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Laboratory
Diagnosis of
Ocular Infections

KIRK R. WILHELMUS, THOMAS J. LIESEGANG,

MICHAEL S. OSATO, and DAN B. JONES

COORDINATING EDITOR
STEVEN C. SPECTER

AMERICAN SOCIETY FOR MICROBIOLOC~

Cumitech 1A Blood Cultures II June 1982
l l

Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987

l l

Cumitech 3A Quality Control and Quality Assurance Practices in Clinical Microbiology

l May 1990 l

Cumitech 4A Laboratory Diagnosis of Gonorrhea April 1993

l l

Cumitech SA Practical Anaerobic Bacteriology December 1991

l l

Cumitech 6A New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide

l l

February 1991
Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infections September 1987
l l

Cumitech 8 Detection of Microbial Antigens by Counterimmunoelectrophoresis

l December 1978 l

Cumitech 9 Collection and Processing of Bacteriological Specimens August 1979

l l

Cumitech 10 Laboratory Diagnosis of Upper Respiratory Tract Infections December 1979

l l

Cumitech 11 Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology
l

Laboratory August 1980 l

Cumitech 12A Laboratory Diagnosis of Bacterial Diarrhea April 1992

l l

Cumitech 13A Laboratory Diagnosis of Ocular Infections September 1994

l l

Cumitech 14A Laboratory Diagnosis of Central Nervous System Infections March 1993
l l

Cumitech 15A Laboratory Diagnosis of Viral Infections August 1994

l l

Cumitech 16 Laboratory Diagnosis of the Mycobacterioses

l March 1983 l

Cumitech 17A Laboratory Diagnosis of Female Genital Tract Infections June 1993
l l

Cumitech 18 Laboratory Diagnosis of Hepatitis Viruses January 1984

l l

Cumitech 19 Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections August 1984

l l

Cumitech 20 Therapeutic Drug Monitoring: Antimicrobial Agents October 1984

l l

Cumitech 21 Laboratory Diagnosis of Viral Respiratory Disease March 1986

l l

Cumitech 22 Immunoserology of Staphylococcal Disease August 1987

l l

Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988

l l

Cumitech 24 Rapid Detection of Viruses by Immunofluorescence

l August 1988 l

Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing

l l

December 1988
Cumitech 26 Laboratory Diagnosis of Viral Infections Producing Enteritis September 1989
l l

Cumifechs should be cited as follows, e.g.: Wilhelmus, K. R., T. J. Liesegang, M. S. Osato, and D. B.
Jones. 1994. Cumitech 13A, Laboratory diagnosis of ocular infections. Coordinating ed., S. C. Specter.
American Society for Microbiology, Washington, DC.

Editorial Board for ASM Cumitechs: Steven C. Specter, Chairman; Mary J. R. Gilchrist, Curt Gleaves,
Janet Hindler, Thomas J. Inzana, Brenda McCurdy, Fredertck S. Nolte, John A. Smith, Alice S. Weissfeld,
and Stephen Young.

The purpose of the Cumitech series is to provide consensus recommendations by the authors as to appropriate
state-of-the-art operating procedures for clinical microbiology laboratories which may lack the facilities for fully
evaluating routine or new methods.
The procedures given are not proposed as “standard” methods.

Copyright 0 1994 American Society for Microbiology

1325 Massachusetts Ave.. N W.
Washington, DC 20005
 LABORATORY DIAGNOSIS OF OCULAR INFECTIONS
KIRK R. WILHELMUS, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030

THOMAS J. LIESEGANG, Department of Ophthalmology, Mayo Clinic Jacksonville, Jacksonville, Florida 32224

MICHAEL S. OSATO, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030

DAN B. JONES, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030

COORDINATING EDITOR
STEVEN C. SPECTER, Department of Medical Microbiology and Immunology, University of South Florida, Tampa,
Florida 33612

SYNOPSIS MATERIALS AND MEDIA FOR

OCULAR SPECIMENS
Presentation of the materials n eeded to per- The materials and media suggestedfor the
form laboratory diagnosis for ocular infections. collection, transport, and culture of clinical spec-
Descriptions of acute and chronic ocular dis- imensmust be assembledand maintained(Table
eases caused by viruses, bacteria, parasites, and 1). Storagein a specialtray in the office or hospital
fungi, including dermatoblepharitis, infections treatment room will simplify ready access.Com-
of the orbit and lacrimal apparatus, marginal monly usedsolidmediacan be obtainedfrom the
blepharitis, conjunctivitis, keratitis, endophthal- clinical microbiology laboratory and should be
mitis, and retinitis. refrigerated at 4°C in plasticbags.Whether in the
Association of symptoms, etiologic agents, clinic, surgicalsuite, or hospitalroom, immediate
specimens, their methods of collection, and inoculation of ocular specimensfrom the patient
laboratory diagnosis for the various infections. to culture media is preferred whenever possible.
Recommendations on specimen transport to Transport containersare availablefor biopsyspec-
the laboratory and proper specimen handling imens,biomaterials,and other specialcases.Ma-
for intraocular fluids, tissues, and materials terials to be shippedelsewhereare securedin a
that cause eye infections, such as contact doublemailingpackagewith a biohazardlabel and
sentaccordingto current postalregulations.Com-
lenses, foreign bodies, and contaminated eye
municationnetworks are maintainedbetweenof-
drops. fice and laboratory personnelso that fresh, ade-
Overview of processing and interpretation quate materialswill be available and test results
of laboratory diagnostic techniques, including will be rapidly accessible.
smears, immune and molecular assays, cultures,
antimicrobial susceptibility testing, and serol- Collection Materials
WY* Fundamentalconsiderationsin the optimal col-
lection of ocular specimensare asfo llows.
The eye and associatedstructuresare uniquely
predisposedto infection by a variety of microor- l Sufficient material must be collected from the
ganisms.The indicationsand techniquesfor mi- infected site with minimal contaminationfrom
crobiologicalinvestigationsare determinedby the adjacentfluids, secretions,or structures.
site of ocular or periocular infection, its severity l An appropriatecollectiondevice,transport me-
and pace of progression,and knowledge of the dium, and culture media mustbe used.
l Material shouldbe collectedearly in the course
responsibleorganisms.Specialproceduresare re- of infection and in the absenceof recent anti-
quired to collect and processthe smallquantity of microbial treatment.
material that can be obtained from ocular tissues
and fluids. Efficient handlingof ocular specimens Specimen collection devices
involves the use and interpretation of various The Kimura spatula(Storz, St. Louis, MO.) is
laboratory proceduresin infectious eye diseases usefulfor obtainingmaterialfrom the conjunctiva
(35). The appendixlistscommonstainsusedin the and cornea (Fig. l), although an alcohol lamp is
ocular microbiology laboratory and a glossaryof neededfor heat sterilization. The tip coolsrapidly
clinical terms pertaining to ocular infections and after flaming and is slightly flexible for efficient
related inflammatory diseases. sampling.Disposablebladesor needlescan also
2 WILHELMUS ET AL. CUMITECH 13A

Orbital Space
Fornix
Conjunctiva: Bulbar
Lid: Anterior Margin

...**.*
:..
Lid: Posterior Margin
Preocular Tear Film
:.:
:
...*..
Cornea1 Epithelium
\k.‘::..*
‘..‘+
/:..,-.*.*.:
..*..
*‘;*a
.’:.
.. . . .. .

..*.*.*
..-.
*Vitreous
Cornea
Anterior Chamber
*
+...“.
..I -..
r :..&.A
Iris Lens
Posterior
Chamber
Conjunctiva: Tarsal
Orbital Septum

Cilia
Conjunctiva: Tarsal
Lacrimal Gland
Cornea
Corneoscleral Tissue
Canthus: Medial

Lacrimal Sac
Canaliculi
Puncta
Conjunctiva: Bulbar
Canthus Lateral
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 3

TABLE 1. Materials for ocular specimen collection

Collection devices Laboratory supplies Fixatives
Calcium alginate swabs with Glass slides with frosted ends and Methanol (95%)
plastic shaft etched circles
Calcium alginate swabs with Microslide holders Acetone (100%)
aluminum shaft
Cotton swabs with wood shaft Pencil Formalin (10%)
Dacron or rayon swabs with Wax pencil Glutaraldehyde (2%) or Trump’s
plastic or wire shaft solution (paraformaldehyde
[4%] and glutaraldehyde [I%])
Kimura platinum spatula Labels Coplin staining jars
l&gauge needles Rapid diagnostic kits for HSV
and C. trachomatis
30-gauge needles
l-ml syringes
Proparacaine hydrochloride
(0.5%) topical solution
Alcohol pads or swabsticks
Alcohol lamp with lighter or
matches

be used (76). The availability of several spatulas Anesthetics

expedites specimen collection. Preservative-freepreparationsare lessantisep-
Cotton, polyester, and calcium alginate swab tic than multiusetopical anesthetics(5). Propara-
tips are suitablefor the recovery of most bacteria Cainehydrochloride (0.5%) solution is preferred
and fungi, althoughcotton-tipped swabsmay con- for obtaining specimensfrom the ocular surface
tain fatty acids that inhibit bacterial growth. for culture and doesnot interfere with cytological
Moistened calcium alginate (Calgiswab; Spec- techniques.Sedation may be needed for young
trum, Houston, Tex.) can be dissolvedin a fixed children and uncooperativeadults.
volume of culture medium with hexametaphos-
phate solutionfor semiquantitativeculturesof the
lid margin and conjunctiva (11). Cotton- or poly- Slidesand fixatives
ester-tippedplastic swabsare preferred for viral Glassslideswith frosted endsand etchedcircles
cultures,and dacron or rayon swabson plastic or (FluorescentAntibody Slides;Becton Dickinson,
wire shafts are used for obtaining chlamydial Lincoln Park, N.J.) facilitate labeling and recog-
specimens.Swabs without transport medium nition of smeared material. Slides should be
should not be used for transporting specimens. cleansedto remove debristhat can absorbstains.
Prepackagedtransport tubeswith sterileswabsare Smearedslidesshouldbe immersedin 95% meth-
availablefor collectingmaterialfor bacterial,viral, anol in a Coplin jar for approximately 5 min for
and chlamydialcultures. fixation before staining. Cover slips are placed
Aspiration with a needle and syringe is the over wet mountswithout fixation and immediately
preferred method for collecting fluid or exudate examined.Slidesare sentto the laboratory in slide
for anaerobicculture. All air bubblesare expelled, holders(Control, Friendswood,Tex.) in the order
and the needletip is stuck into a rubber stopper.
that they were obtained.
Cold acetoneis often recommendedfor prepar-
ing slides for immunofluorescent staining, al-
though methanolcan be just as effective. Bouin’s
fixative is usedfor Papanicolaoustaining.Forma-
lin (10%) and glutaraldehyde (2%) should be
availableto fix histopathologicalmaterial for light
microscopyand electron microscopy,respectively.

