MICROBIOLOGY OF OCULAR INFECTIONS

Microbiology Department/Faculty of Medicine

LEARNING OBJECTIVES
 Normal bacterial Flora
 Pathogenesis and immunology of ocular infections
 Etiology of ocular infections
 Microbiological diagnostic of ocular infections
 Therapeutic principle of ocular infections
Flora normal
ANATOMY OF THE EYE
 Sterile sites
ANATOMY OF THE EYE
MICROBIAL FLORA OF NORMAL EYE
 AEROBIC BACTERIA
 Gram positive cocci
 Staphylococcus epidermidis
 Staphylococcus aureus
 Micrococcus sp.
 Streptococcus pyogenes
 Streptococcus pneumonia
Streptococcus viridans
 Gram negative cocci
 Moraxella catarrhalis
 Gram positive bacilli
 Corynebacterium sp
 Gram negative bacilli
 Haemophilus influenzae
 Klebsiella sp.
 Escherichia coli
 Pseudomonas aeruginosa
 Moraxella sp.
MICROBIAL FLORA OF NORMAL EYE
 ANAEROBIC  FUNGUS
 Propionibacterium sp.  These are transient and
are those found in the
 Peptostreptococcus environment.
 Bacteroides sp.

 Lactobacillus sp.  VIRUS

 Clostridium sp.  These are not normal
residents.
EXTERNAL OCULAR INFECTIONS
INFECTION OF THE EYELID
LACRIMAL APARATUS INFECTIONS
CONJUNCTIVITIS (BACTERIAL, VIRAL)
KERATITIS ( BACTERIAL, FUNGAL, VIRAL)
INFECTIONS OF THE EYELID / EYELID GLANDS (1)
 Blepharitis
 S.aureus is the most
 Hordeolum common causative
 Meibomitis/diffuse agent
meibomitis
 Chalazion
 Folliculitis
 Cannaliculitis
INFECTIONS OF THE EYELID / EYELID GLANDS (2)

 STAPHYLOCOCCAL BLEPHARITIS
 Chronic inflammation of
eyelid margins
 Staphylococcus aureus and
Staphylococcus epidermidis
 Pathogenesis: cell-mediated
immune response with
hypersensitivity to S. aureus

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LACRIMAL APPARATUS INFECTIONS
 DACRYOADENITIS
 Inflammation
of the lacrimal gland
 Most common cause:
 Nocardia, Actinomyces, anaerobic bacteria, or a mixed
flora of both gram positive and gram negative bacteria

Nocardia Actinomyces
CONJUNCTIVITIS (1)
 Benign & often self limiting if not diagnosed and
treated properly ocular morbidity of devastating
consequences

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Conjungtivitis bacterial
Conjungtivitis viral
Conjungtivitis chlamydia
Conjungtivitis chlamydia Conjungtivitis gonorrheoe
Allergic conjungtivitis
BACTERIAL CONJUNCTIVITIS (1)
 Types of Bacterial
Conjunctivitis:
 Hyperacute
(Ophtalmia neonatorum)
 Neisseria gonorrhoeae
 Acute
 Staphylococcus aureus
 Streptococcus pneumoniae
 Haemophilus influenzae
 Chronic (>4 weeks)
 Staphylococcus aureus
 Moraxella lacunata
 Streptococcus
pneumoniae
 Haemophilus influenzae
 Haemophilus aegyptius
 BACTERIAL CONJUNCTIVITIS (2)
 DIPHTERIC (MEMBRANOUS) CONJUNCTIVITIS
 necrosis and sloughing of conjunctival epithelium due to
diffusible toxins produced by the bacterium
Should be differentiated with severe
form of pseudomembranous
conjunctivitis (Streptococcus pyogenes,
Neisseria gonnorhoeae, Haemophilus
influenzae, and rarely Staphylococcus
aureus and Streptococcus pneumoniae)

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BACTERIAL CONJUNCTIVITIS (3)
 CHLAMYDIA CONJUNCTIVITIS (1)
 Chlamydia trachomatis, a non-motile, Gram negative
intracellular bacterium

Immunoflorescence staining showing the reticulate bodies of

Chlamydia trachomatis grown on McCoy cell line culture.

