Bacterial Vaginosis Associated with Increased Risk of Female-to-Male HIV-1 Transmission: A Prospective Cohort Analysis among African Couples
et al. (2012) Bacterial Vaginosis Associated with Increased Risk of Female-to-Male HIV-1
Transmission: A Prospective Cohort Analysis among African Couples. PLoS Med 9(6): e1001251. doi:10.1371/journal.pmed.1001251
Bacterial Vaginosis Associated with Increased Risk of Female-to-Male HIV-1 Transmission: A Prospective Cohort Analysis among African Couples
Craig R. Cohen
Jairam R. Lingappa
Jared M. Baeten
Musa O. Ngayo
Carol A. Spiegel
Elizabeth A. Bukusi
Heather Watts, NICHD, United States of America
Background: Bacterial vaginosis (BV), a disruption of the normal vaginal flora, has been associated with a 60% increased risk of HIV-1 acquisition in women and higher concentration of HIV-1 RNA in the genital tract of HIV-1-infected women. However, whether BV, which is present in up to half of African HIV-1-infected women, is associated with an increase in HIV-1 transmission to male partners has not been assessed in previous studies. Methods and Findings: We assessed the association between BV on female-to-male HIV-1 transmission risk in a prospective study of 2,236 HIV-1-seropositive women and their HIV-1 uninfected male partners from seven African countries from a randomized placebo-controlled trial that enrolled heterosexual African adults who were seropositive for both HIV-1 and herpes simplex virus (HSV)-2, and their HIV-1-seronegative partners. Participants were followed for up to 24 months; every three months, vaginal swabs were obtained from female partners for Gram stain and male partners were tested for HIV-1. BV and normal vaginal flora were defined as a Nugent score of 7-10 and 0-3, respectively. To reduce misclassification, HIV-1 sequence analysis of viruses from seroconverters and their partners was performed to determine linkage of HIV-1 transmissions. Overall, 50 incident HIV-1 infections occurred in men in which the HIV-1-infected female partner had an evaluable vaginal Gram stain. HIV-1 incidence in men whose HIV-1-infected female partners had BV was 2.91 versus 0.76 per 100 person-years in men whose female partners had normal vaginal flora (hazard ratio 3.62, 95% CI 1.74-7.52). After controlling for sociodemographic factors, sexual behavior, male circumcision, sexually transmitted infections, pregnancy, and plasma HIV-1 RNA levels in female partners, BV was associated with a greater than 3-fold increased risk of female-tomale HIV-1 transmission (adjusted hazard ratio 3.17, 95% CI 1.37-7.33). Conclusions: This study identified an association between BV and increased risk of HIV-1 transmission to male partners. Several limitations may affect the generalizability of our results including: all participants underwent couples HIV counseling and testing and enrolled in an HIV-1 prevention trial, and index participants had a baseline CD4 count $250 cells/mm3 and were HSV-2 seropositive. Given the high prevalence of BV and the association of BV with increased risk of both female HIV-1 acquisition and transmission found in our study, if this association proves to be causal, BV could be responsible for a substantial proportion of new HIV-1 infections in Africa. Normalization of vaginal flora in HIV-1-infected women could mitigate female-to-male HIV-1 transmission. Trial Registration: ClinicalTrials.com NCT00194519 Please see later in the article for the Editors' Summary.
Funding: This study was supported through research grants from the Bill & Melinda Gates Foundation (grant 26469) and the US National Institutes of Health
(grant R01 AI-083034). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: ART, antiretroviral therapy; BV, bacterial vaginosis; HSV, herpes simplex virus; IQR, interquartile range; STI, sexually transmitted infection.
Worldwide, an estimated 33.3 million people are infected with
HIV-1, 60% in sub-Saharan Africa, where women account for the
majority of those infected . Antiretroviral therapy (ART),
through reducing HIV-1 plasma  and genital HIV-1 RNA
concentrations , has been associated with .90% reduction in
HIV-1 transmission in observational studies  and a recent trial
of earlier ART initiation . However, only about half of HIV-1
infected adults qualify for ART initiation per current country
guidelines, and only 37% of those qualifying for ART in Africa
received treatment . Thus, new HIV-1 prevention strategies
that will reduce HIV-1 risk for those not on ART remain an
Bacterial vaginosis (BV) is a common disorder characterized by
changes in vaginal flora in which normally predominant
Lactobacillus species are replaced by potential pathogens including
Gardnerella vaginalis, genital Mycoplasma, and fastidious anaerobic
bacteria [6,7]. For unknown reasons, BV is considerably more
common among women in sub-Saharan Africa and other
resource-poor countries than in developed countries, affecting up
to 55% of women in some studies . BV has been associated
with a 60% increased risk of HIV-1 acquisition in women ,
and, among women with HIV-1, with higher HIV-1
concentrations in cervicovaginal fluids . Bacteria associated with BV
can induce viral replication and shedding in the genital tract
[15,16], which may lead to increased HIV-1 infectiousness for
women with BV [17,18]. However, to date, no study has examined
whether BV increases the risk of female-to-male HIV-1
transmission. We hypothesized that HIV-1infected women with BV have
an increased risk of female-to-male HIV-1 transmission than
women with normal vaginal flora. To answer this question, we
prospectively studied a cohort of African heterosexual couples in
which the female was HIV-1 seropositive and the male was
HIV1seronegative who were enrolled in a randomized
placebocontrolled trial of dually HIV-1 and herpes simplex virus (HSV)
type 2seropositive heterosexual African adults, and their HIV-1
The University of Washington Human Subjects Review
Committee, University of California San Francisco Committee
on Human Research, the Kenya Medical Research Institute
(KEMRI) National Ethics Review Committee, and ethics review
boards at each study site reviewed and approved the study
protocol and consent documents.
Population and Procedures
We used data from a cohort of southern and East African
HIV1 serodiscordant heterosexual couples enrolled in a clinical trial
(the Partners in Prevention HSV/HIV Transmission Study)
evaluating HSV-2suppressive therapy with acyclovir 400 mg
bid provided to the HIV-1infected partner to prevent HIV-1
transmission to their HIV-1seronegative partners. As previously
reported, acyclovir decreased plasma HIV-1 levels in the HIV-1
infected partners, but did not reduce HIV-1 transmission risk .
