A Polymerase Chain Reaction–Based Algorithm
to Detect and Prevent Transmission of
Adenoviral Conjunctivitis in Hospital Employees
IRENE C. KUO, COLLEEN ESPINOSA, MICHAEL FORMAN, AND ALEXANDRA VALSAMAKIS
PURPOSE: To devise and implement a practice algo-
rithm that would enable rapid detection and appropriate
furlough of hospital employees with adenoviral conjunc-
tivitis in order to prevent healthcare-associated epidemic
keratoconjunctivitis.
DESIGN: Evaluation of an ongoing quality assurance/
improvement initiative.
METHODS: Employees of Johns Hopkins Hospital with
signs and symptoms of adenoviral conjunctivitis under-
went evaluation by nurse practitioners in Occupational
Health and rapid diagnostic testing by real-time polymer-
ase chain reaction (PCR). Sequencing was used to deter-
mine serotype when adenovirus was detected. Signs,
symptoms, diagnosis, and disposition of employees with
eye complaints as well as PCR and serotype results
were recorded.
RESULTS: Over a 36-month period approximately
18% of initial employee visits were due to unique, eye-
related complaints. Viral conjunctivitis was suspected
in 542 of 858 employees with eye complaints (62%);
adenovirus was detected by PCR in 44 of 542 suspected
viral conjunctivitis cases (8%) or 44 of 858 employees
with any eye concern (5%). Fourteen of the 44 em-
ployees had adenoviral serotypes and clinical presenta-
tion consistent with epidemic keratoconjunctivitis
(type 37 [n [ 6], 8 [n [ 4], 4 [n [ 3], 19 [n [ 1]).
Other serotypes found in individuals with less severe
conjunctivitis were 3 (n [ 5), 4 (n [ 5), 56 (n [ 4),
1 (n [ 2), 42 (n [ 1), and 7 (n [ 1). No healthcare-
associated adenoviral conjunctivitis outbreaks occurred
after algorithm implementation, and fewer employees
required furlough than had clinical diagnosis alone
been used.
CONCLUSIONS: The algorithm is an effective infection
prevention tool that minimizes productivity loss
compared to clinical diagnosis and allows for determina-
tion of prevalence and serotype characterization of adeno-
Supplemental Material available at AJO.com.
Accepted for publication Dec 4, 2015.
From the Wilmer Eye Institute, Department of Ophthalmology, The
Johns Hopkins University School of Medicine (I.C.K.); and the
Division of Occupational and Environmental Medicine (C.E.) and
Division of Medical Microbiology, Department of Pathology (M.F.,
A.V.), The Johns Hopkins Hospital, Baltimore, Maryland.
Inquiries to Irene C. Kuo, Wilmer Eye Institute, 4924 Campbell Blvd
#100, Baltimore, MD 21236; e-mail: ickuo@jhmi.edu
38 2016 BY ELSEVIER INC. ALL
viral conjunctivitis in hospital employees. (Am J
Ophthalmol 2016;163:38–44. 2016 by Elsevier Inc.
All rights reserved.)
