Abstract
Objectives:
Vaginal washing has been associated with reductions in cultivable Lactobacillus and an increased risk of both bacterial vaginosis (BV) and HIV infection. The effect of vaginal washing on the quantity of individual Lactobacillus species is not well characterized. This analysis tested the hypothesis that vaginal washing would be associated with a lower likelihood of Lactobacillus spp. detected by both culture and quantitative PCR (qPCR).
Methods:
We conducted a cross-sectional study of 272 HIV-seronegative women enrolled in an open-cohort study in Mombasa, Kenya. Vaginal washing and sexual risk behaviours were assessed using face-to-face interviews. Vaginal Lactobacillus spp. were detected using cultivation and PCR methods, with L. crispatus, L. jensenii, and L. iners concentrations measured using qPCR assays targeting the 16S rRNA gene. Poisson regression with robust standard errors was used to assess associations between vaginal washing and Lactobacillus detection by culture and qPCR.
Results:
Eighty percent (n=217) of participants reported vaginal washing in the prior week. One-fifth (n=58) of participants had BV by Nugent score. In unadjusted analysis, vaginal washing was associated with a 45% decreased likelihood of Lactobacillus spp. detection by culture (prevalence ratio (PR): 0.55, 95%CI 0.37–0.82). Adjusting for age and condomless sex in the prior week did not change the magnitude of the association (adjusted PR (aPR): 0.56, 95%CI (0.37, 0.85). Vaginal washing was associated with approximately a 40% reduction in L. crispatus detection (aPR: 0.57, 95%CI 0.36–0.92), but was not significantly associated with L. jensenii (aPR: 0.68, 95%CI 0.42–1.09) or L. iners detection (aPR: 1.03, 95%CI 0.92–1.15).
Conclusions:
Vaginal washing in the prior week was associated with a significantly reduced likelihood of detecting cultivable Lactobacillus and L. crispatus by qPCR. Given associations between Lactobacillus detection and improved reproductive health outcomes, these results provide motivation for additional study of vaginal washing cessation interventions to improve vaginal health.
Keywords: Vaginal washing, Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus iners, Vaginal microbiota
BACKGROUND
Bacterial vaginosis (BV) is a polymicrobial condition characterized by a shift from a Lactobacillus-predominant vaginal microbiota to one dominated by anaerobes [1]. Lactobacillus species have been associated with an optimal vaginal environment [1,2], and are hypothesized to foster this environment by producing lactic acid, hydrogen peroxide (H2O2), bacteriocins, as well as other mechanisms [3]. L. crispatus and L. jensenii are most consistently associated with vaginal health and are purported producers of H2O2. Studies suggest, however, that H2O2 production is not the central mechanism through which vaginal lactobacilli suppress BV-associated bacteria [3]. The role of other vaginal Lactobacillus, such as L. gasseri and L. iners, is debated. L. iners is more frequently identified in women with intermediate microbiota and BV, but is also present in women without BV [2]. Absence of vaginal lactobacilli is associated with adverse reproductive outcomes, including increased HIV risk [4], sexually transmitted infections (STIs) [1], and preterm birth [5].
Vaginal washing has also been associated with an increased risk of BV [6,7], decreased likelihood of vaginal Lactobacillus detected by culture [6–8], and increased HIV acquisition [9]. However, women engaging in intravaginal practices often do not know the potential risks, and report engaging in vaginal washing to promote hygiene, remove discharge, odour, or menstrual blood, and prevent disease [10].
Evidence of the effect of vaginal washing on Lactobacillus is predominantly based on cultivation studies [6–8]. Molecular methods involving amplification of the 16S ribosomal RNA gene using polymerase chain reaction (PCR) assays are used to assess the presence and concentrations of vaginal bacteria, including fastidious or cultivation-resistant species [2]. Together, molecular and culture-dependent methods allow for detection and quantification of Lactobacilli, and assessment of viability. The effect of vaginal washing on the quantity of individual Lactobacillus species is not well characterized. The objectives of this analysis were to assess the association between vaginal washing and Lactobacillus colonization using culture and species-specific quantitative PCR (qPCR) in high-risk, Kenyan women, and to examine the concordance of culture-dependent and molecular methods of detection. We hypothesized that vaginal washing would be associated with a lower likelihood of Lactobacillus species detected by both methods.
METHODS
Study design and population
This cross-sectional study utilized data from the Mombasa Cohort, an open, prospective cohort study of high-risk women in Mombasa, Kenya [11]. Women must be ≥16 years old, report transactional sex, and provide written informed consent to enrol. This analysis only included HIV-seronegative women who had Lactobacillus culture and qPCR data available from a single study visit. The Kenyatta National Hospital/University of Nairobi Ethics and Research Committee and University of Washington Institutional Review Board approved the study.
