Davies, Stephen C. MM(ven), FAChSHM*; Taylor, Janette A. MPhil†; Sedyaningsih-Mamahit, Endang R. MD, DrPH‡; Gunawan, Suriadi MD‡; Cunningham, Anthony L. MD, FRACP, FRCPA†; Mindel, Adrian FRCP, MD§
INFECTION WITH HERPES SIMPLEX VIRUS type 2 (HSV-2) is the most common cause of recurrent genital ulcers in most areas of the world, including Asia.1,2 However, most HSV-2 infections are asymptomatic and the introduction of HSV type-specific serologic tests has revealed that HSV-2 infections are far more common than previously suspected.3 Furthermore, in a large population-based survey in the United States, the overall seroprevalence of HSV-2 was 16.4% in samples taken in 1976, rising to 21.7% in samples taken in 1989 to 1990.4 Seroprevalence in people reporting higher numbers of lifetime sexual partners, especially attendees of sexually transmitted infection (STI) clinics and female sex workers (FSWs), has generally been much higher. In FSWs, HSV-2 seroprevalence has been reported in the range of 75% to 95%.5 However, increasing seroprevalence over time has not been seen universally and seroprevalence in comparable risk populations has varied by geographic region.5
The importance of determining the prevalence and risk factors for HSV-2 in populations has increased for a number of reasons. Asymptomatic genital shedding is infectious and therefore prevalence and risk factors need to be established in asymptomatic as well as symptomatic individuals.6 Genital HSV-2 reactivation is associated with enhanced acquisition and shedding of human immunodeficiency virus (HIV), an increase in plasma HIV viral load, and HIV infection leads to enhanced HSV-2 shedding.7 This provides a biologic explanation for the role of coincident HSV-2 in facilitating the sexual transmission of HIV. Also, HSV-2 vaccine development and vaccination policy require accurate country-specific estimation of prevalence.
In Indonesia, STIs are common in some communities, particularly FSWs, and HIV is increasing. In 2003, UNAIDS estimated that there were 90,000 to 130,000 people infected with HIV in Indonesia with the majority of infections concentrated in high-risk groups of injecting drug users, gay men, and FSWs.8 Of great concern is the large rise in reported infections over the last 4 years. Data from the Indonesia Ministry of Health to July 2000 were 961 cumulative cases of HIV infection (non-AIDS) and 380 cases of AIDS. By September 2004, there were 5,701 HIV cases in Indonesia, 2,563 of which were AIDS cases.9 With the fifth largest population in the world, Indonesia has the ingredients for a potentially explosive HIV epidemic with high levels of STIs in high-risk groups, an extensive prostitution industry, frequent use of FSWs by men from most strata of society, and low condom use by FSWs.10–13
We aimed to determine the seroprevalence of HSV-2 in a range of communities in Indonesia and to establish risk factors associated with antibodies to HSV-2.
The Australian International Development Agency (AusAID) commissioned The Indonesian HIV/AIDS and STD Prevention and Care Project to work in partnership with Indonesian authorities to help prevent an HIV epidemic in Indonesia. As part of this project, people were recruited to studies of the prevalence of STIs in 1999 to 2000.
Complete details of recruitment and testing for STIs have been described elsewhere.12,14 Briefly, every second woman attending either of 2 maternal and child health (MCH) clinics in Bali was offered participation. At obstetrics and gynecology clinics at 2 hospitals in Makassar, every woman was offered participation. Similarly, all men and women attending for sexual health reasons to the dermatovenereology clinics of 2 hospitals in Makassar and of 2 hospitals in Denpasar, Bali, were offered participation. At all of these sites, less than 5% declined participation. In Kupang, FSWs were approached at their place of work or at 2 nongovernment organization clinics to participate in the study, and no woman declined participation. Recruitment at all sites occurred over an approximately 6-month period in 1999 to 2000.
All participants provided informed consent and completed a standardized questionnaire on social demographics and sexual behavior. Clinical histories, examinations, and specimen collections were standardized. No financial reward was provided and ethics committees in Australia and Indonesia approved the study. Subjects had to be 15 years of age or over.
Clinical Samples and Laboratory Tests
Participants had genital samples taken and tested for Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), and Trichomonas vaginalis (TV), and sera were screened for syphilis and HIV, as previously described.12,14,15 To summarize, urethral swabs (for men) and endocervical swabs (for women) were directly inoculated onto modified Thayer-Martin medium in a standard Petrie dish incubated on site in a candle-extinction jar at 36°C to 37°C until transfer to the laboratory for standard culture and identification procedures to identify NG. CT was identified by the PACE 2 DNA assay performed at a reference laboratory from a urethral swab in men or endocervical swab in women. TV was identified in women from a vaginal swab using the TV Inpouch culture system. Sera were screened for treponemal infection by rapid plasma reagin and positive results confirmed by Treponema pallidum hemaglutination assay. HIV antibody testing was performed by enzyme-linked immunosorbent assay (ELISA) and immunoblotting on each specimen. Any specimen positive by either test was submitted to Western blotting at a reference laboratory for confirmation.
