Microbiologic and Histopathologic Assessment of

Corneal Biopsies in the Evaluation of Microbial Keratitis

JARED R. YOUNGER, R. DUNCAN JOHNSON, GARY N. HOLLAND, JON P. PAGE,
RICHARD L. NEPOMUCENO, BEN J. GLASGOW, ANTHONY J. ALDAVE, FEI YU, JASON LITAK,
AND BARTLY J. MONDINO, ON BEHALF OF THE UCLA CORNEA SERVICE
PURPOSE: To investigate the utility of corneal biopsy in

the evaluation of infectious keratitis; to compare results

of culture and histopathologic examination of the same
specimens; to investigate potential factors related to
positive biopsy results.
DESIGN: Retrospective, observational case series.
METHODS: We reviewed medical records for all patients who underwent corneal biopsy because of infectious keratitis at the Jules Stein Eye Institute from June
1989 through June 2009. In general, biopsy specimens
were both cultured and examined histopathologically.
Lesion size, lesion progression, and interval from presentation to biopsy were analyzed as possible predictors of
positive biopsies.
RESULTS: Organisms were identified in 20 of 48 (42%)
consecutive biopsies (positive cultures in 9 of 47 cases
[19%]; positive histopathologic examination in 19 of 47
cases [40%]). Culture and histopathologic results were
concordant in 30 of 46 biopsies (65%) for which both
techniques were performed; 10 of the 16 discordant cases
were culture-negative/histopathology-positive, while the
remaining 6 had positive but discordant results for the 2
techniques (cultures all showed bacteria; histopathologic
examination showed fungi or cysts consistent with Acanthamoeba sp.). Corneal biopsy revealed microorganisms
in 12 of 27 patients (44%) with negative cultures of
corneal scrapings obtained at presentation. None of the
potential risk factors was statistically associated with
positive biopsies.
CONCLUSIONS: Corneal biopsy can be useful for identifying the cause of infectious keratitis in selected cases.
Culture and histopathologic examination can provide
complementary information, but discordant results may
occur. Acanthamoebic and fungal infections are more
likely to be identified by histopathologic examination.
(Am J Ophthalmol 2012;154:512519. 2012 by
Elsevier Inc. All rights reserved.)
Accepted for publication Mar 7, 2012.
From the Ocular Inflammatory Disease Center, Jules Stein Eye Institute (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.,
B.J.M.), and the Departments of Ophthalmology (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., B.J.M.) and Pathology and
Laboratory Medicine (B.J.G.), David Geffen School of Medicine at
UCLA, University of California Los Angeles, Los Angeles, California.
Inquiries to Gary N. Holland, Jules Stein Eye Institute, 100 Stein Plaza,
UCLA, Los Angeles, CA 90095-7003; e-mail: holland@jsei.ucla.edu

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DENTIFYING THE CAUSAL ORGANISM IN CASES OF INFEC-

tious keratitis can be difficult. Corneal biopsy has been

recommended for cases in which keratitis persists or
progresses, despite antimicrobial therapy.1 6 Biopsies provide more sample for culture than initial scraping of the
ocular surface, and they provide access to deeper stromal
infections than possible with superficial scraping alone.
They also provide an opportunity for histopathologic
analysis, although there has been conflicting information
in the literature as to whether culture or histopathologic
examination of a corneal biopsy specimen is more useful.4,6
Corneal biopsies are not without risk; they may result in
corneal perforation, stromal scarring, and irregular astigmatism. It is therefore important to establish the indications
for biopsy, understand the limitations of the procedure, and
determine the most appropriate processing of biopsy specimens.
It has been routine practice at the Jules Stein Eye
Institute to divide corneal biopsy specimens and send
portions both to the Clinical Microbiology Laboratory for
culture and to the Ophthalmic Pathology Laboratory for
histopathologic examination. We have performed a comprehensive review of corneal biopsies performed at UCLA
over a 20-year period to assess the utility of this procedure.
Specifically, we undertook this project with the following
goals: to compare results of culture and histopathologic
examination on the same specimen; to determine whether
results of these techniques differed on the basis of the type
of infectious agent; to identify factors that predicted
positive biopsy results; to compare biopsy results to initial
cultures of corneal scrapings; and to determine the clinical
relevance of a negative corneal biopsy.

METHODS
WE REVIEWED MEDICAL RECORDS FOR ALL PATIENTS WITH

suspected infectious keratitis who underwent corneal biopsies at the Jules Stein Eye Institute from June 1989
through June 2009. Cases were identified from a registry of
specimens in the Ophthalmic Pathology Laboratory; included were those specimens identified as being associated
with corneal biopsy or diagnostic keratectomy. For
patients who underwent multiple biopsies, data from first
biopsies were used for analyses, unless otherwise indicated.