A
DENOVIRAL EYE INFECTION IS A PUBLIC HEALTH
concern, with manifestations ranging from self-
limited conjunctivitis to prolonged ocular
morbidity in the case of epidemic keratoconjunctivitis
(EKC). Some adenoviral infections, however, may not be
easily distinguishable from EKC at presentation; the 3
other clinical scenarios are nonspecific follicular conjunc-
tivitis, pharyngeal conjunctival fever, and acute hemor-
rhagic conjunctivitis. EKC is most commonly caused by
serotypes 8, 19, and 371; less typical serotypes associated
with EKC are 4,2 53, and 54.3 EKC is more clinically
obvious and induces more morbidity than the other adeno-
viral eye conditions and is associated with a longer disease
course and significant injection, chemosis, and keratitis
that can impair vision.4,5
Outbreaks in hospitals and eye clinics result in substantial
morbidity and lost productivity.6–12 In response to
healthcare-associated EKC outbreaks,6–12 one of which
resulted in the temporary closure of a large eye institute,12
a ‘‘red-eye room,’’ where persons with potentially infectious
conjunctivitis could be triaged and isolated, was established
in 1990 in the Wilmer Eye Institute Emergency Room (ER),
Department of Ophthalmology, at the Johns Hopkins Hos-
pital (JHH).13 This arrangement was part of an infection
prevention program consisting of ophthalmic instrument
decontamination, hand hygiene, use of single-dose eye
drops, and employee furloughs.14 When institutional
changes led to the permanent closure of the Wilmer ER
and the red-eye room at the end of January 2008, a multidis-
ciplinary team composed of a cornea specialist and stake-
holders in infection prevention, employee health, and the
clinical virology/molecular infectious disease clinical diag-
nostic laboratory was convened in order to devise and imple-
ment a new practice algorithm for preventing healthcare-
associated transmission of adenoviral eye disease. Twenty-
month pilot data have been published.14 Here we report
36-month data on adenovirus detection rates and the results
of adding serotype determination to this infection preven-
tion practice algorithm as applied to healthcare workers
with red eye.
RIGHTS RESERVED.
0002-9394/$36.00 http://dx.doi.org/10.1016/j.ajo.2015.12.007

METHODS
THE INSTITUTIONAL REVIEW BOARDS OF JOHNS HOPKINS
Medicine ruled that publishing of an evaluation of this
quality improvement initiative did not require approval
as it ‘‘does not involve human subjects research under the
Department of Health and Human Services or Food and
Drug Administration regulations.’’
The new red-eye employee practice algorithm, which is
in effect at all times, is shown in Figure 1. It consists of
initial evaluation by nurse practitioners in the Occupa-
tional Health clinic and rapid diagnostic testing by real-
time polymerase chain reaction (PCR) in individuals
with signs and symptoms consistent with adenoviral
conjunctivitis. The Occupational Health clinic sees em-
ployees with health concerns that arise during work hours
and may impinge on their ability to work. In order to limit
the possibility of transmission within the clinic, employees
seeking red-eye evaluation are required to call ahead to
make an appointment.
A corneal specialist taught nurse practitioners in the
Occupational Health clinic to recognize signs and symp-
toms of probable viral conjunctivitis (vs conditions like
suspected corneal abrasion or subconjunctival hemor-
rhage) and how to collect swab specimens of the inferior
conjunctival fornix.14 The nurse practitioners were
instructed to swab only when viral conjunctivitis was
suspected. Swabs (polyester, Dacron, or rayon with plastic
or aluminum shafts) were placed in M4 medium and sent
at room temperature for PCR testing performed daily at
the JHH Clinical Virology/Molecular Infectious Disease
Diagnostic Laboratory. Screening criteria included dura-
tion of symptoms, presence of viral prodrome, discharge
or tearing, and unilateral onset. Employees with suspected
viral conjunctivitis were evaluated, swabbed, and
discharged home within 30 minutes of intake. Nurse prac-
titioners notified employees of PCR results and furlough
status by 8 PM if specimens were received in the labora-
tory by 3 PM. If adenovirus was detected by PCR, a
2-week furlough was invoked. Direct sequencing of spec-
imens found to contain adenovirus DNA was performed
retrospectively for epidemiologic purposes—to determine
the proportion of employees infected with EKC-
associated adenoviral serotypes (most typically types 8,
19, and 37).15–21
Toward the end of the furlough, the nurse case manager
in the Occupational Health clinic contacted employees
with adenoviral conjunctivitis by phone to assess symptoms
and to schedule a follow-up examination at Occupational
Health that was required before return to duty. Furlough
was continued if symptoms and signs of conjunctivitis
persisted, given probable infectious potential. Employees
were sent to the Department of Ophthalmology for consul-
tation for conditions the nurse practitioners deemed an
emergency; symptoms including but not limited to persis-
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tent redness, tearing, pain, and blurred vision; or condi-
tions in which the nurse practitioners were unsure of
diagnosis.