Sera used for testing for HSV antibodies were stored at −20°C in provincial laboratories and then sent on dry ice to the Westmead Millennium Institute, Westmead Hospital in Sydney. These sera were tested for HSV-2 antibodies using the HerpesSelect 2 ELISA IgG (Focus Technologies) and performed blinded to population group and all other data. The manufacturer suggests that a positive result should have an index value >1.10. However, we16,17 and others18 have found that using this cutoff value yields a high proportion of false-positive results. To overcome this problem, a cutoff index 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 Western blotting.19
A subset of the sera (25%) was randomly selected and tested for antibodies to HSV-1. Depending on the HSV-2 serostatus of these sera, they were tested by one of 2 methods. If HSV-2 antibody-negative, sera were tested using the Enzygnost anti-HSV IgG ELISA (Behring) for detection of total antibody to HSV. If HSV-2 antibody-positive, sera were tested using HSV-1 Western blotting. This strategy was used as a result of the lack of a low cost, sufficiently sensitive ELISA for antibody to HSV-1.17
Information collected was entered into an EpiInfo 6.04 (CDC, Atlanta, GA) database. Analyses were performed in SPSS 12.0 for Windows (SPSS Inc., Chicago, IL). Not all cases contained complete information, and therefore records with missing data for variable(s) under specific analysis were excluded with resulting variation in denominators. Differences in proportions were assessed with the Pearson χ2 or Fisher exact test. Differences in means were assessed with the Mann-Whitney nonparametric test. To adjust for multiple risk factors, multivariate analysis was performed; variables associated with HSV-2 seroprevalence on univariate analysis at a significance level of 0.1 or less were included in a backward selection logistic regression model to test for independently significant associations with HSV-2 antibodies.
Of the 1,940 people recruited to the original prevalence studies of STIs, 710 (36.6%) had sera available for HSV testing. Table 1 lists the populations recruited into the STI prevalence studies, the number who had HSV testing performed, and the prevalence of HSV-2 antibodies. There were no significant differences (results not shown) between the groups who had HSV tests and the total populations for sociodemographic and sexual behavior characteristics or prevalence of specific STIs (NG, CT, TV, or syphilis), except for one population. In the original prevalence studies of STIs in women attending sexually transmitted disease (STD) clinics in Makassar, 13 of 170 (7.6%) had a positive culture for NG compared with 7 of 27 (25.9%) of the subset of these women who had sera available for HSV-2 serology (P = 0.01). No other significant differences were detected within this group. In particular, 73 of 180 (40.6%) women in the Makassar STD clinic original population were FSWs compared with the subset with available sera for HSV-2 testing in whom 9 of 27 (33.3%) were FSWs (P = 0.47). No participant had HIV infection detected.
FSWs in Kupang had the highest prevalence of HSV-2 antibodies at 90.3%. For all the FSWs, 86.9% (95% confidence interval [CI], 81.0–91.5) of 176 were positive for HSV-2 antibodies compared with 18.7% (95% CI, 15.0–22.7) of 418 nonsex worker women (odds ratio [OR], 29.0; 95% CI, 17.5–47.9; P <0.0001). This significance level remained when stratified by age (age groups 15–19, 20–25, 26–30, 31–35, >35).
The combined HSV-2 seroprevalence in the 2 male populations was 21.6% (95% CI, 14.5–30.1). Sex with FSWs was common among the men: 30 of 114 (26%) reported that sexual debut had been with an FSW and 48 of 109 (41%) reported commercial sex within the preceding month. In the 107 men for whom there were complete data, the seroprevalence of HSV-2 among men who had known contact with FSW (either sexual debut and/or commercial sex with the preceding month) was 25.0% compared with 15.7% for those who did not have any such sex with FSWs. However, this difference was not significant (OR, 1.79; 95% CI, 0.68–4.71; P = 0.24).
Table 2 lists sociodemographic and sexual behavior characteristics and prevalence of STIs in the populations. The women recruited from MCH clinics in Bali and from the obstetrics and gynecology clinics in Makassar had a similar profile with a mean number of lifetime sexual partners of 1.1, nearly all were married, they had a later age of sexual debut, and most completed high school education compared with the FSWs, most of whom had disrupted relationships, nearly half had not completed even primary school education, and who had a younger age of sexual debut (although sexual debut was typically with their husband or husband-to-be). The men and women attending STD clinics had intermediate levels of education, age of sexual debut, and lifetime numbers of sexual partners. Five (5.2%) of the Bali men and 3 (15.0%) of the Makassar men said they were men who had sex with men.
