Introduction
HIV-infected women outnumber men in many sub-Saharan African countries [1]. Novel
female-initiated preventive methods have, so far, proved elusive [2],[3], so identifying
new modifiable factors that affect women's vulnerability to HIV might help in the
development of new preventive interventions [4]. Women use a wide range of products,
applied in a variety of ways inside the vagina, to manage their sexual relationships,
menstruation, and to improve wellbeing [5].
It has been hypothesised that some intravaginal practices could increase the risk
of HIV infection by causing physical abrasions [4] or by disrupting the vaginal epithelium
and increasing the occurrence of bacterial vaginosis (BV) [6]–[8]. An association
between BV and HIV has been shown in cross-sectional and prospective studies [9],[10]
and, more recently, intermediate grades of vaginal flora have also been associated
with an increased risk of HIV acquisition [11]. However, the evidence linking intravaginal
practices, BV, and HIV infection is currently inconclusive [8],[12],[13], as is evidence
of associations between these practices and BV [12]. A recent systematic review found
that few prospective studies recorded intravaginal practices consistently and that
there was substantial heterogeneity between studies reporting associations between
intravaginal practices and HIV [12]. Even large individual studies lack statistical
power to examine the effects of specific intravaginal practices on HIV acquisition.
Combining individual participant data from different studies might overcome some of
these problems because data can be analysed consistently across studies and statistical
power and precision can be increased [14]. Our overall aim was to determine whether
specific vaginal practices increase the risk of a woman acquiring HIV infection by
facilitating disturbances in vaginal flora or vaginal epithelial disruption. The primary
objective of this study was to pool individual participant data from prospective longitudinal
studies to investigate the association between intravaginal practices and acquisition
of HIV infection among women in sub-Saharan Africa. Secondary objectives were to investigate
associations between intravaginal practices and disrupted vaginal flora; and between
disrupted vaginal flora and HIV acquisition.
Methods
The study protocol specified hypotheses, inclusion criteria, and methods of analysis,
and is available at http://www.ispm.ch/uploads/media/VP_IPD_protocol__final_090205_01.pdf.
The review was reported according to the guidelines of the Preferred Reporting Items
for Systematic Reviews and Meta-Analyses (PRISMA) group (Text S1).
Study Selection
Potentially eligible studies were cohort studies and randomised controlled trials
conducted in sub-Saharan Africa that had collected data prospectively about both intravaginal
practices and incident HIV infection in females aged 10 y or older. We did not consider
studies examining associations between disrupted vaginal flora and HIV unless data
on intravaginal practices were also collected. We excluded studies in which participants
were asked not to use intravaginal practices during the study or where the primary
vaginal practice was female genital cutting or genital surgery. We also excluded studies
involving vaginal microbicides or placebo products, tampons, or other devices to deliver
medication. However, the control group in such studies was eligible for inclusion
if they did not receive these interventions.
We identified studies using the results of a systematic review of published studies,
which has been described elsewhere [12]. We obtained published reports of all studies
included in the previous review and asked experts in the field to identify additional
studies that had collected data about both intravaginal practices and incident HIV
infections. We tried to contact the corresponding author or principal investigator
of all potentially eligible studies by e-mail to determine whether relevant data had
been collected and to invite them to contribute to the individual participant data
meta-analysis. We included eligible studies for which we had received both a signed
agreement to participate and a dataset by 16th March 2009. All included studies were
approved by relevant country-specific and institutional ethical review boards and
all participants within each study provided written informed consent for the original
studies.
Data Collection
We used protocols, questionnaires, and publications from included studies, and information
from investigators to determine whether requested variables had been collected or
could be derived. The final variable list included: HIV infection, intravaginal practices,
vaginal flora status, herpes simplex virus infection (HSV-2) status, age, education,
religion, marital status, employment, age at first sex, numbers of sex partners, sex
in exchange for money, pregnancy and contraception, and general condom use (consistent,
inconsistent, or never). A named investigator for each included study provided access
to the dataset, answered questions about the study procedures and coding of variables,
and contributed to interpretation of results and revising the manuscript. For most
studies we obtained raw data and recoded these in house (MFC, KS, or SCF). For two
studies, a data manager provided a dataset, coded according to our predefined scheme.
Outcome and Exposure Measures
The primary outcome was HIV infection diagnosed within the first 2 y, using the diagnostic
criteria defined by each individual study. We included women with a negative HIV test
at the baseline visit and at least one follow-up HIV test and estimated the date of
HIV infection as the midpoint between the last negative and the first positive test.
Intravaginal practices were the main exposures and were based on self-reported data
collected in face-to-face interviews in all but one study, which instead used audio
computer-assisted self-interviewing [15]. Definitions were based on a published classification
system [6]. After examining the data available in individual studies, we defined five
separate intravaginal practices (Box 1). We used baseline data about current practices
(that is, those from the 1 to 3 mo preceding the start of the study), since there
were too few studies reporting repeated measures of these variables to consider changes
in practices over time. The reference group was women not at increased risk of acquiring
HIV infection through intravaginal practices, and included women using no intravaginal
practice or water.
Box 1. Definitions of intravaginal practices used in this study
Intravaginal Practice Definition
Cleaning with water Cleaning inside the vagina, beyond the introitus, with water as
the only product. Can be with or without specific mention of fingers, other materials,
or douching devices to introduce water inside the vagina.
Cleaning with soap Cleaning inside the vagina, beyond the introitus, with generic
“soap” or “household soap,” or named proprietary bath soaps. Can be with or without
specific mention of fingers, other materials, or douching devices to introduce soap
lather inside the vagina.
