In this large, prospective study of HIV-1-uninfected Kenyan women, we found that biologic and behavioral factors were associated with isolation of vaginal Lactobacillus species, a key component of healthy vaginal ecology. The identification of modifiable biologic factors that influence vaginal Lactobacillus colonization may have important implications for the design of vaginal health strategies to prevent HIV-1 acquisition in women.
Intravaginal practices such as douching or wiping inside the vagina with water or antiseptic preparations have been associated with increased risk for HIV-1 acquisition [6
]. We previously reported elevated HIV-1 risk among women who performed vaginal washing: adjusted hazard ratio (aHR) 2.64 (95% CI 1.00–6.97) for those who used water and aHR 3.84 (95% CI 1.51–9.77) for those who used soap, compared with those who did not vaginally cleanse [16
]. An unadjusted preliminary analysis demonstrated decreased Lactobacillus
colonization associated with vaginal washing [17
]. Another report from our cohort found that Lactobacillus
colonization was associated with decreased HIV-1 incidence [4
]. The results of the present study bridge and reinforce these earlier findings, suggesting that decreased Lactobacillus
colonization as a result of vaginal washing might be an important factor mediating the relationship between intravaginal practices and HIV-1 risk in this population. One limitation of our study was that vaginal washing was measured at enrollment and practices may have changed during follow-up; however, our previous report, associating vaginal washing with HIV-1 risk, used this same measure [16
]. Our findings parallel the results of studies in U.S. women, which have also found douching to be associated with BV and decreased vaginal colonization with H2
-producing lactobacilli [18
Antibiotic use has been reported as a risk factor for loss of H2
-producing lactobacilli [20
], and our results confirm this association. We also found decreased Lactobacillus
isolation after metronidazole use and increased H2
isolation after antifungal treatment, although these did not achieve statistical significance in multivariate analysis. These latter associations may reflect confounding by indication, as metronidazole was very frequently used to treat BV and T. vaginalis
infections in the cohort, while the antifungals were used to treat VC. These vaginal conditions were associated with decreased (BV and T. vaginalis
) and increased (VC) identification of Lactobacillus
, and perturbations of the vaginal flora may persist or recur after treatment.
The VC results are consistent with a recent analysis of women from this cohort who participated in a clinical trial of oral periodic presumptive treatment of vaginal infections between 2003 and 2006 [12
]. Among women in the control arm, concurrent colonization with H2
was associated with increased risk of symptomatic VC (adjusted OR 3.31, 95% CI 1.09–10.08), while prior metronidazole treatment was associated with a trend towards increased risk of symptomatic VC (adjusted OR 6.41, 95% CI 0.68–60.25) [21
]. Given that BV, trichomoniasis, and VC have all been associated with increased HIV-1 risk [22
], new approaches to treating vaginal pathogens need to investigate methods to support Lactobacillus
colonization without trading one vaginal infection for another.
HSV-2 seropositivity was associated with an ~30% decrease in isolation of H2
-producing lactobacilli in this cohort, consistent with other studies [23
]. Subclinical HSV-2 reactivation is frequent in seropositive persons (~20% of days) and leads to long-lasting immunologic changes to the genital mucosa [25
]. HSV-2 seropositivity has been associated with a >3-fold increased risk of HIV-1 acquisition [26
], including in this cohort [10
]. While the HSV-2/HIV-1 association has generally been hypothesized to be mediated through changes to mucosal integrity and mucosal immune cell populations, it is interesting to consider that an HSV-2 effect might be mediated in part through changes in vaginal ecology.
Older age was associated with a step-wise decrease in Lactobacillus
isolation, potentially reflecting lower estrogen levels among older women. However, hormonal contraceptive use was not associated with Lactobacillus
isolation. We and others have previously shown that hormonal contraception decreases BV risk [23
]. Taken together these data suggest that hormonal contraception may reduce the level of disturbances of the vaginal flora, without promoting Lactobacillus
We were surprised to find that a higher number of sex partners was associated with detection of Lactobacillus
(though not with H2
-producing lactobacilli). Previous studies have found lactobacilli to be less common and BV more common among women with a higher number of sexual partners [19
]. The reason for the association observed in our study is not clear, but could relate to incompletely controlled differences in sexual risk (e.g., reduced partner number when women had symptomatic vaginal discharge).
There are several strengths to this study. The large number of women and total number of visits makes this the largest study of correlates of vaginal Lactobacillus colonization to date. Longitudinal and frequent follow-up permitted careful analysis of time-dependent factors like age, antibiotic use, contraception, and sexual behavior.
We found a low colonization rate of vaginal Lactobacillus
, especially H2
-producing lactobacilli. The low isolation rates may be attributed to our population (Kenya vs. US, where most studies of vaginal flora have been conducted) or isolation methods (culture versus PCR [28
]). Some Lactobacillus
species, like L. iners
(which is common among African women [28
]), do not grow on Rogosa agar, and our colonization rates may be underestimates. The high prevalence of vaginal washing and high incidence of BV in our study population also likely contributed to low Lactobacillus
isolation rates. Finally, the results of this study may not be generalizable to all populations – they may be most directly relevant to women at high risk for HIV-1; HIV-1 incidence in this cohort was >8% per year during the period included in this analysis [10
Our findings suggest that modifiable biologic and behavioral correlates – intravaginal practices, antimicrobial use, and genital tract infections – influence vaginal colonization with Lactobacillus
among African women at high risk for HIV-1. Increasing attention to vaginal health interventions as an HIV-1 prevention strategy [12
] requires continued study of risk factors for vaginal pathogens and correlates of healthy vaginal flora. New interventions aimed at improving vaginal Lactobacillus
colonization have the potential to alter HIV-1 susceptibility.