The Outcomes of Primary Pediatric Keratoplasty in
Singapore
JIN RONG LOW, ARUNDHATI ANSHU, ANNA CHENG SIM TAN, HLA MYINT HTOON, AND
DONALD TIANG HWEE TAN

PURPOSE: To evaluate the long-term corneal graft sur-
vival and risk factors for graft failure in pediatric eyes.

DESIGN: Retrospective, interventional consecutive
case series.

METHODS: Unilateral eyes of 105 patients aged 16
years and below were included from the Singapore
Corneal Transplant Study between April 4, 1991 and
April 4, 2011. Corneal graft survival was calculated using
Kaplan-Meier survival analysis, and survival distributions
were compared using log-rank test.

RESULTS: Mean recipient age was 8.38 ± 5.63 years
(range 0.18–15.92 years). Mean follow-up time was
34.16 ± 39.10 months. Main diagnoses were corneal
scar (22.9%), limbal dermoid (21.9%), anterior segment
dysgenesis (15.2%), and keratoconus (14.3%). Forty-
four eyes (41.9%) underwent penetrating keratoplasty
(PK), 37 (35.2%) underwent anterior lamellar kerato-
plasty (ALK), 22 (21.0%) underwent lamellar corneal
patch graft, and 2 (1.9%) underwent Descemet stripping
automated endothelial keratoplasty (DSAEK). Kaplan-
Meier survival rates for PK were 92.8% at 1 year,
88.9% at 2–4 years, and 80.9% at 5–16 years; survival
rates for ALK were 88.0% at 1 year and 84.3% at 2–7
years; survival rates for corneal patch graft were 100%
at 1–3 years and 90% at 4–10 years; these were not sta-
tistically significant (P [ .362). Deep corneal vasculari-
zation (P [ .012), preexisting active inflammation (P [
.023), preexisting glaucoma drainage device (P [ .023),
and preexisting ocular surface disease (P [ .037) were
associated with reduced graft survival in a univariate
analysis.