Procedures
At Mombasa Cohort enrolment and during monthly follow-up visits, participants completed a face-to-face interview to ascertain vaginal washing and sexual behaviour characteristics. Women underwent a pelvic examination with collection of genital specimens for STI testing at each visit; samples are not collected if women were menstruating. Syndromic STI treatment was provided per Kenyan National Guidelines [12]. Vaginal swabs were collected for Gram stain and Nugent scoring for BV [13]. Vaginal swabs were inoculated onto Rogosa and Colombia 5% blood agars to detect cultivable Lactobacillus. Isolates grown on Rogosa and/or Colombia agars were sub-cultured on tetramethylbenzidine agar containing horseradish peroxidase to assess H2O2 production [11]. Neisseria gonorrhoeae and Chlamydia trachomatis were detected using the Aptima Combo-2 CT/NG Detection System (Hologic, San Diego, CA). Trichomonas vaginalis was assessed by saline microscopy. For this cross-sectional study, an additional vaginal swab was collected between October 2011-June 2012 at a regular cohort study visit (enrolment or monthly) from each HIV-seronegative woman for molecular detection of Lactobacillus.
Vaginal microbiota swabs were transported on dry ice to the Fred Hutchinson Cancer Research Center (Seattle, WA) for species-specific qPCR assays. The MoBio BiOstic Bacteremia DNA Isolation kit was used to extract and purify DNA (Carlsbad, CA), utilizing bead beating and chaotropic lysis to recover DNA free of PCR inhibitors. Sham extraction controls were included to monitor contamination from extraction reagents and collection swabs. All samples were subjected to two quality control assays: 1) Samples were evaluated for PCR inhibitors using exogenously added DNA (aequorin plasmid) and an aequorin gene qPCR [14]; 2) We verified that microbial DNA had been collected by employing a broad-range bacterial 16S rRNA gene qPCR assay to measure total bacterial load in each sample [15]. Bacterium-specific qPCR assays were performed using purified DNA and targeted L. crispatus, L. jensenii, and L. iners [16,17]. No-template water controls were included with all PCR runs to monitor contaminants from reagents.
Statistical methods
Participant characteristics were compared by vaginal washing status using the chi-squared, Fisher’s exact, or Wilcoxon-rank sum test, as appropriate. To assess the association between vaginal washing and detection of Lactobacillus by both culture (Rogosa and/or Colombia agar) and qPCR, we utilized Poisson regression with robust standard errors to estimate prevalence ratios (PR) and 95% confidence intervals (CI) [18]. For the species-specific models, we classified qPCR samples as either below or above the lower limit of detection (LLD) (125 copies/swab). This decision was made because of the high proportion of samples at undetectable levels and to facilitate comparison with culture-dependent results. We also assessed associations between vaginal washing method and frequency of washing in prior week Lactobacillus detection. Models were adjusted a priori for age (continuous) and report of any condomless sex (no, yes) in the prior week for their known independent associations with vaginal washing and Lactobacillus [6,19,20]. We assessed for other potential confounders, but none were associated with both the exposure and outcome so were not included in the adjusted models (Table 1, Supplementary Tables 1–5). As other STIs may impact the vaginal microbiota, we conducted a sensitivity analysis excluding women with C. trachomatis or N. gonorrhoeae.
Table 1.
