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51WE REVIEWED MEDICAL RECORDS FOR ALL PATIENTS WITH

suspected infectious keratitis who underwent corneal bi-opsies at the Jules Stein Eye Institute from June 1989through June 2009. Cases were identified from a registry ofspecimens in the Ophthalmic Pathology Laboratory; in-cluded were those specimens identified as being associatedwith corneal biopsy or diagnostic keratectomy. Forpatients who underwent multiple biopsies, data from first

cepted for publication Mar 7, 2012.rom the Ocular Inflammatory Disease Center, Jules Stein Eye Insti-e (J.R.Y., R.D.J., G.N.H., J.P.P., R.L.N., B.J.G., A.J.A., F.Y., J.L.,.M.), and the Departments of Ophthalmology (J.R.Y., R.D.J., G.N.H.,.P., R.L.N., B.J.G., A.J.A., F.Y., J.L., B.J.M.) and Pathology andoratory Medicine (B.J.G.), David Geffen School of Medicine atLA, University of California Los Angeles, Los Angeles, California.nquiries to Gary N. Holland, Jules Stein Eye Institute, 100 Stein Plaza,Microbiologic and Histoporneal Biopsies in the Eval

JARED R. YOUNGER, R. DUNCAN JOHNSRICHARD L. NEPOMUCENO, BEN J. GLASGOW

AND BARTLY J. MONDINO, ON BEH

URPOSE: To investigate the utility of corneal biopsy inevaluation of infectious keratitis; to compare resultsculture and histopathologic examination of the samecimens; to investigate potential factors related tositive biopsy results.ESIGN: Retrospective, observational case series.ETHODS: We reviewed medical records for all pa-

nts who underwent corneal biopsy because of infec-us keratitis at the Jules Stein Eye Institute from June89 through June 2009. In general, biopsy specimensre both cultured and examined histopathologically.sion size, lesion progression, and interval from presen-ion to biopsy were analyzed as possible predictors ofsitive biopsies.ESULTS: Organisms were identified in 20 of 48 (42%)nsecutive biopsies (positive cultures in 9 of 47 cases%]; positive histopathologic examination in 19 of 47es [40%]). Culture and histopathologic results werencordant in 30 of 46 biopsies (65%) for which bothhniques were performed; 10 of the 16 discordant casesre culture-negative/histopathology-positive, while theaining 6 had positive but discordant results for the 2hniques (cultures all showed bacteria; histopathologicamination showed fungi or cysts consistent with Acan-moeba sp.). Corneal biopsy revealed microorganisms12 of 27 patients (44%) with negative cultures ofrneal scrapings obtained at presentation. None of thetential risk factors was statistically associated withsitive biopsies.ONCLUSIONS: Corneal biopsy can be useful for iden-ying the cause of infectious keratitis in selected cases.lture and histopathologic examination can providemplementary information, but discordant results maycur. Acanthamoebic and fungal infections are moreely to be identified by histopathologic examination.bioLA, Los Angeles, CA 90095-7003; e-mail: holland@jsei.ucla.edu

2012 BY ELSEVIER INC. ALL2hologic Assessment oftion of Microbial Keratitis

, GARY N. HOLLAND, JON P. PAGE,THONY J. ALDAVE, FEI YU, JASON LITAK,OF THE UCLA CORNEA SERVICE

DENTIFYING THE CAUSAL ORGANISM IN CASES OF INFEC-

tious keratitis can be difficult. Corneal biopsy has beenrecommended for cases in which keratitis persists orgresses, despite antimicrobial therapy.16 Biopsies pro-e more sample for culture than initial scraping of theular surface, and they provide access to deeper stromalections than possible with superficial scraping alone.ey also provide an opportunity for histopathologicalysis, although there has been conflicting informationthe literature as to whether culture or histopathologicamination of a corneal biopsy specimen is more useful.4,6

rneal biopsies are not without risk; they may result inrneal perforation, stromal scarring, and irregular astig-tism. It is therefore important to establish the indicationsbiopsy, understand the limitations of the procedure, and

termine the most appropriate processing of biopsy speci-ns.It has been routine practice at the Jules Stein Eyetitute to divide corneal biopsy specimens and sendrtions both to the Clinical Microbiology Laboratory forlture and to the Ophthalmic Pathology Laboratory fortopathologic examination. We have performed a com-hensive review of corneal biopsies performed at UCLAer a 20-year period to assess the utility of this procedure.ecifically, we undertook this project with the followingals: to compare results of culture and histopathologicamination on the same specimen; to determine whetherults of these techniques differed on the basis of the typeinfectious agent; to identify factors that predictedsitive biopsy results; to compare biopsy results to initialltures of corneal scrapings; and to determine the clinicalevance of a negative corneal biopsy.psies were used for analyses, unless otherwise indicated.