The present report is a secondary analysis of data from the subset
of 2,236 couples from this prospective cohort in which the HIV-1
infected partner was female .
HIV-1infected partners were required to be seropositive for
HSV-2, with a CD4 count $250 cells/mm3, and without history
of AIDS-defining conditions; couples were followed for up to
24 mo. HIV-1infected women were seen monthly and
underwent a pelvic examination at enrollment and every 3 mo to collect
a vaginal swab for Gram stain for evaluation of BV. Enrollment
vaginal swabs were collected on all participants. Quarterly vaginal
swab collection was performed as part of a protocol modification
implemented at each site once approved by the site institutional
review board; vaginal Gram stain results were not obtained prior
to site-specific approval of the protocol modification.
Plasma for HIV-1 RNA quantification was collected at baseline,
3-, 6-, and 12-mo visits, and at study exit; CD4 counts were
performed at baseline and every 6 mo. HIV-1infected partners
who met national guidelines for initiation of ART during
followup were referred to local HIV-1 care clinics, and those who
became pregnant were referred to antenatal clinics for prevention
of mother-to-child transmission services. HIV-1infected women
underwent a speculum pelvic examination at a visit 6 mo after
enrollment, during which an endocervical Dacron swab for HIV-1
RNA quantification was obtained; swabs were not collected at a
defined time in the menstrual cycle, although women usually
deferred sampling during menstruation. HIV-1uninfected men
were seen quarterly for HIV-1 serologic testing.
Participants received comprehensive HIV-1 prevention
including HIV-1 risk-reduction counseling (both individual and as a
couple), quarterly sexually transmitted infection (STI) symptom
assessment with syndromic treatment of STIs, and provision of
free condoms. All participants provided written informed consent.
Laboratory Methods for Diagnosis of BV
Vaginal swabs collected at enrollment and quarterly follow-up
visits were rolled onto glass slides, air dried, and methanol fixed at
the study site and subsequently Gram stained at the Center for
Microbiology Research Laboratory at KEMRI. Vaginal flora was
evaluated using Nugents criteria : normal vaginal flora,
intermediate flora, and BV categories were defined by Nugents
scores of 03, 46, and 710, respectively. Each slide was
doubleread by two technologists. A digital image of approximately every
tenth slide was sent electronically to one of the investigators (CAS)
for external quality control (EQC). Our target for concordant
results between the laboratory and EQC was $90%. A discordant
result was defined as a difference in the Nugents score $1, which
also caused a change in flora category (e.g., a score difference of 3
to 4, which changes the diagnosis from normal vaginal flora to
intermediate flora). External quality control performed on 1,722
(8.7%) of 19,882 slides (the total number of slides included HIV-1
positive women included in this study, and HIV-1negative
women evaluated for a separate analysis) demonstrated an overall
concordance of 92.2% (K = 0.84); the concordance surpassed our
predefined value of $90% based on expected inter-observer
agreement in other studies [20,21].
Other Laboratory Procedures
HIV-1 serologic testing was by dual rapid HIV-1 antibody tests
performed locally, with positive results confirmed by HIV-1
Western blot at the University of Washington . For couples in
which the initially HIV-1uninfected male partner seroconverted
to HIV-1 seropositive, analysis of HIV-1 env and gag gene
sequences from both members of the couple were used to evaluate
transmission linkage within the partnership . Serologic testing
for HSV-2 and nucleic acid amplification testing for STIs
(specifically Chlamydia trachomatis, Neisseria gonorrhoeae, and
Trichomonas vaginalis) was done at study enrollment . CD4
quantification was performed using standard flow cytometry. All laboratory
procedures followed Good Laboratory Practices, and laboratories
were enrolled in External Quality Assessment programs.
HIV-1 RNA was quantified from plasma at baseline, at months
3, 6, and 12, and at study exit; and from the 6-mo endocervical
swab specimen (collected at the same visit as the 6-mo plasma
specimen) with the COBAS AmpliPrep/COBAS TaqMan
realtime HIV-1 RNA assay version 1.0 (Roche Diagnostics).
Endocervical swabs were eluted in 1,000 ml of GUSCN lysis
buffer, eluted for 15 min, vortexed briefly, and microfuged for 5 s
at 14,000g to pellet debris before removal of fluid for testing. A
final dilution step with 106 PBS was used to achieve sufficient
volume for the COBAS AP/TM assay, with a lower limit of
quantification of 240 copies (per milliliter for blood plasma and per
swab for endocervical samples). Plasma and genital HIV-1 RNA
concentrations were log10-transformed to approximate normality.
Samples below the limit of quantification were assigned values at
half that limit.
The primary outcome was female-to-male HIV-1 transmission,
defined as those HIV-1 seroconversion events that were genetically
linked within the partnership. Male partners who acquired HIV-1
from an outside partner contributed follow-up time up to HIV-1
seroconversion and were censored thereafter. Follow-up for men
was also censored after their HIV-infected partner initiated ART.
The primary exposure was vaginal flora status, as measured at
the quarterly study visit prior to each HIV-1 test, in order to
represent vaginal flora status during the time of potential HIV-1
exposure to the male partner. If the result at the visit 3 mo prior to
HIV-1 testing was expected but missing, the result 6 mo prior was
used; if the results at both the 3 and 6 mo prior to HIV-1 testing
were expected but missing the period was excluded from analysis.
We analyzed vaginal flora in three categories: BV (Nugent score
$7) and intermediate flora (Nugent score 46), each compared
with normal flora (Nugent score #3). We performed two
sensitivity analyses to assess the robustness of our vaginal flora
exposure: first, we analyzed vaginal flora at the visit concurrent
with HIV-1 serologic testing, and second, we analyzed vaginal
flora based on the most severe exposure (highest Nugent category)
occurring at either the prior or current visit.
Association between vaginal flora and time-varying covariates
was assessed using logistic regression for each of intermediate and
BV compared to normal flora, with GEE methods to account for
correlation between visits. HIV incidence rates and confidence
intervals were computed using Poisson rates; absolute rate
differences were calculated .