Media
Culture and transport mediarecommendedfor
ocular infections are listed in Table 2. Media are
FIG. 1. Platinum (Kimura) spatula. The thin tip (in- chosenaccordingto the likely etiology and incu-
set) is ideal for obtaining material from the surface of bated at an appropriate temperature and atmo-
eyelid lesions, conjunctiva, and cornea. sphere.
4 WILHELMUS ET AL. CUMITECH 13A

TABLE 2. Culture and transport media for ocular specimens

Storage Incubation
Medium Purpose Incubation temp (“C)
temp (“C) duration
Routine media
Tryptic soy broth Saturation of swabs 4
Blood agar plate Aerobic and facultative 4 35 (10% COZ) 5-7 days
anaerobic bacteria,
fungi
Chocolate agar plate Aerobic and facultative 4 35 (10% CO,) 5-7 days
anaerobic bacteria,
Neisseria and
Haemophilus spp.
Thioglycolate broth Aerobic and anaerobic 25 35 7 days
bacteria
Brucella agar plate Anaerobic bacteria 4 35 (anaerobic system) 7 days
Sabouraud dextrose agar Fungi 4 25 4 wks
with gentamicin
Special media
Blood agar plate Fungi, mycobacteria 4 25 4-6 wks
Thayer-Martin medium Neisseria spp. 4 35 (10% COZ) 7 days
Lowenstein-Jensen or Mycobacteria and 4 35 6 wks
Middlebrook 7HlO agar Nocardia spp.
slant
BHI broth with gentamicin Fungi 25 4 wks
Nonnutrient agar plate Acanthamoeba spp. 30 and 35 4 wks
with E. coli overlay
Antimicrobial removal Remove antibiotics in 25 to 35 (until inoculation) 7 days
device specimen
Blood culture bottle Aerobic or anaerobic 35 7 days
bacteria, fungi
Transport media
Viral transport medium Viruses 4 4 (until inoculation) 4 wks
Chlamydial transport C. trachoma tis 4 4 (until inoculation) 2 wks
medium

Bacterial culture media For anaerobiccultures, specimensare usually

Soybean-casein digest(tryptic soy) broth (TSB) inoculated into a liquid medium (75). Thioglyco-
is useful for moisteningswabsfor specimencol- late broth enriched with vitamin K, and hemin
lection. TSB can serveasa general-purposenutri- (BBL, Becton Dickinson, Cockeysville, Md.) is
ent broth medium for cultures of aerobic and recommended.Prereduced, anaerobically steril-
facultatively anaerobicbacteriaand canbe usedto ized choppedmeat broth and prereduced,anaer-
collectviral cultures,althougha transport medium obically sterilized brain heart infusion (BHI)
containingantibioticsis preferred. A sterile, non- broth without antibiotics can also be used for
nutritive balancedsalt solution (e.g., BSS; Alcon anaerobic incubation. Inoculated tubes are
Laboratories,Fort Worth, Tex.) can be used for cappedand incubatedat 35 to 37°C.An enriched,
the interim holding of ocular tissues. fresh or prereduced brucella, Schaedler,Colum-
Defibrinated 5% horse or sheepblood agar is bia, or Brewer agarbaseis usedfor an anaerobic
suitablefor the growth of mostbacteria aswell as plate. After inoculation the plate should not be
fungi andAcanthamoeba spp.A MacConkey agar exposed to air any longer than necessaryand
plate or eosin-methyleneblue agar plate can also shouldbe incubatedin an anaerobicchamberor a
be inoculated if enteric gram-negativerods are heat-sealed envelope (GasPak Pouch [Becton
suspected.A chocolate agar plate enriched with Dickinson, Cockeysville,Md.] and Pouch System
vitamins and other supplementssupportsgrowth [Difco, Detroit, Mich.]) with a gasgenerator and
of Haemophilus, Neisseria, and Moraxella species. resazurinindicator.
Thayer-Martin mediumor oneof its modifications If an antibacterialor antifungal agenthasbeen
favors growth in suspectedgonococcalor menin- usedbefore specimencollection, an antimicrobial
gococcal infections. Blood and chocolate agar removal devicemight improve microbialrecovery.
plates should be incubated at 35 to 37°C in a Approximately 3 ml of the solution and resin is
candlejar or inside ziplock bagswith a carbon decanted into test tubes, the ocular specimenis
dioxide generator to create an atmospherecon- introduced into the medium, and aliquots are
taining 3 to 10% C02. inoculated onto solid and liquid culture media.
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 5

Specimensfor mycobacterialand nocardial cul- transport medium containing penicillin or other

tures should be inoculated onto Lowenstein- inhibitory antibiotics.
Jensenor Middlebrook 7HlO agar and incubated
at 35 to 37°C for up to 6 weeks. Laboratory Safety and Quality Assessment
Specimensneed to be transportedto the labo-
Fungal culture media ratory in a sturdy, leakproof container with a lid.
A blood agarplate can be usedto isolatefungi, Shippingrequirementsmandatethat material be
but a nutritionally deficient mediumsuch asSab- containedwithin a screw-cappedcontainer sealed
ouraud dextroseagar containinggentamicinat 40 with adhesivetape, wrapped in absorbentmate-
pg/ml (Emmons modification) should also be rial, enclosedin a watertight container, placed
used. Cycloheximideshould not be included be- inside a padded shippingcontainer, and labeled
causeit inhibits the saprophytic fungi principally with an appropriate biohazardnotice. Laboratory
responsiblefor ocular infections.Platesshouldbe personnelmust comply with guidelinesthat pro-
slightly thicker than usual (35 to 40 ml per petri mote biological, chemical,radiological,and phys-
dish)and are partially sealedor placedin a bag to ical safety (12).
reduce desiccationduring prolongedincubation. The clinical microbiology laboratory should
BHI broth containing gentamicin at 50 @ml maintain a quality-assuranceprogram that moni-
helpsto dilute naturally occurring fungal inhibi- tors equipment and supplies,ensurestechnical
tory substances.The inoculatedvial is incubated proficiency, and adheres to requisite biosafety
on a rotary shakerto enhanceaeration. All fungal practices (4). A schedulefor monitoring speci-
isolation media are incubated in a room-temper- mensand laboratory testsmust be individualized
ature incubator at 25 to 30°C for up to 4 weeks. for the needsand resourcesof each institution.
Indicators of adequatespecimencollectionare the
cytologic pattern of smears(e.g., number of epi-
Acanthamoeba culture media thelial cellsin a conjunctival specimen),growth of
Free-living amoebaecan be isolated on blood contaminantsapart from the inoculationmarkson
agar plates and other aerobic media. Minimal solid culture media, and transport time between
nutrient agar preparedwith an overlay of live or specimenacquisition and receipt in the labora-
dead microorganisms(such as Escherichiucoli) tory. Parametersof laboratory quality control are
enhancesrecovery. An alternative medium for daily temperature records of incubators and re-
primary isolationof amoebaeis bufferedcharcoal- frigerators and periodic conduct of performance
yeast extract agar, originally designedto isolate standardsfor stains,reagents,media, and suscep-
Legion& species.Duplicate plates can be incu- tibility tests.The laboratory should seekto mini-
bated at 30 and 35OC.Pageamoebasalinecan be mize the turnaround time for reporting growth
usedfor transportation of clinical specimens(55). andsusceptibilitydata sinceophthalmologistsmay
Subculturesof isolatedamoebaeshouldbe made be reluctant to alter initial empiric therapy, even
to cation-supplementedpeptone-yeast extract- when laboratory resultssuggestlesstoxic or less
glucoseagar for axenation(55). costly agents.Laboratory recordsshouldbe prop-
erly organized for surveillanceof trends in the
Viral and chlamydial transport media epidemiologyof ocular infectionsand for analysis
Hanks’ and Leibovitz-Emory media are com- of cumulative antimicrobialresistancepatterns.
monly availablefor transportingviral specimens.
Salt solutionsaloneshouldnot be used.Swab-tube METHODS FOR COLLECTION OF
combinations(Viral Culturette [Becton Dickin- OCULAR SPECIMENS
son,Cockeysville,Md.], Virocult [Medical Wire & Specimensshould be obtained from the in-
Equipment, Sparta, N.J.], and M4 [multipurpose fected site when safely accessible.A similar plan
transportmedium][Microtest, Snellville, Ga.]) are for collection and inoculation of material can be
available, as well as transportation deviceswith appliedto mostocular infections (Table 3). Spec-
cell culture (Transporter; Bartels Immunodiag- imencontainersmustbe properly labeledwith the
nostics,Bellevue, Wash.). Viral transport media patient’s name, identification number, collection
should be sent immediately for transfer onto date, and source.Information shouldbe provided
appropriate cell monolayers.Specimensshould about the clinical diagnosisand any recent useof
not be frozen at -2OOCandshouldnever be put in antimicrobial agents.Universal precautionsmust
a frost-free freezer of a standardrefrigerator. be followed during the collection and processing
Sucrose-phosphate-glutamatetransport me- of all clinical specimens.
dium is preferred for Chlamydiatrachomatisand
can also be used as a viral transport medium. Dermatoblepharitis
Prepackagedtransportation tubes are available Infections of the eyelid skin include viral erup-
(Transtube; Medical Wire & Equipment). C. tra- tions,primary bacterialpyodermas,and secondary
chomatis should not be transported in a viral infections complicatingpreexistingskin lesions.
6 WILHELMUS ET AL. CUMITECH 13A

TABLE 3. Guide to specimen collection for common ocular infections

Stains and other Media
Clinical entity Source of material Principal organisms
testsa
Preseptal cellulitis Drainage from S. pyogenes, S. aureus, Gram Blood agar, chocolate
wound or abscess H. influenzae agar, thioglycolate
(children)
Acute orbital Drainage or biopsy S. aureus, streptococci, Gram Blood agar, chocolate
cellulitis from abscess or H. injkenzae agar, thioglycolate,
sinus (children), anaerobes anaerobic agar,
Sabouraud agar,
brain heart infusion
Canaliculitis Expressed material Actinomycetes, Gram Blood agar, chocolate
streptococci agar, thioglycolate,
anaerobic agar
Acute dacryocystitis Drainage from Streptococci, S. aureus Gram Blood agar, chocolate
lacrimal sac agar, thioglycolate
Acute vesicular Vesicle fluid or lid HSV, VZV IF or EIA, Viral transport
blepharitis margin scraping Giemsa medium
Acute marginal Lid margin S. aureus Gram Blood agar, chocolate
blepharitis swabbing agar
Acute purulent Conjunctival S. aureus, S. Gram Blood agar, chocolate
conjunctivitis swabbing pneumoniae, H. agar
influenzae, N.
gonorrhoeae
Acute follicular Conjunctival Adenovirus, HSV IF or EIA Viral transport
conjunctivitis scraping medium
Chronic follicular Conjunctival C. trachomatis Giemsa, IF Chlamydial transport
conjunctivitis scraping medium
Neonatal Conjunctival C. trachomatis, N. Gram, Giemsa, IF Blood agar, Thayer-
conjunctivitis swabbing gonorrhoeae, S. aureus Martin agar,
chlamydial
transport medium
Epithelial keratitis Cornea1 HSV EIA or IF Viral transport
debridement or medium
impression
Suppurative Cornea1 scraping S. aureus, P. aeruginosa, Acridine orange, Blood agar, chocolate
keratitis S. pneumoniae, Gram, agar, thioglycolate,
Moraxella spp., calcofluor anaerobic agar,
enteric gram-negative white, acid-fast Sabouraud agar
rods, C. albicans,
Fusarium spp.
Suppurative Scleral scraping P. aeruginosa, S. Acridine orange, Blood agar, chocolate
scleritis pneumoniae, S. aureus Gram, acid-fast agar, thioglycolate,
anaerobic agar,
Sabouraud agar,
Lowenstein-Jensen
Endophthalmitis Aqueous humor S. epidermidis, S. aureus, Acridine orange, Blood agar, chocolate
tap, vitreous tap, streptococci, P. acnes, Gram agar, thioglycolate,
vitrectomy H. in.uenzae, gram- anaerobic agar,
washing negative rods, Bacillus Sabouraud agar,
spp., C. albicans brain heart infusion
Necrotizing retinitis Vitrectomy washing, CMV, VZV, HSV, C. IF, DNA probe Viral transport
retinal biopsy albicans, T. gondii medium
a IF, immunofluorescence; EIA, enzyme immunoassay.

