Anemii 2011

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 Cumitech 2A Laboratory Diagnosis of Urinary Tract Infections March 1987 Cumitech 3A Quality Control and Quality Assurance Practices in Clinical Microbiology May 1990 Cumitech 4A Laboratory Diagnosis of Gonorrhea April 1993 Cumitech SA Practical Anaerobic Bacteriology December 1991 Cumitech 6A New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide February 1991 Cumitech 7A Laboratory Diagnosis of Lower Respiratory Tract Infections September 1987 Cumitech 8 Detection of Microbial Antigens by Counterimmunoelectrophoresis December 1978 Cumitech 9 Collection and Processing of Bacteriological Specimens August 1979 Cumitech 10 Laboratory Diagnosis of Upper Respiratory Tract Infections December 1979 Cumitech 11 Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology Laboratory August 1980 Cumitech 12A Laboratory Diagnosis of Bacterial Diarrhea April 1992 Cumitech 13A Laboratory Diagnosis of Ocular Infections September 1994 Cumitech 14A Laboratory Diagnosis of Central Nervous System Infections March 1993 Cumitech 15A Laboratory Diagnosis of Viral Infections August 1994 Cumitech 16 Laboratory Diagnosis of the Mycobacterioses March 1983 Cumitech 17A Laboratory Diagnosis of Female Genital Tract Infections June 1993 Cumitech 18 Laboratory Diagnosis of Hepatitis Viruses January 1984 Cumitech 19 Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections August 1984 Cumitech 20 Therapeutic Drug Monitoring: Antimicrobial Agents October 1984 Cumitech 21 Laboratory Diagnosis of Viral Respiratory Disease March 1986 Cumitech 22 Immunoserology of Staphylococcal Disease August 1987 Cumitech 23 Infections of the Skin and Subcutaneous Tissues June 1988 Cumitech 24 Rapid Detection of Viruses by Immunofluorescence August 1988 Cumitech 25 Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing December 1988 Cumitech 26 Laboratory Diagnosis of Viral Infections Producing Enteritis September 1989 l

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Cumifechs should be cited as follows, e.g.: Wilhelmus, Jones. 1994. Cumitech 13A, Laboratory diagnosis of ocular American Society for Microbiology, Washington, DC.

K. R., T. J. Liesegang, infections. Coordinating

Editorial Board for ASM Cumitechs: Steven C. Specter, Chairman; Janet Hindler, Thomas J. Inzana, Brenda McCurdy, Fredertck S. Nolte, and Stephen Young.

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

Copyright

0 1994 American 1325 Massachusetts Washington,

Mary John

which

Society for Microbiology Ave.. N W. DC 20005

M. S. Osato, and D. B. ed., S. C. Specter.

J. R. Gilchrist, Curt Gleaves, A. Smith, Alice S. Weissfeld,

by the authors as to appropriate may lack the facilities for fully

LABORATORY DIAGNOSIS OF OCULAR INFECTIONS KIRK

R. WILHELMUS,

THOMAS MICHAEL DAN

Cullen

J. LIESEGANG, S. OSATO,

B. JONES,

Department Cullen

Cullen

Eye Institute,

College of Medicine,

of Ophthalmology,

Eye Institute,

Eye Institute,

Baylor

Baylor

Baylor

Mayo

COORDINATING STEVEN Florida

C. SPECTER, 33612

Department

of Medical

Houston,

Florida

32224

Texas 77030

Texas 77030

and Immunology,

SYNOPSIS

The eye and associatedstructuresare uniquely predisposedto infection by a variety of microorganisms.The indicationsand techniquesfor microbiologicalinvestigationsare determinedby the site of ocular or periocular infection, its severity and pace of progression,and knowledge of the responsibleorganisms.Specialproceduresare required to collect and processthe smallquantity of material that can be obtained from ocular tissues and fluids. Efficient handlingof ocular specimens involves the use and interpretation of various laboratory proceduresin infectious eye diseases (35). The appendixlistscommonstainsusedin the ocular microbiology laboratory and a glossaryof clinical terms pertaining to ocular infections and related inflammatory diseases.

Houston,

Jacksonville,

EDITOR

Microbiology

Presentation of the materials n eeded to perform laboratory diagnosis for ocular infections. Descriptions of acute and chronic ocular diseases caused by viruses, bacteria, parasites, and fungi, including dermatoblepharitis, infections of the orbit and lacrimal apparatus, marginal blepharitis, conjunctivitis, keratitis, endophthalmitis, and retinitis. Association of symptoms, etiologic agents, specimens, their methods of collection, and laboratory diagnosis for the various infections. Recommendations on specimen transport to the laboratory and proper specimen handling for intraocular fluids, tissues, and materials that cause eye infections, such as contact lenses, foreign bodies, and contaminated eye drops. Overview of processing and interpretation of laboratory diagnostic techniques, including smears, immune and molecular assays, cultures, antimicrobial susceptibility testing, and serolWY*

Texas 77030

Clinic Jacksonville,

College of Medicine,

College of Medicine,

Houston,

University

of South Florida,

MATERIALS AND MEDIA OCULAR SPECIMENS

Tampa,

FOR

The materials and media suggestedfor the collection, transport, and culture of clinical specimensmust be assembledand maintained(Table 1). Storagein a specialtray in the office or hospital treatment room will simplify ready access.Commonly usedsolidmediacan be obtainedfrom the clinical microbiology laboratory and should be refrigerated at 4°C in plasticbags.Whether in the clinic, surgicalsuite, or hospitalroom, immediate inoculation of ocular specimensfrom the patient to culture media is preferred whenever possible. Transport containersare availablefor biopsyspecimens,biomaterials,and other specialcases.Materials to be shippedelsewhereare securedin a doublemailingpackagewith a biohazardlabel and sentaccordingto current postalregulations.Communicationnetworks are maintainedbetweenoffice and laboratory personnelso that fresh, adequate materialswill be available and test results will be rapidly accessible. Collection

Materials

Fundamentalconsiderationsin the optimal collection of ocular specimensare asfo llows. l

l l

Sufficient material must be collected from the infected site with minimal contaminationfrom adjacentfluids, secretions,or structures. An appropriatecollectiondevice,transport medium, and culture media mustbe used. Material shouldbe collectedearly in the course of infection and in the absenceof recent antimicrobial treatment.

Specimen collection

devices

The Kimura spatula(Storz, St. Louis, MO.) is usefulfor obtainingmaterialfrom the conjunctiva and cornea (Fig. l), although an alcohol lamp is neededfor heat sterilization. The tip coolsrapidly after flaming and is slightly flexible for efficient 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 Cornea Anterior Chamber Iris Conjunctiva: Tarsal Orbital Septum

*Vitreous Lens Posterior Chamber

:.: ...**.* ..*.. :.. : . \k.‘::..* ‘..‘+

/:.. ,-.*.*.: ..*.. *‘;*a .’:. ..*.*.* ..-. * -.. +...“. ..I r :..&.A

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

Lacrimal Sac Canaliculi Puncta Conjunctiva: Bulbar Canthus Lateral

.. . . .. .

CUMITECH

DIAGNOSIS

13A TABLE

Collection

devices

OF OCULAR

INFECTIONS

3

1. Materials for ocular specimen collection Laboratory

supplies

Calcium alginate swabs with plastic shaft Calcium alginate swabs with aluminum shaft Cotton swabs with wood shaft Dacron or rayon swabs with plastic or wire shaft

Glass slides with frosted ends and etched circles Microslide holders

Kimura platinum spatula l&gauge needles

Labels Rapid diagnostic kits for HSV and C. trachomatis

Pencil Wax pencil

Fixatives

Methanol (95%) Acetone (100%) Formalin (10%) Glutaraldehyde (2%) or Trump’s solution (paraformaldehyde [4%] and glutaraldehyde [I%]) Coplin staining jars

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 expedites specimen collection.

of several spatulas Anesthetics

Cotton, polyester, and calcium alginate swab tips are suitablefor the recovery of most bacteria and fungi, althoughcotton-tipped swabsmay contain fatty acids that inhibit bacterial growth. Moistened calcium alginate (Calgiswab; Spectrum, Houston, Tex.) can be dissolvedin a fixed volume of culture medium with hexametaphosphate solutionfor semiquantitativeculturesof the lid margin and conjunctiva (11). Cotton- or polyester-tippedplastic swabsare preferred for viral cultures,and dacron or rayon swabson plastic or wire shafts are used for obtaining chlamydial specimens.Swabs without transport medium should not be used for transporting specimens. Prepackagedtransport tubeswith sterileswabsare availablefor collectingmaterialfor bacterial,viral, and chlamydialcultures. Aspiration with a needle and syringe is the preferred method for collecting fluid or exudate for anaerobicculture. All air bubblesare expelled, and the needletip is stuck into a rubber stopper.

FIG. 1. Platinum (Kimura) spatula. The thin tip (inset) is ideal for obtaining material from the surface of eyelid lesions, conjunctiva, and cornea.

Preservative-freepreparationsare lessantiseptic than multiusetopical anesthetics(5). ProparaCainehydrochloride (0.5%) solution is preferred for obtaining specimensfrom the ocular surface for culture and doesnot interfere with cytological techniques.Sedation may be needed for young children and uncooperativeadults. Slidesand fixatives Glassslideswith frosted endsand etchedcircles (FluorescentAntibody Slides;Becton Dickinson, Lincoln Park, N.J.) facilitate labeling and recognition of smeared material. Slides should be cleansedto remove debristhat can absorbstains. Smearedslidesshouldbe immersedin 95% methanol in a Coplin jar for approximately 5 min for fixation before staining. Cover slips are placed over wet mountswithout fixation and immediately examined.Slidesare sentto the laboratory in slide holders(Control, Friendswood,Tex.) in the order that they were obtained. Cold acetoneis often recommendedfor preparing slides for immunofluorescent staining, although methanolcan be just as effective. Bouin’s fixative is usedfor Papanicolaoustaining.Formalin (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 chosenaccordingto the likely etiology and incubated at an appropriate temperature and atmosphere.

4

WILHELMUS

ET AL. TABLE

2. Culture and transport media for ocular specimens

Medium

Routine media Tryptic soy broth Blood agar plate Chocolate agar plate

Thioglycolate broth Brucella agar plate Sabouraud dextrose agar with gentamicin Special media Blood agar plate Thayer-Martin medium Lowenstein-Jensen or Middlebrook 7HlO agar slant BHI broth with gentamicin Nonnutrient agar plate with E. coli overlay Antimicrobial removal device Blood culture bottle Transport media Viral transport medium Chlamydial transport medium

Bacterial

13A

CUMITECH

Storage temp (“C)

Purpose

Saturation of swabs Aerobic and facultative anaerobic bacteria, fungi Aerobic and facultative anaerobic bacteria, Neisseria and Haemophilus spp. Aerobic and anaerobic bacteria Anaerobic bacteria Fungi Fungi, mycobacteria Neisseria spp. Mycobacteria and Nocardia spp.

5-7 days

4

35 (10% CO,)

5-7 days

25

35

7 days

4 4

35 (anaerobic system) 25

7 days 4 wks

4 4

25 35 (10% COZ) 35

4

spp.

Viruses

4 tis

culture media

Soybean-casein digest(tryptic soy) broth (TSB) is useful for moisteningswabsfor specimencollection. TSB can serveasa general-purposenutrient broth medium for cultures of aerobic and facultatively anaerobicbacteriaand canbe usedto collectviral cultures,althougha transport medium containingantibioticsis preferred. A sterile, nonnutritive balancedsalt solution (e.g., BSS; Alcon Laboratories,Fort Worth, Tex.) can be used for the interim holding of ocular tissues. Defibrinated 5% horse or sheepblood agar is suitablefor the growth of mostbacteria aswell as fungi andAcanthamoeba spp.A MacConkey agar plate or eosin-methyleneblue agar plate can also be inoculated if enteric gram-negativerods are suspected.A chocolate agar plate enriched with vitamins and other supplementssupportsgrowth of Haemophilus, Neisseria, and Moraxella species. Thayer-Martin mediumor oneof its modifications favors growth in suspectedgonococcalor meningococcal infections. Blood and chocolate agar plates should be incubated at 35 to 37°C in a candlejar or inside ziplock bagswith a carbon dioxide generator to create an atmospherecontaining 3 to 10% C02.