ADENOVIRAL DETECTION AND SEROTYPE DETERMINA-
14
TION: As described in the pilot study, PCR for adeno-
virus in conjunctival specimens of employees was
developed and validated in the Johns Hopkins Hospital
molecular microbiology diagnostic laboratory. ‘‘Total
nucleic acid was isolated from conjunctival specimens
(processed volume, 400 microliters) using automated
instrumentation (BioRobot M48, using Virus Mini Proto-
col, version 1.1 software and MagAttract Virus Mini M48
reagents [Qiagen, Germantown, MD];’’14 elution volume,
125 mL). Adenovirus DNA was detected by real-time
PCR (primers: 59 -GCC ACG GTG GGG TTT CTA
AAC TT-39 , 59 -GCC CCA GTG GTC TTA CAT
GCA CAT C-39 ; fluorogenic probe FAM 59 -TGC ACC
AGA CCC GGG CTC AGG TAC TCC GA-39
TAMRA)22 using 7500 Real Time PCR instruments
(Life Technologies, Carlsbad, California, USA). The
analytical sensitivity (95% detection rate or limit of detec-
tion) is 300 copies/mL, and the assay detects at least 16
adenovirus serotypes, including strains from 6 of 7 adeno-
virus serogroups (A-F). When adenoviruses were detected,
serotype was determined by nested PCR of the hexon gene
hypervariable regions 1–623 using previously extracted to-
tal nucleic acid (outer primers: AdhexF1 (19135-19160)
59 -TIC TTT GAC ATI CGI GGI GTI CTI GA-39 /
AdhexR1 (20009-20030) 59 -CTG TCI ACI GCC TGR
TTC CAC A-39 ; inner primers: AdhexF2 (19165-19187)
59 -GGY CCY AGY TTY AAR CCC TAY TC-39 /
AdhexR2 (19960-19985) 59 -GGT TCT GTC ICC CAG
AGA RTC IAG CA-39 ) followed by bidirectional Sanger
sequencing using AdhexF2/AdhexR2 as sequencing
primers in reactions containing BigDye Terminator v.3.1
(Life Technologies). Sequencing was performed on either
a 3100 or a 3500 Genetic Analyzer (Life Technologies).
These reagents correctly identified 50 prototype strains; se-
rotypes 15 and 29 have identical sequences in the queried
region, cannot be distinguished,23 and therefore are re-
ported as 15/29. The ability to identify serotypes associated
with ocular disease (8, 19, 37) and other serotypes that
commonly cause ocular disease (3, 4, 7, 11) was confirmed
in house using acquired strains (ATCC, Chantilly, Vir-
ginia, USA).14
RESULTS
FROM NOVEMBER 22, 2011 TO OCTOBER 31, 2014, 858 OF 4883
initial employee Occupational Health visits (18%) were
due to unique, eye-related complaints. The number of em-
ployees seen with eye concerns ranged from 6 to 36 per
month (Figure 2). Most employees complained of red eye.
HOSPITAL EMPLOYEES
INPHTHALMOLOGY MARCH 2016
39
FIGURE 1. Schema of red-eye employee triage system at the Johns Hopkins Hospital with employee totals from November 2011
through October 2014. EKC [ epidemic keratoconjunctivitis; NP [ nurse practitioner; PCR [ polymerase chain reaction. In
all cases, employees are required at the end of their furlough to return to Occupational Health for clearance before return to duty.

Of the 858 employees with eye concerns, 542 (62%) under-
went conjunctival swabbing and adenovirus PCR testing
(Figure 1). Forty-four employees (8% of suspected adeno-
viral cases, or 5% of all employees with eye concerns)
were positive for adenovirus by real-time PCR. Adenovirus
serotype could be determined for 32 employees. Overall, 14
employees had serotypes commonly associated with EKC
and/or signs and symptoms consistent with EKC. EKC-
associated serotypes that were detected included type 37
(n ¼ 6), 8 (n ¼ 4), and 19 (n ¼ 1). Three of 8 employees
infected with serotype 4 had signs and symptoms consistent
with EKC. The 5 other employees infected with serotype 4
had less severe conjunctivitis. Other serotypes found were 3
(n ¼ 5), 56 (n ¼ 4), 1 (n ¼ 2), 7 (n ¼ 1), and 42 (n ¼ 1).