Viral dermatoblepharitis vesicular scrapings),immunofluorescence studies,

Primary or recurrent herpes simplex virus viral culture, and electron microscopycanbe done
(HSV) dermatoblepharitisand primary or recur- (45, 54).
rent varicella-zostervirus (VZV) dermatoblepha-
ritis producea vesicularskin rashwith preauricu- Impetigo
lar iymphadenopathy. Fresh vesicles can be Superficial vesiculopustularor crusting infec-
aspiratedor scrapedwith the edgeof a spatula.A tion of the skin with preauricular lymphadenopa-
Tzanck test (preparinga Giemsa-stained smearof thy, usually in children, is causedby group A
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 7

streptococci and Staphylococcus aureus. Vesicles Erysipelas

shouldbe aspiratedor unroofed. Exudate is sent Superficial cellulitis of the eyelid with well-
for smearsandfor culture on blood and chocolate defined margins and lymphatic involvement is
agar plates. causedby S. pyogenes, other streptococci, or S.
aureus. The characteristicred edemaspreadsfrom
Granulomatous or pustular dermatoblepharitis a site of trauma, local infection, or prior skin
Granulomatouseyelid lesionscan occur from lesion. Swabbingsof intact skin are not reliable.
chancroid, anthrax, actinomycosis,tuberculosis, The etiology is difficult to establishsince sur-
candidiasis,blastomycosis,leishmaniasis, and my- rounding orbital edemamay be due to bacterial
iasis. A dental burr or cornea1microdrill can exotoxins rather than direct invasion. The tech-
securesamplesfrom an ulcerative lesion.Part of nique of injecting and aspirating sterile saline
the specimencan be inoculatedonto aerobic and within the subcutaneoustissuesfrom the advanc-
anaerobicculture media, including a Sabouraud ing edge is controversial. Blood and throat cul-
agar plate and Lowenstein-Jensen agar. To iden- tures can be obtained.Although conjunctival cul-
tify Mycobacterium leprae, slit 2 mm deep into tures may be misleading,material obtained by
pinchedskin and scrapethe lesiononto a slidefor swabbingof the inferior conjunctival fornix of
acid-fast staining. An excisional biopsy can be both eyes can be inoculated onto a blood agar
obtainedfor histopathologicalexamination.Leish- plate.
mania spp.can sometimesbe isolatedon a blood
agar plate, but promastigotesgrow better on
specialmedia such as Schneiderinsect medium Necrotizing fasciitis
andNovy-MacNeal-Nicollemediumat 22 to 26OC. Gangrene of the eyelids following trauma or
Extracted insect larvae can be placed on a blood necrotizing fasciitis associatedwith alcoholismor
agarplate or on raw meatwith moistsandin a jar another debilitating condition is usuallydue to S.
pluggedwith cotton.
pyogenes, either alone or in combination with
other bacteria such as S. aureus. Exudate from
Cellulitis of the Eyelids
ruptured bullae shouldbe smearedand cultured
Preseptal cellulitis (73). Other evidenceof streptococcalinfection is
Infections of the subcutaneouseyelid tissue an acutely elevated anti-DNaseor antihyaluroni-
anterior to the orbital septum may be due to daseantibody titer.
trauma,spreadfrom the upper respiratory tract or
middle ear in children, or spreadfrom infected
skin or adjacent structures (36). Posttraumatic Infections of the Lacrimal System
infections are usually due to S. aureus and Strep- Dacryoadenitis
tococcus pyogenes, although anaerobic and poly- Acute infections of the lacrimal gland are un-
microbial infections can occur. Nonsuppurative common. Typical clinical features include ery-
preseptalcellulitis in susceptiblechildren is com- thema andswellingof the outer third of the upper
monly causedby Haemophilus influenzae or Strep- eyelid, protrusion of the palpebrallobe, conjunc-
tococcus pneumoniae. Secondaryinfection of viral
dermatoblepharitis is uncommon. A ruptured tival discharge,and preauricular lymphadenopa-
hordeolum,lacrimalsac,or other infected adnexal thy. The route of inoculation may be exogenous
structure may causestaphylococcalor streptococ- (by way of the ductules in the superotemporal
cal cellulitis. Infantile gingivitiswith S. aureus and conjunctivalfornix or from penetratingtrauma) or
other bacteriamay lead to facial cellulitis. endogenous(during hematogenousspread).
Drainagefrom an openwound or eyelid abscess The most common exogenouspathogens of
shouldbe sentfor smearsandfor inoculation onto acute purulent dacryoadenitis are S. aureus, S.
blood agar, chocolate agar, brucella agar, and pneumoniae, and S.pyogenes. A calcium alginate
thioglycolate. To obtain material from an eyelid or cotton applicator should be swabbedin the
abscess,the skin is cleaned with an antiseptic superotemporalfornix over the surface of the
agentand dried. The point of maximalfluctuation palpebral lobe and inoculated onto blood and
isincisedparallelto the lid margin,andthe wound chocolate agar plates. The uninvolved contralat-
is spreadopen. If direct inoculation of media is era1conjunctiva shouldbe cultured for compari-
not feasible,aspiratedmaterialin a syringeshould son.Conjunctival smearsare obtainedby instilling
be free of air bubbles, the needle should be 0.5% proparacaine hydrochloride, scraping the
capped,and the material shouldbe immediately conjunctiva in the superotemporalfornix with a
transportedto the laboratory or injected into an spatula,and spreadingthe materialwith a circular
anaerobictransportvial. Blood culturesshouldbe motion onto a glassslide.Needleaspirationof the
obtained. Conj”unctiva1cultures are sometimes lacrimalglandis contraindicatedunlessa localized
helpful. abscess is identified.
8 WILHELMUS ET AL. CUMITECH 13A

Hematogenous acute dacryoadenitis is a rare and prevents reflux. Drainage from the lacrimal
complication of infectious mononucleosis, mumps, sacshouldbe swabbedor aspiratedfor smearsand
influenza, and gonorrhea. Laboratory diagnosis is cultures. Transcutaneousaspiration or incision
based on methods to detect the systemic infection through the wall of the sacis usuallynot indicated,
and is not generally aided by conjunctival cultures although an external fistula can provide a source
or smears. Serologic testing for suspected viral of material. Nasal swabbingunder the inferior
syndromes includes antibody titers for Epstein- turbinate may be attempted.Conjunctivalcultures
Barr virus (EBV) and mumps virus. shouldbe obtained in the standardmanner.
Chronic inflammation of the lacrimal gland may Chronic dacryocystitisproducesepiphora,with
be caused by sarcoidosis, syphilis, tuberculosis, or without pain or discharge.Colonization of the
leprosy, and certain fungal and helminthic infec- lacrimal sacpresumablyoccursfrom conjunctival
tions. Serologic tests are available for some of flora or by retrograde migration from the nose.A
these conditions. If a conjunctival or lacrimal variety of organismsmay contaminate the ob-
gland biopsy is performed, the specimen can be structed sacwithout producing cellulitis. Aerobic
bisected. One half is submitted for histopathologic and anaerobicbacteria, fungi, and mycobacteria
examination, and the other half should be ground have been isolated. Purulent material should be
aseptically and suspended for inoculation onto inoculated onto blood agar, chocolate agar, an
blood agar and Sabouraud agar. Smears should be anaerobicmedium, and Sabouraudagar. Smears
prepared for dark-field examination and staining shouldbe prepared for standardstains.A dacry-
by conventional methods. olith should be bisected and submitted for his-
topathologic and microbiologicstudies.
Canaliculitis Neonateswith congenital dacryostenosis are at
Infections of the lacrimal puncta and canaliculi risk for developing chronic dacryocystitis.Acute
are rare. Canaliculitisis characterizedby hypere- infection may lead to a dacryocystocele,a blue-
mia and edema at the medial canthus and a gray lacrimal sac distention filled with viscous,
dilated, pouting punctumwith mucopurulentdis- translucent mucus.Probing is frequently unsuc-
charge. Inflammation may involve the inferior cessfulin drainageof neonateswith dacryocystitis
punctum or both canaliculi and is frequently as- becauseof compressionof the commoncanalicu-
sociatedwith an obstructedcommoncanaliculus. lus. S. aureus, streptococci, and gram-negative
Most infections are due to anaerobessuch as rods are the most commoncausativeorganisms.
Propionibacterium propionicum and Actinomyces Dacryocystitis is a possiblecomplication of neo-
species(9). Mixed aerobicand anaerobicbacterial natal conjunctivitis due to Neisseria gonowhoeae.
infectionsmay occur. Smearsand cultures should be obtained as de-
Purulent material can usually be expressedby scribedabove.
compressionof the lid and canaliculus.A spatula
shouldbe usedto transfer this material to media Orbital Cellulitis
and to prepare smears.As theseinfections typi-
cally producelarge diverticula that contain cheesy Acute orbital cellulitis
concretions, a sterile spud or small chalazion The routes of infection in orbital cellulitis are
curette shouldbe usedto scrapethe canaliculusto spreadfrom infected ethmoid andother paranasal
obtain additional material. Incision into the hori- sinuses,spread from other adjacent structures
zontal portion of the canaliculusmay be neces- (e.g., dental infection, dacryocystitis,and orbital
sary.“Sulfur granules”and other particulate mat- fracture), exogenousinoculation from trauma or
ter shouldbe crushedonto a glassslide to obtain surgery,and hematogenousseedingduring sepsis.
a thin smearfor staining. The mostcommoncausesof acuteorbital cellulitis
associatedwith sinusitisare streptococci,S. au-
Dacryocystitis reus, and anaerobes.H. influenzae sinusitisand
Infection of the lacrimalsacis generallya result orbital cellulitis may occur in unvaccinatedchil-
of an obstructednasolacrimalduct. Acute dacryo- dren. Mixed aerobicand anaerobicinfectionsmay
cystitis is characterized by epiphora, pain, and occur. S. aureus is a frequent causeof posttrau-
distensionof soft tissue at the inner canthus, matic and postsurgicalorbital cellulitis, and poly-
typically just below the medial canthal ligament. microbial infections are common.
Localizedinflammationmay progressto a dacryo- Blood cultures are often positive during acute
cystopyoceleor orbital cellulitis. Responsiblemi- orbital cellulitis.Smearsandculturesareprepared
croorganismsare S. aureus, S. pneumoniae, S. from purulent material in the noseduring sinus-
pyogenes, H injluenzae, and, lesscommonly, aer- related disease,from swabbings or excisedtissueif
obic or facultative gram-negativerods or anaer- a traumatic or surgicalwound is present,or from
obes. Pressureon the lacrimal sac may express an infected lacrimal sacor other adnexalsource.
purulent discharge“from the puncta, but infection In the presenceof an openwoundor drainagesite,
often obstructsthe distal portion of the canaliculi material shouldbe obtained bv aspirationwith a
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 9