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RIGHTS RESERVED.

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

Although this study was not prospectively designed,

most cases were managed in a typical manner, per protocols of the UCLA Cornea Service, as described below. At
presentation, superficial corneal scrapings are obtained
with a spatula or surgical blade and inoculated directly
onto blood, chocolate, and Sabouraud dextrose agar, and
into Middlebrook or Lowenstein-Jensen and thioglycollate
transport media. Material from the scraping is also placed
on at least 2 glass slides for staining and microscopic
examination. Plates, tubes, and slides are transported
immediately to the UCLA Clinical Laboratories. A specimen is also sent for inoculation onto non-nutrient agar
with Escherichia coli overlay for culture of Acanthamoeba sp.
Indications for biopsy include: 1) lack of clinical response to initial antimicrobial therapy chosen on the basis
of initial culture or smear results (or to broad-spectrum
antibiotics in those cases for which cultures and smears are
negative); or 2) progression of lesions (eg, enlargement of
stromal infiltrates or corneal thinning), despite treatment,
even if there are some other signs of response. Prior to
biopsy, antimicrobial therapy is generally discontinued for
approximately 24 hours.
Biopsies are performed in a minor procedure room using
topical anesthesia. In most cases, a partial-thickness incision is made with a 2-mm or 3-mm trephine at a leading
edge of the corneal infiltrate, incorporating both infected
and adjacent, clear cornea. In selected cases, an alternative, free-hand method is used, in which a sharp blade
creates a keratotomy at the edge of the infiltrate, after
which a surgical blade is used to create a lamellar incision
into the infiltrate and the overlying tissue is removed with
scissors. Biopsy specimens are bisected; half is placed in
unpreserved saline for transport to the UCLA Clinical
Laboratories for culture, while the other half is placed in
10% buffered formalin for transport to the Ophthalmic
Pathology Laboratory. Material for culture is ground with a
mortar and pestle and mixed with sterile trypticase soy
broth; this suspension is placed onto appropriate media for
isolation of bacteria, fungi, mycobacteria, and Acanthamoeba sp. Material for histopathologic examination is processed routinely for paraffin embedding, and 5-m-thick
sections are cut at 250-m-step intervals. Sections are
prepared with hematoxylin-eosin, Gomori methenamine
silver, periodic acidSchiff, Ziehl-Neelsen, and Gram
stains for identification of organisms.
In general, patients are treated topically at presentation
with 2 fortified antibiotics for broad-spectrum coverage
until results from initial scrapings are obtained, at which
time treatment is individualized, based on those results.
Subsequent treatment decisions are based on best medical
judgment. Therapy is changed on the basis of biopsy results
when initial antimicrobial agents are deemed not appropriate treatment for the organisms identified by the biopsy;
if culture and histopathologic examination provide conflicting results (see definition of discordant biopsy results
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TABLE 1. Demographic Factors, Corneal Lesion

Characteristics, and Corneal Biopsy Results for 48
Patients With Infectious Keratitis
Characteristic

Demographic factors
Male sex, n (%)
Age
Mean SD
Median (range)
Corneal lesion characteristics
Size (largest dimension; n36)a
Mean SD (n 36)a
Median (range) (n 36)a
Location (n 38)a
Central
Peripheral
Intervals
Median interval from onset of
keratitis to corneal scraping at
initial UCLA examination (range)
(n 37)a
Median interval from scraping to
biopsy (range) (n 40)a
Positive culture of initial corneal
scrapings (n 42)a
Biopsy-related factors
Indication for biopsy (n 45)a
Lack of improvement
Progression
Otherb
Positive resultsc
Number of second biopsiesd
Perforation during procedure (n 53)e

Summary Values

21 (44%)
55.5 15.8 years
57 (29 to 89) years

5.1 2.3 mm
5.0 (1.1 to 10) mm
28 (74%)
10 (26%)
15 (1 to 240) days

19 (0 to 275) days
15 (36%)

23 (55%)
19 (45%)
3
20 (42%)
5
3 (6%)

a
Number of cases for which information was available, if less
than 48.
b
Two cases of amputation of LASIK flap for confirmation of
diagnosis; 1 case to confirm report of positive fungal isolate from
outside facility.
c
Either positive culture or organisms seen on histopathologic
examination.
d
Three 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.
e
Includes 48 first and 5 second biopsy procedures.

below), we have generally chosen antimicrobial agents on

the basis of histopathologic examination results.
DATA COLLECTION:

Age and sex were identified for

each patient; no other demographic data were collected.
The following data about corneal infections were collected: greatest diameter of corneal stromal infiltrates;
location of the infiltrates (central, peripheral); and change
in clinical signs during follow-up (enlargement of infiltrates;
new foci of infiltration). Culture results for corneal scrapings
obtained at presentation (initial evaluation at UCLA) were

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TABLE 2. Biopsy Results by Category of Infectious Agent

for 48 Patients With Infectious Keratitis

Organisms Identified by
Biopsy

Bacteria
Mycobacteriab
Fungus
Acanthamoeba sp.c
Total

Culture of
Biopsy
Specimen
(n 47)

6 (12.8%)
2 (4.3%)
1 (2.1%)
0
9 (19.1%)

Histopathologic
Examination of
Biopsy Specimen
(n 47)

4 (8.5%)
1 (2.1%)
6 (12.8%)
8 (17.0%)
19 (40.4%)

TABLE 3. Relationship Between Culture and

Histopathologic Examination Results for 46 Corneal
Biopsy Specimens
Histopathologic Examination

P
Valuea

Positive
Culture Results

.53
NE
.025
NE
.002

Positive
Negative

NE not evaluated.
McNemar 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.
b
One of the 2 culture-positive cases was the same case for
which mycobacteria were identified on histopathologic examination. The other culture-positive case was not examined histologically; 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.
c
Results 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.

Negative

2
NA

6
10

0
28

DEFINITIONS:

A biopsy was considered to be positive if

organisms were identified by either culture or histopathologic examination. Concordant biopsy results were those
in which there was agreement between culture results and
histopathologic examination results (both negative or both
positive and consistent with the same organism). Discordant biopsy results were those in which either culture or
histopathologic examination results (but not both) were
positive or those with positive culture and positive histopathologic examination results that suggested different
organisms. Healing was defined as resolution of infiltrates.
A therapeutic PK was one required before healing because
of perforation, impending perforation, or progression with
risk of scleral infection.

DATA ANALYSIS AND STATISTICAL TECHNIQUES:

To
evaluate whether the 2 techniques (culture vs histopathologic
examination) were different in their abilities to identify each
type of organism, we compared the distribution of positive
results, categorized by organism type, using the McNemar
test, which assesses the symmetry of the distribution for each
technique, as a measure of the relative ability of each to
identify organisms. It does not address the accuracy of either
technique. Agreement between culture and histopathologic
examination results was assessed by kappa statistics. The
Kruskal-Wallis test was used to evaluate 2 potential risk
factors for positive biopsies (time from presentation to biopsy;
lesion size [greatest diameter of the stromal infiltrate]) and to
compare times from biopsy to resolution based on biopsy
results. The Fisher exact test was used to evaluate indications
for biopsy as a risk factor. For patients who underwent
multiple biopsies, results of positive second biopsies were used
to calculate intervals from biopsy to outcomes, if first biopsies
were negative.

recorded. We did not consider the results of microscopic

examination of smears made of the material from corneal
scrapings at presentation.
We recorded the following treatment information: antimicrobial therapy prior to initial evaluation at UCLA
and initial antimicrobial therapy at UCLA. We identified
the interval from initial evaluation at UCLA to biopsy and
the indication for each biopsy (progression [enlargement of
infiltrate, new focus of infiltration], lack of response,
other).
We evaluated each case for the following outcomes after
primary biopsy: perforation at the time of biopsy; change in
medical therapy; repeat scraping for culture; repeat biopsies; other surgical intervention (evisceration, penetrating
keratoplasty [PK]); and healing of ulceration. The intervals
from biopsy to each of these outcomes were determined.
For patients who underwent PK, we determined whether
organisms had been identified in the host corneal tissue
removed at the time of surgery.
AMERICAN JOURNAL

Discordantb

NA not applicable.
a
Both culture and histopathologic examination identified organisms; morphologic and staining characteristics on histopathologic examination were consistent with culture results.
b
Both culture and histopathologic examination identified organisms; however, morphologic and staining characteristics on
histopathologic examination were not consistent with culture
results.

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TABLE 4. Biopsy Results for 42 Patients With Infectious Keratitis who Underwent Corneal Scraping for Culture at Presentation
Biopsy Results
Culture (n 41)

Histopathologic Examination (n 41)

Initial Scrapinga

Bacteria

Fungus

Acanthamoeba sp.

Mycobacteria

Negative

Bacteria

Fungus

Acanthamoeba sp.