Serotype could not be determined by sequencing for 12 em-
ployees. Although adenovirus DNA was detected by real-
time PCR from these 12 samples, no PCR products were
obtained with the less sensitive nested PCR reaction used
for serotype determination. A semiquantitative analysis
of cycle thresholds at which adenovirus DNA amplification
was detected suggested that samples that could not be sero-
typed contained approximately 1000-fold less DNA than
samples for which serotype was obtained. The median
PCR cycle at which adenovirus DNA was detected by
real-time PCR was 41 for samples that could not be sero-
typed, compared to 33 for samples for which a serotype
was obtained. None of the 12 patients whose samples could
not be serotyped had EKC.
More than half (469/858; 55%) of employees had condi-
tions that were determined to be noninfectious after
consultation by a Wilmer or private ophthalmologist;
allergic conjunctivitis was most common. The other diag-
noses included corneal or conjunctival foreign body,
pinguecula, episcleritis, scleritis, corneal abrasion, contact
lens overuse, iritis, dacryocystitis, and preseptal cellulitis.
The remainder (389/858; 45%) had adenoviral infection
confirmed by PCR (44 listed above) or were diagnosed
with ‘‘viral’’ or ‘‘bacterial’’ conjunctivitis by an ophthalmol-
ogist (either at Wilmer or in the community), a primary
care provider, or a doctor at an urgent care center.
Conjunctival cultures were not reported for any of these
employees, implying that ‘‘viral’’ or ‘‘bacterial’’ was a clin-
ical diagnosis. The majority of employees seen by primary
care providers and urgent care doctors were started on
topical antibiotic therapy.
To compare the prevalence of laboratory-diagnosed
adenoviral conjunctivitis among healthcare employees
with eye concerns with the prevalence of this condition
among general ophthalmology patients as diagnosed by
clinical examination and/or culture, we examined billing
data from the Wilmer General Eye Service. Visits with
billing diagnoses consistent with adenoviral conjunctivitis
(including International Classification of Diseases, 9th
revision [ICD-9] codes 372.00, 372.03, 372.71, 379.93,
077.1, 077.3, 077.4, and 077.8) were analyzed. In the 2-
year period prior to red-eye room closure (January 2006

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FIGURE 2. Employees seen for eye concerns, adenovirus polymerase chain reaction (PCR) tests ordered, and positive adenovirus
PCR results during the first 36 months of algorithm use.
through January 2008), there were 9609 patient visits with
unique diagnoses (ie, follow-up visits for the same diagnosis
for the patient were excluded). An ICD-9 code consistent
with adenoviral conjunctivitis was attached to 986 visits;
the vast majority of diagnoses were made clinically. There-
fore, the prevalence of clinically diagnosed adenoviral
conjunctivitis in the General Eye Service was 986 of
9609, or 10%.
DISCUSSION
THE NEW TRIAGE ALGORITHM FOR HOSPITAL EMPLOYEES
with red eye has achieved its goal to isolate and furlough
employees with adenoviral conjunctivitis in a rapid
manner, thus preventing spread of disease to patients and
other employees. The causes of red eye in hospital em-
ployees are now recorded systematically at 1 location
(rather than simple description of red eye and varying
levels of evaluation at multiple clinics), and the prevalence
of adenoviral infection, as well as infecting serotypes, can
be monitored for epidemiologic purposes. To our knowl-
edge, there is no other similar institution-wide triage sys-
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tem for adenoviral conjunctivitis, nor has there been any
report of adenovirus serotype or prevalence in hospital em-
ployees, which, by PCR diagnosis, is lower than clinical
diagnosis would indicate.
This algorithm has several potential benefits. First, PCR
offers state-of-the art sensitivity and specificity compared
to other methods, such as culture and antigen detection.