Incubation duration

35 (10% COZ)

Remove antibiotics in specimen Aerobic or anaerobic bacteria, fungi C. trachoma

temp (“C)

4 4

Fungi Acanthamoeba

Incubation

4

4-6 wks 7 days 6 wks

25 30 and 35

4 4

wks wks

25 to 35 (until inoculation)

7 days

35

7 days

4 (until inoculation) 4 (until inoculation)

4 wks 2 wks

For anaerobiccultures, specimensare usually inoculated into a liquid medium (75). Thioglycolate broth enriched with vitamin K, and hemin (BBL, Becton Dickinson, Cockeysville, Md.) is recommended.Prereduced, anaerobically sterilized choppedmeat broth and prereduced,anaerobically sterilized brain heart infusion (BHI) broth without antibiotics can also be used for anaerobic incubation. Inoculated tubes are cappedand incubatedat 35 to 37°C.An enriched, fresh or prereduced brucella, Schaedler,Columbia, or Brewer agarbaseis usedfor an anaerobic plate. After inoculation the plate should not be exposed to air any longer than necessaryand shouldbe incubatedin an anaerobicchamberor a heat-sealed envelope (GasPak Pouch [Becton Dickinson, Cockeysville,Md.] and Pouch System [Difco, Detroit, Mich.]) with a gasgenerator and resazurinindicator. If an antibacterialor antifungal agenthasbeen usedbefore specimencollection, an antimicrobial removal devicemight improve microbialrecovery. Approximately 3 ml of the solution and resin is decanted into test tubes, the ocular specimenis introduced into the medium, and aliquots are 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 Laboratory Safety and Quality Assessment at 35 to 37°C for up to 6 weeks. Specimensneed to be transportedto the laboratory in a sturdy, leakproof container with a lid. Fungal culture media 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 absorbentmatepg/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 proslightly thicker than usual (35 to 40 ml per petri mote biological, chemical,radiological,and physdish)and are partially sealedor placedin a bag to ical safety (12). The clinical microbiology laboratory should reduce desiccationduring prolongedincubation. BHI broth containing gentamicin at 50 @ml maintain a quality-assuranceprogram that monihelpsto 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 speciisolation 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 epiAcanthamoeba 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 laboradead microorganisms(such as Escherichiucoli) tory. Parametersof laboratory quality control are enhancesrecovery. An alternative medium for daily temperature records of incubators and reprimary isolationof amoebaeis bufferedcharcoal- frigerators and periodic conduct of performance yeast extract agar, originally designedto isolate standardsfor stains,reagents,media, and suscepLegion& species.Duplicate plates can be incu- tibility tests.The laboratory should seekto minibated 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 properly organized for surveillanceof trends in the epidemiologyof ocular infectionsand for analysis Viral and chlamydial transport media Hanks’ and Leibovitz-Emory media are com- of cumulative antimicrobialresistancepatterns. monly availablefor transportingviral specimens. METHODS FOR COLLECTION OF Salt solutionsaloneshouldnot be used.Swab-tube OCULAR SPECIMENS combinations(Viral Culturette [Becton Dickinson,Cockeysville,Md.], Virocult [Medical Wire & Specimensshould be obtained from the inEquipment, 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). Speccell 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 Dermatoblepharitis can also be used as a viral transport medium. 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. TABLE

Clinical entity

Preseptal cellulitis Acute orbital cellulitis

Canaliculitis

CUMITECH

3. Guide to specimen collection for common ocular infections

Source of material

Principal organisms

Stains and other testsa

Gram

Blood agar, chocolate agar, thioglycolate

Gram

Expressed material

Actinomycetes, streptococci

Gram

Streptococci, S. aureus

Gram

HSV, VZV

IF or EIA, Giemsa Gram

Blood agar, chocolate agar, thioglycolate, anaerobic agar, Sabouraud agar, brain heart infusion Blood agar, chocolate agar, thioglycolate, anaerobic agar Blood agar, chocolate agar, thioglycolate Viral transport medium Blood agar, chocolate agar Blood agar, chocolate agar

S. aureus S. aureus, S. pneumoniae, H. influenzae, N. gonorrhoeae

Gram

IF or EIA

Acute follicular conjunctivitis Chronic follicular conjunctivitis Neonatal conjunctivitis

Conjunctival scraping Conjunctival scraping Conjunctival swabbing

Adenovirus, HSV

Epithelial keratitis

Cornea1 debridement or impression Cornea1 scraping

HSV

C. trachomatis C. trachomatis, gonorrhoeae,

N. S. aureus

S. aureus, P. aeruginosa, S. pneumoniae, Moraxella spp.,

enteric gram-negative rods, C. albicans, Fusarium spp.

Viral transport medium Giemsa, IF Chlamydial transport medium Gram, Giemsa, IF Blood agar, ThayerMartin agar, chlamydial transport medium EIA or IF Viral transport medium Acridine orange, Gram, calcofluor white, acid-fast

Blood agar, chocolate agar, thioglycolate, anaerobic agar, Sabouraud agar Blood agar, chocolate agar, thioglycolate, anaerobic agar, Sabouraud agar, Lowenstein-Jensen Blood agar, chocolate agar, thioglycolate, anaerobic agar, Sabouraud agar, brain heart infusion Viral transport medium

Suppurative scleritis

Scleral scraping

P. aeruginosa, pneumoniae,

S. S. aureus

Acridine orange, Gram, acid-fast

Endophthalmitis

Aqueous humor tap, vitreous tap, vitrectomy washing

S. epidermidis,

S. aureus,

Acridine orange, Gram

Necrotizing retinitis

Media

Drainage from S. pyogenes, S. aureus, wound or abscess H. influenzae (children) Drainage or biopsy S. aureus, streptococci, from abscess or H. injkenzae sinus (children), anaerobes

Acute dacryocystitis Drainage from lacrimal sac Vesicle fluid or lid Acute vesicular margin scraping blepharitis Lid margin Acute marginal blepharitis swabbing Acute purulent Conjunctival conjunctivitis swabbing

Suppurative keratitis

13A

Vitrectomy washing, retinal biopsy

streptococci, P. acnes, H. in.uenzae, gramnegative rods, Bacillus spp., C. albicans CMV, VZV, HSV, C. albicans,

T. gondii

IF, DNA probe

a IF, immunofluorescence; EIA, enzyme immunoassay. Viral dermatoblepharitis

Primary or recurrent herpes simplex virus (HSV) dermatoblepharitisand primary or recurrent varicella-zostervirus (VZV) dermatoblepharitis producea vesicularskin rashwith preauricular iymphadenopathy. Fresh vesicles can be aspiratedor scrapedwith the edgeof a spatula.A Tzanck test (preparinga Giemsa-stained smearof

vesicular scrapings),immunofluorescence studies, viral culture, and electron microscopycanbe done (45, 54). Impetigo

Superficial vesiculopustularor crusting infection of the skin with preauricular lymphadenopathy, 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 wellfor 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 a site of trauma, local infection, or prior skin Granulomatous or pustular dermatoblepharitis Granulomatouseyelid lesionscan occur from lesion. Swabbingsof intact skin are not reliable. chancroid, anthrax, actinomycosis,tuberculosis, The etiology is difficult to establishsince surcandidiasis,blastomycosis,leishmaniasis, and my- rounding orbital edemamay be due to bacterial iasis. A dental burr or cornea1microdrill can exotoxins rather than direct invasion. The techsecuresamplesfrom an ulcerative lesion.Part of nique of injecting and aspirating sterile saline the specimencan be inoculatedonto aerobic and within the subcutaneoustissuesfrom the advancanaerobicculture media, including a Sabouraud ing edge is controversial. Blood and throat culagar plate and Lowenstein-Jensen agar. To iden- tures can be obtained.Although conjunctival cultify 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.Leishplate. 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 (73). Other evidenceof streptococcalinfection is Preseptal cellulitis Infections of the subcutaneouseyelid tissue an acutely elevated anti-DNaseor antihyaluronianterior to the orbital septum may be due to daseantibody titer. trauma,spreadfrom the upper respiratory tract or middle ear in children, or spreadfrom infected Infections of the Lacrimal System skin or adjacent structures (36). Posttraumatic infections are usually due to S. aureus and StrepDacryoadenitis tococcus pyogenes, although anaerobic and polyAcute infections of the lacrimal gland are unmicrobial infections can occur. Nonsuppurative common. Typical clinical features include erypreseptalcellulitis in susceptiblechildren is com- thema andswellingof the outer third of the upper monly causedby Haemophilus influenzae or Strepeyelid, protrusion of the palpebrallobe, conjunctococcus pneumoniae. Secondaryinfection of viral dermatoblepharitis is uncommon. A ruptured tival discharge,and preauricular lymphadenopahordeolum,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 endogenous(during hematogenousspread). other bacteriamay lead to facial cellulitis. 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 contralatis spreadopen. If direct inoculation of media is era1conjunctiva shouldbe cultured for comparinot 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 abscess is identified. helpful.

8

WILHELMUS

ET AL.

Hematogenous acute dacryoadenitis is a rare complication of infectious mononucleosis, mumps, influenza, and gonorrhea. Laboratory diagnosis is based on methods to detect the systemic infection and is not generally aided by conjunctival cultures or smears. Serologic testing for suspected viral syndromes includes antibody titers for EpsteinBarr virus (EBV) and mumps virus. Chronic inflammation of the lacrimal gland may be caused by sarcoidosis, syphilis, tuberculosis, leprosy, and certain fungal and helminthic infections. Serologic tests are available for some of these conditions. If a conjunctival or lacrimal gland biopsy is performed, the specimen can be bisected. One half is submitted for histopathologic examination, and the other half should be ground aseptically and suspended for inoculation onto blood agar and Sabouraud agar. Smears should be prepared for dark-field examination and staining by conventional methods. Canaliculitis

Infections of the lacrimal puncta and canaliculi are rare. Canaliculitisis characterizedby hyperemia and edema at the medial canthus and a dilated, pouting punctumwith mucopurulentdischarge. Inflammation may involve the inferior punctum or both canaliculi and is frequently associatedwith an obstructedcommoncanaliculus. Most infections are due to anaerobessuch as Propionibacterium propionicum and Actinomyces species(9). Mixed aerobicand anaerobicbacterial infectionsmay occur. Purulent material can usually be expressedby compressionof the lid and canaliculus.A spatula shouldbe usedto transfer this material to media and to prepare smears.As theseinfections typically producelarge diverticula that contain cheesy concretions, a sterile spud or small chalazion curette shouldbe usedto scrapethe canaliculusto obtain additional material. Incision into the horizontal portion of the canaliculusmay be necessary.“Sulfur granules”and other particulate matter shouldbe crushedonto a glassslide to obtain a thin smearfor staining. Dacryocystitis

Infection of the lacrimalsacis generallya result of an obstructednasolacrimalduct. Acute dacryocystitis is characterized by epiphora, pain, and distensionof soft tissue at the inner canthus, typically just below the medial canthal ligament. Localizedinflammationmay progressto a dacryocystopyoceleor orbital cellulitis. Responsiblemicroorganismsare S. aureus, S. pneumoniae, S. pyogenes, H injluenzae, and, lesscommonly, aerobic or facultative gram-negativerods or anaerobes. Pressureon the lacrimal sac may express purulent discharge“from the puncta, but infection often obstructsthe distal portion of the canaliculi

CUMITECH

13A

and prevents reflux. Drainage from the lacrimal sacshouldbe swabbedor aspiratedfor smearsand cultures. Transcutaneousaspiration or incision through the wall of the sacis usuallynot indicated, although an external fistula can provide a source of material. Nasal swabbingunder the inferior turbinate may be attempted.Conjunctivalcultures shouldbe obtained in the standardmanner. Chronic dacryocystitisproducesepiphora,with or without pain or discharge.Colonization of the lacrimal sacpresumablyoccursfrom conjunctival flora or by retrograde migration from the nose.A variety of organismsmay contaminate the obstructed sacwithout producing cellulitis. Aerobic and anaerobicbacteria, fungi, and mycobacteria have been isolated. Purulent material should be inoculated onto blood agar, chocolate agar, an anaerobicmedium, and Sabouraudagar. Smears shouldbe prepared for standardstains.A dacryolith should be bisected and submitted for histopathologic and microbiologicstudies. Neonateswith congenital dacryostenosis are at risk for developing chronic dacryocystitis.Acute infection may lead to a dacryocystocele,a bluegray lacrimal sac distention filled with viscous, translucent mucus.Probing is frequently unsuccessfulin drainageof neonateswith dacryocystitis becauseof compressionof the commoncanaliculus. S. aureus, streptococci, and gram-negative rods are the most commoncausativeorganisms. Dacryocystitis is a possiblecomplication of neonatal conjunctivitis due to Neisseria gonowhoeae. Smearsand cultures should be obtained as describedabove. Orbital Acute orbital

Cellulitis

cellulitis

The routes of infection in orbital cellulitis are spreadfrom infected ethmoid andother paranasal sinuses,spread from other adjacent structures (e.g., dental infection, dacryocystitis,and orbital fracture), exogenousinoculation from trauma or surgery,and hematogenousseedingduring sepsis. The mostcommoncausesof acuteorbital cellulitis associatedwith sinusitisare streptococci,S. aureus, and anaerobes.H. influenzae sinusitisand orbital cellulitis may occur in unvaccinatedchildren. Mixed aerobicand anaerobicinfectionsmay occur. S. aureus is a frequent causeof posttraumatic and postsurgicalorbital cellulitis, and polymicrobial infections are common. Blood cultures are often positive during acute orbital cellulitis.Smearsandculturesareprepared from purulent material in the noseduring sinusrelated disease,from swabbings or excisedtissueif a traumatic or surgicalwound is present,or from an infected lacrimal sacor other adnexalsource. In the presenceof an openwoundor drainagesite, material shouldbe obtained bv aspirationwith a

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sterile syringe and needle for immediate inoculation onto appropriate media or for prompt transportation to the laboratory after either embedding the needle into a sterile rubber stopper or injecting the material into an anaerobic transport vial. Drainage of an infected sinus, subperiosteal abscess, or intraorbital abscess is handled by similar techniques and is processed for smears and for aerobic and anaerobic cultures (34). Blind aspiration of the orbit is not indicated. Zygomycosis Acute rhinoorbitocerebral infection by R&opus and iWucor spp. and other nonseptate fungi of the class Zygomycetes is a potentially lethal disease that primarily occursin patients with diabetic or metabolicketoacidosis,neoplasia,and other alterationsof host defenses.Fungal sporesproliferate within the nasalturbinatesand paranasalsinuses, penetrate arterial walls, and spread by vascular and direct extensionto the orbital apex. Surgical debridementof necrotic sinusand orbital tissue should establishdrainage. Material from a necrotic lesionshouldbe submittedfor frozen and paraffin-embeddedhistopathologic sectionsand for smearsand cultures. Biopsy material is transported to the microbiologylaboratory in a sterile tube or petri dish with sterile balancedsalt solution, emulsified, and inoculated to standard growth media. Smearsare examined by Gram, Giemsa,and calcofluor white stainsto detect the broad, nonseptatehyphae that tend to branch at right angles. Chronic orbital cellulitis Indolent orbital infection may occur after placement of alloplasticmaterial for retinal surgeryor orbital fracture repair. Causesinclude anaerobic bacteria,nontuberculousmycobacteria,actinomycetes, and filamentous fungi. Trauma with retained foreign material may produce draining sinustracksthat simulatechronic microbial cellulitis. Orbital aspergillosisis a slowly progressive granulomatousprocesswith thrombotic vasculitis, usually occurring in otherwise healthy adults in tropical areas. Drainagematerial shouldbe inoculated onto a blood agarplate, Sabouraudagar,BHI broth, and Lowenstein-Jensen agar. Orbital biopsy material shouldbe bisectedfor histopathologicaland microbiologic studies. Smears are prepared with Gram, Giemsa, calcofluor white, and acid-fast stains. Blepharitis Infectionsof the eyelid margingenerallyinvolve the skin, eyelashes,and associated glandsanterior to the gray line or mucocutaneousjunction.