CONCLUSIONS: We report good long-term graft survival
following pediatric keratoplasty for various indications.
Lamellar keratoplasty, when indicated, should be the pro-
cedure of choice in high-risk keratoplasties. (Am J
Ophthalmol 2014;158:496–502. Ó 2014 by Elsevier Inc.
All rights reserved.)
Accepted for publication May 19, 2014.
From Singapore National Eye Centre (J.R.L., A.A., A.C.S.T.,
D.T.H.T.), Singapore Eye Research Institute (H.M.H., D.T.H.T.),
Duke-National University of Singapore Graduate Medical School
Singapore (A.A., A.C.S.T., H.M.H., D.T.H.T.), and Department of
Ophthalmology, Yong Loo Lin School of Medicine, National University
of Singapore (A.A., D.T.H.T.), Singapore.
Inquiries to Prof Donald Tiang Hwee Tan, Singapore National Eye
Centre, 11 Third Hospital Avenue Singapore 168751, Singapore;
e-mail: donald.tan.t.h@snec.com.sg
496 Ó 2014 BY ELSEVIER INC. ALL
P
EDIATRIC KERATOPLASTY IS CONSIDERED HIGH-RISK
keratoplasty and was uncommonly performed before
the 1970s, and was recommended only for pediatric
patients with bilateral corneal involvement.1,2 Over the
past 20 years, there has been increasing frequency and
success rates of pediatric keratoplasty3 owing to advances
in corneal microsurgery, postoperative management, and
better understanding of risk factors that may help predict
and prevent poor outcomes.4,5 Nevertheless, higher graft
failure rates and poorer visual prognosis have been
reported in pediatric keratoplasty compared to adult
keratoplasty.3,6–9 There are many challenges unique to
pediatric keratoplasty. Preoperatively, there is difficult
assessment of visual function and potential in preverbal
children.3,5 Intraoperative challenges include thinner and
less rigid cornea, smaller and more crowded anterior
segment, and decreased scleral rigidity, which require
specialized surgical techniques.3,5,10 Postoperative
problems include difficulty of the child in communicating
postoperative symptoms, difficulty with refractive error
assessments, need for frequent examinations under
anesthesia (EUA) for postoperative follow-up evaluations,
increased fibrinous reactions and possible vitreous pressure,
graft rejection (which occurs both more frequently and
more rapidly, and is less responsive to treatment), increased
infection and rejection risks associated with loose sutures,
and challenges of amblyopia therapy.3,5,10
Although there have been a few studies reporting on pe-
diatric keratoplasty in developed Western countries, there
have been few studies on pediatric keratoplasty in Asian
populations7,11–13 and even fewer evaluating its long-
term outcomes.14 We report the indications, risk factors,
postoperative complications, and long-term graft survival
of primary pediatric keratoplasties performed at the
Singapore National Eye Centre, with a significant referral
base of corneal diseases from less developed countries in
Southeast Asia.
METHODS
THE SINGAPORE CORNEAL TRANSPLANT STUDY IS A LONG-
term ongoing prospective cohort study started in 1991,
tracking all consecutive corneal transplants performed at
a single ophthalmic tertiary referral center in Singapore,
the Singapore National Eye Centre, which provides
RIGHTS RESERVED. 0002-9394/$36.00
http://dx.doi.org/10.1016/j.ajo.2014.05.020
approximately 60% of eye care delivery in Singapore and
performs approximately 80% of all corneal transplants
locally.15,16 Approval was prospectively obtained for this
study from the Singapore Eye Research Institute
institutional review board.
In this retrospective, interventional consecutive case se-
ries, we recruited 168 patients aged 16 years and below at
time of corneal transplant from the Singapore Corneal
Transplant Study cohort between April 4, 1991 and April
4, 2011. After excluding 6 cases with bilateral corneal grafts,
22 cases of repeat corneal grafts, and 35 cases lost to follow-
up, 105 eyes of 105 patients were included for analysis.
Main outcome measure was graft failure, defined as the
irreversible loss of optical clarity, with date of onset of
corneal clouding selected as time point of graft failure. Sur-
vival time of failed grafts was defined as the time between
the date of surgery and recorded date of failure. For
currently surviving clear grafts or cases lost to follow-up,
survival time was calculated as the interval between the
date of surgery and the date when the patient was last
seen. Corneal graft survival time and probabilities were
calculated using Kaplan-Meier survival analysis. Kaplan-
Meier survival distributions were compared for significant
differences using log-rank test. All variables, categorical
and continuous, were individually subjected to univariate
analysis to assess their significance for graft failure. A P
value (2-tailed) of <.05 was considered to be statistically
significant. All analyses were conducted using IBM SPSS
Statistics for Windows, Version 19.0 (IBM Corp, Armonk,
New York, USA).
The pediatric keratoplasty regimen involved team man-
agement by the cornea specialists, pediatric ophthalmolo-
gists, and, if necessary, the pediatric glaucoma specialist
and retina specialists, as well as pediatric anesthetists and
pediatric transplant immunologist for systemic immuno-
suppression in selected cases. We practiced in-depth coun-
seling of parents regarding the importance of compliance
with postoperative follow-up, multiple EUAs, and patch-
ing, especially for patients who are based overseas. A
much closer and more frequent follow-up was carried out
for these patients compared to adult keratoplasty patients.
For instance, the first EUA to start suture removal was as
early as 2 weeks post operation.
RESULTS
A TOTAL OF 105 EYES OF 105 PEDIATRIC PATIENTS UP TO 16
years of age underwent keratoplasty in the 20-year study
period. Of these patients, 67 (63.8%) were boys and 38
(36.2%) were girls. The mean recipient age at surgery
was 8.38 6 5.63 years (range 0.18–15.92 years). The
mean follow-up time was 34.16 6 39.10 months (range
0.26–206.26 months). All pediatric keratoplasties were
performed by 1 surgeon (D.T.).
VOL. 158, NO. 3 PRIMARY PEDIATRIC KERATOPLASTY IN SINGAPORE

INDICATIONS FOR SURGERY: The majority of pediatric
keratoplasties were performed for optical reasons
(67.7%), whereas therapeutic and tectonic indications
accounted for 18.6% and 13.7% of cases, respectively.
The most common diagnoses were corneal scar (22.9%),
limbal dermoid (21.9%), anterior segment dysgenesis
(15.2%), keratoconus (14.3%), and infectious keratitis
(6.7%). Cases of anterior segment dysgenesis comprised Pe-
ters anomaly (62.5%), sclerocornea (25.0%), and Rieger’s
anomaly (12.5%). For penetrating keratoplasty, the most
common indications were anterior segment dysgenesis
(34.1%), keratoconus (22.7%), and corneal scar (18.2%).
For anterior lamellar keratoplasty, the most common indi-
cations were corneal scar (40.5%), limbal dermoid
(18.9%), and keratoconus (13.5%). Most cases that under-
went lamellar corneal patch graft were for limbal dermoid
(68.2%). The proportion of the type of surgery performed
for each diagnosis is shown in Table 1.

