Laboratory
Diagnosis of
Ocular Infections
KIRK R. WILHELMUS,
THOMAS J. LIESEGANG,
MICHAEL S. OSATO, and DAN B. JONES
COORDINATING EDITOR
STEVEN C. SPECTER
Cumitech 1A l Blood Cultures II l June 1982
Cumitech 2A l Laboratory Diagnosis of Urinary Tract Infections l March 1987
Cumitech 3A l Quality Control and Quality Assurance Practices in Clinical Microbiology l May 1990 Cumitech 4A l Laboratory Diagnosis of Gonorrhea l April 1993
Cumitech SA l Practical Anaerobic Bacteriology l December 1991
Cumitech 6A l New Developments in Antimicrobial Agent Susceptibility Testing: a Practical Guide l February 1991
Cumitech 7A l Laboratory Diagnosis of Lower Respiratory Tract Infections l September 1987 Cumitech 8 l Detection of Microbial Antigens by Counterimmunoelectrophoresis l December 1978 Cumitech 9 l Collection and Processing of Bacteriological Specimens l August 1979
Cumitech 10 l Laboratory Diagnosis of Upper Respiratory Tract Infections l December 1979 Cumitech 11 l Practical Methods for Culture and Identification of Fungi in the Clinical Microbiology
Laboratory l August 1980
Cumitech 12A l Laboratory Diagnosis of Bacterial Diarrhea l April 1992 Cumitech 13A l Laboratory Diagnosis of Ocular Infections l September 1994
Cumitech 14A l Laboratory Diagnosis of Central Nervous System Infections l March 1993 Cumitech 15A l Laboratory Diagnosis of Viral Infections l August 1994
Cumitech 16 l Laboratory Diagnosis of the Mycobacterioses l March 1983
Cumitech 17A l Laboratory Diagnosis of Female Genital Tract Infections l June 1993 Cumitech 18 l Laboratory Diagnosis of Hepatitis Viruses l January 1984
Cumitech 19 l Laboratory Diagnosis of Chlamydial and Mycoplasmal Infections l August 1984 Cumitech 20 l Therapeutic Drug Monitoring: Antimicrobial Agents l October 1984
Cumitech 21 l Laboratory Diagnosis of Viral Respiratory Disease l March 1986 Cumitech 22 l Immunoserology of Staphylococcal Disease l August 1987 Cumitech 23 l Infections of the Skin and Subcutaneous Tissues l June 1988 Cumitech 24 l Rapid Detection of Viruses by Immunofluorescence l August 1988
Cumitech 25 l Current Concepts and Approaches to Antimicrobial Agent Susceptibility Testing l December 1988
Cumitech 26 l Laboratory Diagnosis of Viral Infections Producing Enteritis l September 1989
Cumifechs should be cited as follows, e.g.: Wilhelmus, K. R., T. J. Liesegang, M. S. Osato, and D. B. Jones. 1994. Cumitech 13A, Laboratory diagnosis of ocular infections. Coordinating ed., S. C. Specter. American Society for Microbiology, Washington, DC.
Editorial Board for ASM Cumitechs: Steven C. Specter, Chairman; Mary J. R. Gilchrist, Curt Gleaves, Janet Hindler, Thomas J. Inzana, Brenda McCurdy, Fredertck S. Nolte, John A. Smith, Alice S. Weissfeld, and Stephen Young.
The purpose of the Cumitech series is to provide consensus recommendations by the authors as to appropriate state-of-the-art operating procedures for clinical microbiology laboratories which may lack the facilities for fully evaluating routine or new methods.
The procedures given are not proposed as “standard” methods.
Copyright 0 1994 American Society for Microbiology 1325 Massachusetts Ave.. N W.
LABORATORY DIAGNOSIS OF OCULAR INFECTIONS
KIRK R. WILHELMUS, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030THOMAS J. LIESEGANG, Department of Ophthalmology, Mayo Clinic Jacksonville, Jacksonville, Florida 32224 MICHAEL S. OSATO, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030
DAN B. JONES, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030 COORDINATING EDITOR
STEVEN C. SPECTER, Department of Medical Microbiology and Immunology, University of South Florida, Tampa, Florida 33612
Presentation of the materials n eeded to per- form laboratory diagnosis for ocular infections. Descriptions of acute and chronic ocular dis- eases caused by viruses, bacteria, parasites, and fungi, including dermatoblepharitis, infections of the orbit and lacrimal apparatus, marginal blepharitis, conjunctivitis, keratitis, endophthal- mitis, 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 serol- WY*
SYNOPSIS
The eye and associated structures are uniquely predisposed to infection by a variety of microor- ganisms. The indications and techniques for mi- crobiological investigations are determined by the site of ocular or periocular infection, its severity and pace of progression, and knowledge of the responsible organisms. Special procedures are re- quired to collect and process the small quantity of material that can be obtained from ocular tissues and fluids. Efficient handling of ocular specimens involves the use and interpretation of various laboratory procedures in infectious eye diseases (35). The appendix lists common stains used in the ocular microbiology laboratory and a glossary of clinical terms pertaining to ocular infections and related inflammatory diseases.
MATERIALS AND MEDIA FOR OCULAR SPECIMENS
The materials and media suggested for the collection, transport, and culture of clinical spec-
imens must be assembled and maintained (Table
1). Storage in a special tray in the office or hospital
treatment room will simplify ready access. Com-
monly used solid media can be obtained from the clinical microbiology laboratory and should be refrigerated at 4°C in plastic bags. Whether in the clinic, surgical suite, or hospital room, immediate inoculation of ocular specimens from the patient to culture media is preferred whenever possible. Transport containers are available for biopsy spec- imens, biomaterials, and other special cases. Ma- terials to be shipped elsewhere are secured in a double mailing package with a biohazard label and sent according to current postal regulations. Com- munication networks are maintained between of- fice and laboratory personnel so that fresh, ade- quate materials will be available and test results will be rapidly accessible.
Collection Materials
Fundamental considerations in the optimal col- lection of ocular specimens are as fo llows.
l Sufficient material must be collected from the
infected site with minimal contamination from adjacent fluids, secretions, or structures.
l An appropriate collection device, transport me-
dium, and culture media must be used.
l Material should be collected early in the course
of infection and in the absence of recent anti- microbial treatment.
Specimen collection devices
The Kimura spatula (Storz, St. Louis, MO.) is useful for obtaining material from the conjunctiva and cornea (Fig. l), although an alcohol lamp is needed for heat sterilization. The tip cools rapidly after flaming and is slightly flexible for efficient sampling. Disposable blades or needles can also
2 WILHELMUS ET AL. CUMITECH 13A
Orbital Space
Lid: Posterior Margin Preocular Tear Film Cornea1 Epithelium Cornea Anterior Chamber Iris Conjunctiva: Tarsal Fornix Conjunctiva: Bulbar Lid: Anterior Margin
Orbital Septum *Vitreous
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.
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. . . . . . . Lens Posterior Chamber/:..
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.
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-.
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:.
