Hindawi Publishing Corporation
Infectious Diseases in Obstetrics and Gynecology
Volume 2009, Article ID 236919, 6 pages
Detectionof Fastidious VaginalBacteria inWomenwith
Angela M.Caliendo,4,5JaclynnKurpewski,2JessicaIngersoll,4,5and SusanCu-Uvin2
1Department of Obstetrics & Gynecology, University of Washington, P.O. Box 356460, Seattle, WA 98195, USA
2Department of Obstetrics & Gynecology, Brown University, Providence, RI 02912, USA
3Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
4Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA 30322, USA
5Emory Center for AIDS Research, Emory University, Atlanta, GA 30322, USA
Correspondence should be addressed to Caroline Mitchell, firstname.lastname@example.org
Received 6 January 2009; Accepted 6 October 2009
Recommended by Harold Wiesenfeld
of these bacteria in HIV-1 infected women and their relationship with vaginal pH and shedding of HIV-1 RNA. Methods. 64
cervicovaginal lavage (CVL) samples were collected from 51 women. Vaginal microbiota were characterized using 8 bacterium-
and 3 showed a trend to increased HIV-1 shedding (OR 2.59–3.07, P = .14–.17). Absence of Lactobacillus crispatus (P < .005) and
presence of BVAB2 (P < .001) were associated with elevated vaginal pH. BVAB1, 2, and 3 were highly specific indicators of BV in
of BV in HIV-infected women, and BVAB2 may contribute to the elevated vaginal pH that is a hallmark of this syndrome.
Copyright © 2009 Caroline Mitchell et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Bacterial vaginosis (BV) is a vaginal infection characterized
by loss of the normal protective lactobacilli and overgrowth
of diverse anaerobes [1, 2]. This infection is one of the
leading causes of vaginal discharge  and is more prevalent
in HIV-1-infected women compared to uninfected women
. The microbiology of BV is heterogeneous, and culture-
based description of the vaginal microbiota identifies far
fewer organisms than broad range molecular methods [5, 6].
Several fastidious bacteria in the Clostridiales order have
appear to be highly specific markers of BV .
BV has been associated with increased genital HIV
shedding , having high concentrations of Gardnerella
vaginalis and Mycoplasma hominis , but the mechanism
accounting for this association is poorly understood. In
HIV-1-infected women, polymerase chain reaction (PCR)
methods have shown that high concentrations of Gardnerella
vaginalis and Mycoplasma hominis are sensitive indicators
for the diagnosis of BV . It is possible that some
of the proinflammatory vaginal cytokines seen in subjects
with BV [12, 13] increase production of the virus or
stimulate epithelial turnover and microtears in the vaginal
wall that facilitate viral shedding. A second hypothesis is
that loss of hydrogen peroxide-producing lactobacilli and
subsequent increase in vaginal pH results in loss of viral
inhibition . Lactobacilli and G. vaginalis have been
associated with changes in vaginal pH , but little is
known about the effect of the fastidious organisms present
We hypothesize that fermentation products from key
vaginal anaerobes may alter the vaginal pH and facilitate
production and shedding of HIV. We sought to determine
the prevalence of several fastidious vaginal bacteria in HIV-
infected women and to assess the impact of these bacterial
species on (1) vaginal pH and (2) shedding of HIV-1 RNA in
women with and without BV.
2Infectious Diseases in Obstetrics and Gynecology
2.1. Sample Collection and Characterization. This was a
secondary analysis of samples collected as part of a
prospective cohort study of nonpregnant HIV-1-infected
women in Providence, Rhode Island, evaluating the effect
of antiretroviral therapy (ART) on HIV-1 RNA shedding in
the female genital tract . In the parent study, women
were seen at baseline, 2 weeks, one month, and every 6
months for 36 months. All patients gave informed consent
to participate in the study; the study protocol was approved
by the Institutional Review Board of The Miriam Hospital,
Providence, RI. At each visit clinical data and exam findings
were recorded, and blood and cervicovaginal lavage (CVL)
samples were collected. Women were asked not to douche,
have sex, or use any intravaginal product for the 48 hours
prior to the visit. The CVL was collected by infusing 10
cc of normal saline into the vagina with a syringe and
then aspirating back the fluid. For this subanalysis samples
were randomly selected to represent approximately equal
numbers of samples from women with and without BV,
and from women who were on antiretroviral therapy and
those who were not. Not all selected samples were available
for analysis, and so the final numbers in each category are
not exactly equal (e.g., 34 samples from subjects with BV
and 30 samples from subjects without BV). Some women
contributed samples from more than one visit, but each
equations were used in the statistical analysis to account for
Bacterial vaginosis was diagnosed using Amsel’s clinical
criteria : clinicians assessed vaginal discharge, pH, and
presence of clue cells and fishy odor with addition of KOH
and patients with at least 3 of 4 criteria were considered
to have BV. Trichomonas and Candida vaginitis were also
diagnosed clinically using wet mount. Previous studies in
this population had shown that the prevalence of syphilis,
done annually . HIV-1 RNA was quantified using the
nucleic acid sequence-based assay (Nuclisens, bioMerieux,
Durham, NC), with a sensitivity of 400copies/mL. Cells
in the vaginal fluid were classified by an experienced
Medical Technologist as white blood cells, red blood cells,
or nucleated cells (primarily vaginal epithelial cells). Manual
cell counts were performed on a hemocytometer, counting
cells in five 1mm squares and averaging the results.
