cut and a full lamellar cut or as a shaped graft
with combination of cuts. Some surgeons prefer using only the side cut
without the lamellar cut, and then use the groove to inject air and obtain an
Anwar’s big bubble.
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Deep Anterior Lamellar Keratoplasty
With the advent of deep anterior lamellar keratoplasty (DALK), PK is no lon-

ger the first choice for surgical treatment of stromal diseases of the cornea
that havenormal endothelium. In this technique, a donor graft consisting of
epithelium with full thickness donor stroma is transplanted onto the host
bed consisting of only host pre-Descemet’s layer (PDL), Descemet’s mem-
brane and endothelium. Indications of DALK include anterior stromal scars,
keratoconus and ectasias, complications of photorefractive keratectomy or
LASIK, corneal melting disorders, pterygium, interstitial keratitis, descem-
etocele, infectious keratitis, etc. Descemet’s and endothelial scars, especially
if overlying the visual axis and patients with poor endothelial cell counts are
contraindications.
There are various techniques by which DALK can be performed. These
include manual dissection, dry peeling or dissection using viscoelastic and
balancedsalt solution or air. The most preferred technique is the Anwar’s big
bubble DALK technique. The Melles technique is another useful method.
Anwar’s Big Bubble DALK Technique
In this technique, a trephine is used to make a deep groove. A 26-gauge nee-

dle is then used to inject air into the posterior corneal stroma. The air cleaves
a plane that separates the PDL, Descemet’s membrane and the endothelium
on one side from the overlying posterior stroma on the other side to form a
well-defined type 1 bubble. The superficial stroma is then excised and the
bubble space is entered into. As soon as the space is entered into, air escapes
and the bubble space collapses. It is then re-expanded with viscoelastic
solution and the residual thin overlying stroma quadrisected and excised to
expose the host pre-Descemet’s layer. A disk of donor stroma, from which
the Descemet’s membrane and endothelium has been removed, is then
placed over the recipient bed and sutured into place.
Small Bubble Big Bubble Technique
In certain cases of Anwar’s big bubble DALK, it is difficult to ascertain

whether a big bubble is formed or not on injecting air. In this situation, a
small bubble can be placed into the anterior chamber. If the small bubble
appears displaced to the periphery of the anterior chamber, it indicates that
the big bubble has formed. If the small bubble floats up to lie under the cen-
tral cornea, it indicates that the big bubble has not formed (Fig. 19.2A to D).
Complications: The most common intraoperative complication is micro-
perforation. Descemet’s membrane perforation can occur in 10–30% of
cases, however it has a good final outcome in most cases and can be man-
aged by injecting air into the anterior chamber and proceeding carefully. A
Chapter_19.indd 378 06-02-2018 23:08:12

thin layer of stromal tissue may be left undissected above the microperfo-
ration. Fibrin glue may be used to close the perforation. Spontaneous clo-
sure occurs post-operatively in most cases. Other complications that can be
encountered includes macroperforation, second chamber formation, pupil-
lary block, Urrets-Zavalia syndrome, interface scarring, Descemet’s folds
and interface infection.
(A)
(B)
Figs. 19.2A and B: Deep anterior lamellar keratoplasty (DALK): (A) Big bubble formed,
(B) small bubble (yellow arrow) confirms presence of big bubble (white arrow).
Chapter_19.indd 379 06-02-2018 23:08:12

(Contd.)
(C)
(D)
Figs. 19.2C and D: (C) Stromal quadrisection and (D) the host pre-Descemet’s mem-
brane laid bare.
Therapeutic DALK
Therapeutic DALK is performed for removal of infected tissue and to prevent

intra-cameral spread of infection. It is performed in cases where there is no
intraocular spread of infection in order to avoid or replace a therapeutic PK.
This helps decrease the risk of graft rejection, graft failure and secondary
Chapter_19.indd 380 06-02-2018 23:08:12

angle closure glaucoma that can happen with large diameter therapeutic
penetrating grafts.
Advantages of DALK
Advantages of DALK include lesser chances of intraoperative and post-

operative complications as compared to PK. Since DALK is an extra-ocular
procedure, there is a decreased risk of complications that can be associated
with an open-sky procedure, such as expulsive hemorrhage. It does not bear
the risk of endothelial graft rejection. Wound stabilization is faster and there
is less astigmatism as compared to a PK. However, it is a technically difficult
surgery which is time consuming and has a steep learning curve. In cases,
where some deep host stroma has been retained, DALK may be associated
with slightly less good spectacle corrected visual acuity than with an optical
PK. With DALK, a greater number of donor corneas can be used as the donor
endothelial quality is not important. This is, especially important in countries
where donor corneas are difficult to obtain.
POSTERIOR LAMELLAR KERATOPLASTY

OR ENDOTHELIAL KERATOPLASTY
Endothelial keratoplasty (EK) began in 1998. Over the years, it evolved from
PLK to DLEK, DSEK, DSAEK, DMEK and finally to PDEK. Better instrumen-
tation and procedures have provided better postoperative results. Ancillary
techniques, such as the E-DMEK/E-PDEK and the air-pump assisted PDEK
techniques have also helped improve results further.
EK was first performed by Melles et al. in 1998, when it started out as
PLK. The posterior host stroma was manually dissected and replaced with
donor stroma, which was also manually dissected. Terry et al. modified this
procedure slightly and termed it as deep lamellar endothelial keratoplasty
(DLEK). Melles et al. later described descemetorhexis followed by trans-
plantation of manually dissected graft tissue containing posterior stroma,
Descemet’s membrane and endothelium. This was termed as Descem-
et’s Stripping Endothelial Keratoplasty (DSEK) by Price et al. Gorovoy et al.
described the use of the microkeratome to dissect donor graft tissue, thus
eliminating the need for manual dissection, and this technique was termed
as Descemet’s stripping automated endothelial keratoplasty (DSAEK). Ultra-
thin DSAEK (UT-DSAEK) was described by Busin et al., where the micro-
keratome is used to make donor grafts of less than 100–130 mm. Melles et al.
then described transplantation of only donor Descemet’s membrane and
endothelium after performing host Descemetorhexis and this procedure
was termed as Descemet’s membrane endothelial keratoplasty (DMEK).
Agarwal et al. described the transplantation of donor PDL, Descemet’s
membrane and endothelium to replace host Descemet’s membrane and
endothelium, which was termed as pre-Descemet’s endothelial keratoplasty
(PDEK).1
Chapter_19.indd 381 06-02-2018 23:08:13

Indications of EK include eyes with endothelial pathology causing

bullous keratopathy, such as Fuchs’ dystrophy, aphakic and pseudophakic
bullous keratopathy. Since host stroma is retained, eyes with stromal scarring
are not suitable for EK.
Descemet’s Stripping Automated Endothelial Keratoplasty
In this procedure, precut donor tissue can be sourced from the eye bank or
the surgeon may prepare it by using a microkeratome or femtosecond laser.
A donor tissue measuring 150–200 mm is prepared and transplanted into
the eye following host Descemetorhexis. Air is used to help the graft adhere
to the overlying host stroma. The advantage of DSAEK over DMEK is that it
is easier to perform in complex eyes, such as aphakic eyes, vitrectomized
eyes, eyes with drainage shunts. DSAEK has disadvantages of increasing the
pachymetry of the host as well as inducing a hypermetropic shift. The inter-
face between the host and graft may also lead to mild haze formation. The
ideal donor thickness is also unknown. Too thin donors, such as UT-DSAEK,
may be difficult to handle, form striae and have disadvantages similar to
DMEK of more difficult surgery. Too thick grafts may cause increased hyper-
metropic shift as well as may put additional stress on the donor endothelial
pump function, leading to the risk of secondary graft failure and endothe-
lial decompensation. Complications include donor dislocation, retained
viscoelastic, pupillary block, inverted graft, excessive surgical manipulation
leading to primary graft failure, graft rejection, secondary graft failure, IOL
opacification, epithelial downgrowth, etc. (Fig. 19.3).
Fig. 19.3: Descemet’s stripping automated endothelial keratoplasty (DSAEK) graft

being inserted.
Chapter_19.indd 382 06-02-2018 23:08:13

Descemet’s Membrane Endothelial Keratoplasty
The donor Descemet’s membrane and endothelium are transplanted onto

the area of host Descemetorhexis. Once the graft is injected into the anterior
chamber, a combination of gentle tapping on the corneal surface together
with short bursts of fluid is used to unroll the graft. Air is then injected under
the graft to float it up and bring it into apposition against the overlying area
of host stroma, which has been previously denuded of the host Descemet’s
membrane. The advantages of this technique include excellent quality of
vision and decreased chances of graft rejection. The disadvantages of this
technique include the need for very mature surgical skill and expertise
and usage of older donor grafts than PDEK. The DMEK graft is also more
fragile and tears easily as compared to PDEK and is much more difficult
to handle as compared to DSAEK. Unrolling and floating up of the donor
graft accurately is difficult and has a steep learning curve. The DMEK graft
can come as precut graft from the eye bank or the surgeon may prepare it.
Complications include graft detachment and dislocation, complex types of
Descemet’s detachments3, primary and secondary graft failure, graft rejec-
tion, pupillary block or steroid induced glaucoma, interface debris and haze
(Figs. 19.4A and B).
(A) (B)
(C) (D)
Figs. 19.4A to D: (A, B) DMEK: Pre- and postoperative appearance and (C, D) PDEK: Pre-
and postoperative appearance.
DMEK: Descemet’s membrane endothelial keratoplasty; PDEK: Pre-Descemet’s endo-
thelial keratoplasty.
Chapter_19.indd 383 06-02-2018 23:08:13

Pre-Descemet’s Endothelial Keratoplasty
It makes use of the PDL, recently described by Harminder Dua to create

a type 1 big bubble consisting of the PDL, Descemet’s membrane and the
endothelium. Thus, the PDEK graft differs from the DMEK graft (type 2 big
bubble),where the PDL is also transplanted together with the donor Descem-
et’s membrane and endothelium onto the area of host Descemetorhexis. The
PDEK bubble is created by injecting air into the donor corneo-scleral rim.
Like the Anwar’s big bubble, this air cleaves a plane to create one of three
types of big bubbles2 (Figs. 19.4C and D).
Type 1 Big Bubble
Air creates a big bubble that separates the PDL, Descemet’s membrane and
the endothelium on one side from the donor stroma on the other side. This
bubble starts from the center to expand circumferentially outward and has
well-defined sharp edges. It can expand up to a size of 8.5 mm. Advantages
are the more robust nature of this graft as compared to the DMEK graft, which
is more fragile and easily tears. The robustness of the graft allows one to make
use of the air pump-assisted technique, described by the author, to easily
perform the surgery and smoothen the learning curve. It is easy to obtain a
type 1 big bubble even in very young grafts (author has used grafts from a as
young as nine months old donor). It, therefore, has advantages of providing
much larger endothelial cell counts for transplantation as compared to an
older graft as well as expands the donor cornea pool.
Type 2 Big Bubble
Air creates a big bubble that separates the DM and the endothelium from the
PDL and the donor stroma. This is a DMEK graft and, if this type of big bubble
forms, surgery is continued as DMEK. This bubble starts from the periphery
and expands toward the opposite side. It has sloping edges and is larger in
size, however the bursting pressure is low and the graft is more fragile. Type 2
bubble formation is more common in older grafts.
Type 3 Big Bubble
A combination of types 1 and 2 big bubbles forms the same graft. If this
occurs, the type 2 is allowed to expand and the surgery is continued as DMEK
surgery.
Precut PDEK grafts are also now available from eye banks. It decreases
the risk of graft loss from the surgeon’s side. Once the PDEK graft is injected
into the anterior chamber, a combination of gentle tapping on the corneal
surface, together with short bursts of fluid is used to unroll the graft. Air is
then injected under the graft to float it up and bring it into apposition against
Chapter_19.indd 384 06-02-2018 23:08:13

(A) (B)
(C)
Figs. 19.5A to C: (A) Type 1 big bubble, (B) type 2 big bubble and (C) type 3 big bubble
(type 1 seen centrally in combination with type 3 seen peripherally marked out with
arrows).
the overlying area of host stroma, which has been previously denuded of host
DM. PDEK surgery has advantages of being easier to perform using specified
techniques (E-PDEK and air pump-assisted PDEK). The graft is stronger and
does not tear easily unlike DMEK. It also has advantages of being easy to per-
form than DMEK in complicated situations, such as described with DSAEK.
At the same time, it retains the advantages of DMEK of excellent quality of
vision, minimal induced refractive shift and faster recovery rate as compared
to DSAEK (Figs. 19.5A to C).
ANCILLARY TECHNIQUES FOR DMEK AND PDEK

• Endoilluminator-assisted DMEK/endoilluminator-assisted PDEK
(E-DMEK/E-PDEK):4,5 This technique has been described by the author
and utilizes the external endoilluminator for DMEK as well as PDEK. The
technique is termed as endoilluminator-assisted DMEK or E-DMEK,
when used with DMEK and, as endoilluminator-assisted pre-Descemet’s
endothelial keratoplasty (E-PDEK), when applied to PDEK. One of the
major problems with EK is compromised visibility, especially in the pres-
ence of corneal edema. Though visibility may be enhanced preopera-
tively by dehydrating the cornea and intraoperatively by debriding the
Chapter_19.indd 385 06-02-2018 23:08:14

epithelium, staining the graft, and using handheld slit lamp during sur-
gery, yet practically, these are often partly helpful, especially in advanced
cases. Moreover, because both DMEK and PDEK grafts are transparent,
thin and flimsy, it is often difficult to confirm the position, orientation and
morphology even with clearer corneas. The E-DMEK technique allows
identification of graft orientation by enhancing 3-dimensional depth
perception of the graft within the anterior chamber by using oblique light
from the endoilluminator for better visualization. The light bouncing off
the graft folds and edges aids further in this 3-dimensional enhancement
of visualization. The technique has the advantages of the ability to visu-
alize the entire graft even through the hazy cornea. E-DMEK/E-PDEK
makes the surgery easier and faster by allowing better visualization and
better surgeon comprehension of graft morphology and dynamics. It also
decreases graft damage secondary to excessive fluidics and unnecessary
manipulation (Fig. 19.6A and B).
• Air pump-assisted PDEK:6 This technique has been described by the
author, for handling the graft within the anterior chamber. In both DMEK
and PDEK, surgery has a learning curve and is difficult for the beginners.
Graft handling within the anterior chamber still remains a challenge as
the tissue is thinner and behaves differently from a DSAEK which may
result in a variety of complications ranging from inability to unfold the
graft to complex Descemet’s detachment. The air pump-assisted PDEK
technique aids in multiple key steps of PDEK surgery by utilizing contin-
uous pressurized air infusion. It also takes advantage of the robustness
and tear resistance that is offered by the PDL to the PDEK graft.
The technique helps in multiple key steps:
• Descemetorhexis: A continuous, curvilinear Descemetorhexis is easier,
better controlled and better visualized with the DM flat against the over-
lying stroma under air pressure. Uncontrolled flapping of the tearing
(A) (B)
Figs. 19.6A and B: E-DMEK: (A) View under operating microscope and (B) enhanced
view with E-DMEK.
Chapter_19.indd 386 06-02-2018 23:08:14

edge is avoided and better control is obtained over size and centration.
An inverse capsulorhexis, like manoeuvre may be performed to obtain a
continuous, curvilinear Descemetorhexis and the desired size is obtained
using the same principles as that of a capsulorhexis.
• Tamponading of bleed from peripheral iridectomy and intra-ocular ooze
of blood from the conjunctival cul-de-sac: An inferior extreme peripheral
iridectomy (PI) may be performed with needle technique or preferably
with a vitrector. Continuous air infusion tamponades iris bleeding and
prevents hyphema from the PI, which can prolong the surgery. It also
avoids instillation of viscoelastic into the AC while performing PI, which
can also interfere with graft adhesion. The continuous air infusion pre-
vents intra-ocular bleeding and also prevents intra-ocular oozing of
blood from the conjunctival cul-de-sac.
• Graft floatation: With air pump-assisted PDEK, unfolding of the extreme
graft edge and centration need not be as perfect as in a DMEK because
the PDL imparts resistance to tear as well as a splinting action to the graft
that allows these maneuvers to be performed safely even after floatation.
Therefore, floatation can be performed at an earlier stage and maintained
with continuous air infusion.
• Graft centration: With the continuous pressurized air infusion on, the
reverse Sinskey can easily pull the graft into position. The air prevents the
graft from dislocating and the tough, resilient nature of the graft allows
the graft to slide into the stromal undersurface without tearing.
• Graft edge unfolding: Though PDEK gives the advantage of being able to
use young donor tissue with the higher endothelial count, these donors
may also be more difficult to unroll completely to the extreme edges
often remaining curled upwards—a tendency inversely proportional to
age of the donor. Trying to unfold the extreme edge often takes time and a
greater degree of graft manipulation. Floating the largely unrolled PDEK
graft followed by continuous air infusion for posterior support allows
the surgeon to unroll small edge folds without the graft losing support
and detaching. The air infusion holds the graft in place while the reverse
Sinskey hook or a thin, blunt rod introduced through the paracentesis
in a plane between the stroma and the graft can unfold inward rolled
graft edges. Continuous air infusion maintains a well-formed AC despite
some leakage through the side port. Without the air pump, these maneu-
vers would result in dislocation of the graft. This technique also allows
adequate operating space within the AC to insert instruments without
damaging the graft endothelium or pushing on the graft.
• Graft unwrinkling: Wrinkles and creases in the graft can be easily
stretched out with the reverse Sinskey hook by pulling the graft at its
extreme edges.
• Graft adhesion: Earlier and better graft adherence is promoted because
of intraoperative pressurized apposition continuously holding the
Chapter_19.indd 387 06-02-2018 23:08:14

(A) (B)
(C) (D)
Figs. 19.7A to D: Air pump-assisted PDEK: (A) Descemetorhexis performed, (B) non-
hemorrhagic PI done under air, (C) graft unwrinkled and centered. Edge fold being
removed. Only minimal peripheral edge points are utilized and (D) postoperative slit-
lamp photograph.
graft tightly against the overlying stroma. This continuous pressure is

maintained till incisions are sutured and the eye is ready to be closed.
Undesired shallowing of AC, while suturing, is also avoided.
• Third hand: It prevents anterior chamber depth fluctuations and acts as
a third hand supporting the graft and preventing dislocation (Fig. 19.7A
to D).
Repeated graft touch can be minimized when grafting is done with the
posterior air support to the graft. To decrease endothelial cell loss further, all
maneuvers involving graft touch should be performed at single touch points
only at the extreme edges of the graft and kept minimum possible. Though
similar maneuvers may be performed in DMEK with lower infusion pressure,
DMEK grafts have a higher rate of graft wrinkling and graft edge tearing pos-
sibly because of the thinner and more fragile nature of the DMEK graft and
therefore this technique may not be as suitable for DMEK.
FEMTOSECOND-ASSISTED CORNEAL TRANSPLANTATION7