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. &.A
. :
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Cilia Conjunctiva: Tarsal Lacrimal Gland Cornea Corneoscleral Tissue Canthus: Medial Lacrimal Sac Canaliculi Puncta Conjunctiva: Bulbar Canthus LateralCUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 3 TABLE 1. Materials for ocular specimen collection
Collection devices
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 Kimura platinum spatula l&gauge needles 30-gauge needles l-ml syringes
Proparacaine hydrochloride (0.5%) topical solution Alcohol pads or swabsticks Alcohol lamp with lighter or
matches
Laboratory supplies
Glass slides with frosted ends and etched circles
Microslide holders Pencil
Wax pencil
Labels
Rapid diagnostic kits for HSV and C. trachomatis Fixatives Methanol (95%) Acetone (100%) Formalin (10%) Glutaraldehyde (2%) or Trump’s solution (paraformaldehyde [4%] and glutaraldehyde [I%]) Coplin staining jars
be used (76). The availability of several spatulas
expedites specimen collection.
Cotton, polyester, and calcium alginate swab tips are suitable for the recovery of most bacteria and fungi, although cotton-tipped swabs may con- tain fatty acids that inhibit bacterial growth. Moistened calcium alginate (Calgiswab; Spec- trum, Houston, Tex.) can be dissolved in a fixed volume of culture medium with hexametaphos- phate solution for semiquantitative cultures of the lid margin and conjunctiva (11). Cotton- or poly- ester-tipped plastic swabs are preferred for viral cultures, and dacron or rayon swabs on plastic or wire shafts are used for obtaining chlamydial specimens. Swabs without transport medium should not be used for transporting specimens. Prepackaged transport tubes with sterile swabs are available for collecting material for bacterial, viral, and chlamydial cultures.
Aspiration with a needle and syringe is the preferred method for collecting fluid or exudate for anaerobic culture. All air bubbles are expelled, and the needle tip is stuck into a rubber stopper.
FIG. 1. Platinum (Kimura) spatula. The thin tip (in- set) is ideal for obtaining material from the surface of eyelid lesions, conjunctiva, and cornea.
Anesthetics
Preservative-free preparations are less antisep- tic than multiuse topical anesthetics (5). Propara- Caine hydrochloride (0.5%) solution is preferred for obtaining specimens from the ocular surface for culture and does not interfere with cytological techniques. Sedation may be needed for young children and uncooperative adults.
Slides and fixatives
Glass slides with frosted ends and etched circles (Fluorescent Antibody Slides; Becton Dickinson, Lincoln Park, N.J.) facilitate labeling and recog- nition of smeared material. Slides should be cleansed to remove debris that can absorb stains.
Smeared slides should be immersed in 95% meth-
anol in a Coplin jar for approximately 5 min for fixation before staining. Cover slips are placed over wet mounts without fixation and immediately examined. Slides are sent to the laboratory in slide holders (Control, Friendswood, Tex.) in the order that they were obtained.
Cold acetone is often recommended for prepar-
ing slides for immunofluorescent staining, al- though methanol can be just as effective. Bouin’s fixative is used for Papanicolaou staining. Forma- lin (10%) and glutaraldehyde (2%) should be available to fix histopathological material for light microscopy and electron microscopy, respectively.
Media
Culture and transport media recommended for
ocular infections are listed in Table 2. Media are chosen according to the likely etiology and incu- bated at an appropriate temperature and atmo- sphere.
4 WILHELMUS ET AL. CUMITECH 13A
TABLE 2. Culture and transport media for ocular specimens
Medium Purpose Storage
temp (“C) Incubation temp (“C)
Incubation duration
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
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. Fungi Acanthamoeba spp. Remove antibiotics in specimen Aerobic or anaerobic bacteria, fungi Viruses C. trachoma tis 4 4 4 25 4 4 4 25 4 35 (10% COZ) 4 35 4 4 35 (10% COZ) 35 (10% CO,) 35 35 (anaerobic system) 25 25 4 wks 30 and 35 4 wks 25 to 35 (until inoculation) 7 days
35 7 days 4 (until inoculation) 4 (until inoculation) 5-7 days 5-7 days 7 days 7 days 4 wks 4-6 wks 7 days 6 wks 4 wks 2 wks
Bacterial culture media For anaerobic cultures, specimens are usually Soybean-casein digest (tryptic soy) broth (TSB)
is useful for moistening swabs for specimen col- lection. TSB can serve as a general-purpose nutri- ent broth medium for cultures of aerobic and facultatively anaerobic bacteria and can be used to collect viral cultures, although a transport medium containing antibiotics is preferred. A sterile, non- nutritive balanced salt solution (e.g., BSS; Alcon Laboratories, Fort Worth, Tex.) can be used for the interim holding of ocular tissues.
Defibrinated 5% horse or sheep blood agar is suitable for the growth of most bacteria as well as
fungi and Acanthamoeba spp. A MacConkey agar
plate or eosin-methylene blue agar plate can also be inoculated if enteric gram-negative rods are suspected. A chocolate agar plate enriched with
vitamins and other supplements supports growth
of Haemophilus, Neisseria, and Moraxella species. Thayer-Martin medium or one of its modifications favors growth in suspected gonococcal or menin- gococcal infections. Blood and chocolate agar plates should be incubated at 35 to 37°C in a candle jar or inside ziplock bags with a carbon dioxide generator to create an atmosphere con- taining 3 to 10% C02.
inoculated into a liquid medium (75). Thioglyco- late broth enriched with vitamin K, and hemin (BBL, Becton Dickinson, Cockeysville, Md.) is recommended. Prereduced, anaerobically steril- ized chopped meat broth and prereduced, anaer- obically sterilized brain heart infusion (BHI) broth without antibiotics can also be used for anaerobic incubation. Inoculated tubes are capped and incubated at 35 to 37°C. An enriched, fresh or prereduced brucella, Schaedler, Colum- bia, or Brewer agar base is used for an anaerobic plate. After inoculation the plate should not be exposed to air any longer than necessary and should be incubated in an anaerobic chamber or a heat-sealed envelope (GasPak Pouch [Becton Dickinson, Cockeysville, Md.] and Pouch System [Difco, Detroit, Mich.]) with a gas generator and resazurin indicator.
If an antibacterial or antifungal agent has been used before specimen collection, an antimicrobial removal device might improve microbial recovery. Approximately 3 ml of the solution and resin is decanted into test tubes, the ocular specimen is introduced into the medium, and aliquots are inoculated onto solid and liquid culture media.
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 5
Specimens for mycobacterial and nocardial cul- tures should be inoculated onto Lowenstein- Jensen or Middlebrook 7HlO agar and incubated at 35 to 37°C for up to 6 weeks.
Fungal culture media
A blood agar plate can be used to isolate fungi, but a nutritionally deficient medium such as Sab- ouraud dextrose agar containing gentamicin at 40 pg/ml (Emmons modification) should also be used. Cycloheximide should not be included be- cause it inhibits the saprophytic fungi principally responsible for ocular infections. Plates should be slightly thicker than usual (35 to 40 ml per petri dish) and are partially sealed or placed in a bag to reduce desiccation during prolonged incubation.
BHI broth containing gentamicin at 50 @ml helps to dilute naturally occurring fungal inhibi- tory substances. The inoculated vial is incubated on a rotary shaker to enhance aeration. All fungal isolation media are incubated in a room-temper- ature incubator at 25 to 30°C for up to 4 weeks.