2.2. Sample Processing and Testing. The CVL sample was
the supernatant removed. The remaining pellet underwent
DNA extraction with the MoBio UltraClean Soil DNA
Isolation Kit (MoBio,Calsbad, CA) following the manufac-
turer’s instructions. A clean swab was taken through the
DNA extraction process as a negative extraction control.
All extracted DNA samples and the extraction control were
tested in a quantitative PCR using primers targeting the
human 18S rRNA gene to validate that successful DNA
extraction occurred. An internal amplification control PCR
using exogenous DNA from a jellyfish gene was used to
test for presence of PCR inhibitors . The presence of
inhibition is defined as an internal amplification control
qPCR threshold cycle value that was 2cycles higher than that
of the no-template control.
Patient samples were then subjected to eight separate
taxon-directed 16S rRNA gene quantitative PCR assays for
the detection and quantification of individual bacteria: Lac-
Leptotrichia/Sneathia, Megasphaera, and Bacterial Vaginosis-
Associated Bacterium (BVAB) 1, BVAB2, and BVAB3. One
which are closely related. The BVAB are related to bac-
teria in the Clostridiales order and have been found to
be highly specific for BV . Each assay has previously
been validated and proven to be sensitive (to a level of
1–10 DNA copies/reaction, or 150–1500copies/mL) and
specific (does not detect other bacteria at a concentration of
106copies/rxn) . The assays use a TaqMan format and
Foster City, CA). Plasmids containing bacterial 16S rRNA
genes were used to generate standard curves for quantifi-
cation. The standards were generated by cloning bacterial
16S rRNA genes into E. coli and then purifying plasmids.
The plasmids were quantified using a fluorimeter and the
Quant-iT Pico Green assay kit (Invitrogen, Carlsbad, CA) to
determine the number rRNA gene copies per microliter.
2.3. Statistical Analysis. All analyses were carried out using
Stata v9.2 (StataCorp, College Station, Texas). Demographic
variables were compared between groups using the Mann-
Whitney U-test for continuous variables and Independent
samples t -test for categorical variables. Log-transformed
concentrations of bacteria were compared between women
with and without BV using a t-test. The relationships
between presence and absence of bacteria and detection of
HIV and sensitivity and specificity for diagnosis of BV were
modeled using logistic regression and generalized estimating
equations to account for repeat measures. Linear regression
was used to model the relationship between bacterial species
and continuous variables such as quantity of nucleated
cells. For all regression analyses, the method of generalized
estimating equations (GEEs) with robust standard errors
was used to account for residual correlation due to the fact
that some observations were repeated measures on the same
women over time. Our power calculation was based on the
70 samples initially selected, and we estimated an 80% power
to detect a twofold increase in the rate of HIV-1 shedding in
women with detectable BV-associated bacteria.
Sixty-four CVL samples from 51 women were analyzed.
One woman contributed three samples, and eleven women
contributed two samples. When comparing only the first
sample from each subject, 24 women with BV and 27 women
without BV were similar in terms of age, race, time since
HIV-1 diagnosis, antiretroviral treatment status, CD4 count,
and plasma viral load (Table 1). Initially 65 samples were
processed, but one sample did not contain adequate material
Infectious Diseases in Obstetrics and Gynecology3
Table 1: Characteristics of the study population (N = 51§).