Demographic, sexual behaviour, and clinical characteristics at sample collection for 272 women by vaginal washing status in the prior week
| Characteristic* | All women (N=272) | No vaginal washing (N=55) | Any vaginal washing (N=217) | p-value | |||
|---|---|---|---|---|---|---|---|
| Demographic | |||||||
| Age (years) | 34 | (27.5, 41) | 41 | (32, 44) | 33 | (27, 39) | 0.0002 |
| Education (years) | 8 | (7, 11) | 8 | (7,11) | 8 | (7,11) | 0.75 |
| Sexual behaviour | |||||||
| Frequency of sex in last week | 0.02 | ||||||
| 0 | 73 | (26.8) | 20 | (36.4) | 53 | (24.4) | |
| 1–2 | 81 | (29.8) | 20 | (36.4) | 61 | (28.1) | |
| ≥3 | 118 | (43.4) | 15 | (27.3) | 103 | (47.5) | |
| Condomless sex in last week | 67 | (24.6) | 9 | (16.4) | 58 | (26.7) | 0.11 |
| # of sex partners in last week | 0.11 | ||||||
| 0 | 73 | (26.8) | 20 | (36.4) | 53 | (24.4) | |
| 1 | 79 | (29.0) | 17 | (30.9) | 62 | (28.6) | |
| ≥2 | 120 | (44.1) | 18 | (32.7) | 102 | (47.0) | |
| Hormonal contraceptive use† | 0.29 | ||||||
| None/Non-hormonal | 197 | (72.4) | 43 | (78.2) | 154 | (71.0) | |
| Oral contraceptive pills | 12 | (4.4) | 2 | (3.6) | 10 | (4.6) | |
| Injectable | 50 | (18.4) | 10 | (18.2) | 40 | (18.4) | |
| Implant | 13 | (4.8) | 0 | (0) | 13 | (6.0) | |
| Clinical‡ | |||||||
| N. gonorrhoeae | 4 | (1.5) | 1 | (1.9) | 3 | (1.4) | 1.0 |
| C. trachomatis | 13 | (4.9) | 3 | (5.7) | 10 | (4.7) | 0.73 |
| Yeast§ | 35 | (12.9) | 8 | (14.6) | 27 | (12.4) | 0.68 |
| Cervicitis** | 2 | (0.7) | 1 | (1.9) | 1 | (0.5) | 0.36 |
| Vaginal Microbiota | 0.60 | ||||||
| Normal (Nugent 0–3) | 174 | (64.0) | 38 | (69.1) | 136 | (62.7) | |
| Intermediate (Nugent 4–6) | 40 | (14.7) | 6 | (10.9) | 34 | (15.7) | |
| BV (Nugent 7–10) | 58 | (21.3) | 11 | (20.0) | 47 | (21.7) | |
| Self-reported symptoms | |||||||
| Vaginal discharge | 20 | (7.4) | 2 | (3.6) | 18 | (8.3) | 0.38 |
| Vaginal itching | 16 | (5.9) | 2 | (3.6) | 14 | (6.5) | 0.75 |
| Antibiotic or anti-fungal prescription in last 60 days | |||||||
| Metronidazole | 3 | (1.1) | 1 | (1.8) | 2 | (0.92) | 0.49 |
| Anti-fungals | 5 | (1.8) | 2 | (3.6) | 3 | (1.4) | 0.27 |
| Other antibiotics | 9 | (3.3) | 1 | (1.8) | 8 | (3.7) | 0.69 |
Data are presented as n (%) for categorical variables or median (IQR) for continuous variables
None/non-hormonal includes no contraception, condoms, spermicides, diaphragm, hysterectomy, IUD, tubal ligation and other. Contraception data missing for 1 participant.
N. gonorrhoeae results missing for 5 participants. C. trachomatis results missing for 6 participants. No participants in this analysis were diagnosed with T. vaginalis
The presence of yeast buds or hyphae were assessed by saline microscopy with 10% potassium hydroxide.
Cervicitis was defined as an average of ≥30 polymorphonuclear leukocytes in three high-power microscopic fields on a Gram-stained slide of cervical secretions.
Data on the relationship between the quantity of Lactobacillus detected by qPCR and the likelihood that Lactobacillus will be detected by culture are limited. We hypothesize that there is a concentration threshold required for bacterial growth on culture. As a secondary analysis, we explored the quantity of L. crispatus and L. jensenii detected by qPCR that predicts whether Lactobacillus would also be detected by culture (Rogosa and/or Colombia agar). Isolates grown on culture were not speciated, so this analysis was agnostic to the Lactobacillus species grown. To identify a qPCR-threshold predictive of Lactobacillus detection by cultivation, we generated receiver operating characteristic (ROC) curves of the log10 quantity of species by qPCR. For both L. crispatus and L. jensenii, we identified the log10 quantity corresponding to the highest proportion of participants having cultivable Lactobacillus. Similar approaches have been used by our team and others to assess microbial burden and disease risk [21,22]. We repeated our primary analysis utilizing the ROC-derived threshold as the outcome, since bacterial concentrations may be important in mediating susceptibility to adverse outcomes [23]. Statistical significance was p<0.05 for all tests.
RESULTS
Participant characteristics
Culture and qPCR data were available for 272 participants. Median participant age was 34 years (interquartile range (IQR) 28, 41). A fifth (n=58) of participants had BV (Nugent ≥7). (Table 1). Eighty percent of participants (n=217) reported vaginal washing in the prior week, with 58.5% (n=127) of women reporting 14–20 washing episodes in the prior week, and fewer reporting washing <13 (n=40, 18.4%) or more than 20 (n=50, 23.0%). Among women reporting vaginal washing, the majority used water only (n=132, 60.8%) or soap and water (n=75, 34.6%); few washed with other products (n=10, 4.6%). Participants who reported vaginal washing were younger (median: 33 versus 41, p<0.001) and reported a higher frequency of sex in the prior week (≥3 times: 47.5% versus 27.3%, joint p=0.02). Other characteristics were similar between women who reported versus denied vaginal washing.