To assess the risk of HIV-infection we performed multivariable
Cox proportional hazards analysis to adjust for potential
confounding factors, including demographic, medical, and
behavioral characteristics. Variables were selected a priori for inclusion
based on previously published association with HIV transmission,
and included: (1) characteristics from the time of study enrollment:
age (of both partners), region (East versus southern Africa), HSV-2
status of the HIV-1 uninfected male partner, male partner
circumcision status, trial randomization assignment (acyclovir
versus placebo), and laboratory confirmed STIs at enrollment (i.e.,
N. gonorrhoeae, C. trachomatis, and T. vaginalis) of both partners; and
(2) time-dependent variables, including: pregnancy, hormonal
contraceptive use, plasma HIV-1 levels, and CD4 count in the
female HIV-1infected partner, genital ulcer disease in both
partners, and sexual behavior during the month prior to each visit,
as reported by the male HIV-1uninfected partner (analyzed as
any unprotected sexual intercourse with the study partner, any
report of outside partners, and total number of sex acts with the
study partner). Robust standard errors were used to account for
multiple observations from each person in the time-dependent
analyses. Differences in plasma and cervical viral load were
assessed using linear regression methods, adjusted for repeated
observations. Data were analyzed using SAS version 9.2 (SAS
A total of 2,236 couples were included in this analysis (Table 1).
The median age of HIV-1infected female partners was 30 y and
the median age of HIV-1uninfected male partners was 35 y.
Most couples were married and cohabitating. Couples engaged in
sex a median of four times per month, and 30.5% of couples
reported sex that was unprotected by condom use during the
month prior to enrollment. Among the HIV-1infected female
participants, the median CD4 count was 481 cells/mm3
(interquartile range [IQR] 354663) and the median plasma HIV-1
RNA concentration was 3.95 log10 copies/ml (3.244.53).
Follow-up and HIV-1 Incidence
Median follow-up for the HIV-1seropositive female and
HIV1seronegative male partners was 20.8 (IQR 15.324.1) and
19.3 mo (IQR 13.524.0), respectively. Over 3,318 person-years
of follow-up, 90 incident HIV-1 infections among men were
identified, of which 57 (63.3%) were determined by viral
sequencing to be genetically linked within the partnership, for
an incidence of linked transmission of 1.72 cases per 100
personyears (95% CI 1.302.23). Seven HIV-1 infections occurred in
men whose HIV-1seropositive female partner had no vaginal
flora result during the interval when HIV-1 seroconversion
occurred. In four of these seven cases, the vaginal swab collection
was not expected, while in the remaining three, the result was
missing. Thus, 50 HIV-1 incident infections among men with
virologically linked HIV-1 transmissions with their female HIV-1
infected partners for whom BV data were available were included
in this analysis.
BV at Baseline and during Follow-up
Of 12,126 visits expected to have vaginal swabs collected during
the study, 10,232 (84.4%) had vaginal Gram stain data available.
At enrollment, 869 women (41.1%) had BV, 487 (23.0%) had
intermediate flora, and 757 (35.8%) had normal vaginal flora.
Across all quarterly follow-up visits, the median proportion of
women with BV and intermediate vaginal flora was 34.9% (IQR
34.2%36.3%) and 22.8% (IQR 22.0%23.9%), respectively,
while the median proportion of women with normal vaginal flora
was 42.8% (IQR 40.1%44.1%). Of the 2,221 women with at least
one Gram stain result available from the prior 3-mo visit (our main
exposure), 337 (15.2%), 113 (5.1%), and 340 (15.3%) had BV,
intermediate vaginal flora and normal vaginal flora, respectively,
throughout follow-up. An additional 1,151 (51.8%) women had at
least a single episode of BV during follow-up.
During follow-up, HIV-1infected women who had one or
more intervals with BV were slightly younger than women who
had normal vaginal flora and more likely to have an
uncircumcised male partner (Table 2). While periods where unprotected sex
was reported did not differ by vaginal flora, HIV-1infected
women with BV were more likely to report an outside sexual
partner in the last 30 d than HIV-1infected women with normal
vaginal flora. Plasma HIV-1 RNA concentration was slightly
elevated, and mean CD4 count was slightly lower in HIV-1
Hormonal contraceptive use
East Africa (versus southern Africa)
Sexual behavior (prior month)a
n sex acts with study partner
Any unprotected sex with study partner
Any sex with outside partner
HIV-1seropositive female partner characteristics
Plasma HIV-1 RNA, log10 copies/ml
CD4 count, cells/mm3
Randomized to acyclovir (versus placebo)
Median (IQR) or n (%)
infected women during intervals with BV in comparison to
intervals with normal vaginal flora (Table 2).
Effect of BV on Incidence of Female-to-Male HIV
During the study, HIV-1 incidence was 2.91, 1.48, and 0.76 per
100 person-years in men whose female partner in the
seroconversion interval had BV, intermediate vaginal flora, and normal
vaginal flora, respectively (Table 3). In unadjusted analysis, BV
was associated with a 3.62-fold increased risk (95% CI 1.747.52)
and a 2.15 increased attributable rate (95% CI 1.043.25) per 100
person-years of female-to-male HIV-1 transmission in comparison
to women with normal vaginal flora (Table 3). In multivariable
analysis controlling for sociodemographic factors, sexual behavior,
male circumcision, sexually transmitted infections, pregnancy, and
plasma HIV-1 RNA in female partners, men whose HIV-1
infected female partners had BV 3 mo prior to identifying HIV-1
seroconversion had a 3.17-fold increased adjusted risk (95% CI
1.377.33) of female-to-male HIV-1 transmission. Intermediate
flora in comparison to normal vaginal flora was not associated with
an altered risk of female-to-male HIV-1 transmission (Table 4).