The femtosecond laser is a near-infrared laser of 1053 nm wavelength and
a pulse duration of 10-15 sec. It uses the principle of photodisruption by
Chapter_19.indd 388 06-02-2018 23:08:14

creating a shock wave and resultant cavitation, which results in tissue cleav-
age. Keratoplasty by the femtosecond laser eliminates the more difficult and
imprecise manual lamellar dissection. It allows easier, faster and more pre-
dictable surgery with potentially better refractive, topographic and visual
results. Various cut patterns are created by a combination of lamellar, anterior
and posterior cuts. It gives advantages of better healing of epithelium, better
wound apposition and earlier improvement in visual acuity. The shaped pat-
terns can increase wound stability, allow earlier suture removal and decrease
postoperative astigmatism. Disadvantages of femtosecond surgery include an
increase in the cost of surgery and total time taken when compared to manual
surgery by experienced surgeons, decreased BCVA with lamellar cut DALK as
compared to big bubble DALK and also with femtosecond cut DSAEK graft as
compared to microkeratome cut DSAEK graft.
The femtosecond laser can be used to perform:
• Femtosecond-assisted superficial anterior lamellar keratoplasty: This pro-
cedure is performed by turning the hinge option off in LASIK pattern to
program an anterior free cap for ALK.
• Femtosecond-assisted deep anterior lamellar keratoplasty: It can be per-
formed as a conventional graft using a combination of anterior side cut
and a full lamellar cut or as a shaped graft in the form of mushroom cut
or zig-zag cuts using a combination of cuts. Some surgeons also prefer
using only the side cut without the lamellar cut, and then use the groove
to inject air and obtain an Anwar’s big bubble. It has the disadvantages of
the possibility of pathology being left behind in the layers of host stroma
posterior to the lamellar cut and mild interface haze.
• Femtosecond posterior lamellar keratoplasty: The femtosecond laser may
be used to cut the donor cornea. However, too thin grafts can result in
perforated graft, or sometimes endothelial damage. The host cornea
may also be cut to perform a DLEK, if desired. Femtosecond-assisted
Descemetorhexis followed by manual Descemet’s stripping may also be
performed instead to give well-defined and precise margins in the area
of host Descemetorhexis.
GRAFT REJECTION
Graft rejection refers to an immune reaction to donor tissue, which results
in inflammation and resultant non-functioning of a previously functioning
graft. It is seen initially as migration of inflammatory cells from the graft mar-
gin, generally originating from an area of corneal neovascularization. Keratic
precipitates, cells and flare in the anterior chamber and corneal edema are
observed. The classic sign of endothelial rejection is the endothelial rejection
line known as the Khodadoust line. Immune destruction of the endothelial
cells, if irreversed, can result in diffuse edema and opacity of the graft. The
incidence of graft rejection is highest with PK and lower with DSAEK and
DMEK/PDEK, respectively. Risk factors for corneal graft rejection include
Chapter_19.indd 389 06-02-2018 23:08:14

a failed graft, more than two quadrants of corneal neovascularization, large

diameter grafts, eccentrically placed grafts, loose sutures, sutures crossing
the limbus, preexisting inflammation or glaucoma, peripheral anterior syn-
echiae, young age of recipient, etc.
The patient presents with the signs of RSVP: Redness, sensitivity to light,
vision loss and pain, though in mild cases the patient may be asymptomatic.
Patients who have undergone keratoplasty should, therefore, be evaluated
at regular intervals, and in addition should also be educated to seek prompt
medical attention in case of any of the RSVP signs.
Immune graft rejection is an important cause of corneal graft failure
and should be treated as an emergency with high dose topical and systemic
immunosuppresants. Rejection can, in many cases, be reversed if treated
early and adequately.
Patients, who have undergone ALK, can also present with stromal rejec-
tion observed as acute stromal edema. This may proceed from an area of stro-
mal neovascularization. It is also treated with topical and systemic steroids.
The incidence of rejection in ALK is however much lower than in PK.
REFERENCES
1. Agarwal A, Dua HS, Jacob S, et al. Pre-Descemet’s endothelial keratoplasty
(PDEK). Br J Ophthalmol. 2014;98(9):1181-85.
2. Dua HS, Faraj LA, Said DG, et al. Human corneal anatomy redefined: a novel
pre-Descemet’s layer (Dua’s layer). Ophthalmology. 2013:120(9):1778-85.
3. Jacob S, Agarwal A, Chaudhry P, et al. A new clinico-tomographic classifica-
tion and management algorithm for Descemet’s membrane detachment. Cont
Lens Anterior Eye. 2015;38(5):327-33.
4. Jacob S, Agarwal A, Agarwal A, et al. Endoilluminator-assisted transcorneal
illumination for Descemet membrane endothelial keratoplasty: enhanced
intraoperative visualization of the graft in corneal decompensation secon-
dary to pseudophakic bullous keratopathy. J Cataract Refract Surg. 2014;
40(8):1332-26.
5. Jacob S, Agarwal A, Kumar DA. Endoilluminator-assisted Descemet
membrane endothelial keratoplasty and endoilluminator-assisted pre-
Descemet endothelial keratoplasty. Clin Ophthalmol. 2015;9:2123-25.
6. Jacob S, Narasimhan S, Agarwal A, et al. Air Pump-Assisted Graft Centra-
tion, Graft Edge Unfolding, and Graft Uncreasing in Young Donor Graft Pre-De-
scemet Endothelial Keratoplasty. Cornea 2017;36(8):1009-13.
7. Jacob S, Agarwal A. “Femtosecond Laser-assisted Keratoplasty: Lamellar Ante-
rior and Posterior.” Femtosecond Laser Surgery in Ophthalmology. Dick B, et al.
(Ed). New York. Thieme 2017 (in press).
YOUTUBE VIDEOS:
1. Dr. Soosan Jacob YouTube surgical video channel for Endothelial Keratoplas-
ty (ET), Playlist and DALK videos: https://www.youtube.com/channel/UCgQ-
esqB4VuECm4YGKpaZccg
Chapter_19.indd 390 06-02-2018 23:08:14

2. Understanding the PDEK Bubble, Soosan Jacob (audio 33 min): https://

www.youtube.com/watch?v=M4n4bCxApAc&list=PLOLM5nX3h_taUkT5ft-
FzwE_-JmGx6HeWO
3. E-DMEK and E-PDEK with audio, Soosan Jacob (2 min): https://www.youtube.
com/watch?v=4ktkfnOAKmc&list=PLOLM5nX3h_taUkT5ftFzwE_-JmGx6He-
WO&index=3
4. Air pump-assisted PDEK, 11 advantages (audio):https://www.youtube.com/
watch?v=lcIHrzdbDd4&list=PLOLM5nX3h_taUkT5ftFzwE_-JmGx6HeWO&in-
dex=18
5. Descemet’s Detachment Classification & and Management Relaxing Descem-
etotomy and YAG or Surgical Descemetotomy: https://www.youtube.com/
watch?v=nx5M4hibxFs
6. DALK Anwar’s Big Bubble, Soosan Jacob (Audio, 14 min): https://www.you-
tube.com/watch?v=WfYzWVpJ9nc
7. DALK Big Bubble Small Bubble, Soosan Jacob (video, 2.5 min.): https://www.
youtube.com/watch?v=o15Xs4AVqSk
8. DALK Anwar’s Big Bubble, Soosan Jacob (video): https://www.youtube.com/
watch?v=5ovd9sS88Ww
9. DALK Anwar’s Big Bubble, Soosan Jacob (video): https://www.youtube.com/
watch?v=ThnhW3ZhIn0
Chapter_19.indd 391 06-02-2018 23:08:14

20
CHAPTER
Keratoprosthesis
Quresh Maskati
INTRODUCTION
Keratoplasty or corneal transplant is one of the most successful transplants
done in the human body. It comes with a fairly low rejection rate.1 How-
ever, this is only in cases where immunological isolation of the cornea is
preserved. Even today, in the 21st century, there are cases in which kerato-
plasty is doomed to failure. For severe ocular surface disorders with bilat-
eral loss of vision, that is, in cases of severe Stevens-Johnson syndrome (SJS)
with keratinization, severe chemical ocular burns, ocular pemphigus with
significant dry eye, traumatic disorganized anterior segments, bilateral total
stem cell loss or deficiency and severely vascularized recipient eyes, kerato-
plasty does not work. In all these cases, a keratoplasty procedure should not
be attempted.2 Several dozens of KPs have been invented over the years.3-5
However, only a handful number of KPs, which stood the test of time, are
in use.6 Among the available KPs, the Daljit Singh champagne cork KP, also
known as the Worst-Singh KP, is perhaps the highest used KP in India (more
than 5,000 units implanted till date).7 The other varieties of KPs available
are modified osteo-odonto keratoprosthesis (MOOKP)8 and the Pintucci
biointegrated keratoprosthesis (PBIKP).9-11 The Dohlman or Boston KP12 is
useful in less severe eye conditions, such as vascularized corneal opacities,
repeated failed grafts, chemical burns and SJS without keratinization.
For the Dohlman KP (DKP), some degree of tear secretion and a nor-
mal blink mechanism are essential for long-term success. For any KP, it is
essential to have accurate light perception and to rule out preexisting end
stage glaucoma or a retinal detachment.13 The PBIKP and the DKP are the
two procedures in which the author has personal experience. The surgical
techniques of these KPs are described in this chapter.
Keratoprosthesis 393
PINTUCCI BIOINTEGRATED KERATOPROSTHESIS

The current design is a 4 mm diameter cylinder of PMMA with a 0.6 mm thick,
soft Dacron mesh embedded in the centre like a flared Can-Can dancer’s skirt
(Figs. 20.1 and 20.2).
Surgical Technique
The surgical technique is quite similar to the one devised by Dr Pintucci of

Rome. It consists of two stages.
Fig. 20.1: Sketch of a Pintucci keratoprosthesis.
Fig. 20. 2: Pintucci keratoprosthesis.

Stage 1
The lower lip is everted and held with a special clamp, and a large free graft
harvested from the mucosal side (Figs. 20.3 to 20.10). Gauze soaked in hydro-
gen peroxide is used to seal bleeders on the raw donor surface and a tincture
benzoin dressing is applied on the bare area. No sutures are taken. This graft
contains mucosa as well as submucosal connective tissue. The graft is then
Fig. 20.3: Lids are everted.
Fig. 20.4: Buccal mucosal incision (lower lid).

Fig. 20.5: Buccal mucosal graft inner surface.
Fig. 20.6: Buccal mucosal graft outer surface.
placed on a flat surface and visible fibers of the orbicularis oris muscle are
trimmed off. The eye is opened, symblephara are released and a speculum
inserted. The corneal epithelium is scraped off as well as any conjunctival
overgrowth on to the cornea. A 360 degrees peritomy is done. A single 10/0
nylon stitch is taken in the center of the cornea, half thickness depth, to
serve as a marker for the center of the cornea in the second stage. The buccal
Fig. 20.7: Central corneal marker stitch.
Fig. 20.8: Closure of the lower lid wound (orbicularis suturing).
mucosal free graft is placed over the cornea, and sutured with interrupted
polyglycolic acid sutures to the surrounding conjunctiva. Some anchoring
sutures are taken to the recti muscles as well. A conformer is placed in the
eye and left in place for 15 days.
Fig. 20.9: Closure of the skin wound.
Fig. 20.10: End of the first stage.
The PBIKP is removed from its sterile packaging, washed with saline
and placed upside down under the orbicularis oculi muscle after making a
linear incision through skin and orbicularis below the lower lid. This PBIKP
is then left in place for 2 months or more. The orbicularis muscle is sutured
with absorbable suture and the skin with nonabsorbable sutures, which are
removed after 7 to 10 days.
Fig. 20.11: Second stage reflection of buccal mucosa.
Fig. 20.12: Second stage removal of PBIKP from lower lid.
Stage 2
Two months later, the PBIKP is removed after making an incision similar
to the earlier one under the lower lid (Figs. 20.11 to 20.23). The PMMA cylin-
der is cleaned of all connective tissue and the Dacron mesh, (now fully cov-
Fig. 20.13: Cleaned Pintucci biointegrated keratoprosthesis (PBIKP).
(A) (B)
Figs. 20.14A and B: Corneal central button excised.
ered with connective tissue and blood vessels on both surfaces and around
the edge) inspected. The Dacron mesh that was soft and pliable at insertion
is now fairly firm, but still allows passage of vicryl sutures easily. The eye is
then opened, a speculum is inserted and the buccal mucosal flap is reflected
back to be hinged in the upper temporal quadrant. Using the central corneal
suture as a centration point, a 4 mm trephine is used to make a circular groove
in the cornea. Three radial cuts are made on the cornea at 12, 4 and 8 o’clock
positions extending from the circular groove to 1 mm within the limbus. The
central 4 mm button is then excised as done in penetrating keratoplasty, with
a full thickness knife entry, followed by excision with corneal extension scis-
Fig. 20.15: Iris excision.
Fig. 20.16: Intracapsular crystalline lens extraction using cryoprobe.
sors. The iris is held with strong-toothed forceps, after reflecting the three cor-
neal flaps. Multiple cuts are made from the pupil to the iris root and the iris
is removed by cutting segment by segment. The crystalline lens, if present, is
extracted intra-capsularly with cryoprobe. If there is an intraocular lens (IOL)
in place, it is removed. Single port central vitrectomy is done.
The three corneal radial incisions are sealed with 8/0 vicryl. The PBIKP
is, then inserted through the central 4 mm corneal opening. The lower half
Fig. 20.17: Dacron mesh sutured to cornea prior to mucosal flap put back in place.
Fig. 20.18: Central opening in mucosal flap.
of the polymethyl metha acrylate (PMMA) cylinder projects into the vitreous
cavity. The vascularised, colonised, biointegrated Dacron mesh will now sit
on top of the patient’s cornea. This mesh is sutured to the cornea with 6/0 or
7/0 vicryl sutures. The buccal mucosal flap, which had been reflected earlier,
is now brought back and sutured to its original position, after carefully not-
ing the position of the projecting upper half of the PMMA cylinder. The same
(A)
(B)
Figs. 20.19A and B: (A) Second stage surgery completed and (B) 5 years post-op, V/A
6/18.
trephine is used to carefully trephine the central 4 mm of the buccal mucosa.

This allows the PMMA cylinder to project approximately 0.5 mm above the
buccal mucosa. The eye is then patched for 24–48 hours.
Routine postoperative care after stage 1 includes oral fluoroquinolone anti-

biotics for 5 days; oral anti-inflammatory tablets twice a day for 5 days and
Fig. 20.20: Measurements of recipient button taken.
Fig. 20.21: Recipient button trephined.
oral betamethasone 0.5 mg tablets twice a day for 5 days. All patients are
given a liquid diet through a drinking straw for the first 3 days followed a soft,
bland diet for another 4 days. The raw surface on the lower lip heals between
7-10 days.
On removal of the eye patch, the patients are instructed to put antibiotic
drops and steroid drops four times a day for 2 months.
Fig. 20.22: Excision of recipient button.
Fig. 20.23: Donor assembly placed on recipient bed.
After stage 2, they are asked to continue antibiotic drops and steroid
drops four times a day for 1 month and reduced to twice a day in the next
month. The steroid drops are stopped after 2 months. Antibiotic drops, either
twice a day or once a day, are continued for life. All patients are advised to
use lubricating drops as frequently as necessary. Patients are advised to clean
Fig. 20.24: Recipient eye button removed.
Fig. 20.25: IOL is inserted in recipient eye.
their PMMA cylinders using cotton buds moistened with lubricating drops
daily to prevent discharge accumulating on them and reducing their vision.
Reason for Success
The Dacron mesh, which is 0.6 mm thick is porous and allows connective
tissue and blood vessels to grown three-dimensionally into its pores in
Fig. 20.26: Synechiae are separated in the recipient eye.
the 2 months that it is kept under the orbicularis oculi. These mesodermal
elements completely fill each pore from both the anterior and posterior
surfaces as well as from the edges. When this PBIKP is implanted within the
eye, the Dacron mesh, now firm though still pliable, allows blood vessels and
connective tissue from the ocular surface and the buccal mucosa to grow
into the mesh and integrate with the blood vessels and connective tissue
already there. This allows true biointegration. As each pore is filled with liv-
ing tissue, the chances of microbes being able to get into the eye through the
mesh are remote. This ‘living shield’ also acts as a barrier for epithelial migra-
tion from the surface, preventing the possibility of retroprosthetic epithelial
membranes and, therefore, extrusion of the PBIKP.
The incidence of glaucoma postoperatively is relatively low (less than
10% in the author’s series of 75 cases over 12 years; data on file). This is prob-
ably due to the fact that when the iris is excised, the ciliary body is also pulled
upon causing several cyclodialysis clefts, thus causing diminished produc-
tion of aqueous humour.
THE DKP (TYPE 1)

The current design is a three-piece structure with the donor cornea forming
the fourth piece (Fig. 20.24):
1. The front part is a mushroom-shaped structure with a stem (all PMMA).
The front plate is 5 mm in diameter while the stem is 3.35 mm.
2. The back plate, made of PMMA is available as 7.0 mm or 8.5 mm diame-
ter, with a central hole and 16 holes of 1.2 mm each.
3. A titanium-locking ring, which fits posterior to the back plate.
Fig. 20.27: Viscoelastic is injected in the recipient eye.
Two types are available, a DKP for aphakic eyes and one for pseudopha-
kic eyes. Depending on the case, the appropriate one is ordered. It is neces-
sary to share the axial length measurement with the supplier to obtain the
appropriate powered DKP in aphakic eyes.
Surgical Technique
The surgery is performed in a single stage (Figs. 20.25 to 20.37). First, the
donor cornea of 0.5 mm larger than the chosen back plate (donor cornea
9.0 mm for a back plate of 8.5mm and donor cornea 7.5 mm for a back plate
of 7 mm) is punched out as usual in a PK using a Teflon block and a trephine
of one’s choice. A 3 mm hole is made in the center of the donor cornea using a
special disposable dermatological trephine, which is supplied with the DKP.
The DKP is then assembled as follows. The front mushroom plate is kept down
on a flat surface. Viscoelastic is applied to the stem. The donor cornea with
the central hole punched out is then slid onto the stem. The endothelium is
coated with viscoelastic, and then the back plate is pushed into place, with the
central hole accommodating the end of the stem. Finally, the titanium ring is
pushed onto the stem and locks into position with an audible snap. Once the
DKP along with the donor cornea is assembled, it can be placed back into the
MK medium container while attention is now paid to the recipient.
A central corneal button with a diameter 0.5 mm less than the donor
cornea is partially trephined, again with a trephine of the surgeon’s choice.
The anterior chamber (AC) is gently entered with a sharp knife and the
button is excised with right and left corneal scissors as with any penetrating
Fig. 20.28: Insertion of KP under muscle.
Fig. 20.29: Line diagram showing insertion under LL.
keratoplasty. A peripheral iridectomy is performed. If the patient has a

posterior chamber IOL already in place in the bag, it need not be disturbed. All
ill-fitted IOLs are removed. If the eye is Phakic it is made either aphakic (leaving
posterior capsule intact) or pseudophakic with the appropriate IOL inserted
to give emmetropia. If the eye is already aphakic or if vitreous presents
Fig. 20.30: Parts of Dohlman keratoprosthesis.
Fig. 20.31: Donor button in M-K medium.
into the AC during the surgery, a central core vitrectomy is recommended.

