Ocular Microbiology

OCULAR
MICROBIOLOGY
OCULAR
MICROBIOLOGY
PK Mukherjee MS
Consultant Ophthalmologist
Former Professor and Head
Upgraded Department of Ophthalmology
Pt JNM Medical College, Raipur
Chhattisgarh, India
Preeti Bandyopadya MSc PhD

Reader in Microbiology
GD Rungta College of Science and Technology
Bhilai, Chhattisgarh, India
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Jitendar P Vij
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Ocular Microbiology
© 2010, Jaypee Brothers Medical Publishers
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First Edition: 2010

ISBN 978-81-8448-860-9
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PREFACE
Last few decades have witnessed tremendous change in the

field of microbiology in general and ophthalmology in
particular. The behavior of the microbes have undergone
many variations. The two most probable causes being the
changes in the organism and its ability to mutate. Many
organisms have developed newer protective mechanisms
against known antimicrobials. The second cause is the
changed immunity in the hosts.
Some organisms that were thought to be commensals are
proving to be the causes of many unsuspected devastations.
On one end of the scenario is the emergence of newer and
potent drugs that have made management of infections easy,
but on the other end is a dismal picture of drug resistance.
Common organisms have developed mechanisms to
circumvent commonly used older drugs.
Clinical presentation of many of the infections and
infestations are sufficient to reach a tentative diagnosis
which may require simple tests to clinch the presence of the
specific organisms. Unfortunately, this is not always possible
due to a lack of broad based knowledge of ocular micro-
biology in postgraduates and younger teachers. This makes
it imperative that the postgraduates in ophthalmology have
a broad based knowledge of microbiology.
The present book is mainly addressed to those who would
find it handy for day-to-day reference. They are expected to
consult larger books on microbiology for advanced
knowledge.
PK Mukherjee
Preeti Bandyopadya
ACKNOWLEDGMENTS
At the outset I remember with reverence late Dr S Agarwal,

former Professor and Head, Department of Pathology; Dean,
Pt JNM Medical College, Raipur and Director, Medical
Education, Govt of Madhya Pradesh. He was my teacher in
most formative period of medical education, introduced me
to the fascinating world of microbiology. He was happy to
know that I was writing a book on Ocular Microbiology and
encouraged me to go ahead.
I am indebted to Dr Vijaya Sudarshan, Head, Department
of Pathology, Pt JNM Medical College, Raipur, Chhattisgarh
for all the help she has rendered in the project. She not only
supplied me with unending stream of books and journals
but also lent me some useful photographs.
Dr AK Chandrakar, Professor and Head, Department of
Ophthalmology, Pt JNM Medical College, Raipur,
Chhattisgarh; Dr (Prof) ML Garg and Dr Nidhi Pandey,
Associate Professor of Ophthalmology provided a free access
to their personal books. Their generosity is acknowledged
with gratitude.
Dr Santosh Singh Patel, Assistant Professor in
Ophthalmology, Pt JNM Medical College, Raipur,
Chhattisgarh played a major part in writing this book. He
was always available with unending flow of reprints. He
also provided me with good quality photographs. He
deserves a special mention. I thank him for his contributions.
I am thankful to Dr BK Nayak, Editor, Indian Journal of
Ophthalmology, for his permission to use some of the
photographs published in the above journal.
viii OCULAR MICROBIOLOGY
Dr Manik Chatterjee, Associate Professor of Anatomy, Pt

JNM Medical College, Raipur, Chhattisgarh helped me at
every step whenever required. I thank him profusely.
My wife Protima has been the main driving force all
through the writing of this book. She carefully stored, looked
after and kept track of my ever-increasing correspondence.
I am happy that my younger daughter Dr Preeti
Bandyopadya could spare enough time to be the co-author
of this book in spite of her busy teaching and research
schedule.
My elder daughter Protibha Mukherjee Sahukar, both my
sons-in-law, Satyadeep Sahukar and Dr Abir Bandyopadya,
were always ready to go through the manuscript, drawing
figures and fashioning the flow charts. Their contribution is
duly acknowledged with thanks.
Shri Maneesh Dandekar of Hypersoft, was never tired of
typing and re-typing the manuscripts and useful suggestions
in fashioning the book. His efforts are acknowledged with
gratitude.
I am thankful to Shri Jitendar P Vij (Chairman and
Managing Director), Mr Tarun Duneja (Director-Publishing)
and other members of their team in Jaypee Brothers Medical
Publishers (P) Ltd, New Delhi, who extended full cooperation
to publish this book.
CONTENTS
1. General Considerations ................................................ 1

2. Ocular Bacteriology ..................................................... 15
3. Cocci of Ocular Importance ........................................ 25
4. Bacilli of Ocular Interest ............................................. 39
5. Ocular Virology ............................................................ 73
6. Ocular Mycology .......................................................... 99
7. Ocular Parasitology .................................................... 121
8. Outline of Laboratory Tests in
Ocular Microbiology .................................................. 175
Bibliography ................................................................. 192
Index .............................................................................. 197
2 OCULAR MICROBIOLOGY
INTRODUCTION
Infection is an interaction between the host and the micro-

organism. The host tries to minimize the effect of the microbe
by developing a mechanism of resistance called immunity.
Mere presence and multiplication of organism on the host is
called infection that may not result in infectious disease
infectious disease is a state where the invading organism
develops an upper handover the defense mechanism of the
host. Some of the terms used to designate infections are
Primary infection: First infection by a microorganism.
Reinfection: Subsequent infection by the same organism.
Reactivation: The organism lies dormant in a target area, to
become active later under changed condition. The common
examples are herpes zoster, herpes simplex, toxoplasmosis and
histoplasmosis.
Latent period is the period lapsed between the primary
infection and reactivation and the infection is called latent
infection, i.e. herpes simplex and herpes zoster.
Secondary infection: An infection superimposed on a primary
infection that may be by commensal or a pathogen. The ocular
examples are superimposed bacterial infection in viral
keratitis. The reverse is not reported. More common is fungal
infection set-up on bacterial or viral infections.
Cross-infection is a second infection developing in an already
diseased host from either a diseased person or a carrier.
Nosocomial infection is an infection acquired during hospital
stay due to cross-infection from the environment, instruments,
prosthesis, etc.
GENERAL CONSIDERATIONS 3
Iatrogenic infection is a physician induced infection due to

therapy, a common example is hepatitis B infection following
blood transfusion.
Carriers are persons who harbor pathogenic organism
without suffering any adverse effect brought about either due
to partial treatment or immune response.
Various Types of Carriers
1. Temporary carriers, who are capable of infecting for a short

period, i.e. less than six months. The convalescent carrier is
an example of this status.
2. Convalescent carriers are those who have recovered from the
disease and are capable of transmitting disease to others.
3. Chronic carriers: This status lasts for months to years or
even for rest of life.
4. Paradoxical carriers are those carriers who acquire the
disease from another carrier.
Based on the relationship between the microorganism and
the host, the microorganisms have been divided into two
broad groups, i.e.
1. Saprophytes
2. Parasites
The saprophytes are free-living organism that grow on dead or
decaying organic matters. They generally are incapable of
multiplying on living tissue. They are incapable of producing
disease in the host because multiplication of the micro-
organism is one of the essentials of infectious disease.
The position of saprophytes as causative organism for
infectious disease has shown a dramatic change in recent
years due to emergence of immunocompromise in large number
of persons. The disease thus produced is called opportunistic
infection. B. subtilis and B. cereus are emerging as causative

organisms in devitalized tissues. In eyes, they are being
reported frequently to cause traumatic endophthalmitis.
Free-living organisms are those organisms that thrive in soil
and water. Most of the organisms met within nature are free-
living and autotropic, i.e. they are capable of synthesizing
necessary nutrient from water, atmosphere and dead organic
matter. The common examples are P. aeruginosa, B. subtilis,
clostridia, Actinomyces, acanthameba, all of which can cause
systemic as well as ocular infection.
The parasites are those organisms which live on living host
and derive nutrition from the host without any benefit to the
host. Generally, parasites cannot exist independently. They
may be harmless or harmful to the host. The former is called
commensal while the later is called pathogen.
The commensals live in utmost harmony with the host which
is larger than the commensals, without any harm to the host.
The commensals derive nutrition from the host and are in many
ways sheltered by the host. The host does not derive any benefit
from the commensals. The commensals constitute the normal
microbial flora of the body. The examples are Staphylococcus
epidermidis on the skin, E. coli of gastrointestinal tract and
C. xerosis in the eyes. The commensals survive on secretion
and waste products of the host.
Pathogen: A microorganism capable of producing disease is
called pathogen. The pathogens are heterotropic, i.e. they are
not self-sustaining, they require complex organic substances
for nutrition.
As per habitat the parasites can be:
i. Ectoparasite, that lives on the surface of the host, i.e. scabies
or phthiriasis. The disease produced by them are called
infestation.
ii. Endoparasites that live inside the body of the host, i.e.
malarial parasites. The parasites that can not survive
without a host are called obligate parasites, i.e. toxoplasma.
A facultative parasite may exist without a host.
Opportunistic pathogens are commensals and saprophytes
that can produce disease when the body resistance is below
optimal level.
Source of Infection
1. Normal flora
Normal flora may become pathogen due to changed
immunity that may be:
i. Systemic as in AIDS
ii. Biochemical change, i.e. diabetes
iii. Indiscriminate use of antibiotic, steroid
iv. Chemotherapy and radiation.
v. Local ocular conditions like abnormal tear status, ocular
surface defect, contact lens, prolonged uses of antibiotics
or steroids are some of the examples.
2. Human beings are the commonest source of infection to others,
both systemic as well as ocular.
The mode of transmission among humans can be:
i. Inhalation: This method of transmission is commonest
mode in respiratory tract infection. The organisms may
remain suspended in the air and inhaled or may fall on
the ground and inhaled along with dust.
ii. Ingestion: This is the most frequent mode of transmission
of gastrointestinal diseases, may be water borne or food
borne.
iii. Inoculation: The organisms are virtually implanted in the
tissue following breach in the skin. The examples are
tetanus and rabies. The organism can be inoculated in-

advertently by infected needles, syringes, i.e. hepatitis B,
HIV or through blood transfusion. It is a common mode
of transmission in drug addicts.
iv. Autoinoculation: When localized infection is transmitted
to noninfectious part of the same person. The best
example is vaccinia.
v. Contact: This can either be direct contact as sexually
transmitted diseases or indirect through fomites.
Fomites are inanimate objects like clothes, instruments,
toys and cosmetics. The best ocular example of contact
transmission by fomites is trachoma.
vi. Transplacental: These are congenital in nature. The
developing fetus gets the disease from the infected
mother. The examples are rubella, cytomegalovirus,
syphilis, and toxoplasmosis.
vii. Iatrogenic: The infection is transmitted either during
therapy or diagnostic procedure, some of which can be
nosocomial. The examples are meningitis following
lumber puncture, urinary tract infection following
catheterization.
3. Animals: Animals are capable of transmitting diseases, to
humans. The transmitting animals may be symptomatic or
asymptomatic or may act as a carriers. The examples are bite
of dog leading to rabies, ingestion of ova or cyst of parasites
or helminths. The diseases transmitted by animals are
called zoonotic diseases or zoonoses. Some of the birds are
also known to spread disease. These diseases are called
ornithosis, i.e. psittacosis.
4. Insects: The insects that transmit the disease are called
vectors. Common vectors are mosquito, flies, ticks, mites
and lice.
The transmission may be

i. Mechanical transmission: The examples are dysentery and
typhoid which are transmitted from infected stool, water,
etc. to the host.
ii. Biological: The vector acquires the organism at various
stages of development. The pathogens multiply in the body
of the vector and the developmental cycle is completed in
the host. The examples are Anopheles mosquito transmitting
malaria, Aedes aegypti transmitting yellow fever, Simulium
damnosum transmitting river blindness, Chrysops fly (Loa-
Loa), Ixodes-tick ( Lyme disease).
Besides being vectors some of the insects act as reservoir of
the organisms
5. Soil: Some free-living pathogens survive in the soil for long-

time and infect the host either by inoculation, i.e. tetanus
or inhalation Histoplasma capsulatum and Nocardia. Some
of the helminths also survive in the soil and cause
infection, i.e. roundworm and hookworm.
6. Food: Contaminated food material are important source of
avoidable transmission of diseases. The diseases are
typhoid, amebiasis, cysticercosis, toxocariasis.
7. Water: Water transmitted diseases are public health
problem all over the world. The water may be contami-
nated by pathogen that on ingestion cause diseases, i.e.
cholera, infective hepatitis. The water may contain a vector
which harbor the pathogen, i.e. cyclops harboring third
stage larva of guinea worm.
To cause infection, the microorganisms undergo through
following stages:
1. The microorganisms colonize on the surface, they intend to
invade.
2. Adherence: The microorganisms cause diffusion of toxin

and enzyme that leads to next stage, i.e. invasion and
multiplication. The bacteria contain structures called
capsules and pili, they help in adherence.
3. The toxins liberated by bacteria can either be endotoxins or
exotoxins.
Endotoxins are produced by gram-negative bacteria
following lysis or destruction of the cell wall. The action of
endotoxin is similar in all organisms.
Exotoxin: The toxins are liberated extracellular by intact
bacteria. The toxin spreads by bloodstream, may produce
distant or local effect. Exotoxins of different organisms have
different action and invade different tissues. They are active
in minute quantity and are highly antigenic. The exotoxin
when treated with formaldehyde give toxoid which is non-
toxic and is use to immunize persons. The examples are
tetanus and diptheria. They are generally produced by
gram-positive organisms but some gram-negative
organisms also produce exotoxin, they are shigella, vibrio
and E. coli.
4. Enzymes: The common enzymes produced by various
bacteria are Coagulase, collagenase, hyaluronidase, strepto
kinase. Out of these coagulase and collagenase have
significant ocular action.
5. Besides toxins and enzymes, the organisms liberate other
chemicals also. Some of them are hemolysin, fibrinolysin,
leukocidin. The enzymes, toxins and lysins play an
important role in invasiveness of the organism.
6. The term invasiveness means property of organism to invade
the host, multiply locally, spread via lymphatics and
bloodstream. The invasiveness is enhanced by virulence
of the microorganism besides enzymes and toxins.
7. The virulence of a microorganism depends basically on

invasiveness and toxigenicity of the organism which are
constantly opposed by the defense mechanism of the host.
The important factors governing virulence are
i. Infective dose
ii. Presence of capsules and spores in the organism
iii. Ability to produce toxin and enzymes
iv. Communicability of the microorganism.
MICROBIOLOGY IN RELATION TO THE EYES
The principle involved in ocular microbiology is the same as

general clinical microbiology with few differences.
1. There are only a few organisms that cause exclusively
ocular diseases. They are Moraxella lacunata, Haemophilus
aegyptius and Corynebacterium xerosis.
2. Most of the organisms have systemic involvement.
i. The ocular manifestation may develop after the systemic
disease is well-entrenched.
ii. The ocular disease may cause systemic disease, i.e.
meningococci, gonococci.
3. The eyes being small organs, the specimens removed from
them to isolate the organisms are very small that makes
recovery and identification of organisms difficult.
4. The specimens do not withstand transport time.
5. Some of them require immediate staining or microscopic
examination.
Normal Microflora of the Eyes

The eye being exposed to the atmosphere throughout the
waking hours and their continuity with skin, nasopharynx
make them vulnerable to contamination and keep them
contaminated.
The eyes are sterile only for few hours after birth and then get
invaded by microorganisms.
The eyes may be infected during the birth if the labor is

prolonged.
The organisms are found on the skin of the lids, the lid
margins and conjunctiva in abundance. The cornea rarely
yields microorganism unless diseased.
The microorganisms can either be saprophytes or
commensals, both the group of organisms do not produce
disease till the immunity of the host is perfect. Both the groups
have tendency to cause opportunistic infection whenever the
conditions are in favor of the organism and against the host.
Microfloras are generally similar in both eyes at onset, course
and type. Thus, if any organism has been detected in one eye,
the other eye is also expected to harbor the same organism.
This is true only for non-pathogens.
The normal ocular flora is divided in two broad groups—
Resident flora and transient flora.
The resident floras are
1. The same organism grows on repeated culture.
2. They represent true colonization. The organisms are
S. epidermidis, S. aureus, lactobacilli, diphtheroids. Propioni
bacterium is being reported frequently to cause post-IOL
endophthalmitis.
Transient ocular floras are those organisms that are not found
consistently when serial cultures are taken under the same
condition from the same eye. All the cultures do not yield
same microorganism.
The organisms are
i. S. aureus and other species of staphylococci
ii. Pneumococci
iii. Bacillus species

iv. Branhamella catarrhalis
v. E. coli
vi. Klebsiella
vii. Enterobacter
viii. Pseudomonas
ix. Peptococci
x. Clostridia
xi. Many fungi are also found in the conjunctival sac of
persons who live in villages.
Some important observations are:
1. Haemophilus, pneumococci and streptococci are found more
frequently in children.
2. Xerosis is commonest saprophyte.
3. Pseudomonas is recovered more frequently from
hospitalized patients.
4. Keratoconjunctivitis is predisposed to infection by Proteus
and S. aureus.
5. Prolonged use of antibiotics changes the microflora.
6. Viruses are neither resident nor transient flora of the eyes.
Routes of ocular infection can be exogenous or endogenous.

1. Exogenous:
i. Air, water, body fluid, infected drops, instruments,
contact lens, foreign bodies and fomites.
ii. Continuity: Lids, lacrimal sac, conjunctiva to cornea,
meninges, walls of paranasal sinuses.
2. Inoculation: Penetrating injuries, surgical wounds, retained
foreign body, viscoelastic, IOL, irrigating fluids, perforating
corneal ulcers.
3. Endogenous: Blood borne
PROTECTIVE MECHANISM OF THE EYES
Eyes are highly sensitive sensory organs. They have been given
adequate anatomical and physiological protection
The anatomical protection is offered by
1. Walls of orbit: The boney orbital walls surrounds the eyeball
from all sides. The wall is shorter on the lateral side, this
makes the eyes vulnerable to trauma and infection from
the lateral side.
2. Lids: The combination of tarsal plate and orbicularis form
a formidable curtain that can be activated both voluntarily
as well as reflexly. This is the commonest form of protection
from exogenous invasion by microbes.
3. The lashes: The lashes entangle the microorganism and
prevent them from entering the conjunctival sac.
4. The orbital septum prevents infection spreading from
pre-septal area to retroseptal space and vice versa.
5. The conjunctival epithelium itself is resistant to many
organisms. It had good phagotic action as well.
6. The corneal epithelium is resistant to all organisms except
Gonococcus, Meningococcus, Diphtheria, Haemphilus aegyptius
and Listeria.
7. The Descemet’s membrane also restrains organisms to go
deeper but can not stop toxins from passing through it.
8. The tear film has multiple protective functions –
i. Mechanical flushing of the conjunctiva and the cornea.
The tear production is increased manifolds as soon as
an organism finds its way on the conjunctiva or cornea.
ii. Antimicrobial action: The lysozyme and lactoferrin present
in tear have a microstatic action on many organisms. If
the level of these two fall below the critical level, there is
immediate rise of bacterial flora in the conjunctival sac.
iii. The immuglobulins: The tear contains three immuno-

globulins, they are IgA, IgG and IgM. The IgA and IgM
constitute the first line of defense against the microbial
invasion. They help to maintain a certain level of
saprophytes, which in turn prevent the pathological flora
from colonizing on the ocular surface. They also reduce
adherence of bacteria to the epithelium. The
immunoglobulins are thought to neutralize toxins.
The physiological protection are reflex in nature. They are
1. Blink reflex that closes the eye automatically on approach
of any foreign body.
2. Bell’s phenomenon: As the lids close, the eyeball rolls up,
thus more than 2/3 of the cornea goes under the upper lid.
Even in absence of blinking as in lagophthalmos, the Bell’s
phenomenon gives a fairly good protection.
3. Mechanical action of lid movement keeps the conjunctiva moist
and well-lubricated.
4. Reflex tearing increases the tear production to flush out the
microorganism.
Failure of any of the above defense mechanisms predis-
pose ocular infections.
Factors that Predispose Ocular Infections
Rich blood supply of the eye except the cornea and lens
makes the eye predisposed to hematogenous spread of the
infection. The meninges and cerebrospinal fluid (CSF)
transport intracranial infection to the eye. The organism
finds the paper thin walls of paranasal sinuses easy to
pass through. The retained intraocular and intraorbital
foreign bodies are a constant and common source of ocular
infection.
The organisms that are known to cause ocular infections

are
1. Bacteria
2. Chlamydiae
3. Fungi
4. Virus
5. Parasites
i. Protozoa
ii. Metazoa
a. Cestodes
b. Nematodes
6. Arthropods
7. Larvae
MORPHOLOGY OF BACTERIA
Size
The bacteria have variable sizes, the cocci are smaller than
bacilli. The size varies between 0.1 to 50 micron. Common
pathogens have a range between 0.5 to 2.0 micron. The large
bacteria have more complex structure than small bacteria.
The smaller bacteria are deficient in enzymes, hence they
depend more on host cells for growth and multiplication.
Shape
The bacteria are divided into two main groups as per shape,
i.e. the cocci and bacilli. The former are circular, can be spherical,
kidney-shaped or lancet-shaped. The latter are rod-shaped
in between them are Cocco bacilli which are in fact bacilli. The
spirochetes are spiral, vibrios are a comma-shaped. Some are
filamentous, i.e. actinomycetales (Figure 2.1).
Figure 2.1: Shapes of various bacteria: 1. Cocci; 2. Bacilli;

3. spirochete; 4. Cocco bacilli; 5. Diplococci
OCULAR BACTERIOLOGY 17
Arrangement
The cocci can be arranged in clusters, chains, pairs or scattered

(Figure 2.2).
Figure 2.2: Arrangement of cocci: 1. Streptococci; 2. Staphylococci;

3. Gonococci; 4. Meningococci
The bacilli are generally scattered. They can form small

chains—Streptobacilli or may be in pairs—Diplobacilli. When
in pairs, they join each other end to end. The Corynebacterium
diphtheriae are arranged in angular fashion and the
arrangement is called Chinese letter pattern, M. Leprae are
arranged in bundles (Figure 2.3).
Cell Wall
This is a rigid outer layer made of carbohydrates, proteins

and lipids enclosing the cytoplasm. The outer thickened part
of the cytoplasm under the cell wall is called cytoplasmic
membrane. The cell wall and cytoplasmic membrane have
antigenic property. There is differences in composition of
the cell wall in gram-negative and gram-positive organisms.
Figure 2.3: Arrangement of bacilli: 1. In cluster; 2. Chains;

3. Chinese latter pattern
Capsule
Some of the organisms develop a thick outer jelly like covering

called capsule. This is mostly made up of polysaccharide or
polypeptides. It protects the organism against antibacterial
substances and increases the virulence of the organism. It
contains antigen. The common capsulated organisms are S.
pneumonia, Bacillus anthracis, H. influenzae and Cl. welchii
(Figure 2.4).
Flagella
They are responsible for motility of the organism seen only in

bacilli, all bacilli do not have them. They are straight, thread
like structures that protrude through the cell wall.
Fimbriae (Pili)
These are short straight filaments seen only in some gram-

negative bacilli, i.e. Salmonella, Proteus, Shigella. They are
responsible for adherence to cell wall.
Figure 2.4: Capsule of pneumococci stained with Indian ink
Spores
Spores are part of defense mechanism of the bacteria, they are

resistant to antibacterial material and represent dormant stage
of the bacteria. The bacterial spores, which develop inside
the cell wall and called endospores. The fungi have exopore on
the outer cell wall. As per location of the spores, they can be
central, subterminal or terminal (Figure 2.5). The spores are
resistant to boiling, heating and disinfectant. They are best
demonstrated by Gram-stain and modified Ziehl-Neelsen
stain.
The spore forming organisms are B. anthracis, B. subtilis,
Cl. tetanus, Cl. welchii and Cl. botulinum.
Oxygen Requirement
According to effect of oxygen on various microorganisms

they have been divided into—Aerobics and anaerobic
Figure 2.5: Various positions of endospores
organism. The former requires oxygen to grow. The obligate

aerobes grow only in presence of oxygen. The facultative
anaerobes generally require oxygen for growth but may grow
in its absence as well. The obligate anaerobes may be destroyed
in presence of oxygen.
Classification of Bacteria (Figure 2.6)
Besides these the bacteria are classified according to their

Motility, capsule, spore, toxin, enzyme, resistance to antibiotic,
position in relation to cell wall, i.e. intracellular/extracellular,
pleomorphism, resistance to temperature and disinfectants.
Visualization of Bacteria
Bacteria are microscopic and not visible with naked eyes.

They require staining to be visible. The commonly used
microscopes are:
1. Optical or light microscope: They can be bright field, dark
field, ultraviolet or phase contrast microscopes.
2. Electron microscope: Only to see dead and dried tissue.
3. Confocal microscope: To see organisms in living tissue, i.e.
cornea.
Figure 2.6: Classification of bacteria
OCULAR BACTERIOLOGY
21
They can be:

i. Tanden scanning confocal
ii. Slit scanning confocal
The other microscopes used are:
1. Interference microscope
2. Polarization microscope
The bacteria do not show details of their structure under
light microscope as they lack contrast hence they must be
stained to see the structures.
Various staining methods used are:
1. Simple staining: They are not used routinely. The stains
used are methylene blue and basic fuchsin. They give
uniform color to all bacteria.
2. Differential staining: These are the most commonly used
method to examine bacteria and Chlamydiae. They are
i. Gram’s stain (Figures 2.7 and 2.8)
Figure 2.7: Gram-positive cocci

(Courtesy: Dr Santosh Patel)
Figure 2.8: Gram-negative bacilli

ii. Acid-fast stain

The former is used universally to find out if an organism
is gram-positive or negative. On the ability to stain with
Gram’s stain depends many of the clinical presentation
and sensitivity of the organism to antibiotics.
The acid-fast staining is used to identify mycobacteria
mostly in tuberculosis, leprosy and nocardia.
3. Negative staining: This is used to stain the background of
the slide. The bacteria remain unstained.
4. Impregnated methods: The very small bacteria are
impregnated with silver salt to make them visible.
5. Darkground illumination: This is a nonstaining method in
which reflected light is used instead of transmitted light.
The method is used to see slender organisms like spirochete
Figure 2.9: Darkground illumination Treponema pallidum
not visible under ordinary illumination. The organisms

stand out clearly giving an illusion of increased resolution.
The organism appear to be self-illuminating (Figure 2.9).
Both types of cocci, i.e. gram-positive and gram-negative

are known to cause ocular infection.
Gram-positive: Staphylococcus
Streptococcus
Pneumococci
Gram-negative: Gonococci
Meningococci
Branhamella catarrhalis
Acinetobacter
Moraxella
GRAM-POSITIVE COCCI
Staphylococci
Staphylococci are a gram-positive, spherical bacteria

belonging to the family Micrococcaceae (Figure 3.1). The
staphylococci are at the top of the list of the organisms of
ocular importance. They are found constantly on the skin
and mucous membrane. They are both resident as well as
transient flora of the conjunctiva. They are generally seen
in clusters but may be seen single, in pairs or in short chains.
They are nonmotile, noncapsulated, nonsporing, are generally
penicillin sensitive. There is a fast-growing tendency among
the staphylococci to develop resistance to penicillin and
allied antibiotics due to production of beta-lactamase
(Penicillinase). They may develop resistance to multiple
antibiotics at a given time. They are some of most resistant
nonsporing organisms. The resistance to antibiotics and
usual disinfectants make them a potential ocular hazard
especially following surgery.
COCCI OF OCULAR IMPORTANCE 27
Figure 3.1: Staphylococci. Courtesy: Prof V Sudarshan
They are aerobes or facultative anaerobic, generally grow at

30°C both in liquid and solid media. The growth may be
enhanced at 40°C. They are destroyed at 60°C in 30 minutes.
Commonly used media are—blood agar, nutrient agar and
MacConkey’s medium (Figure 3.2). They produce hemolysin
in blood agar and produce pigments ranging from white,
yellow to orange.
Figure 3.2: Staphylococci growing on culture plate.