The two sensitivity analyses looking at vaginal flora at the same
visit as HIV-1 seroconversion detection and the highest category of
vaginal flora between the prior and same visit as HIV-1
seroconversion detection were consistent with these results
(Table 4). Lastly, we did not find evidence of an effect of male
circumcision on the relationship between BV in the HIV-1
seropositive female partner and linked HIV-1 infections in men
Effect of BV on Genital and Plasma HIV-1 RNA
The mean log10 concentration of HIV-1 RNA in genital
secretions and plasma were slightly elevated in participants with
intermediate vaginal flora (log10 difference: 0.21 and 0.16,
respectively) and BV (log10 difference: 0.19 and 0.18, respectively)
(Table 5). After controlling for plasma HIV-1 RNA concentration
at the same 6-mo visit, the mean log10 concentration of HIV-1
RNA in genital secretions was no longer significantly associated
with vaginal flora (Table 5).
In this prospective study of more than 2,200 southern and East
African HIV-1seropositive women and their HIV-1seronegative
male partners with genetic linkage of HIV-1 transmission pairs, we
found that BV was independently associated with a 3-fold
increased risk of female-to-male HIV-1 transmission. The
potential significance of this finding is substantial, given that
35%, 15%, and 52% of women in this study had BV at
enrollment, throughout follow-up, and at least one interval of
BV during the 2 y of follow-up, respectively. This proportion of
HIV-1seropositive women with BV is consistent with prior
studies demonstrating a prevalence of BV ranging from 30%55%
among women in sub-Saharan Africa . Thus, assuming that
the association we report is causal, BV may account for a
substantial population attributable risk percent for new HIV-1
infections in men in Africa.
Although genital HIV-1 RNA predicts female-to-male HIV-1
transmission independent of the HIV-1 RNA concentration in
Table 2. Participant characteristics during quarterly follow-up intervals with BV and intermediate vaginal flora versus normal
Age of HIV-1seronegative partner, years
Children within the partnership
Having at least one child within the partnership
Sexual behavior, HIV-1 uninfected partner (past month)
Any unprotected sex with study partner
Any sex with an outside partner
Number of sex acts with study partner
CD4 count (cells/mm3) in the HIV-1infected partner
Plasma HIV-1 level (log10 copies/ml) in the HIV-1infected partner 4.09 (3.274.69)
HSV-2 serostatus, male partner
Genital ulcer disease, HIV-1infected partner on exam
Genital ulcer disease, HIV-1seronegative partner
Pregnant, female partner (current visit)
Follow-up Intervals for Analysis of HIV-1 Transmission from Women to Mena
(n = 2,236 HIV-1seropositive Women), n (%) or Median (IQR)
aComparisons between BV exposure groups are adjusted for correlation by multiple measures from the same participant using generalized estimating equations. The
number of data points assessed for each cell is total number of visits with each covariate characteristic during study follow-up.
bComparison of BV intervals to normal vaginal flora interval.
cComparison of intermediate vaginal flora intervals to normal vaginal flora intervals.
blood , we found only a modest (0.2 log10) increase in HIV-1
RNA in women with BV in comparison to those with normal
vaginal flora. Thus, it is likely that increased genital HIV-1 RNA
caused by BV only partially explains our results. Most
crosssectional and longitudinal studies have found that women with BV
have higher concentrations of HIV-1 RNA in genital secretions in
comparison to women with normal vaginal flora [12,13,26].
However, two prospective studies did not find differences in genital
HIV-1 RNA concentration associated with BV [14,27].
Differences with previous studies that found an association between BV
and genital HIV-1 shedding could be due to the proportion of
women with BV who were symptomatic, with potentially higher
levels of inflammation associated with symptomatic BV, and the
short duration (i.e., 14 d) after BV treatment in which vaginal
samples were collected in the longitudinal studies to measure
genital HIV-1 RNA, which may have been too soon for decreased
T-cell activation , proinflammatory cytokines , or
reestablishment of lactobacilli predominant flora .
An additional hypothesis to explain our findings is that BV in a
female partner may indirectly increase HIV-1 susceptibility in
men. A growing body of evidence suggests that the female and
male genital microbiota is shared between sexual partners
Seroconversions with missing BV status
Seroconversions with BV status
Normal vaginal flora (Nugent score 03)
Abnormal vaginal flora (Nugent score 410)
Intermediate vaginal flora (Nugent score 46)
BV (Nugent score 710)
Difference (95% CI)
0.72 (20.24 to 1.67)
aFor the primary analytic approach, vaginal flora from the adjacent previous visit (usually 3 mo prior) was used. If this result was missing, the most recent vaginal flora
result from the visit 6 mo prior was used; otherwise the result was considered missing.
More severe BV status between pre- BV versus normal vaginal flora
BV versus normal vaginal flora
Intermediate versus normal vaginal flora
BV versus normal vaginal flora
Intermediate versus normal vaginal flora
Intermediate versus normal vaginal flora
aMultivariable Cox proportional hazards analysis (adjusted hazard ratio [AHR]), adjusting for the following: Fixed covariates: age, geographic region (southern Africa
versus Eastern Africa), HSV-2 status of male partner at study enrollment, male circumcision, randomization treatment assignment, and both female and male sexual
transmitted disease at study enrollment (laboratory confirmed gonorrhea, chlamydia, and trichomonas); Time-dependent covariates (per quarterly visit): pregnancy
(current visit), hormonal contraception (current visit), plasma HIV-1 viral load of female partner (from enrollment and month 3, 6, 12, and study exit), unprotected sex act
with study partner (current visit) (based on reporting from male partner), CD4 count of female partner (6 monthly), outside partners (current visit, based on reporting
from male partner), number of sex acts with their study partner (current visit), and genital ulcer disease (current visit) (based on physical exam from both partners).
[8,29,30]. Recent data from Uganda have demonstrated that male
circumcision reduces the risk of BV in female partners and that
bacterial flora associated with BV commonly colonize the penis
including the distal urethra [30,31]. Anaerobic and other bacteria
increased in male partners of women with BV may cause
inflammation by activating Langerhans cells and CD4+ T-cells,
thereby increasing target cells for HIV-1 and susceptibility to
HIV-1 infection [32,33]. Interestingly, male circumcision did not
affect or modify the relationship between BV and female-to-male
HIV-1 transmission in our study. In comparison to
pre-circumcision abundances of bacterial phylotypes, post-circumcision
abundances of anaerobic bacteria decreased, while abundances
of facultative anaerobic bacteria increased significantly in the
Rakai study . Potential mechanisms need investigation,
including whether the male genital microbiota, in particular
anaerobes, are associated with urethral and penile inflammation
and activation of Langerhans cells and CD4+ T-cells, which could
increase risk of HIV-1 infection in men.