Mycobacteria

Negative

Bacteria (n 13)
Fungus (n 1)
Mycobacteriab
(n 1)
Negativec (n 27)

1
0

0
1

0
0

0
0

12
0

0
0

1
1

0
0

0
0

12
0

0
3d

0
0

0
0

1
1

0
22

3e

15

Category of organisms isolated from corneal scraping material obtained at initial examination.
No histologic examination on subsequent biopsy.
c
One of 27 cases had no culture on subsequent biopsy.
d
One case each of Propionibacterium sp., Pseudomonas stutzeri, and Staphylococcus epidermidis.
e
One case of gram-positive cocci, 1 case of gram-negative rods, and 1 case with findings of both gram-positive and gramnegative
bacteria; in none of these cases was the culture of the biopsy specimen positive.
b

RESULTS
WE IDENTIFIED 48 CONSECUTIVE PATIENTS WHO UNDER-

went at least 1 corneal biopsy. Table 1 shows demographic

factors, corneal lesion characteristics, and corneal biopsy
related factors for this group of patients. Table 2 shows
biopsy results. Organisms were identified by culture or
histopathologic examination in 20 of 48 primary biopsies
(42%). In 1 case, a specimen was not sent for culture; in
another, a specimen was not sent for histopathologic
examination. Cultures were positive in 9 of 47 cases
(19%), while histopathologic examinations were positive
in 19 of 47 biopsies (40%). Histopathologic examination
was more likely to identify fungi and Acanthamoeba sp.
than culture (Table 2). No cultures were positive for
Acanthamoeba sp.; among the 8 cases with presumed
Acanthamoeba sp. on histopathologic examination, some
had only cystic spaces with apparent double walls between
lamellae of corneal stroma (but without nuclei) that were
suggestive, but not diagnostic, of Acanthamoeba sp.
As shown in Table 3, agreement between culture and
histopathologic examination results was poor on a statistical basis (kappa 0.011). Both culture and histopathologic examination results from the same biopsy were
available in 46 cases. Results were concordant in 30 cases
(65%) and discordant in 16 cases. Among the 30 concordant cases, both culture and histopathologic examination
were negative in 28 cases, while both were positive in 2
cases (fungi in 1 case, mycobacteria in the other). Among
the 16 discordant cases, only histopathologic examination
was positive (ie, culture negative) in 10 cases; bacteria
were seen in 4 cases, cysts consistent with Acanthamoeba
sp. in 4 cases, and fungi in 2 cases. In the 6 discordant cases
for which both cultures and histopathologic examinations
were positive, all cultures grew bacteria, while histopathologic examinations revealed cysts consistent with Acanthamoeba sp. (n 4) or fungi (n 2). The bacteria in 4
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of these 6 cases are commonly considered to be contaminants (coagulase-negative staphylococci in 3 cases, Propionibacterium sp. in 1 case); the other 2 positive cultures
grew Pseudomonas stutzeri, which is less virulent than Ps.
aeruginosa but has been reported to cause keratitis,7 and
Acinetobacter lwoffi, an opportunistic pathogen, widely
distributed in nature, that can colonize healthy tissues.
Among the 4 cases with cysts on histopathologic examination and bacteria on culture, 3 were confirmed subsequently to be infected with Acanthamoeba sp. at
therapeutic PK; the other was lost to follow-up after the
biopsy. Of the 2 cases with fungus on histopathologic
examination and bacteria on culture, 1 healed and the
other had no organisms at therapeutic PK. For both,
treatment had been switched to antifungal agents in
response to biopsy results. For each of the culture-negative
discordant cases with Acanthamoeba sp. (n 4) or fungus
(n 2) on histopathologic examination, there were
outcome data (organisms identified on host tissue at
therapeutic PK or healing in response to a change in
therapy) to support the histopathologic diagnoses. There
were no discordant cases in which both culture and
histopathologic examination identified bacteria, but in
which the cultured bacteria were not consistent with the
morphology and staining characteristics of the bacteria
seen on histologic examination (Table 3).
Table 4 shows the relationship between culture results of
corneal scraping at initial UCLA examination and subsequent biopsy results. Results for culture of initial scrapings
were known for 42 cases; results were not available or
scrapings were not performed in 6 cases. Among the 15
cases with positive cultures on initial scrapings, subsequent
biopsies were positive in 3 cases. In 2 of these cases, biopsy
results confirmed the results of the initial cultures (1 case
of mycobacterium and 1 case of fungus); in the third case,
culture of initial scrapings grew Staph. epidermidis, whereas
histopathologic examination of the biopsy specimen iden-