The staphylococci produce many enzymes and toxins

which constitute the virulence factors. Most important
among then is coagulase that clots plasma. Ability of the
organism to cause clotting of the plasma is called coagulase
positive.
The organisms are divided into two broad groups—the
coagulase positive and coagulase negative. The first group has
only one species in the list, i.e. Staphylococcus aureus. The
other group has about 28 species, out of which only S.
epidermidis is of ophthalmic interest. The coagulase positive
are always pathogens, they produce toxins, i.e. hemolysin,
leukocidin, enterotoxin and toxins that cause toxic shock
syndrome and exfoliation.
The coagulase negative organisms are opportunistic
pathogens but more resistant to antibiotics. The coagulase
negative staphylococci have traditionally been thought to
be contaminants in culture media and commensals of
conjunctiva. Now they are emerging as important
opportunistic organisms of ophthalmic importance especially
as a nosocomial infection. They are generally multiresistant and
are commonly seen with prosthetics like IOL and frequently
seen in fluid lines.
Penicillin used to be a preferred antibiotic in Staphylococcal
infection. At present about 80% of the strains are penicillin
resistant but sensitive to methicillin, cephalosporin, ciprofloxacin
and other fluoroquinolone. Resistance to methicillin too is
becoming frequent. The drugs to which they remain sensitive
are vancomycin, ciprofloxacin and rifampin.
The local treatment consists of sulphacetamide, erythro-
mycin, bacitracin, fortified vancomycin 25–50 mg/ml or
cephazolin 50 mg/ml as drops.
Streptococci
These gram-positive cocci are found in respiratory and gastro-

intestinal tract as commensals but can cause widespread
systemic and ocular infections which are more spread out
in contrast to staphylococci that cause mostly localized
infection. Besides systemic infection these organisms are
known to cause serious systemic nonsuppurative compli-
cations like acute rheumatic fever and acute glomerulonephritis.
(Figure 3.3)
Figure 3.3: Streptococci. Courtesy: Prof V Sudershan
The organisms are generally arranged in chains or in pairs.

The chains are longer in liquid media than on solid media
or pus from the lesions. The lengths of the chains have no
relevance to its pathogenicity. The organisms are aerobes or
facultative anaerobes. Growth is more marked in presence of
10 percent CO2.
They are nonsporing, nonmotile and most of the time non-

capsular but may have capsule. The capsule is anti-
phagocytic but not antigenic. The antigenic property lies in
the carbohydrate and protein in outer wall of the organism.
The organism produces various exotoxins and enzymes that
acts as virulence factors. The toxins are:
Hemolysin O and S: These are known as streptolysins and
responsible for zone of hemolysis round the streptococcal
colonies in culture. Following infection by streptococci
antistreptolycin “O” develops in serum. This blocks
hemolysis by Streptolysin O. The other toxin is erythrogenic
toxin.
Some of the enzymes produced are Streptokinase, streptococcal
deoxyribonulease, hyaluronidase and lipoproteinase. The
enzymes streptokinase and hyaluronidase help to spread
the organism. The streptococci are called delicate organisms.
They are killed by heat at 56°C/30 mts and by many
commonly used antiseptics and disinfectants.
Classification of Streptococci
Various types of classifications have been put forwards.

1. The first was on the basis of length of chains of the
organisms later it was found that length of chains was
not related to its pathogenecity.
2. The better classification depends on oxygen requirement
of the organism in culture and the organisms were
divided into aerobes, facultative anaerobes. The last group
consists of only one organism peptostreptococci.
3. The aerobes were further divided into their ability to cause
hemolysis. The organisms causing hemolysis are known
as Hemolytic streptococci. There are three types of Hemolytic
streptococci, i.e. alpha hemolytic (Streptococcus viridance).

Beta-haemolytic streptococcus (St. pyogenes). This is the
commonest pathogen among all streptococci and gamma-
hemolytic also known as Enterococcus.
The beta-hemolytic streptococci were further divided into
serological groups on the basis of polysaccharide C, they
were marked A to V minus I and J. Streptococcus pyogenes
belongs to group A.
The organism is sensitive to many antibiotics. The
antibiotics of choice is penicillin. Some of the strains may be
resistant to penicillin. The other antibiotics to which the
organism is sensitive are sulphonamides, erythromycin,
cefazolin, vancomycin and bacitracin.
Anaerobic Streptococci
Till a few decades ago, these gram-positive organisms which

form chain were not considered to be ocular pathogens and
were only considered to be normal flora of respiratory tract,
intestine and female genital tract.
They have been found to cause not only troublesome
systemic infection but also corneal ulcers and metastatic
endophthalmitis. They are strictly anaerobes, require enriched
media for growth, ferment carbohydrate and produce acid and
gas. Their presence is confirmed by positive culture on fresh
blood agar. They are sensitive to penicillin, chloramphenicol,
clindamycin and metronidazole.
Pneumococci (Streptococcus pneumoniae, Diplococcus

Pneumoniae)
The pneumococci are habitats of human upper respiratory

tract. About forty to fifty percent of conjunctivae harbor
pneumococci as commensals. It is the commonest organism

recovered from normal lacrimal sac.
The organism is genetically related to streptococci hence
called Streptococcus pneumoniae. It resembles morphologically
Streptococcus viridans but differ clinically.
The organism is a gram-positive, lancet (lanceolate) shaped
found most commonly in pairs with the broad sides of each
cocci facing the other. The pairs are surrounded by a
polysaccharide capsule which is demonstrated by Indian ink
preparation. The capsule is responsible for type specific
antigen. The capsule also resists phagocytosis and carries
the virulence factors. The capsule is responsible for the
antigen on the basis of which the organism have been
divided in to 80 serotypes. Less frequently the organisms
are found in small chains.
The organisms are nonmotile, nonsporing. They are aerobic
or facultative anaerobes, grow better in presence of 5-10% CO2.
The organism requires enriched media. They grow within
24 hours and produce a greenish zone of hemolysis round
the growth. On prolonged incubation, they undergo
autolysis. The organism is mistaken as streptococcus viridans
in stained slide. Pneumococci is bile soluble, optochin
susceptible, and ferment inulin but give negative catalase test
which the Streptococcus viridans produces without other
properties pneumococci. The organism either do not produce
toxin or produce very weak toxin.
The organism can be destroyed easily by heat and
commonly used antiseptics and disinfectants.
Pneumococci are sensitive to many common antibiotics.
The commonest being penicillin. Other antibiotics are
erythromycin, chloramphenicol, trimethaprime, cephalosporin,

clindamycin and vancomycin. The organism is less sensitive
to quinolones, i.e. ciprofloxacin.
GRAM-NEGATIVE COCCI
Neisseriaceae
The genus Neisseriaceae to which the organism Gonococcus

belongs, consists of both pathogenic and nonpathogenic gram-
negative organisms. The two pathogenic organisms with
ocular manifestation are N. gonorrhea and N. meningitidis.
The former causes more ocular lesions than the latter. Both
of them can pass through intact corneal epithelium.
Neisseria gonococci
N. gonococci are strict human pathogens. Humans are the only

known reservoir. The organism is gram-negative, bean-shaped
cocci that are mostly seen in pairs. The organisms are mostly
intracellular in polymorphs, extracellular organism are seen
in chronic infection. The organisms are nonmotile, nonsporing,
strict aerobic and oxidase positives. They are capsulated in fresh
specimen. Immunofluorescent staining of smear is better than
gram stain because gonococci are not decolorized well. It
produces endotoxin which increases its virulence. The pili
present on the surface help the organism to be attached to
the mucosal surface. The pili also enhance its virulence. The
pili inhibit phagocytosis. On the basis of presence of pili,
the gonococci have been divided into two groups, i.e. virulent
form—those that possess pili and nonvirulent—those that do
not have pili.
The organism is more difficult to grow than meningococci
in spite of the fact that they are genetically related to each
other. The organism requires enriched media to grow. The

organism grows best in presence of 5-10% CO2 in chocolate
agar, nutrient agar, Thayer-Martin medium and Martin Lewis
medium at 35-36°C at pH 7.2-7.6. It is destroyed in higher
alkaline pH. The gonococci ferment only glucose and not
maltose, lactose and sucrose. It is oxidase positive.
The organism dies quickly outside the human body. It is
killed by heat, drying and by usual antiseptics and
disinfectants.
The organism is sensitive to many antibiotics and
sulphonamides. Most of the strains have developed
resistance to sulpha drugs. Penicillin still remains the drug
of choice. The incidence of B. lactamase (penicillinase)
producing strains are becoming more frequent. The
organism has developed resistance to tetracycline and many
cephalosporins. Cefotaxime and kanamycin have proved to
be the best alternative. Other drugs to which the organism
is sensitive are norfloxacin, ciprofloxacin and ofloxaein.
Meningococcus (N. meningitidis)
The meningococci are better known as causative organism

to produce purulent meningitis especially in children.
Sometimes in epidemics they show morphological and
cultural characteristics of gonococci, i.e. they are gram-
negative diplococci, which do not grow on ordinary media. They
are strict aerobes. They are as delicate as gonococci as far as
resistance is concerned. They do not survive outside the body
for long-time. They are destroyed by heat, change in pH
and usual disinfectants. They can invade the cornea in presence
of intact epithelium. They differ from gonococci
biochemically and in antigenicity. They ferment glucose and
maltose with acid production. They are catalase and oxidase

positive. They do not have affinity for mucous membrane of
genitourinary tract or conjunctiva but may cause
conjunctivitis. No vaccine has been developed against
gonococci but vaccine against meningococci has been
developed against some strains. Rifampin has been advocated
as a chemoprophylactic antibiotic in close contacts during
epidemics.
The organism is sensitive to many antibiotics. The drugs
of choice remain penicillin and sulphonamides. Many strains
have developed resistance to them. The other drugs used
are chloremphenicol, ceftriaxone and ceflazidime.
Neisseria catarrhalis (Branhamella catarrhalis)
It is a commensal of respiratory tract, it is mildly infective

when turns pathogen. It is often confused as Gonococcus. It is
an intracellular, gram-negative, diplobacilli, nonmotile, non-
sporing, grows well on ordinary media in 24 hours. It does not
ferment maltose, glucose or sucrose. It produces oxidase and
catalase. It is not as delicate as gonococci or meningococci as
far as heat is concerned. It is sensitive to many antibiotics,
50% of strains produce β lactamase rendering them resistant
to penicillin and ampicillin.
Acinetobacter
This is a gram-negative, intracellular, Pleomorphic Diplococci

and aerobic that resembles gonococci so much in morphology
and enzyme production that it is called Neisseria mimic or
Acinetobacter mimcae. It also mimics moraxella which is
diplobacillus. It is an oxidase negative that makes it different
from Neisseria and Moraxella, which are oxidase positive. It

does not ferment sugar like Neisseria. It resembles Entero-
bacteriaceae in growth and colony pattern.
The organism was not considered to be an ocular parasite,
however with ever increasing number of HIV infection, it is
emerging as opportunistic organisms. It is also reported to
cause nosocomial infection in immunocompromised persons.
There are frequent reports of strains that are resistant to
antibiotics.
It is sensitive to penicillin, fluoroquinolone, aminoglycosides
and doxycyclin. Imipenem is a preferred drug in cases of
resistance to other antibiotics.
Moraxella lacunata
The organism M. lacunata is pathogenic only to ocular tissues,

was previously included in genus Hemophilus but later on
separated as new genus, Moraxella that has greater
resemblance to Neisseria than any other genus. The genus
Moraxella besides M. lacunata consists of Moraxella liquefaciens,
nonliquefaciens as well.
M. lacunata is the largest gram-negative bacilli to cause ocular
pathogenecity. It is pleomorphic, hence may present as cocco-
bacilli as well which may surprisingly stain gram-positive.
It changes its morphology in culture media, even develops
club-shaped appearance of Corynebacteria.
The organism is nonmotile, nonsporing, aerobe that requires
enriched media to grow. It does not grow on chocolate agar.
The media used most frequently are Loefflers serum slope and
blood agar. It is differentiated from other species of Moraxella
by following features. It is oxidase and gelatine positive. It
reduces nitrate. It is urease and citrate negative. It produces a
proteolytic enzyme that causes maceration of skin. It is

differentiated from other Neisseria by its ability to produce oxidase
and catalase but not fermenting sugar. From the above
description, it is clear that M. lacunata, though a elatively
less important organism is a great mimic. It may resemble
Neisseria, Haemophilus and Corynebacteria.
The organism in spite of its variability is sensitive to many
antibiotics including fluoroquinolones, clindamycin,
erythromycin but not penicillin. A nonantibiotic drug that gives
uniformly good result is zinc sulphate eye drop 0.25-0.5% TDS
or QID. Adding zinc sulphate to antibiotics enhances efficacy
of antibiotics.
The bacilli of ocular importance can either be gram-positive

or gram-negative.
Gram-positive Gram-negative
1. Corynebacterium 1. Enterobacteriaceae
C. diphtheriae E. coli
C. xerosis Klebsiella pneumoniae
Propionibacterium acnes Proteus
2. Bacillus Salmonella
B. anthracis Shigella
B. subtilis Citrobactor
B. cereus Enterobacter
3. Clostridium 2. Pseudomonas
C. tetani 3. Acinetobacter (coccobacilli)
C. welchii 4. Moraxella
C. botulinum 5. Haemophilus
4. Mycobacteria 6. Spirochetes- Syphilis
M. tuberculosis Lyme disease
M. leprae 7. Bordetella pertussis
Atypical mycobacteria
5. Actinomyces
6. Nocardia
GRAM-POSITIVE BACILLI
Corynebacterium
These are thin, club shaped, nonsporing, nonmotile, non-

capsulated organisms. They can be pathogens or saprophytes.
The pathogenic species is Corynebacterium diphtheriae.
The nonpathogenic organism of ocular importance are
C. xerosis and Propionibacterium, which includes C. acnes.
BACILLI OF OCULAR INTEREST 41
Corynebacterium diphtheriae
Corynebacterium diphtheriae is one of the most important

human pathogens that can be fatal even when only the eyes
are involved due to its powerful exotoxin. The exotoxin does
not enter the bloodstream but diffuses fast into the tissue
and is responsible for clinical features. The organism is a
surface saprophyte. Healthy carriers are not uncommon.
The organism is gram-positive but does not stain well, and
is pleomorphic. It stains well with special stains like Albert’s
stain (Figure 4.1) Neisser’s stain or by methylene blue stain. The
organism contains metachromatic granules, which are also
known as volutin meta chromatic granules, polar bodies or
Babes-Earnest granules. The granules are metaphosphates,
meant to store energy. Mere presence of granules is not
diagnostic. The organisms are seen single or in groups
arranged like match sticks and arranged in pattern of Chinese
letters (Figure 4.2).
Figure 4.1: Albert’s stain, C. diphtheriae

Figure 4.2: Arrangement of bacilli: 1. In cluster; 2. Chains;

3. Chinese letter pattern
The organism is aerobic or facultative anaerobe, growing best

37°C in pH 7.2, in media enriched by blood, serum or egg.
The commonly used media are Serum broth, Loefflers slope,
tellurite agar. The organism ferments glucose, maltose, glactose
and dextrose by producing acid but not gas. It does not ferment
mannitol or sucrose. The organism is catalase positive and
oxidase negative. The organism produces an exotoxin which
is heat labile polypeptide, that is destroyed at 70°C. When
exposed for 15 minutes, it is converted to toxoid that sheds
its toxicity but retains antigenecity. When either exposed to
heat at 37°C for four to six weeks or adding 0.4% formalin.
Corynebacterium xerosis
This is also called Diphtheroid but does not produce any

systemic disease. It is a strictly ocular bacterium. It is commensal
of skin and conjunctiva. It can be isolated few hours after
birth from the conjunctiva of the new borne. The source of

infection is most probably from the birth canal. The organism
is one of the most commonly isolated organism from healthy
as well as infected conjunctiva. The organism is short, thick,
pleomorphic, gram-positive bacilli with meta chromatic
granules similar to C. diphtheriae. The organisms are arranged
as Chinese letters in clusters. It is often confused with more
serious organisms like C. diphtheriae, pneumococci and
Streptococcus.
The organism grows more slowly than C. diphtheriae on
blood agar and Loeffler’s media. It ferments glucose and sucrose
by producing acid but no gas. It does not produce any toxin.
Propionibacterium acnes
Propionibacterium acnes is a Corynebacterium that belongs to

genus Propionibacterium. It is also a diphtheroid. The other
species are P. granulosum and P. avidum. Out of these p. acnes
has proved to be an ocular pathogen. It is routinely found on
the skin and mucous membrane including conjunctiva.
Previously, it was thought to be a harmless commensal,
over last two decades it has been proved to be a cause of
delayed postoperative endophthalmitis following IOL implant. The
organism reaches the inner eye by way of contaminated
irrigation fluid to get entrapped in capsular bag and liberated
following YAG laser capsulotomy.
Besides postoperative chronic uveitis and endophthalmitis, it
causes blepharitis, meibomian dysfunction, conjunctivitis, corneal
ulcer and dacryocystitis. The organism can change the lipid
content of meibomian secretion, making it more suitable for
growth of other bacteria.
The organism is gram-positive bacilli, nonsporing, nonmotile,

pleomorphic, anaerobe. It requires enriched media to grow, does
not produce toxin. Vancomycin is the first antibiotic of choice
that is used as fortified drops, 14 mg/ml, it can be used as
intravitreal injection and systemic injection. It is claimed that
there is 100% cure rate of P. acnes endophthalmitis if the capsule
is removed in toto following explanting the IOL.
Bacillus
This group consists of thirty one species and two subspecies,

out of which the following three are of ocular importance—
B. cereus, B. subtilis and B. anthracis. All are gram-positive,
aerobic, sporing, found in water, soil and food. Out of them
the first two are of greater ophthalmic interest. B. anthracis
involves the lid only, is of great systemic importance and is known
as a potent biochemical weapon.
B. cereus
Bacillus cereus is aerobic, gram-positive, spore forming, usually

motile, found in water, soil and food. It has been recognized
as an important cause of food poisoning. It has come out as a
foremost cause of post-traumatic and postoperative endo-
phthalmitis. As an ocular pathogen, it is one of the most
virulent organisms. The virulence is due to its ability to
withstand heat, ultraviolet ray and usual disinfectants. It
produces an exotoxin and a list of enzymes that include
protease, collagenase, phospholipase. All the toxins and enzyme
lead to devastating intra as well as extraocular infection.
The infection can be exogenous following penetrating
injury by plants or due to contamination of irrigation and
viscoelastic fluids used for intraocular surgery. Endogenous

endophthalmitis is seen mostly in intravenous drug
abuses.
The infection should be differentiated from S. epidermidis,
S. aureus, Pseudomonas and Proteus all known to cause severe
endophthalmitis.
The organism does not respond to penicillin. It is sensitive
to ciprofloxacin, gentamycin, vancomycin and clindamycin.
Bacillus subtilis
This is a common laboratory contaminant otherwise found

in soil and water. It is a saprophyte that is gram-positive, aerobic,
spore bearing, noncapsulated, produces hemolysin on blood agar
and liquefies gelatine in stab culture. It is a potent cause of
bacterial corneal ulcer and endophthalmitis in penetrating
injuries.
Bacillus anthracis (Anthrax)
The organism causes limited infection of lids and rarely may

cause corneal ulcer, panophthalmitis or optic atrophy. It is
systemic involvement may be fatal.
The organism is gram-positive, arranged in long chains. The
entire chain is surrounded by a capsule. The organisms are
square or rectangular in shape with central spore and nonmotile.
The organism grows on common media including nutrient
agar or blood agar. Blood agar is used to differentiate
between haemolytic and nonhemolytic strain, the latter is
pathogen. The vegetative forms are destroyed by heat at 60°C
in 30 minutes. The spores are highly resistant to usual physical
and chemical disinfectants.
The ocular involvements are pustules of the lids edema of

the lids and orbital cellulitis.
The organism is sensitive to many antibiotics including
penicillin, synthetic penicillin, cephalosporins, ciprofloxacin,
vancomycin and clindamycin.
Clostridium
The genus Clostridium contains many species but all are not
human pathogen. Only few are opportunistic pathogens. The
important pathogens are C. tetani, C. Welchii and C. botulinum.
All of them have profound systemic involvement. Ocular
involvements are rare and mostly accidental.
The organisms are gram-positive anaerobes which are
destroyed in presence of oxygen. Presence of CO2 enhances
growth. They are spore forming, the positions of the spores
are variable. It can be terminal, subterminal or central. They
are stained with ease. They show slow motility, only few are
capsulated.
All the three organisms produce lethal exotoxin.
Clostridium tetani
Clostridium tetani is found in soil and intestine of humans

and animals. It causes tetanus in both animals and humans.
They are gram-positive rods with terminal spore, are motile
and noncapsulated. They are obligate anaerobes, grow well in
ordinary media, grow better in liquid media. The commonly
used media are Robertson’s cooked meat and thioglycolate media.
The organism is mild proteolytic but does not ferment any sugar.
The spores are relatively resistant to heat than the bacilli. It
requires 15 minutes to 3 hours of boiling or dry heating at
160°C for one hour to destroy the spores. The best method is
autoclaving at 121°C. The spores are also destroyed by iodine
and hydrogen peroxide. The spores are known to survive in
the soil for years.
The organism produces two exotoxin—Tetanolysin and
tetanospasmin, which are responsible for clinical features of
tetanus.
The ocular involvements are secondary to toxic neuritis of
cranial nerves, i.e. blepharospasm, ptosis, various extraocular
muscle palsies, internal ophthalmoplegia, supranuclear gaze palsy
and nystagmus.
Treatment
The disease is fully preventable by tetanus toxoid. The toxoid

should be administered to every child as per National
Program. Once tetanus develops, it is better to get it managed
by physician trained in infectious diseases.
Clostridium welchii (Perfringens)
It is a pleomorphic, gram-positive, non-motile, capsulated, spore

forming, non fastidious anaerobic organism. It is found in soil
and intestine of many animals and humans. It causes gas
gangrene, food poisoning and necrotizing enteritis in human
beings. It causes severe endophthalmitis that terminates in
panophthalmitis. The ocular infections are always due to
penetrating injury of the globe. The organism produces gas
in anterior chamber. The orbit and lids are involved following
craniofacial injury.
The organism grows in Robertson’s cooked media at 45°C
in a day. It also grows on blood agar. It ferments glucose,
lactose, maltose and sucrose.
The organism is destroyed easily by heat except the strains

that cause food poisoning. Autoclaving is the best method
to destroy the spores which are resistant to commonly used
disinfectants.
The organism produces exotoxin which is highly toxic
and invasive.
The drug of choice is IV metronidazole. Other drugs used
are gentamycin and amoxycillin. Passive immunization with
polyvalent anti-gasgangrene serum is used in all cases.
Clostridium botulinum
C. botulinum is a common source of food poisoning. It does not

have any specific ocular involvement except paralysis of
extraocular muscles secondary to systemic involvement.
Botulinum toxin in therapeutic dose is used in various neuro-
opththalmic disorders.
Mycobacteria
The organisms grouped under the heading mycobacteria are

widely distributed throughout the world. They mostly appear
as thin cylinders which occasionally develop branching pattern,
mimicking mycelia of fungi, hence they were given the name
Mycobacterium. The organisms can be either saprophytes or
pathogens. The latter can again be divided into two broader
groups of obligate parasites and opportunistic pathogens. As per
their ability to produce disease, they have been divided into
two groups, i.e. M. tuberculosis and M. leprae. The former causes
tuberculosis in human beings only. The M. leprae shares many
morphological features of M. tuberculosis, which can be
cultured, M. leprae cannot be cultured or grown in animals
except in nine band armadillo. On the basis of cultivability, the

mycobacteria have been divided into
1. Cultivable
2. Noncultivable
The former is divided into two broad groups, i.e.
i. Typical Mycobacterium
a. M. tuberculosis
b. M. bovis
ii. Atypical Mycobacterium
Mycobacterium tuberculosis
The organism is also known as Koch’s bacillus and causes

multisystemic as well as ocular tuberculosis. Ocular tuberculosis
seldom coexist with systemic tuberculosis.
The M. tuberculosis is a gram-positive, straight or slightly
curved bacillus that stains poorly with Gram’s stain but stains
well with Ziehl-Neelsen stain. As the organism once stained
resist decolorization with weak acid, hence they are called
acid fast-bacilli or AFB (Figure 4.3). They are nonmotile, non-
capsulated and nonsporing, intra cellular parasites and obligate
aerobic.
Figure 4.3: M tuberculosis acid-fast stain (Ziehl-Neelsen).

(Courtesy: Prof. V Sudarshan)
The organism grows slowly in culture media. It takes

minimum two weeks to show positive result. The period may
be as long as six to eight weeks. To grow it requires pH
between 6.4-7.0 and optimum temperature of 37°C. It does
not grow under 25°C and above 45°C. The bacilli do not grow
in presence of fatty acid. The M. tuberculosis grows better
than M. bovis in culture media.
The organism is grown in both solid and liquid media.
The solid media commonly used are:
1. Löwenstein-Jensen media (Figure 4.4)
2. Dorset’s egg media
3. Pawlowsky media
4. Brooke’s medium
5. Loffler’s serum slope
6. Tarshi’s medium
Figure 4.4: Löwenstein-Jensen media to culture Mycobacterium.

(Courtesy: Prof. V Sudershan)
The liquid media are:

1. Dubo’s media
2. Sula’s media
3. Sauton’s media
The organism can also be grown on chick embryo and

in tissue culture.
The organism is heat labile but relatively resistant to
chemical disinfectants. The organism dies in 20 minutes
when exposed to 60°C. However, the organism may remain
alive in droplet for 8 to 10 days, if not exposed to sun.
There are various biochemical reactions to differen-
tiate M. tuberculosis from Atypical mycobacteria and
M. bovis.
Atypical mycobacteria and M. bovis are catalase and aryl
sulfatase positive.
Laboratory Diagnosis
The diagnosis is confirmed by:

1. Demonstration of acid-fast bacilli in Ziehl-Neelsen
stain
2. Demonstration of M. tuberculosis in culture
3. Histopathology of lesion that shows typical tubercle
formation with caesation.
4. Demonstration of fluorescent antibody in specific anti-
serum.
5. Specific IgG and IgM.
6. PCR-based ELISA
7. Western blot
8. Demonstration of hypersensitivity to tuberculoprotein –
Mantoux test (tuberculin test)
The tuberculoprotein used in tuberculin test is called
purified protein derivative (PPD). The dose of PPD is expressed
in tuberculin unit (TU). One TU is equivalent to 0.02 µg of
PPD or 0.1 ml of 1 in 10,000 dilution of old tuberculin (OT).
The commercially used tuberculin used for Mantoux test is

available in dilution of 1/10,000, 1/1000 and 1/100 OT. The
above dilutions are available as 0.1 ml.
In Mantoux test, 0.1 ml of PPD is injected in flexor surface
of upper part of forearm intradermal. The results are noted
after 48-72 hours. An induration more than 10 mm
surrounded by a zone of erythema is positive.
Atypical Mycobacteria
These form a large group of bacteria that have:

1. Morphology and cultural characteristics of mycobac-
terium tuberculosis.
2. Produce diseases that resemble tuberculosis.
3. Do not respond to usual antitubercular chemotherapy by
streptomycin and isoniazide.
4. Some strains are sensitive to rifampicin.
5. They are sensitive to amikacin and clofazimine.
They are differentiated from M. tuberculosis by following:
1. They are saprophytes found in soil and water. They are
opportunistic pathogens in immunocompromised persons.
2. Infection by Atypical mycobacterium is rare in areas where
tuberculosis is seen frequently. They are seen commonly
in areas where tuberculosis has been eradicated.
3. They are not known to be transmitted from person to
person.
4. They do not produce progressive disease when injected
in guinea pigs.
5. They are catalase and aryl sulphatase test positive.
6. They give negative niacin and neutral red test.
7. They grow in Löwenstein-Jensen media at temperature below

25°C or above 41°C. M. tuberculosis grows at 37°C.
Classification
As per ability to produce pigment they are classified as:

1. Photochromogens: They produce pigments when exposed
to light—M. Kansassii
2. Scotochromogens: They produce pigments in dark—
M. scrofulaceum
3. Nonphotochromogens: M. intracellulare, M. xenopi,
M. avium
4. Rapid growers: They grow fast, i.e. with in a week.
Organisms of ocular importance are M. fortuitum and M. chelonei
Thus, Atypical mycobacteria are less virulent than

M. tuberculosis. The ocular lesions produced by them are:
1. Post-traumatic corneal ulcer: The trauma may be
accidental or following penetrating keratoplasty or
keratotomy.
2. Stromal keratitis
3. Endophthalmitis
4. Dacryocystitis
5. Preseptal orbital cellulitis
Mycobacterium leprae
The Mycobacterium leprae is found all over the world and cause
leprosy. It is also known as Hansen bacilli.
The organism is gram-positive, stain poorly with Gram’s
stain, is acid-fast but not alcohol fast. The live bacilli can be
differentiated from dead bacilli by their staining character:
1. The live bacilli stain solid and uniformly.