Data on contraception were available for 394 of the non-FSWs. Contraception was used by 52% of these women. Of these, injectable progesterone was used by 41%, intrauterine device (IUD) by 35%, and combined oral contraceptive pill (COCP) by 13%. Among the FSWs, contraception was used by 62% (injectable progesterone in 72%, COCP in 23%, and IUD in only 3%).
Only 1.8% of the FSWs in Kupang had symptoms or clinical signs of genital ulceration compared with 10.5% of the other women, whereas a history of genital ulcer was more common in the FSWs and was uncommon (less than 5% for all the groups) in the other women. The male STD patients were more likely to be symptomatic or have ulceration and also reported a history of genital ulcers more commonly than the female groups.
Prevalence of N. gonorrhoeae and C. trachomatis was highest in the Kupang FSWs and the Makassar STD women, and as expected, was much lower in the screened MCH and antenatal women. When each population group was analyzed for associations of each STI with HSV-2 antibodies, there were 2 significant results. In the women attending the Makassar STD clinic, gonorrhea was more common among those positive to HSV-2 antibodies (3 of 4 women positive for HSV-2 compared with 4 of 23 negative for HSV-2; OR, 14.95; 95% CI, 1.16–178; P = 0.04). In the women attending the Bali STD clinic, CT was detected in 4 of 13 positive for HSV-2 compared with 4 of 53 negative for HSV-2 (OR, 5.44; 95% CI, 1.15–25.8; P = 0.04).
Among the female subjects, we looked for associations with HSV-2 seroprevalence for sociodemographic variables, sexual risk, clinical history, symptoms and signs, prevalence of STIs, and partner sexual risk and clinical symptoms.
Results of univariate associations are listed in Table 3 for selected variables. Because the sexual and sociodemographic characteristics, and seroprevalence of HSV-2 of the FSWs were so different compared with the other female groups, they were analyzed separately for associations with HSV-2 antibodies. Logistic regression analysis was performed to identify independent associations with HSV-2 seroprevalence. In non-FSW populations, the final model found the following significant adjusted associations: any contraceptive use (OR, 2.24; 95% CI, 1.33–3.85; P = 0.003), symptoms or signs of genital ulcer (OR, 2.69; 95% CI, 1.27–5.70; P = 0.01), and younger age at sexual debut (OR, 0.92 per year of age; 95% CI, 0.86–0.99; P = 0.03). For the FSWs, the final model found only increasing age (per year) was significantly associated with HSV-2 antibodies (OR, 1.15; 95% CI, 1.06–1.24; P = 0.001).
Prevalence of HSV-1 antibodies ranged from 75.0% to 91.2% for the female populations in Bali and Makassar, 87.5% to 100% for the 2 male groups, and prevalence was 87.8% for the FSWs in Kupang. Although the number of sera tested for HSV-1 is small, we found no significant associations between HSV-1 and HSV-2 serologic status for any of the populations.
Eighty-seven percent of FSWs were positive for HSV-2 antibodies and in these women, increasing age was the only significant independent risk factor. HSV-2 seroprevalence among nonsex worker females was 19% and significant independent associations were found with contraceptive use, symptoms or signs of genital ulcer, and younger age of sexual debut. Prevalence among male patients of STD clinics was 22%. HSV-1 infection was extremely prevalent occurring in at least 75% of all individuals sampled.
To our knowledge, these are the first published data on seroprevalence to HSV-2 in Indonesian populations. Few data are available on prevalence of HSV-2 in Asian FSW populations.20 Of 500 FSWs in Chiang-Rai, Thailand, 76% were seropositive for HSV-2,21 and among 203 FSWs in Dhaka, Bangladesh, 63% of 203 were seropositive.5,22 The higher proportion in our study population may in part be explained by the older age of the participants. In our study, the mean age for FSWs of 29 was approximately 5 years older than in the Thai study in which the mean age was 24, and in the Bangladesh study, more than 50% of the FSWs were aged 18 to 30.
