HERPES SIMPLEX VIRUSES ARE AMONG the most prevalent sexually transmitted infections (STIs) globally.1–3 Genital herpes usually is associated with herpes simplex virus type 2 (HSV-2), whereas herpes simplex virus type 1 (HSV-1) causes orolabial disease. The latest U.S. National Health and Nutrition Examination Surveys estimates that 23% of women in the United States are infected with HSV-2.3 Consequences of genital herpes include maternal morbidity and neonatal morbidity and mortality, as well as an increased susceptibility to human immunodeficiency virus (HIV).1,4–6 In addition, high-titered transplacentally acquired antibodies to some infectious agents, including HSV-2, have been linked to some forms of neuromental disorders.7
Relatively few studies have measured incident HSV-2 infection.8–18 Four prospective studies have shown that women are at higher risk for acquiring HSV-2 infection than men,10,13,17,18 probably because of anatomical differences that cause male-to-female transmission to be more efficient than that from female to male. Other major risk factors for incident infection have included longer duration of sexual activity, higher number of partners, older partners, greater recent coital frequency, unprotected intercourse, black race, same-sex coitus among men, commercial sex, and earlier or current STI.8–16
We report here on the incidence of HSV-2 infection and correlates of its acquisition and prevention from a prospective cohort study of high-risk, predominantly black women attending a sexually transmitted disease (STD) clinic in Alabama. As noted in a previous report, the prevalence of HSV-2 infection at enrollment was 64%, and no relationship was found between recent use of condoms or spermicide and HSV-2 prevalence.19 Risk factors for seropositivity at enrollment included age, black race, high number of lifetime partners, younger age at first intercourse, history of syphilis, and lack of antibodies to HSV-1.
Materials and Methods
Study Design and Procedures
This analysis is based on a prospective cohort study of the use of male condoms and spermicide conducted among 1122 women attending a public STD clinic in Birmingham, AL, from 1992 to 1995 that has been described elsewhere.19–21 Eligible women were aged 18 to 35 years and could not have had a hysterectomy, be currently pregnant or planning a pregnancy within the next 6 months, or receiving antibiotic therapy. The present analysis of incident HSV-2 infection is restricted to those participants who did not test positive for HSV-2 antibodies at enrollment.
After completing the recruitment and enrollment visits, participants were asked to return for 6 follow-up visits scheduled at monthly intervals. At enrollment and all follow-up visits, study staff interviewed participants on their sexual behaviors and use of condoms, conducted counseling about condoms, and distributed condoms. A study nurse also performed a physical examination and collected endocervical swabs, vaginal secretions, and serum for STI testing at these visits. Those with treatable infections other than HSV received drugs according to Centers for Disease Control and Prevention (CDC) STD Treatment Guidelines.22,23 Participants were asked to complete a diary prospectively between study visits detailing their sexual activity during that interval (including their use of condoms and problems during use such as breakage and slippage). They also were asked to bring empty condom wrappers, unused condoms, and unused spermicide to the follow-up visits. Study staff then reviewed the diaries and the supplies of barrier products with participants to determine whether their self-reported activity and product counts agreed. Intervals were classified as having apparent overreporting of condom use if the frequency of such use reported in the diary entries for the interval was higher than that indicated by the product count and follow-up interview. We used inconsistent condom use (defined as ≤1 coital act without condom use as reported by the participant or identified via the product counts) as the referent for the assessment of the effect of consistent condom use on the risk of infection with HSV-2. Prior research in STD clinic populations supports this comparison (rather than selecting nonusers as the referent) because participants who have any condom use could differ from nonusers in terms of unmeasured confounding related to sexual risk taking.24 Ethical review boards at the University of Alabama at Birmingham, the Alabama Department of Public Health, and the CDC approved the research. All participants gave written informed consent. All names and personal identifiers were removed before researchers at the CDC received the data. The CDC institutional review board determined that the present analysis of anonymous data were exempt from review.