RIGHTS RESERVED. 0002-9394/$36.00http://dx.doi.org/10.1016/j.ajo.2012.03.014

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VOAlthough this study was not prospectively designed,st cases were managed in a typical manner, per proto-ls of the UCLA Cornea Service, as described below. Atsentation, superficial corneal scrapings are obtainedth a spatula or surgical blade and inoculated directlyto blood, chocolate, and Sabouraud dextrose agar, ando Middlebrook or Lowenstein-Jensen and thioglycollatensport media. Material from the scraping is also placedat least 2 glass slides for staining and microscopic

amination. Plates, tubes, and slides are transportedmediately to the UCLA Clinical Laboratories. A spec-en is also sent for inoculation onto non-nutrient agarth Escherichia coli overlay for culture of Acanthamoeba sp.Indications for biopsy include: 1) lack of clinical re-nse to initial antimicrobial therapy chosen on the basisinitial culture or smear results (or to broad-spectrumtibiotics in those cases for which cultures and smears aregative); or 2) progression of lesions (eg, enlargement ofomal infiltrates or corneal thinning), despite treatment,en if there are some other signs of response. Prior topsy, antimicrobial therapy is generally discontinued forproximately 24 hours.Biopsies are performed in a minor procedure room usingical anesthesia. In most cases, a partial-thickness inci-n is made with a 2-mm or 3-mm trephine at a leadingge of the corneal infiltrate, incorporating both infectedd adjacent, clear cornea. In selected cases, an alterna-e, free-hand method is used, in which a sharp bladeates a keratotomy at the edge of the infiltrate, afterich a surgical blade is used to create a lamellar incisiono the infiltrate and the overlying tissue is removed withssors. Biopsy specimens are bisected; half is placed inpreserved saline for transport to the UCLA Clinicalboratories for culture, while the other half is placed in% buffered formalin for transport to the Ophthalmicthology Laboratory. Material for culture is ground with artar and pestle and mixed with sterile trypticase soyth; this suspension is placed onto appropriate media forlation of bacteria, fungi, mycobacteria, and Acantham-a sp. Material for histopathologic examination is pro-sed routinely for paraffin embedding, and 5-m-thicktions are cut at 250-m-step intervals. Sections arepared with hematoxylin-eosin, Gomori methenamineer, periodic acidSchiff, Ziehl-Neelsen, and Gramins for identification of organisms.In general, patients are treated topically at presentationth 2 fortified antibiotics for broad-spectrum coveragetil results from initial scrapings are obtained, at whiche treatment is individualized, based on those results.bsequent treatment decisions are based on best medicalgment. Therapy is changed on the basis of biopsy resultsen initial antimicrobial agents are deemed not appro-ate treatment for the organisms identified by the biopsy;culture and histopathologic examination provide con-

ting results (see definition of discordant biopsy results ob

UTILITY OF CORNEAL. 154, NO. 3low), we have generally chosen antimicrobial agents onbasis of histopathologic examination results.

ATA COLLECTION: Age and sex were identified forh patient; no other demographic data were collected.e following data about corneal infections were col-ted: greatest diameter of corneal stromal infiltrates;ation of the infiltrates (central, peripheral); and changeclinical signs during follow-up (enlargement of infiltrates;w foci of infiltration). Culture results for corneal scrapings

TABLE 1. Demographic Factors, Corneal LesionCharacteristics, and Corneal Biopsy Results for 48

Patients With Infectious Keratitis

Characteristic Summary Values

Demographic factors

Male sex, n (%) 21 (44%)

Age

Mean SD 55.5 15.8 years

Median (range) 57 (29 to 89) years

Corneal lesion characteristics

Size (largest dimension; n36)a

Mean SD (n 36)a 5.1 2.3 mm

Median (range) (n 36)a 5.0 (1.1 to 10) mm

Location (n 38)a

Central 28 (74%)

Peripheral 10 (26%)

Intervals

Median interval from onset of

keratitis to corneal scraping at

initial UCLA examination (range)

(n 37)a

15 (1 to 240) days

Median interval from scraping to

biopsy (range) (n 40)a 19 (0 to 275) days

Positive culture of initial corneal

scrapings (n 42)a 15 (36%)

Biopsy-related factors

Indication for biopsy (n 45)a

Lack of improvement 23 (55%)

Progression 19 (45%)

Otherb 3

Positive resultsc 20 (42%)

Number of second biopsiesd 5

Perforation during procedure (n 53)e 3 (6%)

aNumber of cases for which information was available, if less

than 48.bTwo cases of amputation of LASIK flap for confirmation of

diagnosis; 1 case to confirm report of positive fungal isolate from

outside facility.cEither positive culture or organisms seen on histopathologic

examination.dThree of 5 patients had negative first biopsies; 2 of the

second biopsies were positive by culture or by histopathologic

examination; details are provided in the text.eIncludes 48 first and 5 second biopsy procedures.tained at presentation (initial evaluation at UCLA) were

