Bacterial vaginosis (BV), the main cause of abnormal vaginal discharge, is one of the most common and recurrent lower genital tract infections in women of reproductive age. BV is a sexually associated condition that is common in the general population affecting from 20% to as many as 50% of reproductive-aged women and varies dramatically by race/ethnicity.1 It is characterized by the replacement of lactobacilli, the predominant vaginal flora, with an overgrowth of Gardnerella vaginalis, Mycoplasma organisms, and other anaerobic bacteria. In women without BV hydrogen peroxide producing lactobacilli dominate vaginal flora.2 These lactobacilli are thought to protect against pathogens through the maintenance of a low pH.3 The microorganisms responsible for BV increase mucin-degrading enzymes, which may play a role in the degradation of the gel layer that coats the vaginal and cervical epithelium and endocervical mucus.4–7 BV-associated microorganisms also produce cytokines and inflammatory mediators,8–10 which have been linked to pregnancy complications11 and may increase susceptibility to sexually transmitted infections (STIs). Specifically, in clinical studies BV has been found to be associated with genital tract infections, such as pelvic inflammatory disease,12,13 and the prevalence and incidence of multiple STDs including chlamydia, gonorrhea, herpes, HIV, and trichomoniasis.14–20
Although many studies have attempted to assess the association between BV and HIV, there is little information describing the association between BV and other viral STIs among women in the general population. In this study we describe the prevalence of five viral STIs [human papilloma virus (HPV), HIV, herpes simplex viruses Types 1 and 2 (HSV-1 and HSV-2), and Hepatitis C (HCV)] among women with and without BV between the ages of 20 and 49 years who reside in the United States using the population-based National Health and Nutrition Examination Survey (NHANES). We also examined the association between BV and individual viral STIs.
Design and Study Population
We used data from NHANES samples for the combined intervals 2001–2002 and 2003–2004 to estimate the prevalence of BV among women in the civilian, noninstitutionalized US population. NHANES, conducted by the National Center for Health Statistics at the Centers for Disease Control and Prevention, was designed to obtain nationally representative information on the health and nutritional status of the population of the United States through interviews and direct physical examinations. Methods describing this national survey have been published elsewhere.21 This study submitted to the Washington University School of Medicine Human Subjects Committee for approval was classified as exempted because it is a population-based study devoid of individual identifiers.
For these analyses, women between the ages of 20 and 49 with complete information regarding BV status were used for evaluation. The final sample included data from 2326 women, who when weighted, represent the experience of 55,840,143 noninstitutionalized women residing in the United States.
Female participants in the NHANES study between the ages of 14 and 49 years were tested for BV. Self-collected vaginal swabs were used for the evaluation of BV. Smears were allowed to air dry before shipment to the processing and analysis laboratory at Magee-Women’s Hospital (Pittsburgh, PA). BV score for Gram staining was calculated by Nugent’s method.22 Scores of 7 or higher were considered positive for bacterial vaginosis, whereas those between 4 and 6 were considered intermediate. BV scores from self-collected swabs have been found to be reliable compared with provider collected samples.23–25 Additional details on laboratory procedures have been published elsewhere.26–30 Prevalence was estimated for the 3 levels of BV: positive, negative, and intermediate. For logistic regression analyses, the outcome was defined as BV confirmed (positive) or not (negative and intermediate).
Self-collected vaginal swabs were evaluated for the presence of HPV. The HPV polymerase chain reaction assay used HPV L1 consensus PCR with biotinylated PGMY09/11 primer sets and β-globin as an internal control for sample amplification. The primer mix amplifies essentially all HPV types found in the genital tract. The amplicons were evaluated by gel electrophoresis for the presence of the 450 bp HPV amplicon. Positive samples were typed by hybridization to the Roche protype line probe typing strips followed by colorimetric detection. The strip was a linear array of probes specific for 37 HPV types (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39, and 89) and for the positive β-globin control as well. Types were read by comparing the reaction pattern to the typing template.28,31,32 In addition to examining any HPV in our analyses we also examined common subtypes previously associated with genital warts (6 and 11) and cervical cancer (16 and 18).