Lower socioeconomic status has been associated with higher BV
prevalence [8,34]. Previous studies have also implicated race,
multiple sex partners, trichomoniasis, HIV-1 infection,
intrauterine device use, vaginal douching, recent antibiotic use, and the
absence of vaginal colonization by H2O2-producing lactobacilli as
risk factors for BV [8,9,3438]. Following treatment, BV clinically
recurs in 20%30% of women within 3 mo , and recurs in
approximately 75% of women with symptomatic as well as
asymptomatic BV within 2 mo of treatment . One reason for
the high prevalence of BV and its frequent recurrence may result
from the transfer of potentially pathogenic bacteria between
heterosexual partners through genital, and potentially orogenital
The high prevalence and frequent recurrence of BV has led to
the development of several new strategies including frequent
presumptive antibiotic treatment , and use of probiotic
lactobacilli as an adjuvant or an alternative to antimicrobial
therapy . Recent advances in understanding the
microbiota associated with BV [6,7], including the ability of G. vaginalis
p-Value Versus Normal Vaginal Flora p-Value Versus Normal Vaginal Flora*
Intermediate vaginal flora
Intermediate vaginal flora
and to a lesser degree Atopobium vaginae to form biofilms recalcitrant
to antibiotic treatment [46,47], may eventually lead to therapies
that maintain a lactobacilli-predominant flora in the vagina.
Our study has several strengths starting with its large and
diverse population of HIV-1serodiscordant couples recruited
from across multiple sites in southern and East Africa.
Furthermore, genetic linkage of female-to-male transmitted HIV-1
minimized misclassification in our analysis . One limitation
of our analysis is that we do not know the specific vaginal flora
present at the time of HIV-1 infection since vaginal microbiota
can fluctuate weekly . To address this, we conducted two
sensitivity analyses, the first evaluating vaginal Gram stain results
at the same visit when HIV-1 seroconversion was first noted, and
the second evaluating the severity of vaginal flora between that
and the prior visit. Both evaluations confirmed the results of our
primary analysis. The cohort was a highly selective population
(e.g., all participants underwent couples HIV counseling and
testing, enrolled in an HIV-1 prevention randomized clinical trial,
and index participants had a CD4 count $250 cells/mm3 at
enrollment), which could impact on the generalizability of our
results. Furthermore, all HIV-1infected partners were co-infected
with HSV-2; however, HSV-2 seroprevalence is .80% among
HIV-1infected persons in sub-Saharan Africa  and thus is
unlikely to limit the generalizability of our findings. In addition,
the relatively small number of female-to-male HIV-1 transmissions
(nine among women with normal vaginal flora versus 31 among
women with BV) requires mention. Finally, residual or
unmeasured confounding, which cannot be completely excluded, could
affect the significance of our findings.
This study clearly demonstrates that BV is associated with an
increased risk of female-to-male HIV-1 transmission. BV is a
highly prevalent condition among HIV-1infected women. The
association of BV with increased infectiousness of HIV-1infected
women requires additional research to understand potential
pathogenic mechanisms as well as the etiology, treatment, and
prevention of BV. While a large community randomized
controlled trial that provided presumptive treatment of STIs
including metronidazole for BV failed to reduce HIV-1 incidence
, ongoing studies are evaluating more frequent presumptive
BV therapy , while others are studying naturally occurring and
genetically enhanced probiotics to reduce recurrent BV
[44,45,50,51]. A lactobacillus-predominant vaginal flora might
not only reduce the risk of HIV-1 acquisition in women [9,11], but
also HIV-1 transmission to male partners, and points to the
potential benefits of using the human microbiota to prevent
We gratefully acknowledge the contributions of the HIV-1serodiscordant
couples who participated in this study and the Director, KEMRI for
permission to publish this manuscript. We thank the members of the
Partners in Prevention HSV/HIV Transmission Study Team including:
University of Washington Coordinating Center and Central
Laboratories, Seattle: Anna Wald (protocol co-chair), Mary S. Campbell,
Robert W. Coombs, Lawrence Corey, James P. Hughes, Amalia Magaret,
M. Juliana McElrath, Rhoda Morrow, James I. Mullins
Study sites and site principal investigators:
Cape Town, South Africa (University of Cape Town): David Coetzee;
Eldoret, Kenya (Moi University, Indiana University): Kenneth Fife,
Edwin Were; Gaborone, Botswana (Botswana Harvard Partnership):
Max Essex, Joseph Makhema; Kampala, Uganda (Infectious Disease
Institute, Makerere University): Elly Katabira, Allan Ronald; Kigali,
Rwanda (Rwanda Zambia HIV Research Group, and Emory University):
Susan Allen, Kayitesi Kayitenkore, Etienne Karita; Kisumu, Kenya
Medical Research Institute research staff; Kitwe, Zambia (Rwanda
Zambia HIV Research Group, and Emory University): Susan Allen,
William Kanweka; Lusaka, Zambia (Rwanda Zambia HIV Research
Group, and Emory University): Susan Allen, Bellington Vwalika; Moshi,
Tanzania (Kilimanjaro Christian Medical College, Harvard University):
Rachel Manongi; Nairobi, Kenya (University of Nairobi, University of
Washington): Carey Farquhar, Grace John-Stewart, James Kiarie; Ndola,
Zambia (Rwanda Zambia HIV Research Group, and Emory University):
Susan Allen, Mubiana Inambao; Orange Farm, South Africa (Wits
Institute for Reproductive Health & HIV, University of the
Witwatersrand): Helen Rees; Soweto, South Africa (Perinatal HIV Research Unit,
University of the Witwatersrand): Guy de Bruyn, Glenda Gray, James
McIntyre; Thika, Kenya (University of Nairobi, University of
Washington): Nelly Rwamba Mugo
Conceived and designed the experiments: CRC JRL JMB CAS EAB.
