REVIEW

Risk of Bacterial Vaginosis Among Women With

Herpes Simplex Virus Type 2 Infection: A
Systematic Review and Meta-analysis
Allahna Esber,1 Rodolfo D. Vicetti Miguel,2,3 Thomas L. Cherpes,2,3 Mark A. Klebanoff,4,6 Maria F. Gallo,1 and
Abigail Norris Turner5
1
Division of Epidemiology, 2Department of Microbial Infection and Immunity, 3Department of Obstetrics and Gynecology, 4Department of Pediatrics,
5
Division of Infectious Diseases, Department of Internal Medicine, Ohio State University, and 6Research Institute at Nationwide Childrens Hospital,
Columbus, Ohio

Background. Bacterial vaginosis (BV) is a perturbation of vaginal ora characterized by reduced levels of lacto-
bacilli and concomitant overgrowth of anaerobic bacterial species. BV is highly prevalent and associated with mul-
tiple adverse outcomes, including enhanced human immunodeciency virus transmission. Because recent reports
reveal that herpes simplex virus type 2 (HSV-2) infection may increase BV risk, we initiated a systematic review
and meta-analysis of the link between HSV-2 infection and BV.
Methods. We searched the MEDLINE, EMBASE, and CENTRAL databases to identify articles posted before 1
December 2014. Two screeners independently reviewed the titles and abstracts of all identied articles, reviewed the
full text of articles deemed potentially eligible, and extracted data from 14 cross-sectional and 3 prospective studies.
Using random-effects models, we computed separate pooled estimates for cross-sectional and prospective studies.
Results. The pooled odds ratio for cross-sectional studies was 1.60 (95% condence interval, 1.321.94). Stron-
ger support for the causal effect of HSV-2 infection on BV risk was revealed by the summary relative risk for the
prospective studies, which was 1.55 (95% condence interval, 1.301.84), with minimal heterogeneity (I 2 = 0).
Conclusions. These analyses imply that HSV-2 infection is an important BV risk factor. Pharmacologic HSV-2
suppression may reduce BV incidence and BV-associated adverse events.
Keywords. bacterial vaginosis; herpes simplex virus type 2; meta-analysis; systematic review.

Bacterial vaginosis (BV), a perturbation of vaginal ora
characterized by reduced levels of lactobacilli and over-
growth of Gardnerella, Prevotella, Mobiluncus, and other
anaerobic bacterial species, affects nearly 1 in 3 repro-
ductive-age women worldwide [1]. Clinically, BV is di-
agnosed by a constellation of signs and symptoms that
include malodorous vaginal discharge, increased vagi-
nal pH, and microscopic identication of vaginal epi-
thelial cells dotted with adherent coccobacilli (ie, clue
cells) (Amsel criteria) [2]. For research purposes, BV
Received 19 September 2014; accepted 22 December 2014; electronically pub-
lished 14 January 2015.
Correspondence: Abigail Norris Turner, PhD, Division of Infectious Diseases,
Department of Internal Medicine, The Ohio State University Medical Center, 410
W 10th Ave, Doan Hall, N-1144, Columbus, OH 43210 (ant@osumc.edu).
The Journal of Infectious Diseases 2015;212:817
The Author 2015. Published by Oxford University Press on behalf of the Infectious
Diseases Society of America. All rights reserved. For Permissions, please e-mail:
journals.permissions@oup.com.
DOI: 10.1093/infdis/jiv017
is frequently diagnosed by a Gram stainbased evalua-
tion of vaginal bacterial morphotypes, quantied using
a 10-point scoring system (Nugent score) [3].
BV is associated with numerous adverse outcomes,
including increased human immunodeciency virus
(HIV) transmission and, among pregnant women,
spontaneous abortion and premature delivery [1, 4, 5].
Although not a conventional sexually transmitted infec-
tion (STI), BV is associated with sexual activity and oc-
curs more often in women with greater numbers of sex
partners and higher frequency of intercourse [6]. Other
BV risk factors include nonuse of condoms, introduc-
tion of a new sex partner, history of STIs, and douching
[710]. The strongest predictor of BV in US women is
nonwhite race. Even after adjustment for other BV risk
factors [11], BV prevalence in the United States remains
highly dependent on race (52% and 23% in black and
white women, respectively) [12]. This racial disparity
suggests that an unknown risk factor (or factors) for

