THE CLINICAL IMPORTANCE OF BACTERIAL vaginosis (BV), an important cause of vaginal infection, is the putative association with many adverse obstetrical and gynecological outcomes, including premature rupture of the membranes, chorioamnionitis, prematurity, low birth weight, pelvic inflammatory disease, and HIV acquisition.1,2 Current evidence also suggests that BV, which is a likely risk factor for HIV infection, may be a mediator of the association between intravaginal practices and HIV.3 Knowledge of the risk factors for BV is critical for its prevention and management. However, previous studies of BV risk factors have reported conflicting results.3–5 Two behavioral factors often reported to be associated with BV are intravaginal practices and sexual behaviors.4,6,7 Most previously published studies have been cross-sectional, limiting conclusions about the temporal sequence of events, particularly about the role of intravaginal practices in the development of BV.6,8–10 Furthermore, previous studies have often been conducted in high-risk populations or populations in which intravaginal practices are common and well documented.11–14 This study was a longitudinal investigation of the association between sexual behaviors and intravaginal practices and incident BV among low-risk young Thai women.
Study participants were young (18–35 years) Thai women who sought family planning services in a prospective cohort study, “Hormonal Contraception and the Risk of HIV Acquisition (HC-HIV Study),” from November 1999 to September 2002. The study protocol was approved by the institutional review board responsible for each study site. All women provided written informed consent before study participation. Inclusion criteria included ages 18 to 35 years; use of low dose combined oral contraception (COC) or depo medroxyprogesterone acetate (DMPA) or no hormonal methods for at least 3 months; HIV negative; sexually active (at least 3 coital episodes in the previous 3 months); and at least 4.5 months postpartum, if parous. Women were excluded if they were pregnant or intended to become pregnant during the subsequent 12 months; used an intrauterine contraceptive device in the last month; injected illicit drugs or had a blood transfusion within the previous 3 months; or had a complete hysterectomy. There were seven Thai study sites that included 3 university hospitals and 4 government-affiliated facilities. Sites were located in the northern (Chiang Mai University Hospital and Ministry of Public Health's Health Promotion Center 10), northeastern (Khon Kaen University Hospital and Health Promotion Center 6), central (Rajavithi Hospital in Bangkok), and southern regions (Prince of Songkla University Hospital and Hat Yai Provincial Hospital).
Participants were followed at 3-month intervals for 15 to 24 months. A standardized interview was conducted by a trained nurse at screening, baseline, and follow-up regarding demographic variables, reproductive health history, medical history, current and previous contraceptive history, sexual behavioral variables, STI history (during the previous 3 months at follow-up visits), intravaginal, and other health practices (cigarette smoking, alcohol use, illegal drug use). HIV risk reduction (including condom) counseling, pre- and post-HIV test counseling, and contraceptive counseling and provision were provided at each study visit.
At each visit, participants received a standardized gynecologic examination, including evaluation of vaginal secretions (color, amount, character, consistency); measurement of vaginal pH using indicator strips (Macherey-Nagel, Art-NR.92130); testing of vaginal secretions by wet mount with normal saline for Trichomonas, clue cells, 10% KOH for Candida, and amine odor (whiff test); and endocervical testing for Neurospora gonorrhea and Chlamydia trachomatis DNA by PCR (Roche Amplicor). In this study, BV was diagnosed using Amsel criteria,15 based on the presence of at least 3 of 4 clinical findings, including 1) homogeneous vaginal discharge; 2) vaginal pH of 4.5 or greater; 3) the presence of clue cells; and 4) fishy odor after addition of 10% potassium hydroxide (KOH) to vaginal secretions (amine test). Standard treatment for bacterial vaginosis, trichomoniasis, candidiasis, gonorrhea, and chlamydia was administered without tests of cure. Treatment for BV included oral metronidazole 400 mg twice a day for 7 days for nonpregnant women; and metronidazole (5 g) intravaginally twice a day for 7 days for pregnant women.