Performed the experiments: CRC JRL CAS MON EAB. Analyzed the
data: CRC JRL JMB TH DD. Wrote the first draft of the manuscript:
CRC TH JMB JRL. Contributed to the writing of the manuscript: CRC
JRL JMB TH DD CC EAB CAS SK SD. ICMJE criteria for authorship
read and met: CRC JRL JMB TH DD CC EAB CAS SK SD MON.
Agree with manuscript results and conclusions: CRC JRL JMB TH DD
CC EAB CAS SK SD MON.
Background. Since the first reported case of AIDS in 1981,
the number of people infected with HIV, the virus that
causes AIDS, has risen steadily. By the end of 2010, 34 million
people were living with HIV/AIDS. At the beginning of the
epidemic more men than women were infected with HIV.
Now, however, 50% of all adults infected with HIV are
women and in sub-Saharan Africa, where two-thirds of
HIVpositive people live, women account for 59% of people living
with HIV. Moreover, among 1524 year-olds, women are
eight times more likely than men to be HIV-positive. This
pattern of infection has developed because most people in
sub-Saharan Africa contract HIV through unprotected
heterosexual sex. The risk of HIV transmission for both men and
women in Africa and elsewhere can be reduced by
abstaining from sex, by only having one or a few partners,
by always using condoms, and by male circumcision. In
addition, several studies suggest that antiretroviral therapy
(ART) greatly reduces HIV transmission.
Why Was This Study Done? Unfortunately, in
subSaharan Africa, only about a fifth of HIV-positive people are
currently receiving ART, which means that there is an urgent
need to find other effective ways to reduce HIV transmission
in this region. In this prospective cohort study (a type of
study that follows a group of people for some time to see
which personal characteristics are associated with disease
development), the researchers investigate whether bacterial
vaginosisa condition in which harmful bacteria disrupt the
normal vaginal floraincreases the risk of female-to-male
HIV transmission among African couples. Bacterial vaginosis,
which is extremely common in sub-Saharan Africa, has been
associated with an increased risk of HIV acquisition in
women and induces viral replication and shedding in the
vagina in HIV-positive women, which may mean that
HIVpositive women with bacterial vaginosis are more likely to
transmit HIV to their male partners than women without this
condition. If this is the case, then interventions that reduce
the incidence of bacterial vaginosis might be valuable HIV
What Did the Researchers Do and Find? The researchers
analyzed data collected from 2,236 heterosexual African
couples enrolled in a clinical trial (the Partners in Prevention
HSV/HIV Transmission Study) whose primary aim was to
investigate whether suppression of herpes simplex virus
infection could prevent HIV transmission. In all the couples,
the woman was HIV-positive and the man was initially
HIVnegative. The female partners were examined every three
months for the presence of bacterial vaginosis and the male
partners were tested regularly for HIV infection. The
researchers also determined whether the men who became
HIV-positive were infected with the same HIV strain as their
partner to check that their infection had been acquired from
this partner. The HIV incidence in men whose partners had
bacterial vaginosis was 2.9 per 100 person-years (that is, 2.9
out of every 100 men became HIV-positive per year) whereas
the HIV incidence in men whose partners had a normal
vaginal flora was 0.76 per 100 person-years. After controlling
for factors that might affect the risk of HIV transmission such
as male circumcision and viral levels in female partners
blood, the researchers estimated that bacterial vaginosis was
associated with a 3.17-fold increased risk of female-to-male
HIV transmission in their study population.
What Do These Findings Mean? These findings suggest
that HIV-positive African women with bacterial vaginosis are
more than three times as likely to transmit HIV to their male
partners as those with a normal vaginal flora. It is possible
that some unknown characteristic of the men in this study
might have increased both their own risk of HIV infection
and their partners risk of bacterial vaginosis. Nevertheless,
because bacterial vaginosis is so common in Africa (half of
the women in this study had bacterial vaginosis at least once
during follow-up) and because this condition is associated
with both female HIV acquisition and transmission, these
findings suggest that bacterial vaginosis could be
responsible for a substantial proportion of new HIV infections in
Africa. Normalization of vaginal flora in HIV-infected women
by frequent presumptive treatment with antimicrobials
(treatment with a curative dose of antibiotics without testing
for bacterial vaginosis) or possibly by treatment with
probiotics (live good bacteria) might, therefore, reduce
female-to-male HIV transmission in sub-Saharan Africa.
Additional Information. Please access these Web sites via
the online version of this summary at http://dx.doi.org/10.
N Information is available from the US National Institute of
Allergy and infectious diseases on all aspects of HIV
infection and AIDS and on bacterial vaginosis
N The US Centers for Disease Control and Prevention has
information on all aspects of HIV/AIDS, including specific
information about HIV/AIDS and women; it also has
information on bacterial vaginosis (in English and Spanish)
N NAM/aidsmap provides basic information about HIV/AIDS,
and summaries of recent research findings on HIV care and
treatment, and information on bacterial vaginosis and HIV
transmission (in several languages)
N Information is available from Avert, an international AIDS
nonprofit group on many aspects of HIV/AIDS, including
detailed information on HIV and AIDS prevention, on
women, HIV and AIDS and on HIV/AIDS in Africa (in English
and Spanish); personal stories of women living with HIV are
available; the website Healthtalkonline also provides
personal stories about living with HIV
1 . (UNAIDS) JUNPoHA (2010) Global report: UNAIDS report on the global AIDS epidemic 2010 . New York: UNAIDS.
2. Chaisson RE , Keruly JC , Moore RD ( 2000 ) Association of initial CD4 cell count and viral load with response to highly active antiretroviral therapy . JAMA 284 : 3128 - 3129 .
3. Graham SM , Holte SE , Peshu NM , Richardson BA , Panteleeff DD , et al. ( 2007 ) Initiation of antiretroviral therapy leads to a rapid decline in cervical and vaginal HIV-1 shedding . AIDS 21 : 501 - 507 .
4. Donnell D , Baeten JM , Kiarie J , Thomas KK , Stevens W , et al. ( 2010 ) Heterosexual HIV-1 transmission after initiation of antiretroviral therapy: a prospective cohort analysis . Lancet 375 : 2092 - 2098 .