8 JID 2015:212 (1 July) Esber et al

BV is more common in black women than in white women.
One possible risk factor is genital herpes. Genital herpes is
most commonly caused by infection with herpes simplex
virus type 2 (HSV-2), which has a seroprevalence of 48% in
black women, compared with 16% in white women [13]. Be-
cause newer evidence indicates that HSV-2 infection promotes
loss of normal, lactobacillus-dominated vaginal ora [14, 15],
we conducted a systematic review and meta-analysis to evaluate
whether HSV-2 infection is a risk factor for BV.
METHODS
We included published reports on women who were BV-nega-
tive at baseline in longitudinal studies (observational studies or
randomized trials), which measured incident BV diagnosed via
Amsel criteria or Nugent score, among women who were HSV-
2 seropositive (diagnosed via type-specic serological assays),
compared with incident BV in women who were HSV-2 sero-
negative. We also included published, cross-sectional reports on
the association between HSV-2 serostatus (using type-specic
serological assays) and prevalent BV (determined on the basis
of the Amsel criteria or Nugent score), comparing BV preva-
lence among HSV-2infected women with BV prevalence in
women without HSV-2 infection.
Search Strategy
We initially identied studies by searching the electronic data-
bases MEDLINE via PubMed, EMBASE, and CENTRAL
(Cochrane Central Register of Controlled Trials) for articles
posted between 1 January 1983 (the year the Amsel criteria
were introduced) and 1 December 2014 (see Supplementary
Materials for full search terms) [2]. Two authors (A. E. and
A. N. T.) independently reviewed the titles and abstracts of all
resulting articles. Any article that appeared potentially relevant
by review of the title and abstract was retrieved for full-text re-
view to assess meta-analysis eligibility. To be eligible, studies
had to report a relevant measure of association for the prospec-
tive or cross-sectional association between HSV-2 infection and
BV or include the data necessary to permit its calculation. We
did not restrict inclusion by womens age, HIV status, pregnan-
cy status, or other potentially inuential covariates (eg, sexual
behavior and race), but we made note of these variables during
data extraction. We included studies in which BV status had
been assessed using the Amsel criteria or Nugent scoring. Be-
cause a substantial minority of genital herpes cases are caused
by HSV-1 rather than HSV-2, we included only those studies in
which HSV-2 infection was evaluated by type-specic serologic
assays [16]. We separately examined prospective studies in
which HSV-2 infection status was established at the study
start and incident BV was assessed later in follow-up and
cross-sectional studies in which HSV-2 and BV status were con-
comitantly measured. We hand-searched reference lists of
Systematic Review and Meta-analysis of HSV-2 and BV
included studies, but we did not include conference abstracts
or other reports not published in the peer-reviewed literature.
Data Abstraction
Two authors (A. E. and A. N. T.) independently extracted rele-
vant data from the selected studies into a spreadsheet, using a
piloted, standardized form, including: (1) participant char-
acteristics (ie, age, HIV status, and population [eg, general pop-
ulation or sex workers]), (2) study setting (ie, country, and
community based or clinic based), (3) method of BV diagnosis
(ie, Amsel criteria vs Nugent score), (4) method of HSV-2
diagnosis, (5) study size, (6) study design (ie, cross-sectional
or prospective), (7) frequency of BV evaluation, (8) confounders
included in adjusted models, (9) measure of association (ie, haz-
ard ratio [HR], odds ratio [OR], prevalence ratio [PR], risk ratio,
or incidence rate ratio [IRR]) and 95% condence intervals
(CIs), and (10) funding source. Any inconsistencies in extracted
data were resolved by discussion between the 2 reviewers.
Quality Assessment
We used the Newcastle-Ottawa quality assessment scale to assess
the risk of bias for each study [17]. The scale comprises 3 assess-
ment categories: selection, comparability, and outcome. The scale
includes 4 items to evaluate selection: (1) representativeness of the
exposed cohort, (2) selection of the nonexposed cohort, (3) ascer-
tainment of the exposure, and (4) demonstration that the outcome
of interest was not present at start of study. In our review, studies
were classied as representative on the basis of both eligibility cri-
teria and participation rate. We used 2 items to gauge compa-
rability: whether the study controlled for (a) sexual behavior
(including at least one measure of sexual frequency/partnerships
or condom use), and (b) key patient characteristics including