BV incidence was calculated by dividing the sum of all new episodes of BV by the total follow-up time for all study participants. Women were right-censored from the analysis after being diagnosed with BV. Covariates considered for inclusion in multivariable models included sociodemographic variables (geographic region, age, years of education, marital status, and occupation); contraceptive exposure (COC, DMPA, male condoms, and female tubal resection); participant sexual risks (sex during menstruation, sex while under the influence of alcohol, and having a new sex partner); male partner sexual behavior (circumcision status and reports of the male partner having sex with another woman); other health practices (smoking and alcohol use); and other reproductive tract infections (candidiasis, trichomoniasis, gonorrhea, and chlamydia). Variables related to intravaginal practices included drying or tightening the vagina for sex, cleaning inside of the vagina, and materials and methods used to clean inside the vagina. All data were reported for the 3 months before each study visit, with the exception of sociodemographic data (reported at baseline), and concomitant STIs, which were detected at the same visit as the diagnosis of BV.
Cox proportional hazards regression was used to model time to incident BV.16 We used washout periods of 3 months for COCs and 6 months for DMPA for estimating hormonal contraceptive exposure when switching from a hormonal to a nonhormonal method. Fitting of multivariable models was done by backward selection. Potential confounding variables considered in the multivariable model were variables of interest (sexual behaviors and intravaginal practices, and other variables with P-values of <0.10 in univariate analysis). Because intravaginal practices were of particular interest, it was retained in the final multivariable model.
Of 1576 women recruited for the HC-HIV study, 54 were BV positive at baseline visit and were excluded from this analysis. This yielded an analytic sample of 1522 women. The retention rate of the study cohort was 87.0% throughout the period of follow-up.
At baseline most study participants (68%) were ages 21 to 30 years and about half (55%) had more than 6 years of education (Table 1). The majority of women (89%) reported that they were in a monogamous marriage and slightly more than half (56%) reported unskilled or low-skilled occupations. Contraceptive method use in the previous year included DMPA (42%), COC (40%), and tubal resection (20%). Male condom use in the previous 3 months was 21%. At baseline, 9% of women reported multiple sex partners, 12% reported sex during menstruation, 8% reported a new sex partner, and 7% reported that their male partner had sex with another woman. Nearly half of participants (49%) reported having sex more than 5 times per week. Only 12% of them reported ever having cleaned inside the vagina in the previous 3 months (Table 1).
Prevalence and Types of Intravaginal Practices
The proportion of women who reported using anything to clean the inside of the vagina in the previous 3 months during follow-up ranged from 7.1% to 11.8%, with a median prevalence of 8.0%. Among women who reported cleansing inside the vagina, the majority of them used water (median prevalence 87.0%, range 72.6–89.7%), nearly half (median prevalence 43.0%, range 37.7–47.9%) used soap, and 19.0% (range 14.0–34.6%) used commercially available products. Nearly all of them (98.1%, range 94.3–100%) used their fingers to clean inside the vagina. Only 1.9% (range 0–4.92%) used a douche bag or bulb enema. The median prevalence of practicing dry sex or using an agent to tighten the vagina for sex was only 0.3% (range 0.0–1.5%; n = 25 participants).
Analysis of Incident BV
There were 264 incident cases of BV diagnosed in 2646.9 woman-years of follow-up for a BV incidence rate of 10.0 per 100 woman-years. In univariate analysis, there was no association between age, education, and women's occupation with incident BV. Women residing in the north and in the south were marginally more likely to develop BV (HR, 1.43; 95% CI, 0.94–2.17; HR, 1.43; 95% CI, 0.93–2.22, respectively). Women who were separated, widowed, or divorced were nearly 3 times more likely to develop BV than currently married women (HR, 2.93; 95% CI, 1.70–5.05) (Table 2).
Women's sexual risk behaviors and male sex partners' behaviors in the previous 3 months were significantly associated with incident BV in univariate analysis. This included having engaged in sex work, multiple sex partners, sex during menstruation, sex under the influence of alcohol or drugs, or having new sexual partners. A woman's sex partner having had sex with another woman was also associated with incident BV. Male circumcision status was not associated with incident BV (Table 3).