2. The dead bacilli take fragmented and granular stain.
3. Presence of large number of dead bacilli point towards
efficacy of chemotherapy.
The organisms can be intracellular or extracellular. The
organisms show great morphological variation. They are
generally straight or slightly curved in shape. They sometimes
resemble diphtheroid in morphology. They may even have
coccoid shape. They are found in clumps called cigar bundle.
The masses of bacilli are known as globi. In histopathological
section, the globis are found inside lepra or foamy cells of
Virchow’ which are large undifferentiated histocytes.
The humans are the only known reservoir of the lepra
bacilli and leprosy as a disease is exclusively seen in human
beings.
The organisms cannot be cultured on any known culture
medium. They are grown only on footpads of nine band
armadillo.
The organisms survive outside the body upto 45 days.
They survive exposure to sunlight for two hours and are
destroyed in ultraviolet light in half an hour.
Higher Bacteria
The organisms of this group belong to order Actinomycetale,

which is divided into two families of human pathogens:
1. Actinomycetaceae which is anaerobic and nonacid-fast.
2. Nocardiaces which is aerobic and acid-fast.
These organisms were previously considered to be fungi
because they form mycelial network of branching filaments.
Subsequently, it was found that though they had appearance
of fungi they were in fact bacteria and called higher bacteria
or filamentous bacteria. They are infact related to Mycobacteria

and Corynebacteria.
The characteristics that qualify them to be bacteria are:
1. They have cell wall.
2. The filaments are too slender.
3. They do not have true nuclei. The nuclear material is
scattered in the protoplasm of the cell (Prokaryotic).
4. They respond to many antibiotics.
5. These are:
i. Soil saprophytes
ii. Oral commensals
iii. Potentially pathogens.
Actinomyces
This is gram-positive, nonacid-fast with slender branching

filaments which give a beaded appearance. The branches may
break into coccoid or bacillary shape, are nonmotile, non-
sporing.
The species causing human infection is called A. Israelii.
The organism can be grown both in solid as well as liquid
media.
The common media used are blood agar, brain-heart infusion
agar, Robertson cooked meat media and thioglycolate broth.
The organisms are Facultative anaerobes. They require 5% CO2
at 37°C. They grow slowly which may take few weeks to show
result.
The organisms are best identified by Gram’s stain and
fluorescent antisera. The organism produces sulphur granules
in the tissue, presence of which is almost diagnostic of
actinomycoces. The organism is sensitive to penicillin and
many common antibiotics.
Nocardia
The disease caused by this organism is called Nocardiosis and

the organism is known as Nocardia asteroides due to star like
appearance of its colonies on agar plate.
It is a soil saprophyte, gram-positive, filamentous, nonmotile,
nonsporing, partially acid-fast, staining best by modified Kniyoun’s
method. The filaments break into coccoid and bacillary forms.
It is strict aerobes. It is grow on blood agar, brain-heart
infusion agar and Sabourauds’ dextrose agar and grow
slowly.
GRAM-NEGATIVE BACILLI
The common gram-negative bacilli of ocular importance
belong to genera:
1. Pseudomonas
2. Haemophilus
3. Acinetobacter
4. Moraxella
5. Enterobacteriaceae
6. Brucella
7. Franciscella tularensis
Pseudomonas
Pseudomonas is one of the most dangerous organism to inflict

the eyes. The species most frequently associated with ocular
infection is Pseudomonas aeruginosa.
The organism is saprophyte in soil and water. It is
commensal in human intestine and pathogen at other sites
including eyes. It has come out as a major source of nosocomial
infection. It is a common organism that inhabits contact lenses
and lens fluids. It is also known to contaminate locally prepared

ocular drops. The most important drop to be invaded by the
organism is fluorescein sodium.
The organism is a common cause of infection in post-
operative period and burns. It is known to cause epidemics
among neonates in nurseries, is a major cause of fulminating
corneal ulcer in critically ill persons.
The organism is gram-negative, motile bacillus. It moves
slowly by polar flagella. It is nonsporing, noncapsulated. It is
strict aerobe, grows well in ordinary media.
The commonly used media are nutrient agar, MacConkey’s
agar, blood agar and citrimide agar. The colour of the colonies
vary from yellowish green to brown. In MacConkey’s media,
the colonies are almost colorless.
The organism produces pigments in the media as well as
infected wound. The pigments are pyocyanin, fluorescein
polyrubin and pyoverdin. The pigments are produced best at
room temperatures in day light in fluid media and seen
better in ultraviolet light. About 10% of organisms do not
produce pigment all the time. The organism is bactericidal to
many other bacteria. It is oxidase positive.
The organism produces many enzymes, main among them
are Protease, collagenase, phospholipase. It produces both
endotoxin and exotoxin. The organism also causes hemolysis.
The organism is difficult to destroy. It requires one hour at
55°C to kill the organism. It is resistant to many of commonly
used antiseptics and disinfectants. The organism is known
to grow in the locally prepared antiseptic solution when kept
for long-time.
The organisms ability to produce B. lactamase, makes it
resistant to many antibiotics. The organism is sensitive to
penicillinase resistant penicillin and aminoglycosides. The

commonly used antibiotics are Ciprofloxacilin, ofloxacilin and
tobramycin.
Haemophilus
The genus Haemophilus contains a group of organisms that

thrive in presence of blood or blood constituents in culture
media. They are pleomorphic, gram-negative bacilli that stain
poorly. They are nonmotile, nonsporing. Some of them may be
capsulated. They cause respiratory ocular and urogenital
infections.
Out of many organisms of this genus only Haemophilus
aegyptius also known as Koch-Weeks bacilli is of ophthalmic
importance. Others cause no or very little ocular infection.
Haemophilus aegyptius (Koch-Weeks bacilli)
This is a strictly ocular pathogen. Like any other Haemophilus,

this is a gram-negative bacteria that is difficult to see on stained
slide due to its faint staining and small size. Though it is
classified as bacilli it frequently presents as coccobacillary form.
The organism can be seen single in small chains in clusters or
in pairs. On stained slide, they can either be extracellular or
intracellular. The organism is closely related to H. influenzae
due to similar DNA.
The organism grows on blood enriched media. The
commonly used media are blood agar, chocolate agar and
Levinthal’s medium. It grows better in presence of 5-10% CO2.
One of the characteristics of Haemophilus is Satellitism, i.e.
when grown along with staphylococci i.e. the colonies of
H. aegyptius are larger near the superimposed Staphylococcus
streak and gradually diminish in size away from the

Staphylococcus streak (Figure 4.5).
Figure 4.5: Satellitism of H. aegyptius
H. aegyptius and H. influenzae not only have homologue

DNA, they are related antigenitically as well. The organism
does not produce any toxin. The mode of pathogenecity is
not well understood.
The organism is susceptible to many commonly used
antibiotics.
Acinetobactor
It is an ubiquitous organism that is found in soil and water as
saprophyte. Previously, it was not considered as an ocular
pathogen but there are frequent reports that show it as a
potential cause of endophthalmitis following lens extraction
and penetrating injury. It is an opportunistic organism that
becomes pathogen in immunocompromised persons. It is a
frequent cause of nosocomial infection.
The organism has striking similarity with Neisseria
and Moraxella. There are about seventeen geno species,
out of which the two, i.e. Mima polymorpha and Herellea
vaginicola are ocular pathogens. Its growth pattern and

morphology of colony is similar to those of Enterobacte-
riaceae.
It is gram-negative, nonmotile, pleomorphic. In Gram’s stain,
it looks like Neisseria. It is oxidase negative. It does not ferments
sugar. It is sensitive to many aminoglycosides. It is known to
develop resistance frequently.
Moraxella
Though this is gram-negative bacilli, it has varied morphology

and staining characteristics. It can be coccobacillary. The short
coccobacillary form may stain gram-positive. It resemble
Haemophilus, Neisseriae and sometimes Corynebacterium in
shape and arrangement from which it must be differentiated
by biochemical tests.
Enterobacteriaceae
The family includes a large number of pathogens and

commensals. The commensals are potentially pathogens. The
organisms are aerobes or facultative anaerobe, gram-
negative, grow on simple media, oxidase negative, ferment
many sugars, bile tolerant, most of them are noncapsulated
except Klebsiella, all except Klebsiella are motile Entero-
bacteriaceae of ocular importance are:
Escherichia coli
Escherichia coli commonly referred to as E. coli. It is an

intestinal commensal of human and animal intestine, not
found in soil or water it becomes opportunistic pathogen in
changed environment. Infection of eye by E. coli is relatively
rare but possible. It causes mucopurulent conjunctivitis in

elderly persons, metastatic and exogenous endophthalmitis. It
produces gas in AC.
The organism is gram-negative, straight rod, motile, may
have capsule but do not form spores. The organism is aerobe or
facultative anaerobe grows at optimum temperature of 37°C.
Grows well in ordinary media. Ferment many sugars, except
sucrose produces enterotoxin and hemolysin.
The organism is sensitive to ampicillin, nitrofurantoin,
trimethoprim, sulphamethoxazole and aminoglycosides.
Klebsiella
Klebsiella is a gram-negative, short plump, nonmotile, nonsporing

organism contains a polysaccharide capsule is, a saprophyte and
commensal causes mostly respiratory infection and mild
external ocular infection. It grows in ordinary media, ferments
sugar, producing acid and gas. The organism is being reported
as cause of hospital infection more frequently. The organism
is sensitive to many antibiotics including ampicillin,
amoxycillin and cephalosporins (Figure 4.6).
Figure 4.6: Klebsiella—Gram-negative bacilli

Proteus
Proteus is an enterobacteria found in soil, water and rotten

vegetation, is commensal of mammalian intestine and pathogen
in immunocompromised. It is a frequent nosocomial agent. The
organism is gram-negative, pleomorphic, motile and non-
capsulated. It grows well in ordinary media and said to swarm
the surface of culture plate, may show ripple. The organism
when grown gives a fishy smell. It produces urease. Though
ocular infection by Proteus is less common. It causes post-
traumatic endophthalmitis and panophthalmitis. Such eyes are
invariably lost. The organism is also known to produce corneal
ulcer, gangrene of lid, scleritis.
The organism is sensitive to ciprofloxacin, ofloxacin,
ampicillin and aminoglycosides.
Other less frequent gram-negative bacilli of ocular importance
are:
Salmonella
Intestinal parasite, gram-negative, facultative anaerobic,
motile, nonsporing, grow on ordinary culture media,
ferment glucose, maltose, mannitol but not lactose or sucrose
causes conjunctivitis and corneal ulcer and it is destroyed by
heat at 60°C in 20 minutes, chlorination and pasteurization,
sensitive to ampicillin and ciprofloxacin.
Shigella
Gram-negative, aerobic, nonmotile, nonflagellate, non-
capsulated, grows on ordinary media, ferment glucose, produce
acid but no gas. Produce endotoxin and exotoxin, destroyed
by heat and phenol, sensitive to sulpha and many broad
spectrum antibiotics. Rarely causes dacryocystitis and keratitis.
Francisella tularensis
The systemic infection caused by this organism is called

Tularemia harbored by rodents and birds and transmitted
by ticks.
The organism is gram-negative, non motile, capsulated,
obligate parasite, aerobe, and intracellular Coccobacilli. It
grows in enriched media, cause granuloma of conjunctiva
and sensitive to streptomycin and other aminoglycosides.
Brucella
This causes brucellosis which is a zoonotic disease generally

caused by cattle. The organism is gram-negative,
pleomorphic, cocco bacilli, intracellular aerobic, nonmotile,
noncapsulated, grow on ordinary media. The organism is
killed by heat at a temperature of 60°C in 10 minutes. The
milk of suspected infected animal should be pasteurized
before use, cause anterior uveitis in humans. Diagnosis is
confirmed by positive Brucellin test and ELISA. The organism
is sensitive to aminoglycosides, tetracycline, chloromycetin
and third generation cephalosporins.
Yersinia (Pasteurella pestis) causes plague. It is gram-
negative, short oval, pleomorphic, found in clumps and
Giemsa stain, aerobic or facultative anaerobic. Grows on
ordinary media. Many domestic and wild animals especially
the rodents harbor the disease, may spread by contact with
infected animals or flea.
Besides pasturellapestis, other organisms of the group are
Pseudotuberculosis and P. enterocolitica.
Ocular involvement is rare but can cause conjunctivitis, acute
anterior uveitis, corneal ulcer and endophthalmitis.
The organisms are sensitive to many antibiotics, i.e.

chloramphenicol, streptomycin, kanamycin, tetracyclin.
Bordetella pertussis
These gram-negative coccobacilli were previously thought

to belong to genus Haemophilus genera but as they are
antigenetically different from Haemophilus and do not require
growth factor x and y. They have been separated
from Haemophilus. On morphology, they are similar to
H. influenzae.
The organisms are noncapsulated, nonmotile, nonsporing
aerobes. Optimum temperature for growth is 35°C. It is
oxidase and catalase positive. The organism produces many
endotoxins. It causes whooping cough in children which is a
common cause of subconjunctival hemorrhage in children.
The organism is sensitive to many commonly used
antibiotics. The disease is fully preventable by DPT that
contain vaccines against diphtheria, tetanus and purtussive.
Spirochetes
The spirochetes have distribution world over. Some are free

living saprophytes. Others are obligate parasites. They are
reproduced by transverse fission, may be aerobic, facultative
aerobic or anaerobic, are gram-negative.
The spirochetes belong to order spirochaeles.
Treponemataceae of ocular importance are:
1. Treponema
i. T. pallidum
ii. T. epidemicus
iii. T. petrenue
iv. T. carateum
2. Borrelia
B. Burgdorferi
3. Leptospira
Treponema
Out of the four treponemas named above, only T. pallidum

has extensive ocular involvement.
Treponema pallidum is the cause of Syphilis in humans.
Humans are the only known reservoir.
The organism is a thin, spiral and delicate bacteria. The
biochemical structure of which is more complex than any
other bacteria. It multiplies by transverse fission in contrast
to other bacteria that multiply by binary fission. The organism
is spiral in shape (Figure 2.1) along its long axis while other
bacteria are straight or slightly curved organism.
The organism is gram-negative. It can not be visualized
under light microscope but becomes visible in negative stain
by Indian ink.
Figure 4.7: Dark field microbiology
The other methods to see them are:

1. Dark field microscopy (Figure 4.7)
2. Phase contrast microscopy
3. Fluorescent microscopy
The organism takes pink stain when stained by Giemsa
stain. It also stains with silver impregnation method. The
organism is best seen by Levaditi’s method in tissue section
and Fontana’s method for staining. The organism is actively
motile along its long axis.
The organism does not grow in any culture media. The
organism is destroyed readily by heat. It is destroyed by
heat at 41-42°C in one hour. It does not survive outside the
body hence the disease is not transmitted by fomites. The
disease is spread by intimate body contact. It can pass
through minute abrasion on the skin or mucosa. It also passes
the placental barrier.
The T. pallidum has complex antigen system. It produces
three antibodies.
The disease is confirmed by:

1. Directly demonstrating the organism in dark field
microscopy, phase microscopy and fluorescent
microscopy.
2. Indirectly by serological tests:
i. Nontreponemal tests
a. Conventional VDRL test
b. Modified VDRL test
c. Rapid plasma regain test (RPR)
d. Toludine red unheated serum test.
e. TORCH test (Congenital syphilis)
ii. Treponemal tests
a. Treponema pallidum hemagglutination test
b. Enzyme immunoassay
c. Fluorescent antibody absorbed test FTA-ABS.
The other methods are:
1. Microhemagglutination test
2. Treponema immobilization test
3. Polymerase chain reaction
4. Reiter protein complement fixation test
Almost thirty percent of patients with ocular syphilis may have
negative VDRL hence all nontreponemal test should be confirmed
by treponemal tests. All patients with syphilis should be tested
for HIV infection as well.
The organism is sensitive to penicillin which is still a

preferred antibiotic of choice both congenital and acquired
syphilis of all stages.
Borrelia
The organism of this genera belongs to family Trepone-

malaceae. The three human pathogens are B. recurrentis,
B. vincentii and B. Burgdorferi, they cause multi-systemic

infection including ophthalmic. The disease is otherwise called
Lyme disease. The discovery of the disease as a separate entity
is relatively recent, i.e. 1975 in a place called Lyme in USA
hence eponymed as lyme disease. The cases reported are
mostly from western hemisphere, parts of Europe and
Australia. The disease masquerades many diseases.
The organism like any other Treponema is gram-negative,
motile, stain well with common dyes, is difficult to culture
on common media. The disease is transmitted by bite of ticks,
Ixodes.
1. Immunofluorescent assay titres more than 1 in 256 is
diagnostic.
2. ELISA. Titres more than 1 in 2 is significant
3. PCR
4. Western blot
The organism is sensitive to many antibiotics including
penicillin, tetracycline, amoxycillin, erythromycin. The drugs
have to be administered for two to three weeks.
Leptospira
The organisms of this genera are the smallest pathogenic

Spirochete. There are about 200 serotypes of Leptospira, most
of them are saprophytes only. L. interrogans is human
pathogen. The organism is harbored by many animals. The
disease is zoonotic. The organism is shed in the urine and feces
of the animals which enter the human system through cuts
in the skin. The disease is endemic, which may cause
epidemics.
The organism is gram-negative, motile, can not be seen in

light microscope, best seen in dark ground illumination or
electron microscope.
They stain with Giemsa stain and silver impregnation.
They are grown on liquid or solid enriched media at
25-30°C in pH 7.2-7.5. The Leptospira are killed by heat and
common disinfectants.
The organism is sensitive to penicillin, tetracycline and
doxycyclin.
CHLAMYDIAE
These organisms were previously thought to be viruses
because they are:
1. Intracellular obligate parasites.
2. Filterable.
3. Do not grow on cell free media.
4. Do not grow in culture media.
5. They form inclusion foreign bodies.
It was later realized that as they have following properties
they should be considered as bacteria:
1. They have a cell wall.
2. They possess both DNA and RNA.
3. They have ribosomes
4. They multiply by binary fission.
5. They are susceptible to anti microbial agent.
6. They contain metabolically active enzyme.
The organisms belong to:
Order – Chlamydiae
Family – Chlamydiaceae
Genus – Chlamydia have three species, i.e. C. trachomatis—
causing trachoma, nonspecific urethritis, cervicitis and
lymphogranuloma venerum.
C. pneumoniae causes pneumonia and bronchopneumonia

C. psittaci causes psittacosis and ornithosis in birds.
Out of these three species one C. trachomatis is of utmost
ocular importance.
C. trachomatis is gram-negative, nonmotile, obligate intra-
cellular organism. It is smaller than most of the bacteria and
larger than largest virus. It stains better by Giemsa stain.
Other stains used to see the organism are Castaneda,
Machiavello and Lugols iodine stain. The stained organisms
are visible in light microscope (Figure 4.8). The better
visualization is attained by immunofluorescence using
monoclonal antibody.
Figure 4.8: Reproductive cycle of Chlamydiae
The organism has two forms:

1. Elementary body
2. Reticulate body
The elementary bodies are extracellular infective,
metabolically inert, have rigid cell wall. The reticulate bodies
(initial body) are intracellular replicate forms, the cell wall is

fragile. The reticulate bodies divide repeatedly to form
inclusion bodies in the host cytoplasm. The inclusion bodies
of C. trachomatis are called Halberstaedter-Prowazek bodies
(Figure 4.9).
Figure 4.9: Inclusion body: Halberstaedler-Prowazek body

(HP body) of trachoma
The C. trachomatis is divided into three biovars: 1. TRIC, 2.

LVG and MoPn.
The TRIC has 14 serovars labeled A to K and cause
trachoma and inclusion conjunctivitis. Ocular infection is
typically caused by serovars A, B, Ba and C. The organisms
are basophilic in nature and demonstrable only in early
stages of disease.
Lymphogranuloma venerum
Lymphogranuloma venerum is caused by C. trachomatis serovar

L which are numbered as L1, L2 and L3.
MoPn causes pneumonia in mouse, has no ocular

significance.
1. Serological tests
i. Fluorescein labeled monoclonal antibody
ii. Enzyme immunoassay
iii. Polymerase chain reaction
iv. ELISA
v. DNA probe
2. Isolation of C. trachomatis by yolk sac inoculation.
3. Cytology: Neutrophilic mononuclear infiltrates, presence
of Leber cells and Humbrecht cells.
Presence of submicroscopic organisms as a cause of infection

has been suspected since discovery of bacteria. It is only
about 75 years ago that the presence of viruses was
confirmed and their role in causing disease established. The
viruses cause many diseases in plants, animals and human
beings. In humans, the diseases range from simple coryza
to fatal disease like rabies, smallpox and AIDS. Some of the
viruses have been proved to cause neoplasms, only a few
viruses involve the eyes.
The characteristics of viruses are:
1. They are very small, smaller than smallest bacteria. The
exception being mycoplasma. The viruses are said to
be larger than largest protein molecule.
2. They are submicroscopic, i.e. not visible by light micro-
scope. The exception being pox viruses which are visible
under light microscope with special stain.
3. The viruses do not possess a cellular organization.
4. They do not have a formed nuclei.
5. They possess only one type of nucleic acid either RNA
or DNA. The RNA or DNA can be single, stranded or
double stranded.
6. The viruses are metabolically inert outside the living
cells.
7. They lack enzymes needed to build protein or nucleic
acid.
8. The viruses use the host cells metabolism.
9. They do not contain ribosomes.
10. They do not divide by binary fission.
11. They are insensitive to antibiotics and respond to anti-
viral durgs.
12. The viruses can not be grown in usual media used to
grow bacteria.
OCULAR VIROLOGY 75
13. They grow on living cells only.

14. Some of the viruses grow in the bacterial cells and are
called bacteriophage.
15. The replication of the viruses is inhibited by interferon.
16. They are filterable.
17. The viruses have tendency to mutate, to change the
virulence and antigenicity.
Morphology
The viruses are not visible under light microscopes. They

are seen through electron microscope by which their shapes
can be delineated and size measured. The size varies
between 20 to 400 nm. The viruses have various shapes, i.e.
spherical, cubical, etc.
The extracellular infectious virus particle is called virion.
The virion consists of a nucleic acid core that is surrounded
by a layer of protein called capsid. The capsid and the nucleic
acid core form the nucleocapsid. The capsid protects the core
from nuclease and other toxic matters. The capsid is made up
of polypeptides known as capsomers. The other function of
the capsid is to introduce viral genome in the host cell. Some
of the viruses have envelopes round the nucleocapsid. The
envelopes have antigenic properties and are responsible for
biological and chemical characters of the virus (Figure 5.1).
The virus particle are of three types—the cubical
(icosahedral), helical and complex.
Most viruses are heat labile and destroyed with in few
seconds at 100°C.
The other methods to destroy the viruses are—ultraviolet
radiation, gamma radiation, chlroform, ether, oxidizing
chemicals like halogens, i.e. chlorine and iodine. They are
Figure 5.1: A typical virus
also destroyed by hydrogen peroxide, chlorhexidine, sodium

hypochlorite and many disinfectants.
They remain stable in extreme low temperature and are
stored between –40 to –70°C in lyophilized or freeze dried
state.
Classification of Viruses
1. Clinical presentation: Clinical presentation of some of the

virus infections are so specific that the viruses are named
after the disease, i.e. smallpox, rabies, herpes zoster.
2. Based on tropism (affinity to particular system): The viruses
infecting skin are known as dermotropic, i.e. smallpox,
measles, chickenpox. Poliomyelitis and rabies are put
under neurotropic. Similarly, there are pneumotropic and
viscerotropic. However, they failed to explain
involvement of other systems.
The best classification is based on their chemical structure,
i.e. on the basis of presence of nucleic acid. Thus the viruses
are grouped as:
OCULAR VIROLOGY 77
1. RNA viruses
2. DNA viruses
The RNA viruses of ophthalmic interest are:
1. Measles and mumps
2. Rubella
3. HIV and AIDS
The DNA viruses of ophthalmic interest are:
1. Smallpox, molluscum
2. Herpes simplex, herpes zoster, cytomegalovirus, Epstein-
Barr virus
3. Adenovirus
4. Papovaviruses
Mutation is a characteristic of viruses of all types. It occurs
in vivo as well as tissue culture. The virus changes its
behavior, virulence and antigenic property by mutation.
Multiplication: The viruses do not multiply by binary fission.
As the viruses do not contain biosynthetic enzymes, they
have to use enzyme system of the host cells to form specific
molecule to replicate. The nucleic acid of the virus carries
the genetic information. The replication of virus is a
complicated procedure.
They can be broadly divided into following phase:
1. Adsorption
2. Penetration
3. Uncoating
4. Biosynthesis
5. Maturation
6. Release
Cultivation of the Viruses
The viruses do not grow on inanimate things hence they

cannot be cultured on usual culture media.
The common methods used to cultivate viruses are:

1. Animal inoculation: The animals used commonly are
mice, monkeys, rabbits and guinea pigs, the route of
inoculation may be subcutaneous, intracerebral or
intraperitoneal.
2. Embryonated eggs: The embryonated eggs of hens are
used.
The three common sites used are:
i. Chorioallantoic membrane
ii. Allantoic cavity
iii. Amniotic sac
3. Cell culture: Three types of tissue cultures are available.
i. Organ culture
ii. Explant culture
iii. Cell culture
Virus Infection
The route of infection are varied, they enter the human body
by any of the following routes:
Skin: Through cuts, abrasion
Oral: GI tract, contaminated food and drink.
Respiratory: Droplet infection
Conjunctiva: Droplet or via fomites
Sexually transmitted: HIV, AIDS
Hematogenous: By contaminated needles, bite of vector.
Transplacental: Congenital rubella
OCULAR VIROLOGY 79
Cellular Changes
The cellular changes brought about are varied:

1. They may enter the host body without causing any
cellular damage and may coexist with the host cell.
2. The cellular changes may be minimal and the diseases
pass off without any long standing effect.
3. There is an initial mild symptomatic phase followed by a
period of latency that is followed by a phase of
reactivation (herpes simplex and zoster).
4. Some viruses cause death of the cells or lysis of the cells,
other cause proliferation of the cells as in molluscum or
other oncogenic viruses, they may damage the
chromosome of the host cell.
The important histological changes of viral infection are
formation of inclusion bodies. The inclusion bodies are visible
under light microscope after staining with Giemsa stain or
eosin methyl blue. They can be acidophilic or basophilic in
nature. The inclusion bodies may be seen in cytoplasm
nucleus or both. The inclusion bodies can be either crystalline
aggregates of virion. Presence of some of the inclusion bodies
are diagnostic.
Diagnosis of Viral Disease
Some of the methods used to diagnose viral disease consists

of:
1. Clinical presentation: Some viral infections have so specific
presentation that they do not require any specific test.
The examples are smallpox, herpes zoster, herpes simplex
and molluscum.
2. Serological tests: Rise in titre of antibodies of a virus during

period of disease confirms the diagnosis.
3. IgM specific test
4. ELISA test
5. Microscopy:
i. Presence of elementary bodies in stained slide has given
way to electron microscopy.
ii. Demonstration of viral inclusion bodies has no role in
ocular virology, they are mostly used in rabies.
6. Demonstration of viral antigen in tissue culture.
DNA VIRUSES OF OCULAR IMPORTANCE

Pox Viruses
Pox viruses are the largest human viruses. They belong to

the family Pox viridae. The viruses of ocular importance are
variola (smallpox), vaccinia and molluscum.
Variola
The virion is brick shaped. It is large enough to be seen under

light microscope. It is a DNA virus. The DNA is present in
the nucleoid. The nucleoid is surrounded by a double-
layered membrane.
The virus is stable in dark. It can survive for years when
frozen. The organism is destroyed by ultraviolet rays. They
are killed by oxidizing disinfectants and formalin.
The variola virus shares a single nucleoprotein antigen
with other pox viruses. The organism infects only humans
and some monkeys. The organism is grown on chick embryo
and tissue culture. The diagnosis is confirmed by
demonstrating Guarnieri’s inclusion bodies.
OCULAR VIROLOGY 81
The incubation period is about 12 days. The infection gives

a lifelong immunity. Prophylactic immunization can be
given by vaccine. There is no known antiviral drug against
smallpox.
Vaccinia
Vaccinia is similar to variola virus morphologically but

differs clinically and biologically. They have a broad range
of hosts to infect that includes rabbits and mice besides
primates. It was originally derived from cowpox virus. The
present day vaccinia virus is an orthopox. The organism is
grown on chorioallantoic membrane of chick embryo.
Vaccine prepared from vaccinia virus is used to vaccinate
against smallpox. Though smallpox has been declared
eradicated in 1980, the vaccine is still used routinely in many
countries.
Molluscum Contagiosum
Molluscum contagiosum is another pox virus that is found

world over. It is a double-stranded enveloped DNA virus.
Like other pox viruses, it is a large virus. It replicates in the
cytoplasm of epidermal cells. It causes mildly contagious
disease that can be transmitted through close body contact
including sexual contact. The incidence of the disease is being
reported more frequently with rise in number of immuno-
deficient persons.
The cheesy material expressed from the lesion stains blue
by Giemsa stain. The organism is visible by light microscope
under oil immersion. Inclusion bodies are visible only on
electron microscopy, they are called molluscum bodies.
There is no known prophylaxis or effective antiviral drug

against molluscum infection.
Herpes Virus
There are about 80 viruses in this family of Herpes viridae, out

of which only following are involved in systemic and ocular
infection and are collectively called human herpes viruses (HHV).
They are divided into three subfamilies, i.e. alpha, beta and
gamma virinae.
Classification of herpes virus
The herpes simplex type 1 and 2 along with varicella zoster

are cytolytic. The cytomegalovirus is cytomegalic. Epstein-
Barr, human B herpes virus type 6 and type 7 are lymphoproli-
ferative.
General Properties of Herpes Viruses
1. The genome of HHV is double-stranded DNA

2. The capsid is icosahedral with 162 capsomers.
OCULAR VIROLOGY 83
3. The nucleocapsid is surrounded by an envelope which is

derived from host cell nuclear membrane and is lipid in
nature.
4. The envelope has eight surface spikes.
5. The replication takes place in the host cell nucleus.
6. Inclusion bodies are intranuclear.
7. The HHV are susceptible to ether, chloroform and bile
salt. They are heat labile.
8. Most of the HHV have latency, i.e. after primary infection
they remain dormant and are reactivated periodically.
9. Each type has different antigen except the herpes simplex
type 1 and 2.
Herpes Simplex Virus
Human beings are the natural host of herpes simplex virus

and the disease is seen only in humans.
The disease can be produced experimentally in animals.
The virus belongs to subfamily of alpha herpes virinae. There
are two types of herpes simplex, i.e. type 1 and type 2.
Morphologically, they are similar. The viruses are grown in
cell culture and on chick embryo. The two types can be
differentiated by antigenic differences by using type specific
sera with monoclonal antibodies.
The type 1 generally involves the body above the waist.
The type 2 generally involves the body below the waist and
is called genital herpes. Both types can infect the eyes.
The disease caused by type one spreads by saliva,
respiratory droplets. The type two is mostly spread sexually.
The type one remains latent in trigeminal ganglion while
type two remains latent in sacral ganglion.
Diagnosis
The diagnosis is confirmed by microscopy, virus isolation

and serology.
Microscopy
1. The smear is stained by toluidine blue O for 15 seconds

that shows multinucleotid giant cells.
2. Inclusion bodies: Intranuclear inclusion bodies are stained
by Giemsa stain.
3. Virus particles are seen on electronic microscopy.
4. Fluorescent antibody method is used to demonstrate herpes
virus antigen.
Virus Isolation
Tissue culture is the best method to isolate the virus. The

commonly used tissues are human embryonic kidney,
human amnion and rabbit kidney cells.
Serology is helpful only in primary infection. In recurrent
herpes, there is hardly any change in antibody titre.
Many antiviral drugs are available against herpes
simplex viruses. All do not give uniform result and fail to
stop recurrence. Most commonly used drug is acyclovir.
Varicella Zoster Virus
The virus belongs to the family herpes viridae, subfamily alpha

herpes virinae and known as herpes virus type III.
Morphologically, it is similar to herpes simplex virus but
differs clinically and antigenetically from herpes simplex
virus. Varicella zoster virus can be distinguished from herpes
OCULAR VIROLOGY 85
simplex virus by using highly specific antisera. The varicella

zoster virus does not grow in animals or on chick embryo. It
grows only on human embryonic tissue culture.
The virus causes two clinically different manifestations,
i.e. varicella chickenpox and herpes zoster. The chickenpox occurs
in nonimmune children and gives a lifelong immunity.
Herpes zoster is generally seen in adults (children are not
absolutely immune). Who had contracted chickenpox in
childhood and the organism has remained dormant in root
ganglion and the infection is reactivated later. Contact with
zoster may cause chickenpox but the reverse is not true.
Diagnosis
Clinical diagnosis of either varicella or herpes zoster is not

difficult. In the past when smallpox was prevalent,
sometimes it required differentiation from chickenpox.
The fluid removed from the lesion of varicella zoster shows
intranuclear inclusion bodies similar to those of herpes
simplex. Herpes simplex and zoster can be differentiated by
electron microscopy of typical viral particle. The presence of
immunofluorescent antibodies may help. IgM amd IgG are
found in varicella only, IgG is seen in zoster.
Many antiviral drugs, i.e. acyclovir, valaciclovir have been
found effective against the virus. Active immunization
against chickenpox is available. Its efficacy against herpes
zoster has not been proved.
Cytomegalovirus
This DNA virus belongs to family herpes viridae and sub-

family beta herpes virinae, labeled as herpes virus type V.
It is called cytomegalic because it cause enlargement of
host cells in contrast to herpes simplex and varicella zoster

that are cytolytic. It is differentiated from other herpes
viruses by electron microscopy. The virus is found all over
the world. Like other herpes viruses it shows latency in
children and adults with normal immunity, it does not
cause any symptom unless reactivated. It causes
disseminated disease in neonates and immunocom-
promised persons of all ages.
The cytomegalovirus (CMV) is largest among all herpes
viruses. The CMV is not related to other herpes viruses anti-
genetically. It is found in human beings and many other
body fluids. It causes severe congenital and less symptomatic
acquired disease.
The infection causes prominent intranuclear inclusion
body called Owl’s eye appearance. The inclusion bodies are
so large as to be confused as protozoa in the past. It shows
strict host specificity. The organism is grown in human fibro-
blast culture.
1. Demonstration of cytomegalic cells in centrifuged urine.
2. Recovery of virus from human fibroblast culture after it
has been inoculated with urine or saliva.
3. Antibodies are found only in primary disease.
4. Serological tests employed include ELISA, complement
fixation, immunofluorescence and radioimmunoassay.
5. CMV genome can be detected by PCR.
Epstein-Barr virus
This DNA virus belongs to family herpes viridae and sub-

family gamma herpes viridae and known as human herpes
virus type IV.
OCULAR VIROLOGY 87
The virus is found all over the world, about 80-90%

children become seropositive by the age of five to six years
without showing any signs or symptoms. The virus too has
latency and shows periodic reactivation that lasts for rest of
life. It has affinity for lymphoid. The virus activates
B. lymphocytes and cause secretion of immunoglobulin. The
IgM producing lymphocytes predominate other immuno-
globulins, i.e. IgG3, IgA and IgD are also produced in smaller
quantity. The virus is lymphoproliferative. The disease
spreads by saliva and respiratory secretions.
The virus produces
i. Infectious mononucleosis
ii. Burkitt’s lymphoma
iii. Lymphomas in immunodeficient
iv. Chronic fatigue syndrome
v. Ocular infection
Laboratory tests used to confirm the disease are:
1. Specific antibodies test for Epstein-Barr virus when Paul-
Bunnel test is negative.
2. Paul-Bunnel test
3. IgG virus capsid antigen
No antiviral drugs effective against Epstein-Barr virus.
Adenoviruses
The adenoviruses are DNA viruses, they were first observed

in surgically removed human adenoids hence eponymed
as adenovirus. They were later isolated from washing of
throat of persons suffering from pharyngitis. The virus is
found world over. It has affinity for mucous membrane of
the respiratory tract and gastrointestinal tract. It infects
conjunctiva very frequently with or without involving the
respiratory tract. They are also known to infect urinary tract.

The organism infects human beings, other mammals and
birds. It follows a strict host specificity. More than one type of
virus can present similar signs and symptoms and single
virus can have varied clinical presentation. Each virus gives
immunity against itself.
The family adeno viridae has two genera, i.e. mastadeno-
virus that infects mammals including human beings and
aviadenovirus that infect birds.
All the mastadenovirus are not human pathogen. There
are 47 serotypes of human adenoviruses which are divided
into six sub genera labeled A to F. Some of the adenoviruses
are oncogenic. All human adenoviruses have a common
complement fixation antigen.
As the adenoviruses are host specific. Laboratory animals
cannot be infected by them except the oncogenic adenovirus
that produce malignancy when infected in newborn
Hamsters.
The adenoviruses are DNA viruses that contain
linear double stranded DNA. They do not have envelop.
They are relatively small viruses. The capsid have
252 capsomers.
The viruses can survive for about seven to ten days at
room temperature. They are killed at 50°C.
The diagnosis can be confirmed by:
1. Electron microscopy
2. Complement fixation test
3. ELISA test
4. PCR
5. Virus isolation in specimen collected from throat,
conjunctiva, urine are grown on human embryonic
kidney and epithelial cells.
OCULAR VIROLOGY 89
There are no antiviral drugs that are effective in adeno-

viral diseases.
RNA Viruses of Ophthalmic Interest
RNA viruses are of great public health interest as they

contain some of the important disease producing viruses,
i.e. poliomyelitis, measles, mumps and HIV.
They contain RNA in their genome. The RNA is single,
stranded. Some of them are nonenveloped. The RNA viruses
comprise of following:
1. Picorna viruses
2. Paramyxoviruses
3. Retrovirus
4. Togavirus
Picorna Viruses
These RNA containing viruses are some of the smallest

viruses. The word picorna comprises of two parts, i.e. ‘pico’
meaning small and rna standing for RNA.
The characteristics of picorna viruses are:
1. They are very small in size – 20 to 30 nm
2. The capsid is icosahedral, symmetrical.
3. There are 60 capsomeres in them.
4. They lack envelope
5. They contain single strand RNA.
The picorna viruses are broadly divided into two groups.
1. Those which cause human infection:
i. Enterovirus
ii. Rhinovirus
2. Those that cause infection in animals and birds.
Broad Classification of Picorna Viruses
Of all the picorna viruses only coxsackie, CA 24V and entero-

virus 70 cause acute hemorrhagic conjunctivitis. Both the
viruses are spread by fecal-oral transmission and by fomites.
They have short incubation period of two days. They cause
epidemics all over the world.
Laboratory diagnosis consist of virus isolation by

inoculating the conjunctival discharge in tissure culture. The
coxsackie virus does not grow on tissue culture. It grows in
suckling mice.
Paramyxovirus
The paramyxovirus are relatively larger RNA virus con-

taining single strand RNA. They belong to the family
Paramyxo viridae. The viruses are spherical, pleomorphism is
common, contain hemagglutin envelope.
The family consists of three genera:
OCULAR VIROLOGY 91
1. Paramyxo virus:
i. Mumps
ii. Parainfluenza
iii. New castle disease
2. Morbilli virus
i. Measles virus
3. Pneumovirus—respiratory syncytial virus.
Out of these only mumps and measles have ocular
involvement.
Mumps Virus
Human beings are the only known host. The virus inflicts
mostly children. Children under six months are protected
by maternal antibodies which develop in the mother even
after a subclinical infection. The virus gives a lifelong
immunity hence second attack does not occur. The incubation
period is 16-18 days. Epidemics are common. The virus causes
hemagglutination followed by hemolysis. The organism is
killed at room temperature, formaldehyde and ultraviolet
light. The organism grows on chick embryo and human
amnion.
Diagnosis is simple on the basis of clinical presentation.
Atypical cases may require laboratory tests which consist of:
1. Demonstration of virus by immunofluorescent method.
2. Culture
3. Serology
i. Four-fold rise in antibody titre in paired serum
sample.
ii. Complement fixation test
iii. Hemagglutination inhibition test
iv. ELISA
Measles (Rubeola-Morbilli)
Measles is commonest exanthematous disease of children

world over. It can occasionally be fatal. It is fully preventable
by vaccination. A single attack of measles gives lifelong
immunity.
The organism is a RNA virus belonging to family Paramyxo
viridae and genera moribilli virus. It is an enveloped virus
with coiled helical nucleocapsid. It is moderately large
spherical virus, pleomorphism is common. The envelope is
made up of lipoprotein.
The measles virus grows on human and monkey kidney.
Human amnion culture is a preferred method for primary
isolation.
The virus is heat and ultraviolet labile. It is destroyed by
formaldehyde.
Clinical diagnosis of measles in nonimmunized child is easy.

It is only the atypical form that require laboratory tests.
They consists of:
1. Demonstration of multinucleated giant cells when nasal
discharge is stained by Giemsa stain.
2. Detection of measles antigen by immunofluorescent
method.
3. Positive growth on human kidney and amnion cells.
4. Demonstration of measles specific IgM.
5. Hemagglutination inhibition, complement fixation tests.
There is no known antiviral drug that is effective against
measles. It is fully preventable.
OCULAR VIROLOGY 93
Rubella (German Measles)
Rubella is an important RNA virus that belongs to family

Togo viridae and genus Rubi virus. It is of moderate size is
pleomorphic. Commonest shape being spherical. It is
enveloped virus. The enveloped is lipid in nature. The
envelope has spikes that are responsible for its hemagglutin
property. It is not related to other togo viruses in serology.
It grows on tissue culture that comprises of kidney cells
of specific monkey rabbit kidney and embryonated duck
egg.
The virus can pass the placental barriers during the first
trimester to cause widespread ocular and systemic
congenital anomalies. It produces two distinct clinical
pictures.
1. Acquired: A mild self-limiting infection that is generally
ignored as flu with fever and lymphadenopathy without
any complication in nonpregnant women. In pregnant
mother, the infection is passed to the fetus causing
congenital rubella syndrome. The mother passes the IgG to
the fetus which gradually disappears over next six
months. The IgM does not pass the placental barrier. The
disease gives a lifelong immunity.
2. Congenital: The fetus gets the infection if the mother is
infected during first trimester. Presence of rubella IgM in
infant is conclusive proof of congenital rubella. The live
viruses present in lens for few months after birth.
1. Virus isolation—In throat swab taken within four days

of infection.
2. Tissue culture
3. Immunofluorescent with specific antibody.
4. Serology—Hemagglutination inhibition test in avian
blood.
5. ELISA for IgG and IgM.
The disease is fully preventable by live attenuated MMR vaccine

that consist of viruses of mumps, measles and rubella.
Retrovirus
The retroviruses are RNA viruses. They contain reverse

transcriptase. When the virus infects the host cell, the reverse
tanscriptase changes the virus RNA first into single stranded
DNA and then into double stranded DNA. The double
stranded DNA is called provirus. The retroviruses have lipo-
protein envelop a capsid and genome that contain two
molecules of RNA, reverse transcriptase which is RNA
dependent DNA polymerase.
The retroviruses belongs to family Retro viridae. The viruses
of ophthalmic interest are:
1. Lentivirinae
2. Oncovirinae
Lenti virinae: This subfamily belongs to human immuno-

deficiency virus (HIV) that causes AIDS.
Human immunodeficiency virus: The virus is of moderate size

with iscohedral core. The envelope has spike on the surface
and is made up of lipoproteins. The genome consists of two
similar strands of RNA and reverse transcriptase. Once this
virus enters the host cell, the virus RNA is released and is
transcribed first to single strand DNA and then double
OCULAR VIROLOGY 95
strand DNA by reverse transcriptase. The double strand DNA

also called provirus gets integrated into host cells
chromosomes. The virus becomes dormant for long-time to
be activated. Once activated the virus reproduces. The
structural genes encode various parts of the virus (Figure
5.2). The HIV genome has three structural genes.
Figure 5.2: Structure of HIV
The HIV attacks those cells that have CD4 antigen. These
are helper lymphocytes, monocytes and macrophages.
Diagnosis
The clinical diagnosis is difficult because the HIV infection

has a long course and mimics many other diseases. It requires
high index of suspicion to reach the diagnosis which is
confirmed by laboratory tests.
A person is said to have AIDS if the laboratory tests confirm

to have the following.
1. CD4+ cell count less than 200 cells/cm3
2. Confirmed infection by HIV
3. Infected by at least on opportunistic organism
4. Presence of AIDS related malignancy (Kaposi’s sarcoma)
and lymphoma
The opportunistic organisms that are frequently seen
in AIDS are:
1. Bacteria:
i. Mycobacteria
ii. Atypical mycobacteria
iii. Syphilis
iv. Nocardia
v. Actinomycetes
vi. Salmonella
2. Virus:
i. Cytomegalovirus
ii. Herpes simplex
iii. Herpes zoster
iv. Molluscum
3. Fungi:
i. Candida
ii. Aspergilla
iii. Histoplasm
iv. Cryptococci
4. Parasites:
i. Toxoplasma
ii. Pneumocystis carinii
Besides the above organisms many more nonpathogens
are suspected to cause opportunistic infection in AIDS.
OCULAR VIROLOGY 97
The laboratory tests are:

1. Test for immunodeficiency
2. Specific tests for HIV
3. Tests for other infection
The nonspecific tests consist of:
1. Total and differential leukocyte count. Generally, there is
leukopenia with lymphocyte count less than 2000/cmm.
2. Platelet count shows thrombocytopenia.
3. CD4+ less than 200 cells/cm3
4. Reversal of T4:T8.
5. Raised IgG and IgA.
Specific tests consist of:
1. Antigen detection
2. Antibody detection: The serological tests for anti-HIV
antibodies can be:
a. Screening test: The most common screening test is
ELISA test.
b. Confirmatory test: The most commonly used
confirmatory test is Western blot.
3. Virus isolation
FUNGI
Fungi are ubiquitous organism found in soil and decaying
organic matters. Their importance as cause of disease in
plants and animals was established earlier than bacteria or
viruses. They are generally saprophytes but becomes pathogens
to cause opportunistic infection when there is a decline in the
immunity local or systemic. They are all parasites.
The fungi differ from bacteria and viruses in many ways.
The peculiarities of the fungi are:
1. They are said to be plants of lower order without leaves,
branches and roots.
2. They do not contain chlorophyll, depend upon
decaying organic matter for nutrition.
3. The fungi contain rigid cell wall made up of
polysaccharide and chitin.
4. They have cytoplasmic membrane that contain sterol.
5. They are eukaryotes, i.e. contain true nuclei with
nuclear membrane, paired chromosomes, mitochon-
dria, ribosome and food reserve.
6. They may be unicellular or multicellular.
7. Pleomorphism is common.
8. The cells show various types of specialization.
9. They propagate asexually, bisexually or by combined
methods.
10. The organisms are gram-positive, some are acid-fast.
11. Some fungi show agglutination.
12. Some fungi cause complement fixation reaction.
13. Fungi can produce endotoxin.
14. Hypersensitization to fungi is well established.
15. Fungi are not known to cause epidemics. They cause
chronic diseases both local and systemic. Some of the
systemic infections can be fatal.
OCULAR MYCOLOGY 101
16. The fungi are easy to visualize under light microscope.

Some of them are fluorescent under ultraviolet light.
17. They are difficult to culture, take long-time to grow.
18. Biochemical and serological tests are of less diagnostic
value than direct visualization and positive culture.
19. Antibiotic sensitivity to fungi is not available.
Various methods to demonstrate fungi are:
1. Wet preparation
2. Staining of slides
3. Demonstration in histopathological slide.
Wet Preparation
The specimens are generally skin scrapping, skin snips,

corneal scrapping, conjunctival swab or fluids from the
lesions.
A drop of 10% KOH is put on a clean dry glass slide. The
specimen is added to the fluid by inoculating wire. Steel or
iron wires are not used as they oxidize and produce artefacts.
A thin cover slip is put over the fluid. Press the cover slip
over the fluid to make the specimen flat. Wait for 5 minutes
and then examine under low power. High power may be
needed to see fungal elements that look like hyaline
structures. The hyphae and arthrospores standout well.
Instead of KOH, 10% NaOH can also be used. The
function of the alkali is to digest organic material other than
fungi.
Other less commonly used wet mount is to use glycerine
along with KOH. To make a wet mount, 10 ml of glycerine
and 20 gm of KOH are taken, to this 90 ml distilled water is
added. Presence of glycerine makes the fluid remain wet
longer.
No stains are required while using alkaline wet

preparation. The wet mounts where stains are used are:
1. Lactophenol cotton blue
2. Indian ink.
The first contains lactic acid, glycerine, phenol, distilled
water and cotton blue dye. The lactic acid destroys the tissue
other than the fungal element. The method is used to
visualize both moulds and yeasts. The fungal elements stain
pale to dark blue. The mount can be used as a permanent
slide by staining the edges of the cover slip by nail polish.
Pelikan Indian ink is used to identify Cryptococcus
neoformans that causes meningitis. The stain does not color
the fungus or its capsule, results in negative staining of the
organism that stands out prominently from other organisms
present in the slide.
Other Methods
1. Calcoflour white can be added to KOH mount and seen

under fluorescent microscope.
2. Fluorescent antibody staining is used when few fungi are
available in the specimen. It requires specific antisera.
3. Giemsa stain helps to identify both yeast and hyphae.
4. Periodic acid-Schiff (PAS) and methanamine sliver stains
are used to see fungi in histopathological slide.
Culture of Fungi
Some of the fungi are common contaminants in bacterial

culture. Otherwise fungi require special culture media at
room temperatures and long-time to grow. All the fungi are
aerobes.
OCULAR MYCOLOGY 103
Commonly used culture media for fungal growth are:

1. Sabouraud dextrose with or without antibiotics.
2. Sabouraud dextrose agar broth (SAB)
3. Blood agar with or without antibiotic.
4. Brain heart infusion
5. Corn-meal agar
6. Potato dextrose agar
Out of these Sabouraud media are the most commonly
used media.
CLASSIFICATION OF FUNGI
The fungi belong to Phyllum thallophyta. The phyllum
contains two groups, i.e. algae and fungi. The former contain
chlorophyll and are not known to cause human infection.
The fungi are again divided into Pseudomycetes and Eumycetes,
the true fungi. The pseudomycetes are now considered to
be higher bacteria. The eumycetes can either be with septate
hyphae or without septate hyphae.
Morphological Classification of Fungi

As per morphology the fungi are divided into four classes:
1. Yeast
2. Yeast-like fungi
3. Moulds
4. Dimorphic fungi
The yeasts are unicellular, spherical organisms that
multiply by budding. The only pathogenic yeast is
Cryptococcus neoformans that cause fungal meningitis. Rarely
it causes endogenous endophthalmitis.
Yeast-like fungi: Some of them develop partially as yeasts and
others by pseudomycellia which are chains of elongated
budding cells joined end-to-end. The example is Candida

albicans.
Moulds: These are mycelial or filamentous fungi. The moulds
consist of cylindrical branches called hyphae. Entangled
mass of hyphae are called mucelium.
Dimorphic fungi: These fungi assume two different shapes at
different temperature in soil and in culture. They are moulds
but in host body they appear like yeast. Most fungi causing
systemic infections are dimorphic.
The fungi may reproduce in three ways, i.e. asexually,
sexually and combined mode. The spores are the
reproductive parts in fungi. The asexual spores are called
conidia or conidio spores. On the basis of spore formation,
the fungi are divided in four classes:
1. Phycomycetes: These are lower fungi, have nonseptate
hyphae. They have both types of reproduction, i.e. asexual
and sexual. They have endogenous spores. The asexual
spores are called sporangio spores. The sexual spores are
either oospores or zygospores.
2. Ascomycetes have exogenous sexual spores called
ascospores, they also reproduces asexually.
3. Basidiomycetes form sexual spores called basidiospores.
4. Deuteromycetes (hypomycetes) also known as fungi
imperfecti. The sexual phase of these organism has not
been elucidated well.
FUNGI OF OCULAR IMPORTANCE

Aspergillus
The genus of Aspergillus contains about 900 species and sub-

species, out of which 21 species are human pathogens.
OCULAR MYCOLOGY 105
All human pathogens are not oculopathogens. The organism is

ubiquitous, found in air, water, soil, annual products as
spores. They are the commonest laboratory contaminants.
The Aspergillus fumigatus is the commonest human
pathogen. Others are A. flavus (yellow), A. niger (black). They
grow well and quickly on commonly used fungal culture
media. The color of the colony specifies the name of the
organism, The A fumigatus produces bluish grey, A. niger
(black) A. flavus (yellowish) green colonies (Figure 6.1).
Figure 6.1: Aspergillus
On lactophenol cotton blue stain, they show hyaline

septate, hyphae. The organisms have terminal conidospores
that liberate spores in air and water. The fungus produces
mycotoxin. They also act as allergen. They give positive
complement test and immunodiffusion test.
The organism is better visualized in Gram’s, Giemsa and

Gomori methenamine silver stain than KOH mount.
The organism is sensitive to local natamycin,
amphotericin B, systemic amphotericin B, itraconazole,
fluconazole and ketaconazole. Endophthalmitis requires
intravitreal antifungal injection.
Blastomyces
Blastomyces is a dimorphic fungus. It is thermal dimorphic, i.e.

its phases changes with temperature. At 25°C, it is mycelial
and at 37°C, it is yeast. It is found mostly in soil. The infection
is spread by inhalation of spores. Lungs are the commonest
primary site and skin is frequent extrapulmonary lesion. In
the eyes, it cause orbital cellulitis, granuloma of the lids,
keratitis, choroiditis and endogenous endophthalmitis. Its
incidence is reported to be high in AIDS.
It is visualized on KOH 10% mount and hematoxylin-
eosin stain, periodic acid schiff (PAS) and Gomori
methenamine silver stain are used to see it in tissue section.
In section, the lesion looks like tuberculosis. It is best cultured
on Sabouraud’s media without antimicrobial. It is also
cultured on blood agar and brain heart infusion.
The organism causing human disease is Blastomyces
dermatitidis which is a spherical double-walled structure.
The organism is sensitive to amphotericin B, itraconazole,
ketaconazole and fluconazole.
Candida (Monilia)
The genus Candida consists of more than hundred species of

dimorphic yeast like fungi. Out of which only eight are
clinically important. Eighty percent of infection is caused
OCULAR MYCOLOGY 107
by Candida albicans. The candida is a saprophytic found in

soil, water and food. It is a commensal of all the mucous
membranes of the human body and skin including
conjunctiva. The candidal infection is so common that
Candida skin test is almost universally positive hence is not
diagnostic but an indicator of cell-mediated immunity
(Figures 6.2 and 6.3).
Figure 6.2: Candida albicans (Courtesy: Prof V Sudarshan)
Figure 6.3: Candida albicans
The Candidiasis is the foremost fungal infection of the eyes. It

reaches the eye in two ways:
1. Exogenous following trauma

2. Endogenous due to hematological spread from systemic
site.
Incidence of candial infection is high in immuno-
compromised persons. Immunocompetent persons are liable
to get the infection when the defense mechanism breaks
down. The predisposing factors are prematurity, prolonged
use of antibiotic and steroids, radiation, diabetes, debility
due to malnutrition, leukemia, lymphoma, person on cancer
chemotherapy and radiation are at higher risk groups, so
are the person with in dwelling catheters and intravenous
catheters.
The Candida albicans is a gram-positive organism, it stains
darker than cocci. It is arranged in irregular clusters or in short
chains. The organism stains with Gram’s stain, Giemsa stain,
Gomori’s methenamine silver stain and periodic acid-Schiff.
It is a common contaminant of bacterial culture. It grows
on Sabouraud dextrose agar with chloramphenicol without
cyclohexamide. Other media used are blood agar and corn-
meal media. Chlamydo spores develop only in media that
are nutritionally poor-like corn meal media at 28°C.
The freshly prepared 10% KOH mount seen under low
power microscopes shows the organism as ovoid or spherical
thick-walled budding yeast cells with Pseudomycelia or as
elongated filamentous cells that look-like hyphae. The
organism produces two types of spores, the chlamydospores
and blastospores.
The Candida albicans must be differentiated from
Staphylococci, Streptococci and fungi other than Candida
albicans by growth characteristics, sugar assimilation, sugar
fermentation and germ-tube formation.
OCULAR MYCOLOGY 109
Serologically, Candida albicans has two sero types—A and B.