L BIOPSIES 513

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51orded. We did not consider the results of microscopicamination of smears made of the material from cornealapings at presentation.We recorded the following treatment information: an-icrobial therapy prior to initial evaluation at UCLAd initial antimicrobial therapy at UCLA. We identifiedinterval from initial evaluation at UCLA to biopsy andindication for each biopsy (progression [enlargement ofltrate, new focus of infiltration], lack of response,er).We evaluated each case for the following outcomes aftermary biopsy: perforation at the time of biopsy; change indical therapy; repeat scraping for culture; repeat biop-s; other surgical intervention (evisceration, penetratingratoplasty [PK]); and healing of ulceration. The intervalsm biopsy to each of these outcomes were determined.r patients who underwent PK, we determined whetheranisms had been identified in the host corneal tissue

TABLE 2. Biopsy Results by Category of Infectious Agentfor 48 Patients With Infectious Keratitis

Organisms Identified by

Biopsy

Culture of

Biopsy

Specimen

(n 47)

Histopathologic

Examination of

Biopsy Specimen

(n 47)

P

Valuea

Bacteria 6 (12.8%) 4 (8.5%) .53

Mycobacteriab 2 (4.3%) 1 (2.1%) NE

Fungus 1 (2.1%) 6 (12.8%) .025

Acanthamoeba sp.c 0 8 (17.0%) NE

Total 9 (19.1%) 19 (40.4%) .002

NE not evaluated.aMcNemar test. This test evaluates whether two techniques

perform differently on the same sample; only those cases for

which both techniques had been used (n 46) were assessed.

Without knowing the true distribution, one cannot assess which

of the reported distributions is more accurate.bOne of the 2 culture-positive cases was the same case for

which mycobacteria were identified on histopathologic exami-

nation. The other culture-positive case was not examined his-

tologically; the lack of other histopathologic examination

positive cases prevented statistical comparison using the

McNemar test. If one assumes a binomial distribution, with the

proportion of cultures positive for mycobacteria among 47

specimens being the same as the results for histopathologic

examination (2.1%), then the probability of obtaining 2 or fewer

positive cultures for mycobacteria would be .92.cResults cannot be compared statistically using the McNemar

test because there were no cultures positive for Acanthamoeba

sp. If one assumes a binomial distribution, with the proportion of

cultures positive for Acanthamoeba sp. among 47 specimens

being the same as the results for histopathologic examination

(17.0%), then the probability of obtaining no positive cultures for

Acanthamoeba sp. would be .0002.oved at the time of surgery. we

AMERICAN JOURNAL OF O4EFINITIONS: A biopsy was considered to be positive ifanisms were identified by either culture or histopatho-ic examination. Concordant biopsy results were thosewhich there was agreement between culture results andtopathologic examination results (both negative or bothsitive and consistent with the same organism). Discor-nt biopsy results were those in which either culture ortopathologic examination results (but not both) weresitive or those with positive culture and positive histo-thologic examination results that suggested differentanisms. Healing was defined as resolution of infiltrates.therapeutic PK was one required before healing becauseperforation, impending perforation, or progression withk of scleral infection.

ATA ANALYSIS AND STATISTICAL TECHNIQUES: Toaluate whether the 2 techniques (culture vs histopathologicamination) were different in their abilities to identify eache of organism, we compared the distribution of positiveults, categorized by organism type, using the McNemart, which assesses the symmetry of the distribution for eachhnique, as a measure of the relative ability of each tontify organisms. It does not address the accuracy of eitherhnique. Agreement between culture and histopathologicamination results was assessed by kappa statistics. Theuskal-Wallis test was used to evaluate 2 potential risktors for positive biopsies (time from presentation to biopsy;ion size [greatest diameter of the stromal infiltrate]) and tompare times from biopsy to resolution based on biopsyults. The Fisher exact test was used to evaluate indicationsbiopsy as a risk factor. For patients who underwentltiple biopsies, results of positive second biopsies were usedcalculate intervals from biopsy to outcomes, if first biopsies

TABLE 3. Relationship Between Culture andHistopathologic Examination Results for 46 Corneal

Biopsy Specimens

Culture Results

Histopathologic Examination

Positive

NegativeConcordanta Discordantb

Positive 2 6 0

Negative NA 10 28

NA not applicable.aBoth culture and histopathologic examination identified or-

ganisms; morphologic and staining characteristics on histo-

pathologic examination were consistent with culture results.bBoth culture and histopathologic examination identified or-

ganisms; however, morphologic and staining characteristics on

histopathologic examination were not consistent with culture

results.re negative.