The DKP assembly is then inserted into the central opening and the donor
cornea is sutured with interrupted 10/0 nylon sutures. The knots are buried.
A large diameter special soft lens that is provided with the DKP, is inserted to
cover the ocular surface. This is from Kontur and is a plano lens with 16 mm
diameter with a 9.8 mm base curve.
Fig. 20.32: Donor button trephination.
Fig. 20.33: Frontal plate.
Dohlman’s group from Boston recommends vancomycin (14 mg/mL)

eye drops at least once a day for life and claims that the incidence of
postoperative endophthalmitis has dropped to almost nil after initiating this
regime. The author has been using vancomycin drops for the first month four
Fig. 20.34: Punching out central hole in donor button.
Fig. 20.35: Donor button is being sutured.
times a day, and then switching to the commercially available fourth genera-
tion fluoroquinolone eye drops tapered to once a day for life.
Topical steroids are used four times a day and tapered off within 1-2
months, unless the eye is inflamed, they may be continued for longer. If there
is glaucoma, steroids are discontinued or are replaced with milder ones.
Fig. 20.36: Donor button suturing in progress.
Fig. 20. 37: A large contact lens is to be placed on donor assembly at the end of surgery.
Antiglaucoma drops may be used if glaucoma is suspected by applying

finger palpation method and confirmed by disc and visual field changes.
In case glaucoma still persists, an Ahmed glaucoma valve surgery may be
necessary.
The contact lens is recommended to be worn as an extended wear lens for
life to be replaced in every 3-6 months or earlier if it has too many deposits.
Fig. 20.38: Patient No. 1: Preoperative status.
Fig. 20.39: Patient No. 1: Postoperative status.
Results
The results of both PBIKP and DKP are encouraging (Figs. 20.38 to 20.43).
Besides improvement in the cosmetic appearance, some of the patients
not only regained useful vision, but also their quality of day-to-day lives is
improved.
Reason for Success
The DKP is performed for less severe indications; chief among them being
failed grafts. Careful case selection, rejecting cases with very dry eyes and
Fig. 20.40: Patient No 2: Preoperative status.
Fig. 20.41: Patient No 2: Postoperative status.
Fig. 20.42: Patient No 3: Preoperative status.

Fig. 20.43: Patient No 3: Postoperative status.
inadequate blinks and those with a keratinized ocular surface, has enhanced
the success rate. The problems with the earlier screwed on back plate have
been eliminated by having a snap-on titanium ring locking the assembly
into place. The use of long-term topical antibiotic and the Kontur lens have
reduced the complication rate considerably.
REFERENCES
1. Gloor P. Complicated penetrating keratoplasty. In: ‘Cornea,’ Krachmer, Mannis:
Holland, Mosby; 1997. p. 1731-811.
2. Kenyon KR, Wagoner MD, Shore JW. Ocular surface transplantation. In:
‘Cornea,’ Krachmer, Mannis: Holland, Mosby; 1997. p. 1887-901.
3. Dohlman CH. Keratoprosthesis. In: ‘Cornea,’ Krachmer, Mannis: Holland, Mosby;
1997. p. 1855-62.
4. Cardona H. Keratoprosthesis; acrylic optical cylinder with supporting intral-
amellar plate. Am J Ophthalmol. 1962;54:284-94.
5. Strampelli B. Osteo-odonto-keratoprosthesis. Ann Ottalmol Clin Ocul.
1963;89:1029-39.
6. Liu C, Tighe B. Striving for the perfect keratoprosthesis. Br J Ophthalmol.
1998;82(1):3-4.
7. Singh D, Bansel DC, Singh A. Keratoprosthesis: a clinical study. Indian J Oph-
thalmol. 1973;21(3):112-6.
8. Geerling G, Liu CS, Collin JR, et al. Cost and gains of complex procedures to
rehabilitate end stage ocular surface disease. Br J Ophthalmol. 2002; 86(11):
1220-1.
9. Pintucci S, Pintucci F. The Dacron felt colonisable KP. Refract Corn Surg.
1993;9:196-7.
10. Pintucci S, Pintucci F, Caiazza S, et al. The Dacron felt colonisable keratopros-
thesis after 15 years. Eur J Ophthalmol. 1996;6:125-30.
11. Pintucci S, Pintucci F, Cecconi M, et al. New Dacron tissue colonizable kerato-
prosthesis: clinical experience. Br J Ophthalmol. 1995;79(9):825-9.
12. Dohlman CH, Terada H. Keratoprosthesis in pemphigoid and Stevens-
Johnson syndrome. Adv Exp Med Biol. 1998;438:1021-5.
13. Yaghouti F, Nouri M, Abad JC, et al. Keratoprosthesis: preoperative prognostic
categories. Cornea. 2001;20(1):19-23.
21
CHAPTER
Cystinosis
Shefali Vyas
INTRODUCTION
Embryonic organogenesis of the kidney and eye has been studied since
nineteenth century but the recent evolution of molecular technologies has
advanced our understanding of mechanics of various genes regulating the
development of eye and the kidney tubules. Recent knowledge in under-
standing the genetic pathways for renal tubular acidosis (RTA) have trig-
gered a renewed interest in variety of congenital and acquired oculo-renal
disorders.
The classic Fanconi syndrome was first described by Abderhalden1 in 1903
in a patient with cystinosis. Fanconi syndrome classically presents with severe
proximal tubular dysfunction, which leads to fluid and electrolyte imbal-
ances. Usually patients have polyuria, failure to thrive and recurrent episodes
of dehydration at presentation. Due to proximal tubular dysfunction, patients
have glucosuria, bicarbonaturia, phosphaturia and loss of potassium, sodium,
low molecular weight proteins, amino acids and uric acid.
Primarily, Fanconi is due to missense mutation in sodium/phosphate-
cotransporter (NaPi-II) of the proximal tubular apical membrane. Usui et al.
reported that the human Na-HCO3-cotransporter (NBC1) gene encodes two
sodium-bicarbonate cotransport proteins, pancreatic-type (pNBC1) and
kidney-type (kNBC1). Both kidney- and pancreatic-type of cotransporters
are present in trabecular meshwork, ciliary and lens epithelium and the cor-
neal endothelium. This mutation, common to both pNBC1 and kNBC1 in the
human NBC1 gene, results in severe oculorenal manifestations. The pheno-
typic presentation of this rare mutation involves the kidneys with proximal
RTA and ocular involvement with cataracts, band keratopathy, glaucoma and
even blindness.2
Chapter_21.indd 416 06-02-2018 19:24:58

Cystinosis 417
Fanconi can also be due to other rare secondary genetic causes like
Lowe’s syndrome, hereditary fructose intolerance, galactosemia, cystinosis,
tyrosinemia, Wilson’s disease, but the most common is cystinosis. In com-
parison to congenital Fanconi, drug-induced RTA is the most common cause
of acquired Fanconi. In this chapter, the topic discussed will be cystinosis and
its ocular manifestations.
GENETICS
Cystinosis is a rare autosomal recessive disorder caused by gene mutations
in cystinosis gene (CTNS), which encodes protein cystinosin, a 367-a mem-
brane protein that transports cystine out of the lysosome and is located on
chromosome 17p13. A number of mutations (> 100) have been described in
the promotor of the cystinosis gene, with the clinical phenotypic clustering.
Cystinosis is a multisystem disorder and characterized by an accumulation
of cystine, a disulfide of the amino acid cysteine, in various organs and tis-
sues. The accumulation of cystine crystals in the lysosomes especially in the
eyes and the kidneys leads to severe organ dysfunction. These effects also
involve other organs and tissues like the thyroid, muscles and even testes and
pancreas.3,4 Contingent on the severity of CTNS gene mutations, three forms
of cystinosis have been described: the infantile (nephropathic) form (MIM
#219800), which is the most severe form and the other two the late-onset
(juvenile) (MIM #219900); and the adult onset with only ocular manifesta-
tions (MIM #219750) are more benign. Nephropathic cystinosis, the most fre-
quent (95% of all cases) and also the most severe of all the three, is estimated
to affect 1 of every 100,000 to 200,000 live births.
CLINICAL MANIFESTATIONS
Renal
In children with nephropathic cystinosis the clinical symptoms start by

6–12 months of age but sometimes diagnosis is not made for many months.
Children are born with normal weight and height but typically by first year of
life develop growth delay and present with failure to thrive. They sometimes
present with recurrent bouts of dehydration due to proximal tubular dys-
function causing electrolyte imbalances and associated polyuria. Develop-
ment of Fanconi results in severe electrolyte imbalances with urinary losses
of sodium, potassium, phosphorus and bicarbonate. Some patients also
have vitamin D-resistant rickets secondary to hypophosphatemia due to
ongoing phosphaturia. Also, the tubular losses of carnitine lead to carnitine
deficiency and myopathy with poor motor tone. If untreated, they can prog-
ress to end stage renal disease by first decade of life due to progressive renal
damage from tubular glomeruli. Presently, treatment with cystine depleting
agent cysteamine has greatly improved the life expectancy and quality of life
Chapter_21.indd 417 06-02-2018 19:24:58

with reduction in end-organ damage. So, it is imperative that the patients

are diagnosed to ensure appropriate treatment with cysteamine as soon as
possible.
Extra Renal Manifestations
Growth Retardation
Typically, infants present with failure to thrive within the first 2 years of life.
Aggressive management with fluid and electrolyte corrections, early treat-
ment with cysteamine and sometimes Growth Hormone Therapy is needed
to treat the failure to thrive and growth problems. Hypophosphatemic rick-
ets due to excessive phosphate losses can cause osteodystrophy. Phosphate
repletion and vitamin D supplementation with orthopedic management for
braces and splints is required to ensure proper limb growth and avoidance of
osteodystrophy. Patients may need gastrostomy tube for feedings and medi-
cations to provide nutrition and access for fluid electrolyte management.
Endocrine
There are anecdotal reports of insulin dependent diabetes in patient on peri-

toneal dialysis and in one patient posttransplant on high doses of corticoste-
roids. Thyroid problems have been reported in the form of hypothyroidism
and usually seen after first decade of life. Thyroid functions have to be moni-
tored closely for early detection and treatment.
Myopathy
Muscle weakness is seen in the beginning due to hypokalemia, hypophos-

phatemia and carnitine deficiency. Carnitine levels need to monitored and
replaced. In the second decade of life, progressive distal muscle weakness
and swallowing difficulties have been described.
Neurology
Most children have normal neurological functions but may develop mild
decline in cognitive function. Increased risk of Chiari I malformation have
been described. More severe neurological problems have been described
after second decade in life.3
Gonads
Delayed puberty and infertility have been reported in males. Testosterone

replacement therapy results in pubertal development in males, but does not
affect infertility. Females have delayed puberty but successful pregnancies
have been reported in females.
Chapter_21.indd 418 06-02-2018 19:24:59

Cystinosis 419
The other two forms of cystinosis are milder, the intermediate or juvenile
form usually diagnosed later in adolescence with less severe renal manifesta-
tions. The adult onset is characterized by the presence of corneal crystals and
photophobia and less often renal disease and hence has been also termed
‘ocular’ or non-nephropathic cystinosis.
PATHOGENESIS
Cystinosis is a lysosomal storage disorder wherein there is excessive accumu-
lation of cystine due to defect in the protein membrane transporter for trans-
porting cystine out of the membrane. This intracellular burden of cystine over
time leads to its clinical manifestation with initially loss of low molecular
weight proteins and amino acids and then steady losses of other electrolytes.
Herein are two pictures with cystine deposits in the eye and renal intersti-
tium: the first picture shows classic refractile crystals in the cornea (Fig. 21.1)
and the second picture shows H&E stain of renal biopsy with birefringent
crystals of cystine in the renal interstitium (Fig. 21.2).
The exact mechanism causing cellular dysfunction from the intracellular
accumulation of cystine is not known.5 The possible hypothesis for proximal
tubular dysfunction are increased apoptosis and cell oxidation. The apop-
totic cell death leads to tubular atrophy and damage and ‘swan-neck’ defor-
mity, which has been well described in cystinotic proximal renal tubules. In
mouse model, it has been noted that there are early losses of expression of
apical proximal tubular receptors megalin/cubilin and SGLT-2 and NaPi-IIa
Fig. 21.1: Numerous needle-shaped cystine crystals are seen in the cornea of a patient
with cystinosis.
Chapter_21.indd 419 06-02-2018 19:25:00

Fig. 21.2: Nephropathic cystinosis is marked by the deposition of birefringent intersti-

tial crystals in the kidney. The H&E stain of renal biopsy here shows birefringent crystals
of cystine in the renal interstitium.
transporters-preceding cell atrophy. Oxidative stress, secondary to ATP

depletion, is another proposed mechanism for dysfunction of tubular resorp-
tion. Research is ongoing on mechanisms of cellular dysfunction and other
alternative pathways leading to apoptosis as only cystine accumulation can-
not explain the cell death.
Pathogenesis Involving the Eye
Burki, in 1941, was the first one to describe crystal accumulation in the con-
junctiva and cornea, which is the pathognomonic, ophthalmic manifestation
of cystinosis. Tsilou et al., in their paper, have described at length the vari-
ous ophthalmic manifestations with review of literature.6 They report that the
corneal crystals are highly reflectile opacities easily examined by slit-lamp
examination and they are needle shaped and appear to be present in the cor-
neal epithelium, stroma and endothelium (as shown in the picture). These
can easily be differentiated from other crystalline keratopathies due to their
characteristic appearance and distribution.
The accumulation of crystals begins in infancy and is seen by 16 months
of age in the cornea. By seven years approximately, the peripheral stroma and
endothelium accumulate crystals and by 20 years of age, crystals can be seen
in the entire corneal stroma. Crystal deposition advances more rapidly in the
periphery and is seen usually to begin in the anterior periphery and progress
posteriorly and centripetally. The numerous depositions of cystine crystals
Chapter_21.indd 420 06-02-2018 19:25:00

Cystinosis 421
result in a hazy cornea and in older untreated cystinosis patients can be easily
seen with naked eye.
DIAGNOSIS AND MANAGEMENT

Early diagnosis of cystinosis is important to start therapy with cysteamine
and prevent end organ damage. Currently, as per the cystinosis-consensus
research group, 50% patients worldwide are diagnosed after one year of age.
The following three tests can confirm diagnosis of cystinosis:
1. Measurement of leukocyte cystine levels (LCL) is the gold standard for
diagnosis of cystinosis;
2. Demonstration of corneal cystine crystals by the slit-lamp examination
is the most easily available test in developing countries but the diagnosis
can get delayed as the corneal crystals do not appear till two years of life
and
3. Genetic analysis of the CTNS gene (95% disease causing mutations).
Cystine crystals in the cornea are present by 18 months of age and can be
easily detected by experience ophthalmologist and is the easily conducted
test. Measuring leucocyte cystine levels requires dedicated laboratories and
the diagnostic levels are > 2 nmol 0.5 cystine/mg protein in affected patients,
whereas normal subjects have LCL < 0.2 nmol 0.5 cystine/mg protein.
Molecular testing for CTNS gene is another confirmatory test as it pos-
itive in 95% patients with cystinosis but take longer to get results than the
leucocyte cystine assay.
Prenatal diagnosis in the first trimester (8–9 weeks) using chorionic vil-
lous biopsy or using cultured amniotic cells at 14–16 weeks of gestation can
be reliably used to detect elevated cystine levels in cells of fetal origin from
amniotic fluid cells or chorionic villi.
TREATMENT
Symptomatic Treatment
The supportive care in all children with cystinosis includes: (1) Maintain
acid–base balance and treat fluid and electrolyte imbalances to ensure
adequate growth, (2) provide adequate nutrition via gastrostomy tube and
growth hormone supplementation for growth, (3) replenish phosphates and
vitamin D to prevent rickets and osteodystrophy, (4) monitor for endocrine
and exocrine disturbances and initiate hormone substitution as needed (thy-
roxine, insulin, testosterone, carnitine) and (5) initiate cystine depleting ther-
apy as early as possible both oral and topical to prevent and delay end organ
damage.
It is crucial to start cystine depleting therapy with cysteamine as no cura-
tive cure is currently available but cysteamine does improve the overall prog-
nosis. The aminothiol cysteamine depletes lysosomal cystine content by a
Chapter_21.indd 421 06-02-2018 19:25:00

disulfide-exchange reaction with cystine. Therapy with cysteamine should be

started early and has to be continued life long, even after renal transplan-
tation as the extra-renal damage will continue to progress. The most widely
used cysteamine preparation is cysteamine bitartrate (cystagon) and was
approved for clinical use in United States since 1994 and in Europe since 1997.
Cysteamine has been shown to delay the renal dysfunction and improves
growth and also has been shown to delay extra-renal complications. Leuco-
cyte cystine level (LCL) is used as a biomarker to monitor the effectiveness
of cystine depletion. The LCL levels reach their initial level after six hours of
cysteamine and so the dosing has to be every six hours, which causes many
compliance issues. Recently, a delayed-release form of cysteamine bitar-
trate was approved in the Food and Drug Administration (FDA) of United
States and European Medicines Agency (EMA) in 2013 (Procysbi). Using a
microspheronized enteric-coated formulation of cysteamine bitartrate, the
delayed release leads to higher plasma concentration and higher area under
the curve allowing 12-hour dosing. The decrease in dosing interval will hope-
fully improve compliance and has reportedly decreased gastrointestinal side
effects.
Ophthalmic Treatment
Topical treatment with cysteamine hydrochloride eye drops is used as oral

cysteamine has no effect on corneal cystine crystals (patients are usually
instructed to use it every waking hour). Most patients, however, apply cys-
teamine eye drops less frequently (4–6 times per day instead of > 10 times), in
part because of the low pH of the solution, they cause burning and thus non-
compliance. In addition, the free thiol can oxidize at room temperature, so
it is recommended to store frozen aliquots. A commercial 0.44% cysteamine
ophthalmic solution (cystaran) has been approved for clinical use in United
States. A 0.55% gel formulation (cystadrops) has also been developed and is
recommended to be used only 4 times a day. Unfortunately, if not treated
with topical cysteamine eye drops, patients can develop severe photophobia
and progress to develop corneal lesions severe enough to require a corneal
transplant. Cystine crystals can reappear in the transplanted cornea due to
invading host cells, if topical cysteamine treatment is not used. But, if used
even after crystals develop, it has been shown to reverse the ocular symptoms
and clear the cornea in few months.
Gene Therapy
Various therapies to cure and control cystinosis are in progress and may be
available in near future. Early studies are now available on hematopoietic
stem cell (HSC) transplantation in a mouse model of cystinosis wherein
adult bone marrow stem cells are used as vehicles to bring wild-type CTNS
to tissues.7 Currently research is focused in developing an autologous HSC
Chapter_21.indd 422 06-02-2018 19:25:01