The other test used is ELISA.
The organism is sensitive to amphotrecin B, natamycin,
miconazole, econazole, ketaconazole and triazole.
Coccidioides
The organism coccidioides belongs to the family of thermal

dimorphic fungi. The organism is a soil saprophyte found in
some parts of western hemisphere in semiarid climate. It
has two phases:
1. The saprophyte phase which is mycelial phase seen in the
soil and culture. The organism in this phase has branching
pattern with septate. They form barrel-shaped arthro-
spores (Arthroconidia). The arthospores are infective and
are liberated in air to settle down in the soil.
2. The parasitic phase is an endospore that develops in the
lung. The endospores are contained in spherules that are
spherical and thick-walled. The spherules rupture to
release the endospores in the tissue.
The fungus is cultured on Sabouraud dextrose agar with
or without antibiotic or cycloheximide. The culture is grown
in test tube for safety of persons handling the culture. The
arthrospores are highly infectious and have greater chance
of infecting persons when grown on a larger surface like
petri dish. The organism grows as mycelia in culture but is
yeast in the tissue. The culture tube is incubated at both room
temperature as well as 37°C. It takes three to four weeks for
the growth to be positive.
The fungus is visualized in 10% KOH mount or calcoflour
white stain. The tissue is stained with periodic acid-Schiff
stain. The aspirated intraocular fluid may be stained by Papa-

Nicolaou stain to see the organism.
The commonest lesion is seen in the lung following
inhalation of the endospores. The lung lesions is generally a
mild and self-limited lesion. It sensitizes the person to the
antigen. The sensitivity is tested by commercially available
Coccidiodin and Spherulin. The test is similar to tuberculin test.
It is not a confirmatory test. The disease is confined by
demonstration of arthosphores or endospores. The initial
infection confers lifelong immunity. The second form of the
disease is disseminated disease which is rare but may be fatal.
The dissemination is hematogenous that may spread the
infection to central nervous system, bones, skin and eyes. The
other form of ocular inflammation is hypersensitization of
the fungal antigen causing phlycten, episcleritis and scleritis.
This is seen during pulmonary infection and subsides with
pulmonary infection. The hematogenous spread leads to
chronic and progressive pan uveitis.
Incidence of coccidioidomycosis is so common with AIDS
that it is considered to be AIDS defining opportunistic
infection in person with HIV.
The organism is sensitive to amphotericin B, fluconazole
and itraconazole.
Dermatophytes
It consists of 40 species of filamentous fungi that infect nails,

hair, skin, rarely cornea and conjunctiva. They have been divided
into three genera, i.e. Trichophyton, Epidermophyton and
Microsporum.
The organisms have different morphology in tissue
and culture. In tissue, they have hyphae and produce
OCULAR MYCOLOGY 111
arthospores. In culture, they have hyphae and asexual spore.

The hyphae in culture are septate. The asexual spores are
Microconidia and Macroconidia on the basis of their relative
sizes. The three genera are differentiated on the basis of
macroconidia.
The dermatophytes grow well in Sabouraud’s dextrose
agar with chloramphenicol. The plate is incubated at room
temperature aerobically for three weeks. The species are
identified as per their colony and appearance under
microscope in KOH mount or stained with lactophenol
cotton blue.
The ocular involvement is mostly in the lids as scaly rash,
dermatitis, madarosis, blepharitis, allergic conjunctivitis.
Corneal ulcer is rarest of all ocular involvements. The fungi
are sensitive to many antifungal chemotherapeutic agents
both local as well as systemic.
Histoplasma
The organisms belonging to this group were earlier thought

to be protozoa, later it was established that histoplasma were
infact fungi.
There are two types of histoplasma according to their place
of occurrence:
1. The American histoplasma
2. African histoplasma.
The former is more common and has extensive systemic
involvement and better known as Histoplasma capsulatum.
The latter is known as Histoplasma duboisii. It only involves
the cutaneous and subcutaneous tissue and not the
respiratory system which is the main target of Histoplasma
capsulatum. The duboisii is morphologically similar to
H. capsulatum. Some authors believe that H. duboissi is just a

variant of H. capsulatum.
H. capsulatum is found all over the world but the disease
caused by it is mostly confined to some state of the USA. It
is a soil saprophyte, found in soil, enriched by droppings of
some birds and bats which normally harbor the organism.
It is a dimorphic fungus. The dimorphism is related to
ambient temperature. It is mycelial at 25°C and yeast at 37°C.
The morphology also varies according to the media in which
it is grown. Though American histoplasm is called Histoplasma
capsulatum, it does not have a formed capsule.
The yeast phase is found in the tissue and blood agar
media at 37°C. In Sabouraud’s media at room temperature,
it is mycelial which has a thick-walled spherical spore called
tubercle with finger like projection radiating from the
spherical structure. Presence of such tubercles in the
organism is diagnostic. The spores are asexual and
unicellular, the large spores are called macroconidia.
The conidia are highly infective and cause the disease
histoplasmosis of the lung following inhalation.
The organism grown on Sabouraud’s media and blood
agar enriched by glucose-cystine and mixed with
chloremphenicol or cycloheximide. The culture is incubated
for minimum four weeks, may require as much as twelve
weeks.
The organism is seen with light microscope under oil
immersion. The organism is stained by Giemsa stain or
Wright’s stain.
The tissue sections are stained by Gomori’s methanamine
silver stain, periodic acid Schiff stain and calcoflour white.
OCULAR MYCOLOGY 113
The organism is found in sputum blood and bone marrow.

Histoplasmin is the antigen found in filtrate of mycelial broth.
Complement fixation test for antibodies to histoplasmin is
found to be positive within two to five weeks after infection.
The titre rises during progressive disease and falls with
decline of the infection.
The other tests used are latex agglutination precipitation
test, enzyme immunoassay.
Skin test: The histoplasmin skin test is similar to tuberculin

test. It becomes positive soon after infection and remains so
for years. It becomes negative in progressive disseminated
disease.
The disease caused by H. capsulatum is called Histoplasmosis
which is basically a disease of reticuloendothelial cells. The
commonest site to be involved is respiratory system. The
respiratory disease simulates tuberculosis of lung from
which it should be differentiated. The other diseases that
have similarity with histoplasmosis are blastomycosis and
toxoplasmosis.
The disease in acute phase does not require any treatment.
Intravenous amphotericin B is required for progressive
disseminated disease. Ocular involvement is known as presumed
ocular histoplasmosis.
Mucor
The fungus belongs to order mucorales genera zygomycetes.

The other two species of this genera are rhizopus and absidia.
Infection by them is collectively called mucor mycosis or
zygomycosis (Figure 6.4).
Figure 6.4: Mucor
The organisms are ubiquitous, found in soil, manure and

fruits. The spores which are infective are found in air and
dust. All the three organisms have similar morphology. They
are moulds and generally referred to as bread moulds. The
organism is grown on Sabouraud’s dextrose agar media
without cycloheximidine but with antibiotic. Sometimes the
organisms grow fast, i.e. within three to four days in room
temperature. They are common laboratory contaminants.
The organism when removed from infected tissue are
difficult to culture. The best method is to demonstrate them
in histopathological slide stained by hematoxylin-eosin,
periodic acid-Schiff, Gomori’s methanamine stain.
The fungus reproduces both sexually and asexually by
zygospores and sporangiospores respectively.
Microscopic examination of biopsy material in KOH
mount shows broad, branched hyphae. There are no specific
serological tests to clinch the diagnosis.
The organism spreads by inhaling the spores in the air. It
cause disease mostly in severe diabetes, leukemia and in
AIDS. The organism colonies to form spores in respiratory
tract, i.e. in the paranasal sinuses and lungs. It may invade the
OCULAR MYCOLOGY 115
brain from the paranasal sinuses and from the orbit. It is invariably
a fatal disease.
Rhinosporidium
The organism rhinosporidium is unique in the sense that its

position in systemic classification of microbes is not clear.
Originally, it was thought to be protozoan.
Later it was classified as a lower fungus.
The similarity of Rhinosporidium with lower fungi are:
1. The sporangial wall contains cellulose.
2. The trophic phase consists fatty material as nutritive
reserve.
3. Formation of spore is heralded by repeated nuclear
division.
4. All nuclei undergo simultaneous mitosis.
5. There is no cytoplasmic residue following repeated
nuclear division.
6. A mucoid substance fills the space in between spores.
7. The wall contains definite pores for the spores to escape.
The argument against its being a fungus are:
1. It does not grow in any known media used to culture
fungus.
2. The organism does not have hyphae or septa.
3. The organism does not cause disease in experimental
animals.
4. The organism does not give any serological test.
5. The organism is not known to be sensitive to any known
antifungal drug.
6. No recurrence occurs following complete surgical
removal.
The organism has been put in the class of Phycomycetes

which consists of fungi of lower class.
The organism is a water saprophyte. It is also found less
commonly in air and soil. It has not been isolated from
normal individual. It is known to infect cattle and horses.
The exact life cycle of the organism too has not been
established beyond doubt.
Traditionally, the development of the organism is divided
into three stages.
1. Stage of maturation
2. Stage of sporangia
3. Stage of spore formation.
The earliest stage of development is called trophocyte stage.
The trophocyte has a cell membrane, clear cytoplasm, a
vesicular nucleus and a nucleolus. The stage is found in free
tissue spaces sometimes becoming intracellular as the cell
matures granules and globules appear in the cytoplasm
(Figure 6.5).
They contain fats and protein that supply nutrition to the
growing organisms. At about 50 µ size the nucleus undergoes
first division. The division is mitotic in nature. Further
divisions result in gradual increase in the size of the organism
with deposition of cellulose inside the cell wall. The cellulose
lining is thinner at places. In places of deficient cellulose, the
pores develop through which the future spores will escape.
Multiple cell divisions is followed by formation of sporangia
that contain endospores. The spores escape from the sporangia
either through the spore or the wall of the sporangia gives
way releasing the spores.
The organism does not grow either in culture media, can
not be produced experimentally and does not stain with
OCULAR MYCOLOGY 117
Figure 6.5: Life cycle of Rhinosporidium
Gram’s or Giemsa stain but stains well in tissue section by

hematoxylin-eosin, periodic acid-Schiff and Gomori’s
methenamine silver stain (Figure 6.6).
The sporangia are visible on KOH wet mount or when
the tissue is soaked in normal saliva.
The mode of infection is not known the most widely
accepted hypothesis is that the disease spreads from cattle
to human via water.
The organism causes chronic granuloma in various mucus
membrane including conjunctiva. It may disseminated to
bones and skin (Figures 6.7 to 6.9).
Figure 6.6: Histopathological section of Rhinosporidiosis.

(Courtesy: Prof V Sudarshan)
Figure 6.7: Rhinosporidiosis tarsal conjunctiva.

(Courtesy: Prof AK Chandrakar)
Figure 6.8: Limbal rhinosporidiosis

OCULAR MYCOLOGY 119
Figure 6.9: Rhinosporidiosis of fornix
There is no medical treatment. The best treatment is

removal of the growth in toto.
INTRODUCTION
Parasites as causative agents of diseases were known from
the days of Hippocrates. The modern study of human
parasitology started with discovery of microscope by Antony
Van Leeunhoek in the year 1683 and study of Echinococcosis
by Hartmann in 1685.
It is essential to know some terms used in parasitology to
understand the life cycle and mode of transmission of
diseases caused by various parasite.
Parasites are the living organism that live in/on a host
that provides nutrition to the organism and protects it also.
The organism that harbors the parasite is called the host.
Parasitism is the association in which the parasite gets the
benefit at the cost of the host. In such a situation, it is the
host that suffers the injury. Parasitism exists in some bacteria,
viruses, protozoa and helminths. The host offers resistance
to the injury done by the parasite and there may be some
adaptation (tolerance) to this.
The term free living parasite denotes a parasite which
lives independent of the host, i.e. Acanthamoeba, Naegleria
and hookworm.
The term mutualism denotes a situation where both the
host as well as the parasite derive benefit from each other.
The arrangement is best seen in plants, i.e. mutualism
between rhizobium bacteria and leguminous plants.
Lichens are formed by mutualism between algae and
fungi. This process is not seen in human parasitology.
In symbiosis, the host and the parasite both depend on
each other to thrive, one can not live without the other. If
the parasite derives benefit without causing harm to the host,
the condition is called commensalism. The parasites involved
are called commensals. They live on superfluous fluid or
OCULAR PARASITOLOGY 123
solid in the host. The commensals can be turned into the

pathogens. The pathogens are the organisms that inflict
injury to the host.
Types of Hosts
The hosts are divided into two types, each with a definite
purpose:
1. Definitive host is a host that harbors the adult stage of
parasite or where the parasite uses the sexual method of
reproduction. Man is the definitive host in malaria,
Echinococcus, Toxoplasma gondi.
2. Intermediate host is the host in which the larval form lives
or the asexual replication takes place. In some parasites
the larval developments are completed in two
intermediate hosts which are called first and second
intermediate hosts.
Vectors are arthropods that transmit the parasites to host.
The vectors in which the parasites multiply are called
biological vectors in contrast to mechanical vectors in which
the parasites do not multiply, i.e. housefly is a mechanical
vector for amoebiasis and trachoma.
Zoonosis is a parasitic disease that is primarily a disease
of vertebrate animals but can be transmitted to humans to
produce disease. The examples are Leishmaniasis, Try-
pansomiasis and Echinococcus.
Zoonanthroponosis is an infection where human beings
are incidental hosts and serve as an essential link in the life
cycle of the beef or the pork tapeworm.
Anthroponosis refers to parasitic infection that are solely
seen in humans, i.e. filarial.
Reservoir
A reservoir is a host, may be human or animal in which the

parasite usually resides or an animal that harbors the parasite
in absence of human host. In most of the parasitic infection,
man is the main reservoir. In endemic area, infection is
continued in the population by reservoir. A reservoir is called
a carrier if the parasite acquires resistant form that helps to
disseminate the disease. Though humans are the main carriers
of disease, the animals can also act as carriers.
Types of Parasites
The parasites can be:

1. Obligate parasites, i.e. those that cannot live without a
host.
2. Facultative parasites that can exist independently.
3. Accidental parasites, i.e. that parasite which infects an
unusual host.
The parasites can also be divided into two classes as per
the site of disease produced by them.
1. Ectoparasites that live on the external surface (skin) of
the host and is called ectoparasites. The disease caused
by them is referred to as infestation.
2. Endoparasites are the parasites that live inside the body
of the host, that cause infection.
Route of Entry of Parasites

Parasites find their way into human body by various portals.
1. Ingestion: Oral route is the commonest form of infection
by parasites.
i. Ingesting contaminated food
ii. Consuming undercooked food.
iii. Eating raw vegetables (salads)

iv. Drinking water contaminated by intermediate host, i.e.
Cyclopes that harbor larval form of D. medinensis.
v. Fecal oral transmission, i.e. threadworm.
2. Transcutaneous: A duodenale enters the body through the
sole if a persons walks barefeet on soil contaminated by
the parasite.
3. Bite of insect host
Plasmodia – Mosquito
Trypanosoma gambiense – Tsetse fly
Trypansoma cruzi – Reduviid
Leishmania – Sandfly
Wuchereria – Mosquito
4. Hematogenous: This is the commonest mode of spread
from primary site.
5. Waterborne: Persons may get acanthamebic infection from
swimming pool. Contact lens users get infection from
contaminated contact lens fluid.
6. Deposition of eggs: Flies may deposit eggs in the
conjunctival sac or orbital wound which develop into
larvae in ocular myiasis. Some larvae from the paranasal
sinuses may invade the orbit that may become intraocular
(Myiasis interna). Eggs of thelazia may be deposited in
the conjunctival sac from where they may penetrate the
limbus or sclera to become intraocular.
Laboratory tests employed in diagnosis of human
parasitic disease of ocular interest
Specimen Parasites
a. Blood i. Plasmodium (Malaria) in RBC
ii. L. Donovani (Kala-azar) in monocytes
iii. Trypanosoma in plasma
iv. Wuchereria—Microfilaria in plasma
b. Stool i. Amoebiae—Trophozoites or cysts

ii. Hookworm—Eggs and adultworm
iii. Ascariasis—Eggs and adultworm
iv. Taeniasis—Eggs and segments of
adultworms
v. Threadworm—Eggs and adultworms
are found in perianal region
c. Urine i. Eggs of Schistosoma haematobium is
found in urine of patients suffering
from schistosomiasis.
ii. Chyluria is present in W. bancrofti,
microfilaria may be present.
d. Sputum i. Trophozoites of E. histolytica may be
present if a liver abscess bursts in lungs.
ii. Scolex and hooklets of E. granulosus
can be seen in sputum if a hydatid cyst
of lung ruptures.
e. Aqueous May show microfilaria and thelazia in
AC, rarely cysticercus may be seen in AC.
f. Vitreous Vitrectomy may reveal Toxocara canis,
Echinococcus.
g. Other blood i. All helminthic infections cause
leukocytosis with eosinophilia.
ii. Gamma globulin is raised in visceral
larval migrans and Gambian trypano-
somiasis.
iii. Complement fixation test is done in
Toxoplasmosis, Filariasis, hydatid
disease, Chaga’s disease.
iv. Hemagglutination test is done in
hydatid disease, toxoplasmosis,
visceral larval migrans.
v. Specific dye test—Sabin-Feldman dye

test for toxoplasmosis
vi. Other tests—PCR, ELISA, indirect
fluorescent antibodies, IgG, IgM
antibody.
h. Intradermal test: Following intradermal injection with
parasitic antigen a positive reaction is suggestive of
diagnosis. The test is done in many diseases. Common
ocular diseases where they are used are hydatid disease,
Toxoplasmosis and Trypanosomiasis.
i. X-ray of chest, orbit may reveal hydatid cyst and neuro-
cysticercosis.
j. USG, CT and MRI are more reliable than X-ray.
Immunological Changes in Parasitic Infection
All infective agents including parasites induce specific

immune changes in the host. This is a part of defense
mechanism. The immune reaction by protozoa and
helminths are not so effective as by bacteria and viruses.
The immune reaction heralds antibody response and cell-
mediated response via effector T cells. The immune reaction
is species specific. The immune response to parasite
is associated with production of immunoglobulin IgG
and IgM. The protozoal infection is associated with IgA
while helminthic infection is associated with production
of IgE.
The antibodies reaction is brought about by:
1. Agglutination of parasites by IgM.
2. Opsonization: The antibody acts as opsin increasing
chances of clearance by phagocytosis.
3. Antibody reacting with surface antigen.
4. Antibody dependent cell mediated cytotoxicity.

5. Neutralization: The antibodies neutralize the toxins and
enzyme by binding to them.
Classification of Ocular Parasites
PROTOZOA
Acanthameba
Acanthameba is an ubiquitous free-living ameba found in

fresh water, air and soil. Many species are normal
commensals of oropharynx. Large number of persons with
acanthameba develop immunity to it. Incidence of infection
in immunocompromised is very high.
The organism belongs to Phylum—Sarcomastigophora
Family—Acanthamebida
Genus—Acanthameba
There are many species of the organism, out of which

A culbertsoni is most important ocular pathogen. Commonest
ocular involvement is Acanthamebic keratitis. Other ocular
involvement is iridocyclitis. The organism causes meningo-
encephalitis which can be fatal.
The organism is found in two forms in the infected tissue.
Both the forms are infective. The forms are trophozoites and
cysts (Figure 7.1). The trophozoite is converted to cyst in
unfavorable conditions. The cyst remains dormant until the
condition changes to its favor than it comes out of the cyst.
The mechanism is called excystation.
Figure 7.1: Gross structure of Acanthamebae
The trophozoite is a sluggishly motile, nonflagellated

ameboid structure. The outer wall has five-pointed, slender
projections called acanthopods. These are micropseudopods. The
cytoplasm is foamy with contractile vacuoles and a nucleus
that is visible only on stained preparation. The nucleus
contains a dark karyosome. The trophozoite multiplies by
binary fission. Each developing into independent
trophozoite (Figure 7.2).
In unfavorable conditions the trophozoites are converted
into inactive cysts which are resistant to antiacanthamebic
therapy. The typical cyst is circular or ovoid double-walled
structure with foamy cystoplasm and nucleus with well-
Figure 7.2: Life cycle of Acanthamebiae
stained karyosome. The inner wall is called endocyst. The outer

wall is called ectocyst. The inner wall intersects the outer wall
at places and the contact points are called ostioles or pores
through which excystations occurs.
Both the trophozoites and the cyst forms are visible as
refractile spots in confocal microscope. The organism is also
visible in bright field microscope and phase contrast microscope.
The organisms are visible in 10% KOH wet film. They are
seen in Hematoxilin eosin, stain in tissue. They are also visible
in Gram’s and Giemsa stain. Calcoflour white stain requires
fluorescent microscope.
The organism is cultured aerobic in nutrient agar, blood
agar and chocolate agar overlaid with dead E coli or Klebsiella.
The culture is incubated overnight.
The growth is seen as areas of clearing among the area
seeded by the bacteria.
Little is known about the antigenic property of the
organism.
The organism is transmitted through:
1. Contaminated water of swimming pool, they are not

destroyed by usual methods of chlorination hence are
present in drinking water.
2. They may be inhaled or get entry through minor cuts on
the skin.
3. The most important source of ocular infections are:
i. Contact lens, contact lens storage cases, contact lens
solution especially the homemade solution.
ii. Trauma de novo or by contact lens predispose keratitis.
iii. Patients with AIDS are at higher risk than others.
The exact incubation period is not known, it varies
between few weeks to few months.
The organisms are not susceptible to usual antiamebic
chemotherapy. They are sometimes susceptible to anti-
fungal, i.e. imidazoles. They are also sensitive to
aminoglycocides and surprisingly to antiseptic biocides like
Chlorohexidine, and Polyhexamethylene biguamides. The
medical treatment is protracted and frustrating because there
are no effective drugs available against cyst. The drugs are
more effective against trophozoites than cysts.
Toxoplasma
The organism is an ubiquitous coccidian protozoa belonging to:

Phylum Apicomplexa
Family Sacrocystidae
Class Sporozoea
Genus Toxoplasma
Species Gondi
The genus Toxoplasma has only one species, i.e. T. gondi
(Gondi is the name of North American rodent in which the
organism was first detected).
The organism is found in soil and water. It is also found in

many vertebrates including humans and birds. About 50%
of population in developed countries have serological and
clinical evidence of the disease. The prevalence is higher in
underdeveloped countries. The organism can cause disease
at any age in both the sexes. It can either be congenital or
acquired. The acquired form may be a fresh infection or
reactivation of congenital lesion or may be recrudescence of
healed acquired disease.
The disease can be acute or chronic in nature. The chronic

disease has a protracted course. It generally produces
systemic disease. The organism has predilection for brain
and retina.
The ocular disease is either congenital or secondary to
systemic disease. The ocular diseases are generally milder
than systemic disease. The congenital infection may be fatal
in new born. The immunocompromised are a higher risk of
fatal outcome.
The organism is an obligate intracellular parasite with a
complex life cycle.
The organism occurs in three stages:
1. Trophozoite or tachyzoites (They multiply rapidly inside
the cells) They cause acute infection.
2. Tissue cysts or bradyzoite (cystozoite) (They multiply
slowly in the tissue cell)
3. Oocytes (Sporozoites)
The trophozoites are intracellular, crescent-shaped and

sometimes oval, seen in hanging drop and smear, are motile.
The one end of the organism is pointed and the other end is
rounded, found in various tissues and organs. The
trophozoits contain cytoplasm and a nucleus. The nucleus
is relatively large and occupies the rounded end of the
organism. The nucleus has a nucleolus. The trophozoite stain
with Giemsa and Wright. The cytoplasm stains blue in
Giemsa stain. The nucleus stain red by the same stain.
Sometimes, there may be some extracellular trophozoites
as well in the same slide (Figure 7.3).
Figure 7.3: Trophozoite of Toxoplasma gondii
The organism does not survive in extracellular state in

the host. The organism dies by freezing and drying.
The trophozoites multiply by a process known as
endodyogeny. This is an asexual division in which two
daughter cells are formed in each parent cell. These are called
tachyzoites due to their fast multiplication. The tachyzoites
can infect all the intracellular cells and the organs except
the RBCs. The intracellular collection of multiple tachyzoites
(endozoites) are called pseudocysts. The endozoits keep on
increasing in number till the cell wall ruptures releasing
endozoites (tachyzoites). The released tachyzoites keep on

multiplication in various tissues. Their presence heralds
immune reaction to eliminate many of them. The remaining
tachyzoites are converted into next stage, i.e. tissue cyst
called cystozoites (Figure 7.4).
Figure 7.4: Two forms of T. gondii in humans
Tissue Cysts (Cytozoites)
The tissue cysts are formed within the host cell and contain
thousands of spore like organisms. As they multiply slowly,
they are called bradyzoites. They stain with periodic acid-
Schiff stain (PAS). The cyst wall stain with silver stain. They
persist in every tissue including brain, muscles, liver and
ingested by carnivores, they get infected. There is hardly any
difference in morphology of tachyzoites and bradyzoites. The
conversion from tachyzoites to bradyzoites is a defense
mechanism. The latter linger in the host for years. They cause
chronic infection and can transmit the disease. The cyst wall
is destroyed by digestive fluid after the cyst has been ingested.
Oocytes (Sporozoites)
They are formed only in feline intestine. The cat is the only
animal in which the organism has sexual cycle comprising
of microgametes (male) and macrogametocytes (female) leading
to formation of zygote that is surrounded by wall of the
oocyte. The cat sheds the oocytes for about 5-7 days. Only,
after which there are no oocytes in cat feces. The freshly passed
oocytes are not infective. They develop infectious status in soil
and water after few days. The freshly passed oocyte in cats
feces contains a sporoblast that develops into the sporocytes.
A mature oocyte contains two sporocytes, each contain four
sporozoites (Figure 7.5).
Figure 7.5: Development of sporozoites of T. gondii