sterile syringe and needle for immediate inocula- Viral blepharitis

tion onto appropriate media or for prompt trans- Vesicular or erosive marginal blepharitis is
portation to the laboratory after either embedding caused by HSV or VZV. Vaccinia virus was
the needle into a sterile rubber stopper or inject- formerly a causein individualsrecently vaccinated
ing the material into an anaerobic transport vial. against smallpox. One or more clear vesicles
Drainage of an infected sinus, subperiosteal ab- should be selectedfor sampling.The surface of
scess, or intraorbital abscess is handled by similar the vesicleis cleansedwith a moistenedapplicator
techniques and is processed for smears and for or wiped with an alcohol pad. After puncturing
aerobic and anaerobic cultures (34). Blind aspira- the intact vesicle with a sterile needle, fluid is
tion of the orbit is not indicated. aspirated with a sterile tuberculin syringe or a
capillary hematocrit tube. The base of opened
vesiclesand of ulcerative erosionsis scrapedwith
Zygomycosis a spatulaand/or swabbed.Conjunctival scrapings
Acute rhinoorbitocerebral infection by R&o- and/or swabbingscan also be collected. Smears
pus and iWucor spp. and other nonseptate fungi of are prepared on glassslides.Giemsastaining of
the class Zygomycetes is a potentially lethal disease herpesvirusinfections usually showsmultinucle-
that primarily occursin patients with diabetic or ated cells.Intranuclear Cowdry type A bodiescan
metabolicketoacidosis,neoplasia,and other alter- sometimesbe found with Papanicolaoustaining.
ationsof host defenses.Fungal sporesproliferate Scrapingsof the lid lesion can also be smeared
within the nasalturbinatesand paranasalsinuses, onto a slide for immunofluorescenceor trans-
penetrate arterial walls, and spread by vascular ferred to an enzyme immunoassaysystem.Mate-
and direct extensionto the orbital apex. Surgical rial for viral isolation is placed into a viral trans-
debridementof necrotic sinusand orbital tissue port mediumthat is chilled or refrigerated at 4°C
should establishdrainage. Material from a ne- until it is processed.Acute- and convalescent-
crotic lesionshouldbe submittedfor frozen and phaseantibody titers can be obtained to identify
paraffin-embeddedhistopathologic sectionsand seroconversiondiagnosticof a primary viral infec-
for smearsand cultures. Biopsy material is trans- tion.
ported to the microbiologylaboratory in a sterile Molluscum contagiosum produces dome-
tube or petri dish with sterile balancedsalt solu- shapednodulesthat becomeumbilicated. Single
tion, emulsified, and inoculated to standard or multiple lesionsare located on the skin or
growth media. Smearsare examined by Gram, margin of the eyelids, occasionallyaccompanied
Giemsa,and calcofluor white stainsto detect the by follicular conjunctivitis and punctate cornea1
broad, nonseptatehyphae that tend to branch at erosions.The ulcerated area of the lesioncan be
right angles. scrapedwith a spatula,or the cheesycore can be
expressedwith a comedoextractor. Giemsaand
Chronic orbital cellulitis other stains(Sedi-Stain;Clay Adams, Parsippany,
Indolent orbital infection may occur after place- N.J.) can show myriad tiny dark particles and
ment of alloplasticmaterial for retinal surgeryor intracytoplasmicinclusions(Henderson-Patterson
orbital fracture repair. Causesinclude anaerobic bodies) within epidermal keratinocytes that are
bacteria,nontuberculousmycobacteria,actinomy- composedof poxviruses,as can be seenby elec-
cetes, and filamentous fungi. Trauma with re- tron microscopy or immunofluorescentstaining
tained foreign material may produce draining (20). Cytology of the conjunctival dischargetypi-
sinustracksthat simulatechronic microbial cellu- cally showsmononuclearcells.Incompletevirions
litis. Orbital aspergillosisis a slowly progressive are found from conjunctival specimens.Viral
granulomatousprocesswith thrombotic vasculitis, DNA can be detectedfrom molluscumcontagio-
usually occurring in otherwise healthy adults in sumlesions,but serialpropagationin cell culture
tropical areas. is usuallynot successful. An excisedlesionshould
Drainagematerial shouldbe inoculated onto a be fixed in formalin for light microscopy or in
blood agarplate, Sabouraudagar,BHI broth, and glutaraldehydefor electron microscopyto demon-
Lowenstein-Jensen agar. Orbital biopsy material strate dumbbell-shaped viral particles. His-
shouldbe bisectedfor histopathologicaland mi- topathologicalidentification can be facilitated by
crobiologic studies. Smears are prepared with overnight digestionwith DNase (to degradecel-
Gram, Giemsa, calcofluor white, and acid-fast lular DNA but not viral DNA protected by its
stains. protein capsid)andthen stainingwith the Feulgen
techniqueand light green counterstaining.
An eyelid marginwart usuallybeginsasa single,
Blepharitis sessile,gray nodule. It may graduallydevelop into
Infectionsof the eyelid margingenerallyinvolve a gray-yellow lobulated, hyperkeratotic excres-
the skin, eyelashes,and associated
glandsanterior cencethat may be predunculatedor filiform. The
to the gray line or mucocutaneousjunction. excisedverruca is fixed in 10% formalin. His-
10 WILHELMUS ET AL. CUMITECH 13A

trates with vascularization.A chalazionis a sterile

lipogranulomathat doesnot require microbiolog-
ical investigation.The roles of lipophilic coryne-
bacteria, Propionibacterium acnes, Malassezia fir-
fur, andDemodex folliculorum (Fig. 3A) in chronic
blepharitis have not been established(68).
In chronic blepharitis, lid margin cultures are
usuallydeferred unlessthe patient is not respond-
ing to standardmeasuresand antibacterial resis-
tance is suspected.Cultures are obtained in the
FIG. 2. Schema for inoculation of conjunctival and same manner as for acute blepharitis, possibly
lid cultures. For conjunctival cultures, horizontal and with the inclusionof Sabouraudagar. Smearsof
vertical streaks are made for right and left conjunctivae, the eyelid marginsare usually not helpful, and
respectively. Conjunctival and lid cultures (“R” and “L” smearsof any conjunctival dischargeassociated
patterns are made for right and left lid margins) from with blepharitis predominantly show neutrophils
both eyes can be inoculated onto a single agar plate. with few microorganisms.Scrapingsor swabbings
of conjunctival or cornea1lesionsassociatedwith
topathological examination can show typical in- chronic staphylococcalblepharitis are usuallynot
tranuclearinclusionbodieswithin epidermalcells needed.A biopsy shouldbe consideredfor atypi-
that contain papovaviruses.Eosinophiliccytoplas- cal, chronic lesionsof the lids sinceneoplasmscan
mic inclusions(Guarnieri bodies)occur in poxvi- simulatechronic unilateral blepharoconjunctivitis.
rus (vaccinia, cowpox, and orf) infections.
Lice infestation
Bacterial blepharitis The pubic crab louse (Pthirus pubis) can infest
Acute infection of the eyelid marginwith ulcer- the eyelashes(16), presumablybecausethe spac-
ation and cellulitis is causedpredominantlyby S. ing of the cilia correspondsto the graspingspanof
aureus and Staphylococcus epidemtidis and rarely the adult’s hindlegs.Both adult lice and nits are
by gram-negativebacteria(32). Staphylococciand readily detectable by slit-lamp biomicroscopy.
streptococcimay superinfectviral blepharoderma- One or more affected cilia can be epilated and
titis. Hyperemia,desquamation,and ulceration of examinedwith a wet mount or oil preparation by
the lateral canthus(angular blepharitis) may be light microscopy(Fig. 3B).
causedby Morclxellainfection or can be a form of
eczematoidblepharitiswith staphylococcalcoloni- Conjunctivitis
zation. The conjunctiva is predisposedto infection by
In acutebacterialblepharitis,a sterile cotton or diverse microorganisms.The principal routes of
calcium alginate swabis moistenedwith TSB or inoculation are airborne droplets, hand-to-eye
sterile saline, scrubbed along both upper and contact, and spreadfrom the ocular adnexa, in-
lower eyelid marginsof the closedeye, and then cluding the lacrimal system,nose, and paranasal
rolled acrossthe surfaceof blood and chocolate sinuses.Microbiologic studiesof the normal con-
agar plates, avoiding the edgesof the plate. This junctival flora are influencedby multiple environ-
procedure is repeated for the other eye. For mental and host factors. Interpreting conjunctival
convenienceandeconomy,materialfrom both the cultures requiresfamiliarity with the relative fre-
right and left eyelid marginsis streakedonto the quency of various bacteriacolonizingthe conjunc-
surfaceof the sameplate in the shapeof an “R” tiva (Table 4). Becausethe normal flora of both
and “L” to designateright and left lids, respec- eyes is usually the same,bilateral bacterial cul-
tively (Fig. 2). Smearsare usually not helpful. tures are helpful in evaluatingunilateral conjunc-
Chronic staphylococcalblepharitisis character- tivitis.
ized by bilateral crusting and thickening of the Acute bacterial, viral, and chlamydialconjunc-
anterior lid margins,loss and whitening of the tivitis can often be distinguishedby the history and
eyelashes,recurrent hordeola, conjunctival hy- clinical signs,thereby simplifying the selectionof
peremia, conjunctival and limbal phlyctenules, diagnosticprocedures.Distinctive signsare often
punctate cornea1erosions,and marginal cornea1 not present in infants, and specimensfrom neo-
infiltrates, Chronic staphylococcalblepharitismay nates with conjunctivitis should aim to identify
coexistwith dry eye syndromeand atopic derma- several likely organisms.Chronic microbial con-
titis. Chronic staphylococcalblepharitisshouldbe junctivitis must be differentiated from various
differentiated from meibomiangland dysfunction toxic and allergic processes.Different cytologic
and rosacea characterized by inspissationand patterns occur during various infectiousand non-
dilatation of the meibomian glands, foam and infectious processes(Table 5), and conjunctival
scurf on the lid margin, recurrent chalazia, con- cytology canbe usedto supporta clinical diagnosis
iunctival hyperemia, and marginal cornea1infil- (43 .
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 11

FIG. 3. Parasites of the eyelid. (A) Demodex folliculorum mite from an epilated eyelash (wet mount, x400). (B)
pthirus pubis louse grasping an eyelash (wet mount, x400).
12 WILHELMUS ET AL. CUMITECH 13A