Lactobacillus detection by culture and qPCR
Lactobacillus was detected by culture for 26.8% (n=73) of women (Table 2). The proportion of women with cultivable Lactobacillus was lower in those reporting vaginal washing versus non-washers (23.0% versus 41.8%; p=0.005). Only 13.6% (37/272) of women had H2O2-producing Lactobacillus detected by culture, and detection was lower in women reporting versus denying vaginal washing (11.5% versus 21.8%, p=0.047). By qPCR, 21.3% (n=58) of participants had detectable quantities of L. crispatus, 25.4% (n=69) had L. jensenii detected, and 89.7% (n=244) had L. iners detected. Among samples above the LLD, the mean log10 concentration (copies/swab) was 6.18 (standard deviation [SD] ±2.27) for L. crispatus, 6.18 (SD ±1.36) for L. jensenii, and 7.76 (SD ±1.22) for L. iners. The mean log10 concentration of L. crispatus and L. jensenii were significantly higher in women denying washing compared to those reporting washing (Table 2). Of the 73 women with cultivable Lactobacillus, 4 (5.5%) were not colonized by L. crispatus, L. jensenii, or L. iners.
Table 2.
Lactobacillus detection by culture and qPCR by vaginal washing status
| Lactobacillus detection* | All women (N=272) | No vaginal washing (N=55) | Any vaginal washing (N=217) | p-value | |||
|---|---|---|---|---|---|---|---|
| Cultivable | |||||||
| Any Lactobacillus† | 73 | (26.8) | 23 | (41.8) | 50 | (23.0) | 0.005 |
| H2O2-producing Lactobacillus | 37 | (13.6) | 12 | (21.8) | 25 | (11.5) | 0.047 |
| Detection by qPCR‡ | |||||||
| L. crispatus | 58 | (21.3) | 18 | (32.7) | 40 | (18.4) | 0.02 |
| L. jensenii | 69 | (25.4) | 17 | (30.9) | 52 | (24.0) | 0.29 |
| L. iners | 244 | (89.7) | 47 | (85.5) | 197 | (90.8) | 0.25 |
| Mean log10 concentration (copies/swab) (SD)§ | |||||||
| L. crispatus | 6.18 | (2.27) | 7.17 | (2.16) | 5.74 | (2.20) | 0.02 |
| L. jensenii | 6.18 | (1.36) | 6.77 | (0.75) | 5.99 | (1.46) | 0.04 |
| L. iners | 7.76 | (1.22) | 7.70 | (1.52) | 7.77 | (1.14) | 0.74 |
Abbreviations: qPCR=quantitative PCR
Data are presented as the number of women with the characteristic (%).
Rogosa and/or Colombia agar
Categorized as <lower limit of detection and >lower limit of detection.
Including only women with samples above the LLD
Association between vaginal washing and Lactobacillus
In unadjusted analysis, vaginal washing in the prior week was associated with a 45% decreased likelihood of Lactobacillus detection by culture (PR=0.55, 95% Confidence Interval (CI) 0.37–0.82). When adjusting for age and condomless sex in the prior week, the magnitude of the association did not change (aPR=0.56, 95%CI 0.37–0.85). All washing methods were associated with a similar 45–55% reduction in detection of cultivable Lactobacillus (Table 3). Women who reported vaginal washing were also less likely to have H2O2-producing Lactobacillus detected by culture (aPR=0.50, 95%CI 0.26–0.94). In addition, there was a larger reduction in detection of cultivable Lactobacillus (trend: p<0.01) and H2O2-producing Lactobacillus by culture (trend: p<0.05) with increasing washing frequency (Table 4).