Women who had trichomoniasis had a very high likelihood of BV acquisition, although the 95% CIs were wide (HR, 15.19; 95% CI, 4.82–47.87). Chlamydial infection was associated with a marginally increased risk of BV (HR, 1.92; 95% CI, 0.98–3.75), but neither candidiasis nor gonococcal infection were associated with incident BV. Among contraceptive methods evaluated, male condom use was the only method found to be associated with incident BV but in an inverse direction: women who reported no condom use had a lower risk of developing BV (HR, 0.68; 95% CI, 0.50–0.93) than women who reported condom use. There was no association between incident BV and the use of COC, or tubal resection. DMPA use was marginally associated with a decreased risk of BV (Table 3).
Among the 7.2 woman-years of follow-up for women who reported having used anything to dry or tighten their vagina for sex, there was no incident BV. Cleaning inside of the vagina was not associated with incident BV in univariate analysis (HR, 1.30; 95% CI, 0.8–2.01). However, other health practices including cigarette smoking (HR, 2.56; 95% CI, 1.61–4.05) and alcohol drinking (HR, 1.39; 95% CI, 1.05–1.85) significantly increased the risk for BV acquisition.
In multivariable analysis, women residing in both the north (HR, 1.76; 95% CI, 1.14–2.71) and the south (HR, 1.55; 95% CI, 1.00–2.41) were more likely to develop BV when compared with women in the central region (Table 4). Women who had sex during menstruation had nearly twice at risk of acquiring BV (adjusted HR, 1.8; 95% CI, 1.11–2.92) as were women who reported that their primary partner had sex with other women (adjusted HR, 2.3; 95% CI, 1.45–3.65). After controlling for other factors, trichomoniasis remained the highest risk factor of BV incidence in this cohort (adjusted HR, 15.68; 95% CI, 4.95–49.68). Among the other health behaviors assessed, cigarette smoking nearly doubled the risk of developing BV (adjusted HR, 1.79; 95% CI, 1.08–2.98). However, intravaginal practices remained unassociated with incident BV in multivariate analysis (adjusted HR, 1.2; 95% CI, 0.74–1.93).
In this study of young adult Thai women, the median prevalence of BV from 3 to 24 months of follow-up was 2.5%. The BV incidence was 10.0 per 100 woman-years. Risk factors for incident BV identified in our study were primarily related to recent sexual behaviors. Our study did not find an association between intravaginal practices (which were not common in this Thai cohort) and incident BV.
The BV prevalence found in our study was quite low relative to previous studies. For example, BV prevalence in African countries has been found to be much higher than in our study (44%).10 A study in Swedish family planning clients17 also using Amsel criteria to diagnose BV reported a BV prevalence of 13.7% much higher than that we found in Thailand. The low BV prevalence in our study may have been due to the low-risk sexual behavior profile of the cohort, ethnic differences between Thai women and African and European women, the fact that women with prevalent BV at baseline were excluded from the analysis, the use of hormonal contraceptive methods by a majority of our study population, and the use of a very insensitive test for BV diagnosis (Amsel as opposed to Nugent criteria).18 In contrast to BV prevalence, our study found a higher BV incidence in Thai women (10.0 per 100 woman-years) compared with the BV incidence found in US women (2.3 per 100 woman-years), the only other study to our knowledge that has reported BV incidence.14 This illustrates that BV occurs frequently in young Thai women.
Although many risk sexual behavior factors were found to be associated with BV incidence in univariate analysis, few remained significant in the adjusted analysis. Significant factors were sex during menstruation, partners having had sex with other women, and being infected with Trichomonas vaginalis.
Few studies concerning risk factors of BV have found sex during menstruation as a potential risk factor. A study to evaluate the relationship of BV and douching11 showed that women who had sex during menses had higher BV prevalence whereas in a study of female sex workers in Kenya the prevalence of BV was not higher among women who had sex while menstruating.13 However, there was no report on whether sex during menses was significant in multivariable models in these 2 studies. A study that examined the occurrence of BV over the menstrual cycle revealed a high degree of fluctuation in patterns of vaginal flora.19 Among women with a low frequency of BV, the highest BV frequency was found in the first week of the menstrual cycle, with spontaneous resolution occurring after day 14. This finding of low resistance to BV during the first week of the menstrual cycle might partially explain the higher risk of BV incidence we found in women who had sex during menstruation.