Cleaning with other household products
Cleaning inside the vagina, beyond the introitus, with products that include: generic
“household cleaners”; named proprietary products such as “Omo”; antiseptic solutions;
vinegar; lemon juice. Can be with or without specific mention of fingers, other materials,
or douching devices to introduce liquid inside the vagina.
Cloth to wipe out vagina or apply products
Use inside the vagina, beyond the introitus, of materials such as cloth, tissue, paper,
cotton wool to wipe out vaginal secretions or to apply products. Includes specific
practices described as “cleaning with cloth” without any other product and named products
introduced with cloth or other material. Does not include use of medication, tampons,
or removal of menstrual blood.
Insertion of products to dry or tighten vagina
Pushing or placing mostly nonliquid products inside the vagina (including powders,
creams, herbs, tablets, sticks, stones, leaves, “traditional products”) regardless
of the duration. Some questions ask specifically about the use of this practice before
sexual intercourse. The intention is to achieve a sensation described as dry or tight.
Any (or no) current practice
Includes all positive (or negative) responses to general questions about the use of
an intravaginal practice, or to specific questions about practices described above.
Time period is that asked about at the baseline visit, usually past 1–3 mo.
Categories are not mutually exclusive. Definitions of intravaginal cleaning and insertion
adapted from classification developed by the WHO Gender, Sexuality and Vaginal Practices
Study Group (GSVP Study Group) [6]. Additional definitions based on specific questions
used in individual studies.
Vaginal flora status was considered as both an exposure and an outcome, and assessed
using scores from Gram-stained vaginal smears [16],[17], or Amsel clinical criteria
[18]. We used the Nugent score if results from more than one method were available
[16]. BV was defined as a Nugent score of 7–10, Ison-Hay grade III, or the presence
of three or more Amsel criteria. Intermediate vaginal flora was defined as a Nugent
score of 4–6 [11] or Ison-Hay grade II. Two studies used Amsel criteria only and could
not be included in analyses that included intermediate vaginal flora as an exposure
or outcome [19],[20].
Assessment of the Risk of Bias
We assessed the potential for bias in each cohort study, arising from prespecified
methodological domains [21]: description of participation and evidence of bias; definitions
of diagnostic criteria; blinding of outcome assessment; conduct of follow-up; missing
data; and measurement of main confounders.
Statistical Analysis
Association between intravaginal practices and HIV infection
For the primary objective we included all women and used Cox proportional hazards
models to examine associations in each study between each intravaginal practice at
baseline and HIV acquisition, and expressed these as the hazard ratio (HR) and 95%
confidence intervals (CIs). Follow-up time was measured until the first of: estimated
date of HIV infection; the last follow-up visit; the end of the study; or after 2
y of follow-up. We pooled data if the level of between-study heterogeneity was mild
or moderate, defined as a value from the I
2 statistic below 50% [22]. We used two methods [14]: for all study objectives we
used random effects meta-analysis to combine effect estimates from individual strata
(two-stage method); for analyses with HIV acquisition as the outcome we also used
stratified, fixed effects Cox regression, with studies as the strata (one-stage method).
Both methods gave very similar results. The proportional hazards assumption, tested
on the basis of Schönfeld residuals, was not violated in any model. All statistical
analyses were conducted using Stata (version 10, Stata Corporation).
We looked for statistical evidence of confounding of the association between intravaginal
practices and HIV infection in each individual study and for each intravaginal practice
by comparing the univariable HR with the HR from bivariable models including baseline
measures of each of the following prespecified factors: marital status; numbers of
sex partners; condom use; contraception; educational level; religion; employment;
age at first intercourse; having received goods or money in exchange for sex; pregnancy;
and HSV-2 status. If inclusion of the variable resulted in the HR changing by more
than 10% in one or more individual studies we considered it for inclusion in multivariable
analyses [23]. We controlled for age in all multivariable models. We present results
from models that also controlled for marital status and number of sex partners during
the previous 3 mo as recorded at cohort entry because these were the variables that
fulfilled our criteria most often. The results were very similar to those from models
that controlled for a larger common set of variables, or controlled for different
variables in each study.
We then examined the role of BV. We aimed to control for time-varying confounding
by BV [24], but there were too many differences between studies in the frequency and
timing of vaginal specimen collection to use fully time-updated measures. We therefore
conducted two exploratory analyses in multivariable models to see, qualitatively,
whether there was attenuation of the HR; using either the result of the baseline test
or the last available test for BV before the estimated date of HIV infection, or before
the date of censoring.
Associations between intravaginal practices and changes in vaginal flora status
To examine associations between intravaginal practices and short-term changes in vaginal
flora status we included women who had normal vaginal flora at baseline, assessed
using Gram-stain criteria, and a follow-up assessment within the first year of enrolment.
We categorised vaginal flora status at follow-up as normal, intermediate, or BV. We
used ordered logistic regression with a proportional odds model [25]. The assumption
of the model is that the odds ratio (OR) associated with an intravaginal practice
for the odds of intermediate vaginal flora or BV compared with normal vaginal flora
is equal to the OR for BV compared with normal or intermediate flora. The model therefore
estimates a single OR from the data.
Association between disrupted vaginal flora and HIV acquisition
To examine the association between disrupted vaginal flora and HIV acquisition we
included all women with vaginal flora assessed by Gram-stain at baseline. We considered
vaginal flora status as a three-level ordered exposure and used Cox proportional hazards
regression to estimate the HR (and 95% CI) for incident HIV infection in the first
2 y of follow-up.