HIV (if both HIV-positive and HIV-negative women were eligible
to participate) and race (if the study population was multi-racial).
We assessed the nal Newcastle-Ottawa evaluation category, out-
come, by examining the measurement approach to detect the out-
come (BV via Nugent score or Amsel criteria), the length of
follow-up (at least 1 month, with HSV-2 and BV assessed at base-
line and at least 1 additional time point), and the adequacy of the
duration of follow-up (at least 80% retention).
Data Analysis
We analyzed the data using Stata 13 (Stata, College Station, Texas).
We ran separate analyses for the prospective and cross-sectional
studies. We used DerSimonianLaird random effects models,
which allow for between-study heterogeneity, to produce sum-
mary estimates. CIs were calculated using the Woolf method. Het-
erogeneity was assessed using I 2 [18]. Among the prospective
studies (n = 3), 1 reported a HR [14], 1 reported an IRR [15],
and 1 reported a risk ratio [19]. Similar to the approach used by
others (such as Atashili et al [20]), because these estimates are
all from multiplicative-scale models, we combined them with no
JID 2015:212 (1 July) 9
additional transformations [21], and the pooled estimate of the
prospective studies is reported as a summary relative risk. The
key measure of association for all included cross-sectional studies
was the OR, and the pooled estimate is a summary OR. We eval-
uated publication bias in the cross-sectional studies by using a fun-
nel plot and the Begg and Egger tests [22, 23].
Sensitivity Analyses
To assess the robustness of our ndings, we performed 5 sen-
sitivity or subgroup analyses on the pooled estimate of cross-
sectional studies. First, we used inuence analyses to estimate
the impact of each study on the summary estimate. Studies
were individually removed for subsequent recalculation of a
pooled estimate with a 95% CI; individual studies were deemed
excessively inuential if the magnitude of the recalculated sum-
mary estimate fell outside the 95% CI of summary estimates in
which it was included [24]. Second, we stratied the results by
study population (ie, clinic vs community based) to determine
whether the association between HSV-2 and BV varied by pop-
ulation type. Third, we included only studies that provided
adjusted estimates of association, to determine whether con-
founding had affected the pooled estimate in the main analy-
sis. Fourth, we restricted our analysis to studies found to be of
higher quality according to the Newcastle-Ottawa assessment
scale. Finally, because the Amsel criteria are less sensitive
than the Nugent score for BV diagnosis (70% vs 89%) [25],
we restricted our evaluation to the studies that used the Nugent
score, to examine the impact of diagnostic sensitivity on our
main analysis.
Figure 1. Flow of information through the different phases of the systematic review. Abbreviations: HSV-1, herpes simplex virus type 1; HSV-2, herpes
simplex virus type 2; OR, odds ratio.
10 JID 2015:212 (1 July) Esber et al
RESULTS
The results of this systematic review and meta-analysis are pre-
sented according to PRISMA (Preferred Reporting Items for
Systematic Reviews and Meta-analyses) guidelines [26].
Characteristics of Eligible Studies
The initial MEDLINE, EMBASE, and CENTRAL searches iden-
tied 404 studies; 66 were duplicates, and 225 were excluded for
nonrelevance to the research question, following review of their
title and abstract (Figure 1). We retrieved full-text articles for
the remaining 113 publications and excluded a further 96 arti-
cles that did not report a relevant measure of association or
include the data necessary to calculate one. From the 17 remain-
ing articles, 1 prospective study also presented a relevant cross-
sectional estimate of the HSV-2BV association using baseline
data, and 1 cross-sectional study provided 2 estimates from dif-
ferent populations. Ultimately, from the 17 eligible studies, we
extracted 19 measures of association [14, 15, 19, 2740]: 16 were
cross-sectional estimates, and 3 were prospective estimates. The
association between BV and HSV-2 was a primary outcome in
all but 1 included studies.
In the 3 prospective estimates [14, 15, 19], the mean time for
follow-up ranged from 8.5 months to 2.1 years, with follow-up
visits occurring every 3, 4, or 6 months. The population and lo-
cation of these studies also varied: Kaul et al [15] enrolled 443
female sex workers in Kenya, Cherpes et al [19] evaluated 773
sexually active women in 3 US clinics, and Nagot et al [14] in-
cluded 273 female sex workers in Burkina Faso (Table 1).
 Table 1. Summary of Studies of the Association Between Herpes Simplex Virus Type 2 and Bacterial Vaginosis (BV)