Acanthamoeba culture media
Free-living amoebae can be isolated on blood agar plates and other aerobic media. Minimal nutrient agar prepared with an overlay of live or
dead microorganisms (such as Escherichiu coli)
enhances recovery. An alternative medium for primary isolation of amoebae is buffered charcoal- yeast extract agar, originally designed to isolate Legion& species. Duplicate plates can be incu-
bated at 30 and 35OC. Page amoeba saline can be
used for transportation of clinical specimens (55).
Subcultures of isolated amoebae should be made
to cation-supplemented peptone-yeast extract- glucose agar for axenation (55).
Viral and chlamydial transport media
Hanks’ and Leibovitz-Emory media are com- monly available for transporting viral specimens. Salt solutions alone should not be used. Swab-tube combinations (Viral Culturette [Becton Dickin- son, Cockeysville, Md.], Virocult [Medical Wire & Equipment, Sparta, N.J.], and M4 [multipurpose transport medium] [Microtest, Snellville, Ga.]) are available, as well as transportation devices with cell culture (Transporter; Bartels Immunodiag- nostics, Bellevue, Wash.). Viral transport media should be sent immediately for transfer onto
appropriate cell monolayers. Specimens should
not be frozen at -2OOC and should never be put in a frost-free freezer of a standard refrigerator.
Sucrose-phosphate-glutamate transport me-
dium is preferred for Chlamydia trachomatis and can also be used as a viral transport medium. Prepackaged transportation tubes are available (Transtube; Medical Wire & Equipment). C. tra- chomatis should not be transported in a viral
transport medium con inhibitory antibiotics.
taining penicillin or other
Laboratory Safety and Quality Assessment
Specimens need to be transported to the labo- ratory in a sturdy, leakproof container with a lid. Shipping requirements mandate that material be
contained within a screw-capped container sealed
with adhesive tape, wrapped in absorbent mate- rial, enclosed in a watertight container, placed inside a padded shipping container, and labeled with an appropriate biohazard notice. Laboratory personnel must comply with guidelines that pro- mote biological, chemical, radiological, and phys- ical safety (12).
The clinical microbiology laboratory should
maintain a quality-assurance program that moni-
tors equipment and supplies, ensures technical proficiency, and adheres to requisite biosafety practices (4). A schedule for monitoring speci- mens and laboratory tests must be individualized for the needs and resources of each institution. Indicators of adequate specimen collection are the cytologic pattern of smears (e.g., number of epi- thelial cells in a conjunctival specimen), growth of contaminants apart from the inoculation marks on solid culture media, and transport time between specimen acquisition and receipt in the labora- tory. Parameters of laboratory quality control are daily temperature records of incubators and re- frigerators and periodic conduct of performance standards for stains, reagents, media, and suscep- tibility tests. The laboratory should seek to mini- mize the turnaround time for reporting growth and susceptibility data since ophthalmologists may be reluctant to alter initial empiric therapy, even when laboratory results suggest less toxic or less costly agents. Laboratory records should be prop- erly organized for surveillance of trends in the epidemiology of ocular infections and for analysis of cumulative antimicrobial resistance patterns.
METHODS FOR COLLECTION OF OCULAR SPECIMENS
Specimens should be obtained from the in-
fected site when safely accessible. A similar plan for collection and inoculation of material can be applied to most ocular infections (Table 3). Spec- imen containers must be properly labeled with the patient’s name, identification number, collection date, and source. Information should be provided about the clinical diagnosis and any recent use of antimicrobial agents. Universal precautions must be followed during the collection and processing of all clinical specimens.
Dermatoblepharitis
Infections of the eyelid skin include viral erup- tions, primary bacterial pyodermas, and secondary infections complicating preexisting skin lesions.
6 WILHELMUS ET AL. CUMITECH 13A
TABLE 3. Guide to specimen collection for common ocular infections
Clinical entity Source of material Principal organisms Stains and other
testsa Media
Preseptal cellulitis Drainage from wound or abscess S. pyogenes, S. aureus, H. influenzae (children) S. aureus, streptococci, H. injkenzae (children), anaerobes
Gram Blood agar, chocolate agar, thioglycolate Acute orbital
cellulitis
Drainage or biopsy from abscess or
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
sinus
Canaliculitis Expressed material Actinomycetes, streptococci
Gram
Streptococci, S. aureus Gram HSV, VZV S. aureus IF or EIA, Giemsa Gram S. aureus, S. pneumoniae, H. influenzae, N. gonorrhoeae Adenovirus, HSV Gram C. trachomatis C. trachomatis, N. gonorrhoeae, S. aureus IF or EIA Giemsa, IF Gram, Giemsa, IF Acute dacryocystitis Acute vesicular blepharitis Acute marginal blepharitis Acute purulent conjunctivitis Drainage from lacrimal sac Vesicle fluid or lid
margin scraping Lid margin swabbing Conjunctival swabbing Acute follicular conjunctivitis Chronic follicular conjunctivitis Neonatal conjunctivitis Epithelial keratitis Conjunctival scraping Conjunctival scraping Conjunctival swabbing Viral transport medium Chlamydial transport medium
Blood agar, Thayer- Martin agar, chlamydial transport medium Viral transport medium Cornea1 debridement or impression Cornea1 scraping HSV EIA or IF Suppurative keratitis S. aureus, P. aeruginosa, S. pneumoniae, Moraxella spp., enteric gram-negative rods, C. albicans, Fusarium spp. P. aeruginosa, S. pneumoniae, S. aureus Acridine orange, Gram, calcofluor white, acid-fast
Blood agar, chocolate agar, thioglycolate, anaerobic agar, Sabouraud agar
Suppurative scleritis
Scleral scraping Acridine orange, Gram, acid-fast
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 Endophthalmitis Aqueous humor
tap, vitreous tap, vitrectomy washing
S. epidermidis, S. aureus,
streptococci, P. acnes, H. in.uenzae, gram- negative rods, Bacillus
spp., C. albicans
CMV, VZV, HSV, C.
albicans, T. gondii
Acridine orange, Gram
Necrotizing retinitis Vitrectomy washing, retinal biopsy
IF, DNA probe
a IF, immunofluorescence; EIA, enzyme immunoassay.
Viral dermatoblepharitis vesicular scrapings), immunofluorescence studies, Primary or recurrent herpes simplex virus viral culture, and electron microscopy can be done (HSV) dermatoblepharitis and primary or recur- (45, 54).
rent varicella-zoster virus (VZV) dermatoblepha-
ritis produce a vesicular skin rash with preauricu- Impetigo
lar iymphadenopathy. Fresh vesicles can be Superficial vesiculopustular or crusting infec-
aspirated or scraped with the edge of a spatula. A tion of the skin with preauricular lymphadenopa-
CUMITECH 13A
streptococci and Staphylococcus aureus. Vesicles should be aspirated or unroofed. Exudate is sent for smears and for culture on blood and chocolate agar plates.