The organism goes through three distinct stages and three

cycles. The cats and other members of the cat family are the
definite host, i.e. the organism reproduces sexually in cats.
The cycle in cat is called feline cycle. The cycle in other
vertebrates is called nonfeline cycle.
The various combinations of the cycles can be:
1. The first cycle is limited to definitive host, i.e. cat.
2. The second cycle takes place partly in definitive host, i.e.
cat and the cycle is completed in intermediate host, i.e.
man or other vertebrates.
3. The third cycle takes place entirely in two intermediate
hosts and not the cat.
The cats are also called homologous hosts and the phase
in cat is called enteric phase. The intermediate hosts are called
heterologus hosts and the phase is called extraintestinal or
tissue phase (Figure 7.6).
Figure 7.6: Life cycle of T. gondii

Route of Infection in Humans
1. Ingestion of contaminated food: Ingestion of food and drink

contaminated by oocytes.
2. Transfer of oocyte from cats by human hands direct to
mouth, food or drink.
3. Consumption of tissue cysts via undercooked meat of
infested vertebrate.
4. Through blood transfusion or organ transplant.
5. Transplacental from mother to developing fetus.
6. Through skin abrasion
Diagnosis
Diagnosis of acquired Toxoplasmosis is difficult and

presumptive. It consists of:
1. Clinical impression
2. Exclusion of other diseases that mimic toxoplasmosis
3. Demonstration of parasite in body fluid and tissue
4. Serological tests
Differential Diagnosis
The diseases that need to be excluded in ocular

toxoplasmosis are syphilis, bacterial and Candidal retinitis,
Behçet’s retinitis and subacute neuroretinitis.
The commonly used serological tests are:
1. Complement fixation test
2. Sabin methylene blue dye test: This is till to day the
standard diagnostic test.
3. Rapid agglutination test
4. Indirect hemagglutination test
5. Indirect immunofluorescent test
6. ELISA IgM
7. IgM immunosorbent agglutination test
8. Immuno Blot
9. Western Blot
10. PCR
11. TORCH
Demonstration of Organism
The organism takes Giemsa stain, CSF or aqueous is used

to see the organism. The trophozoite has a crescent shape,
the cytoplasm of which stains blue while the nucleus
stains dark.
The organism is isolated from peritoneal fluid following
intraperitoneal inoculation of infected tissue in white mice.
The infected animal develops acute infection. The organism
is sensitive to many antimicrobials of different nature. They
are sulpha drugs, antibiotics and antimalarial.
Commonly used drugs are:
1. Triple sulpha
2. Clindamycin
3. Spiramycin
4. Pyrimethamine
The two nonantitoxoplasmic drugs administered con-
currently with above drugs are steroid and folinic acid.
Steroids are given to control inflammation. Folinic acid is
given with pyrimethamine to prevent folic acid depletion.
Microsporidia
Microsporidia is an ubiquitous spore forming Protozoa that

belongs to Phylum microspora. It is an obligate intracellular
organism. It is mostly an opportunistic protozoa hence infects
immunocompromised persons more than immunocompetent

persons. It also infects many animals, birds, insects and
worms. The exact reservoir of the organism has not been
established. It is a gram-positive organism, stains with PAS,
Gomori’s methanamine silver stain, Giemsa stain,
hematoxylin eosine stain. It is an acid-fast organism. It has
not been cultured. Its life cycle is also not known. There are
about 100 genera and 1000 species of the organism, only a
few of them are human pathogens. The common human
pathogens are Encephalitozoon, Enterocytozoon, Nosema,
Pleistophora. All the human pathogens are not ocular
pathogens, the ocular pathogens are enterocytozoon and nosema.
The organism has three stages of development. The stages are:
1. Infective stage
2. Proliferative stage
3. Sporing stage
The microsporidial spores are the infective forms.
The microsporidia are eukaryotic, without mitochondria
or Golgi’s membrane. They contain 70s ribosome. The micro-
spora has a thick double-layered cell wall with the nuclei
polaroplast which is a coiled polar tube. The organism
multiplies by binary fission as well as multiple fission inside
the host cell. The host cell makes it resistant to anti-
microsporidial treatment and defense mechanism of the host.
The exact mode of transmission of the disease is not well
established. It is most probably by ingestion or droplet. The
most common route is orofecal. The ocular infection spreads
by fingers contaminated by patients urine and stool. The
predisposing factors are immunosuppression, trauma, close
proximity with infected animals.
The ocular involvements are punctate epithelial keratitis,
keratoconjunctivitis and stromal keratitis.
Diagnosis is confirmed only on:

1. Demonstration of organism of biopsy specimen by light
microscope.
2. Conjunctival scrapping is sufficient only to show gram-
positive and intracellular organism.
3. Other methods used are electron microscopy, fluorescent
microscopy and confocal microscopy.
Treatment
No effective treatment is known for systemic infection.

Drugs of diverse actions have been used. They are
metranidazole, trimethoprime, thiabendazole and
albendazol. Local treatment consists of instillation of
fumagilline 70 µg/ml or propamidine isothionate drops.
Pneumocystis carinii
The taxonomy of this unicellular organism is uncertain.

Previously, it was thought to be a fungus. Analyses of
ribosomal RNA, mitochondrial protein, chitin in cell wall and
presence of enzymes go in favor of its being a fungus. Points
in favor of its being a protozoa are morphology, sensitivity to
antiprotozoal drugs, resistance to antifungal drug and failure
to grow on any media. The organism has been cultivated
in vitro on primary embryonic epithelial lung cells of chick.
The organism has low virulence and slow proliferation. It
thrives well in presence of another microbe. The organism
has a worldwide distribution. It is found as commensal in
many animals. It is an opportunistic organism for humans.
The person gets the infection most probably in infancy and
childhood. They become symptomatic or remain asympto-
matic carriers. The organism is not known to spread from
animals to humans. In humans, the spread is either due to a

reactivation of old lesion or fresh infection by inhalation. The
organism has affinity to respiratory epithelium. The exact life
cycle of the organism and the specific reservoir is also
unknown. The organism is found in three forms, i.e. the
trophozoites, cysts and sporozoites. The trophozoites are
converted into cysts which ruptures to release sporozoites
that give rise to trophozoites. The cycle continues as:
Trophozoites → Cyst → Sporozoites (Figure 7.7).
The trophozoites multiply by binary fission. There is an
immature percyst stage that is followed by fully developed
cyst. A mature cyst contains eight daughter cells or
sporozoites. Some of the sporozoites will be converted to
trophozoites, other will be released in the lung tissue.
Figure 7.7: Development of Pneumocystis carinii

The commonest disease caused by P. carinii is pneumonia

in immunocompromised persons, prematures and neo-
nates. About 80-85% persons with AIDS develop P. carinii
pneumonia.
Ocular Involvement
The only known ocular involvement is choroiditis which is

due to hematogenous spread from respiratory infection.
Diagnosis is confirmed by demonstration of tropozoites
in sputum or bronchoalveolar lavaged fluid. The histo-
pathological sections are stained by Gomori’s methanamine
silver stain, toludine blue, PAS and calcoflour white.
The organism is not stained by hemotoxyline-eosin
stain. The sporozoites are stained by Wrights and Giemsa
stain.
Immunofluorescence using monoclonal antibodies are
very sensitive tests so is PCR.
Treatment: There is no specific treatment. Trimethoprin,
sulphamethoxazole and pentamidine are effective.
Recurrence is common.
Metazoa
Phylum metazoan contains all multicellular animals
including helminths.
The helminths are:
1. Multicellular.
2. Bilaterally symmetric.
3. Have three germ layers.
The helminths that cause diseases in humans are:
1. Nematodes (Roundworm)
2. Cestodes (Flatworms)
3. Trematodes
Only nematodes and cestodes cause ocular lesions.
Nematodes
The nematodes are classified in two groups:

1. Intestinal nematodes
2. Tissue (somatic) nematodes
Intestinal nematodes involve the human eyes less
frequently than tissue nematodes.
Some characteristics of nematodes are:
1. They are nonsegmented cylindrical in shape. The body
tapers at both the ends.
2. They have a tough hyaline cuticle (skin) which is shed
three to four times in life.
3. They have well-developed digestive system.
4. They have body cavity.
5. Sexes are separate and have developed reproductive
organs. The females are larger than the males.
6. The females may be viviparous, oviparous or vivi
oviparous.
7. They possess rudimentary nervous system.
8. They are symmetrical in structure.
9. They do not have separate locomotive organ. They
move by muscular contraction.
10. Man is the optimum host.
11. Most of the nematodes except the filarioidea and
dracanculoidea pass their life in single host.
12. In filariea, the second host is an insect vector in which
the larvae develop.
Mode of Infection by Nematodes

1. Ingestion: This is the commonest mode of infection.
It can follow:
i. Ingestion of embryonated eggs in food or drink or
ii. An intermediate host that harbors growing embryo, is
swallowed, i.e. cyclope which when swallowed in water
cause dracunlosis. Encysted cyst of T. spiralis is ingested
with pork.
2. Penetration of skin, i.e. hookworm infection.
3. Bite of blood-sucking vectors.
4. Inhalation of dust particles containing embryonated eggs,
i.e. threadworm.
The intestinal nematodes that cause ocular diseases are:
i. Toxocara
ii. Thelazia
iii. Bayliascaris
Toxocara
This is the most commonly reported nematode to cause

ocular diseases. The nematode toxocara is an ascarid of
dogs and cats respectively and are known T. canis and T.
catis. The role of T. catis as a pathogen in humans has been
questioned. The T. canis produces a zoonotic disease in
humans mostly in children. Systemic involvement has
world-wide distribution and ocular involvements are
common.
The cats and dogs are the definitive hosts. Humans are not
the natural host. They may be considered as accidental hosts
in whom the nematode develops to third stage larvae but
does not lay eggs.
The organism has two types of life cycle, i.e. in dogs and in
humans. In dogs, the stage differ in adult dogs/bitches and
puppies.
The adult dogs get the infection in two ways:
1. Ingestion of ova in contaminated food
2. Ingestion of larva in infected meat.
In adult dogs the ova develops in third stage larvae in the
intestine. The third stage larvae are liberated in intestine
and penetrate intestinal musculature to reach the circulation.
After some months the larvae get encysted and lie dormant
without developing further.
In pregnant bitches the picture is different. The encysted
larvae are activated to migrate through the placenta to the
developing puppy. By the time the puppies are borne, they
already have third stage larvae. The larvae pass into the gut
and pass the ova in feces.
The life cycle in humans is similar to those seen in adult
dogs. The third stage larvae in the gut penetrate the mucosa
of the intestine and are carried to distant organs by blood,
i.e. eyes, liver, lungs, brain and subcutaneous tissue. The
organism encysts in the tissue and forms a granuloma which
is the main pathological feature of the disease.
The nematode larva may wander aimlessly in various
tissues causing a condition known as larval migrans that could
be cutaneous larval migrans or visceral larval migrans.
Nematodes other than toxocara also produce cutaneous
larval migrans. Toxocara generally causes visceral larval
migrans. The term ocular larval migrans is used to denote
ocular involvement.
The other causes of cutaneous larval migrans are:
1. Human ankylostoma
2. Nonhuman ankylostoma (hookworm infection)

3. Strongyloides infection
4. Anisakis species
5. Ganthostoma
6. Creeping larvae of some flies (cutaneous myiasis)
The organisms that produce visceral larval migrans (VML)
are:
1. Toxocara catis and T. canis
2. Ascaris lumbricoides
3. Strongyloid stercoralis
4. Ganthostoma
5. Anisakis
6. Angiostrongylus cantonensis
7. Trichinella spiralis
The organisms that cause ophthalmic larval migrans (OLM)
are:
1. Baylisascaris
2. Toxocara
3. Ankylostoma canninum
4. Strogyloidis
Diagnosis
Systemic Toxocariasis is difficult to diagnosis. The diagnosis
is based on exclusion and high index of suspicion. Ocular
Toxocariasis and visceral larval migrans do not co-exist.
Ophthalmic larval margins (OLM) is seen in older children,
they may have history of visceral larval migrans (VML) few
years ago. These children have eosinophilia due to lack of
active systemic infection. The diagnosis is confirmatory.
ELISA for toxocara antigen a serum titre of 1:32 is suggestive
of VLM while a titre of 1:8 is suggestive of OLM Antibody
titre is higher in aqueous than in serum.
Ocular toxocariasis should be differentiated from other

causes of white reflex in papillary area by indirect
ophthalmoscopy, ultrasonography, X-ray, CT and MRI. Stool
examination is negative.
Treatment
Systemic toxocariasis is treated by oral albandazole,
thiabendazole.
Thelazia
Thelazia is a small, thin nematode, life cycle and mode of
transmission is not well established. The two common species
that have ocular manifestations are Thelazia californiensis and
Thelazia callipaeda. The adultworm usually resides in the
conjunctival sac, lacrimal sac and lacrimal duct of many
animals and birds. The definite hosts are suspected to be dogs,
cats, horses. The intermediate host is fly of genera mucosa
and fannia. Human beings are accidental hosts. The probable
mode of transmission is by flies which lay the eggs in the
conjunctival sac where they mature to become adultworms
and can be seen moving in the conjunctival sac or in the
subconjunctival space. They may penetrate the cornea or
sclera and become intraocular. The intraocular parasite may
be seen freely moving in the aqueous and mistaken for other
worms. They must be differentiated from Dirofilaria,
Burgiamalayi and Wuchereria bancrofti. Death of the worm
causes severe allergic reaction, hence it is better to remove
the live worm from AC than to use antihelmenthic drugs.
Trichinella
Trichinella is a intestinal nematode of pigs and many other
domestic and wild animals found all over the world. It is an
ascarid. It has six species, out of which only T. spiralis is known

to cause ocular pathology. It is one of the smaller nematodes
to cause disease in humans. It exists in two forms, i.e. adult
and larva. Sexes are separate. The male is smaller than the
female. The females are viviparous.
The disease caused by the nematode is known by many
names, i.e. Trichinosis, Trichinellosis and Trichimiasis.
The disease is acquired by ingesting under cooked
contaminated pork or beef. The animal host sheds the adultworm
and larvae in the feces in acute phase. The nematode has a
short stay in the gut. The larvae and adultworms are found
in the droppings of the pig for a short period.
The pigs are the definitive host and human beings are the
accidental host. Both the adults and larvae are found in the
same host. The infective stage is encysted larva in pork.
The encysted larvae are ingested by humans in under
cooked pork. The cyst wall is digested in the small intestine
within 48 hours of ingesting, liberating adultworms. The
male fertilizes the female and dies. The fertilized female
burrows in the intestinal wall and deposits the larvae which
are carried to distant parts by systemic blood circulation,
mostly the striated muscles where encystment takes place.
This is the dead end of the cycle. The female dies in about
four months. The encysted larvae may live in the muscles
for many years without symptoms. The larvae during their
journey may cause visceral larval migrans, Myocarditis,
Pneumonitis and Encephalitis.
Ocular involvements: The nematode invades the orbicularis
as well as extraocular muscles and cause periorbital edema. It
can cause proptosis. In rare instances, it causes retinal
hemorrhage and optic neuritis.
1. Examination of stool is of limited value for a short period

following ingestion of undercooked pork.
2. Eosinophilia is present in about 80-90% of cases.
3. Serological tests are most useful laboratory investigation.
They are:
i. ELISA for T. spiralis
ii. Complement fixation test
iii. Indirect hemagglutination
iv. Immunofluorescence test
4. The most confirmatory investigation is demonstration of
encysted larvae in striated muscle by histopathology.
5. X-ray may reveal calcified encysted larva.
6. CT, MRI and USG may be helpful.
Treatment
Presently available antihelmenthic drugs are useful only

during the intestinal phase of the nematode. The commonly
used drugs are thiabendazole and mebendazole given orally.
The drugs are not effective against the encysted form.
Baylis ascaris
Baylis ascaris procyonis is a roundworm of raccoon, a wild

animal that has a tendency to come and live near the human
habitats. Sometimes, they are kept as pets. The humans are
the accidental hosts. The humans acquire the disease by
ingestion of food contaminated by eggs of the nematode.
About 75% of raccoons are known to harbor the nematode
and shed the eggs in feces.
The nematodes reach the eyes and the brain by blood-
stream. They cause visceral larval migrans (VLM) and ocular
larval migrans (OLM). In the eyes, the live organism can be

seen moving in the retina causing retinitis, diffuse unilateral
subacute neuroretinitis (DUSN), optic neuritis and retinal
vasculitis.
Presence of motile nematode in the retina confirms the
diagnosis which is otherwise difficult to establish. The motile
nematode is best visualized by Goldman fundus contact lens.
Other tests available are serology for antibodies to
B. procyonis and ELISA for it.
Antinelminintics have limited success in management.
The best management is destruction of the nematode by
laser.
Tissue Nematodes
The tissue nematodes that have ocular involvement are:

i. Onchocerca volvulus
ii. Loa-Loa
iii. Dirofilaria
Onchocerca
Onchocerca volvulus belongs to phylum nemathelminthes

nematode class, family filariodea, all members of this order
are Ovo viviparous. The exact reservoir of the worm is not
known but some cattles and antilopes are thought to be so.
The organism is found all over the world. It has local
distribution in tropical Africa, tropical South America and
Arabian peninsula in these areas the disease is endemic. It is
estimated that about 3 million people are infected by the
disease called Onchocerciasis on onchocercosis and river
blindness. One fifth of persons suffering from the disease
have ocular involvements. Onchocerciasis is second most

common cause of infective blindness. It is one of the major
causes of blindness in the world. The blindness is mostly
preventable by chemoprophylaxis, vector control and change
in life style. Due to magnitude of blindness caused by
onchocerca, it is also called blinding filarial. The blindness
caused is called river blindness because it is seen only in the
viscinity of rapid flowing clear water bodies.
Humans are the definitive hosts. The black fly (buffalo gnat)
Simulium damnosum is the intermediate host.
The life cycle comprises of following stages:
1. Adults
2. Microfilariae (embryo)
3. Larvae—three stages
Out of them adults, and microfilarial are found in
humans. The larvae are found in intermediate host. The
black fly bites in the day. Bites anywhere in the body can
cause the disease. Bites near the eyes are more common and
more frequently associated with ocular involvement.
The adultworm have separate sexes. The females are
larger and live longer than the males. A gravid female may
live as long as 18 years. Both males and females are generally
found in subcutaneous tissue, nodules, in channels, formed
by lymph vessels. The worms generally gather in knots.
The worm is Ovo viviparous. A gravid female produces
thousands of microfilariae in about one year.
The microfilariae are dermotrophic. They are found
mostly in skin, eyes and lymph glands. They are rarely seen
in liver or spleen, most probably spread via lymphatics. The
inguinal lymph nodes are most commonly involved nodes.
The microfilariae are rarely found in blood, sputum or urine.
They are not effected by light. They are nonsheathed.
LARVAE
When the fly bites an infected warm blood host it sucks the
microfilariae. The microfilariae stay in the gut of the fly for
about 20 hours after that they enter the thorax from where
they go to the mouth parts of the fly and await a chance to
be deposited in the definitive host. An infected fly may have
200-250 third stage larvae in it.
The female fly S. damnosum lays eggs on the rocks or plants
in running, rapid flowing well oxygenated clear water where
they are converted to puppa, cocoon and adult fly in
succession in two to three weeks. An adult fly has lifespan
of about four weeks during which it feeds on warm blood
four to five times. Only the female flies cause and spread
the disease.
Life Cycle of Onchocerca volvulus (Figure 7.8)
1. The adult female worms lays about 2000 microfilariae in

the subcutaneous tissue.
2. The microfilariae are picked up by the black fly during a
daytime bite.
3. The microfilariae undergoes Ecdysis in the thoracic
muscles.
4. They are converted into third stage larvae which are
infective.
5. The infective larvae migrate to the mouth parts of the fly.
6. The infective stage of larvae are deposited in humans by
bite of the black fly.
7. The larvae moult twice in the subcutaneous tissue and
develop into adultworm in about one year.
8. The average lifespan of an adultworm is eight to nine
years. A gravid female may live upto 18 years. As the
worm lives long, multiple infections are common. The

adultworm does not cause much reaction. The patho-
genesis is entirely due to antigen of microfilariae. The
incubation period is many months.
Figure 7.8: Life cycle of Onchocerca volvulus
Diagnosis
Diagnosis is simple in endemic area due to typical

presentation. The diagnosis is confirmed by laboratory tests.
1. Demonstration of microfilariae
i. In the anterior chamber on slit-lamp examination.
ii. In skin biopsy under dissecting microscope that shows
nonsheathed microfilariae.
iii. Adultworms are visible in histological slides of biopsied
subcutaneous nodule.
2. Provocative test: A single does of 50 mg of diethyl

carbamazine is administered orally. If the microfilariae
are present, this causes intense itching in 24 hours. It is
not a very reliable test. The reaction is called Mazzoti
reaction.
3. Serological tests:
i. Compliment fixation test
ii. Immunofluorescent antigen test
4. Blood tests:
i. Microfilariae are absent in peripheral smear
ii. High eosinophilia is very common.
Management
The disease is preventable.

The management consists of:
1. Vector control: Keeping the body covered as far as possible,
avoiding peak bite time and use of repellent.
2. Chemoprophylaxis by ivermectin
3. Chemotherapy ivermectin, diethyl carbamazine orally.
4. Nodulectomy
Loa Loa
Filaria loa causes Loasis in human beings. This is endemic in

equatorial Africa. About 15 million people are infected by the
disease. Those persons who are diagnosed to have loases
have definite history of stay in the endemic area and
contracted the disease. The disease is neither life-threatening
nor blinding. It involves mostly subcutaneous tissue and
conjunctiva. The cornea and intraocular structures are not
involved. As it is confined to a part of Africa with prominent
ocular features, it is also called African eye worm.
The monkeys are thought to be its reservoir. The organism

requires two hosts. Humans are the definitive host. The
chrysops fly or mangrove fly or deer fly are the intermediate
hosts. The fly is diurnal, i.e. it bites only in day light but
prefers shade. All flies of this genus do not contain the worm
about 50% of flies cause infection. The larval form in the
intermediate host is infective.
The worm is found in three forms, i.e. adults, micro-
filariae and larvae.
The adult filariae live in the subcutaneous tissue,
lymphatics and subconjunctival tissues in humans. They move
freely in these spaces. The sexes are separate. The female is
larger than the male. The female is viviparous.
The microfilariae are seen in the peripheral blood during
daytime. They are enveloped and sheathed.
The larvae are seen in the intermediate host. The micro-
filariae are sucked by the chrysops fly during blood meal on
an infected person and pass through the developmental
stages in the thorax of the fly to be converted into larva. The
larvae are the infective forms which are transmitted to humans
following bite of the infected female fly.
The larvae develop into adults in the subcutaneous tissue
and take about six to twelve months to mature and mate the
gravid female, gives birth to microfilariae. The microfilariae
are diurnal with peak periodicity in the mid day.
Diagnosis
Diagnosis is easy in endemic areas. The patient have typical

congested conjunctiva under which live worm can be seen to move.
They also have shifting subcutaneous swelling, i.e. the
Calabar swelling in the body. Eosinophilia is the rule.
The laboratory diagnosis consist of:

1. Demonstration of:
i. Microfilariae in peripheral smear between 11 am and
1 pm.
ii. Extraction of worm from the subconjunctival space.
2. Serological tests are not conclusive.
Simultaneous infection by onchocerca volvulus is a common
combination.
Management
1. Vector control.
2. Use of fly repellents.
3. Covering the body as far as possible.
4. Chemotherapy by diethyl carbamazine which is
filariaecidal as well as larvaecidal.
5. The subconjunctival worm is removed under local
anesthesia by snipping the conjunctiva over the worm.
Wuchereria bancrofti
Wuchereria bancrofti is the commonest filaria to infect humans

mostly in south east Asia, far east, Africa, south and central
America. The disease has far reaching systemic manifestation
with only few ocular involvements. The humans are the definitive
hosts and the mosquitos are the intermediate host. Out of culex,
aedes and anoheles mosquitoes, the first is the commonest
mosquito to cause Wuchereriasis or filariasis. It is the female
mosquitoes that is the vector (Figures 7.9A and B).
The organism is found in three forms, i.e. adults, micro-
filariae and larvae (Figure 7.10).
A B
Figures 7.9A and B: Histopathological section showing Wuchereria
bancrofti in high and low power. (Courtesy: Prof V Sudarshan)
Figure 7.10: Life cycle of filaria
The mosquito ingests the microfilaria during blood meal.

The sheath of the microfilaria is shed off in the stomach of
the mosquito and it penetrates the stomach wall and
migrates to the thoracic muscles where they moult to form
first, second and third stage larva. The third stage larva is
the infective form. Each microfilaria gives rise to one larva.
It takes about two weeks for the larva to change from first to
third stage.
The adultworms of both the sexes live in the lymphatics

coiled together in small nodules. The males are smaller than
the females. The average life span is 4 to 5 years.
When the infected mosquito bites the human beings, it
injects the filiform larvae in the blood stream. The larvae
reach the lymphatics and lymph nodes to develop into
adultworms. The adult male fertilizes the female to produce
larvae.
Ocular Involvements
1. They are few.

2. Microfilaria has been reported in AC.
3. Iridocyclitis (commonest).
The microfilaria are mostly nocturnal in peripheral blood
smear.
The diagnosis is not difficult in endemic areas. It is
confirmed by:
1. Demonstration of microfilaria in peripheral smear.
2. Adultworm can be seen in biopsied lymph node in
histopathological sections.
3. Eosinophilia is suggestive of the disease.
4. Immunological tests are:
i. Fluorescent antibody test.
ii. ELISA test.
iii. Skin test.
Management
1. Vector control.
2. Protection from mosquito bite by use of mosquito nets,
use of mosquito repellents.
Treatment
1. Diethylcarbamazine is effective against adults as well as

microfilaria.
2. Ivermectin kills microfilaria but not the adults.
Dirofilaria
Dirofilaria is a nematode that belongs to sub-family Dirofilariinae

and genus Dirofilaria, is a nematode of dogs and other small
animals, i.e. cats, racoon, etc. The worm has localized
distribution in countries bordering Pacific Ocean. It has been
rarely reported from other countries. The animals are the
definitive hosts, humans are accidental hosts. The humans are
the dead end in the life cycle of the dirofilaria. The mosquito is
the vector. Human infection is relatively rare. Most of the human
infections are asymptomatic, when symptomatic it cause
pulmonary lesions. The other lesions are subcutaneous nodules
in the lid and round the eyes (Figures 7.11A and B ).
A B
Figures 7.11A and B: Dirofilaria—histopathological section showing
parasite in tissue and parasite seen in high power magnification.
(Courtesy: Reproduced with permission of editor IJO)
There are no laboratory tests available for diagnosis.