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Viral blepharitis Vesicular or erosive marginal blepharitis is caused by HSV or VZV. Vaccinia virus was formerly a causein individualsrecently vaccinated against smallpox. One or more clear vesicles should be selectedfor sampling.The surface of the vesicleis cleansedwith a moistenedapplicator or wiped with an alcohol pad. After puncturing the intact vesicle with a sterile needle, fluid is aspirated with a sterile tuberculin syringe or a capillary hematocrit tube. The base of opened vesiclesand of ulcerative erosionsis scrapedwith a spatulaand/or swabbed.Conjunctival scrapings and/or swabbingscan also be collected. Smears are prepared on glassslides.Giemsastaining of herpesvirusinfections usually showsmultinucleated cells.Intranuclear Cowdry type A bodiescan sometimesbe found with Papanicolaoustaining. Scrapingsof the lid lesion can also be smeared onto a slide for immunofluorescenceor transferred to an enzyme immunoassaysystem.Material for viral isolation is placed into a viral transport mediumthat is chilled or refrigerated at 4°C until it is processed.Acute- and convalescentphaseantibody titers can be obtained to identify seroconversiondiagnosticof a primary viral infection. Molluscum contagiosum produces domeshapednodulesthat becomeumbilicated. Single or multiple lesionsare located on the skin or margin of the eyelids, occasionallyaccompanied by follicular conjunctivitis and punctate cornea1 erosions.The ulcerated area of the lesioncan be scrapedwith a spatula,or the cheesycore can be expressedwith a comedoextractor. Giemsaand other stains(Sedi-Stain;Clay Adams, Parsippany, N.J.) can show myriad tiny dark particles and intracytoplasmicinclusions(Henderson-Patterson bodies) within epidermal keratinocytes that are composedof poxviruses,as can be seenby electron microscopy or immunofluorescentstaining (20). Cytology of the conjunctival dischargetypically showsmononuclearcells.Incompletevirions are found from conjunctival specimens.Viral DNA can be detectedfrom molluscumcontagiosumlesions,but serialpropagationin cell culture is usuallynot successful. An excisedlesionshould be fixed in formalin for light microscopy or in glutaraldehydefor electron microscopyto demonstrate dumbbell-shaped viral particles. Histopathologicalidentification can be facilitated by overnight digestionwith DNase (to degradecellular DNA but not viral DNA protected by its protein capsid)andthen stainingwith the Feulgen techniqueand light green counterstaining. An eyelid marginwart usuallybeginsasa single, sessile,gray nodule. It may graduallydevelop into a gray-yellow lobulated, hyperkeratotic excrescencethat may be predunculatedor filiform. The excisedverruca is fixed in 10% formalin. His-

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trates with vascularization.A chalazionis a sterile lipogranulomathat doesnot require microbiological investigation.The roles of lipophilic corynebacteria, Propionibacterium acnes, Malassezia firfur, andDemodex folliculorum (Fig. 3A) in chronic blepharitis have not been established(68). In chronic blepharitis, lid margin cultures are usuallydeferred unlessthe patient is not responding to standardmeasuresand antibacterial resistance 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 atypithat 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

Acute infection of the eyelid marginwith ulceration and cellulitis is causedpredominantlyby S. aureus and Staphylococcus epidemtidis and rarely by gram-negativebacteria(32). Staphylococciand streptococcimay superinfectviral blepharodermatitis. Hyperemia,desquamation,and ulceration of the lateral canthus(angular blepharitis) may be causedby Morclxellainfection or can be a form of eczematoidblepharitiswith staphylococcalcolonization. In acutebacterialblepharitis,a sterile cotton or calcium alginate swabis moistenedwith TSB or sterile saline, scrubbed along both upper and lower eyelid marginsof the closedeye, and then rolled acrossthe surfaceof blood and chocolate agar plates, avoiding the edgesof the plate. This procedure is repeated for the other eye. For convenienceandeconomy,materialfrom both the right and left eyelid marginsis streakedonto the surfaceof the sameplate in the shapeof an “R” and “L” to designateright and left lids, respectively (Fig. 2). Smearsare usually not helpful. Chronic staphylococcalblepharitisis characterized by bilateral crusting and thickening of the anterior lid margins,loss and whitening of the eyelashes,recurrent hordeola, conjunctival hyperemia, conjunctival and limbal phlyctenules, punctate cornea1erosions,and marginal cornea1 infiltrates, Chronic staphylococcalblepharitismay coexistwith dry eye syndromeand atopic dermatitis. Chronic staphylococcalblepharitisshouldbe differentiated from meibomiangland dysfunction and rosacea characterized by inspissationand dilatation of the meibomian glands, foam and scurf on the lid margin, recurrent chalazia, coniunctival hyperemia, and marginal cornea1infil-

The pubic crab louse (Pthirus pubis) can infest the eyelashes(16), presumablybecausethe spacing of the cilia correspondsto the graspingspanof the adult’s hindlegs.Both adult lice and nits are readily detectable by slit-lamp biomicroscopy. One or more affected cilia can be epilated and examinedwith a wet mount or oil preparation by light microscopy(Fig. 3B). Conjunctivitis

The conjunctiva is predisposedto infection by diverse microorganisms.The principal routes of inoculation are airborne droplets, hand-to-eye contact, and spreadfrom the ocular adnexa, including the lacrimal system,nose, and paranasal sinuses.Microbiologic studiesof the normal conjunctival flora are influencedby multiple environmental and host factors. Interpreting conjunctival cultures requiresfamiliarity with the relative frequency of various bacteriacolonizingthe conjunctiva (Table 4). Becausethe normal flora of both eyes is usually the same,bilateral bacterial cultures are helpful in evaluatingunilateral conjunctivitis. Acute bacterial, viral, and chlamydialconjunctivitis can often be distinguishedby the history and clinical signs,thereby simplifying the selectionof diagnosticprocedures.Distinctive signsare often not present in infants, and specimensfrom neonates with conjunctivitis should aim to identify several likely organisms.Chronic microbial conjunctivitis must be differentiated from various toxic and allergic processes.Different cytologic patterns occur during various infectiousand noninfectious processes(Table 5), and conjunctival cytology canbe usedto supporta clinical diagnosis (43 .

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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).

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4. Normal conjunctival flora” Relative frequency (%)

Organism

75-90 20-75 50-70 10-30 2-10 2-5 2-5 Gram-negative rods ...................................... o-5 Fungi .............................................................. o-5

Staphylococcus epidermidis ........................... Corynebactetium spp ..................................... Propionibacterium acnes ............................... Staphylococcus aureus ................................... Streptococcus spp . ......................................... Moraxella spp . ............................................... Haemophilus injluenzae ................................

a Compiled from multiple sources. Viral conjunctivitis

The principal causesof acuteviral conjunctivitis are adenovirusand HSV. Adenovirus is the most commoncauseof acute, bilateral keratoconjunctivitis and ishighly contagious.It is ableto survive for several days on inanimate objects and in solutions.Acute conjunctivitisis alsoa component of several viral syndromesincluding influenza, infectiousmononucleosis, measles,and Newcastle disease.Epidemicsof acute hemorrhagicconjunctivitis due to enterovirus70 or coxsackievirusA24 have been predominantly limited to the coastal tropics and to travelers from theseareas. Clinical signsof viral conjunctivitisinclude eyelid edema, preauricular lymphadenopathy, mucoid conjunctival discharge,papillary and follicular conjunctivitis with petechiae, and punctate epithelial keratitis. Adenoviral conjunctivitis in toddlers can produce a pseudomembranethat mimics severe bacterial conjunctivitis. Without cutaneousor lid marginlesions,HSV conjunctivitis may be indistinguishablefrom adenoviral infection. Viral cultures should be obtained before the instillation of a topical anesthetic.A dry cotton or polyester swabshouldbe rubbed over the upper and lower tarsal conjunctiva and fornix of the TABLE Finding

Predominance of polymorphonuclear

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involved eye and then agitated and squeezedinto viral transport medium. To avoid a potential inhibitory effect, the tip of the swabis not broken into the medium. In bilateral conjunctivitis, pooled samplesfrom both eyesmay be inoculated into one vial. The suspectedresponsiblevirus should be indicated on the request form to simplify processingby the laboratory. Swabbingsfrom the nasopharynx or throat can also be done. Specimen containers should be placed on ice (4°C) during transit but never frozen in a standard refrigerator ( - 20°C). If prolonged delay before cell culture is anticipated (greater than 3 days), the specimencan be frozen at -70°C in dry ice or an ultralow freezer. Giemsa-stainedsmearsare obtained by scraping the everted tarsal conjunctiva, gently blanching the vesselsby the spatula.A cytobrushusedto collect conjunctival cells is dipped into a buffer solution for filtration or centrifugation. Cytology during viral conjunctivitis generally showsa predominantly lymphocytic or mixed mononuclear and polymorphonuclear inflammatory cell pattern. Multinucleated giant cells or intracellular inclusionsare usuallynot detectedin conjunctival material by Giemsacytology. Rapid diagnostictechniquessuch as immunofluorescenceand enzyme immunoassayare becomingcommerciallyavailablefor detectingHSV and adenovirusantigensin conjunctival scrapings. One or two dropsof a topical anestheticshouldbe instilled, and a spatulashouldbe scrapedover the upper and lower tarsalconjunctivae.The material is smearedonto a cleanglassslide and then fixed with acetone,methanol, or spray fixative. In unilateral conjunctivitis, it is helpful to obtain material from the contralateral conjunctiva as a referencecontrol. Serologictesting for suspectedviral conjunctivitis is not an expedient considerationin clinical practice. Adenovirus, primary HSV, and other

5. Cytology of Giemsa-stained conjunctival scrapings Implication

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

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viral conjunctivitis syndromes induce type-specific antibody responses that can be measured on paired serum samples by several techniques. A diagnostic rise in antibody titer typically does not occur after recurrent herpes simplex conjunctivitis.

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ice or should be temporarily stored at 4°C until cell culture inoculation. Nasopharyngealswabbingscanalsobe obtainedto increasethe recovery rate. Standard serologictests are generally reserved for population screening.Becauseof a high background positivity rate, a singletiter usually is not helpful. Serodiagnosisrequires seroconversion, Chlamydial conjunctivitis C. trachomatis producestwo principal forms of from no detectable titer to a positive convaleskeratoconjunctivitis: 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 conjunctivitis 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 hyinsectvectors and commonfomites. Acute chla- peremiawith chemosisand petechiae,and mucomydial conjunctivitis is related to sexually trans- purulent conjunctival discharge,sometimesleadmitted 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 conjunctiand pleomorphicpunctate keratitis. vitis, but only a few typically produce severe, For cytology, scrapingsshouldbe obtainedfrom purulent infection. The principal causesof comthe inferior and superior tarsal conjunctivae of munity-acquiredbacterial conjunctivitis are S. auinvolved 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 pretival epithelial cells. septal cellulitis. N. gonowhoeae must be considImmunofluorescence and enzymeimmunoassay ered in anypatient with rapidly progressive,purukits 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 immuable elementarybodies (66). The swabis rolled nocompromisedhost, following prolonged hospionto 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 conjuncpiece of celluloseacetate filter paper (Millipore, tiva and tear film (25). Calciumalginate applicaBedford, 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 exumarked 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 reand 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 contransport 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

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margins are unnecessary except in severe staphylococcal blepharoconjunctivitis. Special circumstances, such as alteration of host factors or concurrent infection of the ocular adnexa, may warrant inclusion of Sabouraud agar or an anaerobic medium. Smears should be obtained in all cases of severe, purulent conjunctivitis. Stained smears may not be reliable in establishing the causative microorganism in nonsevere disease. Organisms can be more readily detected from scrapings from the conjunctival surface than from swabbings of the conjunctival discharge. After one or two drops of proparacaine hydrochloride, a spatula is gently scraped across the lower tarsal conjunctiva so as not to induce bleeding, and the material is smeared in a l-cm-diameter circular area on a clean glass slide. Scraping the bulbar or superior tarsal conjunctiva is generally unnecessary. Smears for Gram stain should be obtained from both conjunctivae. Slides should be immersed in 95% methanol for 5 min. Giemsa-stained conjunctival smears are also helpful in distinguishing the cytologic pattern of several forms of infectious, allergic, and irritative conjunctivitis. Acute bacterial conjunctivitis is characterized by a polymorphonuclear inflammatory cell response. Papanicolaou cytology is helpful in the detection of various neoplastic lesions of the conjunctiva that may simulate chronic infectious conjunctivitis. Conjunctivitis

in neonates

Conjunctivitisof the newborncan occur before birth by ascendinginfection from the vagina and cervix following premature rupture of membranes,during delivery from genitourinary secretions, or after birth by contact with contaminated materialsor persons.Unlike adult conjunctivitis, clinical signs are not reliable in guessingthe responsibleorganism. The principal infectious causesare C. trachomatis, N. gonorrhoeae, S. aureus, Streptococcus species,and gram-negative bacteria (70). Neonatal chlamydial conjunctivitis may be accompaniedby nasopharyngitisand pneumonitis.Pseudomonas conjunctivitis is a rare, but potentially fatal, infection of premature infants. Conjunctival cultures and smearsshould be obtained. Smears are made from conjunctival scrapingsfor Gram stain,Giemsaor Papanicolaou staining,andantigendetection. Giemsacytology is more likely to showinclusionsin neonatal chlamydial conjunctivitis than in adult disease.Swabbingsare inoculatedto blood agar,chocolateagar and/or Thayer-Martin medium, and chlamydial transport medium. If neonatal HSV infection is suspectedbecauseof facial or generalized skin lesionswith keratoconjunctivitis,conjunctival material shouldbe obtainedfor direct immunofluorescenceor similar antigen detection and for