Specimens were tested for HIV-1 and HIV-2 antibodies using the synthetic peptide enzyme immunoassay (EIA; Genetic Systems HIV-1/HIV-2 Peptide EIA). Any specimens that reacted in the initial test were duplicated. Specimens that were reactive in both tests were tested with the Cambridge Biotech HIV-1 Western Blot Kit (Calypte Biomedical Corporation, Rockville, MD). If the EIA was negative and the Western Blot positive, the specimen was coded as positive. If the EIA was positive and the Western Blot negative, the specimen was coded as negative. If the EIA was positive or indeterminate and the Western Blot was indeterminate, the specimen was coded as indeterminate.29
HSV-1 and HSV-2.
Viral glycoproteins specific for HSV-2 (designated gG-2) and HSV-1 (designated gG-1) have been identified. Monoclonal antibodies and affinity chromatography were used to purify these glycoproteins and thus provide antigens for type-specific herpes serologic assays. Solid-phase enzymatic immunodot assays were used to detect antibodies reactive to these antigens,33 which has demonstrated good sensitivity and discriminates HSV-1 and HSV-2.34 Serum samples testing positive for HSV-2 in the immunodot assay were further confirmed by a gG-2 monoclonal antibody inhibition assay.35
Human antibodies directed against hepatitis C virus (anti-HCV) in human serum or plasma were measured using direct solid-phase enzyme immunoassay with the anti-HCV screening ELISA. Results were expressed as “positive,” “negative,” or indeterminate for anti-HCV. Positive specimens are repeated in duplicate according to the same procedure. Repeatedly positive specimens were tested supplementally using the Chiron RIBA Processor System (Chiron Corporation, Inc.). Samples where the RIBA result was positive were reported as confirmed positive for antibody to HCV. Samples where the recombinant immunoblot assay result was negative or indeterminate were reported as negative or indeterminate, respectively.30
Self-reported sociodemographic characteristics included in these analyses included age, race/ethnicity, highest level of education received, and poverty/income ratio. Age was categorized by decade (20–29, 30–39, and 40–49). Race/ethnicity was categorized as white non-Hispanic; black non-Hispanic, Mexican American; or other race/other Hispanic. Three levels of education were evaluated: less than High School, completed High School (or GED), or more than High School. Poverty/income ratio (PIR) was the ratio of the individual’s family household income to the federal poverty level and was categorized as less than the federal poverty level (PIR <1), at or above the federal poverty level (PIR 1 to <2), or twice the federal poverty level (PIR ≥2).
Reproductive and Sexual History
In this analysis, we examined a number of reproductive history variables. Age at first menstruation was categorized as 7 to 11 years, 12 to 14 years, 15 years or later, or unknown. Douching within the past 6 months was defined as yes, no, or unknown.
Women between the ages of 14 and 49 also completed a sexual history questionnaire that included questions on history of sexual intercourse, and number of lifetime and last year sexual partners. However, because of confidentiality concerns, sexual history data for individuals between the ages of 14 and 19 years are only available at the National Center for Health Statistics Research Data Center. Therefore, all analyses were limited to women between the ages of 20 and 49 years.
Categorical data were compared using χ2 tests. Crude and adjusted odds relative risks were estimated using logistic regression for rare STIs (HPV subtypes, HIV and HCV) and Poisson regression for common STIs (HPV, HSV-1, and HSV-2).36 All analyses adjusted for the complex sampling design of NHANES. Specifically, using the svyset command in Stata we specified the individual weight, primary sampling unit, and stratum. The 2-year individual weights estimated by the National Center of Health Statistics and made available as part of the NHANES dataset are adjusted to the entire US population based on 2000 Census information. To accommodate the joining of 2001–2002 and 2003–2004 datasets—each adjusted to the US population—the weight for each individual was divided by 2 to provide a single estimate for the entire US population. Statistical analyses were conducted using Stata (version 9.2).37
One third of the women in this sample were positive for BV at the time of their NHANES examination. BV was significantly more common among black and Mexican American women compared with white women (53.7% and 31.9% vs. 24.3%, respectively), among women living below or at the federal poverty level compared with those living at greater than two times the poverty level (36.1% and 29.7% vs. 24.4%, respectively), and slightly more common among women with earlier ages at first intercourse. The number of lifetime sexual partners was associated with BV prevalence and ranged from a low of 15.6% among women with no reported sexual partners to a high of 35.5% among those with 11 or more lifetime sexual partners. BV prevalence was also associated with number of partners in the past year and recent douching (see Table 1).