Granulomatous or pustular dermatoblepharitis
Granulomatous eyelid lesions can occur from chancroid, anthrax, actinomycosis, tuberculosis, candidiasis, blastomycosis, leishmaniasis, and my- iasis. A dental burr or cornea1 microdrill can secure samples from an ulcerative lesion. Part of
the specimen can be inoculated onto aerobic and
anaerobic culture media, including a Sabouraud agar plate and Lowenstein-Jensen agar. To iden- tify Mycobacterium leprae, slit 2 mm deep into pinched skin and scrape the lesion onto a slide for acid-fast staining. An excisional biopsy can be obtained for histopathological examination. Leish- mania spp. can sometimes be isolated on a blood agar plate, but promastigotes grow better on special media such as Schneider insect medium and Novy-MacNeal-Nicolle medium at 22 to 26OC. Extracted insect larvae can be placed on a blood agar plate or on raw meat with moist sand in a jar plugged with cotton.
Cellulitis of the Eyelids Preseptal cellulitis
Infections of the subcutaneous eyelid tissue anterior to the orbital septum may be due to trauma, spread from the upper respiratory tract or middle ear in children, or spread from infected skin or adjacent structures (36). Posttraumatic infections are usually due to S. aureus and Strep- tococcus pyogenes, although anaerobic and poly- microbial infections can occur. Nonsuppurative preseptal cellulitis in susceptible children is com- monly caused by Haemophilus influenzae or Strep- tococcus pneumoniae. Secondary infection of viral dermatoblepharitis is uncommon. A ruptured hordeolum, lacrimal sac, or other infected adnexal structure may cause staphylococcal or streptococ- cal cellulitis. Infantile gingivitis with S. aureus and other bacteria may lead to facial cellulitis.
Drainage from an open wound or eyelid abscess should be sent for smears and for inoculation onto blood agar, chocolate agar, brucella agar, and thioglycolate. To obtain material from an eyelid abscess, the skin is cleaned with an antiseptic agent and dried. The point of maximal fluctuation is incised parallel to the lid margin, and the wound is spread open. If direct inoculation of media is not feasible, aspirated material in a syringe should be free of air bubbles, the needle should be capped, and the material should be immediately transported to the laboratory or injected into an anaerobic transport vial. Blood cultures should be
obtained. Conj”unctiva1 cultures are sometimes
helpful.
DIAGNOSIS OF OCULAR INFECTIONS 7
Erysipelas
Superficial cellulitis of the eyelid with well- defined margins and lymphatic involvement is caused by S. pyogenes, other streptococci, or S.
aureus. The characteristic red edema spreads from a site of trauma, local infection, or prior skin lesion. Swabbings of intact skin are not reliable. The etiology is difficult to establish since sur- rounding orbital edema may be due to bacterial exotoxins rather than direct invasion. The tech- nique of injecting and aspirating sterile saline within the subcutaneous tissues from the advanc- ing edge is controversial. Blood and throat cul- tures can be obtained. Although conjunctival cul- tures may be misleading, material obtained by swabbing of the inferior conjunctival fornix of both eyes can be inoculated onto a blood agar plate.
Necrotizing fasciitis
Gangrene of the eyelids following trauma or necrotizing fasciitis associated with alcoholism or another debilitating condition is usually due to S.
pyogenes, either alone or in combination with other bacteria such as S. aureus. Exudate from ruptured bullae should be smeared and cultured (73). Other evidence of streptococcal infection is an acutely elevated anti-DNase or antihyaluroni- dase antibody titer.
Infections of the Lacrimal System Dacryoadenitis
Acute infections of the lacrimal gland are un- common. Typical clinical features include ery- thema and swelling of the outer third of the upper eyelid, protrusion of the palpebral lobe, conjunc- tival discharge, and preauricular lymphadenopa- thy. The route of inoculation may be exogenous (by way of the ductules in the superotemporal conjunctival fornix or from penetrating trauma) or
endogenous (during hematogenous spread).
The most common exogenous pathogens of acute purulent dacryoadenitis are S. aureus, S. pneumoniae, and S. pyogenes. A calcium alginate
or cotton applicator should be swabbed in the superotemporal fornix over the surface of the palpebral lobe and inoculated onto blood and chocolate agar plates. The uninvolved contralat- era1 conjunctiva should be cultured for compari- son. Conjunctival smears are obtained by instilling 0.5% proparacaine hydrochloride, scraping the conjunctiva in the superotemporal fornix with a spatula, and spreading the material with a circular motion onto a glass slide. Needle aspiration of the lacrimal gland is contraindicated unless a localized abscess is identified.
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 Epstein- Barr virus (EBV) and mumps virus.
Chronic inflammation of the lacrimal gland may be caused by sarcoidosis, syphilis, tuberculosis, leprosy, and certain fungal and helminthic infec- tions. 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. Canaliculitis is characterized by hypere- mia and edema at the medial canthus and a dilated, pouting punctum with mucopurulent dis- charge. Inflammation may involve the inferior punctum or both canaliculi and is frequently as- sociated with an obstructed common canaliculus. Most infections are due to anaerobes such as
Propionibacterium propionicum and Actinomyces
species (9). Mixed aerobic and anaerobic bacterial infections may occur.
Purulent material can usually be expressed by compression of the lid and canaliculus. A spatula should be used to transfer this material to media and to prepare smears. As these infections typi- cally produce large diverticula that contain cheesy concretions, a sterile spud or small chalazion curette should be used to scrape the canaliculus to obtain additional material. Incision into the hori- zontal portion of the canaliculus may be neces- sary. “Sulfur granules” and other particulate mat- ter should be crushed onto a glass slide to obtain a thin smear for staining.
Dacryocystitis
Infection of the lacrimal sac is generally a result of an obstructed nasolacrimal duct. Acute dacryo- cystitis is characterized by epiphora, pain, and distension of soft tissue at the inner canthus, typically just below the medial canthal ligament. Localized inflammation may progress to a dacryo- cystopyocele or orbital cellulitis. Responsible mi- croorganisms are S. aureus, S. pneumoniae, S. pyogenes, H injluenzae, and, less commonly, aer-
obic or facultative gram-negative rods or anaer- obes. Pressure on the lacrimal sac may express purulent discharge “from the puncta, but infection often obstructs the distal portion of the canaliculi
CUMITECH 13A
and prevents reflux. Drainage from the lacrimal sac should be swabbed or aspirated for smears and cultures. Transcutaneous aspiration or incision through the wall of the sac is usually not indicated, although an external fistula can provide a source of material. Nasal swabbing under the inferior turbinate may be attempted. Conjunctival cultures should be obtained in the standard manner.
Chronic dacryocystitis produces epiphora, with or without pain or discharge. Colonization of the lacrimal sac presumably occurs from conjunctival flora or by retrograde migration from the nose. A variety of organisms may contaminate the ob- structed sac without producing cellulitis. Aerobic and anaerobic bacteria, fungi, and mycobacteria have been isolated. Purulent material should be inoculated onto blood agar, chocolate agar, an anaerobic medium, and Sabouraud agar. Smears should be prepared for standard stains. A dacry- olith should be bisected and submitted for his- topathologic and microbiologic studies.
Neonates with congenital dacryostenosis are at risk for developing chronic dacryocystitis. Acute infection may lead to a dacryocystocele, a blue- gray lacrimal sac distention filled with viscous, translucent mucus. Probing is frequently unsuc- cessful in drainage of neonates with dacryocystitis
because of compression of the common canalicu-
lus. S. aureus, streptococci, and gram-negative rods are the most common causative organisms. Dacryocystitis is a possible complication of neo- natal conjunctivitis due to Neisseria gonowhoeae.
Smears and cultures should be obtained as de- scribed above.