Presence of worm in the biopsy of the nodule is confirmatory.
Dracunculus medinensis (Guinea worm)
This is the largest thread worm. The female measures up to

1 meter, the male is about one-fourth of the size of female.
The infection is found in many parts of the tropical and sub-
tropical countries. The humans are the definitive hosts while
cyclops are the intermediate hosts.
The larvae are the infective agent. Digestive tract is the portal
of entry in human beings. The disease is acquired by
drinking water, frequented by cyclops. The cyclops thus
ingested are destroyed by gastric juice, releasing larvae
which travel to retroperitoneal tissue where they mature and
mate, the male dies after fertilizing. The female is viviparous,
it releases the embryo outside the human body. The sites
selected to release the embryos are the skin surfaces that come
in contact with water for long time like backs of water carriers,
hands and feet of washer men. The female first secretes a
fluid under the skin, this fluid is toxic in nature and causes
a blister at the site. The blister bursts to form an ulcer. The
female protrudes through the base of the ulcer releasing large
number of coiled embryos. As the person comes in contact
with water, the embryos get mingled with water to be
swallowed by the cyclops present in the water.
The ocular involvements are few and uncommon. The eyes
are directly not involved. The worm is lodged under the
skin or in the orbit causing embryo discharging sinuses or
proptosis. The dead worm may be calcified under the skin
or in the retrobulbar space.
Diagnosis is simple in endemic areas where patient gives

history of presence of worm.
Management
Drinking filtered water in endemic areas reduces chances

of infection. Straining drinking water through fine cloth is
sufficient to separate the cyclop from water. There is no
definite anti-dracunculus chemotherapy, however metro-
nidazole 250 mg TDS for seven days, thiabendazole 25 mg/kg
BD for two days, have been found to be effective. Hetrazan
is also used for cure and prophylaxis. The most widely used
method is to remove the worm surgically.
Gnathostoma spinigerum
The nematode is found in eastern and far eastern Asian

countries. It is a zoonotic disease. Humans are the parasitic
hosts, i.e. they keep the parasite viable in their body without
further development. The feline group of animals are the
definitive host. It has two intermediate hosts, i.e. cyclop fish
or snail. Men acquire the disease by swallowing cyclop
with drinking water or undercooked fish. It causes visceral
larval migrans and cutaneous larval migrans in humans
(Figure 7.12).
The ocular involvements are rare and few. Only known
ocular involvement is orbital cellulites and cutaneous larval
migrans of the lids and conjunctiva.
The Cestodes
The cestodes are helminthes also known as tapeworms

because they are flat and ribbon like. The cestodes have
segmented body.
Figure 7.12: Third stage larva of Gnathostoma spinigerum.

(Courtesy: Reproduced with permission of editor IJO)
They are broadly divided into two groups:
1. Intestinal cestodes.
2. Tissue cestodes.
General characteristics of the cestodes are the same in
both the groups except that the adultworms of intestinal
cestodes are found in gut. The larval form of the tissue
cestodes are found in various tissues of humans.
Some of the characteristics of the cestodes are:
1. Dorsoventrally flattened.
2. They are ribbon like.
3. They are segmented.
4. They are of various length ranging from few centimeters
to 15 meters.
5. The cestodes have three parts:
i. Scolex (head)
ii. Neck
iii. Strobila (body).
The scolex is the organ of attachment, it may have
suckers, grooves or hooks to attach to the tissue.
The neck is very short. It is the germinal center from
which the segments (proglottid) are formed. The
newly formed segments are proximal. They push the

mature segments distally.
The strobila is composed of variable number of
segments. The oldest segment is at the tail end of the
worm.
According to sexual maturity the proglottid are:
a. Immature: The reproductive organs are not developed.
b. Mature: Both the sexes are well-developed
c. Gravid: The uterus is full of eggs.
6. The body wall is well developed and three layered.
7. There is no body cavity.
8. The reproductive system is well developed. A mature
proglottid contains fully developed male and female.
9. The cestodes are hermaphrodites.
10. The fertilization may be:
i. Self-fertilization or
ii. Cross-fertilization.
11. The excretory system and nervous systems are fairly
well-developed.
12. The cestodes have no alimentary canal.
The cestodes that have ocular involvement are:
Cystode Disease
1. Taenia solium - Cysticercosis
2. Taenia echinococcus - Hydatid disease
3. Taenia multiceps - Coenurosis disease
Cysticercus
Out of the three infection caused by the a cestodes the

cysticercosis is most frequent cestode infection of the eyes,
this is caused by tapeworm Taenia solium, the tapeworm of
pigs. It is common belief that cysticercosis is a disease of pork
eaters only. On the contrary, it is also seen in persons who

do not eat pork. They acquire the disease by consuming
green vegetables contaminated by human feces in the form
of gravid proglottid (Figure 7.13).
Figure 7.13: Life cycle of T. solium
Humans are the definitive host harboring adultworms. The

pigs are the intermediate host, however humans may become
accidental intermediate host, when the worm reaches the dead
end in human tissue as cysticercus cellulosae.
The other ways of which the eggs of T. solium reach the
human gastrointestinal tract are:
1. Autoinfection by unhygienic condition.
2. Reverse peristatis in the intestine.
The worm T. solium exists in two forms:
1. The adultworm that live in human intestine.
2. Larval form that lives in the tissue of pigs and human

beings.
The adultworm may be as large as three meters with a small,
globular scolex. The neck is short. An adultworm has about
1000 proglottids. The sexes are not separated. Each proglottid
has both male and female reproductive systems in them. The
fertilization takes place between two segments, the process is
called self-fertilization or there may be cross-fertilization, i.e.
fertilization takes place between the two adultworms.
Generally, only one worm is present in the intestinal tract. The
adultworm lives up to twenty five years in the intestine.
The larva is called Cysticercus celluosae. It mostly develop
in tissue of pigs. It can also develop in humans. The larva is
an oval cyst containing fluid. The scolex is intracystic with a
ring of hooks and suckers. The lifespan of the larva is short,
it lives for about 8 months in the tissue and then dies. The
dead cysticercus gets calcified (Figure 7.14).
Figure 7.14: Cysticercus

The human beings are infected following ingestion of larva,

i.e. Cysticercus cellulosae. The scolex is released in the intestine
to be attached to the intestinal wall. Progressive strobilization,
i.e. formation of proglottids leads to development of
adultworm. The tapeworm matures in two to three months
following ingestion of the larva by humans. The proglottid
becomes gravid following fertilization. The gravid proglottids
get detached and pass in the feces. The gravid segment
contains eggs. The eggs survive for months in the soil.
The life cycle in pig consists of ingestion of eggs in human
faeces. In the pigs intestine the embryos are released which
penetrate the musculature of the pigs intestine and travel to
muscles. The embryo is converted to cysticercus in about
two months. The life cycle is completed in humans who
consume under cooked pork with cysticercus.
Human Cysticercosis
The adultworms in the intestine are mostly asymptomatic,

however sometimes they may cause pain in the abdomen
and irregularity of bowel.
The larval form causes many types of systemic
cysticercosis which include nodules under the skin and in
the muscles.
The two serious types of human cysticercosis are neuro-
cysticercosis and ocular cysticercosis (Figures 7.15 and 7.16).
The laboratory tests to diagnose consist of:
1. Examination of stool for:
i. Gravid proglottid
ii. Eggs in stool.
The test is useful only in intestinal cysticercosis.
Figure 7.15: Cysticercus removed from subconjunctiva
Figure 7.16: Histological section of Cysticercus cellulosae.

2. Eosinophilia is seen in early stages. This is not

confirmatory.
3. X-ray skull or limbs may show calcified cysts.
4. CT and MRI of skull, orbit and eyeball.
5. Biopsy of subcutaneous or subconjunctival cysticercosis.
6. Fine needle aspiration cytology has limited value only in
subcutaneous cysticercosis.
7. Immunoblotting and ELISA are helpful in diagnosis.
Treatment
Albandazole is the drug of choice for all forms of cysticercosis

except ocular cysticercosis where the best treatment is
surgical removal.
Echinococcus (Dog Tapeworm)
This is one of the smaller cestodes that produce large larvae. It is

prevalent world over. Most commonly seen in persons
involved in sheep raising where there is a close relationship
among men, sheep and dogs. The dog is the definitive host. The
sheep, goats, cattle and human beings are the intermediate
hosts. The adultworm is found in the intestine of dogs and
man harbours the larval form. The life cycle of echinococcus
come to a dead end in human beings. Out of four species of
echinococci only E. granulosus has ocular involvement and
is reported for most of the hydatid diseases (Figure 7.17).
Figure 7.17: Hydatid cyst
E. granulosus is seen in two forms, i.e. adult and larva.

The adultworm is small in size and found in the intestine
of dogs (or other canine). It’s lifespan is also short, i.e.
6 months to 20 months. The adultworm has a head (scolex), a

neck and strobila (proglottids). Adult generally has three
proglottids and rarely there may a fourth. The proglottid
nearest the neck is immature. The next is mature, the third
is gravid. The gravid proglottid is largest of the three.
There is similarity in morphology of eggs of all
tapeworms. The egg contains a Hexacanth embryo with
hooks. The eggs are the infective form for intermediate hosts.
The larva is the hydatid cyst in the larval form of the worm.
It develops in the tissues of the intermediate host. The liver
is the commonest site (60-70%) where the cysts develop. The
cysts once formed slowly increase in size. It is estimated that
the growth rate is between 1 to 5 cm/year. The cysts are
known to last for years without symptoms if they are not
causing sufficient pressure over organs. This gives the
distinction of producing largest larva (hydatid cyst) by any
tapeworm. The cyst is double walled. The outer is called
ectocyst. It is white in color, thin and homogeneous, hyaline
in nature without nucleus. This is lined by endocyst which is
thinner but made up of nucleated cells. This is the germinal
layer. Its function is to produce the fluid of the cyst, the brood
capsule and the ectocyst. The hydatid fluid is toxic and
causes severe antigen antibody reaction when leaked. On a
longrun, the body grows a fibrous coat round the hydatid
cyst, this is called the pericyst. The peri cyst may get calcified,
cutting off the nutrition to the parasite and killing it.
Life Cycle
The life cycle is in two hosts, i.e. the definitive hosts are dogs
and other canines and the intermediate hosts are sheep, goat,
cattle or other herbivorous animals. The humans are accidental
intermediate hosts in whom the parasite reaches a dead end.
The eggs of Echincocci are the infective stage for the human
beings.
Human beings acquire the infective form from the dogs
either as in contaminated food or drink or through
contaminated hands that have been fondling infected dogs.
The ingested eggs release the embryo in the duodenum
of humans. The embryos penetrate the intestinal mucosa to
reach the portal circulation and reach the liver. Most of the
embryos are filtered in the liver. The next organs where the
embryos settle are the lungs. Other organs are also seeded
by the embryos. The embryos are converted to hydatid cyst
in these organs. It takes few months to years for the cyst to
develop in these organs.
Ocular Involvement
Only one percent of persons with Echinococcosis develop ocular

involvements.
The commonest site being the orbit causing gradual
unilateral proptosis. The cyst may erode the orbital roof to
invade the brain. Other ocular sites to develop the cyst are
subretinal space, vitreous and anterior chamber.
The sheep are infected by consuming grass contaminated
with dog feces. The progress in the sheep and humans is
identical. The sheep may get hydatid cyst in any organ.
The dogs are infected by eating the infected tissue of the
herbivorous animals.
In the dog, the scoleces are released from the consumed
hydatid cysts, in the small intestine. The scolex gets
evaginated to get attached to mucosa of the intestine and
storbilize to form adultworm in two months. The gravid
segment ruptures to release eggs in the intestine which are
expelled in feces to contaminate human food, water, hands

or vegetables to be consumed by sheep, etc. Thus, the life
cycle continues.
Diagnosis
The diagnosis is mostly presumptive where the cyst may be

visualised by chance on an X-ray, ultrasonography or CT taken
for other purpose.
The human stool does not contain ova or cyst.
The serological tests used are ELISA, indirect hemag-
glutination, fluorescent antibody test and immuno-
electrophoresis test.
The casonis test is a skin test for type I hypersensitivity. It
is positive only in 60% cases.
Management
Surgical removal of the cyst in toto is the treatment of choice

when the cyst is accessible. Otherwise medical treatment is
given a try, this consists of oral administration of large dose
of albendazole for long period with drug free gaps in between
the two courses. The usual regimen is 400 mg BD for 28 days
repeated four times with a gap of two weeks in between.
Taenia multiceps (Coenurus)
This is yet another dog tapeworm, less prevalent than either

cysticercus or Echinococcus. The larval form resemble both
cysticercus and hydatid cyst to some extent. It differs from
cysticercosis because it contains multiple scoleces. The
cysticercus has only one scolex. It does not contain brood
capsule or daughter cysts like hydatid cysts. It is longer than
both cysticercus or Echinococcus. The larval form is Coenurus.
The dogs or other canines are the definitive hosts, the sheep
and other herbivores are intermediate hosts. Humans beings
are accidental intermediate hosts.
Life cycle resemble that of cysticercus. The intermediate host
acquires the disease by feeding on grass contaminated by
feces of dogs that contains the infective eggs. Human beings
get infected accidentally.
The Ovo onchospores hatch in the gut of the sheep and
penetrate the gut wall to reach systemic circulation that
carries them to distant organs including the eyes. The
common sites are brain and the spinal cord where they
develop into coenurus or the bladder that has multiple
scoleces. The carnivore which is definitive host gets the
infection by eating dead herbivore with coenurus. The
systemic presentation of coenurosis is that of space
occupying lesion of the brain or the spinal cord.
The ocular manifestations are similar to cysticercosis but
less frequent. They include subconjunctival cysts, cyst in AC
or vitreous, and orbit. There may be associated eosinophilia.
Brain scan shows intracranial cyst. CT, MRI and ocular ultra-
sonography helps to locate intraorbital and intraocular
coenurus. The dead coenurus causes uveitis.
There is no specific treatment. Praziquantel 50 mg/kg/
day in divided dose for a fortnight may be tried. Surgical
removal when possible is the best option.
Ocular Infection Caused by Arthropods
Arthropods belong to phylum arthropoda. They are small

animals generally referred to as insects though all arthropods
do not belong to class insecta. They have segmented bodies which
are bilaterally symmetrical. They have joined appendages. Many
are winged. The number of legs vary between six to ten. When
winged they may have single pair of wings or may have two
wings on each side. Most of them have antennae.
Classification of Arthropods
The arthropods can be divided into three classes.

i. Insecta
ii. Arachnida
iii. Crustacea
Modes of Transmission by Arthropods
1. Mechanical transmission:
Trachoma, infective conjunctivitis by domestic flies, the
vectors involved is not a part of life cycle of the parasite.
2. Biological transmission:
The vector is an integral part of life cycle of the parasite.
i. Only multiplication in vector, i.e. plague (no ocular
involvement)
ii. Development in vector—Wuchereria in mosquito
iii. Multiplication and development in vector
iv. Depositing eggs in the conjunctiva or wound, i.e.
myiasis.
INTRODUCTION
Many of the microbial ocular infections have clinical features

sufficient to clinch the diagnosis. However sometimes
identical clinical features are presented by different organisms
which require different antimicrobial treatment, i.e. in early
stages of Ophthalmia neonatorum it is difficult to say if the
causative organism is gonococcus or the infection is non-
gonococcal especially when the disease is nonbacterial.
Similarly, there are many causes of membrane formation of
conjunctiva but isolation of Diphtheria bacilli requires urgent
management by physician trained in infectious diseases. It is
very common for a bacterial corneal ulcer to be confused as
fungal ulcer and vice a versa. Presence of multiple organisms
sensitive to different antibiotics is yet another problem.
Resistant strains are becoming more frequent, making it
difficult to eradicate the disease.
The above factors make it essential to isolate and identify
the causative organism and its sensitivity to antimicrobials.
The procedures employed are:
1. Collection of specimen
2. Preparation of slide
3. Culture of organism in suitable media
4. Biochemical test to identify the organism in pure form
5. Find out the antimicrobial to which it shows maximum
sensitivity.
COLLECTION OF SPECIMENS
The specimens available from ocular infection are very small,

likely to be destroyed early and contaminated by organisms
OUTLINE OF LABORATORY TESTS 177
from other structures not directly involved in the pathology.

The commonest source of contamination is the lid margin.
The commonly used specimens are:
1. Swab from the lids
2. Conjunctival swab
3. Corneal scrapping
4. Aqueous
5. Vitreous
6. Explanted IOL
7. Infected scleral bands, etc.
8. Contact lens fluids
9. Infected corneal button
10. Regurgitated material from canaliculi/sac
11. Discharge from orbital and lacrimal sinuses/fistula.
The specimens should be collected separately from
different sites, i.e. conjunctival swab and corneal scrapping
should not be taken by same swab. There should either be an
arrangement to make the slides in the clinic or its near vicinity
or the specimen should be transported to microbiological
laboratory as soon as possible.
1. Swabs from the lids are collected by:
i. By moving a sterile cotton swab mounted on a sterile
stick kept in an autoclaved, closed test tube.
• The autoclaved swab is moistened by sterile
distilled water.
• The moistened swab is moved along the whole
length of the lid.
• The cotton swab after it has been rubbed against
the lid margin, is quickly replaced in its original
autoclaved container.
• Moistening the swab with calcium alginate removes
the fatty material present in the swab and gives
better visual impression.
ii. The lid margin is pressed sufficiently to cause mild

ectropion just enough to expose the lid margin. A swab
is moved along the lid margin. Squeezing the lid
between the thumb and the index finger exposes the
meibomian secretion which may be included in a
separate swab.
iii. Crusts from the lid margin are loosened by sterilized
blunt needle and picked up by sterilized forceps.
These are dropped straight in the culture fluid.
iv. In case of conjunctival membrane, the lid is everted to
expose the membrane and the swab is taken from the
membrane and adjoining conjunctiva.
2. Conjunctival swab: The lid margins are cleaned, removing
all discharge. The lower lid is pulled down, exposing the
lower fornix and a swab is taken from the fornix. The lower
fornix yield maximum organism. The conjunctival swab
should be collected before the lacrimal sac is pressed. The
conjunctiva need not be anesthetized.
3. Corneal scrapping: Organisms isolated from cornea may not
be the same as those from conjunctiva or lacrimal sac.
The corneal scrapping requires:
i. Staining
ii. Anesthesia.
Corneal scrapping is best done under slit lamp or operating
microscope.
The cornea is first stained by fluorescein and then
anesthetized. A Kimura spatula is the best device to collect
corneal sample. If it is not available, a number 15 BP knife
may be used. Cotton swabs are not recommended.
To scrap the cornea, the lids are separated by the index
finger and thumb of the examiner. The debris and discharges
are removed. The patient is assured that the procedure is

mandatory and it is not painful. The patient is asked to keep
both eyes open and fix at a distant object.
Multiple scrappings are done from the margin and bed of
the ulcer. The material from the margins are examined
separately. To prepare a slide, the scrapping is spread over
clear autoclaved slides for staining and wet preparation. As
the material is very small it is better to demarcate an area of
the slide by a wax pencil and the material is placed in the
center of the demarcated area. The wax pencil is used on the
reverse side of the slide.
Culture of the Material
To culture the material both solid and liquid media are used.
The solid media are inoculated by spreading the material in
shape of letter C, which stands for cornea, at the same time
letters R or L may be streaked away from C to demarcate the
side R for right and L for left.
Traditionally, any growth outside these letter are considered
to be the contaminants. The solid media are streaked lightly
without cutting the surface. In case of liquid media the spatula
containing the material is swirled in the liquid.
Most commonly used stains are Gram’s stain and Giemsa
stains.
Gram’s stain is used for bacteria, fungi and Acanthameba.
Giemsa stain is used for bacteria and Acanthameba. It is
also used for inclusion bodies of Chlamydia.
Less commonly used stains are:
• Ziehl-Neelson for modified kinyouns media
• Gomori’s methenamine silver stain

• Wright’s stain.
Gram’s stain helps to differentiate between gram positive and
gram negative both cocci and bacilli. It also tells if the organism is
intracellular or extracellular. Mycobacteria, Actinomycetes and
Nocardia stains poorly by Gram’s stain. It also stains Acanthamebae.
Gram’s stain has greater importance in selecting antibiotic than
other stain, i.e. some antibiotics are more effective against gram
positive than negative. Hence staining characteristics can
determine initial choice of antibiotic.
Giemsa stain is used to stain all bacteria and fungi. It stains
fungi better than Gram’s stain. Some bacteria, i.e. H. Aegyptius
(Koch-Weeks), H. Influenzae, Neisseria and Mraxella. It given better
cellular details and is used to demonstrate inclusion bodies of
Chlamydal infection. It also stain Acanthameba.
The other stain used are:
Name of the stain Organism
Ziehl-Neelsen AFB
Modified Kinyoun’s stain Atypical mycobacteria
Gomori methanamine Acanthamebae,
stain The cyst wall of Acanthamebae also
stain black with green background.
Fungi, fungi wall and septae.
The septate stain black with green
background.
Albert’s stain Corynebacterium diphtheria.
Neisser stain C. xerosis.
Calcoflour white The slide is examined under fluores-
cence microscope.
It delineates the fungal wall and cyst

wall of Acanthamebae.
It does not stain trophozoites,
nocardia or actinomycosis.
Lactophenol cotton Fungus and Acanthameba.
blue stain
Potassium hydroxide does not stain any organism. The KOH
is used as 10-20% solution in water. The alkali digests the
debris rendering to fungal element prominent. It clearly shows
the fungal filaments and Acanthameba. The slide should be
examined fresh under low intensity. Adding 10% glycerol
makes the slide clearer and the slide can be preserved for four
to six months.
Culture for Microbes
In ocular microbiology, commonly used culture media are:

1. Blood agar: For aerobic and anaerobic culture. The latter is
done by excluding or reducing O2 by various methods, i.e.
creating vacuum, displacing O2 by other gases, i.e. CO2,
absorption of O2 by chemical (Figures 8.1A to C).
A B C
Figures 8.1A to C: Various agar plates.

2. Chocolate agar
3. Nutrient agar
4. Brain-heart infusion broth
5. Thioglycollate broth
6. Sabouraud’s agar
7. Potatodextrose agar
8. Lowenstein-Jensen medium (Figure 8.2).
Figure 8.2: Lowenstein-Jensen medium to culture Mycobacterium.

Most of the organisms grows at 35°C, but brain heart

infusion for aerobic culture, Sabouraud’s media and non-
nutrient agar media are incubated at 25°C. Some may
have to be incubated at 37°C or higher temperature.
Dimorphic fungi may show different forms at different
temperature.
The culture media are examined every day to observe
growth of colonies. Most of the bacteria grow between seven
to fourteen days. Some bacteria may grow even in shorter
period. Early growth sometimes may not be the real organism

causing the disease but contaminant. After which they may
be declared as culture negative, the exceptions are
mycobacteria and fungi. The mycobacteria may take as 4 to 6
weeks to grow. Similarly, the fungi also take more than one
week to show growth. Acanthamebae are grown on non-
nutrient agar with an overlay of Eschericha coli and incubated
for 7 day. The organisms that require more than a week to
grow are:
1. Mycrobacterium – 4 to 6 weeks
2. Fungi – More than 2 weeks
3. Acanthameba – More than a week.
It is not unusual for more than one organism to grow in the
same media.
The two possibilities are:
1. The infection is really multiorganismal.
2. The other organisms are contaminants.
Once the culture shows a positive growth, the organisms are
subcultured to identify individual organism by chemical
examination or enzyme tests. The most important test is coagulase
test for Staphylococci on the basis of their ability to coagulate
blood plasma. Coagulase is an enzyme produced almost
exclusively by S. aureus this test is used to differentiate S. aureus
from sepidermidis. Other enzyme tests are catalase and oxidase.
The Staphylococci are catalase positive. The Streptococci are
catalase negative. Neisseria, Branhamella, Pseudomonas are oxidase
positive. Hemolysis test differentiated hemolytic from non-
hemolytic Streptococci.
Ocular Microbial Evaluation
Choice of Stain in Ocular Microbial Infection

Stain Bacteria Fungus
Gram’s Gram positive/negative May show protoplasm
intracellular/extracellular. False positive
Morphology of the Not recommended
organism shows cellular response.
May show spores of microsporidia.
Highly recommended
Giemsa No specific characteristics Stain protoplasm in some
like positive or negative. but not cell wall better
All bacteria stains deep blue. than Gram’s.
Recommended as ancillary
test with Gram’s stain.
It highlights cellular response
and stains Acanthameba
KOH wet Not used Recommended as first
preparation test for fungal infection.
Methenamine Stains bacteria black. Good response, fungal

silver stain Generally not used wall stain black,
background transparent
green.
Acid-fast Used only for Mycobacteriae Not used
Classification and Characteristics of Cocci of Ocular Importance

Organisms that stain poorly with Gram’s stain

• Mycobacteria
• M. tuberculosis
• M. Leprae
• Treponema
• Leptospira
• Borellia
• Chlamydia
Classification and Characteristics of Bacilli of

Ocular Importance
Staining Characteristics of Bacteria of Ophthalmic Interest
Scheme of Examination of Extraocular Microbial Specimen
Microbiological Examination of Aqueous and Vitreous
Both the samples are collected under utmost aseptic conditions

preferably in operation room under microscope with adequate
anesthesia.
The aqueous tap is obtained by introducing a fine needle

mounted on an autoclaved 1 ml syringe in AC, 1 mm inside
the limbus avoiding blood vessels. The piston of the syringe is
kept at the lowest level. The intraocular pressure pushes the
piston of the syringe back, otherwise gentle pull on the piston
will draw the shaft back. About 0.2 ml aqueous is drawn. The
needle is not detached from the syringe. One drop of aqueous
is dropped in a clean slide for KOH examination and second
drop is used for Gram’s stain. The needle is plunged in a
small autoclaved rubber block to prevent slow flow of fluid
from the needle. A cotton swab is not used as it does not
prevent leak from the needle, moreover the swab may itself
draw the fluid. The syringe, with needle buried in the rubber
stopper is transferred to a larger autoclaved syringe, the mouth
of which is closed by a dry autoclaved cotton plug and about
2 cm of the test tube are secured by an autoclaved aluminium
foil. The whole assembly is transported to microbiological
lab without delay where the liquid is used for smear, wet
preparation and culture (Figure 8.3).
Figure 8.3: Transporting AC fluid
Vitreous is obtained either by a pars plana puncture or the

fluid from the vitrectomy cassettes. The method of transport
is similar to that is employed to transport aqueous. In some
centers, the vitreous is passed through 0.2 µ millipore filter.