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TABLE 5. Assessment of Potential Risk Factors for Positive Corneal Biopsy Results Among
48 Patients With Infectious Keratitis

Interval from initial evaluation to biopsy

Mean SD
Median (range)
Indication for biopsy
Lack of response (n 23)
Progression (n 19)
Infiltrate size (greatest diameter)
Mean SD
Median (range)

Positive Resultsa

Negative Results

80.4 99.1 days

22 (0 to 275) days

20.5 17.9 days

12.5 (0 to 57) days

12 (52.2%)
5 (26.3%)
5.7 2.0 mm
5.0 (3.2 to 10.0)

P Value

.22b
.12c

11 (47.8%)
14 (73.7%)
4.6 2.4 mm
5.2 (1.1 to 9.0)

.41b

SD standard deviation.
a
Positive results defined as identification of organisms by either culture or histopathologic
examination.
b
Kruskal-Wallis test.
c
Fisher exact test.

tified a fungus (biopsy culture was discordant, also growing

Staph. epidermidis, which was believed to be a contaminant). Of the 27 cases with negative cultures of initial
scrapings, 12 biopsies were positive. Of the 26 cases with
negative cultures of initial scrapings that also had culture
of biopsy specimens, only 4 biopsy cultures were positive (3
bacteria and 1 mycobacterium). Of the 26 cases with
negative cultures of initial scrapings that also had histopathologic examination of biopsy specimens, 12 were
positive, 9 of which were nonbacteria. Of the 3 cases with
bacteria on histopathologic examination only, 1 showed
gram-positive bacteria; 1 showed gram-negative bacteria;
and 1 had substantial crush artifact and findings of both
gram-positive and gram-negative bacteria, none of which
were intracellular. Of these 3 cases, 1 had been using
topical antibacterial and antifungal drugs before the time
of initial scraping, 1 had been using only an antifungal
drug, and 1 had not been treated. Viewed another way,
among the 9 cases with positive cultures of biopsy material,
7 had undergone cultures of initial scrapings. In 3 cases,
biopsy cultures revealed the same organism as initial
scrapings (1 each of Fusarium sp., M. chelonae, and Staph.
epidemidis). The initial cultures had been negative in the
other 4 cases (biopsy positive for bacteria in 3 cases, for M.
chelonae in 1 case).
Table 5 shows data associated with potential risk factors
for positive biopsies. Although cases with positive biopsies
had larger infiltrates at initial UCLA examination (5.7
2.0 mm diameter) than negative biopsies (4.6 2.4 mm),
the difference was not significant (P .41). Mean time
from initial UCLA examination to biopsy was actually
longer for those with positive biopsies (80.4 99.1 days)
than for those with negative biopsies (20.5 17.9 days),
but the difference was not statistically significant (P
.22). The indication for initial corneal biopsy was documented for 45 cases, including progression of disease in 19
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AMERICAN JOURNAL

cases and lack of response to topical antimicrobial therapy

(without lesion enlargement or new foci) in 23 cases
(Table 1). Progression of disease was not more likely to be
associated with a positive biopsy than lack of response
alone (P .12).
There were 3 perforations at the time of biopsy procedures (6% of 53 first and second biopsies). One case
required immediate PK, while the other 2 were managed
adequately with cyanoacrylate glue. A review of these 3
cases revealed no unique features to explain the perforations. None had descemetocoeles, and 2 of 3 were peripheral ulcers. Gram-positive bacteria were identified in 2
cases; biopsy was negative in the other. Each biopsy was
performed by a different surgeon. Thinning was not quantitated in most cases, and thus could not be evaluated as a
risk factor for perforation.
Antimicrobial therapy was changed after biopsy results
in 18 of 48 cases (37.5%) (13 of 20 [65%] positive biopsies;
5 of 28 [18%] negative biopsies; P .002). Outcomes were
known for 43 patients. Evisceration was performed on 1
eye while keratitis was active; biopsy in that case had been
negative. A therapeutic PK was performed in 7 of 19 cases
(37%) with positive corneal biopsies and in 4 of the 24
cases (21%, including the evisceration) with a negative
corneal biopsy (P .31). Among those with positive
biopsies who underwent therapeutic PK, the same organism was identified by culture or histopathologic examination of the corneal button in 4 cases; in contrast, for those
with negative biopsies, no organisms were found by culture
or histopathologic examination of host tissue removed at
time of therapeutic PK. The interval from biopsy to
surgical intervention was not significantly different between those patients with positive biopsies (94.9 112.8
days [median 17 days, range 5285 days], n 7) and those
with negative biopsies (22.8 12.9 [median 21 days, range
6 41 days], n 5, P .94). Six of 13 patients (46%)
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whose treatment was changed following positive biopsy

