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Risk Factors for Infection With Herpes Simplex Virus Type 2:

Role of Smoking, Douching, Uncircumcised Males, and Vaginal Flora

CHERPES, THOMAS L. MD*†; MEYN, LESLIE A. MS; KROHN, MARIJANE A. PhD; HILLIER, SHARON L. PhD†‡

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Sexually Transmitted Diseases: May 2003 - Volume 30 - Issue 5 - p 405-410
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GENITAL HERPES is one of the most prevalent sexually transmitted diseases worldwide. 1 It is more frequently caused by herpes simplex virus type 2 (HSV-2), but HSV-1 can also produce the disease. 2 Genital herpes infections typically occur when the virus is inoculated onto moist epithelium during intimate sexual contact. Although genital herpes infections are rarely fatal, they are a significant public health concern. 3 Primary acquisition can cause painful genital ulceration and systemic manifestations that include headache and fever. Infection is considered life-long, as the virus becomes latent in sacral nerve ganglia, and may result in recurrent genital lesions as well as substantial psychological distress. 4 Intrapartum transmission of HSV can cause neonatal death or permanent neurologic damage. 5 There is also evidence that HSV infection facilitates sexual transmission and acquisition of HIV. 6,7

The development of serologic methods that can accurately diagnosis HSV-1 and HSV-2 infections has enhanced the understanding of genital herpes epidemiology. An ongoing United States population–based study, the National Health and Nutrition Examination Survey (NHANES), documented an increase of more than 30% in the seroprevalence of HSV-2 from the late 1970s through the early 1990s. 8 Demographic characteristics associated with HSV-2 infection include female sex, black race, older age, lower income, and less education. 8–10 In the national serosurvey, women were found to be about 45% more likely than men to be infected with HSV-2. 8

It has been postulated that the increased seroprevalence of HSV-2 in women is the result of more efficient male-to-female transmission of virus, anatomical differences in susceptibility to infection, or the tendency of women to choose sex partners who are older than themselves. 11,12 Identified behavioral risk factors in women include lifetime number of sex partners, years of sexual activity, younger age of sexual debut, and a history of another sexually transmitted disease. 13–15

It is of concern that the promotion of safe sex practices in many countries has not slowed the promulgation of the genital herpes epidemic. Particularly alarming is the shift toward acquisition of HSV-2 infection earlier in life. 8 It is crucial to identify new factors associated with HSV-2 that might represent opportunities to intervene with either primary or secondary prevention maneuvers. The purpose of this investigation was to examine the demographic and behavioral characteristics associated with HSV-2 seroprevalence in young, nonpregnant women.

Methods

Study Population

Participants were recruited from three Pittsburgh-area health clinics (the University of Pittsburgh Student Health Clinic, the Allegheny County Health Department Clinic, and the Family Health Council Clinic of Aliquippa) for an investigation of risk factors associated with vaginal acquisition of group B Streptococcus (GBS). Women between the ages of 18 and 30 years who sought medical care that required a gynecologic examination were eligible for participation. Exclusion criteria included known pregnancy, current vaginal bleeding, or the recent (prior 24 hours) use of any intravaginal products. Approval for the subsequent use of data collected from the GBS study was obtained from the Institutional Review Board of the Magee–Womens Hospital of Pittsburgh.

Over a 2-year period between 1998 and 2000, 1248 women were enrolled. After informed consent was obtained, demographic and behavioral interview data were obtained via a standardized questionnaire administered by trained research personnel. Serum samples were collected from 1207 of the participants and linked to the questionnaire by a numerical code. Sera were frozen and stored at −70°C until testing was performed. Rectal and vaginal swab specimens for bacterial culture and a vaginal smear specimen were also obtained from each woman at enrollment.