Second, the initial evaluation of healthcare workers at a
single site by a select group of trained providers maximizes
the likelihood of adherence to EKC prevention policies
and decreases the possibility of healthcare-associated
spread. Prior to this algorithm, numerous different clini-
cians evaluated employees, adenovirus cultures were infre-
quently obtained, adenoviral conjunctivitis was often
clinically diagnosed, and identification of EKC-related se-
rotypes was not performed. Moreover, many ophthalmolo-
gists are reluctant to examine cases of probable viral
conjunctivitis, necessitating this algorithm. Third, given
that clinical diagnosis of this disease is imperfect and that
initial screening by nurse practitioners at Occupational
Health is expected to yield false-positive cases, adjunctive
use of PCR testing in employees with suspected adenoviral
conjunctivitis facilitates judicious use of furlough for infec-
tion prevention. In the findings above, there was almost a
HOSPITAL EMPLOYEES
MARCH 2016
41
10-fold difference between diagnostically confirmed and and whether signs and symptoms are consistent with
clinically suspicious cases (44 employees with serotype EKC. The nurse practitioners call employees 5–6 days after
and disease course consistent with adenoviral conjuncti-
vitis vs 542 with clinically suspicious findings); furlough
of over 500 employees over a 3-year period would have
been impractical.
Determination of adenovirus prevalence and delinea-
tion of serotypes in hospital employees with red eye
became important objectives once the triage algorithm
was implemented. Prevalence of adenoviral conjuncti-
vitis among hospital personnel with eye concerns
(5%) was less than half that seen in General Eye Service
patients (10%) in whom the infection was clinically
diagnosed. Possible explanations for this difference
include different prevalence of adenoviral conjunctivitis
between the general public and hospital employees or
the comparatively lower specificity of clinical diagnosis
compared with PCR testing. Most likely some of the
clinically diagnosed cases in the General Eye Service
were not truly adenoviral, which is supported by findings
of the current study.
Retrospective molecular serotyping showed that approx-
imately 30% of the employees with adenoviral conjuncti-
vitis were infected with an EKC-associated strain, which
raised the question of whether the algorithm should
include prospective serotype determination to tailor
furlough duration. For example, employees with EKC-
associated serotypes could be assigned a 2-week work
furlough while those with other serotypes could be
furloughed for a shorter duration until resolution of clinical
symptoms; clinical clearance by Occupational Health
nurse practitioners would be required to return to work in
either situation. The 2-week furlough commonly recom-
mended for patients with presumed or definite adenoviral
conjunctivitis may have arisen in part from a report of a
large outbreak of EKC in an ophthalmology department
in which adenovirus was isolated from conjunctival sur-
faces up to 2 weeks after the onset of clinical illness.12 In
spring 2014, however, based on the low prevalence of
EKC in employees suspected of having viral conjunctivitis
and a suspicion that many employees with adenoviral
conjunctivitis recovered well in advance of the 2-week
mark, the ophthalmologist involved in developing this pol-
icy suggested that the nurse case manager call furloughed
employees at the 1-week mark. Most employees with
non-EKC serotypes reported not having any tearing,
redness, or discomfort at that time point. Compilation of
such reports after 2 months suggested that a 2-week
furlough might be unnecessary for the majority of em-
ployees with adenoviral conjunctivitis. Therefore, in April
2014 the red-eye policy was further modified. In this
manner, serotyping is done prospectively (with results in
2–5 days) and furlough length is tailored depending on
whether serotype is typical for EKC (types 4, 8, 19, 37)

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presentation to Occupational Health to inquire of their of a clinical trial to define the clinical indication for the
symptoms. If the employee is still symptomatic, the test and the clinical performance characteristics relative
furlough continues. If the employee is relatively asymptom-
atic, the nurse practitioner asks the employee to return to
Occupational Health to be cleared for work; the vast ma-
jority of employees with non-EKC serotypes are able to
work after a 7-day furlough. As before, in all cases, em-
ployees require clearance by Occupational Health in order
to return to work.
Detection of adenovirus by PCR does not imply the eye
is infectious, and adenoviral conjunctivitis can span from
mild disease to EKC. Though there are other infectious
conjunctivitides (eg, enteroviral), which typically resolve
within 5–7 days,24 adenoviral conjunctivitis is more com-
mon and potential complications (such as chronic visual
symptoms from keratitis) are more severe. For these rea-
sons, the focus of this PCR-based algorithm was adenovirus
detection.