Previous studies have found sexual risk behaviors to be associated with increased BV risk including exposure to a new sexual partner,3,6,14,20 multiple sexual partners,6,8,17,21–26 and higher number of lifetime sexual partners.17 In addition to sex during menses the only other sexual risk behavior that we found significantly associated with incident BV (when controlling for other factors) was male partner having had sex with other women. To our knowledge there have been no previous reports in the literature on whether male partners having sex with other women is a predictor of incident BV. The fact that we did not identify other sexual risk factors in this study might be the result of low statistical power due to low BV incidence and the low frequency of high-risk sexual behaviors in this cohort. Women who had no knowledge whether her husbands had sex with other women also had a greater risk of BV acquisition. Evidence from previous HIV risk factors among Thai women have revealed that extramarital relationship of male partners is the leading cause of sexually transmitted HIV infection in women within stable partnerships.27
Our study confirmed the result of previous research that concurrent STIs, including trichomaniasis,10,13,28,29 chlamydial infection,13,26,30 and gonorrhea,6 were associated with development of BV. BV might facilitate STIs including trichomoniasis by decreasing local secretory leukocyte protease inhibitor levels.30 Although we found significant associations for gonorrhea and chlamydia infection only in univariate analysis, trichomoniasis remained highly statistically significant in the multivariable model (despite having wide confidence intervals).
This study did not detect an association between the use of COCs and incident BV. The protective effect of DMPA against BV was marginally significant. Studies among Kenyan prostitutes and Swedish women attending family planning clinics found protective effect against BV of oral contraceptive pills.31,32 Kenyan prostitutes using DMPA also had significantly decreased risk of BV.31 In addition, among HC-HIV study cohorts in Uganda and Zimbabwe, use of both COCs and DMPA was associated with a decreased risk of both prevalent and incident BV (van de Wijgert, personal communication).
One possible explanation for the finding that condom use failed to demonstrate a protective effect for BV acquisition is that condom use might have been inconsistent. The prevention efficacy of condoms was not seen when controlling for other factors. Cigarette smoking was the only risk health practice found to be associated with BV in our study. This is consistent with other studies.11,33,34 The association between changes in vaginal flora35 or elevated sexual behaviors36,37 and smoking could explain this finding.
The association between intravaginal practices, particularly vaginal douching, and BV have been demonstrated in many studies.3,6,8,11–13,38–41 Those studies were conducted in the United States,3,6,11 Europe,8,21 and Africa,9,13,39 places where practices such as vaginal douching are prevalent. Some study populations were at high risk of STIs such as HIV-infected women,13 STD clinic attendees,12,20 or sex workers.13 We believe that we were unable to detect an association between intravaginal practices and BV acquisition in this cohort of women because of the low prevalence of intravaginal practices and the low behavioral risk, leading to insufficient statistical power. Important reasons for vaginal douching in other populations have been perceived personal hygiene or disease prevention42–44; women in our study might have perceived that they had low risk of acquiring STIs, so intravaginal practices were not deemed necessary or useful.
Strengths of our study include the large study cohort and its prospective nature that allowed for the temporal investigation of sexual risk behaviors, STI, intravaginal practices, and incident BV. Moreover, women in the study were recruited from many regions of Thailand. Our study also had several limitations. Data used for the analysis in this study was from the HC-HIV study. Eligibility criteria set in that study somewhat limited the generalizability of this analysis. In addition, the use of a less sensitive method (Amsel criteria) to diagnose BV can lead to misclassification bias. This bias could dilute the strength of any association between vaginal practices and BV in our study. Finally, the interviews relied on women's self-reports, and there is no assurance that self-report of sexual activities or intravaginal practices were valid.
In conclusion, our study found that BV was associated with sexual risk behaviors but not with intravaginal practices. Further investigation is needed to determine the effect of intravaginal practices on BV in high-risk populations in Thailand and other southeast Asian countries.
