Peroxide-producing Lactobacilli Research Articles

Fighting polymicrobial biofilms in bacterial vaginosis.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge and is often associated with other health consequences mainly in pregnant women. BV is described by an imbalance in the vaginal microbiota where strictly and facultative anaerobic bacteria outgrow the lactic acid- and hydrogen peroxide-producing Lactobacillus species. The species involved in BV are capable to grow and form a polymicrobial biofilm in the vaginal epithelium. The treatment of BV is usually performed using broad-spectrum antibiotics, including metronidazole and clindamycin. However, these conventional treatments are associated with high recurrence rates. The BV polymicrobial biofilm may have an important role on the treatment outcome and is accounted as one of the factors for treatment failure. Other possible reasons for treatment failure include the presence of species resistant to antibiotics or the chance of reinfection after treatment. Therefore, novel strategies to increase the rates of treatment have been studied namely the use of probiotics and prebiotics, acidifying agents, antiseptics, plant-based products, vaginal microbiota transplantation, and phage endolysins. Although some of them are still in an initial phase of development with very preliminary results, they show great perspectives for application. In this review, we aimed to study the role of the polymicrobial nature of BV in treatment failure and explore a few alternatives for treatment.

PREVALENCE OF BACTERIAL VAGINOSIS AMONG SEXUALLY ACTIVE WOMEN ATTENDING THE CDC CENTRAL CLINIC TIKO, SOUTH WEST REGION, CAMEROON.

Background:Bacterial vaginosis (BV) is a polymicrobial, superficial vaginal infection involving a reduction in the amount of hydrogen peroxide-producing Lactobacillus and overgrowth of anaerobic bacteria. Common symptoms include increased fishy smelling vaginal discharge which is usually white or gray in color. Burning with urination may occur and itching is uncommon. Risk factors include douching, new or multiple sex partners, antibiotics, and use of intrauterine device among others.Materials and Methods:This cross-sectional study assessed the prevalence of bacterial vaginosis among sexually active women aged 15-45 years. Vaginal swabs were obtained with the use of sterile swab sticks which were later smeared on clean glass slides and then Gram stained. The stained smears were observed for bacterial morphotypes with the X100 oil immersion objective and the Nugent scoring system was used to determine BV. Data were analyzed using the Statistical Package for Social Scientists (SPSS) version 17.0 and were considered significant at p < 0.05.Results:A total of 100 women participated in the study with the overall prevalence of BV rated 38%. The prevalence of BV with respect to associated factors was also investigated and it was observed that BV was more prevalent in the age groups 20-25 (48.1%) and 25-29 (44.4%), those who had attained only primary education (60.5%), married women, (68.4%), pregnant women (71.0%), and women who practiced vaginal douching, (97.4%). However, no statistical significant difference was observed in the prevalence between these parameters (P > 0.05).Conclusions:Conclusively, the prevalence of bacterial vaginosis in our study population is 38% and highest among women aged between 25 and 34 years, pregnant women, married women, less educated women and women who practiced poor vaginal hygiene.

Hydrogen Peroxide-Producing Lactobacilli Are Associated With Lower Levels of Vaginal Interleukin-1β, Independent of Bacterial Vaginosis.

The presence of hydrogen peroxide (H2O2)-producing lactobacilli in the vagina is associated with decreased rates of preterm birth and HIV acquisition. We hypothesize that this is due to immunomodulatory effects of these species. Concentrations of interleukin (IL)-1β, IL-6, IL-8, secretory leukocyte protease inhibitor, and human β-defensin 2 were quantified from vaginal swabs from 4 groups of women: women with and without bacterial vaginosis (BV) by Nugent score, further stratified by detection of H2O2-producing lactobacilli by semiquantitative culture. Ten quantitative polymerase chain reaction assays characterized the presence and quantity of select Lactobacillus and BV-associated species in each group. Levels of immune markers and bacteria were compared between the 4 groups using analysis of variance, Kruskal-Wallis, Mann-Whitney U, or χ tests. Swabs from 110 women from 4 groups were included: 26 had a normal Nugent score (BV-), and no H2O2-producing lactobacilli detected (H2O2-); 47 were BV-, H2O2+; 27 BV+, H2O2-; and 10 BV+, H2O2+. The groups were similar in age, marital status, and reproductive history, but not ethnicity: the BV-, H2O2- group had more white participants (P = 0.02). In women with and without BV, IL-1β was lower in the H2O2+ groups. Human β-defensin 2 was lowest in BV+ H2O2- women and highest in BV-, H2O2-. Secretory leukocyte protease inhibitor was lower in women with BV and did not differ by the presence of H2O2-producing lactobacilli. In regression analysis, higher quantities of Lactobacillus crispatus were associated with lower quantities of IL-1β. Detection and quantity of BV-associated species by quantitative polymerase chain reaction was significantly different between women with and without BV, but not between women with and without H2O2-producing lactobacilli within those groups. The presence of H2O2-producing lactobacilli is associated with lower levels of some vaginal proinflammatory cytokines, even in women with BV.