Orbital Cellulitis Acute orbital cellulitis
The routes of infection in orbital cellulitis are
spread from infected ethmoid and other paranasal
sinuses, spread from other adjacent structures (e.g., dental infection, dacryocystitis, and orbital fracture), exogenous inoculation from trauma or surgery, and hematogenous seeding during sepsis. The most common causes of acute orbital cellulitis associated with sinusitis are streptococci, S. au- reus, and anaerobes. H. influenzae sinusitis and orbital cellulitis may occur in unvaccinated chil- dren. Mixed aerobic and anaerobic infections may occur. S. aureus is a frequent cause of posttrau- matic and postsurgical orbital cellulitis, and poly- microbial infections are common.
Blood cultures are often positive during acute orbital cellulitis. Smears and cultures are prepared from purulent material in the nose during sinus- related disease, from swabbings or excised tissue if a traumatic or surgical wound is present, or from an infected lacrimal sac or other adnexal source. In the presence of an open wound or drainage site, material should be obtained bv aspiration with a
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 9
sterile syringe and needle for immediate inocula- tion onto appropriate media or for prompt trans- portation to the laboratory after either embedding the needle into a sterile rubber stopper or inject- ing the material into an anaerobic transport vial. Drainage of an infected sinus, subperiosteal ab- scess, or intraorbital abscess is handled by similar techniques and is processed for smears and for aerobic and anaerobic cultures (34). Blind aspira- tion of the orbit is not indicated.
Zygomycosis
Acute rhinoorbitocerebral infection by R&o- pus and iWucor spp. and other nonseptate fungi of
the class Zygomycetes is a potentially lethal disease that primarily occurs in patients with diabetic or metabolic ketoacidosis, neoplasia, and other alter- ations of host defenses. Fungal spores proliferate within the nasal turbinates and paranasal sinuses, penetrate arterial walls, and spread by vascular and direct extension to the orbital apex. Surgical debridement of necrotic sinus and orbital tissue should establish drainage. Material from a ne- crotic lesion should be submitted for frozen and
paraffin-embedded histopathologic sections and
for smears and cultures. Biopsy material is trans- ported to the microbiology laboratory in a sterile tube or petri dish with sterile balanced salt solu- tion, emulsified, and inoculated to standard growth media. Smears are examined by Gram, Giemsa, and calcofluor white stains to detect the broad, nonseptate hyphae that tend to branch at right angles.
Chronic orbital cellulitis
Indolent orbital infection may occur after place- ment of alloplastic material for retinal surgery or orbital fracture repair. Causes include anaerobic bacteria, nontuberculous mycobacteria, actinomy- cetes, and filamentous fungi. Trauma with re- tained foreign material may produce draining sinus tracks that simulate chronic microbial cellu- litis. Orbital aspergillosis is a slowly progressive granulomatous process with thrombotic vasculitis, usually occurring in otherwise healthy adults in tropical areas.
Drainage material should be inoculated onto a blood agar plate, Sabouraud agar, BHI broth, and
Lowenstein-Jensen agar. Orbital biopsy material
should be bisected for histopathological and mi- crobiologic studies. Smears are prepared with Gram, Giemsa, calcofluor white, and acid-fast stains.
Blepharitis
Infections of the eyelid margin generally involve the skin, eyelashes, and associated glands anterior to the gray line or mucocutaneous junction.
Viral blepharitis
Vesicular or erosive marginal blepharitis is caused by HSV or VZV. Vaccinia virus was formerly a cause in individuals recently vaccinated against smallpox. One or more clear vesicles should be selected for sampling. The surface of the vesicle is cleansed with a moistened applicator 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 vesicles and of ulcerative erosions is scraped with a spatula and/or swabbed. Conjunctival scrapings
and/or swabbings can also be collected. Smears
are prepared on glass slides. Giemsa staining of herpesvirus infections usually shows multinucle- ated cells. Intranuclear Cowdry type A bodies can
sometimes be found with Papanicolaou staining.
Scrapings of the lid lesion can also be smeared onto a slide for immunofluorescence or trans-
ferred to an enzyme immunoassay system. Mate-
rial for viral isolation is placed into a viral trans- port medium that is chilled or refrigerated at 4°C until it is processed. Acute- and convalescent- phase antibody titers can be obtained to identify seroconversion diagnostic of a primary viral infec- tion.
Molluscum contagiosum produces dome- shaped nodules that become umbilicated. Single or multiple lesions are located on the skin or margin of the eyelids, occasionally accompanied by follicular conjunctivitis and punctate cornea1 erosions. The ulcerated area of the lesion can be scraped with a spatula, or the cheesy core can be
expressed with a comedo extractor. Giemsa and
other stains (Sedi-Stain; Clay Adams, Parsippany, N.J.) can show myriad tiny dark particles and
intracytoplasmic inclusions (Henderson-Patterson
bodies) within epidermal keratinocytes that are composed of poxviruses, as can be seen by elec- tron microscopy or immunofluorescent staining (20). Cytology of the conjunctival discharge typi- cally shows mononuclear cells. Incomplete virions are found from conjunctival specimens. Viral
DNA can be detected from molluscum contagio-
sum lesions, but serial propagation in cell culture is usually not successful. An excised lesion should be fixed in formalin for light microscopy or in glutaraldehyde for electron microscopy to demon- strate dumbbell-shaped viral particles. His- topathological identification can be facilitated by overnight digestion with DNase (to degrade cel- lular DNA but not viral DNA protected by its protein capsid) and then staining with the Feulgen technique and light green counterstaining.
An eyelid margin wart usually begins as a single, sessile, gray nodule. It may gradually develop into a gray-yellow lobulated, hyperkeratotic excres- cence that may be predunculated or filiform. The excised verruca is fixed in 10% formalin. His-
10 WILHELMUS ET AL.
FIG. 2. Schema for inoculation of conjunctival and lid cultures. For conjunctival cultures, horizontal and vertical streaks are made for right and left conjunctivae, respectively. Conjunctival and lid cultures (“R” and “L” patterns are made for right and left lid margins) from both eyes can be inoculated onto a single agar plate.
topathological examination can show typical in- tranuclear inclusion bodies within epidermal cells that contain papovaviruses. Eosinophilic cytoplas- mic inclusions (Guarnieri bodies) occur in poxvi- rus (vaccinia, cowpox, and orf) infections.
Bacterial blepharitis
Acute infection of the eyelid margin with ulcer- ation and cellulitis is caused predominantly by S.
aureus and Staphylococcus epidemtidis and rarely by gram-negative bacteria (32). Staphylococci and streptococci may superinfect viral blepharoderma- titis. Hyperemia, desquamation, and ulceration of the lateral canthus (angular blepharitis) may be caused by Morclxella infection or can be a form of eczematoid blepharitis with staphylococcal coloni- zation.
In acute bacterial blepharitis, a sterile cotton or calcium alginate swab is moistened with TSB or sterile saline, scrubbed along both upper and lower eyelid margins of the closed eye, and then rolled across the surface of blood and chocolate agar plates, avoiding the edges of the plate. This procedure is repeated for the other eye. For
convenience and economy, material from both the
right and left eyelid margins is streaked onto the surface of the same plate in the shape of an “R” and “L” to designate right and left lids, respec- tively (Fig. 2). Smears are usually not helpful.