TABLE 4. Normal conjunctival flora” involved eye and then agitated and squeezedinto
Relative viral transport medium. To avoid a potential
Organism frequency (%) inhibitory effect, the tip of the swabis not broken
Staphylococcus epidermidis ........................... 75-90 into the medium. In bilateral conjunctivitis,
Corynebactetium 20-75
spp ..................................... pooled samplesfrom both eyesmay be inoculated
Propionibacterium acnes ...............................50-70 into one vial. The suspectedresponsiblevirus
Staphylococcus aureus ................................... 10-30 should be indicated on the request form to sim-
Streptococcus spp . ......................................... 2-10 plify processingby the laboratory. Swabbingsfrom
Moraxella spp . ............................................... 2-5 the nasopharynx or throat can also be done.
Haemophilus injluenzae ................................ 2-5 Specimen containers should be placed on ice
Gram-negative rods ...................................... o-5
Fungi .............................................................. o-5 (4°C) during transit but never frozen in a standard
refrigerator ( - 20°C). If prolonged delay before
a Compiled from multiple sources. cell culture is anticipated (greater than 3 days),
the specimencan be frozen at -70°C in dry ice or
Viral conjunctivitis an ultralow freezer.
The principal causesof acuteviral conjunctivitis Giemsa-stainedsmearsare obtained by scrap-
are adenovirusand HSV. Adenovirus is the most ing the everted tarsal conjunctiva, gently blanch-
commoncauseof acute, bilateral keratoconjunc- ing the vesselsby the spatula.A cytobrushusedto
tivitis and ishighly contagious.It is ableto survive collect conjunctival cells is dipped into a buffer
for several days on inanimate objects and in solution for filtration or centrifugation. Cytology
solutions.Acute conjunctivitisis alsoa component during viral conjunctivitis generally showsa pre-
of several viral syndromesincluding influenza, dominantly lymphocytic or mixed mononuclear
infectiousmononucleosis, measles,and Newcastle and polymorphonuclear inflammatory cell pat-
disease.Epidemicsof acute hemorrhagicconjunc- tern. Multinucleated giant cells or intracellular
tivitis due to enterovirus70 or coxsackievirusA24 inclusionsare usuallynot detectedin conjunctival
have been predominantly limited to the coastal material by Giemsacytology.
tropics and to travelers from theseareas. Rapid diagnostictechniquessuch as immuno-
Clinical signsof viral conjunctivitisinclude eye- fluorescenceand enzyme immunoassayare be-
lid edema, preauricular lymphadenopathy, mu- comingcommerciallyavailablefor detectingHSV
coid conjunctival discharge,papillary and follicu- and adenovirusantigensin conjunctival scrapings.
lar conjunctivitis with petechiae, and punctate One or two dropsof a topical anestheticshouldbe
epithelial keratitis. Adenoviral conjunctivitis in instilled, and a spatulashouldbe scrapedover the
toddlers can produce a pseudomembranethat upper and lower tarsalconjunctivae.The material
mimics severe bacterial conjunctivitis. Without is smearedonto a cleanglassslide and then fixed
cutaneousor lid marginlesions,HSV conjunctivi- with acetone,methanol, or spray fixative. In uni-
tis may be indistinguishablefrom adenoviral in- lateral conjunctivitis, it is helpful to obtain mate-
fection. rial from the contralateral conjunctiva as a refer-
Viral cultures should be obtained before the encecontrol.
instillation of a topical anesthetic.A dry cotton or Serologictesting for suspectedviral conjuncti-
polyester swabshouldbe rubbed over the upper vitis is not an expedient considerationin clinical
and lower tarsal conjunctiva and fornix of the practice. Adenovirus, primary HSV, and other

TABLE 5. Cytology of Giemsa-stained conjunctival scrapings

Finding Implication
Predominance of polymorphonuclear leukocytes ...........................Bacterial conjunctivitis
Severe viral or chlamydial conjunctivitis
Allergic conjunctivitis
Chemical or irritative conjunctivitis
Contact lens or prosthetic-induced giant papillary
conjunctivitis
Predominance of lymphocytes and monocytes ................................Adenoviral conjunctivitis
Herpes simplex viral conjunctivitis
Chronic drug-induced or allergic conjunctivitis
Mixed polymorphonuclear and mononuclear leukocytes ..............Chlamydial conjunctivitis
Adenoviral conjunctivitis
Large macrophages (Leber cells), plasma cells, and
blastoid or stem cells ......................................................................Chlamydial conjunctivitis
Eosinophils ...........................................................................................Allergic conjunctivitis
Keratinized epithelial cells .........................*....................................... Tear dysfunction states
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 13

viral conjunctivitis syndromes induce type-specific ice or should be temporarily stored at 4°C until
antibody responses that can be measured on cell culture inoculation. Nasopharyngealswab-
paired serum samples by several techniques. A bingscanalsobe obtainedto increasethe recovery
diagnostic rise in antibody titer typically does not rate.
occur after recurrent herpes simplex conjunctivi- Standard serologictests are generally reserved
tis. for population screening.Becauseof a high back-
ground positivity rate, a singletiter usually is not
Chlamydial conjunctivitis helpful. Serodiagnosisrequires seroconversion,
C. trachomatis producestwo principal forms of from no detectable titer to a positive convales-
keratoconjunctivitis: trachoma, causedpredomi- cent-phasespecimen,or a fourfold or greater rise
nantly by serovarsA, B, Ba, and C, and acute in titer.
conjunctivitis (formerly called inclusion conjunc-
tivitis or paratrachoma), causedby serovarsD, Bacterial conjunctivitis
Da, E, F, G, H, I, J, and K. Active trachoma Adult bacterialconjunctivitis ischaracterizedby
occursfrom repeatedeye-to-eye spreadaided by unilateral or bilateral lid edema,conjunctival hy-
insectvectors and commonfomites. Acute chla- peremiawith chemosisand petechiae,and muco-
mydial conjunctivitis is related to sexually trans- purulent conjunctival discharge,sometimeslead-
mitted genital tract infection. Chlamydialconjunc- ing to membraneor pseudomembrane formation.
tivitis is often bilateral and is characterized by Preauricular lymphadenopathy is usually not a
gradualonset,preauricularlymphadenopathy,fol- feature except with gonococcalconjunctivitis.
licular conjunctivitis, mucopurulent discharge, Multiple bacterial speciescausemild conjuncti-
and pleomorphicpunctate keratitis. vitis, but only a few typically produce severe,
For cytology, scrapingsshouldbe obtainedfrom purulent infection. The principal causesof com-
the inferior and superior tarsal conjunctivae of munity-acquiredbacterial conjunctivitis are S. au-
involved eyesandfixed in 95% methanol.Giemsa- reus, S. pyogenes, S. pneumoniae, H. influenzae, N.
stainedsmearsare examinedfor a characteristic gonorrhoeae, Neisseria meningitidis, and Moraxella
inflammatory cell pattern (88) and the diagnostic species(80). In children under 3 years old, H.
intracytoplasmicbasophilicinclusionsin conjunc- influenzae producessevereconjunctivitis and pre-
tival epithelial cells. septal cellulitis. N. gonowhoeae must be consid-
Immunofluorescence and enzymeimmunoassay ered in anypatient with rapidly progressive,puru-
kits are commercially available for chlamydial lent conjunctivitis and is one of the few bacteria
detection and are often more reliable than Gi- that can penetratean intact cornea1epitheliumto
emsacytology (14). For immunofluorescentstain- produce suppurative keratitis. Pseudomonas spp.
ing, conjunctivalswabbingresultsin epithelial cell and members of the family Enterobacteriaceae
disruptionthat canincreasethe numberof detect- rarely produce conjunctivitis except in the immu-
able elementarybodies (66). The swabis rolled nocompromisedhost, following prolonged hospi-
onto a glassslide in an area 8 to 10 mm in talization, or in the presenceof a cosmeticscleral
diameter,usingall swabsurfaces.After air drying, shell. Corynebacterium diphtheriae infection is no
methanolor acetonefixative is applied. The slide longer encountered. Anaerobic bacteria rarely
is analyzedimmediatelyor refrigerated. If a delay causeacute conjunctivitis.
of morethan 24 h is anticipated,it is frozen at -20 Conjunctivalculturesshouldbe obtainedbefore
or -70°C. the instillation of a topical anesthetic, as these
Conjunctival impressioncytology can be per- agentsand preservativesmay interfere with the
formed for infectious conjunctivitis but is more recovery of certain organisms.Swabbingsmay be
usefulfor other disordersof the ocular surface.A done to evaluate the ocular flora of the conjunc-
piece of celluloseacetate filter paper (Millipore, tiva and tear film (25). Calciumalginate applica-
Bedford, Mass.) is placed onto the bulbar con- tors are preferred becauseof their solubility in
junctiva, pressedlightly, peeled off, and put into liquid media. Moistening the swabwith TSB is
fixative (42, 83). The paper should be cut or unnecessaryin the presenceof conjunctival exu-
marked to indicate the side on which cells are date. The swabis rubbed over the inferior tarsal
collected. conjunctiva and fornix of the right eye and rolled
Conjunctivalculturesshouldbe obtainedbefore on the surface of a blood agar plate. The same
the instillationof a topical anesthetic.A dry rayon swabcan be used to inoculate a chocolate agar
or dacron swabshouldbe rubbed over the upper plate. This swabbingprocedure should be re-
and lower tarsal conjunctivae and fornix and peatedfor the left eye. Since it is convenient and
squeezedinto chlamydialtransport medium.TSB economicalto inoculate material from both eyes
can be substitutedfor transportation, but a viral to the sameplate, the swabfrom the right con-
transport mediumthat containsinhibitory antibi- junctiva is inoculatedin horizontal streaksand the
otics cannot. The vial shouldbe delivered imme- left is inoculated in vertical streaksto designate
diately to the laboratory in a chilled container on the specimensources(Fig. 2). Cultures of the lid
14 WILHELMUS ET AL. CUMITECH 13A