P3.363 Inflammatory Soluble Immune Mediators and Pathogenic Vaginal Bacteria Impact E. Coli Bactericidal Activity in Female Genital Tract Secretions

BackgroundIn low-risk women, in vitro inhibition of E. coli by genital tract secretions is associated with Lactobacillus crispatus and jensenii proteins. However in at-risk populations, HIV seroconversion was associated with...

S16.2 Recurrent BV

Bacterial vaginosis (BV) is a common cause of vaginitis and increases women’s risk of pelvic inflammatory disease, adverse pregnancy outcomes, and risk of STD/HIV acquisition. The aetiology of BV is unclear, though it is believed to involve loss of vaginal hydrogen peroxide-producing lactobacilli and acquisition of complex bacterial communities that include many fastidious BV-associated bacteria (BVAB) that have recently been detected using PCR methods. Treatment failure (persistence) is common, and may be facilitated by unprotected sex. Potential contributions to BV and BV persistence include (1) sexual partners as a reservoir for BVAB; (2) specific sexual practises, including male partners’ condom use; and (3) the composition of the vaginal microbiota involved in BV. Specific BVAB in the Clostridiales Order may predict BV persistence when detected pre-treatment, and have been detected in men whose female partners have BV. BVAB may be associated with unprotected sexual behaviour and failure of BV to resolve in women, supporting the hypothesis that BVAB colonisation of male genitalia may serve as a reservoir for re-infection of female partners. Moreover, specific sexual practises may favour vaginal colonisation with certain BVAB that have been associated with persistence. This session will provide background on BV, and discuss the epidemiologic and microbiologic data to support a role for acquisition of BVAB and how this process might differ among subsets of women.

Editorial Commentary: Sexual Networks, Sex Hormones, and Recurrent Bacterial Vaginosis: Not Such Strange Bedfellows

In women of reproductive age, a vaginal environment that is quantitatively dominated by hydrogen peroxide–producing Lactobacillus species typically has a pH considered to be normal (<4.7) and has consistently been associated with healthy pregnancy outcomes, lack of abnormal vaginal symptoms, and reduced risk for acquiring several sexually transmitted pathogens, including human immunodeficiency virus (HIV). The most commonly isolated Lactobacillus species associated with this healthy environment are L. crispatus and L. jensenii. In addition to the direct antimicrobial effects of lactic acid and the consequent acidic pH these bacteria engender, the natural defense mechanisms of the vagina include production of endogenous defensins (including HNP1-3), secretory leukocyte protease inhibitor, cytokines, and endocervical mucous. Bacterial vaginosis

Detection of hydrogen peroxide-producing Lactobacillus species in the vagina: a comparison of culture and quantitative PCR among HIV-1 seropositive women

BackgroundThe presence of hydrogen peroxide (H2O2) producing Lactobacillus in the vagina may play a role in controlling genital HIV-1 shedding. Sensitive molecular methods improve our ability to characterize the vaginal microbiota; however, they cannot characterize phenotype. We assessed the concordance of H2O2-producing Lactobacillus detected by culture with quantitative PCR (qPCR) detection of Lactobacillus species commonly assumed to be H2O2-producers.MethodsSamples were collected as part of a prospective cohort study of HIV-1 seropositive US women. Cervicovaginal lavage specimens were tested for L. crispatus and L. jensenii using 16S rRNA gene qPCR assays. Vaginal swabs were cultured for Lactobacillus and tested for H2O2-production. We calculated a kappa statistic to assess concordance between culture and qPCR.ResultsCulture and qPCR results were available for 376 visits from 57 women. Lactobacilli were detected by culture at 308 (82%) visits, of which 233 of 308 (76%) produced H2O2. L. crispatus and/or L. jensenii were detected at 215 (57%) visits. Concordance between detection of L. crispatus and/or L. jensenii by qPCR and H2O2-producing Lactobacillus by culture was 75% (kappa = 0.45).ConclusionsAmong HIV-1 seropositive women, there was a moderate level of concordance between H2O2-producing Lactobacillus detected by culture and the presence of L. crispatus and/or L. jensenii by qPCR. However, one-quarter of samples with growth of H2O2-producing lactobacilli did not have L. crispatus or L. jensenii detected by qPCR. This discordance may be due to the presence of other H2O2-producing Lactobacillus species.