Chronic staphylococcal blepharitis is character- ized by bilateral crusting and thickening of the anterior lid margins, loss and whitening of the eyelashes, recurrent hordeola, conjunctival hy- peremia, conjunctival and limbal phlyctenules, punctate cornea1 erosions, and marginal cornea1 infiltrates, Chronic staphylococcal blepharitis may coexist with dry eye syndrome and atopic derma- titis. Chronic staphylococcal blepharitis should be differentiated from meibomian gland dysfunction and rosacea characterized by inspissation and dilatation of the meibomian glands, foam and scurf on the lid margin, recurrent chalazia, con- iunctival hyperemia, and marginal cornea1 infil-
CUMITECH 13A
trates with vascularization. A chalazion is a sterile
lipogranuloma that does not require microbiolog-
ical investigation. The roles of lipophilic coryne- bacteria, Propionibacterium acnes, Malassezia fir-
fur, and Demodex folliculorum (Fig. 3A) in chronic blepharitis have not been established (68).
In chronic blepharitis, lid margin cultures are usually deferred unless the patient is not respond- ing to standard measures and antibacterial resis- tance is suspected. Cultures are obtained in the same manner as for acute blepharitis, possibly with the inclusion of Sabouraud agar. Smears of the eyelid margins are usually not helpful, and smears of any conjunctival discharge associated with blepharitis predominantly show neutrophils
with few microorganisms. Scrapings or swabbings
of conjunctival or cornea1 lesions associated with chronic staphylococcal blepharitis are usually not needed. A biopsy should be considered for atypi- cal, chronic lesions of the lids since neoplasms can simulate chronic unilateral blepharoconjunctivitis.
Lice infestation
The pubic crab louse (Pthirus pubis) can infest the eyelashes (16), presumably because the spac- ing of the cilia corresponds to the grasping span of 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 examined with a wet mount or oil preparation by light microscopy (Fig. 3B).
Conjunctivitis
The conjunctiva is predisposed to infection by diverse microorganisms. The principal routes of inoculation are airborne droplets, hand-to-eye contact, and spread from the ocular adnexa, in- cluding the lacrimal system, nose, and paranasal sinuses. Microbiologic studies of the normal con- junctival flora are influenced by multiple environ- mental and host factors. Interpreting conjunctival cultures requires familiarity with the relative fre- quency of various bacteria colonizing the conjunc- tiva (Table 4). Because the normal flora of both eyes is usually the same, bilateral bacterial cul- tures are helpful in evaluating unilateral conjunc- tivitis.
Acute bacterial, viral, and chlamydial conjunc- tivitis can often be distinguished by the history and clinical signs, thereby simplifying the selection of diagnostic procedures. Distinctive signs are often not present in infants, and specimens from neo- nates with conjunctivitis should aim to identify several likely organisms. Chronic microbial con- junctivitis must be differentiated from various toxic and allergic processes. Different cytologic patterns occur during various infectious and non- infectious processes (Table 5), and conjunctival cytology can be used to support a clinical diagnosis (43 .
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 11
FIG. 3. Parasites of the eyelid. (A) Demodex folliculorum mite from an epilated eyelash (wet mount, x400). (B) pthirus pubis louse grasping an eyelash (wet mount, x400).
CUMITECH 13A
12 WILHELMUS ET AL.
TABLE 4. Normal conjunctival flora”
Organism frequency Relative (%)
Staphylococcus epidermidis ... Corynebactetium spp ... Propionibacterium acnes ... Staphylococcus aureus ... Streptococcus spp . ... Moraxella spp . ... Haemophilus injluenzae ... Gram-negative rods ... Fungi ... 75-90 20-75 50-70 10-30 2-10 2-5 2-5 o-5 o-5 a Compiled from multiple sources.
Viral conjunctivitis
The principal causes of acute viral conjunctivitis are adenovirus and HSV. Adenovirus is the most common cause of acute, bilateral keratoconjunc- tivitis and is highly contagious. It is able to survive for several days on inanimate objects and in solutions. Acute conjunctivitis is also a component of several viral syndromes including influenza, infectious mononucleosis, measles, and Newcastle disease. Epidemics of acute hemorrhagic conjunc- tivitis due to enterovirus 70 or coxsackievirus A24 have been predominantly limited to the coastal tropics and to travelers from these areas.
Clinical signs of viral conjunctivitis include eye- lid edema, preauricular lymphadenopathy, mu- coid conjunctival discharge, papillary and follicu- lar conjunctivitis with petechiae, and punctate epithelial keratitis. Adenoviral conjunctivitis in
toddlers can produce a pseudomembrane that
mimics severe bacterial conjunctivitis. Without cutaneous or lid margin lesions, HSV conjunctivi- tis may be indistinguishable from adenoviral in- fection.
Viral cultures should be obtained before the instillation of a topical anesthetic. A dry cotton or polyester swab should be rubbed over the upper and lower tarsal conjunctiva and fornix of the
involved eye and then agitated and squeezed into viral transport medium. To avoid a potential inhibitory effect, the tip of the swab is not broken into the medium. In bilateral conjunctivitis,
pooled samples from both eyes may be inoculated
into one vial. The suspected responsible virus should be indicated on the request form to sim- plify processing by the laboratory. Swabbings from 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 specimen can be frozen at -70°C in dry ice or an ultralow freezer.
Giemsa-stained smears are obtained by scrap-
ing the everted tarsal conjunctiva, gently blanch- ing the vessels by the spatula. A cytobrush used to collect conjunctival cells is dipped into a buffer solution for filtration or centrifugation. Cytology during viral conjunctivitis generally shows a pre- dominantly lymphocytic or mixed mononuclear and polymorphonuclear inflammatory cell pat- tern. Multinucleated giant cells or intracellular inclusions are usually not detected in conjunctival material by Giemsa cytology.
Rapid diagnostic techniques such as immuno- fluorescence and enzyme immunoassay are be- coming commercially available for detecting HSV and adenovirus antigens in conjunctival scrapings. One or two drops of a topical anesthetic should be instilled, and a spatula should be scraped over the upper and lower tarsal conjunctivae. The material is smeared onto a clean glass slide and then fixed with acetone, methanol, or spray fixative. In uni- lateral conjunctivitis, it is helpful to obtain mate- rial from the contralateral conjunctiva as a refer- ence control.
Serologic testing for suspected viral conjuncti- vitis is not an expedient consideration in clinical practice. Adenovirus, primary HSV, and other
TABLE 5. Cytology of Giemsa-stained conjunctival scrapings
Finding Implication
Predominance of polymorphonuclear leukocytes . . . Bacterial conjunctivitis
Severe viral or chlamydial conjunctivitis Allergic conjunctivitis
Chemical or irritative conjunctivitis
Contact lens or prosthetic-induced giant papillary conjunctivitis
Predominance of lymphocytes and monocytes . . . Adenoviral conjunctivitis Herpes simplex viral conjunctivitis
Chronic drug-induced or allergic conjunctivitis Mixed polymorphonuclear and mononuclear leukocytes . . . Chlamydial conjunctivitis
Adenoviral conjunctivitis Large macrophages (Leber cells), plasma cells, and
blastoid or stem cells . . . Chlamydial conjunctivitis Eosinophils . . . Allergic conjunctivitis Keratinized epithelial cells . . . ..*... Tear dysfunction states
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 13
viral conjunctivitis syndromes induce type-specific 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 conjunctivi- tis.