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inoculation into viral transport medium for viral culture. Oculoglandular

syndrome

Granulomatous conjunctivitis with regional lymphadenopathyis most commonly due to cat scratch disease.Other bacteria and fungi are rarely encountered.Material obtainedby an excisionalbiopsy shouldbe bisected.The half sentfor histopathologicstudiesshouldbe stainedwith the Warthin-Starry silver impregnation stain. The other half is ground for attempted culture, usinga blood agarplate, thioglycolatebroth, a Sabouraud plate, a lysis-centrifugationtube, and tissue-cell culture. Other media that are sometimesusedin special casesare Lijwenstein-Jensenagar and glucose-cysteine-telluriteagar. Keratitis

Intracellular organismssuchascertain viruses, chlamydiae,and protozoa may infect the cornea1 epithelium. Bacteria and fungi usually directly invade the stromaand stimulatean inflammatory responsethat producessuppurativekeratitis. Endogenousdiseasesthat can produce interstitial keratitis by infectiousor immune-mediatedmechanismsinclude systemicinfections causedby spirochetes. Viral epithelial

keratitis

Viral infection of the cornea1epithelium can occur during acute conjunctivitis causedby HSV, VZV, and adenovirus. Becauseof the limited amount of infected cornea1material that can be obtained and the discomfort and potential complications associatedwith removal of intact corneal epithelium, laboratory diagnosisof acute superficialkeratoconjunctivitisis accomplishedby obtaining conjunctival material. Laboratory confirmation of viral epithelial keratitis without conjunctivitis is supported by collecting a cornea1 epithelial specimen. Recurrent HSV epithelial keratitis usually appearsas linear or macroulcerativeepithelial keratitis. Wiping debridementof a dendrite can remove infected epithelial cellsfor culture, smears, and antigen and DNA detection. HSV is more difficult to isolatein geographicepithelial keratitis and when prior antiviral therapy hasbeen used. A topical anestheticsuchas0.5% proparacaine hydrochloride is instilled, and the abnormalepithelium is removedwith a swabor spatula,being careful not to damageBowman’smembrane.The material is inoculatedinto a vial of viral transport medium. Since swabscan adsorbviral particles and reduce the inoculum, the swab tip is compressedand twisted in the transport mediumand then discarded.If the specimencannot be processedwithin a few hours, it shouldbe placedin a refrigerator at 4°C. Additional material shouldbe

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smeared onto a glass slide and fixed with acetone and stromal ulceration, dense stromal suppuraor 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 cerMultinucleated cells can be demonstrated by tain genera of the family Enterobacteriaceae (EnGiemsa-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 anaerobicbacbodies surrounded by a clear halo with clumping teria may be more commonthan previously recof the basophilic chromatin on the nuclear mem- ognized. Nocardia speciesand nontuberculous brane. mycobacteria(Mycobacterium chelonae andMycoA nitrocellulose acetate membrane (Biopore; bacterium fortuitum) are lesscommonly encounMillipore, Bedford, Mass.) may be blotted onto tered. A nonsuppurativeform of bacterial keratithe 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 comfected cells (30). For impression cytology, the monly causedby viridans streptococci. membrane is placed onto the cornea1 lesion, genFungal 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 transmay 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 (MoniliPatients with AIDS may develop persistent zoster aceae) causingfungal keratitis are Fusarium, Asdendrites. Cornea1 epithelial scrapings or swab- pergillus, Acremonium, Paecilomyces, Penicillium, bings can reveal VZV by cell culture or immunoand Pseudallescheria spp. Important pigmented fluorescence. fungi (Dematiaceae) include Curvularia, AltemaRecurrent 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 deviral 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 detified 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 specimencollecbuttons 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.Specicord blood lymphocytes that are then processed mens should be collected directly from the infor immunofluorescence or nucleic acid hybridizafected 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 simplifiespreciseplacelaboratory diagnosis of EBV keratitis is usually ment for subsequentrecognition of microbialcolbased 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 neefeatures 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 alcoholzoa. lamp flame. The metallic spatula is heated to a Bacterial keratitis is characterizedby epithelial bright orange and allowed to cool. An assistant

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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 severalspatulasready for useeachtime the cornea is scraped. The eyelidsare held widely apart to reduce inadvertent contaminationby the lid marginsor eyelashes,and adherent exudate is removed. Ahernate collection methodsincludecornea1swabbing (8) or rubbingwith a cellulosesponge.Swabbing collectsmore bacteriathan scraping,but it can be contaminatedby normal flora in the tear film. The cooled spatulais scrapedover the surface of the area of suppuration by a seriesof short, moderately firm strokesin one direction, taking care not to touch the lashesor lids. Both the central and peripheral marginsof the infiltrated area are sampledto obtain material for smears and for inoculation onto various media. Each scrapingshould be used to inoculate only one mediumor to prepare one smear.Viable organismsmaybe presentthroughout the inflamedarea or localizedio the advancingmargin or the ulcer crater. After eachset of scrapings,materialis smeared onto a clean glassslide or is inoculated into a culture medium. Smearsare prepared from all representativeareasof suppuration,and multiple samplesare obtained for each culture medium. Rescrapingsare done after reflaming and cooling the spatula to obtain additional material from multiple areasof cornea1suppuration.Notations should be made to identify which inoculation markswere collectedfrom different areas.If material is limited, high priority shouldbe given to inoculating the blood agar and chocolate agar plates and to preparing one or two smears.A purposefulorder of specimencollection (Table 6) requiresabout 20 separatesetsof cornea1scrapings. Inoculation directly onto growth media is preferred, but a reduced transport medium at room temperaturecan be usedif it is delivered to the microbiologylaboratory within one-half hour. Smearsare prepared and fixed in the same manneras for conjunctival scrapings.Material is transferredfrom the spatulato an alcohol-cleaned glassslide over an area approximately 1 cm in

FIG. 4. Schema for inoculation of cornea1 scrapings to an agar plate. Each row of C-shaped marks represents a separate sample.

diameter. Placing the samplewithin an etched circle or a wax-pencil mark avoids needless searchingfor the specimen’slocation by laboratory personnel. Gelatin-coated glass slides are preferred for the modified methenaminesilver stain. Prompt flooding or immersionin methanol in a Coplin jar for approximately5 min is preferable to heat fixation sinceit reducesany alteration of the morphology and stainingcharacteristicsof the microorganisms.At least two smearsare prepared for standardstains,and additional smears can be held in reserve for specialstainspending review. A potassiumhydroxide preparation is not necessaryfor detecting fungi in cornea1specimens. Scrapingsare plated directly onto culture media capableof supportinggrowth of mostmicroorganismsknown to causebacterial andfungal keratitis. Multiple samplesare obtained for each solid culture medium. Samplesare obtained from all representative areas of suppuration since the depth and extent of viable organismsvary. It may be necessaryto limit the number of samplesin casesof focal, nonseverekeratitis and in threatened cornea1perforation. Agar plates are inoculated by lightly streaking both sides of the spatula over the surface to produce a row of separateinoculation marks (“C streaks”). This systemof inoculation provides a way to distinguishvalid growth from plate contamination (Fig. 4). Care should be taken not to penetrate the agar sincerecognition and isolation of microorganismsin cut streaks of agar are difficult and-diagnosis may thusbe delayed-Liquid media suchasthioglycolate broth shouldbe inoculated by transferring the materia1 from the spat-

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ula to a TSB-saturatedcalcium alginate swab(or by premoistening a swab in thioglycolate and brushing it acrossthe surface of the ulcer) and pushingthe swabto the bottom of the tube. The shaft of the swabshouldbe graspednear the end, and that portion shouldbe discardedbefore insertion into the tube. Saturatingthe swabreducesthe amount of air deliveredto the bottom of the tube and might enhancethe growth of anaerobicbacteria. BHI broth is inoculated by agitating the spatuladirectly in the broth. Perforating cornea1trauma may lead to a deep focusof cornea1suppuration.Opening the wound is usually safer than obtaining a retrocorneal specimen.For infections confined to the deep cornea, a vertical or oblique incision can allow samplingwith a needle or minispatula.Another method for obtaining material from the deeper stroma is to passa 6-O silk suture through the involved area that is then cut into separatesections for inoculation onto appropriate culture media. Deep lamellar excisioncan be performed to reach a focal cornea1abscess (10). In the absenceof accessiblecornea1suppuration, a cornea1biopsy can be done with a disposable skin punch or small cornea1trephine (29). After marking and incisingthe superficialcornea, the incision is deepenedwith a surgicalblade to approximately0.2 mm. Lamellardissectionis performed with a sharp, spatulalikeknife or microsurgicalscissors.The biopsy is then carefully excisedwith fine-toothed forceps to avoid crushing the tissue. The intact specimenis placed in a sterilepetri dishfor sectioning.Additional cornea1 scrapingscan be obtained from the base of a partial-thicknesscornea1biopsy. A razor blade or microsurgicalscissors can also be usedto excisecornea1fragmentsfor inoculation to solid and liquid media.A freehand lamellar dissectionwith a diamondblade or superficial keratectomycan excisea singlespecimenor small fragmentsthat can be placedonto culture media. Cornea1tissue obtained by superficial keratectomy or by lamellaror penetratingkeratoplasty is put onto a sterile carrier such as a wet cellulose spongeand bisectedfor microbiologicalprocessing and pathologicalstudy. Cornea1tissueis transported to the microbiology laboratory in a sterile liquid mediumsuchas TSB or sterilesalinewithout preservatives.Tissue grinding, emulsification, or chemical digestion permitsinoculation of multiple mediaasfor routine cornea1scrapings.Small fragments can be inoculated directly into liquid medium. Smears are prepared directly from the suspensionor by crushingtissuefragments. The hypopyon or inflamedaqueoushumor that frequently accompanies microbialkeratitis iscomposedof acuteinflammatorycellsand containsno organisms.Anterior-chamber keratocentesis is

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contraindicatedin mostcircumstancesbecauseof the risk of inoculatingorganismsinto the eye.Two possibleexceptionsare deep stromalsuppuration that cannot be sampledby an anterior approach and infections that have extended into the anterior chamber. Samplesof intraocular fluids are considered if the responsible microorganisms causingsuspectedendophthalmitisare not known. Protozoa1

keratitis

Acartthamoebaspeciesare ubiquitous,free-living protozoa that are an infrequent cause of keratitis. Most casesare characterizedby chronic, multifocal, or ring-like stromal suppurationassociated with contact lenswear or trauma. Cornea1 scrapingsshouldbe collected for direct examination andinoculation onto culture media.If culture plates are not available, Pageamoebasalinecan often be obtained for transport (38). For deeper infections, a cornea1biopsy can be obtained for both histopathologicalexamination and microbiologicaltesting. Scrapingsat the base of the biopsy or superficialkeratectomy site provide additional material for smearsand inoculation of media. Direct incision into the stromaby using a small spatula or similar instrument may allow samplingof a deep focus of inflammation. Keratoplasty specimensof advanced keratitis shouldbe sectionedfor laboratory evaluation. A wet mount of cornea1scrapingsor other material can be preparedusingsalinesolutionfor standard light microscopyor Nomarski interferencemicroscopywith phase-contrastillumination. Smearsare preparedfor stainingby immersionor spray fixation. Trophozoites and cysts can be difficult to distinguishfrom inflammatory cells. Acridine orange,Gram, Giemsa,lactophenolcotton blue, andcertain fluorescein-conjugated lectin stainscan reveal amoebae.Calcofluorwhite helps identify amoebic cysts of Acartthamoeba, l%hZkampfia, and Hartmannella species.Histopathological sectionscan be stainedwith methenamine silver, periodic acid-Schiff, trichrome, and ironhematoxylin-eosin stains. Immunofluorescence and immunoperoxidasetechniquesare available in some laboratories (24). Material for culture isolation can be transported to the laboratory in Pageamoebasaline,but it is preferable to inoculate specimensdirectly onto culture media.Acanthamoeba spp. grow best on blood agar and on nonnutrient agar seededwith an overlay of dead or living E. coli or anothergram-negativerod (48). Specialmethodsare neededfor the classification of Vahlkampfia and Hartmannella spp. (nuclear appearanceduring fission,enzymeprofile, or nucleic acid sequence). Microsporidial keratitis is a rare infection that occursin two distinct clinical forms. Encephalitozoon hellem causespunctate cornea1epitheliopathy with mild conjunctivitisin patientswith AIDS.

