M A J O R A R T I C L E
Clinical and Virologic Manifestations of Primary
Epstein-Barr Virus (EBV) Infection in Kenyan
Infants Born to HIV-Infected Women
Jennifer A. Slyker,1Corey Casper,1,2,3,4Kenneth Tapia,1,5Barbra Richardson,1,4,5Lisa Bunts,4Meei-Li Huang,8
Elizabeth Maleche-Obimbo,6Ruth Nduati,6and Grace John-Stewart1,2,3,7
1Department of Global Health,2Department of Epidemiology,3Department of Medicine, Division of Allergy and Infectious Diseases, University of
Washington,4Divisions of Vaccine and Infectious Disease, Public Health Sciences, and Clinical Research, Fred Hutchinson Cancer Research Center;
5Department of Biostatistics;6Department of Pediatrics and Child Health, School of Medicine, University of Nairobi;7Department of Pediatrics, and
8Division of Laboratory Medicine, University of Washington
(See the editorial commentary by Balfour and Verghese on pages 1787–9.)
associated lymphomas. Characterizing primary infection may elucidate risk factors for malignancy.
Methods.To describe clinical and virologic manifestations of primary EBV infection among infants born to
HIV-infected women, specimens were utilized from a cohort study conducted in Nairobi, Kenya. HIV and EBV
viral loads were measured serially in plasma. EBV serology was performed on EBV DNA–negative infants. Monthly
clinical examinations were performed by pediatricians.
Results. The probability of EBV infection by 1 year of age was .78 (95% CI, .67–.88) in HIV-infected and .49
(95% CI, .35–.65) in HIV-uninfected infants (P<.0001). At 2 years, probability of EBV infection was .96 (95% CI,
.89–.99) in HIV-infected infants. Peak EBV loads were higher in HIV-infected versus HIV-uninfected infants
(median 2.6 vs 2.1 log10copies/mL; P<.0001). The majority of HIV-infected infants had detectable EBV DNA for
>3 months (79%). Primary EBV infection was associated with cough, fever, otitis media, pneumonia, hepatomegaly,
splenomegaly, and hospitalization in HIV-infected infants; conjunctivitis and rhinorrhea in HIV-uninfected infants.
Conclusions. EBV infection occurs early in infants born to HIV-infected women. HIV infection was associated
with more frequent and higher quantity EBV DNA detection.
Human immunodeficiency virus (HIV) infection is a risk factor for Epstein-Barr virus (EBV)–
Keywords.EBV; primary infection; HIV; pediatric; herpesviruses.
Epstein-Barr virus (EBV) infects >95% of the global
population with prevalence varying by sociodemo-
graphics and region. Infection with EBV is uncommon
before the age of 5 in European and American children,
after which seroprevalence increases through adulthood
[1,2]. This epidemiology differs from sub–Saharan
Africa where many children acquire EBV before the age
of 3 [3–6]. EBV infects via the oropharyngeal route,
and is transmitted primarily through saliva. Following
symptomatic primary infection, EBV DNA can be de-
tected in blood from most individuals, and is rapidly
cleared in 1–3 weeks [7,8]; but in saliva, virus may be
detected for months to years [8,9].
EBV is the cause of infectious mononucleosis in ado-
lescents and adults ,but the majority of primary in-
fections in children are asymptomatic or mild .
Symptoms reported in children include pharyngitis,
rash, fever, hepatomegaly, and splenomegaly [12,13],
but are infrequently a cause for hospitalization .Al-
though latent EBV infection is usually silent, EBV is a
major contributor to malignancies worldwide , in-
cluding lymphoma, nasopharyngeal carcinoma, and
gastric cancer. In a large prospective study, the risk of
developing Hodgkin lymphoma was found to peak at
Received 8 October 2012; accepted 21 December 2012; electronically published
14 March 2013.
Presented in part: 19th Conference on Retroviruses and Opportunistic Infections,
Seattle, Washington, March 2012. Abstract S-180.
Correspondence: Jennifer Slyker, PhD, Harborview Medical Center, 325 9th Ave,
Box 359931, Seattle, WA 98104 USA (email@example.com).