Chlamydial conjunctivitis
C. trachomatis produces two principal forms of keratoconjunctivitis: trachoma, caused predomi- nantly by serovars A, B, Ba, and C, and acute conjunctivitis (formerly called inclusion conjunc- tivitis or paratrachoma), caused by serovars D, Da, E, F, G, H, I, J, and K. Active trachoma occurs from repeated eye-to-eye spread aided by insect vectors and common fomites. Acute chla- mydial conjunctivitis is related to sexually trans- mitted genital tract infection. Chlamydial conjunc- tivitis is often bilateral and is characterized by gradual onset, preauricular lymphadenopathy, fol- licular conjunctivitis, mucopurulent discharge, and pleomorphic punctate keratitis.
For cytology, scrapings should be obtained from the inferior and superior tarsal conjunctivae of involved eyes and fixed in 95% methanol. Giemsa- stained smears are examined for a characteristic inflammatory cell pattern (88) and the diagnostic intracytoplasmic basophilic inclusions in conjunc- tival epithelial cells.
Immunofluorescence and enzyme immunoassay
kits are commercially available for chlamydial detection and are often more reliable than Gi- emsa cytology (14). For immunofluorescent stain- ing, conjunctival swabbing results in epithelial cell disruption that can increase the number of detect- able elementary bodies (66). The swab is rolled onto a glass slide in an area 8 to 10 mm in diameter, using all swab surfaces. After air drying, methanol or acetone fixative is applied. The slide is analyzed immediately or refrigerated. If a delay of more than 24 h is anticipated, it is frozen at -20 or -70°C.
Conjunctival impression cytology can be per- formed for infectious conjunctivitis but is more useful for other disorders of the ocular surface. A piece of cellulose acetate filter paper (Millipore, Bedford, Mass.) is placed onto the bulbar con- junctiva, pressed lightly, peeled off, and put into fixative (42, 83). The paper should be cut or marked to indicate the side on which cells are collected.
Conjunctival cultures should be obtained before the instillation of a topical anesthetic. A dry rayon or dacron swab should be rubbed over the upper and lower tarsal conjunctivae and fornix and
squeezed into chlamydial transport medium. TSB
can be substituted for transportation, but a viral transport medium that contains inhibitory antibi- otics cannot. The vial should be delivered imme- diately to the laboratory in a chilled container on
ice or should be temporarily stored at 4°C until cell culture inoculation. Nasopharyngeal swab- bings can also be obtained to increase the recovery rate.
Standard serologic tests are generally reserved for population screening. Because of a high back- ground positivity rate, a single titer usually is not
helpful. Serodiagnosis requires seroconversion,
from no detectable titer to a positive convales- cent-phase specimen, or a fourfold or greater rise in titer.
Bacterial conjunctivitis
Adult bacterial conjunctivitis is characterized by unilateral or bilateral lid edema, conjunctival hy-
peremia with chemosis and petechiae, and muco-
purulent conjunctival discharge, sometimes lead-
ing to membrane or pseudomembrane formation.
Preauricular lymphadenopathy is usually not a feature except with gonococcal conjunctivitis.
Multiple bacterial species cause mild conjuncti- vitis, but only a few typically produce severe, purulent infection. The principal causes of com- munity-acquired bacterial conjunctivitis are S. au- reus, S. pyogenes, S. pneumoniae, H. influenzae, N. gonorrhoeae, Neisseria meningitidis, and Moraxella
species (80). In children under 3 years old, H. influenzae produces severe conjunctivitis and pre- septal cellulitis. N. gonowhoeae must be consid- ered in any patient with rapidly progressive, puru- lent conjunctivitis and is one of the few bacteria that can penetrate an intact cornea1 epithelium to
produce suppurative keratitis. Pseudomonas spp.
and members of the family Enterobacteriaceae
rarely produce conjunctivitis except in the immu-
nocompromised host, following prolonged hospi-
talization, or in the presence of a cosmetic scleral shell. Corynebacterium diphtheriae infection is no longer encountered. Anaerobic bacteria rarely cause acute conjunctivitis.
Conjunctival cultures should be obtained before the instillation of a topical anesthetic, as these agents and preservatives may interfere with the recovery of certain organisms. Swabbings may be done to evaluate the ocular flora of the conjunc- tiva and tear film (25). Calcium alginate applica- tors are preferred because of their solubility in liquid media. Moistening the swab with TSB is unnecessary in the presence of conjunctival exu- date. The swab is rubbed over the inferior tarsal conjunctiva and fornix of the right eye and rolled on the surface of a blood agar plate. The same swab can be used to inoculate a chocolate agar plate. This swabbing procedure should be re- peated for the left eye. Since it is convenient and economical to inoculate material from both eyes to the same plate, the swab from the right con- junctiva is inoculated in horizontal streaks and the left is inoculated in vertical streaks to designate the specimen sources (Fig. 2). Cultures of the lid
14 WILHELMUS ET AL.
margins are unnecessary except in severe staphy- lococcal blepharoconjunctivitis. Special circum- stances, such as alteration of host factors or concurrent infection of the ocular adnexa, may warrant inclusion of Sabouraud agar or an anaer- obic medium.
Smears should be obtained in all cases of se- vere, purulent conjunctivitis. Stained smears may not be reliable in establishing the causative micro- organism 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 conjunc- tival smears are also helpful in distinguishing the cytologic pattern of several forms of infectious, allergic, and irritative conjunctivitis. Acute bacte- rial conjunctivitis is characterized by a polymor- phonuclear inflammatory cell response. Papanico- laou cytology is helpful in the detection of various neoplastic lesions of the conjunctiva that may simulate chronic infectious conjunctivitis.
Conjunctivitis in neonates
Conjunctivitis of the newborn can occur before birth by ascending infection from the vagina and cervix following premature rupture of mem- branes, during delivery from genitourinary secre- tions, or after birth by contact with contaminated materials or persons. Unlike adult conjunctivitis, clinical signs are not reliable in guessing the responsible organism. The principal infectious causes are C. trachomatis, N. gonorrhoeae, S. au- reus, Streptococcus species, and gram-negative bacteria (70). Neonatal chlamydial conjunctivitis may be accompanied by nasopharyngitis and pneumonitis. Pseudomonas conjunctivitis is a rare, but potentially fatal, infection of premature in- fants.
Conjunctival cultures and smears should be obtained. Smears are made from conjunctival
scrapings for Gram stain, Giemsa or Papanicolaou
staining, and antigen detection. Giemsa cytology is more likely to show inclusions in neonatal chla- mydial conjunctivitis than in adult disease. Swab- bings are inoculated to blood agar, chocolate agar and/or Thayer-Martin medium, and chlamydial transport medium. If neonatal HSV infection is suspected because of facial or generalized skin lesions with keratoconjunctivitis, conjunctival ma- terial should be obtained for direct immunofluo- rescence or similar antigen detection and for
CUMITECH 13A
inoculation into viral transport medium for viral culture.