Population (All Measure of Estimate (95%

Study Country Design Women) HIV Status; Age Setting No. Adjustment Variables Effect CI)

Allsworth et al [27] US Cross- Nationally Positive and negative; Community 2326 Race/ethnicity, no. of sex partners in past year, douching OR 1.3 (1.11.6)
sectional representative 2049 y
Chawla et al [28] India Cross- Urban slum and Negative; 1549 y Community 120 Unadjusted OR .6 (.13.0)
sectional middle class
colony
Cherpes et al [29] US Cross- General Not tested; 1830 y Clinic 773 Race, age, smoking, douching, 5 male sex partners, uncircumcised OR 2.2 (1.53.2)
sectional partner, history of BV, gonorrhea, trichomoniasis, group B
streptococcal infection
Evans et al [30] United Cross- General Negative, but positive Clinic 520 Race, age, class, smoking, contraception, coitarche, no. of sex partners OR 3.1 (.910.5)
Kingdom sectional women were (past year and total), anal sex, sex with foreign partner, no nonregular
eligible; 1466 y sex partners, genital herpes, candidiasis, history of genital herpes
or PID, yeast
Kapiga et al [31] Tanzania Cross- Bar and hotel Positive and negative; Community 268 Religion, trichomoniasis, chlamydia, OR 1.8 (1.03.3)
sectional workers 1855 y syphilis
Kaul et al [15]a Kenya Cross- Sex workers Negative; 1852 y Community 443 Unadjusted OR .6 (.41.0)
sectional
Kirakaya- Burkina Cross- Pregnant Positive and negative; Clinic 2019 Age, marital status, contraceptive use, HIV infection, trichomoniasis OR 1.6 (1.02.6)
Samadoulougou Faso sectional 1549 y
et al [32]a
Systematic Review and Meta-analysis of HSV-2 and BV

Kirakaya- Burkina Cross- General Positive and negative; Community 883 Age, marital status, contraceptive use, HIV infection, trichomoniasis OR 3.7 (1.96.3)
Samadoulougou Faso sectional 1549 y
et al [32]a
Kirakaya- Burkina Cross- Pregnant Positive and negative; Clinic 2133 Polygamous status, province, parity, history of abortion, trichomoniasis, OR 1.6 (1.12.6)
Samadoulougou Faso sectional 1449 y candidiasis, HIV infection, syphilis
[33]
Madhivanan et al [34] India Cross- General Not tested; 1530 y Clinic 863 Age, religion, sex partner uses alcohol, sex under influence of alcohol, OR 1.5 (.92.4)
sectional tubal ligation, ever had oral sex, trichomoniasis
Masese et al [40] Kenya Cross- Sex workers Negative; 1850 y Community 406 Age OR 1.3 (1.11.6)
sectionalb
Msuya et al [39] Tanzania Cross- General Positive and negative; Clinic 382 Age, age of sexual debut, no. of sex partners, parity, history of STD, OR 1.3 (.72.3)
sectional 1549 y history of abortion, clinical signs, syphilis, HIV infection
Riedner et al [35] Tanzania Cross- Bar workers Positive and negative; Community 600 Examination round, HIV infection, workplace, duration of work as bar OR 1.0 (.33.5)
sectionalb 1639 y worker, change of residence 12 mo before enrollment
Uma et al [37] India Cross- Sex workers Positive and negative; Community 582 Unadjusted OR 1.9 (1.32.7)
sectional 1840 y
Uma et al [36] India Cross- Low income Positive and negative; Community 487 Unadjusted OR 2.3 (1.43.9)
sectional 1840 y
Wang et al [38] China Cross- Sex workers Positive and negative; Community 345 Age, duration of work as female sex worker, oral or vaginal sex with the OR 5.6 (1.226.9)
sectional 16 y last client, HIV infection

JID 2015:212 (1 July)

Cherpes et al [14] US Prospective General Not tested; 1830 y Clinic 773 Race, smoking, vaginal sex in past 4 months, vaginal sex after anal sex, HR 1.7 (1.32.3)
uncircumcised partner, lack of lactobacilli detected at prior visit, lack of
H202-producing lactobacilli detected at prior visit
Kaul et al [15]a Kenya Prospective Sex workers Negative; 1852 y Community 443 Sexual risk taking, antibiotic use IRR 1.4 (1.11.8)
Nagot et al [19] Burkina Prospective Sex workers Positive and negative; Community 273 No. of sex partners in preceding week, douching, hormonal RR 1.7 (1.12.7)
Faso 1654 y contraception, trichomoniasis, HIV infection