Laboratory Methods

Bacterial vaginosis (BV) is characterized by the replacement of the normally predominant lactobacilli with anaerobic bacteria, genital mycoplasmas, and Gardnerella vaginalis, and the vaginal smears were gram-stained for evaluation of the flora. On the basis of standardized criteria, a normal vaginal flora received a score of 0 to 3; intermediate flora, 4 to 6; and BV, 7 to 10. 16 A score of ≥4 was used to define abnormal vaginal flora. Sera were tested for type-specific antibodies to HSV-1 and HSV-2 with purchased enzyme-linked immunoassays (ELISAs; Focus Technologies, Cypress, CA). Both assays use baculovirus recombinant glycoprotein G (gG) constructs, are FDA approved for testing among sexually active adults, and were used according to the manufacturer’s instructions. The methods used for the culture and identification of GBS and yeast from the vaginal and rectal swabs and of Lactobacillus from the vaginal swabs have been previously described. 17 For a subset of women (n = 308), the results from clinical laboratory testing for Chlamydia trachomatis and Neisseria gonorrhoeae were available.

Statistical Analysis

The enrollment data were analyzed with SPSS statistical software, release 10.1.4 (SPSS, Inc., Chicago, IL). Univariate associations of baseline characteristics with HSV-2 seroprevalence were tested with Pearson’s chi-square test, a chi-square test for linear trend, or Fisher exact test, where appropriate. All statistical tests were evaluated at the 0.05 level of significance. Variables with P values less than 0.1 were considered for inclusion in a logistic regression model. Models were developed with use of forward stepwise regression based on the likelihood ratio test statistic. Variables were retained in the model if the Wald chi-square test statistic had a P value of 0.05 or less.

Results

Of the 1207 women analyzed, 747 (61.9%) described themselves as white, 412 (34.1%) as black, and 48 (4.0%) as Hispanic, Asian, Native American, or multiethnic. The latter 48 women were combined into one racial category, referred to as “other” for analysis. The HSV-2 seroprevalence among all participating women was 24.9%, a similar value to that obtained for women in the most recent NHANES survey. 8

By univariate analysis, HSV-2 seroprevalence was associated with a number of demographic variables, including enrollment from the county health department or family health council clinics, black race, older age, and less education (Table 1). About behavioral risks, HSV-2 seroprevalence was higher among those women who reported a greater lifetime number of sex partners, greater parity, a history of intercourse with an uncircumcised partner, more frequent intercourse, a history of vaginal intercourse after anal intercourse, and douching (Table 1).

TABLE 1
TABLE 1:
Prevalence of HSV-2 Antibodies, by Selected Demographic and Behavioral Characteristics, in Women Aged 18 to 30 Years From Three Pittsburgh Recruitment Sites (N = 1207)

There were no statistically significant differences in HSV-2 seroprevalence among women who drank alcohol or smoked marijuana in the 4 months before testing versus those who did not, but HSV-2 seroprevalence was higher among those who had smoked tobacco in the preceding 4 months. Women with antibody to HSV-1 had a higher HSV-2 seroprevalence at enrollment than did those who were HSV-1 antibody–negative.

Univariate analysis of the self-reported histories of reproductive tract infections showed HSV-2 seroprevalence was higher among those who reported a history of a yeast infection, BV, trichomoniasis, chlamydial infection, gonorrhea, syphilis, or pelvic inflammatory disease (Table 2). The seroprevalence of HSV-2 among women who reported a history of genital herpes was 81.1%, whereas that among women who did not report such a history was 22.4%. Seven of the 10 women without antibody to HSV-2 who reported a history of genital herpes had antibody to HSV-1, while the other three women had antibody to neither virus. The sensitivity of a self-reported history of genital herpes for the presence of HSV-2 antibody was 14.3% overall and did not differ significantly between white and black women (12.9% versus 14.4%).

TABLE 2
TABLE 2:
Prevalence of HSV-2 Antibody, by Reported History of Reproductive Tract Infections

The frequency of HSV-2 infection was lowest among women with normal vaginal flora (14.5%) and was significantly higher among women with BV (41.8%) (Table 3). Similarly, HSV-2 seroprevalence was lowest among women with vaginal Lactobacillus that produced hydrogen peroxide (17.8%), intermediate among women colonized by Lactobacillus that did not produce hydrogen peroxide (33.7%), and highest among women with no vaginal Lactobacillus (38.5%). By univariate analysis, HSV-2 seroprevalence was also found to be associated with vaginal GBS.