results underwent penetrating keratoplasty, while 6 of 30
patients (20%) whose therapy was not changed because of
positive biopsy results underwent penetrating keratoplasty
(P .14). A change in therapy following positive biopsies
was not associated with fewer therapeutic PK (P .14) or
with shorter times to healing (P .42) than all other cases
in our series (data not shown).
For those patients who did not require surgical intervention while infection was active (therapeutic PK or evisceration, n 31), the interval from biopsy to healing was
shorter among those with negative biopsies (mean 85.7
110.7 days [median 49 days, range 13 468 days], n 19)
than for those with positive biopsies (mean 98.6 47.4
days [median 85.5 days, range 39 185 days], n 12),
although the association was weak (P .062). Among the
24 patients with negative biopsies who had follow-up, 7
(29%) eventually had evidence of active corneal disease (5
with clinical progression of corneal infiltrates that required
surgery; 1 with a repeat biopsy positive for fungus; and 1
who was eventually diagnosed with necrotizing herpetic
stromal keratitis on the basis of a positive viral culture).
Second biopsies were performed on 5 patients: 3 were
performed because of negative first biopsies and 2 were
performed because of disease progression after positive first
biopsies, to confirm or rule out persistent infection (1
positive for mycobacteria; the other for presumed Acanthamoeba sp.). Second biopsies were performed from 1 week
to 2 months after first biopsies. Only 1 of the 3 patients
with negative first biopsies had a positive second biopsy;
results were consistent with a fungal infection, and upon
switching therapy to antifungal medications the patients
keratitis healed. One of the 2 patients with positive first
biopsies had a positive second biopsy; the patient had cysts
consistent with Acanthamoeba sp. on histopathologic examination of both biopsies. One of the 5 second biopsies
was complicated by perforation at the time of the procedure; it was managed successfully with cyanoacrylate glue.
We found no evidence of secular trends over the 20-year
study period in terms of the proportion of positive biopsies
or the organisms identified (data not shown).

DISCUSSION
A NUMBER OF REPORTS HAVE DESCRIBED THE SUCCESSFUL

diagnosis of mycobacterial, fungal, and acanthamoebic

keratitis with corneal biopsy,3,4,6,8 13 but the indications
for corneal biopsy remain undefined, and there has been no
consensus on the most appropriate processing of corneal
biopsy specimens. Our series supports the utility of corneal
biopsies. We were able to identify causal agents in 42%
(20/48) of cases that underwent biopsy. Biopsy can provide
diagnostic information in some cases that have negative
results on culture of corneal scrapings at presentation;
among cases for which no microorganisms were seen on
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initial scrapings, 44% (12/27) had positive biopsies. Furthermore, in some cases, biopsy results changed diagnoses
that have been based on positive culture results of initial
scrapings. Our results also support the value of corneal
biopsy for identifying the cause of infectious keratitis in
cases that are refractory to initial antimicrobial therapy;
treatment was changed for 65% (13/20) of our patients
with positive biopsies. Our results indicate that positive
biopsies do not depend on evidence of disease progression,
and that a delay in performing a biopsy does not necessarily
reduce the likelihood of a positive result for patients with
persistent keratitis. Thus, biopsy remains an option, even
in disease that is long-standing.
Our results give some insight into the types of cases that
will eventually need corneal biopsy. The spectrum of
causal organisms identified by corneal biopsy does not
reflect the distribution of all causes of infectious keratitis
seen at UCLA, the majority of which are staphylococci
and Pseudomonas aeruginosa (data not shown). Also, none
of the many cases of infectious keratitis at UCLA for
which Acanthamoeba sp. or gram-negative bacteria were
isolated from initial corneal scrapings later required corneal biopsy. Our results suggest that the cases most likely
to require biopsy are those for which cultures of initial
corneal scrapings are negative or grow gram-positive
bacteria.
We were also able to address the clinical relevance of a
biopsy that is negative by both culture and histopathologic
examination techniques. There was a trend for the mean
time to resolution of active keratitis to be shorter for those
with negative biopsies than for those with positive biopsies. Fewer patients with negative biopsies needed surgery
while corneas were still inflamed, and in the majority of
patients with negative biopsies, the subsequent course was
consistent with eradication of infection. Nevertheless, a
substantial minority of cases with negative biopsies had
subsequent evidence of persistent infection on the basis of
a second scraping, additional biopsy, or examination of
host tissue following PK. Thus, while a negative biopsy
provides some support for the assumption that infection
has been treated adequately, negative biopsies do not rule
out the possibility of persistent infection, and patients
must still be monitored closely for progression of disease.
The results also demonstrate the value of our protocol
for processing specimens, in which both culture and
histopathologic examination of the biopsy specimens is
performed routinely. Most reported series have described
either culture or histopathologic examination of biopsy
specimens, but not both.3 6,13 Among the larger series, Lee
and Green described the identification of organisms by
histopathologic examination in 9 of 42 corneal biopsy
specimens that were performed for diagnosis of infectious
keratitis, but culture of the same specimens was not
performed for comparison.4 Alexandrakis and associates
performed both culture and histopathologic examination
on 11 of 33 biopsy specimens.6 Seven of the 11 biopsies