Results
Description of Studies
We assessed 22 studies for eligibility; 13 prospective studies conducted in sub-Saharan
Africa included in our previous systematic review [8],[19],[20],[26]–[35] and nine
identified through expert meetings [15],[36]–[43]. We excluded five studies for which
we could not determine eligibility because we did not establish contact with the authors
[26]–[28],[31],[33], three studies that did not include data on relevant exposures
or outcomes [30],[32],[34], and four studies for which the investigators declined
to take part, or could not send their agreement to participate or dataset by 16th
March 2009 (Figure 1; Table S1) [29],[38]–[40]. We included ten studies [8],[15],[19],[20],[35]–[37],[41]–[43].
We analysed the data as 13 separate studies, stratifying results from three multicentre
studies according to enrolment site [15],[19],[35]. Table 1 shows selected characteristics
of the studies, which were done in six sub-Saharan African countries and included
data from 14,874 women followed for 21,218 woman years, with 791 incident HIV infections
in the first 2 y. The individual studies were generally assessed as having a low risk
of bias (data available on request). Most studies involved women enrolled from community
settings or clinics providing reproductive health services (studies 1, 6, 7, 8, 9,
10). Two studies in Tanzania enrolled women working as food sellers or in bars and
other recreational facilities, amongst whom high prevalences of sexually transmitted
infections and HIV have previously been reported (studies 4, 5). Two studies in Kenya
enrolled only women who were self-identified sex workers (studies 2, 3). The mean
age at enrolment was 29.4 y (standard deviation [SD] 8.7, range 15.2–67.0 y). The
incidence of HIV infection in individual studies ranged from 1.6 (95% CI 1.2–2.0,
study 1 Uganda) to 15.1 (95% CI 10.5–21.8, study 9) per 100 woman years. The median
time from enrolment to the estimated date of HIV infection was 250 d (interquartile
range [IQR] 130–415 d).
10.1371/journal.pmed.1000416.g001
Figure 1
Flow diagram of included studies.
10.1371/journal.pmed.1000416.t001
Table 1
Characteristics of participants of individual cohort studies.
Study Number; Country [Reference]
Enrolment Settings
Study Population
Planned Study Durationa
Planned Frequency of Follow-upa
Dates of Enrolmenta
n Includedb
Age at Enrolment (y), Mean (SD)
Follow-up per Woman (mo), Median (IQR)
Percent Followed for 12 moc
n Incident HIV Infections
HIV Incidence, per 100 Woman Years (95% CI)
1; Uganda [35]
FPC and other health services
Women attending clinics
15–24 mo
3 Monthly
11/99–03/02
2,201
24.9 (4.5)
23.7 (21.4–24.0)
97.0
63
1.6 (1.2–2.0)
2; Kenya [37]
Randomised trial (all women); peer-leaders network
Self-identified female sex workers
24 mo
6 Monthly
05/98–01/02
414
29.1 (8.0)
23.8 (11.2–24.0)
75.4
30
4.9 (3.4–7.0)
3; Kenya [8]
Municipal STI clinic
Self-identified female sex workers attending a STI clinic
Not fixed
Monthly
02/93–12/02
1,270
27.1 (6.3)
14.3 (3.7–24.0)
55.1
164
11.3 (9.7–13.2)
4; Tanzania [42]
Reproductive health clinics in selected guesthouses
Women working in bars, restaurants, guesthouses
12 mo
3 Monthly
08/02–10/03
978
30.0 (8.2)
11.7 (8.7–12.0)
66.0
23
2.8 (1.9–4.3)
5; Tanzania [43]
Randomised trial (all women); mobile health clinics
Women working in bars, restaurants, guesthouses
30 mo
3 Monthly
11/03–01/06
781
27.5 (5.0)
24.0 (15.5–24.0)
91.3
45
3.4 (2.6–4.6)
6; Malawi [19]
FPC, postnatal clinics
Women attending clinics
9 mo
3 Monthly
06/01–08/02
993
27.9 (8.2)
9.0 (8.7–9.1)
67.5c
33
4.9 (3.5–6.9)
6; Zimbabwe [19]
As above
As above
As above
As above
As above
526
29.0 (8.4)
9.0 (8.8–9.2)
74.7c
19
5.2 (3.3–8.1)
1; Zimbabwe [35]
FPC and other health services
Women attending clinics
15–24 mo
3 Monthly
11/99–08/02
2,248
25.9 (4.4)
23.0 (17.3–24.0)
90.7
153
4.2 (3.6–4.9)
7; Zimbabwe, [15]
Randomised trial (control arm only); FPC, well-baby, general health clinics; community
organisations
Sexually active women (average four sex acts per month)
12–24 mo
3 Monthly
09/03–10/05
1,229
28.4 (7.2)
23.5 (17.9–23.9)
96.2
52
2.5 (1.9–3.3)
7; South Africa [15]
As above
As above
As above
As above
As above
1,247
28.9 (8.0)
17.9 (14.4–22.3)
91.9
151
5.5 (4.5–6.7)
8; South Africa [36]
FPC, well-baby, postnatal clinics
Women attending clinics
12 mo
6 Monthly
01/02–01/04
694
24.7 (5.0)
11.0 (10.8–11.4)
74.1
20
3.4 (2.2–5.3)
9; South Africa [41]
FPC, immunisation clinics
Women attending clinics
12 mo
3 Monthly
07/03–07/04
261
29.3 (9.5)
9.5 (6.0–12.0)
42.9
29
15.1 (10.5–21.8)
10; South Africa [20]
Cervical cancer trial; community meetings and health workers
Women never screened for cervical cancer living in Khayelitsha
6–36 mo
3 Monthly
08/01–11/03
2,032
43.2 (6.8)
24.0 (24.0–24.0)
90.9
61
1.7 (1.3–2.2)
Number of incident HIV infections during up to 2 y follow-up (n = 14,874), ordered
geographically from north to south.
a
From study protocol or publication.
b
Includes only women who were HIV negative at start of follow-up and had at least one
follow-up visit.
c
Includes women who attended a follow-up visit at 12 mo ±30 d, except study 6, which
includes women who completed study follow-up at 9 mo.