Oculoglandular syndrome
Granulomatous conjunctivitis with regional lymphadenopathy is most commonly due to cat scratch disease. Other bacteria and fungi are rarely encountered. Material obtained by an exci- sional biopsy should be bisected. The half sent for histopathologic studies should be stained with the Warthin-Starry silver impregnation stain. The other half is ground for attempted culture, using a blood agar plate, thioglycolate broth, a Sabouraud plate, a lysis-centrifugation tube, and tissue-cell culture. Other media that are sometimes used in special cases are Lijwenstein-Jensen agar and glucose-cysteine-tellurite agar.
Keratitis
Intracellular organisms such as certain viruses, chlamydiae, and protozoa may infect the cornea1 epithelium. Bacteria and fungi usually directly invade the stroma and stimulate an inflammatory response that produces suppurative keratitis. En- dogenous diseases that can produce interstitial
keratitis by infectious or immune-mediated mech-
anisms include systemic infections caused by spi- rochetes.
Viral epithelial keratitis
Viral infection of the cornea1 epithelium can occur during acute conjunctivitis caused by HSV, VZV, and adenovirus. Because of the limited amount of infected cornea1 material that can be obtained and the discomfort and potential com- plications associated with removal of intact cor- neal epithelium, laboratory diagnosis of acute superficial keratoconjunctivitis is accomplished by obtaining conjunctival material. Laboratory con- firmation of viral epithelial keratitis without con- junctivitis is supported by collecting a cornea1 epithelial specimen.
Recurrent HSV epithelial keratitis usually ap- pears as linear or macroulcerative epithelial ker- atitis. Wiping debridement of a dendrite can re- move infected epithelial cells for culture, smears, and antigen and DNA detection. HSV is more difficult to isolate in geographic epithelial keratitis and when prior antiviral therapy has been used.
A topical anesthetic such as 0.5% proparacaine hydrochloride is instilled, and the abnormal epi- thelium is removed with a swab or spatula, being
careful not to damage Bowman’s membrane. The
material is inoculated into a vial of viral transport medium. Since swabs can adsorb viral particles and reduce the inoculum, the swab tip is com-
pressed and twisted in the transport medium and
then discarded. If the specimen cannot be pro- cessed within a few hours, it should be placed in a refrigerator at 4°C. Additional material should be
CUMITECH 13A DIAGNOSIS OF OCULAR INFECTIONS 15
smeared onto a glass slide and fixed with acetone
or methanol for immunofluorescence. Cornea1
debridement specimens are generally more likely to reveal HSV than conjunctival scrapings.
Multinucleated cells can be demonstrated by
Giemsa-stained smears. Papanicolaou-stained
scrapings may show Cowdry type A intranuclear inclusions that appear as eosinophilic Lipschutz bodies surrounded by a clear halo with clumping of the basophilic chromatin on the nuclear mem- brane.
A nitrocellulose acetate membrane (Biopore; Millipore, Bedford, Mass.) may be blotted onto the surface of a cornea1 lesion to map specific cell changes and to localize viral antigens within in- fected cells (30). For impression cytology, the membrane is placed onto the cornea1 lesion, gen- tly pressed with a sterile swab to make a replica of the lesion, and peeled off. The material is trans- ferred to a container and sent to the laboratory to be immediately processed or frozen temporarily at - 2o”c.
Dendritic epithelial keratitis caused by VZV is rarely identified during chickenpox but often oc- curs during the early stages of ophthalmic zoster. Patients with AIDS may develop persistent zoster dendrites. Cornea1 epithelial scrapings or swab- bings can reveal VZV by cell culture or immuno- fluorescence.
Recurrent HSV and VZV eye disease may involve the cornea1 stroma, but the role of active viral replication in stromal keratitis has not been established. Infectious virus is typically not iden- tified in cornea1 scrapings from disciform keratitis, necrotizing keratitis, or trophic ulceration. By using cocultivation culture techniques, electron microscopy, and nucleic acid probes, virus has been identified in the cornea1 stroma from cornea1 buttons obtained by penetrating keratoplasty.
EBV can cause small cornea1 epithelial den- drites. Culture requires fresh human umbilical cord blood lymphocytes that are then processed for immunofluorescence or nucleic acid hybridiza- tion. Viral antigens and viral DNA can be de- tected during cornea1 epithelial infection. The laboratory diagnosis of EBV keratitis is usually based upon detecting a serologic response by measuring circulating antibodies.
Bacterial and fungal keratitis
The diagnosis of bacterial and fungal keratitis cannot be established with certainty by the clinical features alone. Specimens collected from the in- fected cornea are processed to isolate the com- mon causes of microbial keratitis, including aero- bic and anaerobic bacteria and fungi. Additional material can also be collected for selective isola- tion of nontuberculous mycobacteria and proto- zoa.
Bacterial keratitis is characterized by epithelial
and stromal ulceration, dense stromal suppura- tion, and iritis. The most common causes are staphylococci, streptococci, Pseudomonas species,
Moraxella species, Haemophilus species, and cer- tain genera of the family Enterobacteriaceae (En- terobacter, Klebsiella, Proteus, Serratia, etc.). Many culture-positive cases are polymicrobial, and mixed infections with aerobic and anaerobic bac- teria may be more common than previously rec-
ognized. Nocardia species and nontuberculous
mycobacteria (Mycobacterium chelonae and Myco- bacterium fortuitum) are less commonly encoun- tered. A nonsuppurative form of bacterial kerati- tis, often associated with steroid use, is known as infectious crystalline keratopathy and is com- monly caused by viridans streptococci.
Fungal keratitis can be caused by filamentous molds and by yeasts. Filamentous fungal keratitis may follow outdoor trauma, especially in warmer climates, or corticosteroid treatment of eyes with chronic epithelial ulceration. Filamentous fungal keratitis is characterized by shallow ulceration, feathery stromal opacities, and multifocal satellite infiltrates that can evolve into a dense suppurative
abscess. Common nonpigmented fungi (Monili-
aceae) causing fungal keratitis are Fusarium, As- pergillus, Acremonium, Paecilomyces, Penicillium,
and Pseudallescheria spp. Important pigmented fungi (Dematiaceae) include Curvularia, Altema- ria, Bipolaris, and Phialophora spp. Yeast keratitis has no geographic predilection and typically de- velops in eyes with preexisting ocular surface disease. Yeast infections are characterized by de- marcated yellow-white stromal suppuration and are caused principally by Candida albicans and related species. Combined bacterial and fungal infections can occur.
A similar, thorough plan for specimen collec- tion and inoculation should be applied to all causes of suspected microbial keratitis to enhance recovery of all potential microorganisms. Speci- mens should be collected directly from the in- fected cornea1 tissue. Direct inoculation onto growth media is preferred because of the small quantities and because it simplifies precise place- ment for subsequent recognition of microbial col- onies. The value of material obtained from the eyelid margins and conjunctiva has not been clearly determined. If available, cultures can be taken from the responsible foreign body, contact lens, or contaminated solution.
A platinum spatula, disposable blade, bent nee- dle, surgical knife, disposable cautery, or similar instrument is used to perform cornea1 scrapings by using a slit-lamp or operating microscope. The rounded, slightly flexible tip of a Kimura platinum spatula is ideal for scraping the cornea, and it cools rapidly after heat sterilization in an alcohol- lamp flame. The metallic spatula is heated to a bright orange and allowed to cool. An assistant