Cystinosis 423
transplantation approach whereby the patient’s own stem cells are gene-
modified using a lentiviral vector and reinjected into the patient to introduce a
functional CTNS copy in tissues. These early reports are exciting as it may pave
road to cure for cystinosis in future.
SUMMARY
The overall prognosis of children with cystinosis has improved with cystine-
depleting therapy and there is ongoing research to find cure with gene ther-
apy. It is critical to identify and treat these patients early to prevent end-organ
damage. The role of ophthalmologists to treat and monitor the ocular mani-
festations of this rare disorder is important part of the multidisciplinary care
needed for patients with cystinosis.
ACKNOWLEDGMENT
I acknowledge Dr Rudolph Wagner, pediatric ophthalmologist, who provided
the picture for the crystals in the cornea.
REFERENCES
1. Abderhalden E. Familiare cystindiathese. Z Physiol Chem. 1903;38:557-61.
2. Usui T, Hara M, Satoh H, et al. Molecular basis of ocular abnormalities as-
sociated with proximal renal tubular acidosis. J Clin Invest. 2001;108(1):
107-15.
3. Niaudet P. Cystinosis. Available at: https:// www.uptodate.com, May 2016.
4. Middleton R, Bradbury M, Webb N, et al. Cystinosis. A clinicopathological con-
ference. “From toddlers to twenties and beyond” Adult-Paediatric Nephrology
Interface Meeting. Manchester 2001. Nephrol Dial Transplant. 2003;18:2492.
5. Gahl WA, Thoene JG, Schneider JA, et al. Cystinosis. N Engl J Med. 2002;
347(2):111-21.
6. Tsilou E, Zhou M, Gahl W, et al. Opthalmic manifestations and histopathology
of infantile nephropathic cystinosis: report of a case and review of the litera-
ture. Surv Opthalmol. 2007;52(1):97-105.
7. Emma F, Nesterova G, Langman C, et al. Nephropathic cystinosis: an inter-
national consensus document. Nephrol Dial Transplant. 2014;29(Suppl 4):
iv87-94.
Chapter_21.indd 423 06-02-2018 19:25:01

22
CHAPTER
Corneal Changes in Contact
Lens Users
Rajib Mukherjee, Gagan Sahni, G Mukherjee
INTRODUCTION
‘DO NO HARM’ is one of the basic principles of medicine. It also applies to
the practice of contact lens (CL) selection, fitting and prescription. The use
of CL may induce problems in patients, and therefore, the CL practitioners
must know corneal changes in CL users and altered corneal physiology after
the wear of CL.
Essentially, corneal pathophysiological changes,1 seen with CL wear,
can be due to the following mechanisms such as, hypercapnia and hypoxia,
allergy and toxicity, mechanical effects and osmotic effects.
HYPOXIA AND HYPERCAPNIA

It is well-known that since the cornea is an avascular structure, the oxygen
needed specially by the corneal epithelium is obtained by diffusion from air
when the eye is open; and from the tarsal conjunctiva, when the eye is closed;
and the use of a CL markedly reduces this oxygen availability to the corneal
tissue. Oxygen deprivation (Hypoxia)2 on using CL depends on the material
of CL and the duration of wear. Along with hypoxia there is also carbon diox-
ide accumulation (hypercapnia) in the cornea. Deprivation of oxygen and
accumulation of carbon dioxide (hypercapnia) in the cornea suppress the
normal corneal metabolism and stimulate anerobic glycolysis. The anerobic
glycolysis is responsible for lowered epithelial metabolic rate and decreased
epithelial mitotic rate. It also increases epithelial lactate production, which
induces an acidic shift in corneal stromal pH.3
Clinically, this gamut of CL induced corneal insult, in this category,
results in:
• Epithelial thinning and epithelial abrasion4 (corneal epithelial defects):
Corneal changes increase susceptibility to injury due to increased
Chapter_22.indd 424 06-02-2018 19:31:28

Corneal Changes in Contact Lens Users 425
Fig. 22.1: Dimple veiling in rigid contact lens (CL) user.
fragility of the corneal epithelium. This may translate in the CL wearer

presenting with signs of ocular discomfort, reduced wearing time,5 con-
tact lens intolerance, watering, etc.
• Epithelial microcysts6-9 (Sattler’s veil): They appear as small translucent
irregularly shaped dots scattered across cornea (Fig. 22.1), which may
disappear within 2 to 12 weeks after discontinuation of CL wear. These
are best seen on high magnification slit-lamp retroillumination evalua-
tion and these patients may complain diminution of vision and seeing
haloes around light.
• Superficial punctate keratopathy (SPK): They are commonly seen due to
compromised epithelial junctional integrity.9 The SPKs show staining
with fluorescein.
• Microbial keratitis:10-14 A devastating complication, which occurs due
to the breakdown of the natural defense and the sick corneal epithelial
integrity. It is seen more commonly in extended wear as compared to
daily wear CL users (Fig. 22.2). Corneal infection associated with RGP
CL occurs less frequently. Pseudomonas aureginosa and Staphylococcus
aureus are the most common organisms responsible for this catastrophe,
while Streptococcus pneumonia, Serratia marcescens and other bacteria
and fungi are less common. Acanthamoeba is another infection, which
may be associated with infectious keratitis in CL users.
• Corneal stromal edema:15 It is caused by stromal acidosis,16 consequent
to lactate and bicarbonate accumulation in the cornea. Apart from over
wear, a tight fit, more so in rigid CL, leads to this complication (Fig. 22.3).
Chapter_22.indd 425 06-02-2018 19:31:29

Fig. 22.2: Contact lens (CL)-induced infective keratitis.
Fig. 22.3: Scleral compression with scleral contact lens (CL).
• Stromal striae:17 The striae appear as fine vertical lines mostly affecting
the posterior stroma. They appear as dark lines in the posterior stroma.
Specular reflection is of great help in their evaluation and on direct illu-
mination, they are seen as white lines. Usually, if stromal edema exceeds
10%, striae are clinically visible.
• Endothelial blebs:18,19 These are sometimes seen in initial CL users as a
transient phenomenon,20 appearing about 30 minutes after CL use and
subsides over several hours. Decreased pH causes patchy endothelial
edema, which appears as defects (black holes) on specular reflection.
Chapter_22.indd 426 06-02-2018 19:31:29

Fig. 22.4: Contact lens (CL)-induced endothelial changes.
• Endothelial polymegathism:3,21 Increased variation in cell size (Fig. 22.4)

is noticed as long-term/chronic changes on corneal endothelium.19 It
has a direct relation of material of CL and duration of wear.
• Corneal warpage: Long-term users of PMMA CL may develop irregu-
lar astigmatism and distortion of central and peripheral cornea. It is
attributed to both mechanical molding and hypoxic influences.22,23 The
various presentations documented on corneal topography are Cen-
tral Corneal Molding Irregularity (Fig. 22.5), Central Corneal Flattening
(Fig. 22.6), Corneal Furrow Depression (Fig. 22.7) and Peripheral Cor-
neal Steepening (Fig. 22.8). These changes tend to disappear within 2 to
14 days of abstinence from contact lens use.
• Corneal hypoesthesia: Alteration in the afferent corneal nervous supply
occurs because of hypoxia and hypercapnia.24
• Superficial vascularization: There occurs dilatation of existing limbal
capillaries due to chronic hypoxia.25,26
ALLERGY AND TOXICITY

The material used in the manufacturing the contact lens is usually biological
inert. However, in certain instances, it may challenge the immune system of
the body. Usually the main causes of reactions are preservatives in contact
lens solution, deposits on contact lens and deposit of debris between cornea
and CL. Complications due to allergy/toxicity are:
• Immobile lens syndrome: It is also referred to as ‘toxic lens syndrome’,
where an inflammatory response is noticed. It is usually due to debris
Chapter_22.indd 427 06-02-2018 19:31:29

Fig. 22.5: Contact lens (CL) warpage, central molding irregularity.
Fig. 22.6: Contact lens (CL) warpage, central flattening.
trapped behind a CL. It is seen mostly in patients using extended wear

CL.27 Unilateral severe pain, photophobia and watering are the usual
presenting features; along with limbal congestion and infiltrates in the
corneal periphery. The corneal epithelium most often remains intact. Ker-
atic precipitates (KPs) may also be seen in some cases. Discontinuation
Chapter_22.indd 428 06-02-2018 19:31:30

Fig. 22.7: Contact lens (CL) warpage, furrow depression.
Fig. 22.8: Contact lens (CL) warpage, peripheral steep.
of CL use and use of lubricants and broad spectrum topical antibiotic

drops usually resolves the condition in 48 to 72 hours.
• Thiomersal hypersensitivity:28 Thiomersal can activate both humoral and
cellular immune responses on the ocular surface and the cornea. By con-
jugating with carrier protein, thiomersal acts as an HAPTEN, in order to
act as a complete antigen. Patients present with bilateral conjunctival
Chapter_22.indd 429 06-02-2018 19:31:30

hyperemia and itching. Corneal epithelial punctate staining and super-

ficial infiltrates are features of thiomersal hypersensitivity. Another
presentation of thiomersal hypersensitivity is superior limbic keratocon-
junctivitis, where one finds congestion of superior bulbar conjunctiva
along with superior punctate epithelial staining.
• Contact lens solution preservative hypersensitivity/toxicity: The com-
mon offending agents in CL solution have been identified as benzal-
konium chloride, alkyl triethanol ammonium chloride (ATAC) and
chlorhexidine gluconate. Principal corneal signs of toxicity are super-
ficial diffuse punctate staining associated with stinging and burning
sensation. Pseudo-dendrite in the cornea may be seen in some cases,
who present raised branching epithelial plaques, that stain very lightly
with fluorescein, accompanied with papillary cum follicular conjunc-
tivitis.29
MECHANICAL EFFECTS
Cornea is a very sensitive tissue, susceptible to injury on contact lens use,
either due contact lenses itself, CL deposits, or during CL manipulations
(wearing, removing, etc.)
• Lens edge imprint: A rigid CL on the cornea may leave an imprint on CL
removal. This is seen on the corneal epithelium as the outline of the infe-
rior rim of the CL. This manifestation is more often seen in ill-fitting or
overnight use of RGP CL.30 Further, it will be prudent to look into any
damage to the contact lens too, altering the dynamics (Fig. 22.9).
Fig. 22.9: Scratches on RGP contact lens (CL).
Chapter_22.indd 430 06-02-2018 19:31:30

• Tight lens syndrome: It is also known as contact lens adhesion, which can
occur due to inadequate wetting, excessive tear evaporation or because
of over wear/overnight wear. The syndrome is mainly due to contact lens
drying/desiccation; causing tightening and adherence to the cornea.31
This usually seen in Soft CL over wear, swimming with CL or sleeping
with CL in the eye. These cases present with photophobia, decreased
vision, irritation, circumcorneal congestion. Key to diagnosis is that on
slit lamp evaluation there is minimal to no movement of the CL on blink-
ing or even no movement on manual intervention by the contact lens
care personal. Remedy is to re-fit the patient with a centrally flatter and
peripherally steeper CL of high Dk value.
• Epithelial wrinkling:32 Wrinkling is seen in PMMA CL users.33 It is asymp-
tomatic and recovers rapidly after CL removal.
• Air bubble dimpling (Fenestrated CLx2): Entrapment of air bubbles under
a PMMA CL is sufficient to cause small indentations on the corneal epi-
thelium (Fig. 22.10). These appear as discrete green dots, because of
pooling, as seen on fluorescein study.34 This is a very significant fea-
ture to be kept in mind during the era of scleral contact lens use. Keep
a watch on patients using rigid Fenestrated contact lenses (with holes)
(Fig. 22.11). If there is any entrapment of air in the interface, may lead to
potential visual problems. However, it can be diagnosed by fluorescein
staining (Fig. 22.12).
• Contact lens and interface deposits (Fig. 22.13): They cause contact lens
intolerance and reduced wearing time.35 The common reasons and
source of these deposits are:
Fig. 22.10: Trapped air bubbles in scleral lens.
Chapter_22.indd 431 06-02-2018 19:31:31

Fig. 22.11: Fenestrated rigid contact lens (CL).
Fig. 22.12: Large air bubble in fenestrated rigid contact lens (CL).
ear deposits:36 Tear lysozyme and mucin deposits occur mostly due
T
to tear component deficiency as well as ocular surface inflammation.
Protein deposits:37 These are more common deposits, which origi-
nate primarily from albumin, globulin and lysozyme in the tears.
They have an opaque, white filmy appearance (Fig. 22.14), and may
Chapter_22.indd 432 06-02-2018 19:31:31

Fig. 22.13: Interface debris in rigid scleral lens.
Fig. 22.14: Protein deposit contact lens (CL) and clear CL.
have cracks when the film is thick. It is found primarily on the front
surface of soft contact lenses and on both surfaces of GP lenses.
Lipid deposits:38 They have a smeared, greasy whitish appearance.
Source of these deposits are the meibomian glands of the eye-lids.
Silicone hydrogel CL materials are most prone to this phenomenon.
Chapter_22.indd 433 06-02-2018 19:31:31

Rubbing and rinsing these lenses with an alcohol-based daily cleaner

normally takes care of the issue.
OSMOTIC EFFECTS
Altered tear film osmolarity on contact lens wear can be attributed to
increased tear evaporation, stimulated reflex tearing and altered blinking
rate. Localized areas of tear film depletion can contribute to epithelial desic-
cation in the cornea.
• Staining at 3 and 9 o’Clock: Usually seen in persons using rigid CL, where
we find fluorescein staining at the nasal and temporal margins of the cor-
nea, always adjacent to the area of lens coverage.39 This phenomenon is
due to the tear film breakdown near the CL edge. Clinical findings in this
situation show a variety of lesions like, punctuate epithelial fluorescein
staining, epithelial microerosions, corneal dellen formation as well as
corneal neovascularization. Contact lens refitting with a smaller diame-
ter CL and with a thinner edge generally solves this problem.
• Coarse punctate erosions: These erosions mostly occur in patient using
thin high-water content hydrogel CL. The coarse punctate lesions are
white in appearance. These have also been described as crumb like
flakes. The theory responsible for this condition is the water loss through
the CL.26
REFERENCES
1. Liesegang, TJ. Physiologic changes of the cornea with contact lens wear. CLAO
J. 2002;28(1):12-27.
2. Harvitt DM, Bonanno JA. Re-evaluation of the oxygen diffusion model for
predicting minimum contact lens Dk/t values needed to avoid corneal
anoxia. Optometry and Vision Science. October 1999.
3. Connor CG, Zagrod ME. Contact lens-induced corneal endothelial polymega-
thism: functional significance and possible mechanisms. Am J Optom Physiol
Opt. 1986;63(7):539-44.
4. Pérez JG, Méijome JM, Jalbert I, et al. Corneal epithelial thinning profile in-
duced by long-term wear of hydrogel lenses. Cornea. 2003;22(4):304-7.
5. Haque S, Fonn D, Simpson T, et al. Corneal and epithelial thickness changes
after 4 weeks of overnight corneal refractive therapy lens wear, measured with
optical coherence tomography. Eye Contact Lens. 2004;30(4):189-93; discus-
sion 205-6.
6. Lambert SR, Klyce SD. The origins of Sattler’s veil. Am J Ophthalmol. 1981;
91(1):51-6.
7. Bourne WM. Soft contact lens wear decreases epithelial microcysts in Mees-
mann’s corneal dystrophy. Trans Am Ophthalmol Soc. 1986;84:170-82.
8. Keay L, Jalbert I, Sweeney DF, et al. Microcysts: clinical significance and differ-
ential diagnosis. Optometry. 2001;72(7):452-60.
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9. Liesegang TJ. Physiologic changes of the cornea with contact lens wear. CLAO
J. 2002; 28(1):12-27.
10. Liesegang TJ. Contact lens-related microbial keratitis: Part I: Epidemiology. Cor-
nea. 1997;16(2):125-31.
11. Eltis M. Contact-lens-related microbial keratitis: case report and review. J Op-
tom. 2011;4(4):122-27.
12. Borazjani RN, Levy B, Donald G. Relative primary adhesion of Pseudomonas
aeruginosa, Serratia marcescens and Staphylococcus aureus to HEMA- type
contact lenses and an extended wear silicone hydrogel contact lens of high
oxygen permeability. Con Lens Anterior Eye. 2004;27(1);3-8.
13. Robertson DM, Cavanagh HD. The clinical and cellular basis of contact lens-re-
lated corneal infections. Clin Ophthalmol. 2008;2(4):907-17.
14. Stapleton F, Carnt N. Contact lens-related microbial keratitis: how have ep-
idemiology and genetics helped us with pathogenesis and prophylaxis. Eye
(Lond). 2012;26(2):185-93.
15. Bonanno JA, Polse KA. Corneal acidosis during contact lens wear: effects of
hypoxia and CO2. Invest Ophthalmol Vis Sci. 1987;28(9):1514-20.
16. McNamara NA, Polse KA, Bonanno JA. Stromal acidosis modulates corneal
swelling. Invest Ophthalmol Vis Sci. 1994;35(3):846-50.
17. Bergmanson JP, Chu LW. Corneal response to rigid contact lens wear. Br J Oph-
thalmol. 1982;66(10):667-75.
18. Campbell R, Caroline P. Contact lens wear ‘Bugged’ by endothelial blebs. Con-
tact Lens Spectrum. December 1997.
19. Chang SW, Hu FR, Lin LL. Effects of contact lenses on corneal endothelium–
a morphological and functional study. Ophthalmologica. 2001;215(3):
197-203.
20. Antti V, Jukka M, Jukka S, et al. Contact lens induced transient changes in cor-
neal endothelium. Acta Ophthalmol (Copenh). 1981;59(4):552-59.
21. Orsborn GN, Schoessler JP. Corneal endothelial polymegathism after the ex-
tended wear of rigid gas-permeable contact lenses. Am J Optom Physiol Opt.
1988;65(2):84-90.
22. Schornack M. Hydrogel contact lens-induced corneal warpage. Con Lens An-
terior Eye. 2003;26(3):153-9.
23. Tseng SS, Hsiao JC, Chang DC, et al. Mistaken diagnosis of keratoconus
because of corneal warpage induced by hydrogel lens wear. Cornea. 2007;
26(9):1153-5.
24. Martin XY, Safran AB. Corneal hypoesthesia. Surv Ophthalmol. 1988;33(1):
28-40.
25. Papas E. On the relationship between soft contact lens oxygen transmissibility
and induced limbal hyperaemia. Exp Eye Res. 1998;67(2):125-31.
26. Bruce AS, Brennan NA. Corneal pathophysiology with contact lens wear. Surv
Ophthalmol. 1990;35(1):25-58.
27. Moshirfar M, Kurz C, Ghajarnia M, et al. Contact lens-induced keratitis resem-
bling central toxic keratopathy syndrome. Cornea. 2009;28(9):1077-80.
28. Wilson LA, McNatt J, Reitschel R, et al. Delayed hypersensitivity to thimerosal in
soft contact lens wearers. Ophthalmology. 1981;88(8):804-9.
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29. Mondino BJ, Salamon SM, Zaidman GW, et al. Allergic and toxic reactions of
soft contact lens wearers. Surv Ophthalmol. 1982;26(6):337-44.
30. Varsha MR, Preeji SM, Srikanth D, et al. Contact lens in Keratoconus. Ind J Oph-
thalmol. 2013;61(8):410-15.
31. Michael AL, Joseph BG. The effects of extended-wear hydrophilic contact lens-
es on the human corneal epithelium. Am J Ophthalmol. 1986;101(3): 274-77.
32. Giese MJ. Corneal wrinkling in a hydrogel contact lens wearer with Marfan syn-
drome. J Am Optom Assoc. 1997;68(1):50-4.
33. Rosenthal JW. Corneal epithelial wrinkling with contact lenses. Am J Ophthal-
mol. 1963;55:138-9.
34. Dixon JM, Lawaczeck E. Corneal dimples and bubbles: under corneal contact
lenses. Am J Ophthalmol. 1962;54(5):827-31.
35. Zhao Z, Naduvilath T, Flanagan JL, et al. Contact lens deposits, adverse respons-
es, and clinical ocular surface parameters. Optom Vis Sci. 2010;87(9): 669-74.
36. Zhao Z, Wei X, Aliwarga Y, et al. Proteomic analysis of protein deposits on worn
daily wear silicone hydrogel contact lenses. Mol Vis. 2008;14:2016-24.
37. Omali NB, Zhao Z, Zhong L, et al. Quantification of individual proteins in sili-
cone hydrogel contact lens deposits. Mol Vis. 2013;19:390-9.
38. Hart DE, Tidsale RR, Sack RA. Origin and composition of lipid deposits on soft
contact lenses. Ophthalmology. 1986;93(4):495-503.
39. van der Worp E, de Brabander J, Swarbrick HA, et al. Evaluation of signs and
symptoms in 3- and 9-o’clock staining. Optom Vis Sci. 2009;86(3):260-5.
Chapter_22.indd 436 06-02-2018 19:31:32