FPC, family planning clinic; SD, standard deviation; STI, sexually transmitted infections.
Frequency of Intravaginal Practices
The percentage of women reporting any current intravaginal practice at baseline ranged
from 18% (study 8) to 95% (study 3, Table 2). Studies done in South Africa tended
to have a low overall prevalence of any current vaginal practice (18%–27%, studies
8–10) and studies in Zimbabwe tended to have a high prevalence (69%–92%, studies 1,
6, 7). Female sex workers in Kenya (76%–95%, studies 2, 3) and high risk women in
Tanzania (67%–76%, studies 5, 4) also reported high levels of any vaginal practice.
Table 2 shows the proportions of women reporting any use of specific practices, i.e.,
whether or not they reported other practices. Cleaning inside the vagina with soap
was the most common practice involving a specified product, and was reported by more
than one-third of women in six studies in the most northern countries in the region
(studies 1–6 Malawi). Reported use of other more abrasive household cleaning products
was uncommon, ranging from 0.7% (study 8) to 7% (study 2). Reported use of cloth or
paper to wipe out the vagina, or apply products, ranged from 0.3% (study 8) to 70%
(study 7 Zimbabwe). Inserting products to dry or tighten the vagina was uncommon;
this was most commonly reported in four studies conducted in South Africa and Zimbabwe,
where the prevalence was 13%–20% (studies 1 Zimbabwe, 7 Zimbabwe, and 8 South Africa).
Cleaning with water, with or without other practices, was reported by more than 60%
of women in all but four studies in South Africa (studies 7 South Africa, 8–10). Where
measurements of intravaginal practices were available at follow-up visits, the majority
of reported practices were consistent with baseline data; 60% reported the same practice
at all study visits at which data were collected, 34% reported either the same practice
or no practice, and 6% reported different practices at all visits.
10.1371/journal.pmed.1000416.t002
Table 2
Baseline prevalence of intravaginal practices and BV in included cohort studies (n = 14,874).
Study Number; Country
n Included
Any Practicea
Specific Practices
n BV (%)
n Yes (%)
n Cleaning with Water Only (%)b
n Cleaning with Water (%)c
n Cleaning with Soap (%)c
n Cleaning with Household Products (%)c
n Use of Cloth, Tissue, Paper (%)c
n Insertion to Dry/Tighten (%)c
1; Uganda
2,201
1,510 (68.6)
500 (22.7)
1,492 (67.8)
982 (44.6)
32 (1.5)
195 (8.9)
30 (1.4)
465 (21.1)
2; Kenya
414
323 (78.0)
Not asked
273 (65.9)
207 (50.0)
30 (7.3)
2 (0.5)
Not asked
197 (47.6)
3; Kenya
1,270
1,204 (94.8)
243 (19.1)
1,066 (83.9)
820 (64.6)
85 (6.7)
187 (14.7)
11 (0.9)
461 (36.3)
4; Tanzania
978
740 (75.7)
372 (38.0)
728 (74.4)
348 (35.6)
8 (0.8)
32 (3.3)
26 (2.7)
446 (45.6)
5; Tanzania
781
523 (67.0)
101 (11.1)
499 (63.9)
397 (50.8)
16 (2.1)
32 (4.1)
33 (4.2)
482 (61.7)
6; Malawi
993
891 (89.7)
182 (18.3)
880 (88.6)
669 (67.4)
23 (2.3)
47 (4.7)
33 (3.3)
77 (7.8)
6; Zimbabwe
526
364 (69.2)
262 (49.8)
356 (67.7)
68 (12.9)
18 (3.4)
29 (5.5)
6 (1.1)
85 (16.2)
1; Zimbabwe
2,248
1,916 (85.2)
1,323 (58.9)
1,724 (76.7)
320 (14.2)
106 (4.7)
1,122 (49.9)
445 (19.8)
639 (28.4)
7; Zimbabwe
1,229
1,127 (91.7)
629 (51.2)
810 (65.9)
166 (13.5)
41 (3.3)
854 (69.5)
154 (12.5)
82 (6.7)d
7; South Africa
1,247
1,123 (90.1)
86 (6.9)
668 (53.6)
582 (46.7)
49 (3.9)
655 (52.5)
164 (13.2)
0d
8; South Africa
694
124 (17.9)
19 (2.7)
44 (6.3)
17 (2.5)
5 (0.7)
2 (0.3)
87 (12.5)
349 (50.3)
9; South Africa
261
68 (26.1)
19 (7.3)
56 (21.5)
36 (13.8)
14 (5.4)
37 (14.2)
8 (3.1)
124 (47.5)
10; South Africa
2,032
546 (26.9)
304 (15.0)
460 (22.6)
103 (5.1)
85 (4.2)
438 (21.6)
154 (7.6)
174 (8.6)
a
Includes any intravaginal practice reported at the baseline visit.
b
Water is the only substance put into the vagina and woman does not use any other intravaginal
practice (this category was grouped with “no intravaginal practice” to form the reference
category for comparative analyses).
c
Woman reports using this practice and may or may not report any other intravaginal
practice.
d
Based on a subset of 257 women tested for BV at baseline in the Zimbabwe study site
only.