1. Koumans EH, Kendrick JS. Preventing adverse sequelae of bacterial vaginosis: A public health program and research agenda. Sex Transm Dis 2001; 28:292–297.
2. Leitich H, Bodner-Adler B, Brunbauer M, et al. Bacterial vaginosis as a risk factor for preterm delivery: A meta-analysis. Am J Obstet Gynecol 2003; 189:139–147.
3. Taha TE, Hoover DR, Dallabetta GA, et al. Bacterial vaginosis and distrubances of vaginal flora: Association with increased acquisition of HIV. AIDS 1998; 12:1699–1706.
4. Holzman C, Leventhal JM, Qiu H, et al. Factors linked to bacterial vaginosis in nonpregnant women. Am J Public Health 2001; 91:1664–1670.
5. Mead PB. Epidemiology of bacterial vaginosis. Am J Obstet Gynecol 1993; 169:446–449.
6. Schwebke J, Desmon RA, Oh MK. Predictors of bacterial vaginosis in adolescent women who douche. Sex Transm Dis 2004; 31:433–436.
7. Moi H. Prevalence of bacterial vaginosis and its association with genital infections, inflammation and contraceptive methods in women attending sexually transmitted diseases and primary health clinics. In J STD AIDS 1990; 1:86–94.
8. Chiaffarino F, Parazzini F, De Besi P, et al. Risk factors for bacterial vaginosis. Eur J Obstet Gynecol Reprod Biol 2004; 17:222–226.
9. Zhang J, Hatch M, Zhang D, et al. Frequency of douching and risk of bacterial vaginosis in African-American women. Obstet Gynecol 2004; 104:756–760.
10. Bukusi EA, Cohen CR, Meier AS, et al. Bacterial vaginosis: Risk factors among Kenyan women and their male partners. Sex Transm Dis 2006; 33:361–367.
11. Ness RB, Hiller SL, Ritchter HE, et al. Douching in relation to bacterial vaginosis, lactobacilli, and facultative bacteria in the vagina. Ostet Gynecol 2002; 100:765–772.
12. Rajamanoharan S, Low N, Jones SB, et al. Bacterial vaginosis, ethnicity, and the use of genital cleaning agents: A case control study. Sex Transm Dis 1999; 26:404–409.
13. Fonck K, Kaul R, Keli F, et al. Sexually transmitted infections and vaginal douching in a population of female sex workers in Nairobi, Kenya. Sex Transm Dis 2001; 77:271–275.
14. Schwebke JR, Desmond R. Risk factors for bacterial vaginosis in women at high risk for sexually transmitted diseases. Sex Transm Dis 2005; 32:654–658.
15. Amsel R, Totten PA, Spiegel CA, et al. Nonspecific vaginitis. Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74:14–22.
16. Harold AK, Christopher TS. Stat Methods Epidemiol 1989; 10:193–202.
17. Nilsson U, Hellberg D, Shoubnikova M, et al. Sexual behavior risk factors associated with bacterial vaginosis and Chlamydia trachomatis
infection. Sex Transm Dis 1997; 24:241–246.
18. Schwebke JR, Hiller SL, Sobel JD, et al. Validity of the vaginal gram stain for the diagnosis of bacterial vaginosis. Obstet Gynecol 1996; 88:573–576.
19. Morison L, Ekpo G, West B, et al. Bacterial vaginosis in relation to menstrual cycle, menstrual protection method, and sexual intercourse in rural Gambian women. Sex Transm Infect 2005; 81:242–247.
20. Hawes SE, Hiller SL, Benedetti J, et al. Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J Infect Dis 1996; 174:1058–1063.
21. Morris MC, Rogers PA, Kingborn GR. Is bacterial vaginosis a sexually transmitted infection? Sex Transm Infect 2001; 77:63–68.
22. Barbone F, Austin H, Louv WC, et al. A follow-up study of methods of contraception, sexual activity and rates of trichomoniasis, candidiasis, and bacterial vaginosis. Am J Obstet Gynecol 1990; 163:510–514.