Table 3:
Association between vaginal washing and detection of Lactobacillus by culture and quantitative PCR
| Type of Lactobacillus detection | Detection n (%) | Unadjusted PR (95%CI) | Adjusted PR* (95%CI) | |||||
|---|---|---|---|---|---|---|---|---|
| Yes | No | |||||||
| Cultivable Lactobacillus† | N=73 | N=199 | ||||||
| Any vaginal washing | 50 | (68.5) | 167 | (83.9) | 0.55 | (0.37, 0.82) | 0.56 | (0.37, 0.85) |
| Type of vaginal washing | ||||||||
| None | 23 | (31.5) | 32 | (16.1) | REF | REF | ||
| Water only | 31 | (42.5) | 101 | (50.8) | 0.56 | (0.36, 0.87) | 0.57 | (0.36, 0.90) |
| Soap and Water | 17 | (23.3) | 58 | (29.2) | 0.54 | (0.32, 0.91) | 0.56 | (0.33, 0.96) |
| Antiseptic, detergent, other | 2 | (2.7) | 8 | (4.0) | 0.48 | (0.13, 1.72) | 0.46 | (0.13, 1.63) |
| Cultivable H2O2-producing Lactobacillus | N=37 | N=255 | ||||||
| Any vaginal washing | 25 | (67.6) | 192 | (81.7) | 0.53 | (0.28, 0.98) | 0.49 | (0.26, 0.94) |
| Type of vaginal washing | ||||||||
| None | 12 | (32.4) | 43 | (18.3) | REF | REF | ||
| Water only | 13 | (35.1) | 119 | (50.6) | 0.45 | (0.22, 0.93) | 0.42 | (0.20, 0.88) |
| Soap and Water | 10 | (27.0) | 65 | (27.7) | 0.61 | (0.28, 1.31) | 0.57 | (0.26, 1.27) |
| Antiseptic, detergent, other | 2 | (5.4) | 8 | (3.4) | 0.92 | (0.24, 3.50) | 0.86 | (0.22, 3.31) |
| L. crispatus | N=58 | N=214 | ||||||
| Any vaginal washing | 40 | (69.0) | 177 | (82.7) | 0.56 | (0.35, 0.90) | 0.57 | (0.36, 0.92) |
| Type of vaginal washing | ||||||||
| None | 18 | (31.0) | 37 | (17.3) | REF | |||
| Water only | 25 | (43.1) | 107 | (50.0) | 0.58 | (0.34, 0.97) | 0.58 | (0.35, 0.98) |
| Soap and Water | 14 | (24.1) | 61 | (28.5) | 0.57 | (0.31, 1.05) | 0.59 | (0.32, 1.09) |
| Antiseptic, detergent, other | 1 | (1.7) | 9 | (4.2) | 0.31 | (0.05, 2.05) | 0.27 | (0.04, 1.76) |
| L. jensenii | N=69 | N=203 | ||||||
| Any vaginal washing | 52 | (75.4) | 165 | (81.3) | 0.78 | (0.49, 1.23) | 0.68 | (0.42, 1.09) |
| Type of vaginal washing | ||||||||
| None | 17 | (24.6) | 38 | (18.7) | REF | REF | ||
| Water only | 35 | (50.7) | 97 | (47.8) | 0.86 | (0.53, 1.40) | 0.75 | (0.45, 1.24) |
| Soap and Water | 15 | (21.7) | 60 | (29.6) | 0.65 | (0.35, 1.18) | 0.56 | (0.31, 1.04) |
| Antiseptic, detergent, other | 2 | (2.9) | 8 | (3.9) | 0.65 | (0.18, 2.38) | 0.53 | (0.15, 1.90) |
| L. iners | N=244 | N=28 | ||||||
| Any vaginal washing | 197 | (80.7) | 20 | (71.4) | 1.06 | (0.94, 1.19) | 1.03 | (0.92, 1.15) |
| Type of vaginal washing | ||||||||
| None | 47 | (19.3) | 8 | (28.6) | REF | REF | ||
| Water only | 121 | (49.6) | 11 | (39.3) | 1.07 | (0.95, 1.21) | 1.04 | (0.93, 1.17) |
| Soap and Water | 66 | (27.1) | 9 | (32.1) | 1.03 | (0.90, 1.18) | 1.00 | (0.87, 1.14) |
| Antiseptic, detergent, other | 10 | (4.1) | 0 | (0) | 1.17 | (1.05, 1.31) | 1.10 | (0.98, 1.23) |
Adjusted a priori for age at visit and any condomless sex in last week
Growth on Rogosa and/or Colombia agar
Table 4.
