Antibody to Herpes Simplex Virus Type 2 as a Marker of Sexual Risk
Behavior in Rural Tanzania
Angela Obasi, Frank Mosha, Maria Quigley,
Zebedayo Sekirassa, Tom Gibbs, Katua Munguti,
James Todd, Heiner Grosskurth, Philippe Mayaud,
John Changalucha, David Brown, David Mabey,
and Richard Hayes
London School of Hygiene and Tropical Medicine and Enteric and
Respiratory Virus Laboratory, Central Public Health Laboratory,
London, United Kingdom; National Institute for Medical Research,
Office of Public Health, Mwanza Municipality, and African Medical
and Research Foundation, Mwanza, Tanzania
A serosurvey was conducted in a random sample of 259 women and 231 men in 12 rural
communities in Mwanza Region, Tanzania, using a type-specific ELISA for Herpes simplex
virus type 2 (HSV-2) infection. Seroprevalence rose steeply with age to ∼75% in women ?25
years old and 60% in men ?30. After adjusting for age and residence, HSV-2 prevalence was
higher in women who were married, in a polygamous marriage, Treponema pallidum hemag-
glutination assay (TPHA)–positive, had more lifetime sex partners, or who had not traveled.
Prevalence was higher in men who were married, had lived elsewhere, had more lifetime
partners, had used condoms, or were TPHA-positive. HSV-2 infection was significantly as-
sociated with recent history of genital ulcer. The association between HSV-2 infection and
lifetime sex partners was strongest in those !25 years old in both sexes. This association
supports the use of HSV-2 serology as a marker of risk behaviorinthispopulation,particularly
among young people.
By the end of 1997, an estimated 30.6 million adults and
children worldwide were living with human immunodeficiency
virus (HIV) infection or AIDS, 20.8 million of them in sub-
Saharan Africa , where heterosexual intercourse is the main
mode of transmission. In view of the HIV epidemic and the
great burden of ill health caused by other sexually transmitted
diseases (STDs) in the region, the study of sexual behavior, and
the design, evaluation, and implementation of interventions to
decrease sexual risk behavior have assumed increasing impor-
tance. However, the study of sexual behavior is problematic.
By its nature it cannot be directly observed, and consequently
only indirect information may be obtainedfromquestionnaires,
interviews, focus group discussions, and other qualitativemeth-
ods. Due to the sensitive nature of the issues raised, thesemeth-
ods are subject to considerable bias and can be difficult to
reproduce [2–4]. An inexpensive and reliable biologic marker
Received 20 April 1998; revised 6 August 1998.
Presented in part: 12th World AIDS Conference, Geneva, Switzerland,
28 June–3 July 1998 (abstract 14117).
Informed consent was obtained from all study participants. Ethics clear-
ance was given by the Tanzanian National Institute for Medical Research
and the London School of Hygiene and Tropical Medicine.
Financial support: Commission of the European Communities (EC) Life
Sciences and Technologies for Developing Countries Programme,EC AIDS
Task Force, United Kingdom Overseas Development Administration and
Medical Research Council, and German Centre for InternationalMigration
Reprints or correspondence: Prof. Richard Hayes, London School of
Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, UK
The Journal of Infectious Diseases
? 1999 by the Infectious Diseases Society of America. All rights reserved.
of sexual activity would be an invaluable tool in HIV and STD
Herpes simplex virus (HSV) type 2 infection is the major
cause of genital herpes worldwide . Because it is almost ex-
clusively sexually transmitted and leads to the production of
lifelong antibodies and because seropositivity is associatedwith
higher risk sexual behavior in selected populations [6, 7], it has
been suggested that HSV-2 antibody could be used as a biologic
marker of risk behavior . HSV-2 is also of interest in sub-
Saharan Africa for other reasons: It increases the risk of HIV
transmission and acquisition , and it is increasingly recog-
nized that its role in the etiology of genital ulceration inAfrican
populations has been underestimated .
Most previous studies of prevalenceandriskfactorsforHSV-
2 infection have been done in selected groups, such as antenatal
or STD clinic attenders and blood donors in industrialized
countries [6, 7, 11]. Few population-based surveys have been
reported [12, 13] and only one in Africa . We are not aware
of any studies that have examined risk factors for HSV-2 in-
fection in a representative sample of an African population.
Research on the epidemiology of HSV-2 in sub-Saharan Af-
rica has been hampered by deficienciesindiagnostictechnology.
Genital herpes is characterized by long periods of latency with
episodic symptoms. Because culture and antigen detectiontech-
niques are insensitive in the absence of ulcers [15, 16], they
cannot be used for large-scale epidemiologic studies. In addi-
tion, HSV-2 shares extensive sequence homology with HSV-1.
This has caused a high level of cross-reactivity and made type-
specific antibody determinations unreliable . There is no
known cross-reactivity between the glycoprotein G (gG) in
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JID 1999;179 (January) HSV-2 Serology in Rural Tanzania17
HSV-1 (gG-1) and that in HSV-2 (gG-2) . Sensitive type-
specific Western blot assays (WBA) developed over the last
decade depend largely on reactivity to gG-2 for the identifi-
cation of HSV-2 antibody . However, WBA is expensive,
cumbersome, not widely available, and difficult to reproduce.