Frozen sera obtained from the enrollment visit were sent to the Emory University School of Medicine (Atlanta, GA), where they were tested for antibodies to HSV-1 and HSV-2 using an immunodot assay that is sensitive, specific, and reproducible for the 2 types.25,26 We also tested sera obtained from the last follow-up visit for HSV-2 infection. If antibodies to HSV-2 were detected, sera from previous follow-up visits were assayed in reverse chronological order until the visit with the first reactive assay was identified to determine the time interval of seroconversion.
Among the subset of participants without prevalent HSV-1 antibodies at enrollment, we tested sera from their last follow-up visit for HSV-1 to assess whether they had become seropositive for HSV-1 during study follow-up. However, because we did not assess interim serum samples for HSV-1 antibodies, the timing of seroconversion is unknown.
We identified Neisseria gonorrhoeae and Chlamydia trachomatis using culture of endocervical specimens from the enrollment and 6 follow-up visits. Modified Thayer-Martin medium was used for culturing N. gonorrhoeae. C. trachomatis was cultured in McCoy cells using microtiter plates incubated in CO2; fluorescein-conjugated monoclonal antibody reagents (Syva Co., San Jose, CA) were used for detection of C. trachomatis. Trichomonas vaginalis was diagnosed by wet mount. We diagnosed bacterial vaginosis (BV) using Amsel criteria.27 Participants were tested for syphilis as previously described.20
Although the time to seroconversion after acquisition of HSV-2 infection is unknown, limited evidence suggests that antibodies in sera become detectable about 20 to 21 days after the first episode of symptomatic HSV-2 genital infection.25,28 To account for this “window period,” we estimated the date of infection as the date of the follow-up visit date that preceded the visit with the first positive assay for HSV-2 antibodies. However, if the interval between visits was greater than 2 months, we excluded only the last 30 days of the follow-up interval. In addition, we excluded from the analysis women who were newly diagnosed with HSV-2 infection on their follow-up visit at 1 month, because these women might have acquired infection before the enrollment visit. For those women who did not test positive during follow-up, we censored their follow-up time 30 days before their last study visit because they might have acquired infection but not yet have seroconverted.
Time at risk for incident HSV-2 infection began with the participant's first negative serologic assay and ended with the estimated infection date for seroconverters or with the date of the follow-up visit that preceded the last negative assay for nonseroconverters. The incidence rate for HSV-2 was calculated as the number of new seroconversions per woman-years at risk of infection during follow-up, with exact 95% confidence intervals (CIs) based on the Poisson distribution. We calculated hazard ratios (HRs) using Cox proportional hazards modeling with time-dependent and time-independent covariates to assess predictors of HSV-2 infection. The time-dependent covariates were assessed for the interval that preceded the estimated date of infection with HSV-2. Potential risk factors were selected a priori based on the existing literature.8,15,17,18 We fit individual hazard models for the analysis of simple associations (i.e., 1 risk factor at a time). We then fit a multiple proportional hazards regression model including all potential risk factors as independent variables and used backward elimination of factors that were not predictive based on a 2-sided test with an α of 0.10. Three factors were significant: adolescent age, BV, and syphilis. We then fit individual models for each of the potential risk factors adjusted for age and BV; we did not adjust for syphilis because the CI for this estimate was very wide. We used SAS 9.1.3 (SAS Institute, Cary, NC) for the data analysis.
Of 2296 eligible women who consented to participate, 1122 (49%) enrolled, completed the initial visit, and were followed up. As previously described, participants seemed to be at higher risk of STD than nonparticipants.20 Participants were excluded for having antibodies to HSV-2 detected at enrollment (n = 722), missing HSV-2 data at enrollment (n = 14), not having any follow-up visits with HSV-2 data (n = 53), or lacking at least 1 month of follow-up before their estimated date of HSV-2 seroconversion or final negative assessment (n = 40). Thus, analyses were based on the remaining 293 participants, who contributed a total of 1406 months of follow-up. At least 5 follow-up intervals were available for study in 68% of the participants (n = 198). The remaining participants contributed 4 (n = 14), 3 (n = 20), 2 (n = 28), and 1 (n = 33) intervals. Most participants (Table 1) were single and not cohabiting (88%), black (87%), and had completed high school (80%). Their mean age was 23.7 years (SD, 4.2; median, 22.7; range, 18.1–34.8).