Abbreviations: CI, confidence interval; HIV, human immunodeficiency virus; HR, hazard ratio; IRR, incidence rate ratio; OR, odds ratio; PID, pelvic inflammatory disease; RR, relative risk.
a
The study by Kaul et al [15] contributed both a cross-sectional estimate and a prospective estimate to the meta-analysis. The study by Kirakaya-Samadoulougou et al [32] contributed 2 distinct estimates (one from a clinical setting and
another from a community setting) to the cross-sectional meta-analysis. As such these studies each appear twice here.

b
11

The overall design of these studies was prospective, but the estimates extracted for meta-analysis were cross-sectional, so these studies are categorized here as cross-sectional.
Figure 2. Forest plot of cross-sectional associations between herpes simples virus type 2 (HSV-2) infection and prevalent bacterial vaginosis (BV). The
study by Kaul et al [15] also contributed a prospective estimate to the meta-analysis, as shown in Table 1. The study by Kirakaya-Samadoulougou et al [32]
contributed 2 distinct estimates (one from a clinical setting and another from a community setting) to the cross-sectional meta-analysis and therefore
appears twice here. Abbreviations: CI, condence interval; OR, odds Ratio.

Among the 16 cross-sectional estimates, 4 were from studies
conducted in India, 3 each were conducted in Burkina Faso
and Tanzania, 2 each were conducted in the United States and
Kenya, and 1 each was conducted in the United Kingdom and
China. Only 2 studies were explicit about the inclusion of preg-
nant women [32, 33], while the others either excluded pregnant
women or did not specify the pregnancy status of participants.
The 4 estimates from studies that enrolled participants in the
United States or United Kingdom had participants from varied
racial backgrounds. One of these was a nationally representative
US sample [27], and the remaining 3 included predominantly
white women (with 70% [14], 73% [29], and 79% [30] of the par-
ticipants self-identifying as non-Hispanic white). All other studies
were conducted in Africa, Southeast Asia, or East Asia, where par-
ticipants were more likely to be from a single race group. Eleven of
the cross-sectional estimates allowed for enrollment of HIV-pos-
itive women [27, 28, 3033, 3639], with HIV prevalences ranging
from 0% [28] to 67% [35]. Two studies expressly excluded HIV-
positive women [15, 40], and the remaining 2 [29, 34] either did
not assess or did not describe assessment of HIV status.
Of the 16 cross-sectional estimates, 12 controlled for a variety of
demographic, sexual, and reproductive factors, with considerable
variation across studies in approaches to control confounding
(Table 1). For the remaining 4 estimates, we computed unadjust-
ed ORs by using the published tabular data. All 3 prospective es-
timates were presented with adjustment for a range of variables:
Kaul et al stratied by sexual risk taking and antibiotic use [15];
Cherpes et al adjusted for race, smoking status, vaginal inter-
course in the past 4 months, vaginal intercourse after anal inter-
course, male sex partner circumcision status, and an intermediate
Nugent score or lack of H202-producing lactobacilli at the prior
visit [14]; and Nagot et al adjusted for number of sex partners in
the preceding week, douching, hormonal contraception, HIV sta-
tus, and Trichomonas vaginalis coinfection [19].
BV Diagnosis
Most of the included studies diagnosed BV on the basis of the Nu-
gent score, with only 4 relying on the Amsel criteria [30, 31, 38, 39].
All 3 prospective estimates used the Nugent score for BV diagnosis.
Cross-sectional Associations Between HSV-2 and BV
The 16 cross-sectional estimates showed moderate to high het-
erogeneity (I 2 = 64%), ranging from an OR implying that HSV-
2 infection was associated with a reduced BV prevalence (0.6;

12 JID 2015:212 (1 July) Esber et al

 Table 2. Quality Assessment Using the Newcastle-Ottawa Scale

Selection Comparability Outcome

Representativeness Selection of Absence of Adjusted for

of the Exposed Nonexposed Exposure Outcome at Sexual Accounted for Outcome Length of Adequacy of
Study Cohort Cohort Ascertainment Baseline Behavior HIV and Race Assessment Follow-up Follow-up
Allsworth et al [27] + + + + + +
Chawla et al [28] + + + +
Cherpes et al [29] + + + +
Evans et al [30] + + + + + +
Kapiga et al [31] + + + + +
Kaul et al [15] (cross-sectional)a + + + +
Kirakaya-Samadoulougou et al + + + + + +
[32] (clinic)a