(MAb)–blocking RIAs that detect type-specific HSV-1 and
HSV-2 antibodies. The HSV-2 antibody assay was validated
against WBA using sera from culture-proven recurrent genital
HSV-2 casesand had equivalentsensitivity(96%).Furthermore,
no HSV-2 antibodies were detected inpatientswithfirst-episode
genital HSV-1 or in children, indicating high specificity. The
assay has been converted to an ELISA format with similar test
characteristics. The HSV-2 antibody ELISA, when validated
against the Chiron assay, had similar sensitivity and specificity
(94% and 92%, respectively) . When compared with the
WBA on 473 sera collected in rural Uganda, sensitivity was
91% and specificity 93%, demonstrating its suitability for use
in seroepidemiologic studies (unpublished data).
A recent community-randomized trial conducted in rural
Tanzania afforded the opportunity to examine the seroepide-
miology of HSV-2 in a representative sample of an African
population using the novel ELISA described. The extensive
data provided by a detailed behavioral questionnaire enabled
us to examine associations between HSV-2 and sexual risk be-
havior and to assess the role of HSV-2 serology as a marker
of sexual risk behavior. The specific objectives were to measure
the seroprevalence of HSV-2 infection in this population by age
and sex and to examine associations between HSV-2 infection
and reported sociodemographic and behavioral factors.
Selection of study subjects.
improved STD treatment services on HIV incidence in a rural Af-
rican population, randomly selected persons were recruited in
Mwanza Region, Tanzania, and followed for 2 years. The study
population, design, and methodology of the trial are described
elsewhere . In brief, 12 communities were randomly assigned
to intervention or comparison groups.The effectoftheintervention
was measured in a cohort of ∼1000 adults, aged 15–54 years, se-
lected from each community by randomclustersampling.Serawere
taken from the cohort at baseline and again after 2 years, and the
incidence of HIV infection was compared between the intervention
and comparison groups .
A random subsample from the trial cohort (1500 adults) was
selected for a detailed behavioral survey  and served as the
controls for a case-control study of risk factors for HIV infection
. The homes of all selected subjects were visited. Subjects were
interviewed concerning demographiccharacteristicsandbehavioral
risk factors using a structured questionnaire.Thequestionnairewas
designed in English, translated into Kiswahili, back-translatedinto
English, and pretested in a pilot study. Of subjects eligible for the
study, 74% participated. Interviews were conducted in May and
June 1993, 6–17 months after the baseline survey.
In order to evaluate the effect of
The random subsample of 1500 subjects formed the sampling
frame for the present study. Rates of HSV-2 infection change most
rapidly among adolescents and young adults. Consequently, the
selection of subjects was stratified by age at baseline survey and
weighted to maximize the number of young people. All 290 subjects
aged 15–19 years, and simple random samples of 100 subjects aged
20–24, 50 subjects aged 25–34, and 50 subjects ?35 were selected
for the present study. Sociodemographic and behavioral data were
taken from the behavioral study questionnaire, and data on re-
ported STD syndromes from the baseline survey of the cohort.
Syphilis and HSV-2 serology were done on sera obtained at the
Syphilis serology at baseline was mea-
sured by the Treponema pallidum hemagglutination assay (TPHA;
Type-specific antibodies to HSV-2 were determined using a
MAb-blocking ELISA. This is a direct modification of the vali-
dated MAb-blocking RIA ; full details of the assay will be
reported elsewhere. In brief, Greiner microtiter plates were coated
with HSV-2–infected cell lysate at 1:25 dilution in PBS overnight
at 4?C. The coated plates were then incubated successively with
detergent (1.5% Triton X-100 and 0.5% Nonidet P40 in PBS) for
30 min at room temperature, with 10% fetal calf serum in PBS for
2 h at 37?C, with a 1:4 dilution of test serum for 1 h at room
temperature, with a 1:1600 dilution of HSV-2–specific MAb and a
1:1000 dilution of peroxidase-conjugated anti-mouse antibody.
Plates were washed three times between each stage using 0.05%
PBS-Tween. Finally, tetramethylbenzadene was added to each well,
and the reaction was stopped as appropriate by the addition of 2
M H2SO4. The absorbance of each well was measured at 450 nm,
and the percentage blocking of each serum specimenwascalculated
by comparison with diluent control and the mean absorbance of
four wells containing positive control sera.
The positive cutoff was determined by fitting a mixture model
to the bimodal distribution of percentage blockings obtained as
previously described . With the cutoff set at 30% blocking, this
gave estimated false-negative and false-positive rates of 0.4%. Fifty
sera were further tested by in-house WBA to confirm thesensitivity
and specificity of the testing strategy.
HSV-2 seroprevalence by age and sex was
estimated using all selected subjects. The distributions of socio-
demographic and behavioral risk factors were examined by age
and sex in the 73% of selected subjects who participated in the
behavioral survey. Because the distribution of risk factors varied
between women and men and because gender was strongly asso-
ciated with risk of infection, data for women and men were ana-
lyzed separately. Odds ratios (ORs) were estimated for each risk
factor and were adjusted for age in years and residence stratum
(rural, island, lake shore, or roadside) using logistic regression.For
significant variables and other behavioral factors of interest, ORs
were further adjusted for variables significantly associated with
HSV-2 infection on univariate analysis. Since HSV-2 infection in
young people is more likely to reflect recent sexual exposure, a
further analysis was conducted to compare the association of se-
ropositivity and reported numbers of partners in those aged !25
and ?25 years. Statistical significance of ORs was assessed using
the likelihood ratio test. The analysis was performed using STATA
software (Stata, East College Station, TX).
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24 Obasi et al. JID 1999;179 (January)
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