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Nosema corneum producesfocal necrotizing stro-

ma1keratitis following trauma in healthy individuals.Scrapingscanreveal microsporidialsporesby Gram, Giemsa, acid-fast, and silver stains (47). Both extracellular and intracytoplasmic spores and meronts can be found. Paraffin-embedded cornea1or conjunctival biopsy and keratoplasty specimenscan be examined by phase-contrast microscopyandwith Brown-Hopps,acid-fast,and periodic acid-Schiff stains. By electron microscopy, classificationis possibleby counting the spirals of the coiled polar filament around the nucleus. Isolation of microsporidia is difficult, even with cell culture. Nonsuppurative

stromal keratitis

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immune-mediatedreaction.An evaluationfor systemic diseaseshould be performed in patients with nonsuppurativediffuse,nodular, or necrotizing scleritis. Suppurative microbial scleritis can occur after scleral trauma, after scleral surgery (such as scleralbuckling procedures,diathermy, and strabismussurgery), after pterygium excisiontreated with irradiation or antimitotic drugssuch asmitomycin, asan extensionof severemicrobial keratitis, as an extension of severe endophthalmitis (panophthalmitis), or from a septic embolism. Causes of exogenous microbial scleritis are Pseudomonas aeruginosa, S. pneumoniae, S. aureus, S. epidemzidis, Proteus species,nontuberculous mycobacteria,Nocardia asteroides, and fila-

Nonulcerativeinterstitial keratitis encompasses mentousand dimorphic fungi. Specimensfor smearsand cultures should be a range of diffuse, focal, and multifocal forms of 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 formay 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 cauterizaious 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 histopathoinflammation. logical evaluation with specialstainsfor identifiHSV eye diseaseis the most commonnontrau- cation of microorganisms.Scleral buckling elematic causeof nonsuppurativestromal keratitis, ments and other material should be aseptically but the differential diagnosisincludesseveralin- divided for inoculation to standardmedia. fectious and immune-mediateddiseases.Other Endophthalmitis virusesthat causestromalkeratitis include VZV, EBV, and mumpsvirus. Bacterial causesof interMicrobial endophthalmitisis the most serious sight-threateningocular infection. The principal stitial keratitis are Treponema pallidurn, Borrelia burgdorferi, Mycobactetium tuberculosis, and 1M. route of intraocular infection is exogenous:folZeprae. Systemicprotozoa1infections that can af- lowing intraocular surgery, associatedwith a filfect 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 condiagnosisof interstitial keratitis. Someorganisms taminants.Endogenousendophthalmitisis an uncan be identified in skin or other nonocular loca- commoncomplicationof sepsis. tions. Serologicaltesting can reveal prior expoThe most common causesof postsurgicalendophthalmitis are S. epidemzidis, S. aureus, S. pneumoniae, other streptococcalspecies,various Microbial Scleritis gram-negativerods such as Proteus and PseudoMost 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,nontubercuImmunogenicscleritisis 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

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in compromised hosts or intravenous drug abusers. The most commonly identified organisms that can seed the uvea or retinal circulation are C. albicans and related species,Aspergillus species, other filamentousand dimorphic fungi, staphylococci, streptococci, Bacillus species,and gramnegativebacteria.Direct invasioninto the globeis uncommon but can occur with fungal keratitis, microbial scleritis,zygomycosisof the orbit, and cryptococcalneuroretinitis. Conjunctival and wound cultures can be obtained by scraping or swabbing,but organisms isolated from the ocular surface, even from an infectedfiltering bleb, may be different from those producing endophthalmitis.For endogenousendophthalmitis, blood cultures and samplesof urine, cerebrospinalfluid, and infected wounds are collected. For endophthalmitisoccurring by extension from microbial keratitis or scleritis, specimensshouldbe collected from the infected eyewall or periocular structures. The laboratory diagnosisof microbial endophthalmitis requires a systematicapproachfor procuring intraocular fluids. Samplesof intraocular fluids shouldbe collectedwheneverpossiblefrom patientswith endophthalmitis,ideally in the operating room or in a well-equippedtreatment room with a surgicalmicroscope.Vitreous cultures are usuallymore likely to yield the responsibleorganism than aqueoussamples.In selectedcasesof delayed-onsetendophthalmitis suggestiveof P. acnes infection, the residual lens material and capsulemay needto be removedand examinedto detect sequesteredbacteria. An aqueoustap is done through a limbal keratocentesis.Local anesthesiais provided, and aspirationis performedwith an operating microscopeor other magnification.A smallneedlemay be inserted directly into the temporal limbusby twistingthe syringewith back-and-forthrotations. A beveledincisionpreparedwith a surgicalknife can easeentry with the needle through the temporal limbus.The needlesize is chosenaccording to the anticipatedviscosityof the fibrinous aqueousfluid. With fixation by toothed forceps at the entrance site, a half-inch 30-, 27-, or 25-gauge needleattachedto a l-ml syringeis inserted into the anterior chamber.Before completeentry, the plunger is pulled slightly to create a negative pressurewithin the syringe to permit free flow; alternatively, the plunger can be removed. The needleisadvancedalongthe planeof the needle’s bevel through the preplaced paracentesis.After enteringthe anterior chamber,the syringeis lowered to elevate the needle point away from the lensand iris during aspiration.Approximately 0.1 to 0.25 ml of aqueousfluid is aspiratedwhile the surgeonobservesthe anterior chamberdepth and the assistantmonitorsthe volume being obtained. As the needleis withdrawn, countertraction with

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toothed forceps at the opposite limbus can prevent inadvertent rotation of the globe. The anterior chamber can be reformed with sterile balancedsalt solution. The needletrack is generally self-sealingand infrequently requiresa 10-Onylon suture. A vitreous specimenis obtained by vitreous aspiration or by a vitrectomy. With an intact posterior capsuleand in phakic patients,the pars plana is entered superotemporallyor superonasally, 3.5 mm from the limbus in pseudophakia and 4 mm in a phakic eye. In an aphakicpatient, the sameparacentesissite usedfor the aqueous tap can be usedby directing the needle through the pupil or a peripheral iridectomy to crossthe anterior hyaloid face. After exposingthe scleraby a conjunctival incision, a 2-mm sclerotomy is prepared parallel to the iimbus. A preplaced mattresssuture is positionedfor needletracks of 22 gaugeor greater. Becausethis is difficult to do in a soft eye,this procedureis usuallydone before the aqueoustap. In an emergencybedsidesituation, a sharp instrument can enter the globe through the conjunctiva. The vitreous cavity is enteredwith a sharpinstrumentsuchas a microvitreoretinal blade. A 51%inch,25- or 23-gauge needleis inserted and angledposteriorly toward the optic nerveheadto locate a pocket of liquid vitreous, visualizingthe tip through the pupil. The needlecan be marked at 5-mmintervals to judge the lengthof insertion.Fluid canbe obtainedfrom the anterior vitreous cavity in an eyethat hashad an anterior vitrectomy. A 20- or 21-gaugeneedle can aspiratevitreous gel, but this risks unnecessary vitreous traction. Approximately 0.5 to 2 ml of vitreous isaspiratedfrom the midvitreouscavity into a 3-ml syringe. A vitreous tap can induce traction at points of vitreoretinal adhesionand may missa focal vitreous abscess.Pars plana vitrectomy with direct visualization is preferred to obtain a vitreous specimen.Iris retraction may be neededif pupillary dilatation is inadequate, and fibrin in the anterior chamber may need to be removed or sweptaway.Suction, appliedby an assistantusing a syringe, can collect 1 to 2 ml of undiluted vitreous prior to turning on the infusion port by connectinga 5-ml syringeto a three-waystopcock or T-valve connector on the aspiration tubing. Vitreous washingscan alsobe collectedin a sterile cassetteduring vitrectomy by usinginstrumentation capableof infusion, suction,and cutting. If a collection trap is not provided with the vitrectomy instrument, the suction line can be disconnected and hooked to a sterile syringefor aspiration. Syringes containing intraocular fluids are capped and taken directly to the microbiology laboratory for smearsand inoculation of culture media.The vitrectomy canisteris sealedand sent for vacuum filtration or centrifugation. If only a

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small amount of fluid is available, an equal amount of TSB can be drawn up into the specimen syringe along with a small air bubble. The syringe is inverted several times to mix the fluids and to prevent clot formation. Smears are prepared by putting one drop of each specimen onto glass slides, spreading each in a circular area 1 cm in diameter with another needle or a spatula to produce a thin smear, and allowing them to air dry. A drop of sterile saline or liquid medium can be admixed with viscous specimens to provide a thin smear that can prevent loss of material during fixation. Smears are fixed in methanol for 3 to 5 min and stained by acridine orange, Gram, and other stains (21). Cytospin preparations can be made from vitreous washings (81). Giemsa-stained smears of intraocular fluids may aid in recognition of conditions that simulate microbial endophthalmitis, such as sterile intraocular foreign bodies, lens-induced uveitis, metastatic neoplasms, and ocular toxocariasis. A single drop of intraocular fluid is placed individually onto the center of each type of solidmedium plate (blood agar, chocolate agar, brucella agar, and Sabouraud agar) and into liquidmedium tubes (thioglycolate and BHI) that have been allowed to warm to room temperature. To avoid inadvertently expressing a large portion of the fluid to any one medium, the plunger should be gently twisted rather than pushed by constant finger pressure. Plates are tipped slightly to allow fluid to run over the surface for 3 to 4 cm without reaching the edge. Inoculation sites are allowed to dry before inverting the plates. The thioglycolate broth is inoculated by putting one or two specimen drops onto a saturated calcium alginate swab and pushing the swab to the bottom of the tube. BHI broth is inoculated by expressing 1 or 2 drops directly into the vial. Vitrectomy washings can be concentrated by a membrane filter or centrifugation system to increase the chance of microbial recovery. If this equipment is not available, vitreous aspirates can be directly injected into two blood culture bottles (e.g., Septi-chek [Roche Diagnostic Systems, Nutley, N.J.], Bactec [Becton Dickinson, Cockeysville, Md.], and Signal [Oxoid, Hampshire, England]) for aerobic and anaerobic incubation (39). Pediatric bottles are preferred for small volumes, but inoculation directly onto culture media speeds and enhances recovery.

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Viral retinitis

Congenitalinfection with rubella virus, rubeola virus, or mumps virus can cause a diffuse or multifocal retinopathy. In neonates,primary infection by HSV type 1, HSV type 2, VZV, or cytomegalovirus(CMV) can produce retinal necrosiswith vasculitisand encephalitis.Retinitis in adults is usually due to hematogenousdissemination during AIDS or other immunosuppression. Human immunodeficiencyvirus (HIV) is associated with a retinal microvasculopathy,and CMV retinitis is characterizedby a hemorrhagicnecrotizing retinitis. Acute retinal necrosisconsistsof necrotizing retinitis, retinal perivasculitis,and vitreitis and is usually causedby VZV, although HSV and CMV have also been identified as causativeagents. While many adults have positive serum antibody titers to herpesviruses,an extremely high titer or rising titers may indicate active infection. Autochthonous antibodiesin the aqueoushumor or vitreous can be measured.By comparing the amount of specific antibody present in the eye with the amount in the serum, local intraocular antibody productioncan be inferred. An increased ratio of aqueousor vitreous titers in comparison with serum titers suggestsocular involvement. Viral antigensin ocular fluids can be assessed by enzyme-linked immunosorbentassays,immunofluorescence, and other techniques (78). Viral DNA sequencescan be detected in intraocular fluids. Diagnosticvitrectomy canestablishthe causeof posterior segmentinfection (37). Viral culturesof intraocular or subretinalfluids can be performed, although culturesmay be negativebecauseof the smallnumberof viral particles or samplingerror. Retinal biopsy is possibleby an external or internal approach to investigate progressiveinflammation involving the peripheral retina near the ora serrata or equator (28, 50). For the external approach, a pars plana vitrectomy is performedwith cauterization or laserphotocoagulation around the biopsy site. A deep partialthicknessscleralflap is fashioned,diathermy demarcatesthe biopsy edges,and the chorioretinal biopsy is cleanly excisedwith a blade and microsurgicalscissors.Endoretinal biopsy of a portion of detachedretina isprocureddirectly through the pars plana sclerotomy.A lo- to 20-mm2strip of 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. OpportuMinced or homogenized tissue is plated on nistic retinal infections occur in people with appropriate cell lines; cocultivation may be reAIDS, transplant recipients, and other immuno- quired. Histopathologicexaminationcan demonsuppressed 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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viral inclusions more evident. Viral particles can occasionally be identified by electron microscopy enhanced by negative staining or immunochemistry techniques. Viral cultures also can be obtained from separated leukocytes, urine, and other fluids.

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measuredby an indirect fluorescent-antibodytest or enzyme-linkedimmunosorbentassay.The presence of immunoglobulinM antibodiesindicates recent infection, and a positive assayat any dilution for immunoglobulin G confirms previous infection. Intraocular antibody production can be Fungal and bacterial chorioretinitis testedby comparingaqueousand serumantibody EndogenousCalzdidainfectionsmay presentas titers. a unilateral or bilateral focal or multifocal necroParasitic retinovitreitis may simulate other tizing granulomatouschoroiditisand retinitis with forms of endogenousmicrobial endophthalmitis vitreitis, most commonly seen during prolonged and necessitateaspiration of intraocular fluid. use of corticosteroid or immunosuppressive Giemsa-stainedsmearsduring ocular toxocariasis agents,prolongedindwellingcatheters,and intra- and degeneratingcysticercosistypically contain venous drug abuse.Cryptococcus neoforrnans can numerouseosinophils.Determination of the speproduce focal or multifocal choroiditis with sub- cific antibody titer in intraocular fluid may be sequent retinitis and vitreitis, and immunosup- helpful in doubtful casesbut is usually not indipressedpatients are susceptibleto cryptococcal cated. meningitis with extension to the eye. The preInfectious Optic Neuritis sumedocular histoplasmosis syndromeconsistsof atrophic choroidalscarsin otherwisehealthy peoThe mostcommondiseaseassociated with optic ple. Aspergillus speciesand other filamentous neuritis is multiple sclerosis.Although uncomfungi are rarely encounteredascausesof endog- mon, infectious causesof optic neuritis are syphenouschoroiditis.Focal and multifocal chorioreti- ilis, tuberculosis,toxoplasmosis, toxocariasis,viral nitis has occurred during syphilis, tuberculosis, illnesses,Lyme disease,and cat scratch disease. and nocardiosis. Patients with AIDS may develop neuroretinitis Diagnosticvitrectomy for cytologic examination from CMV, VZV, or HSV. Cryptococcal meninand culture is consideredwhenvitreous inflamma- gitis and other fungal infections of the central tion is present. Intraocular specimenscan be nervous system or orbit can invade the optic plated onto blood agarand Sabouraudagarplates nerve. Serologic testing is available for someof and stained with Giemsa, calcofluor white, and theseconditions. Diagnostic lumbar puncture or methenaminesilver stains.Blood specimenscan vitrectomy may be indicated in selectedcases. be cultured and measuredfor specific fungal anti,gensby agglutination or enzyme immunoasSpecimens Associated with Ocular say.Cerebrospinalfluid is obtainedduring central or Trauma nervous systeminvolvement for cytologic examination, culture, and antigendetection. Other tests Preoperative cultures The value of routine conjunctival or lid cultures include serologictestsfor syphilis,tuberculin skin prior to intraocular surgery has not been estabtesting, chestX ray, and HIV antibody testing. lished.The number and type of organismsof the normal conjunctiva may vary periodically, and Parasitic retinitis initial culturesmay not reflect the flora presentat Transplacentaltransmission of Toxoplasma gondii during the first trimesterof pregnancycan lead the time of surgery. Routine cultures may also to fetal dissemination,producingan inactive reti- yield organismsthat might be construedasocular nochoroidal scar that may later reactivate as a pathogens and unnecessarilypostpone surgery focal retinitis and vitreitis. The occurrence of (Table 4). In the absenceof ocular inflammation, acquiredtoxoplasmicretinochoroiditis from con- routine preoperative cultures of the noninflamed sumingcontaminatedmeator cat fecesisprobably eye are not necessary. Special consideration uncommon. Toxoplasmic retinochoroiditis and shouldbe given to patientswho have been hospiPneumocystis carinii choroiditis are complications talized for prolongedperiods,who are wearing a cosmeticprosthesisin the contralateral eye, who of AIDS. Toxocariasisis encounteredmainly in children have lacrimal dysfunction, or who have acute or chronic blepharoconjunctivitisor other signsof who ingest nematode ova. A larva of Toxocara canis can lodge in a retinal blood vesseland die, ocular inflammation. Bilateral eyelid margin and producing a focal retinochoroidal granuloma or conjunctival cultures can be obtained from these retinovitreitis. Cysticercosis resultsfrom the inges- patients in the standardmanner. tion of eggsof Taenia solium. Serologicaltesting can detect specificantibod- Cornea1 tran .splant tissue ies. Becausemany healthy people have been ex- preserva tion mediu .m posed,a positive titer provides only presumptive Donor corneas often have surface bacterial evidenceof a specificetiology. Antibodies can be contamination by gram-positivecocci and other