23
CHAPTER
Episcleritis and Scleritis
Parthopratim Dutta Majumder, Jyotirmay Biswas
INTRODUCTION
Episcleritis and scleritis are important causes of acute red eye. Even though
both are inflammation of the outer coat of the eyeball, they are clinically, eti-
ologically and morphologically distinct and different. Therefore, it is essential
to differentiate between these two clinical entities, particularly from treat-
ment point of view.
ANATOMICAL CONSIDERATIONS
The term sclera is derived from Greek word ‘scleros’ meaning ‘hard.’ Sclera
is an opaque, elastic and resilient tissue of the eye. It can be compared with
an incomplete shell comprising approximately 90% (five-sixths) of the outer
coat of the eye. Anteriorly it begins at the limbus and terminates at the optic
nerve canal posteriorly. It is predominantly composed of collagen and some
elastin fibrils and is relatively avascular and acellular.
The episclera is the thin densely vascularized layer of connective tissue
overlying the sclera and situated below the Tenon’s capsule. Apart from the
vessels and unmyelinated nerve fibers, it contains bundles of collagen. Anteri-
orly episclera blends with subconjunctival tissues and Tenon’s capsule 1–3 mm
behind the limbus and it becomes very thin and indistinct posterior to the
equator. Scleritis is always accompanied by an overlying episcleritis. On the
other hand, episcleritis per se is very rarely associated with scleritis. How-
ever, sclera is supplied with nerves particularly near the extraocular muscle
insertions. Direct damage to or the stretch of these nerves is the cause for pain
in scleritis. Scleritis occurs more commonly anterior to the equator because
of the more abundant anterior vascular supply. The circulation overlying the
sclera includes three vascular layers.1,2 The location, configuration of vessels
Chapter_23.indd 437 06-02-2018 19:42:47

Table 23.1: Layers of vessels of episclera and sclera.
Conjunctival Most superficial in Arteries are Can be easily moved on the

plexus conjunctiva tortuous and underlying structure blanch with
veins straight 10% phenylephrine
Superficial Lies at the level of Vessels are In episcleritis, maximal
episcleral Tenon’s capsule straight with a congestion occurs within this
plexus radial vascular plexus
configuration Can be easily moved on the
underlying structure
Blanch with 10% phenylephrine
Deep vascular Lies deep to the Vessels are Maximal congestion in scleritis
plexus Tenon’s capsule arranged in Cannot be moved on the
and directly over crisscross underlying structure
sclera pattern Does not blanch with 10%
phenylephrine
Fig. 23.1: Diagram showing vascular plexuses.
and their clinical significance is tabulated in Table 23.1 and shown in the
Figure 23.1.
NOMENCLATURE AND CLASSIFICATION

The classification system proposed by Watson and Hayreh is widely accepted.3
Scleritis can be divided into anterior and posterior scleritis. Anterior scleri-
tis has further been classified into four subgroups: (1) Diffuse, (2) nodular,
(3) necrotizing with inflammation and (4) necrotizing without inflamma-
tion. Necrotizing scleritis without inflammation is also called scleromalacia
perforans (Flowchart 23.1).
Episcleritis
Episcleritis was first called ‘subconjunctivitis’ by von Graefe, then

subsequently ‘hot eyes’ by Hutchinson. The name ‘episcleritis’ was first used
Chapter_23.indd 438 06-02-2018 19:42:48

Episcleritis and Scleritis 439
Flowchart 23.1: Classification of episcleritis and scleritis
by Fuchs, who described the condition as ‘episcleritis periodica fugax’ in

1895.
Episcleritis is a transient, self-limited disease of adults. It is seen more
frequently in young- to middle-aged women. The chief complaint of patients
with episcleritis is usually ocular redness with or without irritation. The red-
ness typically persists for 24–72 hours and then resolves spontaneously.
Rarely, patients may experience more severe redness and mild pain. Epis-
cleritis occurs most commonly in the exposed zone of the eye and is generally
recurrent in nature. More than one-third of patients have bilateral disease.
Half of the episcleritis cases are idiopathic in nature. Systemic diseases
associated with episcleritis are rheumatoid arthritis, relapsing polychondri-
tis, Cogan’s syndrome, polyarteritis nodosa, etc.4 The majority of patients with
episcleritis recovers completely through treatment with NSAIDs5 and has no
residual changes. However, episcleritis can sometimes be associated with cor-
neal involvement, uveitis and glaucoma.
Episcleritis is diagnosed clinically by the presence of inflamed episcleral
vessels, which typically radiate from the limbus, have a salmon pink color in
natural sunlight, can be moved over the deeper sclera with a cotton tipped
applicator and will blanch with topical 10% phenylephrine.6,7 Episcleritis is
classified as diffuse or nodular. A localized mobile nodule develops in nodular
episcleritis.
Episcleritis can be differentiated from the scleritis on cetain clinical fea-
tures. The distinguishing features between episcleritis and scleritis are tabu-
lated in Table 23.2.
Scleritis
Scleritis is a relatively more severe ocular inflammation than episcleritis. It is

a severe painful inflammatory process characterized by edema and cellular
infiltration of the sclera and episclera. If not treated properly and well in time,
Chapter_23.indd 439 06-02-2018 19:42:49

Table 23.2: Distinguishing features of episcleritis and scleritis.
Episcleritis Scleritis
Generally redness, irritation are the main Severe boring pain is the main presenting
presenting symptoms complaint of the patient
No or minimal tenderness Moderate to severe tenderness
Congested vessels are bright red in color Congested vessels are purple-red in color
and vessels can be moved easily with the and vessels cannot be moved easily with
help of a cotton bud the help of a cotton bud
Blanching of the vessels occurs with 10% Blanching of the vessels does not occur
phenylephrine with 10% phenylephrine
it can be a significant threat to vision. Scleritis affects women more often than
men, with a peak incidence in the fifth decade. It frequently starts in one eye
and become bilateral in more than half of the cases.3,7
Half of the patients with scleritis have evidence of an underlying sys-
temic disease. Scleritis can be a presenting manifestation of a life-threatening
systemic autoimmune disease. Rheumatoid arthritis is the most common sys-
temic condition associated with scleritis. The incidence of rheumatoid arthri-
tis in patients with scleritis ranges from 10 to 33%.3,4,7
Scleritis may be associated with the following systemic diseases:3,4,7-13
• Rheumatoid arthritis
• Relapsing polychondritis
• Systemic lupus erythematosus and antiphospholipid syndrome
• Wegener’s granulomatosis
• Polyarteritis nodosa
• Cogan syndrome
• Juvenile rheumatoid arthritis
• Ankylosing spondylitis
• Ulcerative colitis
• Polymyositis
• Sarcoidosis
• Syphilis
• Tuberculosis
• Herpes zoster ophthalmicus
• Acanthamoeba keratitis
DIAGNOSIS
Diagnosis of scleritis is almost always clinical; however, when the posterior
sclera is involved, clinical signs may be less obvious, and imaging studies are
necessary to confirm the diagnosis. Patients with anterior scleritis presents
with redness and pain. The onset is usually gradual, extending over several
days. Ocular pain is severe and typically dull and boring (piercing) in nature,
exacerbated by eye movement and, occasionally, may worsen at night and
Chapter_23.indd 440 06-02-2018 19:42:50

Perhaps Galileo had sufferered with

scleritis associated with rheumatoid
arthritis. He became totally blind at the
age of 74 years probably due to com-
plcations of sclerits.8
waken the patients from sleep. The pain often radiates to the ear, scalp, face
and jaw.
The sine qua non of scleritis is the presence of scleral edema and conges-
tion of the deep episcleral plexus. Slit-lamp examination using red-free light is
extremely helpful in determining the pattern and depth of episcleral vascular
congestion and engorgement.
In diffuse scleritis (Fig. 23.2), the sclera assumes a violaceous hue in nat-
ural sunlight. It is very important to examine patients in daylight with the
unaided eye to note the subtle color differences of the vessels. Also inflamed
scleral vessels have a crisscross pattern. They are adherent to the sclera and
cannot be moved with a cotton-tipped applicator. Engorged scleral vessels
cannot by blanched with 10% phenylephrine, whereas phenylephrine easily
blanches engorged vessels in the superficial episcleral and conjunctival plex-
uses.6,7,9,10 There is tenderness of the globe.
Nodular anterior scleritis (Fig. 23.3) is characterized by a localized area
of scleral edema and congestion of the scleral vessels. The scleral nodule is
deep red to purple in color, immobile, tender to palpation and separated from
the overlying episcleral tissue, which is elevated by the nodule. These features
Fig. 23.2: Diffuse scleritis.
Chapter_23.indd 441 06-02-2018 19:42:52

Fig. 23.3: Nodular scleritis.
distinguish the nodular scleritis from diffuse episcleritis. The lack of necro-
sis within the nodule and the localization of inflammation within the borders
of the nodule differentiate this form from necrotizing anterior scleritis with
inflammation. All of the vascular layers overlying the nodule are displaced
forward. Sometimes, multiple nodules may be present and there may be an
overlying episcleritis.7-10
Necrotizing anterior scleritis with inflammation (Fig. 23.4) is the most
severe of all the types and is a potential threat to visual loss. It is seen in patients
with rheumatoid arthritis (Fig. 23.5). It is bilateral in 60% cases. The patient
presents with severe pain out of proportion to inflammatory signs. Examina-
tion reveals white, avascular areas of localized scleral edema and congestion;
edges of these lesions are more inflamed than the center. Underlying uveal tis-
sue becomes visible as the sclera becomes thin and translucent. If not treated,
the necrotizing scleritis may spread to the equator and circumferentially and
can involve the entire globe.14,15
Necrotizing Anterior Scleritis without Inflammation
Necrotizing anterior scleritis without signs of inflammation (scleromalacia

perforans), occur predominantly in patients with long-standing rheumatoid
arthritis. There are minimal signs of inflammation and generally no pain
accompanying with this type of scleritis. As the disease progresses, the sclera
progressively thins and the underlying dark uveal tissue becomes visible
(Fig. 23.6). Staphylomas can develop if the intraocular pressure (IOP) is ele-
vated. Though spontaneous perforation is rare, these eyes are prone to rupture
with minimal trauma.14,15 Occasionally, patients may present with blurred
vision because of astigmatism due to thinning and distortion of the globe.
Chapter_23.indd 442 06-02-2018 19:42:53

Fig. 23.4: Necrotizing anterior scleritis with inflammation (necrotizing scleritis). Note the
avascular area (white arrow)
Fig. 23.5: Bilateral necrotizing scleritis in a patient of rheumatoid arthritis.
Posterior Scleritis
Posterior scleritis is defined as an inflammation of the sclera, posterior to the

ora serrata. Posterior scleritis may occur in association with anterior scleritis
or may be isolated. Posterior scleritis, without anterior scleritis, is difficult to
Chapter_23.indd 443 06-02-2018 19:42:54

Fig. 23.6: Necrotizing anterior scleritis without signs of inflammation (scleromalacia

perforans).
diagnose. Patients with posterior scleritis present with pain, tenderness, pro-
ptosis, visual loss and occasionally restricted motility, macular or paramacu-
lar edema (Figs. 23.7 and 23.8) choroidal folds, exudative retinal detachment
(RD), papilledema, and angle-closure glaucoma secondary to choroidal thick-
ening. Most cases of posterior scleritis need ancillary imaging modalities like
ultrasonography (USG) (Fig. 23.9) and a magnetic resonance imaging for con-
firmation of the diagnosis. An infiltration of extraocular muscles in the region
of the posterior scleritis may lead to retraction of the lower lid in the upper
gaze.6,7,16,17
Surgically induced necrotizing scleritis (SINS) can occur after any type
of ocular surgery with excessive scleral manipulation, mostly after cataract
surgery18,19 (Fig. 23.10), but may also be seen after glaucoma, RD, squint or
pterygium surgery. SINS may occur from few days to few years after surgery.
Inflammation is typically localized around the site of the surgical wound or
adjacent to the site of surgery, but may progress to involve the entire sclera.
SINS is mostly necrotizing in nature and sometimes may even be the initial
manifestation of serious systemic disease. So, all patients with SINS need to
be investigated appropriately.
Infectious Scleral Inflammation
Scleritis with purulent exudates or infiltrates should raise the suspicion

of an infectious etiology. Formations of granulomas or fistulas, painful
nodules, conjunctival and scleral ulcers are often seen in infectious scleral
inflammations. Endogenous spread of bacteria (Staphylococci, Haemophilus
Chapter_23.indd 444 06-02-2018 19:42:54

Fig. 23.7: A circular whitish-yellow patch of edema is seen temporal of the macula in
posterior scleritis.
Fig. 23.8: Fundus fluorescein angiography (FFA): Angiography of the patient with pos-
terior scleritis showing punctate leaks at the level of the retinal pigment epithelium and
pooling into the subneurosensory retinal space.
influenzae, Treponema pallidum, Mycobacterium tuberculosis); fungi19

(Aspergillus); viruses (Herpes simplex or Herpes zoster) or parasites (Toxocara,
Toxoplasma, Onchocerca) are reported to cause infective scleritis.6,7 Also, in
Chapter_23.indd 445 06-02-2018 19:42:54

Fig. 23.9: Ultrasonography (USG) scan of posterior scleritis. Note the characteristic
T-sign (white arrow) and thickened sclera.
Fig. 23.10: Surgically induced necrotizing scleritis.
rare instances, infections of adjacent tissues like the conjunctiva, cornea

may involve the sclera by contiguous spread. In chronic cases, possibility of
a foreign body must be ruled out. Scleral infections can often follow buckling
procedures for RD.
Chapter_23.indd 446 06-02-2018 19:42:54

DIAGNOSTIC EVALUATION OF SCLERITIS

As we know that scleritis can occur in association with various systemic
diseases. Sometimes scleral inflammations can be the presenting signs of
an underlying systemic disease. Thus workup of scleritis should include
a thorough physical examination, with attention to the joints, skin and
cardiovascular and respiratory system. The following routine investigations
should be performed:
• Hemoglobin
• White blood cell count and differential count
• Erythrocyte sedimentation rate
• If connective tissue disease is suspected, full immunologic workup
should be undertaken, including levels of immunoglobulins and immu-
nofluorescent studies for autoantibodies (including rheumatoid factor
and antinuclear and anti-ds-DNA antibodies) and circulating immune
complexes. If Wegener’s granulomatosis and polyarteritis nodosa are
suspected, the antinuclear cytoplasmic antibody (ANCA) tests should be
performed. The C-reactive protein is an important laboratory parameter
of an active generalized inflammatory response.4,6
• Serum uric acid
• Full serologic tests for syphilis
• X-ray chest
• Ultasonography
• Ultrasound biomicroscopy (UBM)
B-scan Ultrasonography
B-scan ultrasonography should always be included in the examination of

patients with scleritis. Many patients who were formerly thought to have only
anterior segment disease have been found to have extensive and sight threat-
ening posterior scleritis as well. It also has become known that many patients
with posterior scleritis with minimum symptoms and signs may have much
more extensive disease than had previously been considered possible. The
hallmark features of posterior scleritis seen with B-scan ultrasonography are
helpful in differentiating posterior scleritis from other conditions. B-scan
ultrasonography may reveal the characteristic flattening of the posterior
aspect of the globe due to retrobulbar edema. Abnormally increased thick-
ening of the posterior ocular coats of the globe (> 2 mm), optic disc swelling,
distension of the optic nerve sheath, RDs, and choroidal detachments can be
detected. Fluid can accumulate in the posterior subtenon space and extend
around the optic nerve, forming the characteristic ‘T-sign’ on B-scan.17
Ultrasound Biomicroscopy
This can be valuable for better delineation of scleral thinning and evalua-
tion of scleral edema (Fig. 23.11) and nodules. It is also an important boon
Chapter_23.indd 447 06-02-2018 19:42:55

Fig. 23.11: Ultrasound biomicroscope (UBM) picture of a case of scleritis showing

scleral edema (white arrows).
for ruling out any malignancy. An underlying squamous cell carcinoma or a

medulloepithelioma can extend to the sclera and can masquerade as scleral
inflammation.
Complications of Scleritis
Unlike episcleritis, scleritis is associated with potentially sight threatening

ocular complications. Vision may be limited in scleritis due to keratitis, ante-
rior uveitis, cataract, change of refractive status, macular edema, optic disc
edema, or atrophy, exudative RD. Decreased vision occurs most frequently
with posterior scleritis followed by necrotizing scleritis, nodular scleri-
tis and least often with diffuse anterior scleritis. During any stage of scleral
inflammation, the IOP may be elevated due to several mechanisms, such as
obstruction of the aqueous outflow channels, elevated episcleral pressure,
angle-closure or secondary to a steroid response. Signs of corneal infiltrate,
thinning or stromal keratitis may be present with corneal ulceration. Cataract
formation may be accelerated by long-standing inflammation or secondary
to steroid use.6,7,15
Necrotizing scleritis is a serious diease. Scleral thinning is most com-
mon and it may progress to staphyloma in the presence of raised IOP. Though
increased scleral transparency and thinning is a frequent complication of
necrotizing scleritis but perforation is relatively uncommon Both ocular and
systemic complications are reported in abot 60% of cases and approximately
40% of the patients suffer from serious visual impairment or visual loss. It can
be fetal. About 29% of patients die within 5 years of onset mainly due to com-
plications of vasculitis.
Chapter_23.indd 448 06-02-2018 19:42:55