OVERALL GRAFT SURVIVAL: Overall Kaplan-Meier
mean graft survival time was 174.0 months (95% confi-
dence interval [CI], 154.3–193.7). Kaplan-Meier mean
graft survival times for optical grafts, therapeutic grafts,
and tectonic grafts were 171.9 months (95% CI, 146.5–
197.4), 135.9 months (95% CI, 121.0–150.7), and
93.5 months (95% CI, 59.5–127.5), respectively. Kaplan-
Meier survival rates for optical grafts were 92.3% at 1
year, 88.0% at 2–4 years, and 81.2% at 5–16 years; survival
rates for therapeutic grafts were 94.1% at 1–12 years; sur-
vival rates for tectonic grafts were 90.9% at 1–3 years and
68.2% at 4–10 years (Figure 1). The Kaplan-Meier survival
distributions for optical, therapeutic, and tectonic grafts
were similar and had no statistically significant differences
(P ¼ .556).

GRAFT SURVIVAL BASED ON TYPE OF SURGERY: Forty-
four eyes (41.9%) underwent penetrating keratoplasty
(PK), 37 eyes (35.2%) underwent anterior lamellar kerato-
plasty (ALK), 22 eyes (21.0%) underwent lamellar corneal
patch graft, and 2 eyes (1.9%) underwent Descemet strip-
ping automated endothelial keratoplasty (DSAEK). Of the
eyes that underwent ALK, 28 (75.7%) underwent pre-
Descemetic deep anterior lamellar keratoplasty (manual
dissection technique) (DALKm), 5 (13.5%) underwent
Descemet-bearing deep anterior lamellar keratoplasty (big
bubble technique) (DALKa), and 4 (10.8%) underwent
automated lamellar therapeutic keratoplasty (ALTK).
Kaplan-Meier mean graft survival times for PK, ALK,
and corneal patch grafts were 171.7 months (95% CI,
141.3–202.2), 74.5 months (95% CI, 64.4–84.5), and
115.8 months (95% CI, 101.0–130.5), respectively.
Kaplan-Meier survival rates for PK were 92.6% at 1 year,
88.9% at 2–4 years, and 80.9% at 5–16 years; survival rates
for ALK were 88.0% at 1 year and 84.3% at 2–7 years; sur-
vival rates of corneal patch graft were 100% at 1–3 years
and 90% at 4–10 years (Figure 2). The Kaplan-Meier
497
 TABLE 1. Frequency and Proportion of Infants and Children who Underwent Keratoplasty for Each Primary Clinical Diagnosis

Penetrating Anterior Lamellar Descemet Stripping Automated

Diagnosis Total, n (%) Keratoplasty, n (%) Keratoplasty, n (%) Corneal Patch Graft, n (%) Endothelial Keratoplasty, n (%)

Corneal scar 24 (100.0) 8 (33.3) 15 (62.5) 1 (4.2) 0 (0.0)

Limbal dermoid 23 (100.0) 1 (4.3) 7 (30.4) 15 (65.2) 0 (0.0)
Anterior segment dysgenesis 16 (100.0) 15 (93.8) 0 (0.0) 0 (0.0) 1 (6.3)
Keratoconus 15 (100.0) 10 (66.7) 5 (33.3) 0 (0.0) 0 (0.0)
Infectious keratitis 7 (100.0) 4 (57.1) 3 (42.9) 0 (0.0) 0 (0.0)
Corneal melt or perforation 5 (100.0) 2 (40.0) 3 (60.0) 0 (0.0) 0 (0.0)
Iris tumor 3 (100.0) 1 (33.3) 0 (0.0) 2 (66.7) 0 (0.0)
Pseudophakic or aphakic bullous 2 (100.0) 1 (50.0) 0 (0.0) 0 (0.0) 1 (50.0)
keratopathy
Bullous oculocutaneous disease 2 (100.0) 0 (0.0) 2 (100.0) 0 (0.0) 0 (0.0)
Limbal tumor 2 (100.0) 0 (0.0) 0 (0.0) 2 (100.0) 0 (0.0)
Tube extrusion 2 (100.0) 0 (0.0) 0 (0.0) 2 (100.0) 0 (0.0)
Congenital hereditary endothelial dystrophy 1 (100.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0)
Reis-Bucklers corneal dystrophy 1 (100.0) 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0)
Glaucoma 1 (100.0) 1 (100.0) 0 (0.0) 0 (0.0) 0 (0.0)
Aniridia 1 (100.0) 0 (0.0) 1 (100.0) 0 (0.0) 0 (0.0)