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microorganisms prior to processing by the eyebank, especially those obtained from donors with sepsis or who were connected to a respirator. Microbial growth increases with advanced cadaver time. Quality control procedures include antibiotic or antiseptic irrigation of the globe before retrieval. The whole eye is kept in a moist chamber with antibacterial solution or the excised corneoscleral cap is stored in a preservation medium with antibiotics at 4°C. At the time of surgery, microbial cultures of the remaining globe or corneoscleral rim and of a sample of the preservation medium are recommended. Material is usually transferred to a sterile tube in the operating room and sent to the microbiology laboratory for inoculation to standard culture media to detect aerobic and anaerobic bacteria and fungi (26, 51). The corneoscleral rim can be directly inserted into a liquid medium such as chopped meat broth, and a 2-ml aliquot of storage medium can be immediately injected into thioglycolate broth. The use of an antimicrobial removal device can help isolate microorganisms (46) . Eyebanks routinely screen donors for transmissible diseases. HIV antibody, hepatitis B antigen, and hepatitis C antibody tests are performed before distribution of donor eye tissue. Donors with diseases that can be transmitted by cornea1 transplantation, such as Creutzfeldt-Jakob disease and rabies (rabiesvirus has been found in the cornea1 epithelium by cytology showing cytoplasmic Negri bodies and by direct immunofluorescence), are excluded. Ophthalmic

biomaterials

Intraocular lensesmay be associatedwith pseudophakicendophthalmitis.A keratoprosthesiscan lead to wound and intraocular infections. Retinal reattachmentmay be complicatedby infection of the scleral buckling or other synthetic material. Orbital implantscanlead to cellulitisor abscess. A biofilm can form on thesematerialsto sequester and protect microorganisms. In selectedcircumstances,the implant or explant materialshouldbe removed(74). The device shouldbe inoculated into sterile salineor into a liquid mediumsuchasTSB for transportation to the laboratory, where it can be divided among standardmedia as for other foreign bodies. Removedmaterialcan alsobe examinedby scanning electronmicroscopy.Suturesfrom a wound infection are streaked across agar plates and then insertedinto thioglycolate broth. Contact lensesincriminated in causingcornea1 infection may be submittedfor laboratory evaluation. The contact lensis streakedacrosssolidagar platesand then inoculatedinto TSB or thioglycolate. The lenscan alsobe sectioned,and part can be submittedfor electron microscopy.Intraocular

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lenses,ocular prostheses,and other biomaterials are handled in a similar fashion. Material is collected from contact lens cases and other containers by swabbingthe internal surfaces or caps with a moistened swab (89). Because residual preservatives or disinfectants can inhibit growth of contaminatingbacteria, the specimencanbe either diluted in a broth medium or processedwith an antimicrobialremoval device before inoculation. Multiple media shouldbe inoculated becauseof a possiblehigh microbial density and the likelihood of multiple species. Contact lens solutions should be shaken and then squirtedonto swabsfor inoculatingstandard culture media.Nonnutrient agarfor isolatingfreeliving amoebae should be utilized in selected circumstances.Large volumesof solutionscan be concentratedby filtration or centrifugation. Ophthalmic

medications

Contaminated ophthalmic solutionsand ointments are a potential sourceof ocular infection (15). Drops from a squeezeor pipette container shouldbe plated directly onto media and spread by tilting the agar plate slightly. The insideof the cap should alsobe swabbedfor culture inoculation. Ocular

trauma

Cornea1foreign bodies,particularly wood fragments, can be cultured for microorganismsfollowing removal. A single fragment should be inoculatedinto TSB, choppedmeatbroth, or thioglycolate broth. Multiple fragments should be divided amongthe standardmediausedfor investigation of microbial keratitis. Intraocular foreign bodies are handled in the samemanner as a cornea1foreign body. Wound swabs,samplesof intraocular fluid, and excised necrotic tissuecan also be obtained for culture during the surgicalrepair of a corneosclerallaceration or perforating injury. Specimensare inoculated and smearedin the samemanner as that usedfor suspectedmicrobialendophthalmitis.Ocular tissuespecimensare divided for histopathologic andmicrobiologicstudies.After grinding the specimenin sterile saline,the maceratedtissueis processedto prepare smearsand cultures. PROCESSING

AND INTERPRETATION OF SMEARS

Smearsare prepared from scrapingsor swabbings of ocular tissuesand from aspiration of ocular fluids. An unstained wet mount with a coverslip can demonstrateparasitessuch as lice and mites.Adding an India ink or nigrosin(Harleco, Philadelphia,Pa.) suspension to a wet mount can outline fungal elements(3). Giemsaand other stainsare better than a potassiumhydroxide preparation for detection of fungi in ocular specimens.

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Methanol fixation is preferred to heat for preparing microbial smears. Methanol or acetone is used to prepare specimens for immunofluorescence. Extra unstained smears can be frozen at -70°C. Cellular analysis of infected tissue can be accomplished by enzymatic disaggregation using 0.25% pancreatin and 0.05% collagenase (Sigma, St. Louis, MO.) in buffered saline (6).

DIAGNOSIS

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berstaedter-vonProwazekbodies)are found more often in neonatal than adult chlamydialconjunctivitis. Intranuclear viral inclusionsare difficult to identify but can be detected by usingthe Papanicolaoustain. Cellular inclusionsmustbe differentiated from melanin,nuclear extrusions,and lysosomalbodies. For impressioncytology, the filter paper usedto collect cellsfrom the conjunctival or cornea1surDirect Stains face is fixed in methanolfor 5 to 10 min. Giemsa Giemsaand Gram stainsare widely available. stain, fluorescein-conjugatedantibody, or other The Giemsa stain is used to characterize the stain is applied.The stainedmembraneis rinsed, inflammatory cell responseand other cytological applied to a glassslide, and examined under a details.The Gram stain aidsin bacterial identifi- coverslipwith glycerol. cation. Both stainsare equally effective in detectIn the evaluation of intraocular fluids, pigment ing ocular fungal infection. Smearswith lessthan granulescan be misinterpretedascocciby inexpe10 organismson the slideare indeterminate. riencedmicroscopistsbut shouldbe readily distinguishedby their larger size and faint brown pigGiemsa stain mentation. Melanin granules of the uvea are Freshly prepared Giemsastain rendersleuko- spherical,and pigment granulesfrom the retinal cyte nuclei purple-blue and the cytoplasm light pigment epithelium are ellipsoid. Noninfectious blue againsta light gray background.Bacteria and intraocular inflammationmay be identified by the fungal elementsstain dark blue. Although the type of inflammatory response.Large macroWright stain andother methyleneblue derivatives phagesand other mononuclearcellsarepresentin demonstrateinflammatory cells, these alternate smearsof aqueousfluid in lens-induceduveitis. methodsdo not reveal the intracytoplasmicinclu- Tumor cells may be present in leukemia and sionsseenwith chlamydialconjunctivitis. Normal lymphoma. Numerous eosinophilsin a vitreous conjunctival epithelialcellsare slightly larger than specimensuggests a parasiticinfection. leukocytesand possess a central blue nucleusand homogeneouscytoplasm(Fig. 5). A smearmust Gram stain contain more than 500 epithelial cellsin order to The Gram stain is used to identify the type of determine the status of conjunctival epithelial responsiblebacteria and to recognizefungal elecells and the presenceof intracellular inclusions. ments.Precipitated stain,carbon or talc particles, Smearswithout at least 10 cells in several low- salt crystals,and necrotic debriscan lead to artipower fields have insufficientmaterial. facts. Bacteriaare usuallypresentin areasof many After scanningthe entire specimen,oil immer- neutrophils and necrotic epithelial cells and can sion is usedto count the different cell types in at be detected extracellularly and within leukocytes. least 10 typical high-power fields to determine Gram-positive bacteria are usually more numertheir averagedensity(Table 7). The cellsrecorded ous and easierto detect than gram-negativebacinclude normal and degeneratedepithelial cells, teria. The positive Gram stain characteristicmay degeneratedor keratinized epithelial cells,goblet be lost during antibacterialtreatment. cells, neutrophils, lymphocytes, monocytes and Yeasts and pseudohyphaetypically stain dark macrophages,eosinophils,basophils and mast blue. Fungal hyphae and protozoa have variable cells, plasma cells, large macrophages(Leber stainingcharacteristicssincetheir cell wallsdo not cells),immunoblasts, balloonedor multinucleated stainwell and areusuallyseenasnegativeoutlines epithelial cells, and erythrocytes. Determination (Fig. 7). Conidia and other features that provide of the predominant type of inflammatory cell is cluesto fungal classificationrarely appearin ocuAcanthamoeba spp. can appearas simplifiedby performing a different count, asfor a lar specimens. peripheral blood smear. The inflammatory cell trophozoites and asdouble-walledcysts. pattern and other findingsin Giemsa-stainedconBacteria comprisingthe indigenousmicroflora junctival scrapingsmay suggestspecificgroupsof of the conjunctivaandtear film maybe detectedin ocular surface disease(Table 5), although the small numbersin ocular surfacespecimens,geninflammatory reaction varies during different erally appearingasone or two organismsper oil stagesof viral and chlamydial conjunctivitis. A immersion field in random areas. Yeasts (M. mixed inflammatory reaction of neutrophils and fu@r) may occasionallybe seenfrom lid scraplymphocytes together with plasmacells, macro- ings. The Gram stain may be misleadingduring phages,or multinucleatedepithelial cells can di- nonsevere bacterial conjunctivitis and marginal rect the microscopicsearchfor chlamydialinclu- blepharitis. Gram and acridine orangestainscan sionswithin sheetsof separatedepithelial cells correctly identify the morphologyof the responsi(Fig. 6), althoughthesebasophilicinclusions(Hal- ble organismin approximatelytwo-thirds to three-

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13A

FIG. 5. Conjunctival cytology. (A) Normal conjunctival epithelial cells have a homogeneous cytoplasm with dark, round nuclei and tend to clump together in sheets (Giemsa, X630). (B) Degenerated and keratinized conjunctival epithelial cells (Giemsa, X630).

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13A

DIAGNOSIS

TABLE 7. Density estimation by light microscopy for cells and microorganisms in ocular specimens Grade

Avg no. per high-power

field

None on smear 4 l-4 5-10 x0

0

l+ 2+ 3+ 4+

fourths of casesof microbial keratitis and onethird to one-half of cases of culture-proven microbial endophthalmitis. Acid-fast

stain

Using heat (Ziehl-Neelsentechnique) or a detergent (Kinyoun method),carbol fuchsinbindsto cell wall fatty acids to stain mycobacteria red againsta counterstainedblue-greenbackground. An acid-faststain shouldbe usedwhen nontuberculousmycobacteria,Nocardia spp., or microsporidia are suspected.A rhodamine-auraminestaining technique can be used with fluorescence microscopy,but somenontuberculousmycobacteria may be difficult to detect. Other stains

Lactophenol cotton blue is commonly usedto confirm fungal cultures and can be added to a smear of cornea1scrapings(84). A coverslip is pressedagainstthe slide with filter paper. Fungi and amoebiccystsstain dark blue againsta light blue background. The trichrome stain and a modification of the methenaminesilver stainmay be helpful for identification of fungal elements.Methenaminesilver stainsfungal cell walls and septablack againsta transparent green background. The modified Warthin-Starry silver impregnationstain can reveal smallbacilli in a conjunctival biopsy during cat scratch disease.A modified Dieterle stain is used to demonstrate spirochetesduring Lyme diseaseand syphilis. Fluorochrome

Stains

Fluorescentstainsenablemicrobialrecognition but require examinationwith a UV light filter or fluorescencemicroscope.Several fungi fluoresce on hematoxylin-eosin-stainedhistopathological material. Acridine

orange

Acidic acridine orange binds to polymerized nucleic acids. With UV light, most bacteria, yeasts,fungal hyphae, and amoebic cysts stain bright orange againsta dark green background. Nonviableorganisms,nontuberculousmycobacteria, and amoebictrophozoites may be difficult to distinguish.Degeneratingleukocvtescan nroduce

OF OCULAR INFECTIONS

25

faint yellow-orangegranulesthat can be mistaken for smallbacteria. Acridine orangeis better than the Gram stain for detecting low numbers of organismsfrom cornea1scrapings(33, 40, 85). After examination by fluorescencemicroscopy,Gram stainingcan be performed on the sameslide for further evaluation (Fig. 8). In fluid specimens,acridine orange and Gram stains require at least lo4 and 10’ bacteria per ml, respectively,for consistentdetection. Calcofluor

white

This fluorochromecombineswith P-linked polysaccharidesin the cell walls of yeasts(Fig. 9), pseudohyphae,hyphae,amoebicexocysts,and microsporidialspores(49, 87). Theseorganismsfluorescebright apple green or blue-white, depending on the wavelengthof the excitor light. Smears and tissuesectionscan be subsequentlystained with methenaminesilver and other techniquesto confirm findingsand to provide permanentslides. Calcofluorwhite providesbetter contrastbetween fungi and the backgroundthan lactophenolcotton blue and Giemsastains. Electron

Microscopy

Electron microscopycan aid in the identification of virusesandplaysa role in the classification of certain parasites(41). The selectedspecimenis put directly into cold, electron microscopy-grade glutaraldehyde.Material is then transferred to a coatedcoppergrid and negativelystainedwith 1% phosphotungsticacid or processedfor immunoelectron microscopy.Fluids and tissuesof ocular specimenscan sometimesreveal infectious agents by electron microscopywhen cultures are negative. PROCESSING AND INTERPRETATION MOLECULAR DIAGNOSTIC TESTS

OF

Immunologictestsare availablefor culture confirmation of many organismsand have been applied to the detection of infectious agents in ocular specimens (31, 65). Commerciallyavailable tests can supplement smearsand cultures for certain ocular infections. Somelaboratorieshave accessto fluorescein-conjugated antibodiesor fluorescein-conjugatedlectins (67). Antigens can be identified by light microscopyusing immunoperoxidase(72), avidin-biotin, and other chromogenic procedures.Macroscopiclatex and coagglutination methods may be applicable for certain bacterial and fungal eye infections (77). The sensitivities of antigen detection testsvary for different specimens,infectious agents, commercial sources,and technicians.

