Haddow, Lewis J. MB, ChB*; Sullivan, Elizabeth A. MBBS, MPH, MMed†; Taylor, Janette MPhil‡; Abel, Myriam MPH§; Cunningham, Anthony L. MD, FRACP‡; Tabrizi, Sepehr PhD∥; Mindel, Adrian MD, FRCP*
HERPES SIMPLEX VIRUS TYPE 2 (HSV-2) causes genital herpes and is transmitted during sexual intercourse. Infection causes considerable morbidity and can occasionally cause mother-to-child transmission and death in neonates. It is important to determine the prevalence of HSV-2 in populations for 2 major reasons. First, recent data suggest that prevalent HSV-2 infection is associated with enhanced HIV acquisition1,2 and shedding3,4 and an increase in plasma HIV load,5,6 and conversely that HIV infection leads to enhanced HSV-2 shedding.7 Second, an accurate estimation of the seroprevalence of HSV is needed at the country level for the development of relevant national HIV/sexually transmitted infection (STI) prevention and HSV vaccine policies. There is now the potential availability of a vaccine to reduce HSV-2 infection and perhaps consequent HIV transmission.8
HSV-2 infections are common throughout the world, particularly in parts of Africa.9 There are limited data from the Asia Pacific region. Studies in selected populations, considered to be at high risk for the acquisition of STIs, have shown that HSV-2 occurs in 13% to 75% of patients attending sexually transmitted disease clinics10–13 and 74% to 98% of female sex workers.14,15 In populations considered to be at lower risk for the acquisition of STIs, the prevalence of HSV-2 is generally lower. In women attending antenatal clinics, reported prevalence ranges from 5% to 55%12,16–19 and in women attending family planning clinics 11% to 38%.18,20 Demographic risk factors and behaviors associated with HSV-2 antibodies include increasing age, female gender, ethnic group, previous STIs, young age at first intercourse, lower educational level, and lower social class.
Other workers have shown that adolescents and young adults in the South Pacific Island nation of Vanuatu frequently engage in risky sexual behaviors with low levels of condom use.21–23 Our recent study, conducted of women attending an antenatal hospital clinic in Port Vila, Vanuatu, has shown that STIs are common,24 but HIV has not yet emerged as a major problem.25,26 As a follow-up study, we aimed to estimate the prevalence and correlates of HSV-2 infection in women attending the major urban antenatal clinic in Vanuatu.
Materials and Methods
Study Design and Patients
A cross-sectional survey of pregnant women attending the first-visit antenatal clinic (ANC) at Vila Central Hospital (VCH) was conducted by the Ministry of Health in 1999–2000. This survey of prevalence of STIs in women in Vanuatu has previously been published.24 Serum samples from the previous study were used as specimens for the current study. Vila Central Hospital is located in Port Vila, the capital and largest town in Vanuatu, population approximately 30,000 (Fig. 1). The majority of pregnancy care for women in Vila and the surrounding villages of Éfaté is delivered through the antenatal clinics at VCH.
Ethical approval for the original STI study was granted by the Ministry of Health, Republic of Vanuatu and University of New South Wales, Australia. Further ethical approval for the testing of sera for HSV-2 antibodies was obtained from Western Sydney Area Health Service, Australia, and the Ministry of Health, Republic of Vanuatu. Samples were fully deidentified for the current study and patient consent for HSV-2 testing was not sought.
Consecutive women were invited by midwives to participate in the study until the sample size of 550 was reached. Women attending the ANC for results or follow up were excluded. Informed consent was obtained for voluntary confidential testing of STIs (gonorrhea, chlamydia, trichomoniasis, and syphilis) and for unlinked anonymous testing for HIV infection.
Each woman was interviewed by a midwife using a standardized questionnaire. Information collected included gestational age, maternal age, parity, marital status, occupation, place of residence (whether town or village), and island or country of birth. For the purpose of the study, no information was collected on sexual behavior or clinical signs and symptoms. After this, a study obstetrician conducted the standard first-presentation antenatal assessment of history, physical examination, and specimen collection. Testing, treatment, and follow up of STIs have been described previously.24
Specimen Collection, Transportation, and Testing
Two 5-mL blood samples were obtained from each patient in Serum Separation Gel tubes (Sarstedt, Adelaide, Australia). These were centrifuged and transported to Australia for syphilis and HIV testing as previously described. HIV tests were done on one delinked aliquot of serum. The remainder of this serum was used for HSV antibody detection.