FIGURE 1. Kaplan-Meier survival curves of keratoplasty in infants and children by optical, tectonic, and therapeutic indications.

survival distributions for PK, ALK, and corneal patch grafts
were similar (P ¼ .362).
For optical grafts, the Kaplan-Meier survival rates for PK
were 91.4% at 1 year, 87.0% at 2–4 years, and 77.4% at 5–
17 years; survival rates for DALKm were 88.0% at 1 year
and 78.2% at 2–4 years; survival rates for DALKa were
100% at 1–7 years; and survival rates for ALTK were
100% at 1–3 years.

GRAFT SURVIVAL BASED ON DIAGNOSIS: Kaplan-Meier
survival rates for patients with corneal scar were 85.2% at 1–
11 years; survival rates for patients with limbal dermoid were
95.0% at 1–10 years; survival rates for patients with keratoco-
nus were 92.9% at 1–17 years; survival rates for patients with
anterior segment dysgenesis were 93.8% at 1 year and 78.1%
at 2–4 years; and survival rates for patients with active infec-
tious keratitis were 100% at 1–12 years (Figure 3).

498 AMERICAN JOURNAL OF OPHTHALMOLOGY SEPTEMBER 2014

FIGURE 2. Kaplan-Meier survival curves of keratoplasty in infants and children by type of surgery.

GRAFT SURVIVAL BASED ON AGE AT TIME OF SURGERY:
Kaplan-Meier survival rates for patients aged 0–1 year were
84.8% at 1 year and 74.2% at 2–6 years; survival rates for
patients aged 2–5 years were 94.1% at 1–12 years; survival
rates for patients aged 6–10 years were 94.4% at 1 year, 87.7
at 2–3 years, and 70.2% at 4 years; and survival rates for pa-
tients aged 11–16 years were 94.5% at 1–4 years and 84.0%
at 5–17 years (Figure 4). However, there was no statistically
significant difference in graft survival distributions among
the different age groups (P ¼ .256).

RISK FACTOR ANALYSIS: Seventeen putative risk fac-
tors for graft failure were grouped into patient demo-
graphics and clinical diagnosis, preoperative recipient
corneal status, and intraoperative categories. These
included 6 patient- and disease-related factors (recipient
age, sex, ethnic group, nationality, clinical diagnosis,
and primary indication for transplantation), 8 preopera-
tive risk factors (preoperative corneal vascularization;
previous grafts; preexisting glaucoma; and the presence
of active infection, active inflammation, ocular surface
disease, lid disease, and globe perforation at time of sur-
gery), and 3 intraoperative risk factors (concurrent cata-
ract surgery [intracapsular or extracapsular cataract
extraction or phacoemulsification], concurrent anterior
chamber intraocular lens implantation, and concurrent
anterior vitrectomy). These were the same risk factors as
described in our previous Singapore Corneal Transplant
Study publications.15,16
VOL. 158, NO. 3 PRIMARY PEDIATRIC KERATOPLASTY IN SINGAPORE
Univariate analysis identified 4 risk factors that were
significantly associated with reduced graft survival. They
were deep corneal vascularization (P ¼ .012), active inflam-
mation (P ¼ .023), preexisting glaucoma drainage device
(P ¼ .023), and preexisting ocular surface disease (P ¼ .037).