Systematic Review and Meta-analysis of HSV-2 and BV

Kirakaya-Samadoulougou et al + + + + + +
[32] (community)a
Kirakaya-Samadoulougou [33] + + + +
Madhivanan et al [34] + + + +
Masese et al [40] + + + + + +
Msuya et al [39] + + + + + +
Riedner et al [35] + + + + +
Uma et al [37] + + +
Uma et al [36] + + +
Wang et al [38] + + + + +
Cherpes et al [14] + + + + + + +
Kaul et al [15] (prospective)a + + + + + + + + +
Nagot et al [19] + + + + + + + +

The Newcastle-Ottawa scale includes the following quality-assessment criteria: (1) representativeness of cohort: was detailed eligibility criteria and information on participation rates provided? (2) selection of the
nonexposed cohort: were these individuals selected from the same study population? (3) exposure ascertainment: was herpes simplex virus type 2 (HSV-2) diagnosed by type-specific serological assay? (4) absence
of outcome at baseline: were participants confirmed to be BV negative at enrollment (prospective studies only)? (5) adjusted for sexual behavior: did analysis control for at least 1 measure of sexual behavior, including

sexual frequency, sex partnerships, or condom use? (6) accounted for HIV and race: did analysis measure key population characteristics including HIV (if both HIV-positive and HIV-negative women were eligible for the study)
JID 2015:212 (1 July)

and race (if the study population was multiracial)? (7) assessment of outcome: was BV diagnosed by the Amsel criteria or Nugent score? (8) length of follow-up: were participants followed for at least 1 month, with HSV-2 and
BV both assessed at baseline and at least 1 follow-up visit (prospective studies only)? and (9) adequacy of follow-up: did at least 80% of participants return for at least 1 follow-up visit (prospective studies only)? Cross-
sectional studies could receive a maximum of 6 pluses, and prospective studies could receive up to 9 pluses.
Abbreviations: BV, bacterial vaginosis; HIV, human immunodeficiency virus; HSV, herpes simplex virus; +, criterion was met; , criterion was not met.
a
The study by Kaul et al [15] contributed both a cross-sectional estimate and a prospective estimate to the meta-analysis. The study by Kirakaya-Samadoulougou et al [32] contributed 2 distinct estimates (one from a clinical
setting and another from a community setting) to the cross-sectional meta-analysis. As such these studies each appear twice here.
13
 Quality Assessment
In general, the quality of included cross-sectional estimates was
moderate to high, as assessed using the Newcastle-Ottawa scale
(Table 2). Nine cross-sectional estimates met at least 5 of the spec-
ied criteria (out of 6 items relevant to cross-sectional studies) and
were included in the sensitivity analysis restricted to higher-quality
studies [27, 3032, 35, 3840]. All but 3 of the cross-sectional esti-
mates [28, 34, 35] were deemed to have a low risk of bias stemming
from their funding, as the source was acknowledged or the authors
stated that they had no conicts of interest.

Figure 3. Funnel plot with pseudo 95% condence limits.
95% CI, .13.0) [28] to an OR suggesting that BV prevalence
was >5 times as high in HSV-2positive women, compared
with HSV-2negative women (5.6; 95% CI, 1.226.9) [38].
The pooled analysis of the cross-sectional studies yielded a sum-
mary OR of 1.60 (95% CI, 1.321.94; Figure 2), indicating that
the odds of prevalent BV was 60% greater among HSV-2
positive women, compared with HSV-2negative women.
Publication Bias
The funnel plot of cross-sectional estimates suggests that publi-
cation bias did not meaningfully affect the summary estimate
(Figure 3). The Begg test (P = .82) and the Egger test (P = .23)
similarly demonstrate that publication bias was unlikely to have
had a signicant impact on the cross-sectional analysis.
Sensitivity Analyses
The inuence analysis assessed the impact of each cross-
sectional estimate on the pooled estimate [24]. For each study
that was removed, the resulting estimate remained within the
CI of the original pooled estimate, suggesting that no study
was excessively inuential. Summary estimates in the inuence

Figure 4. Inuence analysis of cross-sectional studies. The solid lines at 1.60, 1.32, and 1.94 represent the primary pooled estimate and 95% condence
interval (CI) for all cross-sectional studies. The open circles indicate the pooled estimate when the named study is omitted and the corresponding lines are
the upper and lower limits of the 95% CI. The study by Kaul et al [15] also contributed a prospective estimate to the meta-analysis, as shown in Table 1. The
study by Kirakaya-Samadoulougou et al [32] contributed 2 distinct estimates (one from a clinical setting and another from a community setting) to the cross-
sectional meta-analysis and therefore appears twice here.