The filter is removed aseptically, cut into smaller pieces and
used for direct inoculation for culture.
Drug Resistance
The pathogenic microorganism specially bacteria are known

to develop resistance to antibiotics or may be insensitive to a
particular antibiotic. Though this can happen with any
bacterium, it is more common with Staphylococci and gram-
negative organism both cocci and bacilli.
Gonococci are developing resistance more often than in the
past. Resistant strains of tuberculosis have become of health
hazard. Nosocomial infections are more resistant than others.
There are two types of drug resistance:
1. Primary: Some bacteria have inborn property of
insensitivity to particular drug, i.e. E. coli does not respond
to penicillin.
2. Acquired resistance is genetic, it could either be due to
mutation or gene transfer. Gene transfer may occur either
in the same species or may involve the other species also.
The main pathways of drug resistance are:
1. Change in permeability: This is most likely to be brought
about by chemical change in the outer membrane.
2. Change in target structure: There may be change in ribosome
or the receptors may be altered.
3. Change in metabolic pathway: This is commonly seen in
sulphonamides where preformed folic acid does not allow
use of PABA.
4. Production of enzyme that makes the drug infective. The
commonest example is ability of Staphylococci to produce
beta-lactamase that makes it penicillin resistant.
Test for Drug Resistance
This is used to find out the most useful antibiotic that destroys
the organism in therapeutic dose in expected time. It also
finds out the drugs to which the organism is absolutely
resistance.
There are two commonly used methods. They are:
i. Disk diffusion method
ii. Tube dilution method.
Disk Diffusion Method
Disk diffusion method is most widely used method, it is easy

to perform, cost effective and reasonably accurate.
It consist of using standard disk of filter paper impregnated
with standard strength of antibiotic under investigation. The
disks are 6 mm in diameter. They can be prepared in the
laboratory itself. The more convenient way is to use
commercially available disks. They are single use, disposable.
The unused disks are stored unopened in refrigerator.
To perform the test, solid media are used in plates. There
are three ways to perform the test. The first consists of
spreading liquid media containing the bacteria over the solid
media in the culture plate. Excess of liquid is drained off.
The other method is to streak the plate with cultured
bacteria. The third method is to spread the liquid specimen,
i.e. aqueous or vitreous over the disk directly.
The next step is to place the medicated disks on the already
inoculated media and incubate the plate in the usual method.
More than one disk, each saturated by different antibiotic
can be used in one test.
After twenty four hours, the zone of inhibition in the

bacterial culture is noted and grades as sensitive, partially
sensitive or resistant, 12-15 mm of inhibition zone is
considered to be sensitive.
Tube Dilution Method
This is mostly employed to titrate the therapeutic dose of
antibiotic.
Lacrimal canaliculi, lacrimal sac and lacrimal fistula: The swab
is put against the puncta and gentle pressure is applied over
the sac.
The material from the lid margin, conjunctival sac and
lacrimal apparatus are collected on swabs.
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JS, Savitri Sharma. Current perspectives in infectious keratitis,
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edited by Fraunfelder FT, Roy FH (Eds)., WB Saunders Company,
Philadelphia 2000;15-16.
3. Anantnarayan R, Jayaram Paniker CK. Texbook of Microbiology
(5th edn) Orient Longman Hyderabad 1996.
4. Arseculeratne SN. Recent advances in rhinosporidiosis and
rhinosporidium seebri. Ind Jr of Medical Microbiology 2002;
20(3):118-31.
5. Barkay S, Garzozi H. Ann Oph 1984;16:164-68.
6. Bartley GB. Blastomycosis of the eye lid. Ophthalmology 1995;
102:2020-23.
7. Basak SK, Sinha TK, Bhattacharya D, Hazra TK Parikhs. Intra
vitreal live guanthostoma.
8. Berglodd J Basser, Feigl B. Ophthalmic manifestation in Lyme
borreliosis. Jr Neuro Oph 1994;14:14-20.
9. Bharathi MJ. Aetiological diagnosis of microbial keratitis in South
India. Ind Jr Med Microbio 2002;20:19-24.
10. Bharathi MJ, Ramkrishna R, Vasu S, Meenakshi R, Palaniappan R.
Epidemiological characteristics and laboratory diagnosis of fungal
keratitis. IJO 2003;51:315-21.
In vitreo efficacy of anti bacterials against bacterial isolates from
corneal ulcers. IJO 2002;50:109-14.
12. Bharathi MJ, Ram Krishnan R, Vasu S, R Meenakshi, Palaniappan
R. Epidemiological characteristics and laboratory diagnosis of
fungal Kt Ind Jr Oph 2003;51:315-21.
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1995.
14. Biswas J, Amala J, Raijada S, Kumarasamy N, Suniti Solomon.
Ophthalmic manifestation of HIV in India. Ind Jr Op 1999;47:
87-93.
15. Biswas J, Lingam G, Nirmala Subramaniam, Badrinath SS: Clinico
pathological study of parasitic lesions of the eye. Insight 1993;
11(2):32-39.
16. Biswas J, Madhvan HN, Badrinath SS. Ocular lesions in AIDS.
Ind Jr Op 1995;43:69-72.
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17. Biswas PN, Thakur SKD, Mukherjee H, Bhaduri G. Ophthal-

momyiasis, JIMA 2005;542-43.
18. Blanksma LJ, Slijper J. Actinomycosis Dacryocystitis ophthal-
mologica 1978;176:145-49.
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bacteriology, (5th ed) Williams and Wilkins, Baltimore 1974.
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Book agency, Kolkata, 1955.
21. Chakraborty P. Textbook of Medical Parasitology, (1st ed) New
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14:502-05.
23. Chhatterjee KD. Parasitology, (4th ed), 1962.
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Butterworth, Oxford, 1994.
25. Cruickshank R, Duguid JP, Marmion BP, Swain RHA. Medical
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26. Das JC, Chaudhuriz Bansal RL, Bhomaj S, Sharma P, Chauhan D.
Visco expression of AC cysticercus cellulosae. Ind Jr Op
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27. Das TP, Dogra MR, Gopal L, Jalali S, Kumar A, Malpani A,
Natrajan S, Rajeev B. Postsurgical endophthalmitis–Diagnosis and
management.
28. Davey RT, Tauber WB. Post-traumatic endophthalmitis emerging
role of bacillus cereus infection Rev Inf Dis 1989;9:110-23.
29. David DB, Kirby GR, Noble BA. Bacillus cereus endophthalmitis,
BJO 1994;78:577-80.
30. Ffytche T. Blindness in Leprosy, Aust Nz Jr Oph 1989;17:257-60.
31. Fogra R, Biswas J. Parasitic infections of the lid, Insight 1997;15:15-
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control. A guide for district health worker, Geneva WHO/PBL/93,
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Saunders Co, Philadelphia 2000.
34. Garcia LS. Diagnostic microbiology (4th edn), ASM press,
Washington, 2001.
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unilateral subacute neuroretinitis. Morphometric serology and
epidemiologic support for basliascariasis as causative agent.
Ophthalmology 1993;100:1695-701.
36. Goodman DF, Gottsch JS. Methicillin resistant S. epidermidis
keratitis treated with vancomycin. Arch Oph 1988;106:1570-71.
37. Harley RD. Pediatric ophthalmology, Vol. 2, (2nd edn) WB

Saunders Co. Philadelphia, 1983.
38. Helen CJ, Holland GN. Ocular tuberculosis. Surv Oph 1993;38:229-
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39. Helene B Fedukowitz. External infections of the eye. (2nd ed)
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40. Hunt KE, Glasgo BJ. Aspergillus endophthalmitis an unrecognized
endemic disease. Ophthalmology 1996;44:19-21.
41. Ingram DL. Neisseria gonorrhea in children. Pediatric Ann 1994;
23:341-45.
42. Isada CM, Kasten BL. Infectious diseases handbook. Lexii – Comp
the Cleveland, 1998.
43. Isenber SJ, Apt L, Wood M. A controlled trial of Povidon iodine as
prophylaxis against ophthalmia neonatorum. N Eng J Med 1995;
332:562-66.
44. Ishibashi Y. Acanthameba keratitis. Ophthalmoplegia 1997;
211:39-44.
45. Isselbacher KJ, Adams PD, Brannwalds E, Petersdorf RG, Wilson
JD: Harrison’s principles of internal medicine, (9th ed) McGraw
Hill Kogakusha, Tokyo, 1980.
46. Jawetz E, Melnick JL, Adelberg EA. The review of medical
microbiology, (2nd edn) 1974.
47. Karyakarte R, Damle A. Medical parasitology, (1st edn) Books
and allied, Kolkota, 2005.
48. Kelkar U, Kelkar S, Bal AM, Sandhya Kulkarni, Kulkarni S.
Microbiological evaluation of various parameters in ophthalmic
operating rooms. IJO 2002;51:171-76.
49. Kim VY, Duker JS. Ocular manifestation of human immuno-
deficiency virus. Semin.oph 1986;93-106.
50. Kinota S, Wong KW, Biswas, J Rao J. Changing pattern of infectious
keratitis Ind Jr Oph 1993;41:3-14.
51. Knight R. Parasitic diseases in men. Churchill Livingstone,
Edinburgh, 1982.
52. Leaser RL. Ocular manifestation of Lyme disease. Am J M 1995;
98:605-25.
53. Lennette EH, Spaulding EH, Truant JP. Manual of clinical
microbiology, (2nd edn) American society for microbiology,
Washington, 1974.
54. Lindsay EM. Practical introduction to microbiology, (1st edn) E &
FN Spon Ltd, London, 1962.
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(5th ed), edited by Fraunfelder FT, Roy FH, WB Saunders
Company, Philadelphia, 2000;45-46.
56. Margo CE, Hameed LM. Ocular syphilis, seminar Oph 1992;
37:203-20.
57. Marsh RJ. Varicella and herpes zoster in Current ocular therapy.
(5th edn), Fraunfelder FT and Roy FH (Eds)., WB Saunders
58. MeHugh D, Fison PN. Ocular erysipelas, Arch Oph 1992;
110:1315.
59. Moa LK, Flynm HW, Miller D, Pffugfelder SC. Endophthalmitis
caused by streptococal species. Arch Oph 1992;110:798-801.
60. Moreria BM, Daum RS. Antimicrobial resistance in staphylococci,
Pedia Clin Nor Am 1995;42:619-48.
61. Mukherjee KL. Medical laboratory technology, Vol. II, Tata
McGraw Hill, New Delhi, 1994.
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International, New Delhi, 2005.
63. Munoz B, West C. Trachoma–The forgotten cause of blindness.
Epidemio Rev 1997;19:205-17.
64. Nithayanandam Suneetha, Jacob MS, Battu RR, Thomas RK,
Correa MA, D’Souza, O. Rhino-orbitp cerebral mucor mycosis.
Ind J Op 2003;51:231-36.
65. Obertynsk H, Dyson C. Clostridium perfringens endophthalmitis,
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66. P Sivamani. Medical mycology, Shiva Publication.
67. Panjarathinam R. Text book of medical parasitology. (1st ed)
Orient Longman, Madras, 1990.
68. Powar CB, Daginawala HF. General microbiology, Vol. I and II
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hospital, Maduri, 1999.
70. Reddy CCM, Gupta VP, Sarada P. Ocular cysticercosis, Ind J Op
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BIBLIOGRAPHY
1. Agrawal V, Biswas J, Madhavan HV, Mangat G, Reddy MK, Saini

JS, Savitri Sharma. Current perspectives in infectious keratitis,
Ind Jr Oph 1994;42:171-79.
2. Ali S Al Katf. Brucellosis in Current Ocular therapy (5th edn)
edited by Fraunfelder FT, Roy FH (Eds)., WB Saunders Company,
Philadelphia 2000;15-16.
3. Anantnarayan R, Jayaram Paniker CK. Texbook of Microbiology
(5th edn) Orient Longman Hyderabad 1996.
4. Arseculeratne SN. Recent advances in rhinosporidiosis and
rhinosporidium seebri. Ind Jr of Medical Microbiology 2002;
20(3):118-31.
5. Barkay S, Garzozi H. Ann Oph 1984;16:164-68.
6. Bartley GB. Blastomycosis of the eye lid. Ophthalmology 1995;
102:2020-23.
7. Basak SK, Sinha TK, Bhattacharya D, Hazra TK Parikhs. Intra
vitreal live guanthostoma.
8. Berglodd J Basser, Feigl B. Ophthalmic manifestation in Lyme
borreliosis. Jr Neuro Oph 1994;14:14-20.
9. Bharathi MJ. Aetiological diagnosis of microbial keratitis in South
India. Ind Jr Med Microbio 2002;20:19-24.
Epidemiological characteristics and laboratory diagnosis of fungal
keratitis. IJO 2003;51:315-21.
In vitreo efficacy of anti bacterials against bacterial isolates from
corneal ulcers. IJO 2002;50:109-14.
12. Bharathi MJ, Ram Krishnan R, Vasu S, R Meenakshi, Palaniappan
R. Epidemiological characteristics and laboratory diagnosis of
fungal Kt Ind Jr Oph 2003;51:315-21.
13. Billore OP. Rhinosporidiosis (1st ed), AITBS Publishers, New Delhi,
1995.
14. Biswas J, Amala J, Raijada S, Kumarasamy N, Suniti Solomon.
Ophthalmic manifestation of HIV in India. Ind Jr Op 1999;47:
87-93.
15. Biswas J, Lingam G, Nirmala Subramaniam, Badrinath SS: Clinico
pathological study of parasitic lesions of the eye. Insight 1993;
11(2):32-39.
16. Biswas J, Madhvan HN, Badrinath SS. Ocular lesions in AIDS.
Ind Jr Op 1995;43:69-72.
BIBLIOGRAPHY 193
17. Biswas PN, Thakur SKD, Mukherjee H, Bhaduri G. Ophthal-

momyiasis, JIMA 2005;542-43.
18. Blanksma LJ, Slijper J. Actinomycosis Dacryocystitis ophthal-
mologica 1978;176:145-49.
19. Buchanan RE, Gibbon NE. Bergey’s manual of determinative
bacteriology, (5th ed) Williams and Wilkins, Baltimore 1974.
20. Chakraborty P. A Textbook of Microbiology (1st edn), New Central
Book agency, Kolkata, 1955.
21. Chakraborty P. Textbook of Medical Parasitology, (1st ed) New
Central book agency, Kolkata, 2004.
22. Chen CJ. Nocardia asteroides endophthalmitis. Oph Surg 1983;
14:502-05.
23. Chhatterjee KD. Parasitology, (4th ed), 1962.
24. Collins CH, Lyne PM, Grange JM. Microbiology methods (7th ed),
Butterworth, Oxford, 1994.
25. Cruickshank R, Duguid JP, Marmion BP, Swain RHA. Medical
microbiology, (12th edn), Churchill Livingston, 1975.
26. Das JC, Chaudhuriz Bansal RL, Bhomaj S, Sharma P, Chauhan D.
Visco expression of AC cysticercus cellulosae. Ind Jr Op
2002;50:133-35.
27. Das TP, Dogra MR, Gopal L, Jalali S, Kumar A, Malpani A,
Natrajan S, Rajeev B. Postsurgical endophthalmitis–Diagnosis and
management.
28. Davey RT, Tauber WB. Post-traumatic endophthalmitis emerging
role of bacillus cereus infection Rev Inf Dis 1989;9:110-23.
29. David DB, Kirby GR, Noble BA. Bacillus cereus endophthalmitis,
BJO 1994;78:577-80.
30. Ffytche T. Blindness in Leprosy, Aust Nz Jr Oph 1989;17:257-60.
31. Fogra R, Biswas J. Parasitic infections of the lid, Insight 1997;15:15-
16.
32. Francis V, Turner V. Achieving community support for trachoma
control. A guide for district health worker, Geneva WHO/PBL/93,
36.
33. Fraunfelder FT, Roy HF. Current ocular therapy, (5th edn) WB
Saunders Co, Philadelphia 2000.
34. Garcia LS. Diagnostic microbiology (4th edn), ASM press,
Washington, 2001.
35. Goldberg MA, Kazacos KR, Boyce WM, Ai E, Katz B. Diffuse
unilateral subacute neuroretinitis. Morphometric serology and
epidemiologic support for basliascariasis as causative agent.
Ophthalmology 1993;100:1695-701.
36. Goodman DF, Gottsch JS. Methicillin resistant S. epidermidis
keratitis treated with vancomycin. Arch Oph 1988;106:1570-71.
37. Harley RD. Pediatric ophthalmology, Vol. 2, (2nd edn) WB

Saunders Co. Philadelphia, 1983.
38. Helen CJ, Holland GN. Ocular tuberculosis. Surv Oph 1993;38:229-
56.
39. Helene B Fedukowitz. External infections of the eye. (2nd ed)
Appleton-Century, New York, 1978.
40. Hunt KE, Glasgo BJ. Aspergillus endophthalmitis an unrecognized
endemic disease. Ophthalmology 1996;44:19-21.
41. Ingram DL. Neisseria gonorrhea in children. Pediatric Ann 1994;
23:341-45.
42. Isada CM, Kasten BL. Infectious diseases handbook. Lexii – Comp
the Cleveland, 1998.
43. Isenber SJ, Apt L, Wood M. A controlled trial of Povidon iodine as
prophylaxis against ophthalmia neonatorum. N Eng J Med 1995;
332:562-66.
44. Ishibashi Y. Acanthameba keratitis. Ophthalmoplegia 1997;
211:39-44.
45. Isselbacher KJ, Adams PD, Brannwalds E, Petersdorf RG, Wilson
JD: Harrison’s principles of internal medicine, (9th ed) McGraw
Hill Kogakusha, Tokyo, 1980.
46. Jawetz E, Melnick JL, Adelberg EA. The review of medical
microbiology, (2nd edn) 1974.
47. Karyakarte R, Damle A. Medical parasitology, (1st edn) Books
and allied, Kolkota, 2005.
48. Kelkar U, Kelkar S, Bal AM, Sandhya Kulkarni, Kulkarni S.
Microbiological evaluation of various parameters in ophthalmic
operating rooms. IJO 2002;51:171-76.
49. Kim VY, Duker JS. Ocular manifestation of human immuno-
deficiency virus. Semin.oph 1986;93-106.
50. Kinota S, Wong KW, Biswas, J Rao J. Changing pattern of infectious
keratitis Ind Jr Oph 1993;41:3-14.
51. Knight R. Parasitic diseases in men. Churchill Livingstone,
Edinburgh, 1982.
52. Leaser RL. Ocular manifestation of Lyme disease. Am J M 1995;
98:605-25.
53. Lennette EH, Spaulding EH, Truant JP. Manual of clinical
microbiology, (2nd edn) American society for microbiology,
Washington, 1974.
54. Lindsay EM. Practical introduction to microbiology, (1st edn) E &
FN Spon Ltd, London, 1962.
BIBLIOGRAPHY 195
55. Louis A Wilson. Microsporidial infection in Current Ocular therapy.

(5th ed), edited by Fraunfelder FT, Roy FH, WB Saunders
56. Margo CE, Hameed LM. Ocular syphilis, seminar Oph 1992;
37:203-20.
57. Marsh RJ. Varicella and herpes zoster in Current ocular therapy.
(5th edn), Fraunfelder FT and Roy FH (Eds)., WB Saunders
58. MeHugh D, Fison PN. Ocular erysipelas, Arch Oph 1992;
110:1315.
59. Moa LK, Flynm HW, Miller D, Pffugfelder SC. Endophthalmitis
caused by streptococal species. Arch Oph 1992;110:798-801.
60. Moreria BM, Daum RS. Antimicrobial resistance in staphylococci,
Pedia Clin Nor Am 1995;42:619-48.
61. Mukherjee KL. Medical laboratory technology, Vol. II, Tata
McGraw Hill, New Delhi, 1994.
62. Mukherjee PK. Pediatric ophthalmology, (1st edn) New Age
International, New Delhi, 2005.
63. Munoz B, West C. Trachoma–The forgotten cause of blindness.
Epidemio Rev 1997;19:205-17.
64. Nithayanandam Suneetha, Jacob MS, Battu RR, Thomas RK,
Correa MA, D’Souza, O. Rhino-orbitp cerebral mucor mycosis.
Ind J Op 2003;51:231-36.
65. Obertynsk H, Dyson C. Clostridium perfringens endophthalmitis,
Am J O 1991;111:654.
66. P Sivamani. Medical mycology, Shiva Publication.
67. Panjarathinam R. Text book of medical parasitology. (1st ed)
Orient Longman, Madras, 1990.
68. Powar CB, Daginawala HF. General microbiology, Vol. I and II
(5th ed) Himalaya Publishing House, Mumbai, 1993.
69. Rajalaskshmi P, Prajna L. Ocular microbiology. Arvind eye
hospital, Maduri, 1999.
70. Reddy CCM, Gupta VP, Sarada P. Ocular cysticercosis, Ind J Op
1980;28-69.
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INDEX
A Carriers 3
Actinomyces 55 Cestodes 161
Adenoviruses 87 Chlamydiae 69
Adherence 8 Clostridium 46
Anthrax 45 Collection of specimens 176
Ascomycetes 104 Commensals 4, 10
Aspergillus 104 Conjunctival swab 178
Atypical mycobacteria 52 Corneal scrapping 178
Corynebacterium 40
B Cross-infection 2
Bacteria 16 Culture for microbes 181
classification 20, 21 Culture of material 179
morphology 16 Cysticercus 163
arrangement 17 Cytomegalovirus 85
capsule 18
D
cell wall 17
fimbriae pili 18 Deuteromycetes 104
flagella 18 Dimorphic fungi 104
shape 16 Dirofilaria 159
size 16 DNA viruses 80
spores 19 pox viruses 80
oxygen requirement 19 variola 80
visualization 20 Dog tapeworm 168
Baylis ascaris 149 Dracunculus medinensis 160
Bell’s phenomenon 13 Drug resistance 189
Blastomyces 106
E
Blink reflex 13
Bordetella pertussis 64 Echinococcus 168
Borrelia 67 Endoparasite 5
Branhamella catarrhalis 35 Endotoxins 8
Enterobacteriaceae 60
C Epstein-Barr virus 86
Candida monilia 106 Escherichia 60
Capsule 18 Fimbriae 18
Flagella 18 Gram-negative cocci 33

Francisella tularensis 63 acinetobacter 35
Free-living organisms 4 lacunata 36
Fungi 100 neisseriaceae 33
classification of fungi 103 Gram-positive bacilli 40
culture of fungi 102 actinomyces 55
fungi of ocular importance bacillus 44
104 B. cereus 44
Blastomyces 106 bacillus anthracis anthrax
Candida monilia 106 45
Coccidioides 109 bacillus subtilis 45
Dermatophytes 110 botulinum 48
Histoplasma 111 tetani 46
Mucor 113 Corynebacterium 40
Rhinosporidium 115 Corynebacterium
wet preparation 101 diphtheriae 41
Corynebacterium xerosis
G
42
Gnathostoma spinigerum 161 mycobacteria 48
Gram’s stain 180 Nocardia 56
Gram-negative bacilli 56 Propionibacterium 43
Acinetobactor 59 tuberculosis 49
Bordetella pertussis 64 Gram-positive cocci 26
Borrelia 67 meningococcus 34
Brucella 63 pneumococci 31
Enterobacteriaceae 60 staphylococci 26
Escherichia 60 streptococci 29
Francisella tularensis 63 anaerobic 31
Haemophilus 58 classification 30
Klebsiella 61 Guinea worm 160
Leptospira 68
Moraxella 60 H
Proteus 62 Herpes virus 82
Pseudomonas 56 general properties 82
Salmonella 62 herpes simplex virus 83
Shigella 62 Higher bacteria 54
Treponema 65 Histoplasma 111
INDEX 199
Human cysticercosis 166 Nematodes 143

Human immunodeficiency Nocardia 56
virus 94 Nosocomial infection 2
I O
Infestation 4 Ocular microbiology 9
Invasiveness 8 Onchocerca 150
Onchocerca volvulus 152, 153
K
Opportunistic 3
Klebsiella 61
P
L
Paramyxovirus 90
Latent period 2 Parasites 122
Latrogenic infection 3 immunological changes 127
Lenti virinae 94 reservoir 124
Leptospira 68 route of entry 124
Loa loa 154 types 124
Lymphogranuloma venerum types of hosts 123
71 Pathogen 4
Phycomycetes 104
M
Picorna viruses 89
Measles Rubeola-Morbilli 92 Pneumococci 31
Meningococcus 34 Pneumocystis carinii 141
Metazoa 142 Primary infection 2
Microbiological examination Propionibacterium 10, 43
187 Protective mechanism 12
Microsporidia 139 Proteus 62
Molluscum contagiosum 81 Protozoa 128
Moraxella 60 Provocative test 154
Moulds 104 Pseudomonas 56
Mucor 113
Mumpus virus 91 R
Mycobacteria 48 Reflex tearing 13
Mycobacterium 182 Reinfection 2
Retrovirus 94
N Rhinosporidium 115
Neisseria catarrhalis 35 RNA viruses 89
Neisseriaceae 33 Rubella German Measles 93
S Treponema 65
Salmonella 62 Trichinella 147
Saprophytes 3, 10 Tube dilution method 190
Secondary infection 2 Type of carriers 3
Shigella 62
V
Source of infection 5
Spirochetes 64 Vaccinia 81
Spores 19 Varicella zoster virus 84
Staining methods 22 Virulence 9
Staphylococci 26 Virus isolation 84
Streptococci 29 Viruses 74
T cellular changes 79
classification 76
Taenia multiceps coenurus 171
cultivation 77
classification 173
mode of transmission 173 diagnosis of viral disease 79
Thelazia 147 infection 78
Toxocara 144 morphology 75
Toxoplasma 131 multiplication 77
demonstration of organism W
138
diagnosis 137 Welchii (perfringens) 47
differential diagnosis 137 Wuchereria bancrofti 156
oocytes sporozoites 135
Y
route of infection 137
tissue cysts cytozoites 134 Yeasts 103

Ocular Microbiology

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Ocular Microbiology

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OCULAR

Preeti Bandyopadya MSc PhD

© 2010, Jaypee Brothers Medical Publishers

First Edition: 2010

Last few decades have witnessed tremendous change in the

At the outset I remember with reverence late Dr S Agarwal,

Dr Manik Chatterjee, Associate Professor of Anatomy, Pt

1. General Considerations ................................................ 1

Infection is an interaction between the host and the micro-

Iatrogenic infection is a physician induced infection due to

Various Types of Carriers

1. Temporary carriers, who are capable of infecting for a short

infection. B. subtilis and B. cereus are emerging as causative

tetanus and rabies. The organism can be inoculated in-

The transmission may be

5. Soil: Some free-living pathogens survive in the soil for long-

2. Adherence: The microorganisms cause diffusion of toxin

7. The virulence of a microorganism depends basically on

MICROBIOLOGY IN RELATION TO THE EYES

The principle involved in ocular microbiology is the same as

Normal Microflora of the Eyes

The eyes may be infected during the birth if the labor is

iii. Bacillus species

Routes of ocular infection can be exogenous or endogenous.

PROTECTIVE MECHANISM OF THE EYES

iii. The immuglobulins: The tear contains three immuno-

Factors that Predispose Ocular Infections

The organisms that are known to cause ocular infections

Figure 2.1: Shapes of various bacteria: 1. Cocci; 2. Bacilli;

The cocci can be arranged in clusters, chains, pairs or scattered

Figure 2.2: Arrangement of cocci: 1. Streptococci; 2. Staphylococci;

The bacilli are generally scattered. They can form small

This is a rigid outer layer made of carbohydrates, proteins

Figure 2.3: Arrangement of bacilli: 1. In cluster; 2. Chains;

Some of the organisms develop a thick outer jelly like covering

They are responsible for motility of the organism seen only in

These are short straight filaments seen only in some gram-

Figure 2.4: Capsule of pneumococci stained with Indian ink

Spores are part of defense mechanism of the bacteria, they are

According to effect of oxygen on various microorganisms

Figure 2.5: Various positions of endospores

organism. The former requires oxygen to grow. The obligate

Classification of Bacteria (Figure 2.6)

Besides these the bacteria are classified according to their

Bacteria are microscopic and not visible with naked eyes.

They can be:

Figure 2.7: Gram-positive cocci

Figure 2.8: Gram-negative bacilli

ii. Acid-fast stain

Figure 2.9: Darkground illumination Treponema pallidum

not visible under ordinary illumination. The organisms

Both types of cocci, i.e. gram-positive and gram-negative

Staphylococci are a gram-positive, spherical bacteria

Figure 3.1: Staphylococci. Courtesy: Prof V Sudarshan

They are aerobes or facultative anaerobic, generally grow at

Figure 3.2: Staphylococci growing on culture plate.

The staphylococci produce many enzymes and toxins

These gram-positive cocci are found in respiratory and gastro-

Figure 3.3: Streptococci. Courtesy: Prof V Sudershan

The organisms are generally arranged in chains or in pairs.

They are nonsporing, nonmotile and most of the time non-

Various types of classifications have been put forwards.

streptococci, i.e. alpha hemolytic (Streptococcus viridance).

Till a few decades ago, these gram-positive organisms which