L&T microbiology research centre

 BACTERIAL CONJUNCTIVITIS (4)
 CHLAMYDIA CONJUNCTIVITIS (2)
 Trachoma
 the largest, single cause of preventable blindness worldwide
 spread by flea, direct contact with infected material
 serovar A, B, Ba, C

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 BACTERIAL CONJUNCTIVITIS (5)
 CHLAMYDIA CONJUNCTIVITIS (3)
 Inclusion conjunctivitis
 serovar D, E, F, G, H, I, J, K, found in genital tract
 have been reported in more than 25% of newborn conjunctivitis
 has a range of clinical features indistinguishable from viral,
baterial and allergic
 conjunctivitis or keratoconjunctivitis and clinical presentation may
be acute or chronic
(a) Normal conjunctiva (b) Follicular trachomatous inflammation (TF). (c) Intense
trachomatous inflammation (TI) (and follicular trachomatous inflammation). (d)
Conjunctival scarring (TS). (e) Trichiasis (TT). (f) Corneal opacity (CO
LABORATORY DIAGNOSIS (1)
 Often number of organisms are small
 In particular, patients with external eye infection often are
on topical antibiotics when referred to for investigation
 Fastidious organisms are frequently encountered, therefore
enriched media are necessary
 Inoculation of media are done at `bedside' and use of
transport or preservative media is avoided particularly for
bacterial isolation.
LABORATORY DIAGNOSIS (2)
 LID MARGIN
 No topical anaesthetic is needed.
 Sterile cotton tipped swab in brain heart infusion broth (BHIB) is rubbed
over the lid margin
 Swab is inoculated directly
 Media:
 Blood Agar (BA)
 Chocolate Agar (CA)
 Brucella Blood Agar (BBA)
 Smears to glass slide, and stained with: Gram
LABORATORY DIAGNOSIS (3)
 CONJUNCTIVAL SWAB
 Swab is inoculated directly
 Media:
 Blood Agar (BA)
 Chocolate Agar (CA)
 Brucella Blood Agar (BBA)
 Smears to glass slide, and
stained with: Gram, Giemsa, ZN,
immunofluorescent
 LABORATORY DIAGNOSIS (4)
 CONJUNCTIVAL SWAB

Gram stained smear of conjunctival swab showing the pus cells Growth of Propionibacterium
(polymorpho leucocytes) and typical coryneform diptheriodes in acnes colonies on Brucella Blood
clusters and in singles and pairs agar (anaerob)
LABORATORY DIAGNOSIS (5)
 CONJUNCTIVAL SCRAPPING
 For detection and isolation of Chlamydia trachomatis and viruses
 Topical anaesthetic is placed over the eye
 Scrap using the Kimura spatula
 Media:
 Blood Agar (BA)
 Chocolate Agar (CA)
 Brucella Blood Agar (BBA)
 Sucrose phosphate broth
 HBSS (for virus)
 Smears to glass slide, and stained with: Gram, Giemsa, ZN, immunofluorescent
Mikroskopis Chlamydia
 Pemeriksaan dalam gelas objek
 diwarnai dengan pewarnaan giemsa atau larutan jodium
 diperiksa dengan mikroskop cahaya biasa.
 Pada pewarnaan Giemsa, Badan Inklusi (BI) terdapat intra
sitoplasma sel epitel akan nampak warna ungu tua,
 sedangkan dengan pewarnaan yodium akan terlihat berwarna
coklat.
 Jika dibanding dengan cara kultur, pemeriksaan mikrosopik
langsung ini sensitifitasnya rendah dan tidak dianjurkan pada infeksi
asimtomatik.
 Pengecatan Giemsa

Typical perinuclear intracytoplasmic

inclusion bodies of Chlamydia in
Pada infeksi chlamydia ditemukan
conjunctival cytologic preparation:
adanya inclusion bodies pada
Giemsa stain. (Photomicrograph
pengecatan giemsa.
courtesy of Dr. Morton Smith.)
VIRAL CONJUNCTIVITIS (1)
 Self limiting disease with low morbidity
 acute follicular conjunctivitis of sudden onset
 often with respiratory and systemic symptoms
 with involvement of the other eye within a week
 preauricular lymphadenopathy
 lid oedema
 conjunctival haemorrhages and corneal changes
VIRAL CONJUNCTIVITIS (2)
 ADENOVIRUS, occuring in sporadic and epidemic forms
 Enterovirus 70
 Coxsackie A24
 HSV
 VZV
 Influenza A virus
 Newcastle disease virus
 Cytomegalovirus
VIRAL CONJUNCTIVITIS (3)
 ADENOVIRAL CONJUNCTIVITIS
 Epidemic keratoconjunctivitis (EKC)
 Incubation period of 8-10 days
 Occurs as sporadic or in clusters and epidemics
 Associated with ADENOVIRUS serotypes 8, 19, 37, less
often 3, 4, 7, 10, 21.
VIRAL CONJUNCTIVITIS (4)
 ADENOVIRAL CONJUNCTIVITIS
 Pharyngo conjunctival fever (PCF)
 Incubation period of 6-9 days
 Characterized by fever, pharyngitis, conjunctivitis and other
systemic symptoms
 Occurs as sporadic in all ages, outbreak in young children
 Associated with ADENOVIRUS serotypes 3, 4, 7 less often 1,
11, 14, 16-19 and 37
VIRAL CONJUNCTIVITIS (5)
 ADENOVIRAL CONJUNCTIVITIS
 Non-spesificconjunctivitis
 Chronic papillary conjunctivitis