PHTHALMOLOGY SEPTEMBER 2012

WE

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VORESULTS

IDENTIFIED 48 CONSECUTIVE PATIENTS WHO UNDER-

nt at least 1 corneal biopsy. Table 1 shows demographictors, corneal lesion characteristics, and corneal biopsyated factors for this group of patients. Table 2 showspsy results. Organisms were identified by culture ortopathologic examination in 20 of 48 primary biopsies%). In 1 case, a specimen was not sent for culture; inother, a specimen was not sent for histopathologicamination. Cultures were positive in 9 of 47 cases%), while histopathologic examinations were positive19 of 47 biopsies (40%). Histopathologic examinations more likely to identify fungi and Acanthamoeba sp.n culture (Table 2). No cultures were positive foranthamoeba sp.; among the 8 cases with presumedanthamoeba sp. on histopathologic examination, somed only cystic spaces with apparent double walls betweenellae of corneal stroma (but without nuclei) that weregestive, but not diagnostic, of Acanthamoeba sp.As shown in Table 3, agreement between culture andtopathologic examination results was poor on a statis-al basis (kappa 0.011). Both culture and histopatho-ic examination results from the same biopsy wereailable in 46 cases. Results were concordant in 30 cases%) and discordant in 16 cases. Among the 30 concor-nt cases, both culture and histopathologic examinationre negative in 28 cases, while both were positive in 2es (fungi in 1 case, mycobacteria in the other). Among16 discordant cases, only histopathologic examination

s positive (ie, culture negative) in 10 cases; bacteriare seen in 4 cases, cysts consistent with Acanthamoebain 4 cases, and fungi in 2 cases. In the 6 discordant caseswhich both cultures and histopathologic examinationsre positive, all cultures grew bacteria, while histopatho-ic examinations revealed cysts consistent with Acan-

TABLE 4. Biopsy Results for 42 Patients With Infectious Keratit

Initial Scrapinga

Culture (n 41)

Bacteria Fungus Acanthamoeba sp. Mycobacteria

Bacteria (n 13) 1 0 0 0

Fungus (n 1) 0 1 0 0

Mycobacteriab

(n 1) 0 0 0 1

Negativec (n 27) 3d 0 0 1

aCategory of organisms isolated from corneal scraping material obtbNo histologic examination on subsequent biopsy.cOne of 27 cases had no culture on subsequent biopsy.dOne case each of Propionibacterium sp., Pseudomonas stutzeri, aeOne case of gram-positive cocci, 1 case of gram-negative rods,

bacteria; in none of these cases was the culture of the biopsy specimmoeba sp. (n 4) or fungi (n 2). The bacteria in 4 his

UTILITY OF CORNEAL. 154, NO. 3these 6 cases are commonly considered to be contami-nts (coagulase-negative staphylococci in 3 cases, Propi-ibacterium sp. in 1 case); the other 2 positive culturesw Pseudomonas stutzeri, which is less virulent than Ps.uginosa but has been reported to cause keratitis,7 andinetobacter lwoffi, an opportunistic pathogen, widelytributed in nature, that can colonize healthy tissues.ong the 4 cases with cysts on histopathologic exami-

tion and bacteria on culture, 3 were confirmed subse-ently to be infected with Acanthamoeba sp. atrapeutic PK; the other was lost to follow-up after thepsy. Of the 2 cases with fungus on histopathologicamination and bacteria on culture, 1 healed and theer had no organisms at therapeutic PK. For both,atment had been switched to antifungal agents inponse to biopsy results. For each of the culture-negativecordant cases with Acanthamoeba sp. (n 4) or fungus 2) on histopathologic examination, there were

tcome data (organisms identified on host tissue atrapeutic PK or healing in response to a change inrapy) to support the histopathologic diagnoses. Therere no discordant cases in which both culture andtopathologic examination identified bacteria, but inich the cultured bacteria were not consistent with therphology and staining characteristics of the bacterian on histologic examination (Table 3).Table 4 shows the relationship between culture results ofrneal scraping at initial UCLA examination and subse-ent biopsy results. Results for culture of initial scrapingsre known for 42 cases; results were not available orapings were not performed in 6 cases. Among the 15es with positive cultures on initial scrapings, subsequentpsies were positive in 3 cases. In 2 of these cases, biopsyults confirmed the results of the initial cultures (1 casemycobacterium and 1 case of fungus); in the third case,lture of initial scrapings grew Staph. epidermidis, whereas

o Underwent Corneal Scraping for Culture at Presentation

iopsy Results

Histopathologic Examination (n 41)

ative Bacteria Fungus Acanthamoeba sp. Mycobacteria Negative

2 0 1 0 0 12

0 0 1 0 0 0

0

2 3e 3 5 1 15

at initial examination.

aphylococcus epidermidis.

1 case with findings of both gram-positive and gramnegative

sitive.thethewehiswhmosee

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51ed a fungus (biopsy culture was discordant, also growingph. epidermidis, which was believed to be a contami-nt). Of the 27 cases with negative cultures of initialapings, 12 biopsies were positive. Of the 26 cases withgative cultures of initial scrapings that also had culturebiopsy specimens, only 4 biopsy cultures were positive (3cteria and 1 mycobacterium). Of the 26 cases withgative cultures of initial scrapings that also had histo-thologic examination of biopsy specimens, 12 weresitive, 9 of which were nonbacteria. Of the 3 cases withcteria on histopathologic examination only, 1 showedm-positive bacteria; 1 showed gram-negative bacteria;d 1 had substantial crush artifact and findings of bothm-positive and gram-negative bacteria, none of whichre intracellular. Of these 3 cases, 1 had been usingical antibacterial and antifungal drugs before the timeinitial scraping, 1 had been using only an antifungalg, and 1 had not been treated. Viewed another way,ong the 9 cases with positive cultures of biopsy material,ad undergone cultures of initial scrapings. In 3 cases,psy cultures revealed the same organism as initialapings (1 each of Fusarium sp., M. chelonae, and Staph.demidis). The initial cultures had been negative in theer 4 cases (biopsy positive for bacteria in 3 cases, for M.lonae in 1 case).Table 5 shows data associated with potential risk factorspositive biopsies. Although cases with positive biopsiesd larger infiltrates at initial UCLA examination (5.7 mm diameter) than negative biopsies (4.6 2.4 mm),difference was not significant (P .41). Mean time