Altogether, 24 women (8.2%) acquired new HSV-2 infection during follow-up (Table 2). The overall incidence of HSV-2 infection was 20.5 per 100 woman-years (95% CI, 13.1–30.5). Women aged 18 to 20 years had a higher incidence than their counterparts aged 21 to 25 years or those aged 26 to 35 years.
Analysis of Simple Associations
In analyses evaluating 1 risk factor at a time, adolescents (aged ≤20 years) had a higher risk of incident HSV-2 infection than older women (unadjusted HR, 2.8; 95% CI, 1.2–6.3; Table 3). Also, women who reported young age at first intercourse (≤16 years) seemed to have a shorter time to HSV-2 infection during follow-up than those who reported an older age at first intercourse, but this result did not attain conventional (i.e., using an α of 0.05) statistical significance (unadjusted HR, 2.4; 95% CI, 0.9–6.5). HSV-1 seroprevalence at enrollment was 75.8% (n = 219), and 2 women were diagnosed with incident HSV-1 during follow-up. Prevalent HSV-1 was not associated with diagnosis of incident HSV-2 infection. Furthermore, no relationship with incident HSV-2 was suggested with other time-independent factors, which included marital or cohabitation status, educational level, income, having had sex for material compensation in past month, number of lifetime partners, and prevalent STI.
Among time-dependent variables, having a diagnosis of BV was associated with a higher unadjusted rate of HSV-2 infection (HR, 2.4; 95% CI, 1.1–5.5). Also, those with syphilis had a higher risk of HSV-2 infection than those without syphilis (unadjusted HR, 50.4; 95% CI, 6.8–374.0), but this estimate was imprecise because of the small number of syphilis infections. Diagnoses of trichomoniasis or C. trachomatis during follow-up were not associated with HSV-2 acquisition, and we could not assess the role of gonorrhea because no incident cases were diagnosed among women with HSV-2 infection.
The self-reported, time-dependent sexual behaviors (i.e., condom use, coital frequency, current partners, and new partners during follow-up interval) reported in the study coital diary were not significantly related to HSV-2 seroconversion. To assess the potential effect of condom use on HSV-2 seroconversion rates, we triangulated self-reports of the frequency of sexual activity and condom use obtained from the coital diaries, with information obtained from counts of returned products and follow-up interviews. Participants seemed to overreport condom use in the coital diaries in 22.8% of the intervals (n = 274) when product count and interview data were used as the standard for comparison. Consistent (100%) condom use without apparent overreporting of use or reports of condom breakage or slippage was not associated with significantly lower rates of HSV-2 acquisition when a comparison was made with consistent use with overreporting of condom use or with inconsistent use (Table 3). This lack of an association was not sensitive to the use of alternative analysis populations or categorizations for condom use. For example, after excluding the 274 intervals with apparent overreporting of condom use, the adjusted HR for consistent condom use without breakage or slippage compared with inconsistent condom use remained 0.8 (95% CI, 0.2–3.1). Similarly, the adjusted HR for this same comparison was 1.0 (95% CI, 0.3–3.7) when the seroconversion date was estimated as the date of the follow-up visit when she tested positive for HSV-2 antibodies.
Multiple Regression Analysis
In multivariable analyses, women who were aged 20 years or younger had a higher adjusted HR for incident HSV-2 infection (2.8; 95% CI, 1.3–6.4) than older women (Table 3). Also, women with prevalent or incident BV had a higher rate of incident HSV-2 than those without this condition (adjusted HR, 2.4; 95% CI, 1.1–5.6). No other factor was significantly associated with incident HSV-2 infection in the multiple regression analysis.