The viruses spread rapidly in the community as a result of respiratory tract to eye, eye
to eye and via infected tissues and clothes and other fomites and contaminated
swimming pools.
VIRAL CONJUNCTIVITIS (6)
 ADENOVIRAL
CONJUNCTIVITIS
 Laboratory diagnosis
 detection of virus by
direct methods and
isolation of the infecting
agent
 isolation by conventional
test tube cell cultures

Microbiology of ocular infection

VIRAL CONJUNCTIVITIS (7)
 BILATERAL FOLLICULAR CONJUNCTIVITIS
(ENTEROVIRUS 70 & COXSACKIE 24)
 Sudden onset
 + conjunctival haemorrhages, transient keratitis
VIRAL CONJUNCTIVITIS (8)
 FOLLICULAR CONJUNCTIVITIS
 Eyelid vesicles, preauricular lymphadenopathy,
ulcerative blepharitis
 Primary infection with herpes simplex virus (HSV) occur
in newborn (ophtalmia neonatorum), children, young
adults
VIRAL CONJUNCTIVITIS (9)
 FOLLICULAR CONJUNCTIVITIS
 VARICELLA ZOSTER VIRUS (VZV)
 Following reactivation of latent endogenous virus
 A papillary, follicular or membranous conjunctivitis may occur
 Distinctive skin eruptions on the eyelid and forehead are
diagnostic

 INFLUENZA A VIRUS, NEWCASTLE DISEASE VIRUS, CMV

 Acute follicular conjunctivitis
VIRAL CONJUNCTIVITIS (10)
 CHRONIC TOXIC CONJUNCTIVITIS
 MOLLUSCUM CONTAGIOSUM
 Pearly white umbilicated nodules on histopatholigcal
examination confirms the diagnosis
KERATITIS
 Infective keratitis is a major ophthalmic problem
often leading to corneal blindness
 A rapid etiological diagnosis may help in initiating
an aggressive specific treatment for a cure or
minimize scar formation
BACTERIAL KERATITIS (1)
 Clinical characteristics of bacterial keratitis caused
by individual bacteria are so much overlapping
 Bacteria by virtue of their toxins, adherence
captivities, invasiveness or strain differences in
within a species may produce different types of
clinical picture
 ULKUS KORNEA

Lekoma (sikatriks) Abses kornea dan

hipopion
Pseudomonas corneal Staphylococcus corneal ulcer
ulcer
BACTERIAL KERATITIS (2)
 Gram-positive bacteria
 Discrete, small abscess like
lessions
 Staphylococcus aureus or
Moraxella sp.
 Indolent corneal ulcers
 Pneumococcal or
Pseudomonas
 Hypopyon
 Gram-negative bacteria
 rapid, diffuse, spreading
necrotic lessions
 marked eyelid oedema and
conjunctival chemosis
 Pseudomonas aeruginosa and
Streptococcus pnumoniae
 acute pain with watering and
rapidly spreading corneal
ulcer
FUNGAL KERATITIS (1)
 30-40% of all cases of culture-positive infectious
keratitis
 70% Aspergillus and Fusarium
 >> trauma with organic matter
FUNGAL KERATITIS (2)
 The ulcers commences insidiously
 Begin at the midperiphery  the ulcers spreads towards the
centre of the cornea (corneal perforation can occur 5-6
days)
 Moderate / cheesy hypopyon
 The ulcer base: wet, raised, soft creamy, grayish-white/
yellowish-white without mucous
 Pigmented fungi: brown/dark pigment covering the ulcer base
 Feathery/ hyphate borders (≈dendritic)  misdiagnosis w/
virus
Fungus corneal ulcer and satelitism
VIRAL KERATITIS (1)
 Herpes simplex virus (HSV), type I >>
 Type II found in 20% infant born w/ HSV infection
 In adults the HSV reccurence rate: 25% w/in 1 y,
33% w/in 2 ys
 Precipitatingfactors: trauma, fever, menstruation,
psychological stress
 VIRAL KERATITIS (2) HSV KERATITIS
 Corneal Epithelial Lesions:
 Dendritic keratitis
 Marginal herpetic keratitis
 Geographic/ amoeboid
herpetic ulcers
 Indolent keratitis
L&T Microbiology Research Centre
 Endothelial Involvement
 Diciform keratitis
 Herpetic Stromal Keratitis
 Superficial keratitis w/
epithelial lesions
 Diffuse Stromal Keratitis
 Classic type Punctal epithelial