COMPLICATIONS: The most common postoperative
complications were ocular surface disease (15.2%) (16 of
105 eyes), such as limbal stem cell deficiency and nonheal-
ing epithelial defects; glaucoma (13.3%) (14 of 105 eyes);
and cataract (10.5%) (11 of 105 eyes) (Table 2). For eyes
that underwent PK, the most common complications
were glaucoma (25.0%) (11 of 44 eyes), cataract (20.5%)
(9 of 44 eyes), and ocular surface disease (18.2%) (8 of 44
eyes); for eyes that underwent lamellar keratoplasty, the
most common complications were ocular surface disease
largely secondary to limbal stem cell deficiency (12.8%)
(5 of 39 eyes), glaucoma (5.1%) (2 of 39 eyes), and late graft
failure (5.1%) (2 of 39 eyes); for eyes that underwent
lamellar corneal patch graft, the most common complica-
tions were ocular surface disease (13.6%) (3 of 22 eyes)
and recurrence of primary disease (13.6%) (3 of 22 eyes).
DISCUSSION
INDICATIONS FOR PEDIATRIC KERATOPLASTY IN DEVELOPED
and developing countries vary according to the different
499
FIGURE 3. Kaplan-Meier survival curves of the most common diagnoses after pediatric keratoplasty.
spectrum of corneal disease in each country.3,5,11,12,17 A
review by Vanathi and associates showed that congenital
causes ranged from 14% to 64%, acquired nontraumatic
causes ranged from 19% to 80%, and acquired traumatic
causes ranged from 6% to 29%.3 Although congenital
corneal opacification and corneal dystrophies are the pri-
mary indications for pediatric keratoplasty in the developed
world, infectious keratitis, corneal scar, and perforation
caused by ulceration are the major indications for pediatric
keratoplasty in the Asia-Pacific region.11–13 In our study,
the majority of the patients had acquired causes (60%),
and corneal scar, limbal dermoid, and keratoconus were
the most common indications for pediatric keratoplasty.
These represent a spectrum of Asian corneal disease from
a combination of patients from an urban developed
population (similar to the West [eg, keratoconus, anterior
segment dysgenesis]) and referrals from more developing
surrounding populations in Southeast Asia (infectious
keratitis and postinfectious scarring).11,12
Comparison of overall corneal graft survival with
different studies in literature is difficult owing to the het-
erogeneity of etiology, as well as varying sample sizes and
differing follow-up periods. Vanathi and associates showed
that graft survival rates of congenital opacities vary widely
in literature: congenital hereditary endothelial dystrophy
(25%–90%), congenital glaucoma (50%), and sclerocor-
nea (50%–83%).3 Acquired opacities were found to have
better visual outcomes compared to congenital opaci-
ties.3,7,18 Of these, traumatic causes had survival rates of
55%–100%, whereas nontraumatic causes had survival
500 AMERICAN JOURNAL OF OPHTHALMOLOGY
rates of 40%–85%.3 Our overall graft survival was better
than that of similar studies.7,11,14,17 This may be
attributable to the majority of our cases being acquired
and the higher mean age at time of transplant of our
cases. In our study, cases that underwent keratoplasty for
keratoconus had an excellent long-term graft survival
rate of 92.9% at 17 years, which was similar to findings in
other studies.3,10,17 Peters anomaly is known to be
associated with a guarded postoperative visual prognosis
and a higher incidence of graft failure.3,19,20 A large
series by Yang and associates reported that the graft
survival rate of Peters anomaly was 39% with a median
follow-up time of 11.1 years.21 Our study, however, showed
good 4-year graft survival rates of 100% for Peters anomaly,
which we attributed to close postoperative monitoring and
follow-up, intensive steroid regimens, and a single, highly
experienced surgeon who performed all keratoplasties.
Another series, by Zaidman and associates, similarly re-
ported good graft survival of 70% at 200 months after PK
in 24 children with Peters anomaly Type I.22
Several risk factors are known to affect long-term corneal
graft survival. In our series, deep corneal vascularization,
active inflammation, preexisting glaucoma drainage device,
and preexisting ocular surface disease affected graft survival
time more than other factors. These risk factors were consis-
tent with those reported in other studies2,7,10,17,18 and were
similar to what we have reported in an earlier study
consisting mainly of adult keratoplasties.15
Literature has shown mixed results for graft survival rela-
tive to age of patient at the time of keratoplasty.5 Two
SEPTEMBER 2014
FIGURE 4. Kaplan-Meier survival curves by recipient age at time of pediatric keratoplasty.