Based on a preliminary analysis, the red-eye algorithm
has been cost effective for our institution. This analysis
was based on Johns Hopkins Hospital absorbing the cost
of PCR performed on employees with clinical suspicion
of adenoviral conjunctivitis (542) and the cost to the hos-
pital of furloughing only PCR-positive employees (44) for
2 weeks vs the cost of furloughing all 542 employees, as
might have been done prior to this algorithm. From sero-
type prevalence and record of employees’ clinical course,
it appears that the 2-week furlough is excessive for most
employees with adenoviral conjunctivitis. If trends
continue, based on the distribution of serotypes and
furlough lengths (one-third of employees with adenovirus
conjunctivitis having EKC-associated serotypes requiring
2 weeks vs two-thirds with non-EKC-related serotypes
requiring 1 week), the number of furlough days potentially
could be reduced by another third. A more flexible policy,
invoking shorter or longer furlough based on community
molecular epidemiologic data of circulating adenovirus se-
rotypes, as begun in April 2014, may be more appropriate
and deserves further investigation.
While PCR for adenovirus is available, there is currently
no commercially available PCR test for the detection of
adenovirus in conjunctival specimens. Therefore, clinical
laboratories must develop their own tests for this use.
Laboratory-developed tests are regulated by a federal law
entitled Clinical Laboratory Improvement Act, which stip-
ulates that laboratories can develop and offer tests clini-
cally if they ascertain the analytical performance
characteristics of the test. For molecular infectious disease
tests, analytical sensitivity is defined as limit of detection,
which is determined in experiments with a dilution series
of concentrations, and is defined as the concentration of
the panel member for which 95% of replicates are detected.
Manufacturers seeking Food and Drug Administration
(FDA) approval to market a test are held to the standard

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to disease, defined by clinical symptoms and the results of a
gold-standard diagnostic test (clinical sensitivity and clin-
ical specificity). Such determinations are not required of
laboratory-developed tests. Demonstrating clinical need
for initiatives like the one described herein may motivate
other centers to adopt some or all parts of the algorithm
to diagnose adenoviral conjunctivitis accurately in order
to limit transmission of disease. The perceived benefit
from such testing also may provide commercial impetus
for development of an FDA-cleared/approved PCR-based
test for adenoviral conjunctivitis that can be used in hospi-
tals, clinics, and other settings.
The inability to sequester a potentially large number of
individuals with possible infectious conjunctivitis in a
designated room in the Department of Ophthalmology
led us to develop an algorithm for rapid diagnosis of adeno-
viral conjunctivitis in hospital employees, who have the
potential to infect patients and other employees alike.
Implementation required a multidisciplinary effort across
several hospital departments and divisions. This compre-
hensive institutional policy has allowed for accurate detec-
tion and tracking of the prevalence of adenoviral
conjunctivitis, and judicious application of furlough based
on objective criteria. The relative simplicity and functional
benefits of this effort suggest that it merits consideration as
a practice model for hospitals and clinics seeking to prevent
healthcare-associated transmission of adenoviral conjunc-
tivitis. The addition of serotyping furthers our understand-
ing of the prevalence of adenoviruses that cause EKC. It
also provides preliminary data to support the development
of tests (such as a carefully designed multiplex PCR test)
that are simpler to perform but as accurate as direct Sanger
sequencing to detect all adenoviruses and to distinguish
those that cause EKC.

FUNDING/SUPPORT: UNRESTRICTED INSTITUTIONAL SUPPORT FROM RESEARCH TO PREVENT BLINDNESS, NEW YORK,
New York. Financial disclosures: The following authors have no financial disclosures: Irene C. Kuo, Colleen Espinosa, Michael Forman, and Alexandra
Valsamakis. All authors attest that they meet the current ICMJE criteria for authorship.

10. Piednoir E, Bureau-Chalot F, Merle C, Gotzmanis A,

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