In bivariate analyses BV was strongly associated with all 5 viral STIs examined, including any HPV, HIV, HSV-1, HSV-2, and HCV (see Table 2). BV was significantly associated with 2 oncogenic subtypes (HPV 16 and HPV 18) and positively correlated, but not significantly so, for 2 common subtypes associated with genital warts (HPV 6 and HPV 11). For all STIs, other than HPV (HPV 16, HPV 18), the prevalence was highest among those positive for BV. The relationship for HPV (HPV 6, HPV 11) and HCV increased with Nugent category, whereas for HIV, HSV-1, and HSV-2 the prevalence was similar for those in the intermediate and negative categories and highest among those positive for BV.
We examined the relative risk of each viral STI adjusting for potential confounders, including sociodemographic characteristics, sexual behavior, and douching practice (see Table 3). In crude analyses, BV was associated with increased risk of HIV, HSV-1, and HSV-2. After adjustment for race/ethnicity, number of sexual partners in the past year, and douching in the past month, only HSV-1 and HSV-2 were significantly associated with the presence of BV. There were positive, yet nonsignificant associations between BV and HPV (subtypes 6 and 11) HIV, and HCV. Adjusting for lifetime number of sexual partners did not alter the findings (data not shown).
Finally, we also stratified by race/ethnicity to evaluate for the presence of effect modification given the documented association with BV and STIs.38 For these analyses, we collapsed intermediate and negative Gram stain scores into a non-BV category. In the combined univariate analyses, BV was systematically associated with viral STIs; however, this association may be modified by race/ethnicity (data not shown). In the stratified analyses, there was little absolute difference in the prevalence of HPV by BV status among white women (27.1% vs. 26.8%, P = 0.07), yet there were substantial, yet nonsignificant differences among black (30.3% vs. 44.0%, P = 0.18) and Mexican American women (19.8% vs. 32.3%, P = 0.12). HSV-2 was more common among women with BV for both white (18.3% vs. 28.6%, P = 0.01) and black (53.3% vs. 66.0%, P = 0.02) women, but this association was not found among Mexican American women (17.5% vs. 18.7%, P = 0.86). Similarly, HSV-1 and HCV were more common among white and black women with BV, but slightly less common in Mexican American women with BV.
In this nationally representative sample, we found an association between HSV-1 and HSV-2 with the presence of BV. The risk of concurrent HSV-2 infection was 32% higher and the risk of concurrent HSV-1 was 8% higher among women with BV than among women without BV after adjusting for race/ethnicity, sexual partners in the past year, and douching in the past month. Whether this association applies across categories of race/ethnicity is not clear. Our crude analyses found the prevalence of HSV-2 was associated with BV only among white, non-Hispanic, and black non-Hispanic women. Among Mexican American women the prevalence was approximately 18% in both the BV categories.