5. Cohen MS , Chen YQ , McCauley M , Gamble T , Hosseinipour MC , et al. ( 2011 ) Prevention of HIV-1 infection with early antiretroviral therapy . N Engl J Med 365 : 493 - 505 .
6. Fredricks DN , Fiedler TL , Marrazzo JM ( 2005 ) Molecular identification of bacteria associated with bacterial vaginosis . N Engl J Med 353 : 1899 - 1911 .
7. Mitchell C , Moreira C , Fredricks D , Paul K , Caliendo AM , et al. ( 2009 ) Detection of fastidious vaginal bacteria in women with HIV infection and bacterial vaginosis . Infect Dis Obstet Gynecol 2009 : 236919 .
8. Bukusi EA , Cohen CR , Meier AS , Waiyaki PG , Nguti R , et al. ( 2006 ) Bacterial vaginosis: risk factors among Kenyan women and their male partners . Sex Transm Dis 33 : 361 - 367 .
9. Martin HL , Richardson BA , Nyange PM , Lavreys L , Hillier SL , et al. ( 1999 ) Vaginal lactobacilli, microbial flora, and risk of human immunodeficiency virus type 1 and sexually transmitted disease acquisition . J Infect Dis 180 : 1863 - 1868 .
10. Sewankambo N , Gray RH , Wawer MJ , Paxton L , McNaim D , et al. ( 1997 ) HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis . Lancet 350 : 546 - 550 .
11. Atashili J , Poole C , Ndumbe PM , Adimora AA , Smith JS ( 2008 ) Bacterial vaginosis and HIV acquisition: a meta-analysis of published studies . AIDS 22 : 1493 - 1501 .
12. Coleman JS , Hitti J , Bukusi EA , Mwachari C , Muliro A , et al. ( 2007 ) Infectious correlates of HIV-1 shedding in the female upper and lower genital tracts . AIDS 21 : 755 - 759 .
13. Sha BE , Zariffard MR , Wang QJ , Chen HY , Bremer J , et al. ( 2005 ) Female genital-tract HIV load correlates inversely with Lactobacillus species but positively with bacterial vaginosis and Mycoplasma hominis . J Infect Dis 191 : 25 - 32 .
14. Wang CC , McClelland RS , Reilly M , Overbaugh J , Emery SR , et al. ( 2001 ) The effect of treatment of vaginal infections on shedding of human immunodeficiency virus type 1 . J Infect Dis 183 : 1017 - 1022 .
15. Hashemi FB , Ghassemi M , Faro S , Aroutcheva A , Spear GT ( 2000 ) Induction of human immunodeficiency virus type 1 expression by anaerobes associated with bacterial vaginosis . J Infect Dis 181 : 1574 - 1580 .
16. Simoes JA , Hashemi FB , Aroutcheva AA , Heimler I , Spear GT , et al. ( 2001 ) Human immunodeficiency virus type 1 stimulatory activity by Gardnerella vaginalis: relationship to biotypes and other pathogenic characteristics . J Infect Dis 184 : 22 - 27 .
17. Clemetson DB , Moss GB , Willerford DM , Hensel M , Emonyi W , et al. ( 1993 ) Detection of HIV DNA in cervical and vaginal secretions. Prevalence and correlates among women in Nairobi, Kenya . JAMA 269 : 2860 - 2864 .
18. Rebbapragada A , Howe K , Wachihi C , Pettengell C , Sunderji S , et al. ( 2008 ) Bacterial vaginosis in HIV-infected women induces reversible alterations in the cervical immune environment . J Acquir Immune Defic Syndr 49 : 520 - 522 .
19. Celum C , Wald A , Lingappa JR , Magaret AS , Wang RS , et al. ( 2010 ) Acyclovir and transmission of HIV-1 from persons infected with HIV-1 and HSV-2 . N Engl J Med 362 : 427 - 439 .
20. Nugent RP , Krohn MA , Hillier SL ( 1991 ) Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation . J Clin Microbiol 29 : 297 - 301 .
21. Forsum U , Jakobsson T , Larsson PG , Schmidt H , Beverly A , et al. ( 2002 ) An international study of the interobserver variation between interpretations of vaginal smear criteria of bacterial vaginosis . APMIS 110 : 811 - 818 .
22. Campbell MS , Mullins JI , Hughes JP , Celum C , Wong KG , et al. ( 2011 ) Viral linkage in HIV-1 seroconverters and their partners in an HIV-1 prevention clinical trial . PLoS One 6 : e16986. doi:10.1371/journal.pone. 0016986
23. Lingappa JR , Baeten JM , Wald A , Hughes JP , Thomas KK , et al. ( 2009 ) Daily acyclovir for HIV-1 disease progression in people dually infected with HIV-1 and herpes simplex virus type 2: a randomised placebo-controlled trial . Lancet 375 : 824 - 833 .
24. Sahai H ( 1996 ) Statistics in epidemiology: methods, techniques, and applications . Sahai H, Khurshid A, editors. Boca Raton (Florida): CRC Press .
25. Baeten JM , Kahle E , Lingappa JR , Coombs RW , Delany-Moretlwe S , et al. ( 2011 ) Genital HIV-1 RNA predicts risk of heterosexual HIV-1 transmission . Sci Transl Med 3 : 77ra29 .
26. Tanton C , Weiss HA , Le Goff J , Changalucha J , Rusizoka M , et al. ( 2011 ) Correlates of HIV-1 genital shedding in Tanzanian Women . PLoS One 6 : e17480. doi: 10.1371/journal.pone. 0017480
27. Moreira C , Venkatesh KK , DeLong A , Liu T , Kurpewski J , et al. ( 2009 ) Effect of treatment of asymptomatic bacterial vaginosis on HIV-1 shedding in the genital tract among women on antiretroviral therapy: a pilot study . Clin Infect Dis 49 : 991 - 992 .
28. Cohen CR , Plummer FA , Mugo N , Maclean I , Shen C , et al. ( 1999 ) Increased interleukin-10 in the the endocervical secretions of women with non-ulcerative sexually transmitted diseases: a mechanism for enhanced HIV-1 transmission? AIDS 13 : 327 - 332 .