TREATMENT
The primary aim of the treatment of scleral inflammation is to control the
inflammatory process to relieve the symptoms and thereby reduce the
damage to the eye. However, the effective management of a case of scleral
inflammation involves timely diagnosis, prevention of complications and
identification of underlying systemic or local cause, if any.
Generally, episcleritis is self-limiting benign inflammation, whether
treated or not, it will resolve in 10–21 days. If the condition is recurrent, med-
ications such as topical nonsteroidal anti-inflammatory drugs (NSAIDs) like
flurbiprofen, bromfenac and nepafenac or topical weaker corticosteroid
(loteprednol, fluorometholone) are often required to control the symptoms
of irritation, foreign body sensation, etc. However, prolonged use of topical
corticosteroid should be avoided because of the side effects. It should be kept
in mind that though simple episcleritis resolves spontaneously and rapidly,
resolution of nodular episcleritis is much slower and may require oral medi-
cations. Systemic medications like oral NSAID and very rarely oral corticoste-
roids are required in the treatment of indolent episcleritis.5,6
Anterior non-necrotizing scleritis readily responds to topical steroid
and systemic NSAIDs. Both non-selective cyclooxygenase (COX) inhibitors
(e.g., flurbiprofen, indomethacin, and to a lesser extent ibuprofen) and the
more selective COX-2 inhibitors have successfully been used. Sustained-re-
lease indomethacin 75 mg twice daily has been found to be very effective in
controlling the inflammation. However, prolonged use of NSAIDs should be
avoided in view of their significant side effects on long-term use.
Corticosteroids are helpful in patients not responding to COX-inhibitors
or those with posterior or necrotizing disease. A starting dose of 1 mg/kg/day
is standard with weekly reduction by 10 mg/week until a dose of 40 mg/day
is reached. After this dose is reached, the rate of reduction is individualized,
according to the clinical findings and patients’ response but is in the order of 5
mg/week until cessation or an acceptable maintenance dose is reached. Intra-
venous methylprednisolone is advocated in cases with threatened scleral or
corneal perforation in necrotizing scleritis, which requires a rapid control of
the inflammation.3,5-7,9
Necrotizing scleritis, particularly associated with autoimmune diseases,
is difficult to treat and almost always requires systemic immunosuppressive
therapy, not only for ocular involvement, but also for life threatening systemic
complications. For example, prompt and effective immunosuppression is
required to control the necrotizing scleritis associated with systemic vascu-
litis like Wegener’s granulomatosis because mortality is higher in this group
of patients because of the systemic complications. This group of patients also
requires a consultation with rheumatologist for their systemic ailments.20
Indications for immunosuppressive therapy in scleral inflammation are:
• Anterior necrotizing scleritis
• Posterior scleritis
• Scleritis associated with a systemic disease
Chapter_23.indd 449 06-02-2018 19:42:55

Various immunosuppressants have been tried for treatment of scleritis

and these include antimetabolites (methotrexate, azathioprine and myco-
phenolate mofetil), alkylating agents (chlorambucil and cyclophosphamide),
T-cell inhibitors (cyclosporine and tacrolimus). Newer biological agents like
tumor necrosis factor-alpha (TNF-a) inhibitors (infliximab or adalimumab),
rituximab are reported to be used for the treatment of scleritis.
Methotrexate is commonly used to treat scleritis not responding to oral
corticosteroid and less sever anterior necrotizing scleritis with inflammation.
Often the drug is used as a first line treatment in patients in whom oral steroid
cannot be started because of systemic ailments. Among steroid sparing
immunosuppressives, methotrexate has gained the most widespread usage
due to its relatively safe profile. Methotrexate, a folic acid analog, inhibits the
enzyme dihydrofolate reductase and thus the production of thymidylate,
which is essential for DNA replication. This results in the inhibition of rapidly
dividing cells, including leukocytes. The drug is used with or without a short
course of oral steroid in tapering dosage. Dosage of methotrexate is 0.1–
0.5 mg/kg/week; low dose therapy is started at a dose of 7.5 mg/week
and it can be increased up to 25 mg/week. Generally, it is given orally
once a week. It has been observed that methotrexate immunosuppressive
therapy is moderately effective. The drug takes months to achieve adequate
tissue concentration for the therapeutic success. Severe side effects
such as hepatotoxicity, cytopenias and interstitial pneumonia are not
uncommon.20,21
Treatment of scleritis associated with necrotizing systemic vasculitis
should be prompt and effective if immunosuppression is required. Treat-
ment in such patients should be guided both by the ophthalmic response and
control of the underlying disease. Cyclophosphamide is an effective immu-
nosuppressive drug used in patients with necrotizing scleritis associated with
systemic vasculitis like Wegener’s granulomatosis (Fig. 23.12A to C), relapsing
polychondritis. Antineutrophil cytoplasmic antibody test is a useful labora-
tory parameter to monitor therapeutic response in patients with Wegener’s
granulomatosis. Concomitant administration of prednisone at a dose of 1
mg/kg/day may be needed. Oral corticosteroids can usually be tapered and
often discontinued over the first 6–12 weeks of cyclophosphamide therapy.
Cyclophosphamide in a dose of 100 mg/day (2 mg/kg/day) orally and tapered
monthly, should be the first choice in treating patients with associated poten-
tially lethal vasculitic diseases, such as Wegener’s granulomatosis or polyar-
teritis nodosa. The patient should drink copious amounts of fluid to prevent
hemorrhagic cystitis. In severe and nonresponsive cases, infusion of 500 mg
of cyclophosphamide (given over 1–2 hours) is often required. Because of
potential life threatening complications, it should be administered under the
supervision of a rheumatologis. Other immunosuppressive agents, including
methotrexate, azathioprine, cyclosporine and newer agents like biologicals
have been successfully used for the treatment of necrotizing systemic vasculi-
tis, but reports available are based on small case series.6,20,22
Chapter_23.indd 450 06-02-2018 19:42:55

(A)
(B)
(C)
Figs. 23.12A to C: Necrotizing scleritis associated with Wegener’s granulomatosis.
Chapter_23.indd 451 06-02-2018 19:42:56

Fig. 23.13: Scleral patch graft.
Treatment of Infectious Scleritis
Systemic treatment with topical antimicrobial therapy is the mainstay of

treatment of scleritis of infectious origin. Differentiating infectious scleritis
from non-infectious scleritis is of paramount importance because cortico-
steroid therapy and immunosuppressive therapy (often used in noninfec-
tious autoimmune scleritis) are contraindicated in active infections. Prior to
microbiological determination of the causative organism and its antibiotic
sensitivity spectrum, vancomycin or cephalosporins in combination with
aminoglycosides usually are chosen. Any foreign body, if present, may need
to be removed before the infection can be brought under control.20,22
SURGICAL TREATMENT
Tectonic surgical procedures rarely may be required to preserve the integrity
of the globe. Scleral grafts from fresh sclera or glycerin-preserved sclera those
are available through eye banks. Grafting (Fig. 23.13) may be performed in
cases of threatening perforation before the effects of systemic immunosup-
pressive agents manifest.23,24
SUMMARY
Scleral inflammations are important causes of red eye and need to be dif-
ferentiated from other causes. Though episcleritis is a benign self-limiting
disease, scleritis is highly associated with potentially sight threatening ocular
complications and serious systemic diseases. Early diagnosis and treatment
Chapter_23.indd 452 06-02-2018 19:42:56

of scleritis is important in preventing and diminishing ocular and systemic

morbidity. Hence, attempts should be made to achieve good long-term prog-
nosis with careful clinical history, detailed ocular examination and use of
immunosuppressant drugs whenever necessary without any delay.
Graphics: Dr Parthopratim Dutta Majumder
REFERENCES
1. Snell R, Lemp M. Clinical Anatomy of the Eye. Malden: Blackwell Scientific Pub-
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2. Remington LA. Clinical Anatomy and Physiology of the Visual System (3rd edi-
tion). Butterworth-Heinemann; 2011. pp. 29-32.
3. Watson PG, Hayreh SS. Scleritis and episcleritis. Br J Ophthalmol. 1976;60(3):
163-91.
4. Pavesio CE, Meier FM. Systemic disorders associated with episcleritis and scleri-
tis. Curr Opin Ophthalmol. 2001;12(6):471-8.
5. Lyons CJ, Hakin KN, Watson PG. Topical ﬂurbiprofen: an effective treatment for
episcleritis? Eye (Lond). 1990;4(Pt 3):521-5.
6. McCluskey P. Scleritis. BMJ Publishing Group; 2001. pp. 39-100.
7. Okhravi N, Odufuwa B, McCluskey P, et al. Scleritis. Surv Ophthalmol. 2005;
50(4):351-63.
8. Germani GM. Rheumatoid disease and the blindness of Galileo Galilei. Osp
Maggiore. 1964;59:193-6.
9. Watson PG. Anterior segment changes in connective tissue disease. Trans Oph-
thalmol Soc U K. 1974;94(3):773-84.
10. Watson PG, Hazelman BL. The Sclera and Systemic Disorders. Philadelphia, PA:
WB Saunders; 1976.
11. Sainz de la Maza M, Foster CS, Jabbur NS. Scleritis associated with rheumatoid
arthritis and with other systemic immune-mediated diseases. Ophthalmology.
1994;101(7):1281-6; discussion 1287-8.
12. McGavin DD, Williamson J, Forrester JV, et al. Episcleritis and scleritis. A study
of their clinical manifestations and association with rheumatoid arthritis. Br J
Ophthalmol. 1976;60(3):192-226.
13. Power WJ, Rodriguez A, Neves RA, et al. Disease relapse in patients with ocu-
lar manifestations of Wegener granulomatosis. Ophthalmology. 1995; 102(1):
154-60.
14. Sainz de la Maza M, Jabbur NS, Foster CS. Severity of scleritis and episcleritis.
Ophthalmology. 1994;101(2):389-96.
15. Tuft SJ, Watson PG. Progression of scleral disease. Ophthalmology. 1991;
98(4):467-71.
16. McCluskey PJ, Watson PG, Lightman S, et al. Posterior scleritis: clinical features,
systemic associations, and outcome in a large series of patients. Ophthalmolo-
gy. 1999;106(12):2380-6.
17. Biswas J, Mittal S, Ganesh SK, et al. Posterior scleritis: clinical profile and imag-
ing characteristics. Ind J Ophthalmol. 1998;46(4):195-202.
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18. Scott JA, Clearkin LG. Surgically induced diffuse scleritis following cataract sur-
gery. Eye (Lond). 1994;8(Pt 3):292-7.
19. Sawant SD, Biswas J. Fungal scleritis with exudative retinal detachment. Ocul
Immunol Inflamm. 2010;18(6):457-8.
20. Thomas AA, Narsing AR, Ronald ES. The diagnosis and management of anterior
scleritis. Int Ophthalmol Clin. 2005;45(2):191-204.
21. Kaplan-Messas A, Barkana Y, Avni I, et al. Methotrexate as a first-line corticoste-
roid-sparing therapy in a cohort of uveitis and scleritis. Ocul Immunol Inflamm.
2003;11(2):131-9.
22. Rachitskaya A, Mandelcorn ED, Albini TA. An update on the cause and treat-
ment of scleritis. Curr Opin Ophthalmol. 2010;21(6):463-7.
23. Nguyen QD, Foster CS. Scleral patch graft in the management of necrotizing
scleritis. Int Ophthalmol Clin. 1999;39(1):109-31.
24. O’Donoghue E, Lightman S, Tuft S, et al. Surgically induced necrotising sclero-
keratitis (SINS)—precipitating factors and response to treatment. Br J Ophthal-
mol. 1992;76(1):17-21.
Chapter_23.indd 454 06-02-2018 19:42:57

Index
Page numbers followed by f refer to figure and t refer to table.
A Acremonium sp. 170

Acridine orange 146
Aberrations, high order 88, 102
ACS See Automated corneal shaper
Aberrometer 137
Acute red eye, causes of 437
Aberropia 32
Adalimumab 450
Ablation-related complications 110 Adenoviral keratoconjunctivitis 159
Absidia sp. 170 diagnosis of 162
Acanthamoeba 71, 72f, 139, 140, 145, 148, Air bubble
152, 156, 202-205, 211, 425
dimpling 431
adhesion 206 in scleral lens, trapped 431f
and hartmanella, stages of 203 large 432f
castellanii 204, 206, 207 Air injection in big bubble technique 302f
classification of 203 Air pump-assisted PDEK 386
cysts 152, 152f, 210, 211 Air-guided deep stromal dissection 300
detection of 212 AK See Astigmatic keratotomy
diagnosis of 211 Albumin 432
double walled cysts of 210f Alcohol dehydrogenase 204
genotypes of 212 ALK See Automated lamellar keratoplasty
genus 148 Alkyl triethanol ammonium chloride 430
in corneal stroma 140 Alkylating agents 450
infection 205, 209 Allergy 427
interactions of 207 Allogenic grafts 366
keratitis 70, 71, 140, 143, 202, 208f, Alport syndrome 253
210f, 440 AM See Amniotic membrane
diagnosis 202, 210 American Society of Microbiology 141
diagnosis molecular methods of 211 Amethocaine eye drops 128
immune-biology of 207 Ametropic eye 78
pathogenesis 202, 205 AMG See Amniotic membrane graft
morphological classification of 203t Amiodarone 75
pathogenicity of 207 Amniotic graft 339
plasma membrane of 206 Amniotic membrane 345, 345f, 350
rhysodes 204 characteristics of 345
species 205 extract 353
treatment of 202 for corneal perforation 350
trophozoites 149f, 206, 209 graft 337, 349f
Acid phosphatase, zymograms of 204 advantages of 352
Acid-fast bacilli 154f multilayered 352f
histology of 345f Autologous conjunctival

inlay 347, 348f transplantation 339
with overlay 348 Autologous limbal grafts 366
overlay 347 Autologous oral mucosal tissue,
patch technique 350 transplantation of 367
preparation of 347 Automated corneal shaper 128
transplantation 196, 344, 345, 349f, Automated lamellar
351, 353f, 359f keratoplasty 80, 107
indications for 346 therapeutic keratoplasty 376
surgical techniques 347 technique 376
wound healing of 345 Autosomal recessive disorder 417
Amphotericin B 179, 183, 347 Avellino corneal dystrophy 243
intracameral injection of 180 Azathioprine 450
AMT See Amniotic membrane
transplantation
AMX See Amniotic membrane extract B
Anerobic glycolysis 424 Bacterial and fungal keratitis 202
Aniridia 357 Bacterial infections 172
Ankylosing spondylitis 440 Bacterial keratitis 169, 175, 184
Antibiotic susceptibility 156 Bacterial susceptibility 156
Antifungal Balamuthia species 212
agents 178 Balanced salt solution 117
drug 182, 183 Barraquer’s disciples 80
classes of 179 Barraquer-Krumeich-Swinger
susceptibility testing 156 technique 80
therapy 72 Basal epithelia of limbus 355f
Antigenic tissues, types of 362 Basal epithelial cells 57f
Antiglaucoma Basal limbal epithelium 355f
agents 252 Basement membrane dystrophy 222f
drops 412 Benzalkonium chloride 321, 330, 430
Anti-inflammatory proteins 345 Best corrected visual acuity 32, 120, 129f
Antimicrobial susceptibility testing 148 Best spectacle corrected visual acuity 285
Antineutrophil cytoplasmic antibody Big bubble technique 304, 306, 378
test 450 Blepharitis 321f
Antinuclear cytoplasmic antibody 447 posterior 320
Antiphospholipid syndrome 440 Blepharoconjunctivitis 188
Anwar’s big bubble 384 Blood agar 144, 147f
DALK technique 304, 378 aerobic 144
Aqueous layer 316 anaerobic 144
Aqueous tear production, assessment Bowman’s layer 68
of 327 absence of 226
Aqueous-deficient dry eye 366 dystrophy 221, 231
Argon-fluoride excimer laser 83, 83f Bow-tie pattern 17f
Aspartyl acid 172 Brain heart infusion 144, 177
Aspergillus 169, 170, 179, 181, 182, 445 broth 144, 145
fumigatus 171, 172, 178 with antibiotic 144
niger 331 Breakup test, noninvasive 325t
strains of 171 Bromfenac 449
Astigmatic keratotomy 38, 41f BSCVA See Best spectacle corrected
Astigmatism 88 visual acuity
Autoimmune diseases 346, 449 Bubble technique, small 304, 378
Index 457
Buccal mucosal Cincinnati procedure 362

Graft Clear lens extraction 78
inner surface 395f Clotrimazole 180
outer surface 395f Coarse punctate erosions 434
incision 394f Cogan’s microcystic epithelial
Bulbar conjunctiva 333f dystrophy 224f
superior 430 Cogan’s syndrome 439, 440
Bullous keratopathy 66, 74, 193, 281, Color-coded scales 15
339, 346 Coma primary 32t
aphakic 382 Confocal microscope 66, 68
chronic 357 advantages of 53
early stages of 66 optics of 54f
pseudophakic 382 Confocal microscopy
Burns employs 53
chemical 299 fundamental of 53
thermal 299 in corneal pathology 61
BUT See Breakup test in SMILE 102
of normal cornea 55
Congenital erythrokeratodermia 357
C Congenital glaucoma, coexisting 255
Calibrated spheres 1 Congenital hereditary endothelial
Candida 175, 178, 179, 181, 182 dystrophy 255, 256, 256f, 257f
albicans 172, 178 Congenital stromal corneal dystrophy 246
keratitis 171 Conidiophores 178
Candidal keratitis 179 Conjunctiva 240, 338
Canthal angle, reconstruction of 339 Conjunctival autograft 339, 340f, 343,
Carbohydrate sulfotransferase 244 343f, 344t
Carbon dioxide 424 efficacy of 340
Carboxymethylcellulose 328, 329 for pterygium 341
Cardiac arrythmias 75 in situ 344f
Carnitine 421 procedure 339
deficiency 418 Conjunctival congestion 172
Conjunctival cul-de-sac 387
Cataract and implant surgery 112
Conjunctival epithelial cells 313
Cataractous lens 137
Conjunctival goblet cells 313
Cellular components, types of 55
Conjunctival granulomas 344
Cellular dysfunction 419
Conjunctival inflammation 366
Cellulose esters 329
Conjunctival lesions 346
Central cornea 38
Conjunctival limbal
Central corneal
allograft, living-related 362
marker stitch 396f
autograft 359, 360, 361f
sensitivity 98
Conjunctival plexus 438
thickness 45f
Conjunctival tissue 361, 362
Cephalosporins 452
Conjunctival transdifferentiation 339
Cephalosporium 182 Conjunctival transplantation 339
Chlorhexidine gluconate 430 procedure of 339
Chlorobutanol 330, 331 Conjunctival tumor, post-excision of 339
Chloroquine 75 Conjunctivochalasis 346
Chocolate agar 144 Contact lens 268, 368, 424, 427f, 428f,
Chronic renal failure 102 430f, 431
Cidofovir 198 assisted corneal collagen
Ciliary nerve, long 56 cross-linking 291
fenestrated rigid 432f Corneal collagen 280