All sera were tested for antibody to HSV-2 using an indirect enzyme-linked immunosorbent assay (ELISA), specifically the HerpeSelect 2 ELISA IgG (Focus Technologies). The manufacturer recommends that an index value of >1.10 is presumptive for the presence of antibody to HSV-2. However, we have found that using this cutoff value yields a high rate of false-positive results (unpublished data). To overcome this problem, a cutoff value of 3.5 was used to determine HSV-2-seropositive sera. Sera that gave an equivocal result in the HerpeSelect 2 ELISA (index values, 0.9–3.5) were resolved using HSV-2 Western blot.27 A randomly selected subset of the sera (25%) was tested for antibodies to HSV-1. Depending on the HSV-2 serostatus of these sera, they were tested by one of 2 strategies. If HSV-2 antibody negative, serum was tested using the Enzygnost anti-HSV IgG ELISA (Behring) for detection of total antibody to HSV. If HSV-2 antibody-positive, the serum was tested using HSV-1 Western blot. This strategy was used as a result of the lack of a low-cost, sufficiently sensitive ELISA for antibody to HSV-1.17,28
Variables collected on each woman in the midwife questionnaire were coded in a database. Syphilis was defined as positive if rapid plasma reagin was positive or equivocal and Treponema pallidum hemagglutination was positive. The presence or absence of an STI (chlamydia, gonorrhea, trichomoniasis, and syphilis) was recorded based on the testing protocols described as well as the woman's HSV-2 serostatus. The number of concurrent STIs was also evaluated.
The Mann-Whitney U test was used to compare the age of HSV-2-negative and -positive women, and χ2 tests were performed for all other variables to test for association with HSV-2 seropositivity (Fisher exact test for variables with 1 degree of freedom or Pearson χ2 for other variables). Variables were included in a logistic regression model and were excluded by stepwise selection in which the P value was greater than 0.1. The data were compiled and analyzed using statistical software SPSS for Windows version 13.0 (Chicago, IL).
In total, 547 women were eligible and all agreed to participate in the study. Valid HSV-2 serology results were obtained on 535 women and HSV-1 results on 134. Women were aged 15 to 46 years with a mean age of 25.8 years. Marital status was given as married by 303 of 530 (57%) women and not recorded in 5. The majority lived in Vila (396 of 530 [75%]) with 113 (21%) living in other villages in Éfaté and 21 (4%) living on other islands. The main islands of birth were Tanna (89 women [17%]), Éfaté (69 [13%]), Ambrym (55 [10%]), Malekula (50 [9%]), Paama (44 [8%]), Pentecost (36 [7%]), and Ambae (36 [7%]). Seven women (1.3%) were born outside of Vanuatu.
Nulliparous women accounted for 184 of 534 (34%). Among parous women, 135 (25%) were parity one, 76 (14%) were parity 2, and 139 (26%) were parity 3 or more. Gestational age was first trimester in 104 of 528 (20%), second trimester in 279 (53%), and third trimester in 145 (27%).
The overall seroprevalence of HSV-2 was 158 of 535 (30%). All 134 (100%) of the serum specimens that were tested for HSV-1 were positive. In the current study, only those women who had both STI and HSV-2 testing were included in the analysis. The prevalence of concurrent STIs was as follows: chlamydia, 115 of 533 (22%); gonorrhea, 32 of 533 (6%); trichomoniasis, 146 of 533 (27%); syphilis confirmed on rapid plasma reagin and T. pallidum hemagglutination, 13 of 535 (2.4%). None of the women were HIV antibody-positive. The number of women with no STIs (other than HSV-2) was 325 of 533 (61%), women with one STI numbered 137 (26%), and 71 (13%) had more than one concurrent STI.