29. Bukusi EA , Thomas K , Nguti R , Cohen CR , Weiss N , et al. ( 2011 ) Topical penile microbicide use by men to prevent recurrent bacterial vaginosis in sex partners: a randomized clinical trial . Sex Transm Dis 38 : 483 - 489 .
30. Gray RH , Kigozi G , Serwadda D , Makumbi F , Nalugoda F , et al. ( 2009 ) The effects of male circumcision on female partners' genital tract symptoms and vaginal infections in a randomized trial in Rakai, Uganda . Am J Obstet Gynecol 200 : 42 e41- e47 .
31. Price LB , Liu CM , Johnson KE , Aziz M , Lau MK , et al. ( 2010 ) The effects of circumcision on the penis microbiome . PLoS One 5 : e8422. doi:10.1371/ journal.pone. 0008422
32. Donoval BA , Landay AL , Moses S , Agot K , Ndinya-Achola JO , et al. ( 2006 ) HIV-1 target cells in foreskins of African men with varying histories of sexually transmitted infections . Am J Clin Pathol 125 : 386 - 391 .
33. de Jong MA , Geijtenbeek TB ( 2009 ) Human immunodeficiency virus-1 acquisition in genital mucosa: Langerhans cells as key-players . J Intern Med 265 : 18 - 28 .
34. Klebanoff MA , Schwebke JR , Zhang J , Nansel TR , Yu KF , et al. ( 2004 ) Vulvovaginal symptoms in women with bacterial vaginosis . Obstet Gynecol 104 : 267 - 272 .
35. Amsel R , Totten PA , Spiegel CA , Chen KC , Eschenbach D , et al. ( 1983 ) Nonspecific vaginitis . Diagnostic criteria and microbial and epidemiologic associations . Am J Med 74 : 14 - 22 .
36. Baeten JM , Strick LB , Lucchetti A , Whittington WL , Sanchez J , et al. ( 2008 ) Herpes simplex virus (HSV)-suppressive therapy decreases plasma and genital HIV-1 levels in HSV-2/HIV-1 coinfected women: a randomized, placebocontrolled, cross-over trial . J Infect Dis 198 : 1804 - 1808 .
37. Eschenbach DA ( 1993 ) History and review of bacterial vaginosis . Am J Obstet Gynecol 169 : 441 - 445 .
38. Greenblatt RM , Bacchetti P , Barkan S , Augenbraun M , Silver S , et al. ( 1999 ) Lower genital tract infections among HIV-infected and high-risk uninfected women: findings of the Women's Interagency HIV Study (WIHS). Sex Transm Dis 26 : 143 - 151 .
39. Bradshaw CS , Morton AN , Hocking J , Garland SM , Morris MB , et al. ( 2006 ) High recurrence rates of bacterial vaginosis over the course of 12 months after oral metronidazole therapy and factors associated with recurrence . J Infect Dis 193 : 1478 - 1486 .
40. Tabrizi SN , Fairley CK , Bradshaw CS , Garland SM ( 2006 ) Prevalence of Gardnerella vaginalis and Atopobium vaginae in virginal women . Sex Transm Dis 33 : 663 - 665 .
41. Verstraelen H , Verhelst R , Vaneechoutte M , . Temmerman M The epidemiology of bacterial vaginosis in relation to sexual behaviour . BMC Infect Dis 10 : 81 .
42. McClelland RS , Richardson BA , Hassan WM , Chohan V , Lavreys L , et al. ( 2008 ) Improvement of vaginal health for Kenyan women at risk for acquisition of human immunodeficiency virus type 1: results of a randomized trial . J Infect Dis 197 : 1361 - 1368 .
43. Anukam KC , Osazuwa E , Osemene GI , Ehigiagbe F , Bruce AW , et al. ( 2006 ) Clinical study comparing probiotic Lactobacillus GR-1 and RC-14 with metronidazole vaginal gel to treat symptomatic bacterial vaginosis . Microbes Infect 8 : 2772 - 2776 .
44. Bolton M , van der Straten A , Cohen CR ( 2008 ) Probiotics: potential to prevent HIV and sexually transmitted infections in women . Sex Transm Dis 35 : 214 - 225 .
45. Hemmerling A , Harrison W , Schroeder A , Park J , Korn A , et al. ( 2010 ) Phase 2a study assessing colonization efficiency, safety, and acceptability of Lactobacillus crispatus CTV-05 in women with bacterial vaginosis . Sex Transm Dis 37 : 745 - 750 .
46. Swidsinski A , Mendling W , Loening-Baucke V , Ladhoff A , Swidsinski S , et al. ( 2005 ) Adherent biofilms in bacterial vaginosis . Obstet Gynecol 106 : 1013 - 1023 .
47. Swidsinski A , Mendling W , Loening-Baucke V , Swidsinski S , Dorffel Y , et al. ( 2008 ) An adherent Gardnerella vaginalis biofilm persists on the vaginal epithelium after standard therapy with oral metronidazole . Am J Obstet Gynecol 198 : 97 e91- 96 .
48. Thoma ME , Gray RH , Kiwanuka N , Aluma S , Wang MC , et al. ( 2011 ) The Short-term Variability of Bacterial Vaginosis Diagnosed by Nugent Gram Stain Criteria Among Sexually Active Women in Rakai, Uganda . Sex Transm Dis 38 : 111 - 116 .
49. Wawer MJ , Sewankambo NK , Serwadda D , Quinn TC , Paxton LA , et al. ( 1999 ) Control of sexually transmitted diseases for AIDS prevention in Uganda: a randomised community trial . Rakai Project Study Group. Lancet 353 : 525 - 535 .
50. Hemmerling A , Cohen CR ( 2011 ) Probiotics: the potential for a live microbicide to prevent HIV . J Acquir Immune Defic Syndr 56 : e98 - 101 .
51. MacPhee RA , Hummelen R , Bisanz JE , Miller WL , Reid G ( 2010 ) Probiotic strategies for the treatment and prevention of bacterial vaginosis . Expert Opin Pharmacother 11 : 2985 - 2995 .