Associations between Intravaginal Practices and HIV Infection
Intravaginal use of cloth or paper to wipe out the vagina or apply products was the
practice most strongly associated with HIV acquisition in univariable analysis and
after controlling for age, marital status, and number of sex partners in the past
3 mo (pooled adjusted HR 1.47, 95% CI 1.18–1.83) (Figure 2). There was also an increased
risk of HIV acquisition in women reporting intravaginal cleaning with soap and water
(pooled adjusted HR 1.24, 95% CI 1.01–1.53) (Figure 3) and insertion of products to
dry or tighten the vagina (pooled adjusted HR 1.31, 95% CI 1.00–1.71) (Figure 4).
One study (study 2) did not ask about insertion practices. The use of household cleaning
products other than soap was much less common; in four studies the HR could not be
estimated (studies 1 Uganda, 4, 5, 8; Figure 5). In the remaining studies the pooled
analyses showed no evidence of an increased risk of HIV acquisition (pooled adjusted
HR 1.11, 95% CI 0.73–1.68). There was little evidence of between-study heterogeneity
in any of these analyses (I
2 values 0%–14%). Results from the fixed effects models were the same or very similar
to those from random effects models (Table S2).
10.1371/journal.pmed.1000416.g002
Figure 2
Association between use of cloth or paper to wipe out vagina or apply products and
HIV acquisition, ordered by country, north to south (n = 10,332).
Individual study results from Cox regression. Pooled unadjusted and aHRs from random
effects meta-analysis. Reference group is women who reported no intravaginal practice
or cleaning with water only. Multivariable models adjusted for age, marital status,
and number of partners in last 3 mo. No estimate possible if there were no events
in one group.
10.1371/journal.pmed.1000416.g003
Figure 3
Association between intravaginal cleaning with soap and HIV acquisition, ordered by
country, north to south (n = 3,071).
Individual study results from Cox regression. Pooled unadjusted and aHRs from random
effects meta-analysis. Reference group is women who reported no intravaginal practice
or cleaning with water only. Multivariable models adjusted for age, marital status,
and number of partners in last 3 mo.
10.1371/journal.pmed.1000416.g004
Figure 4
Association between insertion of products to dry or tighten the vagina and HIV acquisition,
ordered by country, north to south(n = 9,420).
Individual study results from Cox regression. Pooled unadjusted and aHRs from random
effects meta-analysis. Reference group is women who reported no intravaginal practice
or cleaning with water only. Multivariable models adjusted for age, marital status,
and number of partners in last 3 mo. No estimate possible if there were no events
in one group. Stratum excluded if there were no events in either group, or standard
error could not be estimated by model.
10.1371/journal.pmed.1000416.g005
Figure 5
Association between intravaginal cleaning with household cleaners and HIV acquisition,
ordered by country, north to south (n = 8,879).
Individual study results from Cox regression. Pooled unadjusted and aHRs from random
effects meta-analysis. Reference group is women who reported no intravaginal practice
or cleaning with water only. Multivariable models adjusted for age, marital status,
and number of partners in last 3 mo. No estimate possible if there were no events
in one group.
There was no statistical evidence that intravaginal use of water alone increased the
risk of HIV acquisition when compared with no practice in univariable (Table S3, pooled
unadjusted HR 1.02, 95% CI 0.78–1.35, I
2 3.9%) or multivariable analyses (pooled adjusted HR 1.03, 95% CI 0.76–1.40, I
2 16.1%). Women who used only water intravaginally, compared with those using no practice,
were younger (p<0.001), more likely to be currently married (p<0.001), more likely
to have one partner in the last 3 mo (p<0.001), more likely to be using contraception
(p<0.001), more likely to be Catholic or Muslim than Protestant (p<0.001), and less
likely to never use condoms (p = 0.005). Controlling for these variables did not alter
the strength of the associations found in univariable analyses (unpublished results).
Table 3 shows the results of analyses controlling for BV in addition to demographic
and behavioural characteristics. These analyses exclude most participants from study
7, in whom vaginal flora was only assessed in a subset of women from one study site.
Intravaginal use of cloth or paper remained associated with HIV acquisition after
controlling for BV. For cleaning with soap and insertion of products to dry or tighten
the vagina, the associations with HIV acquisition were weakened slightly after adjustment
for demographic and behavioural variables. Adjustment for the presence of BV at baseline
or at the last visit before the estimated date of HIV infection did not have any additional
effect. When vaginal flora status was considered with intermediate vaginal flora and
BV as separate categories, the pattern of results was similar to that seen when BV
was included as a binary variable, but confidence intervals were wider because studies
that used only Amsel criteria for diagnosis were excluded (unpublished results).
10.1371/journal.pmed.1000416.t003
Table 3
Associations between intravaginal practices and HIV acquisition, adjusting for different
measures of disrupted vaginal flora.