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13A

FIG. 6. Conjunctival cytology. (A) Basophilic intracytoplasmic inclusion body within a conjunctival epithelial cell during chlamydial conjunctivitis (Giemsa, X630). (B) Pseudoinclusion (arrow) within conjunctival epithelial cell caused by extruded nucleus (Giemsa, X630).

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13A

DIAGNOSIS

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I

FIG. 7. Smears of cornea1 scrapings from C. albicum keratitis. (A) Gram stain shows negative contrast of nonstained pseudohyphae with an intracellular “box car” appearance of safranin stain (X630). (B) Giemsa stain shows darkly stained pseudohyphae from same smear (X630).

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FIG. 8. Smears of cornea1 scrapings from Moraxeh nonliquefuciens keratitis. (A) Acridine orange highlights multiple rods against dark background (X630). (B) Gram stain of restained smear shows gram-negative diplobacilli with acute inflammatory cells (X630).

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29

FIG. 9. By fluorescence microscopy, calcofluor white reveals yeasts (blastoconidia) and pseudohyphae during C. albicans keratitis (calcofluor white, X400).

Immunofluorescence A specimen for immunofluorescence is placed at the designatedringed areaof a cleanglassslide or into a well of a Teflon-coated slide. Sufficient material must be obtained for adequateevaluation. Stored slides are allowed to reach room temperaturebefore adding freshly constituted reagents. Immunofluorescence staining kits are available for HSV (e.g., HSV MicroTrak, Syva, Palo Alto, Calif.), C. truchomatis (Chlamydia MicroTrak [Syva] and Pathfinder [Kallstad Diagnostics, Chasta,Minn.]), and other infectious agents. The direct immunofluorescencemethod is generally more specific,and the indirect method ismore sensitive. The smearis examinedwith a 60 to 100X oil objective using330- to 380-nmUV excitation and a 420-nm barrier filter. The number of epithelial cells is estimated to check for adequacy of the specimen.Positive smearsshowapple green tluorescenceof specific organismsor infected cells (Fig. 10). A positive smearmusthave at least5 to 10 fluorescent bodies. Extracellular chlamydial elementarybodiesare apple green dots that must be distinguishedfrom background debris. Virusinfected cells showdiffuse or granular stainingin the cytoplasm or nucleus of infected cells (53). Stained slidesare storedfrozen in the dark.

Immunoassays Enzyme immunoassays (62, 86) are applicable to HSV epithelial keratitis (Herpchek; DuPont, Wilmington, Del.), adenoviral conjunctivitis (Adenoclone; Cambridge Bioscience, Hopkinton, Mass.), and chlamydial conjunctivitis (Chlamydiazyme; Abbott, North Chicago, Ill.). The specimen is transferred to the solid-phasereagent. Enzyme-conjugated antibody is added, which is followed by the chromogenicsubstrate.The absorbancevalue of the colored product is readwith a spectrophotometerand compared to a cutoff value. Limulus Lysate Assay The Limulus lysate assaydetects endotoxin by an enzymatic reaction. This test can indicate the presence of gram-negative bacteria in cornea1 scrapingsor vitreous samples.Contact lenscases and solutions can be tested for gram-negative endotoxin by the Limulus lysateassaybut mustbe diluted because of the presence of inhibitory substances(1). False-positive results can occur sincethis test may detect trace amountsof endotoxin present in tap water used to rinse contact lenses.Few cliniciansrequestthis test becauseof difficulties in interpreting a positive reaction. A quantitative test that measuresthe concentration

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FIG. 10. Immunofluorescence staining. (A) Extracellular elementary bodies (arrows) during chlamydial conjunctivitis (direct fluorescent antibody, X630). (B) Fluorescent cornea1 epithelial cell (arrow) during herpes simplex dendritic keratitis (direct fluorescent antibody, x630).

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DIAGNOSIS

of endotoxin in a sample might be useful in the rapid detection of gram-negative bacteria in ocular specimens or in in-use ophthalmic solutions, but a minimal cutoff level that identifies true infection or contamination has not been established. Nucleic Acid Probes

Nucleic acid sequences homologousto thoseof infectious agentsare detectableby Southern blot analysisand other hybridization methods.Amplification of the target (e.g.,by the polymerasechain reaction) or the signal(e.g., by Ql3 replicase)has potential applicationsto ocular infections. DNA and RNA probes have been applied to vitreous specimensfor the diagnosisof infectious retinitis (2, 19,27), to conjunctival specimensfor identifying chlamydial and viral infections (82), and to cornea1 epithelial debridement for confirming HSV keratitis (44) and papillomavirus-related dysplasia.Ocular tissuescan alsobe examinedfor viral nucleic acid sequences (90). PROCESSING

AND INTERPRETATION OF CULTURES

Detecting microbialcomponentsin ocular specimensby stainingand other assaysprovides presumptiveevidenceof eye infection. Culture isolation gives diagnostic confirmation. Media that have been properly inoculatedwith ocular specimensare transportedto the laboratory. The surfaces of the petri plates are faced downward to prevent condensedmoisture from dripping onto the medium.Broth tubesare held upright and not shaken. Media are incubated at appropriate temperatures (Table 2). Placingplatesin a plastic bag or candle jar maintains carbon dioxide levels and adequatehumidity. If a blood culture systemis used, subculture slidescan be attached to the aerobicjar; the anaerobicbottle is left unvented. Tissuesare gently ground with sterile salinein a mortar and pestleor electric grinder, taking care not to raisethe temperatureof the specimen. All plates shouldbe examinedwith magnification daily, and liquid media are observed for turbidity. Anaerobic growth typically beginsas a globular haze around the swab,deep in the thio-

TABLE Grade

8.

Semiqu .antitative growth estimation on solid media Colony

growth

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31

glycolate broth. In vision-threatening infections such as keratitis and endophthalmitis, media shouldbe examinedwithin 12 to 24 h after inoculation. Bacteriologic media are held for at least one week. Standardized procedures should be followed for description of colony morphology, selectionof coloniesfor Gram staining,transferto selectivemedia,and biochemicaltesting. Reports shouldinclude the day that growth of eachmicroorganismfirst appearedand the amountof growth (Table 8). Polymicrobialinfectionscommonlyoccur. All microbial isolates should be noted and identified if possible.Lessthan 10colonieson only one solid mediumor growth only in thioglycolate is equivocalevidenceof ocular infection. Laboratory-criteria for definitive infection includegrowth on two or more media, growth on at least one medium of the sameorganismsidentified on the smear,confluentgrowth at the inoculationsiteson at leastone solidmedium,or anaerobicgrowth in an anaerobicmedium. Interpretation of microbiologic resultsmay not be valid with recent antimicrobial therapy, incomplete specimencollection, or fastidiousorganisms. If the patient has recently received antimicrobial therapy, an antimicrobialremovaldevicecontaining cationic and polymeric resinsin a sodium polyanethol sulfonate-salinesolution can be used to bind antibiotics that may be present in the specimen.A 3-ml volume of the antimicrobial removal device suspensioncan be inoculated as for other liquid media. The liquid portion is inoculatedonto standardgrowth mediaassoonas possible,sinceprolongedexposurecan be toxic to somestreptococciand Haemophilusspp.Thioglycolate broth is added to the remnant and incubated. This techniquecan give a 20% increasein recovery of microorganismsfor endophthalmitis and somewhatlessfor microbial keratitis specimensif antibiotics have been used. Viral specimensare inoculated onto appropriate cell monolayers(Table 9) by direct transfer or by centrifugation (17, 18). Tissuesare homogenized in a small volume of transport medium. Clonesof HeLa-229,pretreated McCoy, and certain other cell lines are usedfor C. trachomatis isolation (7). Cell cultures are maintained with Eagle’sor Dulbecco’smodified essentialmedium at 36 to 37°C and, periodically examined with magnification for cytopathic effects (CPE). Cell lines showing CPE are harvested and concentrated by centrifugation for immunofluorescence or enzyme immunoassay. Laboratory and clinical staff need to establish an ongoingcommunicationnetwork. Ocular infections that may be reportable to the local health agency are chlamydial infection, neonatal conjunctivitis, gonococcal keratoconjunctivitis, epidemic adenoviral conjunctivitis, impetigo, VZV

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TABLE Virus

Vero

13A

9. Cell cultures for ocular viral specimens”

MRC-5

SF

HEK

HEp-2

A549

Usual time to CPE (days)

HSV

+

+

+

+

+

+

4

vzv

+-

+

+

+

-

+

14-28

EBV CMV

+-

+

+

-

-

+

7-21

Adenovirus

+

+

+

+

+

+

14-28

Enterovirus

-

+

+

+

-+

+

3-7

Molluscum contagiosum

-

-

-

-

-

-

-

CPE description

Rapid progression of rounded, granular cells that form syncytia Elongated foci of granular cells with slow progression Compact foci of enlarged cells with slow progression Clusters of enlarged cells Small foci of swollen, granular cells that enlarge and detach -

’ MRC-5, human embryonic lung; SF, human foreskin fibroblasts; HEK, human embryonic kidney; HEp-2, human larynx carcinoma; A549, human lung carcinoma. +, many strains recovered; +, low isolation rate; -, not suitable.

disease,HIV infection, syphilis, Lyme disease, toxoplasmosis,Hansen’sdisease,and any common-sourceoutbreak (13). Isolatesfrom different sourcescan be comparedby serotyping and susceptibility profiles. Epidemiologic investigations may be helped by phenotypic procedures (e.g., phagetyping and protein analysis)andgenotyping (79 . Lid and Conjunctival

Cultures

Interpretation of microbial growth requiresfamiliarity with the normal indigenousmicroflora (Table 4). Selectionof coloniesfor identification and antibiotic susceptibilitytesting are basedon the clinical diagnosisand, in unilateral infections, the type of growth from the contralateral eye. Cornea1 Cultures

Early growth can be detectedwith a dissecting microscopeas small colonies on the C-streak marks.Growth outsidethe inoculation marks on solid media shouldbe disregardedas a contaminant. Indigenousorganismsof the preocular tear film can produce sparsegrowth from cornea1 scrapingsor swabbings. The number of colonies is counted, and the estimatednumberper C streakand the numberof streaksyielding growth are recorded.If anaerobic culture waslimited to placing a swabinto thioglycolate, aerobic and anaerobic subcultures are made whenever turbidity appears.Nutritionally variant streptococcicausinginfectious crystalline keratopathy can be isolated on chocolate agar; special studies with these bacteria use media supplementedwith pyridoxal hydrochloride or platescross-streaked with staphylococci.

Fungal growth is often detected during the first week, but ocular cultures should be kept for at least3 weeks.A germ tube test canbe performed by mixing yeast isolatesin fetal bovine serum, incubating for 3 h, and examining under low power for germ tubes extending from blastoconidia. Filamentousfungi are identified by their colony morphologyand the appearanceof conidia producedon a slideculture with a sealedcoverslip (Fig. 11). Filamentousfungi isolatedfrom fungal keratitis include nonpigmented species (e.g., Fusarium, Aspergillus, Acremonium, Paecilomyces, Penicillium, and Pseudallescheria spp.) and pigmentedspecies(e.g., Cuwularia, Altemaria, Phialophora, and Bipolaris spp.). Acanthamoeba trophozoites produce meander-

ing trails on the surfaceof blood agar and other solid media. Growth can be detected by using oblique illumination to visualize the fine indentation lines(Fig. 12). Serialtransfer isperformed to confirm amoebicisolation,sinceviable leukocytes in ocular specimenscan produce short trails. Acanthamoeba and other protozoa (e.g., some microsporidia and T. gondii) can be isolated on HeLa and other cell cultures.Although the CPE causedby free-living amoebaecanmimic the CPE produced by viruses,cell culture isolation is used only when other recovery methods are unavailable. Intraocular

Cultures

Cultures from direct inoculation of smallvolumesof aqueoushumor (0.1 to 0.2 ml) or vitreous (0.5 to 2.0 ml) are handledin the samemanneras cultures of cornea1specimens.TSB can be added to smallspecimens to yield a volume of at least0.5

CUMITECH

FIG. (inset).