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were culture-positive (3 of which had negative histopathologic examinations), and 5 of the 11 biopsies had positive
histopathologic examination findings (only 1 of which was
culture-negative). On the basis of these results, they
concluded that culture was more sensitive than histopathologic examination. In contrast, Ishibashi and associates reported that histopathologic examination of biopsy
specimens in 4 cases provided information that culture did
not.1,2
Our series provides some additional support for the
notion that histopathologic examination is more valuable
than culture as a means of assessing biopsy specimens. The
proportion of positive results was higher for histopathologic examination than for culture, and when results were
discordant, culture results were often consistent with
contaminant bacteria. In each of these discordant cases for
which follow-up information was available, organisms seen
on histopathologic examination were confirmed subsequently (repeat biopsy, culture of repeat scraping, and
culture or histopathologic examination of host tissue at
therapeutic PK) or treatment directed to the organism
identified on histopathologic examination led to resolution of infection. Histopathologic examination was more
likely to identify fungi and cysts consistent with Acanthamoeba sp. than culture, despite the use of appropriate media.
These observations are consistent with laboratory studies
involving rabbit models of corneal infection, in which
histopathologic examination of biopsy specimens was more
likely to identify the presence of fungi than culture.2 In
particular, histopathologic examination appeared to be
more useful than culture of biopsy material when culture of
initial corneal scrapings was negative. Nevertheless, each
technique can provide complementary and unique information regarding infection; for example, culture can identify the specific species of organism and sensitivities to
antimicrobials, while histopathologic examination cannot.
Unlike other reported series, we had no cases in which
cultures were positive but histopathologic examination was
negative; should that situation occur, our culture data suggest
that clinicians should be wary of possible contaminants.
Previous studies have attempted to relate outcomes of
infectious keratitis to biopsy results. In the series reported
by Alexandrakis and associates, a lower proportion of
patients with positive biopsies eventually underwent penetrating keratoplasty (5 of 27 patients [18.5%]) than did
patients with negative biopsies (5 of 6 patients).6 The
authors hypothesized that positive biopsies were more
likely to lead to correct treatments and thus to a reduced
need for surgery. Because many unrelated factors can
influence decisions about surgery, we chose instead to look
only at the need for penetrating keratoplasty before resolution of active corneal inflammation, as a more meaningful surrogate for ability to control infection. Neither a
positive biopsy nor a change in treatment based on a
positive biopsy predicted the need for penetrating keratoplasty while keratitis was still active.
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Other techniques for the identification of infectious

organisms have become available since the start of this
series, including confocal microscopy.14 Our study does not
permit an assessment of the relative utility of corneal
biopsy vs confocal microscopy; nevertheless, while confocal microscopy holds promise as an adjunctive test, it has
limitations, including lack of widespread availability and
the need for experienced examiners who can obtain and
interpret findings properly. Corneal biopsy remains a relatively simple and more accessible method for evaluating
the cornea in difficult cases, including those with negative
or equivocal confocal microscopy results. In a masked
assessment of patients with infectious keratitis, pathogens
subsequently confirmed by other means have been identified correctly by confocal microscopy in only 30% to 60%
of cases, depending on the experience and skill of the
examiner.14 Future studies are needed to determine the
sensitivity of corneal biopsy vs confocal microscopy.
A major strength of our study is the fact that we
followed a standard approach to the processing of biopsy
specimens throughout the study period, which reduces the
potential for bias in the comparison of culture vs histopathologic examination of specimens. We also studied a
relatively large number of patients, when compared to
most previously reported series. Nevertheless, the number
of cases is still too small for detailed statistical analyses of
many potential influences on biopsy results. Another
limitation of this study was the inability to evaluate the
influence of biopsy location, surgical technique, and specimen size on results. We did not compare biopsy results to
microscopic examination of smears made with material
obtained at the time of initial scrapings; although such
examinations can provide important information about
disease causation when positive, the sensitivity of this
procedure is low, especially for bacterial infections.15
Because we did not know the true cause of keratitis in each
case, we were unable to study specificity or sensitivity of
each technique.
The study includes other limitations typically associated
with retrospective analyses; not all patients were managed
in the same manner, and treatment was uncontrolled.
Also, patients were not randomized to biopsy procedures,
making it difficult to confirm that the information obtained in those cases with positive biopsy results had an
impact on outcomes. Furthermore, as we studied a selected
population, our cases are not necessarily representative of
all patients with infectious keratitis, which limits the
ability to generalize our results; we are not, however,
advocating biopsies on all patients. We believe that simply
the fact that biopsies can identify pathogens not found on
initial evaluation justifies use of this procedure.
In summary, our results indicate the value of corneal
biopsy in the management of selected patients with infectious keratitis, including those with negative cultures of
corneal scrapings at presentation and those with lack of
appropriate response to initial treatment. Delay in perOF