Epithelial Herpetic Keratitis

Report from the Ad Hoc Committee for the Management of Epithelial Herpetic Keratitis. Reviewed by the
Committee November 11, 2012, Chicago, IL
Tzanck smear demonstrating multinucleated giant cells (Giemsa stain,
×1000)
VIRAL KERATITIS (2) HZO KERATITIS
 Herpes Zoster Ophthalmicus (HZO)
 Due to activation of latent varicella zoster virus (VZV)
infection
 Dendrites (coarse, stellate, no terminal bulbs), punctate
keratitis, mucous plaques
 Resolve w/out treatment w/in 1 mo
LABORATORY DIAGNOSIS (1)
 Collection of corneal scrapping (with the aid of slit
lamp)
 Topical anaesthetic is applied
 Avoid eyelashes contamination

 Purulent material is removed

 Sterile Kimura spatula is used

L&T Microbiology Research Centre

LABORATORY DIAGNOSIS (2)
 Material is inoculated directly onto media
 Media :
 Blood agar (aerobic)
 Chocolate agar (CO2)

 Brucella blood agar (anaerobic)

 Sabouraud’s dextrose agar (aerobic)

 Hank’s Balanced Salt Solution viral and chlamydial PCR

LABORATORY DIAGNOSIS (3)
 Smears of about 5-6 are made on clean microscopic slide
 The smears are stained:
 KOH for fungal
 Gram stain
 Giemsa stain
 Calcofluor white (CFW)
 Immunofluorescent, Ziehl-Neelsen
 Periodic Acid Schiff (PAS)/ Gomori Methenamine Silver (GMS) for
fungal
KOH, fungal element
Gram, diplococci Gr +

CFW, septate hyphae

Gram neg bacilli Immunofluorescence

L&T Microbiology Research Centre
INTERNAL OCULAR INFECTIONS
INFECTIOUS ENDOPHTHALMITIS
 Causes mainly by bacteria and fungi
 A rational therapy on the use of antibiotics and steroids
necessitates to determine whether the inflammation is
infectious or sterile
 Classification:
 Mode of entry:
 Exogenous: post-surgical (70%), post-traumatic
 Endogenous
ETIOLOGY
 Gram positive (76-90%)
 S.epidermidis (33-63%), S. aureus, S. pneumoniae, S. viridans and
S.pyogenes.
 Gram negative (3-22%)
 Pseudomonas aeruginosa (>>), Klebsiella pneumoniae, H. influenzae,
Escherichia coli and Enterobacater aerogenes
 Fungi (3-8%)
 Anaerobic bacteria
 Propionibacterium spp., Bacteroides spp., anerobic streptococci
POST-OPERATIVE ENDOPHTHALMITIS
 Sources: > periocular flora (S. epidermidis, S.aureus,
streptococci)
 Acute onset (2-7 days): fulminant, S.aureus, Gram neg
bacili
 Late onset:
 Since surgical: Propionibacterium acnes, S. epidermidis
 Post-operative period: Haemophilus influenzae, Streptococcus
spp
POST-TRAUMATIC ENDOPHTHALMITIS
 Has a relative poor prognosis
 Bacillus spp. >>, Staphylococci, Streptococci, Gram
neg bacili, fungal isolates, anaerob (Clostridial
species)
LABORATORY DIAGNOSIS (1)
 Collection of specimen during operation
 NO TRANSPORT MEDIUM
 Directly inoculated onto:
 BA (incubated aerobically at 37 C)
 Brucella blood agar – BBA (incubated 37 C anaerobically)
 Chocolate agar – CA (incubated 37 C in an atmosphere of 10%
CO2)
 Liquid media - Brain heart infusion broth (BHIB) and Robertson
cooked meat medium (RCM) / thioglycollate broth.
 Sabouraud dextrose agar (SDA) is incubated at 25 C
Aspergillus flavus on BA Fusarium on BA Fusarium on SDA
LABORATORY DIAGNOSIS (2)
 Direct Smears
 3-4 smears are made by cytospin and fixed in methyl
alcohol.
 The smears are stained by : Gram stain, Giemsa stain,
Gomari's Methanamine, silver stain, KOH, Calcofluor
(fungus)
LABORATORY DIAGNOSIS (3)
 Molecular biological technique
 PCR
aWith antibiotics (gentamicin or
chloramphenicol)
but without cycloheximide.
Potato dextrose agar may be used in
addition to Sabouraud dextrose agar
for better sporulation.
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