Intravaginal Practice
Number in Model (Strata/Studies)a
HR (95% CI)
aHR (95% CI)
Unadjusted
I
2% (95% CI)
Demographic/Behavioural Factorsb
I
2% (95% CI)
Demographic/Behavioural Factors + BVc at Baseline
I
2% (95% CI)
Demographic/Behavioural Factors + BV‡ before Seroconversion
I
2% (95% CI)
Cleaning with soap and water
11,387 (12/10)
1.20 (0.97–1.49)
8.2 (0–62)
1.18 (0.94–1.48)
14.3 (0–54)
1.18 (0.94–1.49)
15.8 (0–55)
1.18 (0.94–1.48)
13.6 (0–53)
Cleaning with household products
7,893 (12/10)
1.20 (0.78–1.85)
0.0 (0–58)
1.19 (0.77–1.84)
0.0 (0–60)
1.25 (0.80–1.94)
0.0 (0–60)
1.18 (0.76–1.83)
0.0 (0–62)
Cloth to wipe out vagina or apply products
8,475 (12/10)
1.44 (1.13–1.82)
0.0 (0–58)
1.38 (1.06–1.80)
6.5 (0–61)
1.39 (1.06–1.81)
6.9 (0–61)
1.38 (1.03–1.85)
15.9 (0–56)
Insertion of products to dry or tighten vagina
8,216 (11/9)
1.36 (1.01–1.85)
0.0 (0–60)
1.32 (0.97–1.79)
0.0 (0–60)
1.33 (0.98–1.82)
0.0 (0–60)
1.32 (0.97–1.80)
0.0 (0–62)
HRs from two-stage random effects meta-analysis. Intravaginal practices measured at
baseline; reference category is no vaginal practice or use of water only.
a
Numbers of observations differ from Figures 1–
4 because they exclude those with no BV measurement, mostly from study 7 in which
BV was measured only in a subset at baseline in the Zimbabwe site; model for insertion
of products also excludes study 2, which did not ask about this practice.
b
Adjusted for age, marital status, and reported number of sex partners in last 3 mo,
as recorded at cohort entry.
c
BV as binary variable defined as Nugent score of 7–10, Ison-Hay grade III, or the
presence of three or more Amsel criteria.
Associations between Intravaginal Practices and Disrupted Vaginal Flora
Table 4 shows the associations between intravaginal practices and the development
of intermediate vaginal flora or BV amongst women with normal vaginal flora at the
baseline visit and vaginal specimens examined by Gram-stain criteria at follow-up.
Amongst women who cleaned intravaginally with soap and water, the incidence of disrupted
vaginal flora at the next visit was increased in univariable and multivariable analyses
(pooled adjusted OR from ordered logistic regression 1.24, 95% CI 1.04–1.47). There
was a similar but weaker trend for the insertion of products to dry or tighten the
vagina, but confidence intervals for these estimates were wider (pooled adjusted OR
1.29, 95% CI 0.98–1.71, p = 0.072). There was no evidence of an association between
intravaginal cleaning with household products or use of cloth or paper and development
of disrupted vaginal flora. There was no evidence of between-study heterogeneity (I
2 values 0.0% for all analyses).
10.1371/journal.pmed.1000416.t004
Table 4
Associations between intravaginal practices and disrupted vaginal flora in women with
normal vaginal flora at baseline.
Intravaginal Practicea
Number in Model (Strata/Studies)b
Number Developing Disrupted Florac
Disrupted Vaginal Florab
Unadjusted OR (95% CI)
p-Value
Adjusted OR (95% CI)d
p-Value
Cleaning with soap and water
3,222 (8/7)
1,088
1.27 (1.07–1.50)
0.006
1.24 (1.04–1.47)
0.014
Cleaning with household products
2,045 (7/6)
641
0.95 (0.62–1.44)
0.797
0.89 (0.58–1.36)
0.576
Cloth to wipe out vagina or apply products
2,177 (5/4)
704
1.06 (0.85–1.32)
0.588
1.06 (0.85–1.33)
0.577
Insertion of products to dry or tighten vagina
2,264 (7/6)
735
1.26 (0.96–1.66)
0.099
1.29 (0.98–1.71)
0.072
OR from two-stage random effects meta-analysis based on ordered logistic regression.
a
Baseline category for intravaginal practices is no vaginal practice or use of water
only. Intravaginal practices measured at baseline.
b
Disrupted vaginal flora as a three-level ordered categorical variable: normal vaginal
flora defined as Nugent score 0–3, or Ison-Hay grade I; intermediate vaginal flora
defined as Nugent score 4–6, or Ison-Hay grade II; BV defined as Nugent score 7–10,
or Ison-Hay grade III. Excludes two studies that did not use Gram stain criteria [19],[20].
c
Number with normal flora at baseline who developed disrupted vaginal flora includes
both women using and not using each intravaginal practice.
d
Adjusted for age, marital status, and reported number of sex partners in last 3 mo
as reported at cohort entry.
Associations between Disrupted Vaginal Flora and HIV Infection
Vaginal flora status was associated with HIV incidence in univariable and multivariable
analyses (Table 5). The risk of HIV acquisition was higher in women with BV than with
intermediate vaginal flora. Controlling for potential confounders did not substantially
attenuate the effect estimates. The strength of association between vaginal flora
status and HIV acquisition was slightly weaker when using the vaginal flora status
measured at the visit preceding the estimated date of HIV infection (median 51 d,
IQR 40–147 d between vaginal flora assessment and HIV infection) than when using the
baseline value (median 253 d, IQR 116–426 d).
10.1371/journal.pmed.1000416.t005
Table 5
Association between disrupted vaginal flora and HIV acquisition, stratified Cox regression.