Effect of Sexual Activity on Vaginal Colonization With Hydrogen Peroxide-Producing Lactobacilli and Gardnerella vaginalis

Bacterial vaginosis is associated with sexual activity, but mechanisms for this association are unclear. Cross-sectional analysis of data from women reporting sex with women who provided information on sexual behaviors as part of a study of vaginal bacteria was conducted. Vaginal bacteria were detected by semiquantitative culture. Binomial regression with log-link evaluated associations between detection of bacteria and sexual behaviors reported to occur before enrollment. Linear regression evaluated associations between these behaviors and quantity of bacteria. Of the 320 women, 216 (68%) were colonized with H2O2-producing lactobacilli and 142 (44%) with Gardnerella vaginalis. Colonization with G. vaginalis was associated with >20 digital-vaginal sex acts (risk ratio, 2.01; 1.22, 3.29) or >10 toy-vaginal acts in the past 3 months (risk ratio, 1.76; 1.32, 2.36). Quantity of H2O2-producing lactobacilli was 1.3 log lower in colonized women reporting >10 acts of insertive vaginal sex-toy use in the past 3 months (95% confidence interval: -2.04, -0.56), 1.19 log lower with toy-vaginal sex in past 7 days (-1.75, -0.62), and 0.78 log lower in women sharing toys with a partner (-1.25, -0.31). Vaginal insertive use and sharing of sex toys were associated with decreased quantities of H2O2-producing lactobacilli and higher risk of colonization with G. vaginalis.

Interpreting the epidemiology and natural history of bacterial vaginosis: Are we still confused?

Bacterial vaginosis (BV) is a common cause of vaginitis and increases women’s risk of pelvic inflammatory disease, adverse pregnancy outcomes, and risk of STD/HIV acquisition. The etiology of BV is unclear, though it is believed to involve loss of vaginal hydrogen peroxide-producing lactobacilli and acquisition of complex bacterial communities that include many fastidious BV-associated bacteria (BVAB) that have recently been detected using PCR methods. Treatment failure (persistence) is common, and may be facilitated by unprotected sex. Potential contributions to BV and BV persistence include (1) sexual partners as a reservoir for BVAB; (2) specific sexual practices, including male partners’ condom use; and (3) the composition of the vaginal microbiota involved in BV. Specific BVAB in the Clostridiales order may predict BV persistence when detected pre-treatment, and have been detected in men whose female partners have BV. BVAB may be associated with unprotected sexual behavior and failure of BV to resolve in women, supporting the hypothesis that BVAB colonization of male genitalia may serve as a reservoir for re-infection of female partners. Moreover, specific sexual practices may favor vaginal colonization with certain BVAB that have been associated with persistence. This review provides background on BV, and discusses the epidemiologic and microbiologic data to support a role for acquisition of BVAB and how this process might differ among subsets of women.

Effective of metronidazole to bacterial flora in vagina and the impact of microbes on live birth rate during intracytoplasmic sperm injection (ICSI)

The aim of this study was to investigate the impact of bacterial vaginal flora on life-birth rate during ICSI and influence of metronidazole as antibiotic treatment course before ICSI. We enrolled 71 women who were undergoing ICSI. At embryo transfer (ET), all of the women had quantitative vaginal culture, ET catheter-tip culture, and vaginal Gram stain scored for bacterial vaginosis. The overall live birth rate (LBR) was 36.6% (26/71), and the rate of early pregnancy loss was 13% (4/30). In women with bacterial vaginosis, intermediate flora and normal flora, the conception rates were 35% (9/26), 42% (14/33) and 58% (7/12), respectively (p = 0.06 for trend). Metronidazole effect to bacterial flora in vaginal. The predominant species isolated from the tip of the embryo transfer catheter in negative pregnancy was Staphylococcus epidermidis (7 vs. 15.2%), and Streptococcus viridians (11 vs. 24%). Woman with bacterial vaginosis and with a decreased vaginal concentration of hydrogen peroxide-producing lactobacilli may have decreased conception rates and increased rates of failed pregnancy. A larger prospective treatment trial designed to evaluate the impact on ICSI outcomes of optimizing the vaginal flora prior to ICSI may be warranted.