TABLE 3
TABLE 3:
Vaginal Microbiology of Women Seropositive for HSV-2 Antibody

Because increased sexual activity is associated with HSV-2 and altered vaginal flora, additional analyses were performed to uncover possible confounding of the association between HSV-2 seroprevalence and abnormal vaginal flora by the reported number of sex partners in the previous year. The frequency of BV was higher in individuals with more sex partners; bacterial vaginosis was detected in 16.0% of women reporting no partners in the previous year, 28.3% of women reporting one partner, and 35.4% of women reporting more than one partner (P < 0.001). HSV-2 seroprevalence was also higher among women reporting sexual activity in the previous year. However, at each level of sexual activity, for women with at least one sex partner in the previous year, a higher proportion of women with vaginal flora abnormalities than women with normal vaginal flora were HSV-2-seropositive (Fig. 1).

Fig. 1
Fig. 1:
HSV-2 antibody prevalence, as related to vaginal flora morphology and number of sex partners in the previous year.

To determine the variables independently associated with the presence of antibody to HSV-2, multivariable logistic regression analyses were performed. Originally we did not include variables for N gonorrhoeae or C trachomatis because of the limited number of women for which results were available. The independent predictors of HSV-2 seropositivity included black race, older age, cigarette smoking, douching, a greater lifetime number of sex partners, a history of sexual intercourse with an uncircumcised partner, the presence of vaginal GBS, and altered vaginal flora (Table 4). When only the women for whom C trachomatis and N gonorrhoeae testing results were available were considered, altered vaginal flora remained a statistically significant independent predictor of HSV-2 infection. The adjusted odds ratio for HSV-2 infection associated with altered vaginal flora in this subset of women was 2.2 (1.2–4.0), and it was 1.9 (1.0–3.7) for those with BV.

TABLE 4
TABLE 4:
Summary of Multivariable Logistic Regression Analyses of Factors Associated with HSV-2 Antibody Prevalence

Discussion

Our investigation of the prevalence of genital herpes in young, nonpregnant women detected previously unidentified associations between HSV-2 infection and vaginal colonization with GBS, history of intercourse with an uncircumcised partner, and abnormal vaginal flora. It is also among a limited number of studies that uncovered associations between HSV-2 infection and douching or smoking. The cross-sectional design of our study makes it impossible to determine whether these variables are similarly associated with disease acquisition; longitudinal studies will be necessary to clarify these relationships. Generally, an increased risk for HSV-2 infection is correlated with markers of increased sexual exposure. These markers, seen in our study as well, include a higher lifetime number of sex partners, increased frequency of intercourse, increased age, increased parity, and a history of other sexually transmitted diseases. 15,18

The small number of women aware of their HSV-2 infection was not unexpected. Although nearly 25% of adults in the United States are thought to have genital herpes, less than 20% are cognizant of their infection. 19 Reasons for this lack of awareness include symptoms of disease that are frequently mild, 20 the attenuation of symptoms by a prior HSV-1 infection, 21 and the infrequency with which clinical screening (history and physical examination) can detect infection. 22 The low sensitivity of a self-reported history of genital herpes in our investigation was similar to that reported in the NHANES, but our findings diverged when stratified by race. While we found that sensitivity did not differ significantly between white and black women, the national survey found awareness of infection to be markedly higher among whites than among blacks. 8 Although the reasons for the dissimilar results are not clear, it is possible that in the Pittsburgh area there are fewer differences in barriers to health care and health education information for black versus white women than are observed nationally.

Our findings are consistent with recent publications that suggest antecedent HSV-1 infection does not provide significant protection against the acquisition of HSV-2. 21,23 Several studies published in the 1990s found evidence of a partially protective role of HSV-1. 11,13,24 However, a large, prospective trial, designed to evaluate the efficacy of a HSV-2 glycoprotein-subunit vaccine, showed that previous HSV-1 infection did not reduce the incidence of HSV-2 infection. 21 This has been corroborated by an analysis of the NHANES data that found HSV-2 seroprevalence to be higher among persons with HSV-1 antibody. 25 Similarly, in our study the seroprevalence of HSV-2 among HSV-1-infected women was higher than in those not infected with HSV-1. Since HSV-1 infection is typically acquired earlier in life than HSV-2, if HSV-1 were protective, the seroprevalence of HSV-2 would be expected to be lower among those infected with HSV-1.