Variable
Baseline Vaginal Flora Status (n = 8,452)a
Vaginal Flora Status at Visit before HIV Seroconversion (n = 8,626)a
Unadjusted HR (95% CI)
Adjusted HR (95% CI)b
p-Value
Unadjusted HR (95% CI)*
Adjusted HR (95% CI)b
p-Value
Vaginal flora
<0.001
<0.001
Normal vaginal flora
1 (reference)
1 (reference)
1 (reference)
1 (reference)
Intermediate vaginal flora
1.62 (1.27–2.08)
1.54 (1.20–1.97)
1.51 (1.19–1.91)
1.41 (1.12–1.79)
BV
1.84 (1.48–2.28)
1.69 (1.36–2.10)
1.66 (1.35–2.05)
1.53 (1.24–1.89)
HSV status at baseline
Negative
1 (reference)
1 (reference)
Positive
2.14 (1.70–2.70)
2.29 (1.80–2.90)
<0.001
2.14 (1.70–2.69)
2.31 (1.82–2.91)
<0.001
Age at cohort entry
<0.001
<0.001
>25 y
1.25 (1.04–1.50)
1.37 (1.13–1.65)
1.26 (1.05–1.52)
1.38 (1.14–1.66)
25–34 y
1 (reference)
1 (reference)
1 (reference)
1 (reference)
35 y or older
0.80 (0.56–1.15)
0.80 (0.56–1.15)
0.79 (0.55–1.13)
0.78 (0.54–1.12)
Marital status
<0.001
<0.001
Currently married
1 (reference)
1 (reference)
1 (reference)
1 (reference)
Currently unmarried
1.96 (1.46–2.64)
1.78 (1.32–2.40)
1.96 (1.46–2.62)
1.77 (1.31–2.38)
Number of partners last 3 mo
0.034
0.023
No partner
0.97 (0.48–1.97)
0.94 (0.46–1.91)
0.96 (0.47–1.95)
0.90 (0.44–1.84)
1 partner
1 (reference)
1 (reference)
1 (reference)
1 (reference)
More than 1 partner
2.14 (1.47–3.12)
1.59 (1.09–2.31)
2.15 (1.48–3.13)
1.62 (1.11–2.35)
a
Included in analysis are women with available vaginal flora status measured by Gram
stain criteria: normal vaginal flora defined as Nugent score 0–3, or Ison-Hay grade
I; intermediate vaginal flora defined as Nugent score 4–6, or Ison-Hay grade II; BV
defined as Nugent score 7–10, or Ison-Hay grade III. Excludes two studies that did
not use Gram stain criteria [19],[20].
b
Multivariable model controls for all variables in the table.
Discussion
This study combined individual participant data from ten prospective studies in six
sub-Saharan African countries. Intravaginal use of cloth or paper remained associated
with HIV acquisition after controlling for age, marital status, number of sex partners
in the past 3 mo, and in models that controlled for BV. Insertion of products to dry
or tighten the vagina and intravaginal cleaning with soap were associated with HIV
acquisition in univariable and multivariable analyses controlling for demographic
and behavioural variables, but not in models that controlled for BV. Intravaginal
cleaning with soap was also associated with the development of intermediate vaginal
flora and BV at follow-up in women with normal vaginal flora at baseline. Disrupted
vaginal flora measured at baseline or at the visit before the estimated data of HIV
infection was associated with HIV acquisition in both univariable and multivariable
analyses.
Strengths and Weaknesses
The main strength of this study was the collaboration of investigators from ten different
studies, which allowed the collation of individual participant data from nearly 15,000
women and analysis using consistent definitions of intravaginal practices across studies.
By pooling data we increased the power and precision of our analyses and adjusted
for confounding, which is difficult or impossible in an aggregate data meta-analysis
[14]. There was a striking lack of between-study heterogeneity in results, despite
differences in study populations, designs, and questionnaires, which increases the
robustness of our findings. In addition, we obtained very similar results using different
statistical methods to pool data. Our results might be biased because we did not identify
or include all eligible studies. We did, however, conduct a wide-ranging search and
reasons for exclusion were not related to study results.
Limitations of this study were mainly due to data collection differences that could
not be remedied by recoding. Questions about intravaginal practices were asked in
different ways because there are no agreed upon definitions [5], there is no validated
measurement instrument, and the purposes of the studies differed. We therefore had
to limit the number of specific intravaginal practices from those originally planned
and differences in wording of questions about intravaginal practices between studies
might have affected our results. By grouping exposure categories, we might have masked
harms (or benefits) of particular practices or products. Grouping of categories to
obtain uniformly defined variables for confounding factors, or imprecision in the
measurement of other variables included in the analysis might also have resulted in
residual confounding. In addition, we cannot exclude the possibility of residual confounding
from unmeasured factors, such as sexually transmitted infections, from our reliance
on baseline measures of intravaginal practices that changed over time, or the motivation
for performing certain practices, which can vary according to perceptions of risk
[5].
A further limitation was the difficulty in definitively establishing the temporal
sequence of intravaginal practices and changes in vaginal flora status; intravaginal
practices could promote disruption of vaginal flora but symptoms related to those
changes, such as vaginal discharge or fishy odour, could prompt intravaginal washing
or wiping [24]. We tried to overcome this problem when examining the association between
intravaginal practices and disrupted vaginal flora by including only women with normal
vaginal flora at baseline. We could not, however, consider changes in exposure status
over time because of data collection differences and uncertainty about the effects
of treatment for BV, which was documented in some studies but not in others.
Comparison with Other Studies
This study is likely to be the largest to have examined associations between intravaginal
practices and HIV acquisition and it allowed us to reexamine some previously observed
inconsistencies between published studies. For example, McClelland and colleagues
found a strong association between intravaginal cleaning with soap and incident HIV
infection in Kenya [8] but van de Wijgert and colleagues and Myer and colleagues found
no associations in their studies in Uganda and Zimbabwe [44] and South Africa [20].