Association between frequency of vaginal washing and detection of cultivable Lactobacillus, detection of H2O2-producing Lactobacillus by culture, and detection of L. crispatus, L. jensenii, and L. iners above the lower limit of detection by quantitative PCR
| Type of Lactobacillus detection | Detection n (%) | Unadjusted PR (95%CI) | Adjusted PR* (95%CI) | p-value† (test for trend) | |||||
|---|---|---|---|---|---|---|---|---|---|
| Yes | No | ||||||||
| Cultivable Lactobacillus‡ | N=73 | N=199 | |||||||
| No vaginal washing | 23 | (31.5) | 32 | (16.1) | REF | REF | <0.01 | ||
| <13 times in last week | 10 | (13.7) | 30 | (15.1) | 0.60 | (0.32, 1.11) | 0.64 | (0.34, 1.22) | |
| 14–20 times in last week | 33 | (45.2) | 94 | (47.2) | 0.62 | (0.40, 0.95) | 0.63 | (0.41, 0.97) | |
| ≥21 times in last week | 7 | (9.6) | 43 | (21.6) | 0.33 | (0.16, 0.71) | 0.33 | (0.15, 0.71) | |
| Cultivable H2O2-producing Lactobacillus | N=37 | N=255 | |||||||
| No vaginal washing | 12 | (32.4) | 43 | (18.3) | REF | REF | <0.05 | ||
| <13 times in last week | 6 | (16.2) | 34 | (14.5) | 0.69 | (0.28, 1.68) | 0.62 | (0.24, 1.59) | |
| 14–20 times in last week | 16 | (43.2) | 111 | (47.2) | 0.58 | (0.29, 1.14) | 0.55 | (0.28, 1.07) | |
| ≥21 times in last week | 3 | (8.1) | 47 | (20.0) | 0.28 | (0.08, 0.92) | 0.26 | (0.07, 0.92) | |
| L. crispatus | N=58 | N=214 | |||||||
| No vaginal washing | 18 | (31.0) | 37 | (17.3) | REF | REF | <0.05 | ||
| <13 times in last week | 9 | (15.5) | 31 | (14.5) | 0.69 | (0.34, 1.37) | 0.77 | (0.38, 1.55) | |
| 14–20 times in last week | 21 | (36.2) | 106 | (49.5) | 0.51 | (0.29, 0.87) | 0.51 | (0.30, 0.88) | |
| ≥21 times in last week | 10 | (17.2) | 40 | (18.7) | 0.61 | (0.31, 1.20) | 0.59 | (0.30, 1.19) | |
| L. jensenii | N=69 | N=203 | |||||||
| No vaginal washing | 17 | (24.6) | 38 | (18.7) | REF | REF | 0.18 | ||
| <13 times in last week | 11 | (15.9) | 29 | (14.3) | 0.89 | (0.47, 1.69) | 0.73 | (0.38, 1.40) | |
| 14–20 times in last week | 31 | (44.9) | 96 | (47.3) | 0.79 | (0.48, 1.30) | 0.70 | (0.42, 1.17) | |
| ≥21 times in last week | 10 | (14.5) | 40 | (19.7) | 0.65 | (0.33, 1.28) | 0.56 | (0.27, 1.15) | |
| L. iners | N=244 | N=28 | |||||||
| No vaginal washing | 47 | (19.3) | 8 | (28.6) | REF | REF | 0.80 | ||
| <13 times in last week | 40 | (16.4) | 0 | (0) | 1.17 | (1.05, 1.31) | 1.13 | (1.02, 1.26) | |
| 14–20 times in last week | 114 | (46.7) | 13 | (46.4) | 1.05 | (0.93, 1.19) | 1.02 | (0.91, 1.16) | |
| ≥21 times in last week | 43 | (17.6) | 7 | (25.0) | 1.01 | (0.86, 1.18) | 0.97 | (0.83, 1.13) | |
Adjusted a priori for age at visit and any condomless sex in last week
Non-parametric test for trend
Growth on Rogosa and/or Colombia agar
Any vaginal washing in the prior week was associated with >40% reduction in L. crispatus detection (aPR: 0.57, 95%CI 0.36–0.92) and >30% reduction in L. jensenii detection (aPR: 0.68, 95%CI 0.42–1.09), although the latter was not statistically significant (Table 3). Vaginal washing was not significantly associated with L. iners detection (aPR: 1.03, 95%CI 0.92–1.15). Increasing frequency of washing was associated with larger reductions in L. crispatus detection (trend: p<0.05), but not L. jensenii (trend: p=0.18) (Table 4). When excluding 23 women with C. trachomatis, N. gonorrhoeae, or missing STI results, vaginal washing remained associated with reduced likelihoods of cultivable Lactobacillus and L. crispatus, although the magnitude was attenuated (Supplementary Table 6). The association between vaginal washing and cultivable H2O2-producing Lactobacillus was no longer significant. While not the primary focus of this analysis, we assessed the relationship between additional behavioural and clinical factors and Lactobacillus detection (Supplementary Tables 1–5).