findings in another series of 50 children who underwent

TABLE 2. Frequency and Proportion of Infants and Children PK.16 Others found no difference in graft survival based
who Developed Complications After Keratoplasty on age at the time of transplant.5,6,8
Commonly reported postoperative complications
Penetrating Corneal
Keratoplasty, Lamellar Patch
include infectious keratitis (10%–50%), graft rejection
Complications n (%) Keratoplasty, n (%) Graft, n (%) (22%–43.4%), glaucoma (5%–9%), and cataract (2%–
Ocular surface disease 8 (18.2) 5 (12.8) 3 (13.6)
7%),3,6,7,11 and this is in keeping with the findings in our
Glaucoma 11 (25.0) 2 (5.1) 1 (4.5) study. Complications such as infectious keratitis,
Cataract 9 (20.5) 1 (2.6) 1 (4.5) epithelial defects, band keratopathy, wound leakage,
Late graft failure 4 (9.1) 2 (5.1) 0 (0.0) retrocorneal membrane, cataract formation, secondary
Recurrence of primary 0 (0.0) 1 (2.6) 3 (13.6) glaucoma, and retinal detachment cause challenges to
disease postoperative management and frequently result in
Allograft rejection 4 (9.1) 0 (0.0) 0 (0.0) regrafting.23
Wound dehiscence 3 (6.8) 0 (0.0) 0 (0.0) Unlike most reported series in literature, which
Endophthalmitis 1 (2.3) 0 (0.0) 0 (0.0) evaluated PK as the only modality of pediatric kerato-
Infectious keratitis 0 (0.0) 0 (0.0) 1 (4.5)
plasty,1–11,13,18,20–24 the present study analyzed and
Descemet membrane 0 (0.0) 1 (2.6) 0 (0.0)
compared the outcomes of PK, ALK, and lamellar corneal
detachment
Cystoid macular edema 1 (2.3) 0 (0.0) 0 (0.0)
patch grafts. Although 2 other studies included patients
Retinal detachment 1 (2.3) 0 (0.0) 0 (0.0) who underwent PK and ALK,12,17 none compared the
Suture abscess 0 (0.0) 0 (0.0) 1 (4.5) graft survival outcomes between the different types of
Irregular astigmatism with 0 (0.0) 1 (2.6) 0 (0.0) surgery. ALK may be more technically challenging, but it
corneal scar offers several advantages over PK in terms of lower
incidence of graft rejection and failure.14,25–27 Hence, it
should be performed when indicated and possible,
studies concluded that patients aged less than 5 years have particularly in children who have a higher risk of graft
worse graft survival.7,10 Our study showed a trend toward failure. While our series had only 2 cases that underwent
better graft survival in patients older than 1 year, DSAEK, which limited meaningful analysis, a few series
compared to patients aged less than 1 year, although the have shown promising results following DSAEK in
trend was not statistically significant. This mirrors the pediatric eyes with endothelial diseases.25,26

VOL. 158, NO. 3 PRIMARY PEDIATRIC KERATOPLASTY IN SINGAPORE 501

 The main limitation in this retrospective study is the
relatively small sample size and variable follow-up periods,
although these same limitations are mirrored in many other
published series, simply because pediatric keratoplasty is
not that commonly indicated or performed, as compared
to adult keratoplasties. Nevertheless, this study provided
valuable information regarding the indications, risk factors,
and long-term outcomes of pediatric keratoplasty in Asian
eyes. A larger prospective study is currently being conduct-
ed to evaluate visual acuity outcomes and to discuss the is-
sues of immunosuppression in children with repeat grafts.
Pediatric keratoplasty remains a challenge to most ante-
rior segment surgeons. We report good long-term graft
survival following primary pediatric keratoplasty for both
ALL AUTHORS HAVE COMPLETED AND SUBMITTED THE ICMJE FORM FOR DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
and none were reported. The authors indicate no funding support. Contributions of authors: design of the study (D.T.H.T., A.A.); conduct of the study
(J.R.L., A.C.S.T., A.A., D.T.H.T.); data collection (A.C.S.T., J.R.L., H.M.H., A.A.); analysis and data interpretation (J.R.L., A.A., H.M.H., A.C.S.T.,
D.T.H.T.); preparation and writing of the manuscript (J.R.L., A.A., H.M.H., D.T.H.T.); and review and approval of the manuscript (A.A., D.T.H.T.).
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