23. Larsson P-G, Platz-Christensen J-J, Sundstrom E. Is Bacterial vaginosis a sexually transmitted disease? Int J STD AIDS 1991; 2:362–436.
24. Hart G. Factors associated with trichomoniasis, candidiasis, and bacterial vaginosis. Int J STD AIDS 1993; 4:21–25.
25. Hart G. Risk profiles and epidemiologic interrelationships of sexually transmitted diseases. Sex Transm Dis 1993; 20:126–136.
26. Yen S, Shafer M-A, Moncada J, et al. Bacterial vaginosis in sexually experienced and non-sexually experienced young women entering the military. Obstet Gynecol 2003; 102:927–933.
27. Xu F, Kilmarx PH, Supawitkul S. HIV-1 seroprevalence, risk factors, and preventive behaviors among women in northern Thailand. JAIDS 2000; 25:353–359.
28. Cherpes T, Meyn L, Krohn M, et al. Association between acquisition of herpes simplex virus type 2 in women with bacterial vaginosis. Clin Infect Dis 2003; 37:319–325.
29. Thomason JL, Gelbrt SM, Sobun JF, et al. Comparison of four methods to detect Trichomonas vaginalis
. J Clin Mirobiol 1984; 27:1095–1096.
30. Draper DL, Landers DV, Krohn MA, et al. Levels of vaginal secretory leukocyte protease inhibitor are decreased in women with lower reproductive tract infections. Am J Obstet Gynecol 2000; 183:1243–1248.
31. Baeten JM, Nyange PM, Richardson BA, et al. Hormonal contraception and risk of sexually transmitted disease acquisition: Results from a prospective study. Am J Obstet Gynecol 2001; 185:380–385.
32. Shoubnikova M, Hellberg D, Nilsson S, et al. Contraceptive use in women with bacterial vaginosis. Contraception 1997; 55:355–358.
33. Smart S, Singal A, Mindel A. Social and sexual risk factors for bacterial vaginosis. Sex Transm Infect 2004; 80:58–62.
34. Bradshaw CS, Morton AN, Garland SM, et al. Higher-risk behavioral practices associated with bacterial vaginosis compared with vaginal candidiasis. Obstet Gynecol 2005; 106:105–114.
35. Alnaif B, Drutz HP. The association of smoking with vaginal flora, urinary tract infection, pelvic floor prolapse, and post-void residual volumes. J Low Genit Tract Dis 2001; 5:7–11.
36. Langstrom N, Hanson RK. High rates of sexual behavior in the general population: Correlates and predictors. Arch Sex Behav 2006; 35:37–52.
37. Camenga DR, Klein JD, Roy J. The changing risk profile of the American adolescent smoker: Implications for prevention programs and tobacco interventions. J Adolesc Health 2006; 39:120–130.
38. Aral SO, Mosher WD, Cates W Jr. Vaginal douching among women of reproductive age in the United States: 1988. Am J Public Health 1992; 82:210–214.
39. van De Wijgert JH, Mason PR, Gwanzura L, et al. Intravaginal practices, vaginal flora disturbances, and acquisition of sexually transmitted diseases in Zimbabwean women. J Infect Dis 2000; 181:587–594.
40. Cottrell BH. Vaginal douching practices of woman in eight Florida Panhandle counties. J Obst Gyn Neonat Nurs 2006; 35:24–33.
41. La Ruche G, Messou N, Ali-Napo L, et al. Vaginal douching: Association with lower genital tract infections in African Pregnant women. Sex Transm Dis 1999; 26:191–196.
42. Brown JE, Brown RC. Traditional intravaginal practices and the heterosexual transmission of disease: A review. Sex Transm Dis 2000; 27:183–187.
43. Rosenberg MJ, Phillips RS, Holmes MD. Vaginal douching: Who and why? J Reprod Med 1991; 36:753–758.
44. Annang L, Grimley D, Hook E, et al. Vaginal douching practices among black women at risk: Exploring douching prevalence, reasons for douching, and sexually transmitted disease infection. Sex Transm Dis 2006; 33:215–219.