margins are unnecessary except in severe staphy- inoculation into viral transport medium for viral
lococcal blepharoconjunctivitis. Special circum- culture.
stances, such as alteration of host factors or
concurrent infection of the ocular adnexa, may Oculoglandular syndrome
warrant inclusion of Sabouraud agar or an anaer- Granulomatous conjunctivitis with regional
obic medium. lymphadenopathyis most commonly due to cat
Smears should be obtained in all cases of se- scratch disease.Other bacteria and fungi are
vere, purulent conjunctivitis. Stained smears may rarely encountered.Material obtainedby an exci-
not be reliable in establishing the causative micro- sionalbiopsy shouldbe bisected.The half sentfor
organism in nonsevere disease. Organisms can be histopathologicstudiesshouldbe stainedwith the
more readily detected from scrapings from the Warthin-Starry silver impregnation stain. The
conjunctival surface than from swabbings of the other half is ground for attempted culture, usinga
conjunctival discharge. After one or two drops of blood agarplate, thioglycolatebroth, a Sabouraud
proparacaine hydrochloride, a spatula is gently plate, a lysis-centrifugationtube, and tissue-cell
scraped across the lower tarsal conjunctiva so as culture. Other media that are sometimesusedin
not to induce bleeding, and the material is special casesare Lijwenstein-Jensenagar and
smeared in a l-cm-diameter circular area on a glucose-cysteine-telluriteagar.
clean glass slide. Scraping the bulbar or superior
tarsal conjunctiva is generally unnecessary. Keratitis
Smears for Gram stain should be obtained from Intracellular organismssuchascertain viruses,
both conjunctivae. Slides should be immersed in chlamydiae,and protozoa may infect the cornea1
95% methanol for 5 min. Giemsa-stained conjunc- epithelium. Bacteria and fungi usually directly
tival smears are also helpful in distinguishing the invade the stromaand stimulatean inflammatory
cytologic pattern of several forms of infectious, responsethat producessuppurativekeratitis. En-
allergic, and irritative conjunctivitis. Acute bacte- dogenousdiseasesthat can produce interstitial
rial conjunctivitis is characterized by a polymor- keratitis by infectiousor immune-mediatedmech-
phonuclear inflammatory cell response. Papanico- anismsinclude systemicinfections causedby spi-
laou cytology is helpful in the detection of various rochetes.
neoplastic lesions of the conjunctiva that may
simulate chronic infectious conjunctivitis. Viral epithelial keratitis
Viral infection of the cornea1epithelium can
Conjunctivitis in neonates occur during acute conjunctivitis causedby HSV,
Conjunctivitisof the newborncan occur before VZV, and adenovirus. Becauseof the limited
birth by ascendinginfection from the vagina and amount of infected cornea1material that can be
cervix following premature rupture of mem- obtained and the discomfort and potential com-
branes,during delivery from genitourinary secre- plications associatedwith removal of intact cor-
tions, or after birth by contact with contaminated neal epithelium, laboratory diagnosisof acute
materialsor persons.Unlike adult conjunctivitis, superficialkeratoconjunctivitisis accomplishedby
clinical signs are not reliable in guessingthe obtaining conjunctival material. Laboratory con-
responsibleorganism. The principal infectious firmation of viral epithelial keratitis without con-
causesare C. trachomatis, N. gonorrhoeae, S. au- junctivitis is supported by collecting a cornea1
reus, Streptococcus species,and gram-negative epithelial specimen.
bacteria (70). Neonatal chlamydial conjunctivitis Recurrent HSV epithelial keratitis usually ap-
may be accompaniedby nasopharyngitisand pearsas linear or macroulcerativeepithelial ker-
pneumonitis.Pseudomonas conjunctivitis is a rare, atitis. Wiping debridementof a dendrite can re-
but potentially fatal, infection of premature in- move infected epithelial cellsfor culture, smears,
fants. and antigen and DNA detection. HSV is more
Conjunctival cultures and smearsshould be difficult to isolatein geographicepithelial keratitis
obtained. Smears are made from conjunctival and when prior antiviral therapy hasbeen used.
scrapingsfor Gram stain,Giemsaor Papanicolaou A topical anestheticsuchas0.5% proparacaine
staining,andantigendetection. Giemsacytology is hydrochloride is instilled, and the abnormalepi-
more likely to showinclusionsin neonatal chla- thelium is removedwith a swabor spatula,being
mydial conjunctivitis than in adult disease.Swab- careful not to damageBowman’smembrane.The
bingsare inoculatedto blood agar,chocolateagar material is inoculatedinto a vial of viral transport
and/or Thayer-Martin medium, and chlamydial medium. Since swabscan adsorbviral particles
transport medium. If neonatal HSV infection is and reduce the inoculum, the swab tip is com-
suspectedbecauseof facial or generalized skin pressedand twisted in the transport mediumand
lesionswith keratoconjunctivitis,conjunctival ma- then discarded.If the specimencannot be pro-
terial shouldbe obtainedfor direct immunofluo- cessedwithin a few hours, it shouldbe placedin a
rescenceor similar antigen detection and for refrigerator at 4°C. Additional material shouldbe
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 15

smeared onto a glass slide and fixed with acetone and stromal ulceration, dense stromal suppura-
or methanol for immunofluorescence. Cornea1 tion, and iritis. The most common causesare
debridement specimens are generally more likely staphylococci,streptococci,Pseudomonas species,
to reveal HSV than conjunctival scrapings. Moraxella species,Haemophilus species,and cer-
Multinucleated cells can be demonstrated by tain genera of the family Enterobacteriaceae (En-
Giemsa-stained smears. Papanicolaou-stained terobacter, Klebsiella, Proteus, Serratia, etc.). Many
scrapings may show Cowdry type A intranuclear culture-positive cases are polymicrobial, and
inclusions that appear as eosinophilic Lipschutz mixed infections with aerobic and anaerobicbac-
bodies surrounded by a clear halo with clumping teria may be more commonthan previously rec-
of the basophilic chromatin on the nuclear mem- ognized. Nocardia speciesand nontuberculous
brane. mycobacteria(Mycobacterium chelonae andMyco-
A nitrocellulose acetate membrane (Biopore; bacterium fortuitum) are lesscommonly encoun-
Millipore, Bedford, Mass.) may be blotted onto tered. A nonsuppurativeform of bacterial kerati-
the surface of a cornea1 lesion to map specific cell tis, often associatedwith steroid use,is known as
changes and to localize viral antigens within in- infectious crystalline keratopathy and is com-
fected cells (30). For impression cytology, the monly causedby viridans streptococci.
membrane is placed onto the cornea1 lesion, gen- Fungal keratitis can be causedby filamentous
tly pressed with a sterile swab to make a replica of moldsand by yeasts.Filamentousfungal keratitis
the lesion, and peeled off. The material is trans- may follow outdoor trauma, especiallyin warmer
ferred to a container and sent to the laboratory to climates,or corticosteroid treatment of eyeswith
be immediately processed or frozen temporarily at chronic epithelial ulceration. Filamentousfungal
- 2o”c. keratitis is characterized by shallow ulceration,
Dendritic epithelial keratitis caused by VZV is feathery stromalopacities,and multifocal satellite
rarely identified during chickenpox but often oc- infiltrates that can evolve into a densesuppurative
curs during the early stages of ophthalmic zoster. abscess.Common nonpigmented fungi (Monili-
Patients with AIDS may develop persistent zoster aceae) causingfungal keratitis are Fusarium, As-
dendrites. Cornea1 epithelial scrapings or swab- pergillus, Acremonium, Paecilomyces, Penicillium,
bings can reveal VZV by cell culture or immuno- and Pseudallescheria spp. Important pigmented
fluorescence. fungi (Dematiaceae) include Curvularia, Altema-
Recurrent HSV and VZV eye disease may ria, Bipolaris, andPhialophora spp.Yeast keratitis
involve the cornea1 stroma, but the role of active has no geographicpredilection and typically de-
viral replication in stromal keratitis has not been velops in eyes with preexisting ocular surface
established. Infectious virus is typically not iden- disease.Yeast infections are characterizedby de-
tified in cornea1 scrapings from disciform keratitis, marcated yellow-white stromal suppuration and
necrotizing keratitis, or trophic ulceration. By are causedprincipally by Candida albicans and
using cocultivation culture techniques, electron related species.Combined bacterial and fungal
microscopy, and nucleic acid probes, virus has infections can occur.
been identified in the cornea1 stroma from cornea1 A similar, thorough plan for specimencollec-
buttons obtained by penetrating keratoplasty. tion and inoculation should be applied to all
EBV can cause small cornea1 epithelial den- causesof suspectedmicrobial keratitis to enhance
drites. Culture requires fresh human umbilical recovery of all potential microorganisms.Speci-
cord blood lymphocytes that are then processed mens should be collected directly from the in-
for immunofluorescence or nucleic acid hybridiza- fected cornea1 tissue. Direct inoculation onto
tion. Viral antigens and viral DNA can be de- growth media is preferred becauseof the small
tected during cornea1 epithelial infection. The quantities and becauseit simplifiespreciseplace-
laboratory diagnosis of EBV keratitis is usually ment for subsequentrecognition of microbialcol-
based upon detecting a serologic response by onies. The value of material obtained from the
measuring circulating antibodies. eyelid margins and conjunctiva has not been
clearly determined. If available, cultures can be
Bacterial and fungal keratitis taken from the responsibleforeign body, contact
The diagnosisof bacterial and fungal keratitis lens,or contaminatedsolution.
cannotbe established with certainty by the clinical A platinum spatula,disposableblade,bent nee-
features alone. Specimenscollected from the in- dle, surgicalknife, disposablecautery, or similar
fected cornea are processedto isolate the com- instrumentisusedto perform cornea1scrapingsby
mon causesof microbial keratitis, including aero- using a slit-lamp or operating microscope.The
bic and anaerobicbacteria and fungi. Additional rounded, slightly flexible tip of a Kimura platinum
material can alsobe collected for selectiveisola- spatula is ideal for scrapingthe cornea, and it
tion of nontuberculousmycobacteriaand proto- coolsrapidly after heat sterilization in an alcohol-
zoa. lamp flame. The metallic spatula is heated to a
Bacterial keratitis is characterizedby epithelial bright orange and allowed to cool. An assistant
16 WILHELMUS ET AL. CUMITECH 13A

TABLE 6. Suggested sequence of inoculation of

media and preparation of smears in microbial keratitis
1. Blood agar
2. Smear for acridine orange stain
3. Chocolate agar
4 Sabouraud agar
5 Smear for Gram stain
6 Thioglycolate broth
7 Smear for Giemsa or calcofluor white stain
8 Anaerobic agar
9 BHI broth
10 Smear for reserve
11. Nonnutrient agar, if Acanthamoeba spp. suspected
12. Lowenstein-Jensen agar, if nontuberculous
mycobacteria suspected

can speedthe procedureby having severalspatu-

lasready for useeachtime the cornea is scraped.
The eyelidsare held widely apart to reduce inad-
vertent contaminationby the lid marginsor eye-
lashes,and adherent exudate is removed. Aher- FIG. 4. Schema for inoculation of cornea1 scrapings
to an agar plate. Each row of C-shaped marks represents
nate collection methodsincludecornea1swabbing a separate sample.
(8) or rubbingwith a cellulosesponge.Swabbing
collectsmore bacteriathan scraping,but it can be
contaminatedby normal flora in the tear film. diameter. Placing the samplewithin an etched
The cooled spatulais scrapedover the surface circle or a wax-pencil mark avoids needless
of the area of suppuration by a seriesof short, searchingfor the specimen’slocation by labora-
moderately firm strokesin one direction, taking tory personnel. Gelatin-coated glass slides are
care not to touch the lashesor lids. Both the preferred for the modified methenaminesilver
central and peripheral marginsof the infiltrated stain. Prompt flooding or immersionin methanol
area are sampledto obtain material for smears in a Coplin jar for approximately5 min is prefer-
and for inoculation onto various media. Each able to heat fixation sinceit reducesany alteration
scrapingshould be used to inoculate only one of the morphology and stainingcharacteristicsof
mediumor to prepare one smear.Viable organ- the microorganisms.At least two smearsare pre-
ismsmaybe presentthroughout the inflamedarea pared for standardstains,and additional smears
or localizedio the advancingmargin or the ulcer can be held in reserve for specialstainspending
crater. review. A potassiumhydroxide preparation is not
After eachset of scrapings,materialis smeared necessaryfor detecting fungi in cornea1speci-
onto a clean glassslide or is inoculated into a mens.
culture medium. Smearsare prepared from all Scrapingsare plated directly onto culture media
representativeareasof suppuration,and multiple capableof supportinggrowth of mostmicroorgan-
samplesare obtained for each culture medium. ismsknown to causebacterial andfungal keratitis.
Rescrapingsare done after reflaming and cooling Multiple samplesare obtained for each solid
the spatula to obtain additional material from culture medium. Samplesare obtained from all
multiple areasof cornea1suppuration.Notations representative areas of suppuration since the
should be made to identify which inoculation depth and extent of viable organismsvary. It may
markswere collectedfrom different areas.If ma- be necessaryto limit the number of samplesin
terial is limited, high priority shouldbe given to casesof focal, nonseverekeratitis and in threat-
inoculating the blood agar and chocolate agar ened cornea1perforation.
plates and to preparing one or two smears.A Agar plates are inoculated by lightly streaking
purposefulorder of specimencollection (Table 6) both sides of the spatula over the surface to
requiresabout 20 separatesetsof cornea1scrap- produce a row of separateinoculation marks (“C
ings. Inoculation directly onto growth media is streaks”). This systemof inoculation provides a
preferred, but a reduced transport medium at way to distinguishvalid growth from plate contam-
room temperaturecan be usedif it is delivered to ination (Fig. 4). Care should be taken not to
the microbiologylaboratory within one-half hour. penetrate the agar sincerecognition and isolation
Smearsare prepared and fixed in the same of microorganismsin cut streaks of agar are
manneras for conjunctival scrapings.Material is difficult and-diagnosismay thusbe delayed-Liquid
transferredfrom the spatulato an alcohol-cleaned media suchasthioglycolate broth shouldbe inoc-
glassslide over an area approximately 1 cm in ulated by transferring the materia1 from the spat-
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 17