14 JID 2015:212 (1 July) Esber et al

Figure 5. Forest plot of prospective associations between herpes simplex virus type 2 (HSV-2) infection and incident bacterial vaginosis (BV). The study by
Kaul et al [15] also contributed a cross-sectional estimate to the meta-analysis, as shown in Table 1. Abbreviations: CI, condence interval; RR, relative risk.
analysis ranged from 1.51 to 1.69, values qualitatively similar to
the primary pooled estimate of 1.60 (Figure 4).
Four subgroup analyses were also performed with the cross-
sectional data. For the 6 clinic-based estimates, the pooled OR
was 1.72 (95% CI, 1.412.11; I 2 = 0%), similar in magnitude to
the pooled OR for the 10 community-based estimates (1.55;
95% CI, 1.182.05; I 2 = 74%). The difference in I2 by population
indicates that the heterogeneity in the overall pooled estimate is
driven by variability in the community-based studies. Inclusion
of only adjusted estimates in the cross-sectional analysis elimi-
nated 4 reports, but the summary OR in this sensitivity analysis
(1.65; 95% CI, 1.371.99; I 2 = 51%) was also comparable to that
in the primary analysis. Results of the sensitivity analysis re-
stricted to higher quality cross-sectional studies only (n = 9)
were also very similar to those of the primary analysis (1.61;
95% CI, 1.282.02; I 2 = 54%). Restricting the evaluation to esti-
mates using the Nugent score for BV diagnosis excluded 4 re-
ports, but the summary OR (1.55; 95% CI, 1.251.92; I 2 = 70%)
was again very similar to that from the primary analysis.
Prospective Associations Between HSV-2 and BV
Despite the considerable variability in the studies generating the 3
prospective estimates, including population, sample size, control
for confounding, and other factors, the adjusted estimates were
highly consistent, with an I 2 of 0% (Figure 5). Each demonstrated
that HSV-2infected women were at increased risk for incident
BV, with comparable values of 1.4 (95% CI, 1.11.8) in the study
by Kaul et al, 1.7 (95% CI, 1.32.3) in the study by Cherpes et al,
and 1.7 (95% CI, 1.12.7) in the study by Nagot et al (Table 1).
Pooled analysis of these estimates with a random effects model
yielded a summary estimate of 1.55 (95% CI, 1.301.84; Figure 5).
In other words, compared with HSV-2negative women, HSV-
2infected women had 55% increased risk of BV acquisition.
Systematic Review and Meta-analysis of HSV-2 and BV
All 3 estimates were judged to be of higher quality, based on sat-
isfying at least 7 of the 9 Newcastle-Ottawa scale criteria applica-
ble to prospective studies. All 3 also had low risk of bias due to
funding source or stated conicts of interest (Table 2).
DISCUSSION
In this systematic review and meta-analysis, HSV-2 infection
was signicantly associated with increased BV prevalence and
incidence. Moreover, despite variations in study populations,
BV diagnosis methods, and confounder adjustment, multiple
sensitivity analyses conrmed the robustness of these ndings.
Despite the consistency in our ndings, some limitations per-
sist when combining estimates to create summary pooled mea-
sures. While heterogeneity overall was much greater in the
cross-sectional compared to the prospective estimates, the sensi-
tivity analysis of cross-sectional estimates stratied by population
type suggested that the community-based studies were consider-
ably more heterogeneous than the clinic-based studies. This nd-
ing is not surprising, as diverse populations were recruited in the
community-based projects: 6 enrolled sex workers, and 4 en-
rolled from the general population. Despite these differences,
our inuence analysis conrmed that no study exerted excessive
inuence on the pooled estimate.
A greater number of prospective estimates in the meta-anal-
ysis would have generated a more precise pooled estimate and
would have permitted additional sensitivity analyses regarding
the robustness of our main ndings. Although an assessment of
publication bias is not possible with only 3 prospective reports,
the extremely low heterogeneity and highly comparable mea-
sures of association, despite differences in population and analy-
tic approaches, argue against the presence of signicant bias in the
pooled prospective estimate. Publication bias also appears not
JID 2015:212 (1 July) 15
to have impacted the summary estimate of the cross-sectional
studies. Of course, limitations in the design, conduct, measure-
ment, and analysis of the included reports may have adversely
affected the overall quality of the pooled estimates. For example,
one challenge in BV-related research is that many women cycle
between BV and normal ora on a daily or weekly basis [41]. The
absence of BV at enrollment into a prospective study assessing
the effect of HSV-2 infection on BV risk, while methodological-
ly necessary to measure BV incidence, does not exclude the pos-
sibility that some participants had BV that resolved prior to