fitting for keratoconus 269 cross-linking 270, 276, 283f
induced corneal physiology of 277
molding 48 Corneal confocal microscopy 53, 140
warpage 48 culture methods 145
induced infective keratitis 426f interpretation of microbiology
soft 50f results 151
solution preservative hypersensitivity/ molecular methods 150
toxicity 430 Corneal cross-linking
warpage 23, 428f, 429f technique, steps of 279
wear 357, 434 treatment, indications for 278
Cornea 1, 19f, 78, 338, 395, 419f, 430 Corneal curvature, change in 78
bioengineered 368 Corneal cystine crystals 422
deep vascularization of 358 Corneal decompensation 281
donor 73 Corneal dystrophy 64, 74, 219, 221, 230f
entire circumference of 38 anterior 219
guttata, stage of 250 classification of 219
in keratoplasty surgery, gelatinous drop-like 229
postoperative 46 histopathology 230
in refractive surgery, postoperative 38 inheritance 229
in SMILE, biomechanical properties
management 230
of 100
signs 230
in vivo 53
symptoms 229
irregular 14, 24f
granular 241
lower area of 323f
signs 241
normal 23
symptoms 241
pathological 32
lisch epithelial 221, 228
postsurgery 14
histopathology 229
projection of slit light onto 7
inheritance 229
range of normal 53
management 229
retreated 128
signs 229
sculpting of 79
symptoms 229
shape of 15
management 224
normal 3
steep 115 posterior 219
verticillata 75 posterior amorphous 248
Corneal aberrations 33f histopathology 249
effect of 27 inheritance 248
in normal population 32t management 249
Corneal aberrometry 24 signs 248
Corneal allogenic intrastromal ring symptoms 248
segment 288, 289f posterior polymorphous 253
Corneal asphericity 88 management 255
Corneal astigmatism 344 signs 253
Corneal biopsy tissue 143, 145 symptoms 253
Corneal blindness 337 prevalence of 219, 220t
vision to 222 signs 223
Corneal button 158 subepithelial mucinous 226
section 208f histopathology 227
Corneal cap precision in SMILE 102 inheritance 226
Corneal clouding, diffuse 257f management 227
Index 459
signs 226 Corneal phenotype, marker of 358

symptoms 226 Corneal photoablation 83
symptoms 223 Corneal power, average 22
types of lattice 236 Corneal resistance factor 276
X-linked endothelial 258 Corneal rings in keratoconus,
histopathology 258 indications for 285
management 258 Corneal samples
signs 258 collection of 142
symptoms 258 transport of 142
Corneal eccentricity index 22 Corneal scar 193
Corneal ectasia 276 Corneal scraping 145, 152, 154f, 158, 160f
postoperative 137 collection 143f
procedures for 146t
Corneal edema 66, 254, 281
processing of 143
Corneal endothelial monolayer,
bioengineered 367 stained with gram stain 153f
Corneal endothelium 54, 55, 61, 219, transportation of 142
246, 272 Corneal signs of toxicity 430
disorder of 66 Corneal steepening, inferior 49
Corneal epithelial Corneal stem cells 354
cell 189, 206 Corneal stroma 219, 238f, 298
posterior 378
defects 75, 346, 424
Corneal stromal dystrophy 234
Corneal epithelium 55, 313, 356, 365, 428
inheritance 236
superficial 54
lattice 236
Corneal erosions, recurrent 237
signs 236
Corneal fibers, strengthening of 278f
symptoms 236
Corneal fluorescein staining 98, 323f
Corneal stromal edema 425
Corneal graft 73, 73f
Corneal surface, anterior 3, 99f
Corneal guttata 66
Corneal tensile properties, calculation
Corneal hydrops, acute 61 of 101f
Corneal hypoesthesia 427 Corneal thickness
Corneal impression smear 158 increased 249f
Corneal indexes 21 maps 21
Corneal inlay 78 Corneal tissue 248
Corneal lamellae 175, 240, 298 Corneal topographic patterns, normal 6f
Corneal lesions 75 Corneal topography 1, 3, 5, 10, 16f, 17f
post-excision of 299 in normal right eye 5f
Corneal leukomas 73 maps, interpretation of 13
Corneal limbal epithelial stem quantitative descriptors of 21
cells 356t, 365f uses of 49
Corneal maps 18 Corneal transplant 392
Corneal measurement 1 Corneal ulcer 140, 150f
Corneal melting 281 infectious 351
Corneal myoring with central management of 141
aplannation 21f with serrated and immune ring 174f
Corneal nerves 53, 56 Corneal warpage 48, 50f, 427
Corneal opacification 233 Corneal wavefront
Corneal pachymetry 55, 127 aberration, measuring 26
alteration in 40f analysis derived 27f
Corneal perforation 117, 193 Corneal/conjunctival swab 158
glue in management of 350 Corneal/corneoscleral ulcers 346
Corneoscleral Dematiaceous fungi 174

junction 354 Dendritic ulcer 189, 194f
limbus 355 Descemet’s folds and interface
perforation 344 infection 379
ulcers 346 Descemet’s membrane 61, 66, 171, 172,
Corticosteroids antibiotics 79 175, 219, 248, 256, 272, 298, 304f,
Corynebacterium species 155 305f, 363, 378
and endothelial dystrophy 221, 249
CRF See Chronic renal failure
endothelial keratoplasty 253, 383
Cryopreservation of corneal lenticules,
technique of 103 lamination of 257
Cryoprobe 400f part of 251
Cryptococcus 179 perforation 306
Descemet’s stripping automated endo
neoformans 172
thelial keratoplasty 252, 382, 382f
Curvularia sp. 170, 181
Descemetorhexis 386
Cycloheximide 177
performed 388f
Cyclophosphamide 450
Diabetes 279
Cyclosporin A 195, 334
Diffuse lamellar keratitis 68, 70f, 86,
Cyclosporine 450 119, 119f
Cystadrops 422 DLK See Diffuse lamellar keratitis
Cystaran 422 DMEK See Descemet’s membrane
Cysteamine 422 endothelial keratoplasty
hydrochloride eye drops, treatment DMEK and PDEK, ancillary techniques
with 422 for 385
ophthalmic solution 422 Dohlman keratoprosthesis, parts of 409f
Cysteine, intracellular burden of 419 Doughnut-shaped flap 115, 116f
Cystinosis 416, 417 Dry eye 319
Cystinosis cases of 97
clinical manifestations 417 diagnosis of 321
diagnosis of 421 forms of 326
early diagnosis of 421 inflammation and 319
gene 417 patients 325
genetics 417 severe 339
management 421 status 311
pathogenesis 419 syndrome 321
treatment 421 treatment of 327
gene therapy 422 Dry eye disease 311, 312, 316, 318, 320
ophthalmic 422 and allergies 319
symptomatic 421 and blepharitis 320
and conjunctivitis 320
D and eyedrops 320
causes of 333
Dacron mesh sutured to cornea 401f diagnosis of 328t
DALK See Deep anterior lamellar epidemiology of 312
keratoplasty induction of 319
Debulking after air injection, anterior 303f prevalence 312
Deep anterior lamellar keratoplasty 247f, scale of problem of 312
272, 298, 309f, 378, 379f vicious cycle of 319f
advantages of 381 Dry Weck-Cel sponge 302
ondications of 299 DSAEK See Descemet’s stripping
Deep lamellar keratoplasty 298 automated endothelial keratoplasty
Dehydrating agents 252 Dystrophy
Dellen formation 344 epithelial 220, 222
Index 461
map-dot-fingerprint 222 Epithelial keratitis 187, 191f, 194, 197

subepithelial 220, 222 Epithelial layers distinctly 55
Epithelial microcysts 425
Epithelial microerosions 434
E
Epithelial thinning 424
EBMD See Epithelial basement Epithelial wrinkling 431
membrane dystrophy Epithelium 53, 55
Econazole 180, 181 in keratoconus, superficial 64f
Eczema, severe 191f Erosion dystroph 225f
EK See Endothelial keratoplasty epithelial recurrent 225
ELISA See Enzyme-linked histopathology 226
immunosorbent assay
management 226
Embryonic organogenesis 416
signs 226
Endocrine deficiencies, multiple 357
symptoms 225
Endoilluminator-assisted
Escherichia coli 147, 149f, 211
DMEK 385
Eukaryotic and heterotrophic
PDEK 385
organisms 169
Endophthalmitis 92, 272
Ex vivo stem cell expansion 363
Endothelial blebs 426
Excimer laser 81
Endothelial cell 61, 63f, 73, 74f
pulse 82f
epithelialization of 255
tissue interaction 83
hexagonal 62f
treatment, customized 88
loss of 73, 91, 309
Exogenous sources 74
Endothelial corneal dystrophy,
X-linked 258 Eye
Endothelial decompensation 249f of immunocompetent 171
Endothelial keratiits 189, 197 pathogenesis involving 420
Endothelial keratoplasty 192, 193f, Eyeball 437
252, 298, 381 Eyelid 433
Endothelial polymegathism 427 scarring 339
Endothelium 53, 61, 63f, 378
Enzyme-linked immunosorbent assay 161
Eosinophilic hyaline deposits,
F
deposition of 65 Fabry’s disease 74
Epidermal keratinocytes 365 Fanconi syndrome 416
Epikeratophakia 107 Fehr corneal dystrophy 244
Episclera 437 Femtosecond laser 87, 90, 92, 288
Episcleral plexus, superficial 438 eliminating 79
Episcleritis 437-440, 440t, 448, 449 intrastromal lenticular
classification of 439 implantation 103
Epithelial abrasion 424 LASIK 100
Epithelial basement membrane platform 102
dystrophy 222, 223f technique 286
Epithelial cell 315 Femtosecond lenticule extraction 99, 101
intermediate 57f Femtosecond posterior lamellar
proliferating 86 keratoplasty 389
shape of superficial 64 Femtosecond-assisted
superficial 56, 56f corneal transplantation 388
Epithelial inclusion cyst 344 deep anterior lamellar keratoplasty 389
Epithelial ingrowth 122, 123f lamellar keratoplasty 377
management of 123 LASIK 96, 97, 101f
superficial anterior lamellar principles of therapy 182

keratoplasty 389 risk factors 170
Fenestrated rigid contact lens 432f surgical treatment 184
Fibrin glue 344f treatment for 182, 183
Fibrin substrate 367 Fungal species, identification of 148
Filamentous fungi 170 Fungal ulcer, typical 172f
Flap Fungi
complications 108 classification of 169
decentered 117f detection of 152
problems 85 dimorphic 170
striae 120f nonreplicating, nonreplicating 175
Fleck corneal dystrophy 247 Fusarium sp 169-171, 181, 182
histopathology 248
inheritance 247
management 248 G
signs 248
GCD See Granular corneal dystrophy
symptoms 248
Gene mutations in cystinosis gene 417
Fleshy pterygium 340f
Genetic locus 222
FLEX See Femtosecond lenticule
extraction Giant cell, multinucleated 159f
Fluconazole 180, 181, 183 Giemsa stain 144, 146, 159, 163, 177
Flucytosine 182, 183 Glaeseria 203
Fluorescein 322 Glaucoma
staining 322 coexisting 255
tear breakup test 326 congenital 257
Fluorinated pyrimidines 182 incidence of 406
Fluorometholone 449 intractable 368
Flurbiprofen 449 medications, multidose 331
Food and Drug Administration 422 secondary 272
Forme fruste keratoconus 123 steroid induced 344
Fornix reconstruction 339 Globulin 432
Foscarnet 198 Glucose phosphate isomerase 204
Free flap 116f Gold fish analogy for functions of
FS-LASIK See Femtosecond-assisted tear layers 317f
LASIK Gomori methenamine 177
Fuchs’ dystrophy 66, 249f, 250, 382 silver stain 211
Fuchs’ endothelial Graft
corneal dystrophy 249 adhesion 387
dystrophy 66 centration 387
Fundus fluorescein angiography 445f edema 344
Fungal filaments 176f edge unfolding 387
in gram staining 177f failure, primary 377f
Fungal hyphae 72f floatation 387
Fungal keratitis 71, 139, 140, 169, 184 hemorrhage 344
clinical features 172 inversion 344
diagnosing 72 necrosis in inverted graft 344
epidemiology 169 rejection 308, 389
incidence of 169 retraction 344
laboratory diagnosis 176 unwrinkling 387
medical treatment 178 Gram stain 144, 146, 176
pathogenesis 171 techniques 177
Index 463
Gram-negative bacteria 152 Herpetic keratitis 71, 187, 193

Gram-positive bacteria 152 clinical presentation 189
Granular corneal dystrophy 242f, 243 complications 193
Granular dystrophy 65 epidemiology 187
Grayson-Wilbrandt corneal etiology 188
dystrophy 221, 234 management 194
histopathology 234 manifestations of 187
inheritance 234 prevention 197
signs 234 risk factors 190
symptoms 234 High-hyperopic ablation 45f
Homogeneous hexagonal cells 61
HSV See Herpes simplex virus
H Human amniotic membrane 365f
Haemophilus influenzae 444 Human anterior lens capsular scaffold 367
HAM See Human amniotic membrane Human immunodeficiency virus 347
Hank’s balanced salt solution 158 infection 362
Hansatome microkeratome 128 Human leukocyte antigen 375
Hartmanella 203 Hydroxychloroquine 75
species 212 Hydroxyethyl-cellulose 329
Harvesting conjunctival autograft 342 Hypercapnia 424
Hazy cornea 256f Hyperlipidemia 74
HBSS See Hank’s balanced salt solution Hyperopia 103, 112
Healthy cornea surgical correction of 107
in high magnification 62f Hyperopic LASIK, complications of 108
in low magnification 62f Hyperreflective cellular stroma 68
Healthy endothelial cells, number of 73 Hypertonic sodium chloride 224
Hematopoietic stem cell 422 Hyphe 170
Hemosiderosis 74 Hypokalemia 418
Hepatitis Hypophosphatemia 418
B 347 Hypoxia 424
C 347 Hypromellose 329
Hereditary corneal stromal dystrophies,
types of 300
Herpes keratitis 279 I
Herpes simplex virus 160f, 161, 445
keratitis 159f, 160f, 187-192, 196 Iatrogenic ectasia 87
classification of 189t Iatrogenic keratectasias 87
prevention of recurrent 197 ICL See Implantable contact lens
risk factors for 191t ICRS 284, 285, 292
type of 189 Imidazoles 180
necrotizing keratitis 351 Immobile lens syndrome 427
vaccination 198 Immunological disorders 357
Herpes zoster 445 Immunoperoxidase technique 211
ophthalmicus 440 Immunosuppressive therapy 452
Herpetic disciform keratitis 196 Implantable and prosthetic devices 368
Herpetic endothelitis 196 Implantable contact lens 91
Herpetic epithelial keratitis, treatment In situ keratomileusis 80
of 194 In vivo confocal microscopy 71f, 72, 72f
Herpetic eye disease 187 of cornea 210f
study 188 Infection, initial 207
Herpetic infection 346 Infectious scleritis, treatment of 452
Infectious suppurative keratitis, cause disciform 189, 190, 196f

of 169 fungal etiology of 211
Infective scleritis, causes of 445 infectious 70, 88, 346, 425
Inflammatory response 207 linear 189
Infliximab 450 medicamentosa 330f
Insulin 421 microbial 139, 143, 190, 425
Intacs regular segment 284 non-necrotizing 195
Interblink interval 325 role in 70
Intracapsular crystalline lens type of 197
extraction 400f Keratoconic cornea 62, 270
Intracorneal deposits 74 Keratoconjunctivitis sicca 311, 323f, 324f
sources of 74 Keratoconus 15, 23, 32, 47f, 61, 253,
Intracorneal ring segments 269 285, 289
types of 284t advanced 65f
Intracytoplasmic lysosome-like cases of 62
lamellar inclusion 75 early 33f
Intraepithelial edema 66 optical effects of 268
Intraocular lens 1, 400 prediction index 23
Intraocular pressure 301, 366, 442 progressive nature of 268
accurate 110 surgical management of 271
reduction of 115 suspect 23, 34, 87
Intraocular surgery 92 topography pattern 35f
Intrastromal corneal ring segments 282 treatment options for 268
advantages of 284 Keratocyte 53, 246
complications 288 depletion 207
contraindications 284 nuclei 59
disadvantages of 285 Keratoglobus 36, 253
indications for 283 Keratolimbal
intraoperative complications 288 allograft 359, 363, 364f
postoperative complications 288 graft 363f
structure of 283 Keratometer 1
surgical techniques 285 Keratometric index, standard 2
types of 282 Keratometry reading
Iris excision 400f minimum 22
Itraconazole 180, 181 simulated (SimK values) 21
Keratomileusis 79
experience of 87
J
literally 79
Juvenile rheumatoid arthritis 440 Keratopathy, drug-induced 75
Keratoplasty 220t, 252, 309, 338, 392
advances in 375
K
classification advances in 375
KCS See Keratoconjunctivitis sicca conductive 112
Keratconus, management of 268 indications of 74
Keratectomy, postphotorefractive 40 manual 375
Keratic precipitates 428 procedure 392
Keratitis 160f, 183, 357 repeat penetrating 377f
cause of 171 superficial 338
diagnosis of nonviral 144t, 146t, 150 Keratoprosthesis 392
diagnostic procedures in infectious 139 Keratorefractive procedures 32
diffuse 189 Keratoscopy 2
Index 465
Keratotomy in hyperopia 107, 110

hexagonal 107 indications of 89
overcorrected radial 112 infections 122
photorefractive 40, 68, 79, 83, 95, interface debris 121
99, 107, 114 intraoperative
Ketoconazole 180, 181, 183 bleeding 118
Kinyoun method 154 complications 114
Kinyoun’s modification of acid-fast keratectasia 123
stain 146 late postoperative complications 122
Kontur lens 415 limitations of 89
Krypton fluoride excimer laser 83f management 114
overcorrected myopic 112
L partial flap 114, 115
primary 135
Lacrimal gland 313, 318, 319, 337 regression and haze 125
atrophy, cause of 318 retinal complications 125
Lactophenol cotton blue 146 surgery 127
Lamellar keratoplasty 272, 298, 375, 376 wavefront-guided 134
anterior 376 LASIK See Laser-assisted in situ
posterior 376, 381 keratomileusis
superficial anterior 376 Lasiodiplodia sp. 170
LASEK See Laser epithelial keratomileusis Lattice corneal dystrophy 237, 238, 238f
Laser cavity 82f Leaking blebs 346
Laser correction for myopia 284, 285 Lens
Laser epithelial keratomileusis 85, 107 and cornea
Laser lenticular extraction 103 distance between front 55
Laser thermal keratoplasty 44, 46f, 112 front 54
for hyperopia 46f edge imprint 430
Laser-assisted in situ keratomileusis 42, 67, supplementary 78
69f, 70f, 78, 84, 95-103, 109f, 114, 127 syndrome, tight 431
ablation 43, 135 Lenticular problems 92
birth of 79 Lenticule creation 102
central islands 124 Leucine aminopeptidase 204
complications 85, 90, 114 Lid 394f
contraindication for 110 lower 394f
decentered Lignocaine hydrochloride 141
ablations 124 Limbal allograft 339
flap 117 rejection 362
early postoperative complications 119 Limbal autograft 339
epithelial transplantation 360
defect 118, 119 Limbal cell transplantation, surgical
ingrowth 122 techniques for 359
flap Limbal dermoid 346
displacements 121 Limbal epithelium 313, 356
wrinkling/striae 120 Limbal stem cell 355, 367
free cap 116 cultivated 363
group 97 deficiency 338, 356, 359f
hyperopic 108, 109f, 111, 111f, 112 causes of 357t
ablation-related complications 110 total and partial 357f
flap complications 108 location of 354f
management of complications 110 transplantation 339, 354, 366
Limbal tumors 357 Mucoepidermal junction of lid 313