ula to a TSB-saturatedcalcium alginate swab(or contraindicatedin mostcircumstancesbecauseof

by premoistening a swab in thioglycolate and the risk of inoculatingorganismsinto the eye.Two
brushing it acrossthe surface of the ulcer) and possibleexceptionsare deep stromalsuppuration
pushingthe swabto the bottom of the tube. The that cannot be sampledby an anterior approach
shaft of the swabshouldbe graspednear the end, and infections that have extended into the ante-
and that portion shouldbe discardedbefore inser- rior chamber. Samplesof intraocular fluids are
tion into the tube. Saturatingthe swabreducesthe considered if the responsible microorganisms
amount of air deliveredto the bottom of the tube causingsuspectedendophthalmitisare not known.
and might enhancethe growth of anaerobicbac-
teria. BHI broth is inoculated by agitating the Protozoa1 keratitis
spatuladirectly in the broth. Acartthamoebaspeciesare ubiquitous,free-liv-
Perforating cornea1trauma may lead to a deep ing protozoa that are an infrequent cause of
focusof cornea1suppuration.Opening the wound keratitis. Most casesare characterizedby chronic,
is usually safer than obtaining a retrocorneal multifocal, or ring-like stromal suppurationasso-
specimen.For infections confined to the deep ciated with contact lenswear or trauma. Cornea1
cornea, a vertical or oblique incision can allow scrapingsshouldbe collected for direct examina-
samplingwith a needle or minispatula.Another tion andinoculation onto culture media.If culture
method for obtaining material from the deeper plates are not available, Pageamoebasalinecan
stroma is to passa 6-O silk suture through the often be obtained for transport (38).
involved area that is then cut into separatesec- For deeper infections, a cornea1biopsy can be
tions for inoculation onto appropriate culture obtained for both histopathologicalexamination
media. Deep lamellar excisioncan be performed and microbiologicaltesting. Scrapingsat the base
to reach a focal cornea1abscess (10). of the biopsy or superficialkeratectomy site pro-
In the absenceof accessiblecornea1suppura- vide additional material for smearsand inocula-
tion, a cornea1biopsy can be done with a dispos- tion of media. Direct incision into the stromaby
able skin punch or small cornea1trephine (29). using a small spatula or similar instrument may
After marking and incisingthe superficialcornea, allow samplingof a deep focus of inflammation.
the incision is deepenedwith a surgicalblade to Keratoplasty specimensof advanced keratitis
approximately0.2 mm. Lamellardissectionis per- shouldbe sectionedfor laboratory evaluation.
formed with a sharp, spatulalikeknife or micro- A wet mount of cornea1scrapingsor other
surgicalscissors.The biopsy is then carefully ex- material can be preparedusingsalinesolutionfor
cisedwith fine-toothed forceps to avoid crushing standard light microscopyor Nomarski interfer-
the tissue. The intact specimenis placed in a encemicroscopywith phase-contrastillumination.
sterilepetri dishfor sectioning.Additional cornea1 Smearsare preparedfor stainingby immersionor
scrapingscan be obtained from the base of a spray fixation. Trophozoites and cysts can be
partial-thicknesscornea1biopsy. difficult to distinguishfrom inflammatory cells.
A razor blade or microsurgicalscissors can also Acridine orange,Gram, Giemsa,lactophenolcot-
be usedto excisecornea1fragmentsfor inocula- ton blue, andcertain fluorescein-conjugated lectin
tion to solid and liquid media.A freehand lamel- stainscan reveal amoebae.Calcofluorwhite helps
lar dissectionwith a diamondblade or superficial identify amoebic cysts of Acartthamoeba, l%hZ-
keratectomycan excisea singlespecimenor small kampfia, and Hartmannella species.Histopatho-
fragmentsthat can be placedonto culture media. logical sectionscan be stainedwith methenamine
Cornea1tissue obtained by superficial keratec- silver, periodic acid-Schiff, trichrome, and iron-
tomy or by lamellaror penetratingkeratoplasty is hematoxylin-eosin stains. Immunofluorescence
put onto a sterile carrier such as a wet cellulose and immunoperoxidasetechniquesare available
spongeand bisectedfor microbiologicalprocess- in some laboratories (24). Material for culture
ing and pathologicalstudy. isolation can be transported to the laboratory in
Cornea1tissueis transported to the microbiol- Pageamoebasaline,but it is preferable to inocu-
ogy laboratory in a sterile liquid mediumsuchas late specimensdirectly onto culture media.Acan-
TSB or sterilesalinewithout preservatives.Tissue thamoeba spp. grow best on blood agar and on
grinding, emulsification, or chemical digestion nonnutrient agar seededwith an overlay of dead
permitsinoculation of multiple mediaasfor rou- or living E. coli or anothergram-negativerod (48).
tine cornea1scrapings.Small fragments can be Specialmethodsare neededfor the classification
inoculated directly into liquid medium. Smears of Vahlkampfia and Hartmannella spp. (nuclear
are prepared directly from the suspensionor by appearanceduring fission,enzymeprofile, or nu-
crushingtissuefragments. cleic acid sequence).
The hypopyon or inflamedaqueoushumor that Microsporidial keratitis is a rare infection that
frequently accompanies microbialkeratitis iscom- occursin two distinct clinical forms. Encephalito-
posedof acuteinflammatorycellsand containsno zoon hellem causespunctate cornea1epitheliopa-
organisms.Anterior-chamber keratocentesis is thy with mild conjunctivitisin patientswith AIDS.
18 WILHELMUS ET AL. CUMITECH 13A

Nosema corneum producesfocal necrotizing stro- immune-mediatedreaction.An evaluationfor sys-

ma1keratitis following trauma in healthy individ- temic diseaseshould be performed in patients
uals.Scrapingscanreveal microsporidialsporesby with nonsuppurativediffuse,nodular, or necrotiz-
Gram, Giemsa, acid-fast, and silver stains (47). ing scleritis.
Both extracellular and intracytoplasmic spores Suppurative microbial scleritis can occur after
and meronts can be found. Paraffin-embedded scleral trauma, after scleral surgery (such as
cornea1or conjunctival biopsy and keratoplasty scleralbuckling procedures,diathermy, and stra-
specimenscan be examined by phase-contrast bismussurgery), after pterygium excisiontreated
microscopyandwith Brown-Hopps,acid-fast,and with irradiation or antimitotic drugssuch asmit-
periodic acid-Schiff stains. By electron micros- omycin, asan extensionof severemicrobial kera-
copy, classificationis possibleby counting the titis, as an extension of severe endophthalmitis
spirals of the coiled polar filament around the (panophthalmitis), or from a septic embolism.
nucleus. Isolation of microsporidia is difficult, Causes of exogenous microbial scleritis are
even with cell culture. Pseudomonas aeruginosa, S. pneumoniae, S. au-
reus, S. epidemzidis, Proteus species,nontubercu-
Nonsuppurative stromal keratitis lous mycobacteria,Nocardia asteroides, and fila-
Nonulcerativeinterstitial keratitis encompasses mentousand dimorphic fungi.
a range of diffuse, focal, and multifocal forms of Specimensfor smearsand cultures should be
inflammatory (disciform) edemaand necrotizing collected from patients with suppurativescleritis
infiltration. Two specialpatterns are subepithelial as for those with suppurative keratitis. Acid-fast
infiltrates (e.g., viral or chlamydialepithelial ker- staining and inoculation to Lowenstein-Jensen
atitis) and marginal keratitis. The route of entry culture medium should be considered.Any for-
may be from an exogenoussource,through endo- eign material should be removed for culture. A
genousspreadof a systemicdisease via the limbus, scleral biopsy can be obtained if scrapingsare
or by extensionfrom the adjacent sclera or iris. negative.The conjunctivaand Tenon’scapsuleare
Replicatingmicrobesand their antigenselicit var- incised and dissected.Using minimal cauteriza-
ious immunogenicand inflammatory reactions. tion, the necrotic sclera is excisedand bisected.
The individual’s immune responseoften deter- Half is homogenizedfor smearsand culture, and
minesthe presenceand severityof cornea1stromal the other half is placed in fixative for histopatho-
inflammation. logical evaluation with specialstainsfor identifi-
HSV eye diseaseis the most commonnontrau- cation of microorganisms.Scleral buckling ele-
matic causeof nonsuppurativestromal keratitis, ments and other material should be aseptically
but the differential diagnosisincludesseveralin- divided for inoculation to standardmedia.
fectious and immune-mediateddiseases.Other
virusesthat causestromalkeratitis include VZV, Endophthalmitis
EBV, and mumpsvirus. Bacterial causesof inter- Microbial endophthalmitisis the most serious
stitial keratitis are Treponema pallidurn, Borrelia sight-threateningocular infection. The principal
burgdorferi, Mycobactetium tuberculosis, and 1M. route of intraocular infection is exogenous:fol-
Zeprae. Systemicprotozoa1infections that can af- lowing intraocular surgery, associatedwith a fil-
fect the cornea are leishmaniasis and trypanoso- tering bleb, or after perforating injury (especially
miasis.Onchocercalkeratitis is causedby a nem- with an intraocularforeign body). Microorganisms
atode in parts of the developingworld. causing exogenousendophthalmitis may come
Cornea1specimensare not indicated in the from the conjunctival flora or from outside con-
diagnosisof interstitial keratitis. Someorganisms taminants.Endogenousendophthalmitisis an un-
can be identified in skin or other nonocular loca- commoncomplicationof sepsis.
tions. Serologicaltesting can reveal prior expo- The most common causesof postsurgicalen-
dophthalmitis are S. epidemzidis, S. aureus, S.
pneumoniae, other streptococcalspecies,various
Microbial Scleritis gram-negativerods such as Proteus and Pseudo-
Most nontraumaticinflammationsof the sclera monas spp.,and certain anaerobes(61). Infection
are associatedwith rheumatoid arthritis, systemic with organismsof low virulence (e.g., P. acnes, S.
vasculitis,and other immune-mediateddisorders. epidewrtidis, Corynebacterium species,nontubercu-
Immunogenicscleritisis also associatedwith cer- lous mycobacteria, actinomycetes, and some
tain infectiousdiseases, includingophthalmiczos- fungi) may not becomeevident for severalweeks
ter andsyphilis.Scleralinflammationhasoccurred after surgery. Bleb-associatedendophthalmitisis
during active HSV keratitis,Acanthamoeba kera- usually due to streptococci,H. injluenzae, or S.
titis, toxoplasmic retinochoroiditis, and ocular aureus. Posttraumaticendophthalmitiscan be due
toxocariasis.Scleritis during syphilis, Lyme dis- to staphylococci,Bacillus species,other aerobic
ease,tuberculosis,leprosy, and mumpsmay be and anaerobic bacteria, and fungi. Endogenous
due to direct invasionor may be the result of an microbialendophthalmitisoccursmostcommonly
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 19