The annual incidence of HSV-2 infection among adult women attending this STD clinic during the mid-1990s was 20.5 cases per 100 women, which is higher than estimates from previous studies of adult women attending STD clinics in the United States during this time period. Gottlieb et al.13 found an incidence rate of 14.8 per 100 woman-years among women aged 14 years or older attending 5 public STD clinics in the United States, and Wald et al.12 reported a rate of acquisition of 5.7 per 100 woman-years among high-risk adult women from 22 STD clinics during approximately the same time period. As expected, the estimates of incident HSV-2 infection in the current study are substantially higher than those previously reported from non-STD clinic populations. For example, modeling that used prevalence findings from 2 waves of the U.S. National Health and Nutrition Examination Surveys yielded an annual HSV-2 incidence estimate of 1.0 per 100 adult women in the general U.S. population.29 For adolescent women, smaller studies of attendees at teen clinics and health centers in the United States have found HSV-2 incidence rates ranging from 4.4 to 8.2 per 100 woman-years.11,14,16
The higher incidence of HSV-2 infection observed in the current study than in other studies conducted in STD clinics could reflect the methodological differences in calculating time at risk as well as differences in geographic locations or other characteristics of the populations. For example, the populations studied by Gottlieb et al.13 and Wald et al.12 included fewer black participants (52% and 32%, respectively), and populations of black women have been found to have a higher prevalence of HSV-2. For example, the odds of HSV-2 seropositivity at enrollment in the current study were roughly twice as high among black women as white women (odds ratio, 1.9; 95% CI, 1.2–3.0).19 Also, the women in the Wald et al. study population were older (median age, 27 years) than in the current study (median age, 24 years), which could contribute to the difference in incidence detected. Furthermore, women in the current study could have been exposed to a pool of sex partners with HSV-2 prevalence higher than other populations. This is supported by a study from Sizemore et al.,30 which found a higher prevalence (45%) of antibodies to HSV-2 in men attending the same STD clinic in Birmingham, AL, than that detected, for example, among men in the Gottlieb et al.31 study at baseline (32%).
Correlates of prevalent HSV-2 antibodies at enrollment among female participants in the current study have been described previously.19 Many of the correlates of incident HSV-2 infection in the present analysis were consistent with those observed in the analysis of HSV-2 prevalence (in both magnitude and direction) with the notable exception of age. Older age was associated with less incident HSV-2 infection during follow-up but with more prevalent infection at baseline, which could be an expected pattern given the lack of curative treatment for the infection. Other significant predictors of prevalent infection (black race, young age at first intercourse, number of lifetime partners, HSV-1 infection, and syphilis) might not have achieved significance in the current analysis of incident HSV-2 infection because of the small sample and the small absolute number of HSV-2 events.
Although recent research suggests that prevalent HSV-2 infection could have led to BV among female sex workers in Bukina Faso,32 the previous report based on this study population did not find an association between prevalent HSV-2 infection and the occurrence of BV.19 However, a diagnosis of prevalent or incident BV was associated with incident HSV-2 in the current study. This finding is consistent with other research showing an association between BV and both incident HSV-233 and increased genital shedding of HSV-2.34 Several mechanisms by which BV could lead to the acquisition of HSV-2 have been proposed.35 First, BV is characterized by an elevated pH level, which could facilitate the growth and survival of HSV-2. Another mechanism could be that the reduction in lactobacilli associated with BV decreases the production of antimicrobial substances, such as lactic acid and hydrogen peroxide, which help to regulate a healthy microenvironment. Finally, BV-related, anaerobic Gram-negative rods produce enzymes possibly capable of weakening the epithelium's protective mucus.
Although data are limited, other studies conducted in high-risk populations have yielded mixed results regarding the association between self-reported condom use and risk of incident HSV-2 among women.12,15 A study by Wald et al.15 of HSV-2 discordant couples participating in a vaccine trial found that women reporting in coital diaries condom use for >25% of acts had reduced acquisition of HSV-2 (adjusted HR, 0.09; 95% CI, 0.01–0.67) when they were compared with those reporting condom use in less than 25% of acts. Another study of STD clinic patients found no association between increasing levels of condom use (0%–25%, 25%–75%, and >75% of acts) and rates of HSV-2 infection in women, although such an association was reported for men.12 In the current study, we also did not demonstrate a significant protective effect of self-reported condom use against incident HSV-2 acquisition among female STD clinic patients. Of note was that 5 of the 24 incident HSV-2 infections that were detected were diagnosed during intervals in which study participants reported using condoms consistently, without apparent overreporting of condom use or reports of breakage or slippage.