Analysis of ROC-derived quantitative thresholds
The concordance between qPCR detection of L. crispatus and L. jensenii above the LLD and culture was 78.3% (kappa=0.41) and 78.7% (kappa=0.45), respectively. Concordance between qPCR detection of L. crispatus (81.3%, kappa=0.36) and L. jensenii (80.9%, kappa=0.40) and detection of H2O2-producing Lactobacillus by culture were similar. In the analysis employing ROC curves to examine the quantity of species-specific Lactobacillus DNA detected by qPCR and the likelihood of cultivable Lactobacillus detection, we identified a threshold of log10 4.41 copies/swab for L. crispatus and log10 6.14 copies/swab for L. jensenii (Supplementary Figure 1). Forty-one (15.1%) women had concentrations above the ROC-thresholds for each species
Similar to the primary analysis, vaginal washing was associated with a statistically significant 55% reduction in L. crispatus detection above the ROC-threshold (aPR=0.46, 95%CI 0.25–0.82) (Supplemental Table 3). There was a non-significant 45% reduction in L. jensenii above the ROC-threshold (aPR=0.54, 95%CI 0.29–1.02) (Supplemental Table 4).
DISCUSSION
In this cross-sectional study utilizing both culture and molecular methods for detecting vaginal Lactobacillus among high-risk Kenyan women, vaginal washing was associated with a 45% reduced likelihood of detecting Lactobacillus by culture and L. crispatus by qPCR. In contrast, there was a non-significant 30% reduction in L. jensenii detection and no association with L. iners detection. These results suggest that vaginal washing may have the greatest impact on L. crispatus, the species most consistently linked to optimal vaginal health and reductions in risk of adverse reproductive health events [2,24].
Our vaginal washing and Lactobacillus culture findings are consistent with a prospective study of the correlates of cultivable Lactobacillus in the Mombasa Cohort from 1995–2003 that found a 40% reduction in Lactobacillus positive culture among women reporting vaginal washing [6]. Both of these Mombasa Cohort results are consistent with studies in the US [7,8]. Moreover, in this analysis, reductions in Lactobacillus detection generally became larger with increasing frequency of vaginal washing, suggesting a dose-response effect. Mean concentrations of L. crispatus and L. jensenii, but not L. iners, were also lower among women who reported vaginal washing. Bacterial concentrations of L. iners tend to be higher than other Lactobacillus species [21], including in this study where the mean concentration was 1.5 log10 higher than L. crispatus and L. jensenii. The effect of vaginal washing on L. iners concentrations may therefore need to be stronger to reduce L. iners concentrations to undetectable.
The lack of difference in Nugent score category between vaginal washing groups exemplifies the motivation for this analysis specifically interrogating Lactobacillus detection. Since Nugent score classifies bacteria by morphotype, misclassification is possible since the approach is not highly sensitive. Molecular and culture-dependent methods are complimentary methods of characterizing the microbiota. The former is highly sensitive, detecting bacteria at low levels, but may detect non-viable bacteria. Culture-dependent methods, while less sensitive, detect growth, a sign of viable bacteria. Few studies have utilized both culture and molecular methods for independent characterization of vaginal Lactobacillus colonization [25,26]. In a study of US HIV-seropositive women conducted by our group, there was a lack of concordance between samples with cultivable H2O2-producing Lactobacillus and detection of L. crispatus and L. jensenii, two purported H2O2-producing species. The discrepancy may be due to growth of other H2O2-producing Lactobacillus, such as L. gasseri, and/or quantitative detection of non-functional or non-H2O2 producing strains [26]. In a small study in Belgium, researchers noted no or low H2O2-production by Lactobacillus on culture when concentrations of L. crispatus were low [25]. In our study of HIV-seronegative women, there was moderate concordance between L. crispatus and L. jensenii by qPCR and cultivable Lactobacillus. This also likely reflects growth of other Lactobacillus species or presence of non-viable bacteria detected by qPCR, highlighting the joint importance, and complimentary nature, of culture and molecular methods. The ROC analysis to ascertain qPCR thresholds of L. crispatus and L. jensenii predictive of Lactobacillus detection by culture demonstrated that vaginal washing reduced the proportion of women with detectable L. crispatus and L. jensenii. Furthermore, vaginal washing reduced concentrations of Lactobacillus below the optimal concentration needed to predict growth on culture. Of note, vaginal washing was not significantly associated with a reduction in L. jensenii detection above the LLD or ROC-derived threshold. We are not aware of any known biological reason for this difference between L. crispatus and L. jensenii. Further research may be needed to discern the distinct roles of these species.