in compromised hosts or intravenous drug abus- toothed forceps at the opposite limbus can pre-
ers. The most commonly identified organisms that vent inadvertent rotation of the globe. The ante-
can seed the uvea or retinal circulation are C. rior chamber can be reformed with sterile bal-
albicans and related species,Aspergillus species, ancedsalt solution. The needletrack is generally
other filamentousand dimorphic fungi, staphylo- self-sealingand infrequently requiresa 10-Onylon
cocci, streptococci, Bacillus species,and gram- suture.
negativebacteria.Direct invasioninto the globeis A vitreous specimenis obtained by vitreous
uncommon but can occur with fungal keratitis, aspiration or by a vitrectomy. With an intact
microbial scleritis,zygomycosisof the orbit, and posterior capsuleand in phakic patients,the pars
cryptococcalneuroretinitis. plana is entered superotemporallyor superona-
Conjunctival and wound cultures can be ob- sally, 3.5 mm from the limbus in pseudophakia
tained by scraping or swabbing,but organisms and 4 mm in a phakic eye. In an aphakicpatient,
isolated from the ocular surface, even from an the sameparacentesissite usedfor the aqueous
infectedfiltering bleb, may be different from those tap can be usedby directing the needle through
producing endophthalmitis.For endogenousen- the pupil or a peripheral iridectomy to crossthe
dophthalmitis, blood cultures and samplesof anterior hyaloid face. After exposingthe scleraby
urine, cerebrospinalfluid, and infected wounds a conjunctival incision, a 2-mm sclerotomy is
are collected. For endophthalmitisoccurring by prepared parallel to the iimbus. A preplaced
extension from microbial keratitis or scleritis, mattresssuture is positionedfor needletracks of
specimensshouldbe collected from the infected 22 gaugeor greater. Becausethis is difficult to do
eyewall or periocular structures. in a soft eye,this procedureis usuallydone before
The laboratory diagnosisof microbial endoph- the aqueoustap. In an emergencybedsidesitua-
thalmitis requires a systematicapproachfor pro- tion, a sharp instrument can enter the globe
curing intraocular fluids. Samplesof intraocular through the conjunctiva. The vitreous cavity is
fluids shouldbe collectedwheneverpossiblefrom enteredwith a sharpinstrumentsuchas a micro-
patientswith endophthalmitis,ideally in the oper- vitreoretinal blade. A 51%inch,25- or 23-gauge
ating room or in a well-equippedtreatment room needleis inserted and angledposteriorly toward
with a surgicalmicroscope.Vitreous cultures are the optic nerveheadto locate a pocket of liquid
usuallymore likely to yield the responsibleorgan- vitreous, visualizingthe tip through the pupil. The
ism than aqueoussamples.In selectedcasesof needlecan be marked at 5-mmintervals to judge
delayed-onsetendophthalmitis suggestiveof P. the lengthof insertion.Fluid canbe obtainedfrom
acnes infection, the residual lens material and the anterior vitreous cavity in an eyethat hashad
capsulemay needto be removedand examinedto an anterior vitrectomy. A 20- or 21-gaugeneedle
detect sequesteredbacteria. can aspiratevitreous gel, but this risks unneces-
An aqueoustap is done through a limbal ker- sary vitreous traction. Approximately 0.5 to 2 ml
atocentesis.Local anesthesiais provided, and of vitreous isaspiratedfrom the midvitreouscavity
aspirationis performedwith an operating micro- into a 3-ml syringe.
scopeor other magnification.A smallneedlemay A vitreous tap can induce traction at points of
be inserted directly into the temporal limbusby vitreoretinal adhesionand may missa focal vitre-
twistingthe syringewith back-and-forthrotations. ous abscess.Pars plana vitrectomy with direct
A beveledincisionpreparedwith a surgicalknife visualization is preferred to obtain a vitreous
can easeentry with the needle through the tem- specimen.Iris retraction may be neededif pupil-
poral limbus.The needlesize is chosenaccording lary dilatation is inadequate, and fibrin in the
to the anticipatedviscosityof the fibrinous aque- anterior chamber may need to be removed or
ousfluid. With fixation by toothed forceps at the sweptaway.Suction, appliedby an assistantusing
entrance site, a half-inch 30-, 27-, or 25-gauge a syringe, can collect 1 to 2 ml of undiluted
needleattachedto a l-ml syringeis inserted into vitreous prior to turning on the infusion port by
the anterior chamber.Before completeentry, the connectinga 5-ml syringeto a three-waystopcock
plunger is pulled slightly to create a negative or T-valve connector on the aspiration tubing.
pressurewithin the syringe to permit free flow; Vitreous washingscan alsobe collectedin a sterile
alternatively, the plunger can be removed. The cassetteduring vitrectomy by usinginstrumenta-
needleisadvancedalongthe planeof the needle’s tion capableof infusion, suction,and cutting. If a
bevel through the preplaced paracentesis.After collection trap is not provided with the vitrectomy
enteringthe anterior chamber,the syringeis low- instrument, the suction line can be disconnected
ered to elevate the needle point away from the and hooked to a sterile syringefor aspiration.
lensand iris during aspiration.Approximately 0.1 Syringes containing intraocular fluids are
to 0.25 ml of aqueousfluid is aspiratedwhile the capped and taken directly to the microbiology
surgeonobservesthe anterior chamberdepth and laboratory for smearsand inoculation of culture
the assistantmonitorsthe volume being obtained. media.The vitrectomy canisteris sealedand sent
As the needleis withdrawn, countertraction with for vacuum filtration or centrifugation. If only a
20 WILHELMUS ET AL. CUMITECH 13A

small amount of fluid is available, an equal Viral retinitis

amount of TSB can be drawn up into the specimen Congenitalinfection with rubella virus, rubeola
syringe along with a small air bubble. The syringe virus, or mumps virus can cause a diffuse or
is inverted several times to mix the fluids and to multifocal retinopathy. In neonates,primary in-
prevent clot formation. fection by HSV type 1, HSV type 2, VZV, or
Smears are prepared by putting one drop of cytomegalovirus(CMV) can produce retinal ne-
each specimen onto glass slides, spreading each in crosiswith vasculitisand encephalitis.Retinitis in
a circular area 1 cm in diameter with another adults is usually due to hematogenousdissemina-
needle or a spatula to produce a thin smear, and tion during AIDS or other immunosuppression.
allowing them to air dry. A drop of sterile saline or
Human immunodeficiencyvirus (HIV) is associ-
liquid medium can be admixed with viscous spec- ated with a retinal microvasculopathy,and CMV
imens to provide a thin smear that can prevent retinitis is characterizedby a hemorrhagicnecro-
loss of material during fixation. Smears are fixed tizing retinitis. Acute retinal necrosisconsistsof
in methanol for 3 to 5 min and stained by acridine necrotizing retinitis, retinal perivasculitis,and vit-
orange, Gram, and other stains (21). Cytospin reitis and is usually causedby VZV, although
preparations can be made from vitreous washings HSV and CMV have also been identified as
(81). Giemsa-stained smears of intraocular fluids causativeagents.
may aid in recognition of conditions that simulate While many adults have positive serum anti-
microbial endophthalmitis, such as sterile intraoc-body titers to herpesviruses,an extremely high
ular foreign bodies, lens-induced uveitis, meta- titer or rising titers may indicate active infection.
static neoplasms, and ocular toxocariasis. Autochthonous antibodiesin the aqueoushumor
A single drop of intraocular fluid is placed or vitreous can be measured.By comparing the
individually onto the center of each type of solid- amount of specific antibody present in the eye
medium plate (blood agar, chocolate agar, bru- with the amount in the serum, local intraocular
cella agar, and Sabouraud agar) and into liquid- antibody productioncan be inferred. An increased
medium tubes (thioglycolate and BHI) that have ratio of aqueousor vitreous titers in comparison
been allowed to warm to room temperature. To with serum titers suggestsocular involvement.
avoid inadvertently expressing a large portion of Viral antigensin ocular fluids can be assessed by
the fluid to any one medium, the plunger should enzyme-linked immunosorbentassays,immuno-
be gently twisted rather than pushed by constant fluorescence, and other techniques (78). Viral
finger pressure. Plates are tipped slightly to allow
DNA sequencescan be detected in intraocular
fluid to run over the surface for 3 to 4 cm withoutfluids.
reaching the edge. Inoculation sites are allowed to Diagnosticvitrectomy canestablishthe causeof
dry before inverting the plates. The thioglycolate posterior segmentinfection (37). Viral culturesof
broth is inoculated by putting one or two specimen intraocular or subretinalfluids can be performed,
drops onto a saturated calcium alginate swab and although culturesmay be negativebecauseof the
pushing the swab to the bottom of the tube. BHI smallnumberof viral particles or samplingerror.
broth is inoculated by expressing 1 or 2 drops Retinal biopsy is possibleby an external or
directly into the vial. internal approach to investigate progressivein-
Vitrectomy washings can be concentrated by a flammation involving the peripheral retina near
membrane filter or centrifugation system to in- the ora serrata or equator (28, 50). For the
crease the chance of microbial recovery. If this external approach, a pars plana vitrectomy is
equipment is not available, vitreous aspirates can performedwith cauterization or laserphotocoag-
be directly injected into two blood culture bottlesulation around the biopsy site. A deep partial-
(e.g., Septi-chek [Roche Diagnostic Systems, Nut- thicknessscleralflap is fashioned,diathermy de-
ley, N.J.], Bactec [Becton Dickinson, Cockeysville,marcatesthe biopsy edges,and the chorioretinal
Md.], and Signal [Oxoid, Hampshire, England]) biopsy is cleanly excisedwith a blade and micro-
for aerobic and anaerobic incubation (39). Pedi- surgicalscissors.Endoretinal biopsy of a portion
atric bottles are preferred for small volumes, but of detachedretina isprocureddirectly through the
inoculation directly onto culture media speeds pars plana sclerotomy.A lo- to 20-mm2strip of
and enhances recovery. tissueis withdrawn without crushingand placed
onto moistenedsterilepaper. Sectionsare divided
for culture, histopathologicexamination,electron
Infectious Retinitis microscopy,immunopathologicalstaining,and/or
Endogenousinfectionscan spreadto the retina nucleic acid hybridization (63).
by the retinal and choroidal circulation. Opportu- Minced or homogenized tissue is plated on
nistic retinal infections occur in people with appropriate cell lines; cocultivation may be re-
AIDS, transplant recipients, and other immuno- quired. Histopathologicexaminationcan demon-
suppressed individuals,especiallywhenthe CD4+ strate intracellular viral inclusions.Fixatives other
lvmohocvtecount is lessthan 50 cellsper mm3. than formalin, suchasBouin’sfixative, mav make
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