13A

11. Cottony

DIAGNOSIS

colony

on a C streak

of Fusarium

ml for smears and cultures. Any foreign body, suture, or filtering bleb samples are cultured and identified separately. Plate contaminants can be recognized as colony growth away from the fluid impression zones. Diluted vitreous washings are concentrated by centrifugation or filtration. Sonication of vitreous specimens prior to culture inoculation can release organisms trapped within phagocytes. Hyaluronidase can be added to digest thick vitreous gel. For centrifugation, a portion of the specimen is aseptically transferred to a centrifuge tube and spun at 1,000 X g (1,000 to 2,000 rpm) for 10 to 15 min. The supernatant is aspirated with a micropipette and can be tested for antibodies and other soluble inflammatory factors. A sterile pipette can

soluni

producing

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INFECTIONS

multiseptate

33

macroconidia

collect the sediment and approximately 1 ml of the supernatant for smears and culture inoculation. Residual material can be sent for immunocytology on a gelatinized slide or for other cell-sorting techniques. For filtration, the specimen is transferred to a sterile membrane filter apparatus and suctioned onto filter paper with a 0.45 pm pore size (Nalgene 245-0045, Rochester, N.Y.; Falcon 7102, Oxnard, Calif.). Smears are prepared by aspirating aliquots of the partially suctioned concentrate or by scraping the surface of the filter paper. The filter paper is cut from the unit with sterile instruments and divided into wedges for culture inoculation to standard media. Solid media are inoculated by placing the filter paper wedge face down

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FIG. 12. Indentation marks produced on blood agar plate by excysted Acanthamoeba migrating from a C streak.

on the agar surface, dragging it across the plate, then placing it face up in the center of the plate. The residual vitreous fluid can be processed for soluble factors. Acidification of the filtrate may be necessary to retard evaporation of fatty acids and other volatile substances.

ANTIMICROBIAL

13A

trophozoites (inset)

SUSCEPTIBILITY TESTING

Standard disk diffusion or microdilution techniquesshouldbe utilized for antimicrobialsusceptibility testing of bacterial ocular isolates(56-60, 69). The choiceof antimicrobialagentsfor susceptibility testingof ocular isolatesrequiresselecting representativesfrom different antibiotic classes. Tissue or Biopsy Culture Resultsof agar disk diffusion testsrelate to levels Specimenscan be ground aseptically with a of drug in serumrather than to concentrationsof mortar and pestle or in a sterile tissuegrinder to antibacterial agents achievable in the preocular which 2 ml of sterile salineis added.Drops of the tear film and ocular tissues.Estimating the MIC suspensionare inoculated as for liquid ocular by broth microdilution, using buffered cationspecimens. adjusted Mueller-Hinton broth in a prepared Samplesof donor corneasand cornea1preser- panel (Sensititre [Westlake, Ohio] and Scepter vation medium are inoculated to thioglycolate [Becton Dickinson, Sparks,Md.]), provides more broth, chopped meat broth, or BHI broth with useful information for ocular infections than rebeef extract to reduce antibacterial activity in the porting an isolate as sensitiveor resistant.Antispecimens.The use of an antimicrobial removal microbial susceptibility testing is usually considdevice may also enhance recovery. Microbial ered optional for blepharitisand conjunctivitis but growth occurs from approximately 5 to 15% of is often requestedfor more seriousinfectionssuch preserveddonor corneasand must be correlated asbacterial keratitis and endophthalmitis. The quantitative MIC can be comparedto the with clinical status.

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TABLE 10. Approximate sustained antimicrobial levels in an inflamed eye after usual dosea

Antibiotic

Beta-lactams Cefazolin Ceftazidime Penicillin G Aminoglycosides Amikacin Gentamicin Tobramycin Vancomycin Fluoroquinolones Ciprofloxacin Ofloxacin Bacitracin Chloramphenicol Erythromycin Polymyxin B Tetracycline Sulfadiazine Amphotericin B Natamycin

Cornea1 level achieved by repeated topical administration b%k)

Vitreous level achieved by single intravitreal administration bg/ml)

20-50 20 100-500

50-100 50-100

20 20-50 20-50 100-200

10-20 20-40 20-40 25-100

5 10 20-50 20-50 10 50 5-10 5 l-10 50

l-5

u Compiled from multiple sources.

antimicrobialconcentrationexpectedat the siteof infection. For microbial keratitis and endophthalmitis, approximate drug levels are basedon experimentaldata after treatment by topical and intravitreal routes, respectively (Table 10). The measurementof antimicrobial drug levels is not usedin clinical practicebecauseof the difficulty in safelyobtainingocular fluids or tissuefor analysis. Becauseof uncertaintiesregardingthe actual drug concentration obtained for sufficient duration in patientswith different ocularinfections,resistance breakpoints for ocular isolates have not been determined. An organismmight be considered susceptibleif its MIC is lessthan the anticipated sustainedantimicrobiallevel in the infected ocular compartment,but there are no generallyaccepted cutoff points. Qualitative interpretation of susceptibility resultsis a compositeof expectedpharmacokinetics of each antimicrobial agent and the overall susceptibilitydistribution of similar isolates. Subculturing those MIC tubes or wells that showinhibition can determinethe minimalbactericidal (lethal) concentration. The fractional inhibitory concentrationcanbe measuredby checkerboard susceptibility testing to determine the interaction of two antimicrobial agents. Broth microdilution with Schaedlerbroth or agar dilution with Wilkins-Chalgrenagar (Difco Laboratories) is usedfor antimicrobic susceptibilitytesting of anaerobicand other fastidiousbacteria. Com-

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mercial panelsare also available.Nitrocefin-containing disks (BBL Microbiology Systems,Cockeysville, Md.) are used to test for P-lactamase production. The role of antifungal susceptibilitytesting has not been establishedfor ocular fungal infections. A broth dilution assayis proposed for certain yeasts,but a comparablemethod for filamentous fungi is not standardized.Determination should include the antifungal agentsof potential value in ocular infections:amphotericinB, natamycin,miconazole,ketoconazole,itraconazole, and fluconazole. Assaysfor measuringdrug susceptibilityof freeliving amoebaeare not widely available. Serial drug dilutions can be addedto calibratedtrophozoite suspensions to estimatethe minimaltrophozoiticidal concentration. The minimal cysticidal concentration for Acanthamoeba spp. can be determined by a related technique by assessing excystment,but resultsmay not correlate with clinical efficacy (23). SEROLOGICAL TESTING OCULAR INFECTIONS

OF

Antibodies in serum and ocular fluids are detectable with precipitin reactions, agglutination, indirect immunofluorescence, and enzymeimmunoassays.The diagnosisof primary infection generally requires two separate serum samplesto detect a fourfold or greater rise in antibody titer. Seroconversioncan be used in the diagnosisof primary HSV eye disease,varicella, adenoviral keratoconjunctivitis, first-episodechlamydial keratoconjunctivitis, and early EBV infection (52). A single positive titer identifies prior exposure. While it cannot substantiatean etiology, serological testing is useful in the diagnosisof ocular syphilis, toxoplasmosis(64), toxocariasis (71), Lyme disease,late EBV infection, and HIV infection. A lack of measurableantibody helpsexclude a specificcause. Tear fluid for antibody analysiscan be collected by glasscapillary tubes, cellulose sponges,and filter paper strips.Reflex tearing during collection can dilute solublefactors in the tear film, and thus the least irritating method should be used. Intraocular fluids are obtained by aspiration. The presenceof antibodiesin ocular fluids may indicate local production or diffusion from the blood (22). A high titer of specificimmunoglobulin A in tearsoccursduring active infectionsof the ocular surface.Antibody levelswithin the aqueous humor or vitreous can be comparedto the serum by calculating the antibody coefficient: [(inverse titer of specificserum antibody) X (total serum antibody)]/[ (inverse titer of specific ocular antibody) X (total ocular antibody)]. A high antibody coefficient suggestsintraocular antibody production, although leakageacrossa disrupted blood-

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GLOSSARY OF TERMS ocular barrier could lead to local sequestration. The role of intraocular antibody assessment is limited but has been used in the evaluation of Abscess, orbital. Focal purulent inflammation within the orbital cavity, usually subperiosteal. infectious retinitis and certain uveitis syndromes. Blepharitis. Inflammation of the margin of the eyelids. ACKNOWLEDGMENTS Blepharoconjunctivitis. Inflammation of the marNettie M. Robinson helped develop many of our gin of the eyelid and palpebralconjunctiva. recommended procedures in ocular microbiology. Work Blepharodermatitis. Inflammation of the margin in this Cumitech is from the Sid W. Richardson Ocular of the eyelid and adjacentskin. Microbiology Laboratory, Houston, Tex. Canaliculitis. Inflammation of the punctum and canaliculus portions of the lacrimal outflow system. APPENDIX: STAINS Cellulitis, orbital. Inflammation within the bony Giemsa Stain cavity containingthe eyeball and related structures. 1. Add 1 ml of Giemsa stain (Matheson, Cellulitis, preseptal. Inflammation of the subcuColeman, and Bell Manufacturing Chemists, taneous tissue of the eyelids anterior to the Norwood, Ohio) and 2 ml of buffer (PS1166; orbital septum and within the limits of the Paragon C & C, Bronx, N.Y.) to 47 ml of orbital rims. distilledwater in a Coplin stainingjar. Chalazion. Lipogranulomaof a meibomiangland 2. Immersefixed slidein stainsolutionfor 45min. within the eyelid. 3. Rinseby dipping twice rapidly in 95% ethanol Chemosis. Edema of the conjunctiva. and air dry. Choroiditis. Inflammation of the posterior uveal tract. Acridine Orange Stain Conjunctivitis. Inflammation of the tissuelining the eyelids (tarsal or palpebralconjunctiva) and 1. Flood slidewith acridine orange stain (Difco) eyeball (bulbar conjunctiva). for 2 min. Inflammation of the lacrimal Dacryoadenitis. 2. Rinsewith distilled water and air dry. gland. Dacryocystitis. Inflammation of the lacrimal sac. Calcofluor White Stain Dermatoblepharitis, Inflammation of the skin of 1. Flood slide with 0.1% calcofluor white and the eyelids. 0.1% Evans blue counterstain (Fungi-fluor; Endophthalmitis. Inflammation of the intraocular Polysciences, Warrington, Pa.) or a liquid nontissues(uvea and retina) and fluids (aqueous bleachdetergentcontainingfabric whitener for humor and vitreous humor). 1 min. Epiphora. Overflow of tearsdueto faulty drainage 2. Apply coverslip. by the lacrimal outflow structures. Episcleritis. Diffuse or nodular inflammationbeKinyoun Acid-Fast Stain tween the conjunctiva and sclera. Hordeolum. Inflammationof a gland of the eyelid 1. Flood fixed smearwith carbol fuchsin for 2 margin. min. Hypopyon. Accumulation of inflammatory exu2. Rinsewith distilled water. date in the anterior chamber,typically produc3. Decolorize with acid-alcohol (or 1% sulfuric ing a fluid level in the inferior portion. acid for Nocardia spp.). Iridocyclitis. Inflammation of the iris (iritis) and 4. Counterstainwith methyleneblue for 20 s. ciliary body (cyclitis); anterior uveitis. 5. Rinsewith distilled water and air dry. Keratitis. Inflammation of the cornea. Keratitis, epithelial. Inflammation of the surface Direct Fluorescent-Antibody Stain layer (epithelium) of the cornea, usually characterizedby punctate or linear clustersof swol1. Fix smearin methanolor acetone. len diseasedcells. 2. Prepare control slide to verify test perforKeratitis, stromal. Inflammation of the connecmance. tive tissueof the cornea. 3. Reconstituteantibody reagent at an appropriate dilution to avoid nonspecificreactionsand Keratocentesis. Surgicalpuncture of the corneato obtain intraocular fluids. allow to equilibrate for 30 min. Keratoconjunctivitis. Inflammation of the ocular 4. Add antibody reagentto smear. surface. 5. Let sit in moist chamberfor 15 min. 6. Removereagent, rinse slide in distilled water, OD. Right eye (RE). OS. Left eye (LE). and air drv.

CUMITECH

13A

DIAGNOSIS

OF OCULAR INFECTIONS

37

1982. Diagnosing and treating Phthirus pubis palpebrarum. Inflammation of all intraocular Surv. Ophthalmol. 26:219-225. tissuesand fluids of the eye and the eye wall 17. Darougar, S., P. Walpita, U. Thaker, N. Viswalingham, and (cornea and sclera). M. S. Wishart. 1984. Rapid culture test for adenovirus Proptosis. Forward displacementof the eyeball. isolation. Br. J. Ophthalmol. 68:405-408. Retinitis. Inflammation of the retina. 18. Darougar, S., R. W. Woodland, and P. Walpita. 1987. Value and cost effectiveness of double culture tests for diagnosis of Retinochoroiditis. Inflammation of the retina and ocular viral and chlamydial infections. Br. J. Ophthalmol. underlying choriocapillarisand choroid. 71:673- 675. Retinovitreitis. Inflammation of the retina and r9 De La Paz, M. A., and L. H. Y. Young. 1993. Acute retinal area of the vitreous immediatelyin front of the necrosis syndrome. Semin. Ophthalmol. 8:61-69. 20 Denis, J., D. Chauvaud, M. Savoldelli, and Y. Pouliquen. retina. 1978. Fine structure of palpebral molluscum contagiosum Scleritis. Inflammation of the sclera. and its secondary conjunctival lesions. Albrecht von Graefes Uveitis. Inflammation of the iris, ciliary body, Arch. Klin. Exp. Ophthalmol. 208:207-216. and/or choroid. 21 Durfee, K., and J. P. Smith. 1982. Use of Gram stain to Vitrectomy. Surgical excision of the fluid or gel predict vitreous fluid infection. Am. J. Med. Technol. 48: 525-529. within the vitreous cavity of the globe. Vitreitis. Inflammationwithin the vitreous cavity. 22 Dussaix, E., P. M. Cerqueti, F. Pontet, and E. Bloch-Michel.

Panophthalmitis.

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