Small studies of vaginal washing cessation interventions have consistently shown no effect on BV even when there were reductions in self-reported intravaginal practices [10,27–30], with some exceptions [27,29]. In a pilot study in the US, while the reduced odds of BV in the washing versus washing cessation phases was not significant, when excluding samples taken during and shortly after menstruation, there were significant reductions in BV episodes [27]. There was also a significant reduction in BV in the subgroup of women who reported engaging in washing for menstrual hygiene purposes. Results of a Zambian trial found a moderate reduction in abnormal microbiota (Nugent ≥4) among women participating in a group session on healthy vaginal practices (p=0.09) [29]. Two of the cessation intervention studies also assessed detection of cultivable Lactobacillus [10,28]. In one study of 23 women, the cessation intervention demonstrated a 3.7-fold increased odds of detection of cultivable Lactobacillus (95%CI 0.72–18.76) [10]. The overall null effect observed in these studies could be explained by social desirability bias affecting self-report of vaginal washing, small sample sizes (n=23–128), and short follow-up that may not be long enough to elicit lasting behaviour change and/or allow for recolonization with sufficient quantities of Lactobacillus. These important limitations highlight the need for designing studies with larger sample sizes powered to detect changes in BV and Lactobacillus, and longer follow-up that can adequately test vaginal washing cessation interventions while accounting for other factors associated with BV (e.g., condomless sex).
The major strength of this analysis was utilization of both culture and molecular methods for Lactobacillus detection. In addition, Lactobacillus were cultured on both Rogosa and Columbia agars, increasing the range of detectable Lactobacillus species, including L iners. Together, this allowed for a thorough assessment of vaginal Lactobacillus, providing rigorous assessment of presence, quantity, and viability of Lactobacillus species. However, the analysis is limited by its cross-sectional design; we were unable to assess the temporality of the association between vaginal washing and absence of Lactobacillus. While it is possible women were engaging in vaginal washing as a result of a disrupted vaginal microbiota, few women in this study reported vaginal symptoms associated with BV. In addition, sexual behaviours were only reported for the prior week, so residual confounding may be present. Moreover, we did not speciate the lactobacilli detected by culture so cannot confirm which Lactobacillus species were present. Similarly, we did not use molecular methods to detect specific BV-associated bacteria, so our findings are limited to the targeted species. Furthermore, the high proportion of participants who reported vaginal washing (80%) and with L. iners (90%) provided limited power to detect small differences between vaginal washers and non-washers. Lastly, we did not control for multiple comparisons so some statistically significant findings may be a result of chance.
Vaginal washing in the last week was associated with reductions in likelihood of both cultivable Lactobacillus and L. crispatus by qPCR. In contrast, there were no significant associations between vaginal washing and detection of L. jensenii and L. iners, suggesting that vaginal washing may have the most negative impact on L. crispatus. Given data highlighting the association between the optimal, Lactobacillus-dominated vaginal microbiota, and increased concentrations of L. crispatus specifically, and reduced risk of HIV [4,24], these results provide motivation for future study of vaginal washing cessation interventions that could promote vaginal colonization with L. crispatus. This may include randomized trials with larger sample sizes, longer follow-up time, and molecular methods to detect quantitative changes in Lactobacillus species.
Supplementary Material
Key messages:
Vaginal washing was common among women at high risk for HIV and other sexually transmitted infections.
Vaginal washing was associated with reductions in detection of Lactobacillus by culture and qPCR, including species associated with a reduced risk of adverse reproductive outcomes.
The magnitude of reductions in detection was greater among women who reported washing more frequently, highlighting the importance of interventions to reduce vaginal washing frequency.
ACKNOWLEDGEMENTS:
The authors would like to thank the dedicated study participants for their engagement in this research and to express gratitude toward the research, clinical, laboratory, outreach, and administrative staff in Mombasa, Kenya, and Seattle, Washington for making this study possible.
FUNDING: This research was funded by grants from the National Institutes of Health [R37 AI38518, P01 HD64915, EML by T32 AI07140, K24 HD88229 to RSM for mentoring). Infrastructure and logistical support for the Mombasa research site was provided by the University of Washington’s Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027757) which is supported by the following research centers: NIAID, NCI, NIMH, NIDA, NICHD, NHLBI, NCCAM. The content of this paper is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.
Footnotes
PRESENTATION INFORMATION: This research was presented in part as a poster (#P3.059) at the 20th Meeting of the International Society for Sexually Transmitted Diseases Research (ISSTDR) held in Vienna, Austria on July 14–17, 2013.
CONFLICTS OF INTEREST: R.S.M. receives research funding, paid to the University of Washington, from Hologic, Inc (Marlborough, Massachusetts). J.E.B. received honoraria for consulting from Lupin Pharmaceuticals (Mumbai, India). All other authors declare they have no conflicts of interest to report.
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