The relationship between BV and HSV-2 may be bidirectional. A recent clinic study found that current, but not past, BV was found to predict disease HSV-2 seropositivity.20 The presence of BV has also been linked to increased frequency of genital tract shedding of HSV-2 and its reactivation from latency.39 Conversely, HSV-2 seropositivity has been found to be associated with the incidence of BV,40,41 and BV has been found to increase acquisition of HSV-2 in comparison with those with normal vaginal flora.42
BV was associated with a modest, but statistically significant, increase in the risk for HSV-1. Given that HSV-1 was determined through serologic testing that does not allow for the isolation of infection site or transmission pathway, the current estimate is likely an underestimate of the true association. Although HSV-1 has been traditionally considered to result from nonsexual transmission, recent studies have found that an increasing proportion of genital herpes infections, particularly among adolescents and those in industrialized countries, are attributable to HSV-1.43–47 Roberts et al.43 examined the incidence of genital herpes attributable to HSV-1 among college students and detected a significant shift from 31% of infections in 1993 to 78% in 2001. Similarly, a study from an academic medical center that included participants of all ages reported that the proportion of genital herpes infections attributable to HSV-1 increased from 24% to 45% from 1994 to 1999.45 As with HSV-2, there is some evidence that HSV-1 seropositivity may also increase the acquisition of BV.41 A longitudinal study by Cherpes et al. examined whether women with serum antibodies for HSV-1 were more likely to develop BV and found a modest association in crude analyses, but no effect after adjusting for potential confounders.
We found that BV was associated with a 5-fold higher risk of HIV among women with BV; however, these findings were attenuated significantly after adjusting for race/ethnicity, recent sexual partners, and douching in the past month (RR = 2.10, 95% CI, 0.48–9.15). Previous studies have correlated the presence of BV with increased HIV infection and shedding of HIV,18,48–50 but it is possible that the lack of significant finding in this sample is the result of inadequate sample size resulting from the low prevalence of HIV. When additional waves of NHANES data become available this finding can be confirmed.
This study did not confirm other studies that have found a correlation between BV and increased risk of HPV infection. A recent study found that BV is associated with an increased risk of prevalent and incident HPV infection, but not with duration or development of squamous intraepithelial lesion even when adjusted for lifetime and current partners.51 Another study performed a time lag analysis of HPV DNA detection and clinical diagnosis of BV and found that HPV DNA detection usually preceded the presence of BV.52 Although we did not find a significant association for either any HPV type or HPV (HPV 6, HPV 11) the adjusted relative risk for HPV (HPV 16, HPV 18) was approximately 2.7, suggesting a potentially important risk, which should be investigated further. Whether this lack of statistical association is due to inadequate sample size or some other influence, such as spontaneous clearance of HPV infection,53 is not known.
There are a number of strengths of the current analysis. Clinical studies of BV and STIs are limited in generalizability to comparable patient populations. This nationally representative sample of noninstitutionalized women of reproductive age, however, is generalizable to women in the general population. Moreover, the NHANES sample is not subject to many of the selection forces that limit participation in clinical studies including ability to pay for health care and proximity to health care facilities. Finally, the measurement of BV and viral STIs use objective biologic specimens with well regarded testing procedures.
There are a number of limitations that should be considered when evaluating these findings. First, the viral STIs studied here are not currently curable and may persist in the body for long periods. Second, for HSV-1, HSV-2, and HCV there are sexual and nonsexual modes of transmission, and for HSV-1 and HSV-2 serologic results also reflect nongenital infections. Collapsing sexual and nonsexual modes of transmission and genital and nongenital infections may have led us to underestimate the association between BV and sexually transmitted genital HSV. Moreover, the use of NHANES data limited the information about confounding variables, and the sample size limited the analyses that could be conducted including multivariable models stratified by race/ethnicity potentially resulting in incomplete confounding, especially by sexual activity. Finally, this is a cross-sectional study and does not imply causality or ensure temporality. Moreover, it is plausible that BV increases risk of viral infections and that viral infections may increase the risk of BV40,41; therefore, it is not possible in a cross-sectional study to disentangle or quantify the possible bidirectional relationship. Additional prospective research is needed to estimate the relative contribution of BV to viral STIs.
In this study, we found a statistically significant association between HSV-1, HSV-2 and BV among women in the general population. Whether this finding can be extended to all race/ethnicity groups is not known, but should be explored in future studies. Given that BV may be an important risk factor for STIs, additional research exploring whether these findings are a reflection of differences in biologic susceptibility, differences in risk behaviors, or a consequence of preceding STI needs to be elucidated. Additional prospective research is needed to estimate the relative contribution of BV to the incidence of viral STIs.
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