The Journal of InfectiousDiseases2013;207:1798–806
© The Author 2013. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
1798 • JID 2013:207 (15 June) • Slyker et al
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2.4 years following infectious mononucleosis . EBV is also
the most common cause of cancer among children in equatorial
Africa, endemic Burkitt lymphoma [17,18]. Peak Burkitt lym-
phoma incidence in African children occurs in childhood,
highlighting the potential importance of primary infection in
the risk of malignancy [19,20]. EBV additionally causes non–
Hodgkin lymphoma in the context of human immunodefi-
ciency virus (HIV). HIV-infected children are at an estimated
>40-fold increased risk for cancer  and many of these ma-
lignancies are associated with EBV. Risk factors for HIV-
associated malignancy include a diagnosis of AIDS, low CD4
lymphocyte cell count, unsuppressed HIV replication, short
antiretroviral therapy (ART) duration, and high EBV viral
We hypothesized that primary HIV infection may result in
semination,which couldin turn elevate achild’s long-term riskof
developing cancer. In a cohort of HIV-infected and uninfected
infants born to HIV-infected mothers, we describe the incidence
and correlates of EBV infection, the kinetics of EBV viremia,
and the clinical manifestations of primary EBV infection.
This longitudinal study utilized repository specimens and data
from an HIV-1 transmission study in Nairobi, Kenya [26,27].
The current study leveraged this historic observational cohort to
compare EBV detection and viral loads between HIV-infected
and -uninfected infants. Sample size calculations were based on
an earlier paper reporting 68% EBV seroprevalence in Nigerian
children screened at 6–16 months of age . With a sample
size of 50 HIV-infected and 50 HIV-uninfected infants, we esti-
mated the study would be powered to detect a hazard ratio
(HR) of ≥2.2 for the effect of HIV on EBV detection. We later
modified our sample size to all eligible HIV-infected infants
(n =75) to increase statistical power further. For EBV viral
load, we estimated 80% power to detect a minimum 0.6 log10
mean difference in peak EBV viral load between HIV-infected
and -uninfected infants, assuming a standard deviation (SD) of
1.0 log10. Both power calculations assumed 2-sided tests with
80% power and α= 0.05. All sample size calculations and statis-
tical analyses were planned a priori, with the exception of the
clinical manifestations, which were performed post hoc after
observing the high incidence of early EBV infection in the
Inclusion criteria were survival and follow-up to ≥3 months
and well-defined timing of HIV infection. Because maternal
antibody is highly protective, we assumed few EBV infections
would occur before 6 months of age. All eligible HIV-infected
infants were included in the study. To meet the sample size of
50 HIV-uninfected infants, a random sample was drawn from
the 338 HIV-uninfected infants satisfying inclusion criteria.
This was done by assigning a uniformly distributed set of
pseudo-random numbers between zero and 1 to each infant ID
number, sorting by random number, and selecting the first 50
The study was approved by the University of Washington Insti-
tutional Review Board and the Kenyatta National Hospital
Ethics and Research Committee. Mothers provided written in-
formed consent; recruitment, enrollment, and follow-up are de-
tailed elsewhere [26–28]. At enrolment, women provided a
detailed medical history and sociodemographic data. HIV-
seropositive pregnant women were enrolled between 1999–
2003 and provided with zidovudine from 32 weeks’ gestation
for the prevention of mother-to-child HIV transmission
(PMTCT) . The study was conducted before ART became
widely available in Kenya; participants in this study received no
ART other than PMTCT. HIV-infected infants were followed
for 2 years, while HIV-uninfected infants exited the study at 12
As part of the historic cohort study, mothers and infants at-
tended monthly clinic visits in which a study pediatrician col-
lected information regarding infant illness, and conducted a
physical examination [26,27].Infant symptoms were systemati-
cally assessed at each study visit with the aid of a standardized
questionnaire, which included measurement of vital signs;
examination of the head, chest, abdomen, and skin; and assess-
symptoms. Symptoms were recorded separately as maternally
reported since previous visit, maternally reported at current
visit, or physician-observed at current visit.
Specimen Collection and Infant HIV Diagnosis and Viral Load
Blood was collected at 32 weeks’ gestation, delivery, 1 and 3
months postpartum, and every 3 months thereafter from
mothers and infants. Infant HIV was diagnosed by polymerase
chain reaction (PCR) detection of HIV gag DNA from dried
blood spots  or HIV RNAviral load in plasma , which-
ever was detected first.
Measurement of EBV Viral Load
EBV was measured in the same plasma specimens used for
HIV viral loads, using a real-time PCR assay. DNA was extract-
ed from 200µL of plasma specimens using QIAamp 96 DNA
Blood Kit (Qiagen, Valencia, CA) and eluted into 100µL
Qiagen AE buffer. BALF5 primers (forward 5′-CGGAAGC
GAATG-3′) and probe (FAM 5′-TGT ACA CGC ACG AGA
AAT GCG CC-3′-TAMRA) were used to detect EBV genome
in 10µL of the extracted DNA . Each 30µL PCR reaction
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