Cervicovaginal Shedding of HIV Type 1 Is Related to Genital Tract Inflammation Independent of Changes in Vaginal Microbiota

We examined the relationship of proinflammatory vaginal cytokines and secretory leukocyte protease inhibitor (SLPI) with genital HIV-1 shedding after controlling for genital coinfections. Fifty-seven HIV-1-infected women in Seattle, WA (n = 38) and Rochester, NY (n = 19) were followed every 3-4 months for a total of 391 visits. At each visit, plasma and cervicovaginal lavage (CVL) were tested for HIV-1 RNA using qPCR. Vaginal samples were tested for bacterial vaginosis, yeast, hydrogen peroxide-producing Lactobacillus colonization, Trichomonas vaginalis, Neisseria gonorrhea, Chlamydia trachomatis, CMV, and HSV shedding. CVL interleukins (IL)-1β, IL-6, IL-8, and SLPI were measured using ELISA. Linear regression with generalized estimating equations examined effects of cytokine concentrations on CVL HIV-1 RNA, adjusted for plasma HIV RNA, and measured coinfections. CVL IL-1β and IL-8 were significantly associated with CVL HIV-1 RNA. This persisted after adjusting for plasma HIV-1 RNA. Higher levels of IL-1β were associated with higher concentrations of HIV-1 RNA in CVL (β = 0.25, 95% CI 0.09, 0.42), as were higher levels of IL-8 (β = 0.34, 95% CI 0.17, 0.50). Adjusting for the presence of the coinfections described, this relationship was attenuated for IL-1β (β = 0.16; 95% CI -0.01, 0.33) but still significant for IL-8 (β = 0.29; 95% CI 0.13, 0.45). The proinflammatory cytokines IL-1β and IL-8 are associated with higher cervicovaginal HIV-1 RNA concentrations, even after controlling for plasma viral load and vaginal microbial cofactors. This association suggests that there may be additional, noninfectious causes of inflammation that increase cervicovaginal HIV-1 shedding.

Candidate vaginal microbicides with activity against Chlamydia trachomatis and Neisseriagonorrhoeae

Vaginal microbicides with activity towards organisms that cause sexually transmitted infections have been proposed as a strategy to reduce transmission. Small-molecule inhibitors of Chlamydia trachomatis serovar D belonging to the class of salicylidene acylhydrazides (INPs) have been shown to work through a mechanism that involves iron restriction. Expanding on this work, ten INPs were tested against a lymphogranuloma venereum strain of C. trachomatis (serovar L2), Neisseria gonorrhoeae, and hydrogen peroxide-producing Lactobacillus crispatus and Lactobacillus jensenii. Seven INPs had minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations of <50 μM towards C. trachomatis L2. Three INPs had a MIC <12.5 μM against N. gonorrhoeae. Inhibition was reversed by iron, holo-transferrin and holo-lactoferrin but not by the iron-poor forms of these compounds. The compounds exhibited no bactericidal activity toward Lactobacillus. The INPs were not cytotoxic to HeLa 229 cells. When INP 0341 was tested in a mouse model of a Chlamydia vaginal infection there was a significant reduction in the number of mice shedding C. trachomatis up to 4 days after infection ( P < 0.01). In summary, select INPs are promising vaginal microbicide candidates as they inhibit the growth of two common sexually transmitted organisms in vitro, are active in a mouse model against C. trachomatis, are not cytotoxic and do not inhibit organisms that compose the normal vaginal flora.

Individual and co-operative roles of lactic acid and hydrogen peroxide in the killing activity of enteric strain Lactobacillus johnsonii NCC933 and vaginal strain Lactobacillus gasseri KS120.1 against enteric, uropathogenic and vaginosis-associated pathog

The mechanism underlying the killing activity of Lactobacillus strains against bacterial pathogens appears to be multifactorial. Here, we investigate the respective contributions of hydrogen peroxide and lactic acid in killing bacterial pathogens associated with the human vagina, urinary tract or intestine by two hydrogen peroxide-producing strains. In co-culture, the human intestinal strain Lactobacillus johnsonii NCC933 and human vaginal strain Lactobacillus gasseri KS120.1 strains killed enteric Salmonella enterica serovar Typhimurium SL1344, vaginal Gardnerella vaginalis DSM 4944 and urinary tract Escherichia coli CFT073 pathogens. The cell-free culture supernatants (CFCSs) produced the same reduction in SL1344, DSM 4944 and CFT073 viability, whereas isolated bacteria had no effect. The killing activity of CFCSs was heat-stable. In the presence of Dulbecco's modified Eagle's minimum essential medium inhibiting the lactic acid-dependent killing activity, CFCSs were less effective at killing of the pathogens. Catalase-treated CFCSs displayed a strong decreased activity. Tested alone, hydrogen peroxide triggered a concentration-dependent killing activity against all three pathogens. Lactic acid alone developed a killing activity only at concentrations higher than that present in CFCSs. In the presence of lactic acid at a concentration present in Lactobacillus CFCSs, hydrogen peroxide displayed enhanced killing activity. Collectively, these results demonstrate that for hydrogen peroxide-producing Lactobacillus strains, the main metabolites of Lactobacillus, lactic acid and hydrogen peroxide, act co-operatively to kill enteric, vaginosis-associated and uropathogenic pathogens.