In this analysis, estimates from the same studies were close to the published data;
when pooled with the other studies, using the same definitions and multivariable model,
the results were consistent with the overall finding of a modest increase in the risk
of HIV acquisition (Figures 1–
4). Differences in definitions of intravaginal practices and methods of analysis in
individual studies make it difficult to compare findings directly and to synthesise
results across studies. These differences meant that in our systematic review of aggregated
published data we were limited to examining associations with intravaginal cleaning
and insertion of products and could not draw conclusions about any specific practice
[12], illustrating the advantages of individual participant data meta-analysis.
Our findings about the associations between disrupted vaginal flora and increased
risk of HIV are consistent with those of other studies [10],[11]. Given the rapid
fluctuations that occur in vaginal microflora [11],[45], the intervals between assessment
of vaginal flora status and HIV acquisition in our analyses, and those of previous
studies, likely resulted in substantial misclassification. However, these analyses
captured some of the increase in risk of HIV infection for women who had BV identified
on at least one occasion. A disadvantage of our analysis is that this was a secondary
objective and our search strategy did not include all studies addressing these associations.
Nevertheless, we included more prospective studies than the previously published systematic
review examining the links between BV and risk of HIV infection [10] and we were able
to conduct both univariable and multivariable analyses across all studies.
Interpretation of Study Findings
Our findings suggest an increase in the risk of acquiring HIV infection amongst women
who use cloth or paper to wipe out the vagina or apply products, insert products intended
to dry or tighten the vagina, or clean with soap intravaginally. Whilst effects of
this magnitude could result from residual confounding or bias, there are also plausible
biological mechanisms for these associations. Use of cloth or paper to wipe out the
vagina was associated with HIV acquisition after controlling for BV and was not associated
with the development of disrupted flora. Use of cloth might increase the risk of HIV
if removal of protective vaginal mucus exposes existing micro-trauma or causes inflammation
or micro-trauma [4], especially if used frequently, as reported in some regions [5].
Insertion of products into the vagina could also directly cause micro-trauma and/or
inflammation. Since the products used are often intended to dry or to tighten the
vagina in preparation for sexual intercourse [5], viral entry through breaks in the
cervico-vaginal epithelium could be facilitated during or after sex [4]. We found
only indirect support in this study for the hypothesis linking intravaginal cleaning
with soap, disruption of vaginal flora, and HIV acquisition [12],[20]. Amongst women
with normal vaginal flora at baseline, those who reported cleaning with soap were
slightly more likely to develop intermediate vaginal flora and BV, possibly because
an alkaline pH might promote the growth of BV-associated bacteria. The presence of
both intermediate vaginal flora and BV were also associated with an increased incidence
of HIV in this study, confirming recent observations [11]. We could not examine the
effect of BV in a causal model, as planned. In exploratory analyses, adjustment for
BV in addition to demographic and behavioural variables in a standard regression model
did not further alter the association between intravaginal cleaning with soap and
HIV. The adjusted effect size will not have been estimated precisely in this model,
however, because BV is on the hypothesized causal pathway. Contrary to expectation
[4], use of household cleaners, vinegar, or lime juice did not increase HIV risk in
this study. Lime juice has been reported to cause vaginal epithelial damage in clinical
studies [46]. These practices were, however, reported infrequently so our study might
have lacked statistical power to answer this question, or measurement error might
have reduced our ability to detect modest effects despite pooling data from multiple
studies.
Implications for Research and Policy
It is becoming increasingly important to understand the distribution, motivations
for, and health effects of intravaginal practices [12],[47], particularly since a
randomised controlled trial has, for the first time, shown that a vaginal microbicide
can reduce acquisition of HIV infection [48]. Abdool Karim and colleagues found that
the incidence of HIV in women using the antiretroviral agent tenofovir was 39% (95%
CI 6%–60%) lower than in women using placebo gel [48]. Whilst reported to be acceptable,
there are reasons why intravaginal practices might reduce the effectiveness of microbicides.
Women might wash or wipe out microbicides, even when advised not to use habitual intravaginal
practices during trials. In Tanzania, about half of women who washed intravaginally
reported doing so within 2 h of intercourse [47]. Women who use intravaginal practices
might adhere less to vaginal gels, as observed in a trial of the effectiveness of
diaphragms and lubricant gel [49]. Alternatively, products inserted into the vagina
might react with microbicides, making them inactive or potentially harmful [6]. New
female-initiated interventions also need to be developed despite the challenges involved
in measuring the impact on preventing HIV acquisition. Behavioural interventions that
have been successful in helping young US women to stop vaginal douching [50] might
be adapted for women in sub-Saharan Africa to encourage less harmful practices [7]
such as use of water alone, which was not associated with an increased risk of HIV
acquisition. This study provides evidence to suggest that some intravaginal practices
increase the risk of HIV acquisition but a direct causal pathway linking intravaginal
cleaning with soap, disruption of vaginal flora, and HIV acquisition has not yet been
demonstrated. More consistency is needed in definitions and measurements of intravaginal
practices so that the effects of specific intravaginal practices and products can
be further elucidated.
Supporting Information
Table S1
Studies excluded from individual participant data meta-analysis, reasons for exclusion,
alphabetical order.
(0.05 MB DOC)
Click here for additional data file.
Table S2
Sensitivity analysis for comparison between random and fixed effects models, comparing
incidence of HIV in women using intravaginal practices with women using no practice
or water only.
(0.03 MB DOC)
Click here for additional data file.
Table S3
Sensitivity analysis for associations between intravaginal practices and HIV acquisition,
with different reference groups.
(0.03 MB DOC)
Click here for additional data file.
Text S1
PRISMA checklist.
(0.13 MB PDF)
Click here for additional data file.