Limbic keratoconjunctivitis, superior 322 Mucor 170
Limbitis, chronic 357 Mucosal epithelium, biopsy of 365
Limbus 338 Mucosal flap, central opening in 401f
Lipid deposits 433 Mueller-Hinton agar 148, 150f
Lipid layer 316 Mycobacterium chelonae keratitis 154f
LK See Lamellar keratoplasty Mycobacterium tuberculosis 150, 445
Local tangential curvature map 20 Mycophenolate mofetil 450
Loteprednol 449 Mycotic keratitis 71
Lowenstein-Jensen medium 144 diagnosis of 177
LSCD See Limbal stem cell deficiency Myopia 282, 284
LTK See Laser thermal keratoplasty high 78, 99f
Lycoprotein 270 low 89
Lymphoproliferative syndromes 319 moderate 98f
Lysophospholipase 206 treatment of 95
Lysosomal enzymes 209 Myopic ablation 43f, 84f
Lysosomal storage disorder 419 decentered 46f
Lysozyme 432 Myopic corrections 41
Myopic eyes 87
M Myopic laser in situ keratomileusis 17f
Myopic LASIK 108
Macula in posterior scleritis 445f
Macular corneal dystrophy 244, 245f, 300
Macular dystrophy 246, 247f N
Macular edema 92 Natamycin 179, 183
Malate dehydrogenase 204 Near-Descemet’s deep lamellar
Mannose-binding protein 205 keratoplasty 299
Matrix metalloproteinases 270, 351 Necrotizing anterior scleritis 442, 444f
Matted eyelashes 321f with inflammation 442, 443f
Maumenee corneal dystrophy 227, 256 without inflammation 442
histopathology 228 Necrotizing keratitis 195
management 228 Necrotizing scleritis 438, 442, 443f, 448,
signs 228 449, 451f
symptoms 227 surgically induced 444
Meibomian gland 313, 433 Nephropathic cystinosis 417, 420f
dysfunction 320 Nerve
obstructed 321f fibers, small 58
Meniscus height 326 growth factor 100
Metaherpetic ulcer 193f plexuses, sub-basal 58
healing 348f plexuses, subepithelial 58
Methotrexate 450 Neurotrophic keratitis 171
dosage of 450 Neurotrophic keratopathy 193, 346
Miconazole 180, 183 Neurotrophic ulcer 349f
Microbial keratitis 140 healed 353f
diagnosis of 141 Neutrophils 171
Microkeratome-assisted LASIK 96 kill ameba 209
Moire deflectometry-based systems 7 Newer biological agents 450
Molecular methods 156 Nipkow disk 54
Mooren’s ulcer 357 Nocardia species 154
Mucin in goblet cells 358 Nodular scleritis 442, 442f
Mucin layer 316 Non-amniotic substrate 367
Index 467
Noninflammatory ectatic disorder 61 Orbscan II system 8f

Non-nutrient agar 143, 144 Orbscan, postoperative 133f
Nonpreserved drugs 331 OSD See Ocular surface disorders
Non-Sjögren’s syndrome 319 Osmotic effects 434
Nonsteroidal anti-inflammatory drugs 449 OSR See Ocular surface reconstruction
Nonviral corneal ulcer 143 OSSN See Ocular surface squamous
Nonviral keratitis 140, 144f, 157, 211 neoplasia
NSAIDs See Nonsteroidal Oxychloro complex, stabilized 331
anti-inflammatory drugs
Nuclear sclerotic cataract 137
Nystatin 180 P
Pachymetry 89
O Paecilomyces 182
Ocular Pallikaris 84
adnexa 337 Papanicolaou stain 159
chemical injury 346 Papillary cum follicular conjunctivitis 430
cicatricial pemphigoid 347, 357 PBIKP See Pintucci biointegrated
disease, active 279 keratoprosthesis
Ocular protection index 325 PBS See Phosphate buffered saline
calculating 325t PDA See Potato dextrose agar
Ocular surface 313, 313f, 338 PDEK See Pre-Descemet’s endothelial
comprises 337 keratoplasty
damage 317 Pellucid marginal corneal
disease 97, 299 degeneration 292
disorders 337 Pellucid marginal degeneration 35
dysfunction of 316 Penetrating keratoplasty 48f, 271, 375, 376
epithelium 354 Perilimbal injection 362
equivalents, bioengineered 363 Perinuclear hypodense rings 56
inflammatory reactions of 318 Periodic acid-schiff stains 177, 211
multiple 357 Peripheral cornea 38, 355f
reconstruction 337 Peripheral iridectomy 387
indications for 339t Peripheral ulcerative keratitis 357
squamous neoplasia 299, 339, 346 Phakic lens 78, 91
resection 352f
Phosphate buffered saline 158
staining 322, 323t
Phosphoglucomutase 204
transplantation, evolution of 338
Phospholipase A 206
Oculopalpebral and reconstructive
surgery 347 Photoablation related complications 85
Oil glands, assessment of 327 Photokeratoscope raw image 14f
Onchocerca 445 Photokeratoscopy 2
Opaque corneal stroma 305 Photophobia 256, 356, 428, 431
Ophthalmic practice 70 Phototherapeutic keratectomy 125
Ophthalmometer 1 Pigmented fungal ulcer 175f
Optical coherence tomography 11f Pintucci biointegrated keratoprosthesis
Optics 54 392, 393, 399f
Optimum refractive treatment 49 surgical technique 393
Oral ketoconazole 183 Pintucci keratoprosthesis 393f
Oral steroids, starting 191f Piramidal aberrometry 26
Orbicularis Placido’s disk 7
muscle 397 method 7
suturing 396f system 5
Placido’s targets, types of 7 Pseudoanterior chamber 307

Placido’s rings 3f, 14 Pseudokeratoconus 49
Plano refraction 136 Pseudomonas aeruginosa 147f, 425
Pleomorphism 73f isolated 150f
PMMA See Polymethyl methacrylate Pseudomonas keratitis 156
Polyarteritis nodosa 439, 440, 447 Pseudomonas spp 143
Polyenes 179 Pterygium 38, 299, 339, 346, 357
Polyglycolic acid 396 excision 340f, 342, 343f
Polymegathism 73f surgery 343
Polymerase chain reaction 151, 158 complications of 344t
Polymethyl methacrylate 282, 368, 401 Punctate epithelial staining, superior 430
Polymorphous dystrophy, posterior 66 Punctate keratitis, superficial 321
Polymorphous membranous dystrophy, Punctum patch 333f
posterior 254f Pupillary block glaucoma 307
Polymyositis 440 Pupillometer 89
Polyphaga 204 Pure placido disk technology 18
Polyvinyl alcohol 329 Pythium insidiosum 148
Polyvinyl pyrrolidone 329
Postastigmatic keratotomy 38
R
Postintrastromal corneal rings
implantation 45 Rabbit corneas 83f
Postlaser in situ keratomileusis 42 Ray tracing system 26
Postlaser thermal keratoplasty 44 Reactive oxygen species 277
Post-LASIK Recipient eye 406f, 407f
ectasia 282, 289, 292 button removed 405f
eye 137 Rectus muscle 396
surgery 154f, 276 disinsertion 344
Post-PRK ectasia 282 Reflection of buccal mucosa, second
Postradial keratotomy 38 stage 398f
cornea 40f Refractive lens surgery 89
Post-refractive keratectasia 276, 280 Refractive lensectomy 78
Potassium hydroxide 144, 176, 176f Refractive map 20
preparation 146 Refractive outcome 308
Potato dextrose agar 144, 145 Refractometer, spatially resolved 26
Potential visual acuity 22 Reis-Bucklers corneal dystrophy 231, 232f
Povidone 329 histopathology 232
Preclude iris 256f inheritance 231
Precorneal tear film, layers of 316t management 233
Pre-Descemet’s endothelial signs 232
keratoplasty 381, 383, 384 symptoms 232
Prednisolone 308 Relapsing polychondritis 439, 440
Preocular tear film 314 ReLEx technique 90
PRK See Photorefractive keratectomy Renal dysfunction 422
Prokera 353 Renal manifestations, extra 418
Prolate surface 4 endocrine 418
Propionibacterium gonads 418
acnes 150 growth retardation 418
species 155 myopathy 418
Propionyl esterase 204 neurology 418
Protein deposit 432 Renal tubular acidosis 416
contact lens 433f Residual hyperopia 135
Index 469
Residual pachymetry 131 diagnosis of 440

Residual stroma, thickness of 87 diagnostic evaluation of 447
Retinal detachment 92 diffuse 441f
Retreated corneas 127 posterior 438, 443, 444, 446f, 447, 449
RGP lenses See Rigid gas treatment of 450, 452
permeable lenses Scleromalacia perforans 438, 442
Rheumatoid arthritis 440, 442, 443f Scotopic pupils, large 88
Rheumatoid factor 447 SDA See Sabouraud dextrose agar
Rhizopus sp. 170 Securing conjunctival autograft 342
Riazole antifungal agents 182 Septate fungal filaments 152f
Riboflavin acts 270 Serratia marcescens 425
Rigid contact lens 425f Serum uric acid 447
Rigid gas permeable lenses 48, 116, Sexual hormones 335
268, 269
Shack-Hartmann method 26
Rigid scleral lens, interface debris in 433f
Sheep blood agar 145, 177
Robertson’s cooked meat broth 144
chocolate 145
Root mean square 32t
Sheimpflug-based noncontact
Rose bengal 324
tonometer 100
staining of conjunctiva 324f
Shield ulcer 346
RTA See Renal tubular acidosis
Silver sulfadiazine 183
Sirius system 9
S Sjögren’s syndrome 171, 319
Sabouraud dextrose agar 144, 145 primary 319
Sahara syndrome 86 secondary 319
SAI See Surface asymmetry index Slice of cornea, computer-generated 53
Sands of Sahara 119 Small-incision lenticule extraction 89, 91,
syndrome 68 96-99, 101, 101f
Sarcoidosis 440 advantages of 97
Satellite lesions 173 all-femtosecond 91
multiple 174f surgery, enhancements after 103
Sattler’s veil 425 technique 96
Scedosporium 182 SMILE See Small-incision lenticule
Scheimpflug camera 9 extraction
Scheimpflug photography, principle of 8 SMILE ReLEx, future of 90
Scheimpflug technology 15 Sodium perborate 331
Scheimpflug-based topographer 9f Spherical aberration 32t
Schirmer’s test 98, 327 primary 32t
Sclera 437 Spherical configuration 38
Scleral contact lens 426f Spherical equivalent 287
Scleral edema 442, 448f SPK See Superficial punctate keratopathy
Scleral inflammation 452 Staphylococcus aureus 425
infectious 444 Staphylococcus epidermidis 155
treatment of 449 Stem cell
Scleritis 437, 439, 440, 440t, 444, 447-449 cultures 347
anterior 438, 443 transplantation 360f, 366
necrotizing 449, 450 Stevens-Johnson syndrome 299, 339,
non-necrotizing 449 357, 368, 392
classification of 439 Stocker-Holt variant encompasses 228
complication of 448 Strabismus, surgical correction for 247
necrotizing 448 Streptococcus pneumonia 149, 152, 425
Stroma 55, 58 inflammatory mediators in SMILE 100

acellular 58 lysozyme 432
cellular 58 meniscus height 98
neurosensory 58 nonpreserved artificial 331
Stromal corneal dystrophy 219, 235, 235t preservation 332
Stromal disease, surgical treatment of 378 preservatives in artificial 329
Stromal dystrophy 221, 234 substitutes, artificial 329t
Stromal edema 254f substitution 328
Stromal herpes simplex keratitis 351 Tear film 313, 313f
Stromal keratitis 175, 187, 189, 190, composition of 315
195, 197 dysfunction of 316
Stromal keratocyte 60f functional unit 313t
nuclei 59 normal 316f
Stromal necrosis 207 osmolarity, altered 434
Stromal nerve fibers 59 regulation 314
Stromal opacities 245f stability, assessment of 325
Stromal striae 426 Tenon’s capsule 437
Subconiunctival hemorrhage 85 Terrien’s marginal degeneration 37
Subconjunctival miconazole 183 Testosterone 421
Subconjunctival tissues 437 Tetraene antibiotic 179
Therapeutic DALK 380
Subconjunctivitis 438
Therapeutic keratoplasty 184
Subepithelial nerve
Thiel-Behnke corneal dystrophy 233
fibers 58f, 69
histopathology 234
plexus 55, 56
inheritance 233
Subneurosensory retinal space 445f
signs 233
Sulfacetamide 183
symptoms 233
Superficial punctate keratopathy 321, 425
Thioglycollate broth 144
Suprachoroidal hemorrhage 92
Thiomersal hypersensitivity 429
Symblephara 395
Threatening infectious keratitis 70
Symblepharon 346
Thyroxine 421
formation 339
Tissue culture methods 161
shell in situ 349f TMH See Tear meniscus height
Syphilis 347, 440 Topical antibiotic drops 308
serologic tests for 447 Topical autologous serum 335
Systemic autoimmune disease 440 Topical corticosteroids 334
Systemic diseases 439 Topographers
Systemic lupus erythematosus 440 curvature-based 5
Systemic tetracyclines 334 elevation-based 7
Topographic indexes, basic 21
Topography
T examination 10
Tacrolimus 450 improvement in 290f
T-cell inhibitors 450 Topography-guided treatment 89
Tear Toxic epidermal necrolysis 346, 357
artificial 328 Toxic lens syndrome 427
breakup time, noninvasive 325 Toxicity 427
deposits 432 Toxocara 445
drainage system, occlusion of 332 Toxoplasma 445
evaporation in ocular surface gondii 150
disease, role of 318f Transient amplifying cells 355f
Index 471
Transporters-preceding cell atrophy 420 Virology results, interpretation of 163

Trauma 357 Visante ocular coherence tomography 307f
Treponema pallidum 445 Visual acuity 308
Triazole 180 assessment 279
water-soluble 181 best corrected 270
Trichiasis 339 corrected distance 103
Trifluridine 195, 198 decreases 243
Trigeminal nerve 314 spectacle-corrected 86
Trophozoites of acanthamoeba 152 uncorrected 131f, 308
Troublesome artefacts 152 Visual disturbances, severe 42
Tscherning aberrometry 135 Visualize corneal guttata 66
Tscherning technique 26 Vogt’s striae 61
Tuberculosis 440 Voriconazole 181
Tubular glomeruli 417 Vortex keratopathy 75
Tumor necrosis factor-alpha 100 VZV See Varicella zoster virus. 161
U
W
Ulcer 173f
Wegener’s granulomatosis 440, 447,
Ulcerative colitis 440
449-451, 451f
Undercorrected hyperopic 111
Wilson’s disease 74
LASIK 111
Witschel dystrophy 246
Unmyelinated nerve fibers 437
Urrets-Zavalia syndrome 379
Y
V Yeasts 170
Valacyclovir 198
Vancomycin 452
drops 410 Z
Varicella zoster virus 159, 161
Zeihl Neelsen acid-fast stain 146
Vascular plexuses 438f
Zeiss achroplan lens 141f
Vessels of episclera and sclera, layers
of 438t Zernike polynomial 27
Videokeratography system 4f expansion 28f
Videokeratoscopes 7 Zernike second order 134
Videokeratoscopy 3 Ziehl-Neelsen
Viral staining 154f
disease 157 technique 154
infections, diagnosis of 156 Zylink software program 128
Viral keratitis 157, 158t Zyoptix enhancement
collection of samples 157 program 132
diagnosis of 157, 163 surgery 129
diagnostic tests for 161t Zyoptix system 127, 135
protocol for 156 Zyoptix wavefront-guided customised
transport of samples 157 ablation 127, 128

Gems of Ophthalmology Cornea and Sclera

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Gems of Ophthalmology Cornea and Sclera

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Gems of Ophthalmology

CORNEA AND SCLERA

Nitin Nema MS DNB

The Health Sciences Publisher

Inquiries for bulk sales may be solicited at: jaypee@jaypeebrothers.com

Vinay Agarwal MS Frank Joseph Goes MD

Charmaine Chai MBBS MMed (Ophth)

G Madhavi DNB N Venkatesh Prajna FRCOphth

Quresh Maskati MS FCPS DOMS FICS

Manotosh Ray MD FRCS

Rajib Mukherjee MS Gagan Sahni OD

Prema Padmanabhan DNB FRCOphth

Radhika Tandon MD DNB FRCS MRCOphth M Vanathi MD

Cornea is one of the important structures of the eyeball. It serves as a window

chances of rejection. The other procedure is Descemet’s stripping automated

2. Corneal Confocal Microscopy 53

4. SMILE versus LASIK 95

5. LASIK in Hyperopia 107

6. LASIK—Complications and Management 114

7. Zyoptix Wavefront-guided Customised Ablation in

8. Diagnostic Procedures in Infectious Keratitis 139

9. Fungal Keratitis 169

10. Herpetic Keratitis 187

11. Acanthamoeba Keratitis—Pathogenesis and Diagnosis 202

12. Corneal Dystrophies 219

13. Management of Keratoconus 268

14. Corneal Collagen Cross-linking 276

15. Intrastromal Corneal Ring Segments 282

16. Deep Anterior Lamellar Keratoplasty 298

17. Dry Eye Disease 311

18. Ocular Surface Reconstructions 337

19. Advances in Keratoplasty 375

20. Keratoprosthesis 392

21. Cystinosis 416

22. Corneal Changes in Contact Lens Users 424

23. Episcleritis and Scleritis 437

FIRST STEPS IN CORNEAL MEASUREMENT

In 1854, Helmhotz described the first true keratometer, which he called

This apparatus is based on the tendency of the anterior corneal surface

Although keratometers are still common in ophthalmology clinics, they do

Keratoscopy and Photokeratoscopy

Fig. 1.1: Placido rings.

When a photographic camera is attached to the keratoscope, we have a

At the beginning of this century, modern corneal topographers were based

FUNDAMENTALS AND TECHNOLOGICAL APPROACHES

Fig. 1.2: Videokeratography system.

astigmatism. Depending on the position of the axes, corneal astigmatism is

CORNEAL TOPOGRAPY: CURRENT TECHNOLOGIES

Systems Based on the Light Reflection on the Cornea:

Placido Disk System

Interferometric Method-based Systems

In essence, a reference surface (or its hologram) is compared to the tested

Moire Deflectometry-based Systems

Systems Based on the Projection of a Slit Light onto the

two-dimensional (2D) cross-sections, it is possible to create a reliable three-

Systems Based on the Principle of Normal Photography

Systems Based on the Principle of Scheimpflug Photography

Fig. 1.5: Orbscan II system.

Fig. 1.6: Scheimpflug-based topographer—Pentacam.

properly compensate for an off-center measurement, a dual Scheimpflug

Systems Based on Optical Coherence Tomography

Optical coherence tomography (OCT) of the cornea and anterior segment is

PERFORMING A GOOD TOPOGRAPHY EXAMINATION

Fig. 1.7: Optical coherence tomography (OCT)-based topographer—CASIA SS-1000.