Only a limited number of studies have revealed an association between HSV-2 infection and douching. 12 Although cross-sectional data cannot reveal a temporal relationship between douching and acquisition of HSV-2, it is plausible that douching increases the susceptibility to genital herpes infection. Women who douche have also been shown to be at an increased likelihood for chlamydial infection or BV. 26,27 Douching might increase the risk for HSV-2 acquisition, either by altering the normal vaginal flora or destroying the protective mucin layer thereby facilitating exposure to the underlying epithelium to virus. Conversely, it is possible that symptoms, such as dysuria, associated with genital herpes infection increase the likelihood that a woman will douche.

Few epidemiologic investigations have reported a greater prevalence of HSV-2 infection among women cigarette smokers. Among middle-aged Mexican women, those with a reported history of smoking had a 90% greater risk for HSV-2 infection than did those who had never smoked. 12 Tobacco smoking has been found to damage cervical epithelium through DNA modification, and this may represent a mechanism for increased susceptibility to HSV-2 infection. 28

It appears that our study is the first to detect an association between HSV-2 infection and the presence of vaginal GBS. A recent investigation showed that frequent sexual intercourse and an increased number of sex partners were independently associated with the vaginal acquisition of GBS. 17 Therefore, the increased prevalence of vaginal GBS in women seropositive for HSV-2 may represent a marker for increased sexual exposure. It is also possible that a genital herpes infection alters the vaginal flora in a way that is more permissive to the growth of GBS in the vagina or that GBS colonization increases susceptibility to HSV-2 infection.

The association in women between HSV-2 infection and a history of intercourse with an uncircumcised partner is novel. There is good evidence of an association between male circumcision status and HIV infection of women, but the relationship between genital herpes infections and circumcision status has been less explored. 29–31 Although many of the studies involving males have not adequately adjusted for confounding factors, uncircumcised men appear to be at a higher risk for the acquisition of genital herpes. 32,33 Therefore, an increased HSV-2 seroprevalence among uncircumcised males could explain the increased HSV-2 infection we found among the women who had intercourse with an uncircumcised male. It is also possible that there is an enhanced efficiency of transmission of HSV-2 from uncircumcised males to their female partners.

In this study, lack of a Lactobacillus-predominant vaginal flora was identified as a risk factor for HSV-2 infection. In multivariable logistic regression analysis, altered vaginal flora remained an independent predictor of HSV-2 seropositivity. Furthermore, the association was not confounded by concurrent infection with N gonorrhoeae or C trachomatis. These results are consistent with those of previous longitudinal and cohort studies that have examined the relationship between bacterial vaginosis and HIV. 34–36

Either the loss of the viricidal effects of Lactobacillus or the presence of abnormal vaginal flora may increase susceptibility to HSV-2 infection. In a healthy vagina, lactobacilli produce antimicrobial substances, such as lactic acid and hydrogen peroxide, which help control the microenvironment and inhibit the overgrowth of potentially pathogenic organisms. In BV, the Lactobacillus-predominant vaginal flora is lost. 37 Hydrogen peroxide and lactic acid, produced by the lactobacilli, are also lost, and host defenses capable of virus inactivation are weakened. 38,39 Furthermore, the anaerobic gram-negative rods associated with BV have been shown to produce mucinases, sialidases, and other enzymes that can degrade the protective mucus layer and facilitate contact and attachment of virus to epithelium. 40

It will be important to ascertain if BV increases the risk of acquisition of HSV-2. The direct morbidity associated with the genital herpes epidemic continues worldwide, and infection also promotes both the transmission and acquisition of HIV. BV is the most prevalent vaginal infection, and treatment of BV is both inexpensive and efficacious. Therefore, in the absence of vaccines that effectively prevent the acquisition of HIV or HSV-2, treatment of BV may represent a cost-effective means of slowing the transmission of these viruses. Although this cross-sectional study cannot show whether the variables found independently associated with HSV-2 increase susceptibility to infection in women, smoking, douching, lack of male circumcision, and the presence of BV may represent alterable risk factors of disease.

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