m initial UCLA examination to biopsy was actuallyger for those with positive biopsies (80.4 99.1 days)n for those with negative biopsies (20.5 17.9 days),t the difference was not statistically significant (P ). The indication for initial corneal biopsy was docu-

TABLE 5. Assessment of Potential Risk Facto48 Patients With In

Pos

Interval from initial evaluation to biopsy

Mean SD 80.4

Median (range) 22 (0

Indication for biopsy

Lack of response (n 23) 12 (5

Progression (n 19) 5 (2

Infiltrate size (greatest diameter)

Mean SD 5.7

Median (range) 5.0 (3

SD standard deviation.aPositive results defined as identification of

examination.bKruskal-Wallis test.cFisher exact test.nted for 45 cases, including progression of disease in 19 6

AMERICAN JOURNAL OF O6es and lack of response to topical antimicrobial therapyithout lesion enlargement or new foci) in 23 casesable 1). Progression of disease was not more likely to beociated with a positive biopsy than lack of responsene (P .12).There were 3 perforations at the time of biopsy proce-res (6% of 53 first and second biopsies). One caseuired immediate PK, while the other 2 were managedequately with cyanoacrylate glue. A review of these 3es revealed no unique features to explain the perfora-ns. None had descemetocoeles, and 2 of 3 were periph-l ulcers. Gram-positive bacteria were identified in 2es; biopsy was negative in the other. Each biopsy wasrformed by a different surgeon. Thinning was not quan-ated in most cases, and thus could not be evaluated as ak factor for perforation.Antimicrobial therapy was changed after biopsy results18 of 48 cases (37.5%) (13 of 20 [65%] positive biopsies;f 28 [18%] negative biopsies; P .002). Outcomes wereown for 43 patients. Evisceration was performed on 1while keratitis was active; biopsy in that case had been

gative. A therapeutic PK was performed in 7 of 19 cases%) with positive corneal biopsies and in 4 of the 24es (21%, including the evisceration) with a negativerneal biopsy (P .31). Among those with positivepsies who underwent therapeutic PK, the same organ-was identified by culture or histopathologic examina-

n of the corneal button in 4 cases; in contrast, for thoseth negative biopsies, no organisms were found by culturehistopathologic examination of host tissue removed ate of therapeutic PK. The interval from biopsy togical intervention was not significantly different be-een those patients with positive biopsies (94.9 112.8ys [median 17 days, range 5285 days], n 7) and thoseth negative biopsies (22.8 12.9 [median 21 days, range

Positive Corneal Biopsy Results Amongous Keratitis

esultsa Negative Results P Value

1 days 20.5 17.9 days

5) days 12.5 (0 to 57) days .22b

.12c

) 11 (47.8%)

) 14 (73.7%)

mm 4.6 2.4 mm

10.0) 5.2 (1.1 to 9.0) .41b

isms by either culture or histopathologicin5 okneyene(37cascobioismtiowiortimsurtwdawi41 days], n 5, P .94). Six of 13 patients (46%)

PHTHALMOLOGY SEPTEMBER 2012

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VOose treatment was changed following positive biopsyults underwent penetrating keratoplasty, while 6 of 30tients (20%) whose therapy was not changed because ofsitive biopsy results underwent penetrating keratoplasty .14). A change in therapy following positive biopsiess not associated with fewer therapeutic PK (P .14) orth shorter times to healing (P .42) than all other casesour series (data not shown).For those patients who did not require surgical interven-n while infection was active (therapeutic PK or eviscer-on, n 31), the interval from biopsy to healing wasrter among those with negative biopsies (mean 85.7 0.7 days [median 49 days, range 13468 days], n 19)n for those with positive biopsies (mean 98.6 47.4ys [median 85.5 days, range 39185 days], n 12),hough the association was weak (P .062). Among thepatients with negative biopsies who had follow-up, 7%) eventually had evidence of active corneal disease (5th clinical progression of corneal infiltrates that requiredgery; 1 with a repeat biopsy positive for fungus; and 1o was eventually diagnosed with necrotizing herpeticomal keratitis on the basis of a positive viral culture).Second biopsies were performed on 5 patients: 3 wererformed because of negative first biopsies and 2 wererformed because of disease progression after positive firstpsies, to confirm or rule out persistent infection (1sitive for mycobacteria; the other for presumed Acan-moeba sp.). Second biopsies were performed from 1 week2 months after first biopsies. Only 1 of the 3 patientsth negative first biopsies had a positive second biopsy;ults were consistent with a fungal infection, and uponitching therapy to antifungal medications the patientsratitis healed. One of the 2 patients with positive firstpsies had a positive second biopsy; the patient had cystsnsistent with Acanthamoeba sp. on histopathologic ex-ination of both biopsies. One of the 5 second biopsiess complicated by perforation at the time of the proce-re; it was managed successfully with cyanoacrylate glue.We found no evidence of secular trends over the 20-yeardy period in terms of the proportion of positive biopsiesthe organisms identified (data not shown).