BV+ (N = 24)
35.7 ± 8.6
BV −(N = 27)
38.2 ± 6.5
Time since HIV diagnosis
CD4 Count (median, IQR) cells/mL
Plasma viral load (median, IQR) copies/mL
Vaginal fluid cells (cells/cc)
Log-transformed bacterial 16S rDNA copies/mL (mean ± SD)
7.4 ± 4.8
6.8 ± 4.0
961 ± 1296
39 ± 41
2.9 ± 5.4
0.8 ± 1.6
1.2 ± 1.8
752 ± 1034
676 ± 2458
0.2 ± 0.6
0.5 ± 1.3
0.3 ± 1.0
6.93 ± 1.74
2.36 ± 3.05
7.21 ± 1.82
3.56 ± 3.66
5.82 ± 3.59
2.06 ± 3.51
4.22 ± 3.61
2.38 ± 3.15
6.71 ± 2.08
2.43 ± 3.30
3.70 ± 3.04
0.56 ± 1.71
2.12 ± 3.23
0.50 ± 1.85
0.48 ± 1.50
0.50 ± 1.50
§For individuals who contributed more than one sample, only data from the first visit are included in this table.
∗Tests are chi-square for categorical variables, and t-test or Mann-Whitney for continuous variables.
Data presented as mean ± SD for continuous variables or n(%) for categorical variables unless otherwise noted.
for analysis. All other samples had detectable DNA extracted,
and no evidence of PCR inhibition.
Sixty (94%) of the 64 samples had any lactobacilli
detected by genus specific PCR, but only 23 (37%) had
Lactobacillus crispatus detected. Gardnerella vaginalis was
detected in 84% of samples, Megasphaera in 31%, Lep-
totrichia/Sneathia in 59%, BVAB1 in 19%, BVAB2 in 41%,
and BVAB3 in 27%. As in previous studies, not all bacteria
were detected in all women with BV, and the novel bacteria
BVAB1, BVAB2, and BVAB3 were detected in some women
without BV though at a lower prevalence. Furthermore,
the concentrations of Leptotrichia/Sneathia, Megasphaera,
BVAB1, BVAB2, and BVAB3 were significantly lower in
women without BV compared to women with BV (Table 1).
After controlling for plasma viral load there were no
significant associations between HIV-1 RNA in the genital
tract and detection of any individual bacteria; however there
were trends toward increased HIV shedding in subjects with
BVAB 1, 2, and 3 (OR 2.59–3.07, p = .14–.17) irrespective
of BV diagnosis (Table 2). The largest odds ratio, while still
not significant, was for an association of vaginal HIV-1
RNA with detection of Gardnerella vaginalis (OR 17.2, 95%
CI 0.48,619). There were no significant differences in the
quantities of individual bacteria between women with CD4
counts >500cells/mL, 200–500cells/mL, and <200cells/mL
(data not shown).
Novel bacteria in the Clostridiales order-designated
of BV in HIV infected women, with specificities of 89%–
93% (Table 3). However, BVAB2 is a more sensitive indicator
of BV (63%) and when detection of either BVAB2 or
Megasphaera is used as a diagnostic criterion sensitivity
improves to 71%. As has been established in other studies
, high concentrations of Gardnerella vaginalis are also
sensitive for the diagnosis of BV and more specific than the
mere presence of the organism.
Higher concentrations of Lactobacillus crispatus were
associated with a lower vaginal pH. Higher concentrations
of Gardnerella vaginalis, Leptotrichia/Sneathia, Megasphaera,
BVAB1, BVAB2, and BVAB3 were all associated with an
increase in vaginal pH (Table 4). In a multivariate model,
quantities of Lactobacillus crispatus, Gardnerella vaginalis,
and BVAB2 are most independently predictive of vaginal pH
(p = .004, p = .047, and p < .001, resp.). The presence
of any Lactobacillus species was associated with a significant
decrease in number of nucleated cells (primarily epithelial
4Infectious Diseases in Obstetrics and Gynecology
Table 2: Odds ratio for having detectable HIV-1 in cervicovaginal lavage when individual bacteria are detected by PCR.
(any versus none)
OR 95% CI
aNumber of women with bacteria detected by qPCR.
bCannot be computed, because only 4 women had no lactobacilli, and all had detectable cervicovaginal HIV shedding (OR would be infinity).
Table 3: Sensitivity and specificity of detection of individual and combinations of bacteria for diagnosis of bacterial vaginosis compared to
Amsel’s clinical criteria.
OR (95% CI)
1.8 (0.15, 20.8)
1.2 (0.39, 3.75)
10.9 (1.3, 94)
6.3 (1.8, 21)
8.3 (2.0, 35)
5.4 (0.99, 29)
13.9 (3.2, 60)
5.0 (1.2, 21)
14.6 (3.7, 58)
13.8 (3.6, 53)
Any Lactobacillus spp.