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forming biopsy does not necessarily eliminate the possibility of positive results. Histopathologic examination of
biopsy specimens was more likely to identify Acanthamoeba
sp. and fungi than culture, but results support the continued practice of processing biopsy specimens for both
culture and histopathologic examination; the 2 techniques
can give complementary information. In our experience,
when discordant positive results occur (culture and histo-

pathologic examination reveal different pathogens), histopathologic examination results are more likely to indicate
the causal organism, but this issue requires additional
study; for instance, in this study, we could not rule out the
possibility that discordant positive results reflect infection
with multiple organisms. Although useful, corneal biopsy
procedures are not without risks; in our series, 3 perforations occurred.

ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF
Interest. 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 Nancy
Cooperman Fellowship (Dr Younger), the David May II Fellowship (Dr Johnson), and the Klara Fleming Fellowship (Dr Page). Dr Younger was an
Allergan Cornea Fellow (20072008). All named fellowships are funded by endowments within the UCLA Department of Ophthalmology. None of the
authors have conflicts of interest with any aspect of this study. Funding entities had no role in the conduction or presentation of this study. Involved
in 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 review and approval of manuscript (all authors). Drs Younger and Johnson contributed equally to preparation of this article. The retrospective review
of medical records was approved by the Institutional Review Board at UCLA prior to commencement of the study.
Participating members of the UCLA Cornea Service included Richard Casey, Sophie X. Deng, and D. Rex Hamilton.

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vs culture of biopsy specimens for the diagnosis of keratomycosis. Am J Ophthalmol 1987;103(5):636 640.
3. Newton C, Moore MB, Kaufman HE. Corneal biopsy in
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7. Brinser JH, Torczynski E. Unusual Pseudomonas corneal
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9. DeVoe AG. Keratomycosis. Am J Ophthalmol 1971;71(1 Pt

2):406 414.
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caused by Mycobacterium gordonae. Am J Ophthalmol
1986;102(4):516 521.
12. Whitehouse G, Reid K, Hudson B, Lennox VA, Lawless
MA. Corneal biopsy in microbial keratitis. Aust N Z J
Ophthalmol 1991;19(3):193196.
13. Allan BD, Morlet N, Dart JK. Microbiologic investigation of
suspected microbial keratitis. Ophthalmology 1996;103(8):
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14. Hau SC, Dart JK, Vesaluoma M, et al. Diagnostic accuracy of
microbial keratitis with in vivo scanning laser confocal
microscopy. Br J Ophthalmol 2010;94(8):982987.
15. Sharma S, Kunimoto DY, Gopinathan U, et al. Evaluation
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647.

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Biosketch
R. Duncan Johnson, MD, is a David May II Fellow in Cornea-External Ocular Disease and Refractive Surgery in the
UCLA Department of Ophthalmology, Los Angeles, California. He has a particular interest in surgical management of
complicated corneal diseases. He has published on a variety of topics related to corneal and external ocular diseases,
including biomechanical properties of corneas undergoing refractive surgery.

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Biosketch
Jared R. Younger, MD, MPH, is a former Clinical Fellow in Cornea-External Ocular Disease and Refractive Surgery in the
UCLA Department of Ophthalmology, Los Angeles, California. He is currently in private practice in Fountain Valley,
California, and continues to be a member of the Medical Staff at the Ronald Reagan-UCLA Medical Center. He also
provides eye care to the underserved population of Santa Catalina Island, off the coast of Southern California, as a
part-time member of the Catalina Island Medical Center (Avalon, CA). Dr Younger is a member of the Board of Directors
of the Orange County (CA) Society of Ophthalmology.

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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.