DISCUSSION

UMBER OF REPORTS HAVE DESCRIBED THE SUCCESSFUL

gnosis of mycobacterial, fungal, and acanthamoebicratitis with corneal biopsy,3,4,6,813 but the indicationscorneal biopsy remain undefined, and there has been nonsensus on the most appropriate processing of cornealpsy specimens. Our series supports the utility of cornealpsies. We were able to identify causal agents in 42%/48) of cases that underwent biopsy. Biopsy can providegnostic information in some cases that have negativeults on culture of corneal scrapings at presentation;

ong cases for which no microorganisms were seen on on

UTILITY OF CORNEAL. 154, NO. 3tial scrapings, 44% (12/27) had positive biopsies. Fur-rmore, in some cases, biopsy results changed diagnosest have been based on positive culture results of initialapings. Our results also support the value of cornealpsy for identifying the cause of infectious keratitis ines that are refractory to initial antimicrobial therapy;atment was changed for 65% (13/20) of our patientsth positive biopsies. Our results indicate that positivepsies do not depend on evidence of disease progression,d that a delay in performing a biopsy does not necessarilyuce the likelihood of a positive result for patients withrsistent keratitis. Thus, biopsy remains an option, evendisease that is long-standing.Our results give some insight into the types of cases thatll eventually need corneal biopsy. The spectrum ofsal organisms identified by corneal biopsy does notect the distribution of all causes of infectious keratitisn at UCLA, the majority of which are staphylococcid Pseudomonas aeruginosa (data not shown). Also, nonethe many cases of infectious keratitis at UCLA forich Acanthamoeba sp. or gram-negative bacteria werelated from initial corneal scrapings later required cor-al biopsy. Our results suggest that the cases most likelyrequire biopsy are those for which cultures of initialrneal scrapings are negative or grow gram-positivecteria.We were also able to address the clinical relevance of apsy that is negative by both culture and histopathologicamination techniques. There was a trend for the meane to resolution of active keratitis to be shorter for thoseth negative biopsies than for those with positive biop-s. Fewer patients with negative biopsies needed surgeryile corneas were still inflamed, and in the majority oftients with negative biopsies, the subsequent course wasnsistent with eradication of infection. Nevertheless, astantial minority of cases with negative biopsies hadsequent evidence of persistent infection on the basis ofsecond scraping, additional biopsy, or examination ofst tissue following PK. Thus, while a negative biopsyvides some support for the assumption that infections been treated adequately, negative biopsies do not rulet the possibility of persistent infection, and patientsst still be monitored closely for progression of disease.The results also demonstrate the value of our protocolprocessing specimens, in which both culture and

topathologic examination of the biopsy specimens isrformed routinely. Most reported series have describedher culture or histopathologic examination of biopsycimens, but not both.36,13 Among the larger series, Leed Green described the identification of organisms bytopathologic examination in 9 of 42 corneal biopsycimens that were performed for diagnosis of infectiousratitis, but culture of the same specimens was notrformed for comparison.4 Alexandrakis and associatesrformed both culture and histopathologic examination

611 of 33 biopsy specimens. Seven of the 11 biopsies

L BIOPSIES 517

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51re culture-positive (3 of which had negative histopatho-ic examinations), and 5 of the 11 biopsies had positivetopathologic examination findings (only 1 of which waslture-negative). On the basis of these results, theyncluded that culture was more sensitive than histo-thologic examination. In contrast, Ishibashi and associ-s reported that histopathologic examination of biopsycimens in 4 cases provided information that culture didt.1,2

Our series provides some additional support for thetion that histopathologic examination is more valuablen culture as a means of assessing biopsy specimens. Theportion of positive results was higher for histopatho-ic examination than for culture, and when results werecordant, culture results were often consistent withntaminant bacteria. In each of these discordant cases forich follow-up information was available, organisms seenhistopathologic examination were confirmed subse-

ently (repeat biopsy, culture of repeat scraping, andlture or histopathologic examination of host tissue atrapeutic PK) or treatment directed to the organismntified on histopathologic examination led to resolu-n of infection. Histopathologic examination was moreely to identify fungi and cysts consistent with Acantham-a sp. than culture, despite the use of appropriate media.ese observations are consistent with laboratory studiesolving rabbit models of corneal infection, in whichtopathologic examination of biopsy specimens was moreely to identify the presence of fungi than culture.2 Inrticular, histopathologic examination appeared to bere useful than culture of biopsy material when culture oftial corneal scrapings was negative. Nevertheless, eachhnique can provide complementary and unique infor-tion regarding infection; for example, culture can iden-the specific species of organism and sensitivities to