enrollment. Prevalent BV may even lead to an increased risk
of HSV-2 acquisition [29], underscoring the complexity in as-
sessing the temporal association between these 2 highly preva-
lent vaginal infections.
While the quality of included studies was generally moderate
to high, we also identied limitations in some studies by using
the Newcastle-Ottawa scale. For example, approximately half
the studies did not meet the criterion for representativeness of
the exposed cohort, typically because the authors failed to pro-
vide data about participation rates, which could reveal how
many potential research subjects were screened and deemed in-
eligible for the research or how many individuals who were
found to be eligible went on to enroll in the study. Yet despite
variations in study quality, both the sensitivity analysis restrict-
ed to the higher-quality projects and the subgroup analysis pre-
senting separate estimates for clinic- and community-based
studies conrmed that the variability across studies did not
have a large impact on the summary estimate.
Our ndings are also limited because of the design of our re-
view. We chose not to include unpublished ndings presented
at conferences (the so-called gray literature). No approach exists
to systematically search the unpublished literature. In addition,
analyses presented in conference abstracts are (by requirement)
brief, providing considerably less opportunity to assess quality
or to extract all relevant information about the population and
analytic methods. Unpublished ndings may remain unpub-
lished because they are of lower quality than published studies,
and their inclusion in a systematic review may introduce bias
[42]. We also chose to limit this review to English-language-
only publications. While this restriction may have led to the ex-
clusion of potentially relevant ndings, prior research illustrates
that excluding nonEnglish-language reports does not lead to
signicant differences in the summary estimates reported in
meta-analyses [43]. We did allow for nonEnglish-language ab-
stracts and titles in our search strategies, and only 3 potentially
relevant articles were identied. We also did not contact study
authors for clarication or missing information, which in theo-
ry could have increased the number of studies included in the
review. However, we were generally able to nd information
missing from a given paper by consulting previously published
papers from the same authors on the same study population.
Given that we searched 3 databases (MEDLINE, EMBASE,
16 JID 2015:212 (1 July) Esber et al
and CENTRAL) and hand-searched reference lists of included
studies, we suggest that the likelihood is low that our summary
estimates are biased because of missing eligible studies.
Our ndings of a signicant association between HSV-2 in-
fection and BV are further supported by additional research not
directly contributing to our meta-analysis calculations. As ex-
amples, active genital shedding of HSV-2 is associated with in-
creased BV prevalence [44]. Compared with HSV-2-negative
women, HSV-2-positive women have reduced vaginal coloniza-
tion with H2O2-producing lactobacilli [45]. Finally, women
with HSV-2 are more likely to lose healthy vaginal lactobacilli
and less likely to successfully respond to standard antimicrobial
BV therapy [44, 46]. After antimicrobial therapy, BV is expected
to recur in about 10% of women overall within 1 month, in 30%
within 3 months, and in 50% within 1 year [47, 48]. However,
among HSV-2infected women, BV recurrence rates within 1
month of treatment appear to approach 90% [46].
Our ndings offer new indication that HSV-2 infection cre-
ates a vaginal environment that promotes loss of vaginal lacto-
bacilli or overgrowth of abnormal ora. Moreover, as the
fundamental characteristic of HSV-2 infection is intermittent
viral reactivation and epithelial shedding, we hypothesize that
local host responses to replicating virus are responsible for cre-
ating a vaginal environment that is inhospitable to healthy ora
and therefore are an underappreciated but important risk factor
for incident BV. If our hypothesis is correct, daily HSV-2 sup-
pressive therapy (a safe, available, and affordable treatment op-
tion) may help normalize vaginal ora, reduce BV incidence,
and improve the efcacy of antimicrobial BV therapy among
HSV-2infected women. Testing this hypothesis through pri-
mary research or secondary use of existing data from HSV-2
suppression trials may uncover a secondary prevention strategy
that could reduce the signicant racial disparities in BV preva-
lence and reduce the incidence of BV-associated adverse events.
Note
Potential conicts of interest. All authors: No reported conicts.
All authors have submitted the ICMJE Form for Disclosure of Potential
Conicts of Interest. Conicts that the editors consider relevant to the con-
tent of the manuscript have been disclosed.
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