Any Lactobacillus crispatus
Any Gardnerella vaginalis
Any Megasphaera spp
Any Megasphaera spp. OR BVAB2
High Gardnerella vaginalis (>median)
quantity of nucleated cells in the CVL and between concentration of bacterial species and pH of the vaginal secretions.
Nucleated cells pH
(model with presence/absence bacterial species)
(model with concentration of bacterial species)
cells) in CVL (p = .001), while Gardnerella vaginalis was
associated with a significant increase (p = .015) (Table 4).
BV in HIV-1-infected women. Detection of the fastidious
bacterium BVAB2 in combination with Megasphaera species
provides a reasonably sensitive marker for the diagnosis of
The impact of bacterial vaginosis on vaginal health may
increase in women with BV  which may cause epithelial
damage or recruit immune cells capable of HIV replication.
Infectious Diseases in Obstetrics and Gynecology5
However, the association between Gardnerella vaginalis and
the presence of more nucleated cells in the vagina suggests
that the BV-associated biofilm, of which G. vaginalis is
a significant component, may cause direct effects on the
epithelial surface and may be a mechanism to facilitate entry
or shedding of HIV.
It is not surprising that the absence of L. crispatus is
associated with higher vaginal pH, but the significant effect
of the BVABs on vaginal pH is a new finding. Given the
strongly significant P values for this finding in spite of the
small sample size, it is likely that this is a true biologic
phenomenon that may be related to fermentation products
produced by these fastidious bacteria as well as G. vaginalis,
Megasphaera, Leptotrichia, and Sneathia spp . Lower pH
has been shown to inactivate HIV-1 ; thus any organism
that increases the normally low vaginal pH may facilitate
replication and proliferation of HIV-1.
We did not see a relationship between quantities of any
individual bacteria and vaginal shedding of HIV, which is in
contrast to a previous study that looked at the relationship
between concentrations of Lactobacillus species, Gardnerella
vaginalis, and Mycoplasma hominis . That study had
significantly more women, all of whom had a log higher
detectable plasma viral load than the women in our study
and none of whom were on highly active ART, thus making
it significantly more likely that they would have detectable
vaginal HIV shedding and power to detect associations. As
our understanding of the bacterial diversity of BV grows,
it seems less likely that one individual bacterium will be
suspect that the loss of protective lactobacilli with a resulting
by anaerobes work together to produce negative effects such
as increased shedding of HIV-1 .
There are several limitations to this study. The small
in individual women result in limited power to detect
relationships between individual bacteria and HIV shedding.
Although individual samples were selected randomly, not
all samples were available and thus this analysis does not
represent a true random sampling of the parent cohort. The
lack of an HIV-1-uninfected control group means that these
results may not be generalizable to women without HIV
infection. However, the prevalence and quantities of bacteria
described in this population are similar to those reported
in an HIV-1 negative population, as is the heterogeneity
between individuals . One difference in this cohort is that
the prevalence and concentration of Lactobacillus crispatus
may have unique features. Additionally, BV was diagnosed
by clinical criteria, while Gram-stain criteria are frequently
used as a gold standard in research. In this type of analysis
we feel that the clinical diagnostic criteria are quite relevant.
Since the Gram stain criteria give a higher score to women
with more G. vaginalis morphotypes , it would not be
surprising to find that women with BV diagnosed by Gram
stain have higher concentrations of G. vaginalis using PCR.
Thus, comparing bacterial qPCR results to clinical criteria
is a more independent comparison. The contribution of
other genital tract infections to these results is difficult to
assess. Coinfection with gonorrhea, Chlamydia, or syphilis
waspreviouslyseento be verylow in this cohort butwas
not measured at each visit. The use of wet mount to diagnose
yeast or Trichomoniasis has low sensitivity [26, 27] and may
have misdiagnosed some women.
Several bacteria found in HIV-1-infected women with
BV may impact HIV shedding through their impact on
promoting turnover of nucleated vaginal epithelial cells
(Gardnerella vaginalis) or by increasing vaginal pH (BVAB2).
There was a trend suggesting that BVAB1, BVAB2, and
BVAB3 may be associated with increased shedding of HIV-
1, but this hypothesis will need to be confirmed or refuted in
This research was supported by NIH/NIAID RO1 AI40350
(SCU), K24AI066884 (SCU), Centers for AIDS Research
at Lifespan/Brown/Tufts Center (P30AI42853) (SCU), and
Emory University (P30AI050409) (AC) and R01AI061628
(DF). Dr. Mitchell is supported by the NICHD Women’s
Reproductive Health Research Career Development Award
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