On univariate analysis (Table 1), HSV-2 seropositivity was significantly associated with increasing age and concurrent chlamydia, trichomoniasis, and syphilis, but not marital status, gonorrhea, parity, gestational age, place of residence, primary occupation, or place of birth. Older women were also more likely to be married and be parous (data not shown). There were statistical associations between all pairs of other STIs (gonorrhea, chlamydia, trichomoniasis, and syphilis) except gonorrhea and syphilis. The rates of HSV-2 were greater in women with greater numbers of concurrent STIs.
Multiple logistic regression was performed, including HSV-2 serostatus as the dependent and age, marital status, and the presence of each other STI as covariates. The number of concurrent STIs was not included because it was a composite variable of all other STI variables. The figures obtained from this final model are shown in Table 2. The independent predictors of HSV-2 infection were age, marital status, and trichomoniasis. Syphilis had the largest odds ratio of 2.89, but the 95% confidence interval crossed 1.0. However, only 13 women had syphilis confirmed on T. pallidum hemagglutination.
This study shows an HSV-2 seroprevalence of 30% among antenatal clinic attendees in Vila, Vanuatu. The study sample was a large group of young pregnant women with high rates of STIs, who were parous in approximately two thirds of cases and married in just over half. They also displayed a high degree of mobility around Vanuatu with most having been born on islands distant from the clinic. Women with HSV-2-positive serostatus were more likely to be older, unmarried, and have concurrent trichomoniasis or syphilis.
There are no published studies of HSV-2 seroprevalence in Vanuatu. There are few studies of STIs in the Pacific island nations. Seafarers in Kiribati have been found to have a 20% HSV-2 prevalence rate and a 9% chlamydia prevalence.29,30 Nearby Papua New Guinea has been studied in more detail with prevalence rates of HSV-2 of 20% to 39%, depending on age and geographic location, and a demonstrable association with HHV-8-seropositive status.31,32
Previous work has shown high rates of other STIs, particularly chlamydia and trichomoniasis, in this population of antenatal women.24 In that study, younger age and nonmarried status were associated with the presence of an STI. HSV-2 is a lifelong infection; therefore, seropositivity is a marker of lifetime sexual risk, whereas curable or transient STIs may be more representative of recent risk. We have no behavioral data in our sample and rely on limited published work on sexual behavior in Vanuatu. Our study also lacks clinical information that might be of interest.
One further weakness of this study is the absence of demographic variables found in other studies to be associated with HSV-2 status such as ethnicity, educational level, and socioeconomic status. However, the ethnicity of these women is likely to have been fairly uniform, with 98% born in Vanuatu, and residence and primary occupation are surrogates for the other factors.
We have found 100% HSV-1 seroprevalence, similar to other developing regions of the world.9 The only vaccine with proven efficacy against genital HSV is ineffective in individuals seropositive for HSV-1; therefore, this vaccine is unlikely to be of use in Vanuatu.8
The 2 most serious health implications of genital herpes infection are neonatal herpes33 and enhanced transmission of HIV. Neonatal herpes is most likely to occur when primary HSV infection occurs during pregnancy, particularly in the third trimester.16,34 With HSV-2 infection prevalent in one third of sexually active women, particularly unmarried women that rises with age, new infections with HSV are likely to be frequent within this population. Therefore, incidence estimates and further studies of men are needed. Incidence could be estimated by longitudinal studies of HSV seroprevalence or possibly by testing for HSV-specific IgM in a large sample.
The transmission of HIV is known to be enhanced in the presence of genital herpes in either the HIV-positive donor or the HIV-negative recipient.35 There were only 2 cases of HIV infection (both AIDS) reported from Vanuatu by December 2004, giving an age-adjusted incidence of 0.8 per 100,000, although surveillance estimates are likely to underestimate the true rates of HIV.25 Our population's high STI rates are notable because there were no cases of HIV identified in over 500 women. Therefore, there is an opportunity for prevention of spread of HIV within this tightly contained community. The rates of HSV-2 that we report here are cause for concern because this disease may contribute to the rapid spread of HIV by sexual transmission.
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