The seroprevalence reported here for the low-risk female populations and the STD clinic male and female populations is higher than most reports for comparable populations elsewhere in Asia.5,20 Reported seroprevalence among low-risk women was 9% in the Philippines,23 12% in Bangladesh,22 and 8% of antenatal clinic women in India and Sri Lanka.24 HSV-2 seroprevalence among higher-risk male military conscripts in Thailand was 15%.25 However, within similar risk populations, most other studies find higher prevalence among women compared with men. In our study, HSV-2 was detected in 22% of men attending STD clinics compared with 18% of women attending STD clinics (and compared with 19% of all the non-FSW women), but these differences were not statistically significant. The men had higher numbers of lifetime sexual partners, more often reported a history of genital ulceration, and were more likely to be symptomatic compared with women, although again these differences were not significant. In addition, contact with commercial sex is a reported risk factor for HSV-2 infection in men,26 and we found 14 of the 22 men who were HSV-2-seropositive to have had sexual debut with a FSW and/or have had commercial sex contact within the past month. However, our data are limited by the relatively small number of men in the study and the relatively crude nature of data on sexual contact with FSWs (which almost certainly underestimates the frequency of sex with FSWs).
Clearly, the FSWs had far more lifetime sexual partners than non-FSW women. Within the FSW group, the number of reported lifetime partners was not significantly associated with HSV-2 antibodies. Perhaps this is because there is a maximum of sex partners that saturates risk for HSV-2 acquisition, above which little further risk occurs.27 If so, years of sex work may be a better marker of risk for FSWs, but unfortunately, we did not collect data on this variable. Our data are also limited by lack of other relevant information such as age at which FSWs entered prostitution and number of sexual partners before commencement of commercial sex, which could be useful in further assessing sex risk associated with number of partners. Also, recall of number of lifetime sexual partners in FSWs is frequently an unreliable variable.28,29
Among the non-FSW women, we did not find an association between older age and HSV-2 antibodies, contrary to most studies.5 We did find a significant association for younger age at sexual debut and for symptoms or signs of genital ulcer, as have other investigators,30 both probably markers of increased sexual activity and/or risk of exposure to STIs. Why contraceptive use should increase risk for HSV-2 infection is more difficult to explain. Genital tract antibody responses appear dependent on estrogen levels,31 and hormonal contraception was an independent predictor of genital tract shedding of HSV-2 in one study.32 However, in a prospective study of women attending STD clinics in London, hormonal contraceptive use was associated with a significantly lower risk of HSV-2 seroprevalence.33 Our results are further complicated by the fact that 35% of those using contraception used an IUD rather than hormonal contraception. We found no significant association when contraception was stratified by type.
This study confirmed that sex work is a major risk for HSV-2 infection and that among the FSWs, older age was the major risk factor. Older age typically represents more years of sexual exposure, although in our study, we did not collect years of sex industry work. In these women in Kupang, age also reflects likelihood of a disrupted marriage and consequent severe socioeconomic disadvantage. These women are frequently married at a young age, have children, and then suffer a disrupted marriage, turning to sex work for economic sustenance.10,12,13 However, we could not confirm the marital or economic status of the sex workers, and it is known that some sex workers will suggest a disadvantaged status to authorities because this is a more socially accepted reason to be a sex worker.34 For example, in some Indonesian brothels, the managers only accept widows or divorcees for sex work, and in some Indonesian cultures, it is considered a disgrace to parents if a woman is not married by a certain age, and a divorced woman may be more valued than an unmarried woman.34
This study emphasizes the small percentage of FSWs infected with HSV-2 who have no recent or history of typical symptoms. Paucity of symptoms from other STIs among FSWs in Indonesia has been previously noted and at least partially blamed on the common practice of vulvovaginal cleansing.35,36 We included questions about vulvovaginal cleansing for the FSWs in Kupang, and all practiced this habit. The most common cleansing agents used were soap, toothpaste, or iodine solution. Given the potential for improving symptom recognition for genital herpes infection,37 health promotion campaigns targeted at FSWs should incorporate symptom recognition of genital herpes as well as those of other common STIs and should warn against inappropriate vulvovaginal cleansing.
This study’s strength is the testing of participants from a range of recognized risk groups recruited regardless of symptoms. However, there is possible bias surrounding which sera were available for HSV testing compared with the original populations recruited into the prevalence studies of STIs and consequent small numbers of sera tested in some groups. The groups surveyed in Makassar had less than 20% sera available from the laboratory. The reason for this is unknown, but possible explanations include insufficient specimen remaining after earlier tests were performed and individual laboratories failing to freeze-store remaining sera. We are unable to determine if any bias may have occurred from these possible reasons.
In the longer term, the availability of an effective HSV-2 vaccine offers the best hope for control of genital herpes infections.38 However, HSV-1 seroprevalence is likely to influence the efficacy of the only available vaccine (manufactured by GSK [Simplirix]) because efficacy has only been demonstrated in women with no prior antibodies to HSV.38
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