In contrast to previous studies,12,15 we were able to more fully assess the condom use status of participants in this evaluation by measuring both consistent (100%) use as well as the occurrence of some, although not all, problems with condom use (such as breakage and slippage), the importance of which has been emphasized by others.36–38 Although measures of condom use are based on self-report and thus subject to error, we found good agreement between diaries, interviews, and counts of condom products at follow-up visits, suggesting that the study methods successfully minimized participant bias in the reporting of condom use. However, even low levels of misreported coital and condom use data could mask a protective effect of condoms, if present.39 Furthermore, we were not able to measure the infectiousness of sex partners at the time of intercourse or even their infection status, the latter having proven important for demonstrating the effectiveness of condoms against a variety of STIs, including HIV, gonorrhea, C. trachomatis, and HSV-2.15,36,40–43 Confounding from differences between condom users and nonusers in exposure to infected partners has been shown in STD clinic populations to underestimate the effectiveness of condoms and obscure any relationship between condom use and acquisition of an STI. Even when the infection status of partners cannot be directly measured, differences in exposure between users and nonusers can be minimized using special techniques such as the case-crossover design in which analyses focus only on those persons known to have infection.44 This case-crossover approach has been applied previously to data from this study to demonstrate that unmeasured confounding in cohort analyses results in underestimation of the effectiveness of condoms against incident gonorrhea and chlamydia.45 Because data were too sparse in this evaluation to apply the case-crossover design, we were unable to conduct a similar comparison of the case-crossover and cohort study designs. The possibility thus remains that the time-to-event approach presented here may have masked any protective effect of condom use against HSV-2 despite our attempts to control confounding in the analysis. Finally, because transmission of HSV-2 involves genital areas that may or may not be covered by condoms, we cannot rule out that condoms might not protect women against this pathogen as well as they protect women against other STIs that are transmitted to and from the male urethra.46 Given the high prevalence of genital herpes among women in the United States, additional research is warranted to better determine the level of protection provided by condom use.
Although the presence of antibodies to either HSV-2 or HSV-1 theoretically could provide cross-protection against infection with the other HSV type, empirical evidence regarding these relationships has been mixed.47 We found no evidence that antibodies to HSV-1 protect against the acquisition of HSV-2.
The current study was limited in that the serological assays cannot distinguish between genital and nongenital HSV-1 infection. Although genital herpes usually is associated with HSV-2, in some settings the disease increasingly is attributed to HSV-1.1,48–53 Consequently, the incidence rates of HSV-2 demonstrated in our study possibly underestimate the overall incidence of genital HSV in this population. A second limitation is that any mistiming in our estimates of the onset of infection could have led to bias in assessing the role of time-dependent risk factors and incident infection. Finally, as previously noted, the small study size and the infrequent occurrence of several covariates (e.g., sex for material compensation and syphilis) resulted in limited power to detect significant associations.
The limitations cited above are offset by several important strengths. First, the frequent and systematic collection of serum samples allowed us to construct refined risk intervals for estimating the onset of incident infection. Second, we used a type-specific HSV serologic assay with high sensitivity and specificity.25,26 Third, the completion of sexual diaries allowed us to prospectively collect detailed, time-varying data on sexual activity and condom use. Finally, our decision to count condom products permitted an independent assessment of the reliability of the frequency of condom use reported in the sexual diaries. To our knowledge, this is the first study to use this methodology.
Most HSV infections are asymptomatic or unrecognized, and yet all infected persons shed virus intermittently in the genital tract, and most infections are transmitted by persons who are unaware that they are infected or who are asymptomatic when transmission occurs.54 Screening using new type-specific HSV serologic tests has been proposed as a potential control strategy, and the CDC's STD Treatment Guidelines specify subgroups for whom serologic testing may be useful.46 Promising areas include ongoing HSV vaccine trials for the prevention of HSV-1 and HSV-2 infection and suppressive therapy among HSV-infected persons to prevent acquisition of HIV infection.1,5,6
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