timicrobials, while histopathologic examination cannot.like other reported series, we had no cases in whichltures were positive but histopathologic examination wasgative; should that situation occur, our culture data suggestt clinicians should be wary of possible contaminants.Previous studies have attempted to relate outcomes ofectious keratitis to biopsy results. In the series reportedAlexandrakis and associates, a lower proportion of

tients with positive biopsies eventually underwent pen-ating keratoplasty (5 of 27 patients [18.5%]) than didtients with negative biopsies (5 of 6 patients).6 Thethors hypothesized that positive biopsies were moreely to lead to correct treatments and thus to a reduceded for surgery. Because many unrelated factors canuence decisions about surgery, we chose instead to lookly at the need for penetrating keratoplasty before reso-ion of active corneal inflammation, as a more meaning-surrogate for ability to control infection. Neither a

sitive biopsy nor a change in treatment based on asitive biopsy predicted the need for penetrating kerato-

sty while keratitis was still active. ap

AMERICAN JOURNAL OF O8Other techniques for the identification of infectiousanisms have become available since the start of thisies, including confocal microscopy.14 Our study does notrmit an assessment of the relative utility of cornealpsy vs confocal microscopy; nevertheless, while confo-microscopy holds promise as an adjunctive test, it hasitations, including lack of widespread availability andneed for experienced examiners who can obtain and

erpret findings properly. Corneal biopsy remains a rela-ely simple and more accessible method for evaluatingcornea in difficult cases, including those with negativeequivocal confocal microscopy results. In a maskedessment of patients with infectious keratitis, pathogenssequently confirmed by other means have been identi-d correctly by confocal microscopy in only 30% to 60%cases, depending on the experience and skill of theaminer.14 Future studies are needed to determine thesitivity of corneal biopsy vs confocal microscopy.A major strength of our study is the fact that welowed a standard approach to the processing of biopsycimens throughout the study period, which reduces thetential for bias in the comparison of culture vs histo-thologic examination of specimens. We also studied aatively large number of patients, when compared tost previously reported series. Nevertheless, the numbercases is still too small for detailed statistical analyses ofny potential influences on biopsy results. Anotheritation of this study was the inability to evaluate theuence of biopsy location, surgical technique, and spec-en size on results. We did not compare biopsy results tocroscopic examination of smears made with materialtained at the time of initial scrapings; although suchaminations can provide important information aboutease causation when positive, the sensitivity of thiscedure is low, especially for bacterial infections.15

cause we did not know the true cause of keratitis in eache, we were unable to study specificity or sensitivity ofh technique.The study includes other limitations typically associatedth retrospective analyses; not all patients were managedthe same manner, and treatment was uncontrolled.so, patients were not randomized to biopsy procedures,king it difficult to confirm that the information ob-ned in those cases with positive biopsy results had anpact on outcomes. Furthermore, as we studied a selectedpulation, our cases are not necessarily representative ofpatients with infectious keratitis, which limits the

ility to generalize our results; we are not, however,vocating biopsies on all patients. We believe that simplyfact that biopsies can identify pathogens not found on

tial evaluation justifies use of this procedure.In summary, our results indicate the value of cornealpsy in the management of selected patients with infec-us keratitis, including those with negative cultures ofrneal scrapings at presentation and those with lack of

propriate response to initial treatment. Delay in per-

PHTHALMOLOGY SEPTEMBER 2012

forming biopsy does not necessarily eliminate the possibil-ity of positive results. Histopathologic examination ofbiopsy specimens was more likely to identify Acanthamoebasp. and fungi than culture, but results support the contin-ued practice of processing biopsy specimens for bothculture and histopathologic examination; the 2 techniquescan give complementary information. In our experience,when discordant positive results occur (culture and histo-

pathologic examination reveal different pathogens), histo-pathologic examination results are more likely to indicatethe causal organism, but this issue requires additionalstudy; for instance, in this study, we could not rule out thepossibility that discordant positive results reflect infectionwith multiple organisms. Although useful, corneal biopsyprocedures are not without risks; in our series, 3 perfora-tions occurred.

ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OFInterest. Publication of this article was supported by funding from the Research to Prevent Blindness (New York, New York) (Drs Holland, Aldave,Mondino), a Horizon Grant to the UCLA Department of Ophthalmology from Allergan, Inc (Irvine, California, USA), the Steven and NancyCooperman Fellowship (Dr Younger), the David May II Fellowship (Dr Johnson), and the Klara Fleming Fellowship (Dr Page). Dr Younger was anAllergan Cornea Fellow (20072008). All named fellowships are funded by endowments within the UCLA Department of Ophthalmology. None of theauthors have conflicts of interest with any aspect of this study. Funding entities had no role in the conduction or presentation of this study. Involvedin study design (G.N.H., R.L.N., F.Y., B.J.M.); data collection (J.R.Y., R.D.J., J.P.P., R.L.N., J.L.); data management and analysis (J.R.Y., R.D.J., G.N.H.,J.P.P., R.L.N., F.Y., J.L., B.J.M.); data interpretation (all authors); preparation of initial draft of manuscript (J.R.Y., R.D.J., G.N.H., R.L.N., J.L., B.J.M.);and n conof m A pr

asey

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7. Brinser JH, Torczynski E. Unusual Pseudomonas corneal

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15. Sharma S, Kunimoto DY, Gopinathan U, et al. Evaluation

VO UTILITY OF CORNEAL. 154, NO. 3ulcers. Am J Ophthalmol 1977;84(4):462466.. Malbran ES, Fernandez Meijide RE, Stefani C. [Diagnosticvalue of biopsy in mycotic abscess of the cornea]. ArchOftalmol B Aires 1970;45(10):419423.

of corneal scraping smear examination methods in thediagnosis of bacterial and fungal keratitis: a survey of eightyears of laboratory experience. Cornea 2002;21(7):643647.review and approval of manuscript (all authors). Drs Younger and Johnsoedical records was approved by the Institutional Review Board at UCL

Participating members of the UCLA Cornea Service included Richard C

REFERENCES

. Ishibashi Y, Kaufman HE. Corneal biopsy in the diagnosis ofkeratomycosis. Am J Ophthalmol 1986;101(3):288293.

. Ishibashi Y, Hommura S, Matsumoto Y. Direct examinationvs culture of biopsy specimens for the diagnosis of keratomy-cosis. Am J Ophthalmol 1987;103(5):636640.

. Newton C, Moore MB, Kaufman HE. Corneal biopsy inchronic keratitis. Arch Ophthalmol 1987;105(4):577578.

. Lee P, Green WR. Corneal biopsy. Indications, techniques, and areport of a series of 87 cases. Ophthalmology 1990;97(6):718721.

. Kompa S, Langefeld S, Kirchhof B, Schrage N. Cornealbiopsy in keratitis performed with the microtrephine. GraefesArch Clin Exp Ophthalmol 1999;237(11):915919.

. Alexandrakis G, Haimovici R, Miller D, Alfonso EC. Cor-neal biopsy in the management of progressive microbialkeratitis. Am J Ophthalmol 2000;129(5):571576.tributed equally to preparation of this article. The retrospective reviewior to commencement of the study., Sophie X. Deng, and D. Rex Hamilton.

DeVoe AG. Keratomycosis. Am J Ophthalmol 1971;71(1 Pt2):406414.Moore MB, McCulley JP, Luckenbach M, et al. Acantham-oeba keratitis associated with soft contact lenses. Am JOphthalmol 1985;100(3):396403.Moore MB, Newton C, Kaufman HE. Chronic keratitiscaused by Mycobacterium gordonae. Am J Ophthalmol1986;102(4):516521.Whitehouse G, Reid K, Hudson B, Lennox VA, LawlessMA. Corneal biopsy in microbial keratitis. Aust N Z JOphthalmol 1991;19(3):193196.Allan BD, Morlet N, Dart JK. Microbiologic investigation ofsuspected microbial keratitis. Ophthalmology 1996;103(8):11651166.Hau SC, Dart JK, Vesaluoma M, et al. Diagnostic accuracy ofmicrobial keratitis with in vivo scanning laser confocalmicroscopy. Br J Ophthalmol 2010;94(8):982987.L BIOPSIES 519

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Duncan Johnson, MD, is a David May II Fellow in Cornea-External Ocular Disease and Refractive Surgery in theLA Department of Ophthalmology, Los Angeles, California. He has a particular interest in surgical management of

mplicated corneal diseases. He has published on a variety of topics related to corneal and external ocular diseases,luding biomechanical properties of corneas undergoing refractive surgery.UTILITY OF CORNEAL BIOPSIESL. 154, NO. 3 519.e1

JarUCCapropaof

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ed R. Younger, MD, MPH, is a former Clinical Fellow in Cornea-External Ocular Disease and Refractive Surgery in theLA Department of Ophthalmology, Los Angeles, California. He is currently in private practice in Fountain Valley,lifornia, and continues to be a member of the Medical Staff at the Ronald Reagan-UCLA Medical Center. He alsovides eye care to the underserved population of Santa Catalina Island, off the coast of Southern California, as art-time member of the Catalina Island Medical Center (Avalon, CA). Dr Younger is a member of the Board of Directorsthe Orange County (CA) Society of Ophthalmology.AMERICAN JOURNAL OF OPHTHALMOLOGY9.e2 SEPTEMBER 2012

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00029394_v154i3_S000293941200205X_main.pdfMicrobiologic and Histopathologic Assessment of Corneal Biopsies in the Evaluation of Microbial ...MethodsData CollectionDefinitionsData Analysis and Statistical Techniques

ResultsDiscussionReferences