A pilot study evaluating the safety of vaginal administration of a multi-particulate pellet formulation

Aim Quantitative evaluation of the effect caused by vaginal administration of gelatin capsules loaded with starch pellets and lyophilized powder, respectively, on vaginal pH and microflora. Method Administration of gelatin capsules loaded with fast-disintegrating starch pellets (group P) or lyophilized lactose/skimmed milk (group L) was compared to no intervention (group C) in a 3-way randomized, double-blinded, parallel study with 18 volunteers. Follow-up visits were at day 6 (immediately after administration), day 14 (pill stop), day 22 (after withdrawal bleeding) and day 35 (midcycle). Vaginal pH was measured and swabs were taken for Gram staining and culture to assess the presence of hydrogen peroxide-producing lactobacilli. Colposcopy was performed to assess the occurrence of adverse effects on the vaginal and ectocervical mucosa. Results No severe adverse events occurred. For all women, vaginal pH and Gram stain were normal from screening until pill stop. Although immediately after withdrawal bleeding, 8 out of 18 women had an elevated pH, a disturbed microflora or lacked hydrogen peroxide-producing lactobacilli, all women had hydrogen peroxide-producing lactobacilli and a normal vaginal pH at midcycle, and all but two had a normal Gram stain. Conclusion No major differences could be observed between the groups, whereby all changes in pH and microflora could be ascribed to withdrawal bleeding, indicating that gelatin capsules, starch pellets and lyophilized powder are acceptable carrier materials for the vaginal delivery of probiotic strains.

Hydrogen Peroxide–Producing Lactobacilli Inhibit Gonococci In Vitro but Not during Experimental Genital Tract Infection

Commensal lactobacilli that produce hydrogen peroxide (H(2)O(2)) inhibit Neisseria gonorrhoeae in vitro, and clinical data suggest that they are associated with a reduced risk of gonorrhea. We precolonized mice with Lactobacillus crispatus and then challenged them with N. gonorrhoeae, to measure the effects of H(2)O(2)-producing lactobacilli on gonococcal infection. We found no difference in the duration of infection or the number of gonococci recovered from untreated mice and mice colonized with L. crispatus. A gonococcal catalase mutant and a catalase, cytochrome C peroxidase mutant exhibited greater susceptibility to L. crispatus in vitro than did wild-type bacteria; however, recovery of these mutants from mice was not affected by L. crispatus. We also found no evidence that utilization of lactobacillus-produced lactate by N. gonorrhoeae balances the detrimental effects of H(2)O(2) during infection. We conclude that the association between lactobacilli and gonococci is complex and may be subject to factors that have not been reproduced in vitro.

Antagonistic Potential against Pathogenic Microorganisms and Hydrogen Peroxide Production of Indigenous Lactobacilli Isolated from Vagina of Chinese Pregnant Women

To investigate the indigenous lactobacilli from the vagina of pregnant women and to screen the isolates with antagonistic potential against pathogenic microorganisms. The strains were isolated from pregnant women's vagina and identified using the API50CH system. The ability of the isolates to produce hydrogen peroxide was analyzed semi-quantitatively using the TMB-HRP-MRS agar. The antagonistic effects of the isolates on pathogenic microorganisms were determined with a double layer agar plate. One hundred and three lactobacilli strains were isolated from 60 samples of vaginal secretion from healthy pregnant women. Among them, 78 strains could produce hydrogen peroxide, in which 68%, 80%, 80%, and 88% had antagonistic effects against Candida albicans CMCC98001, Staphylococcus aureus CMCC26003, Escherichia coli CMCC44113, and Pseudomonas aeruginosa CMCC10110, respectively. The recovery of hydrogen peroxide-producing lactobacilli decreases with the increasing pregnant age and time. The most commonly isolated species from vagina of Chinese pregnant women are Lactobacillus acidophilus and Lactobacillus crispatus. Most of L. acidophilus and L. crispatus produce a high H2O2 level.

A Randomized Trial of Metronidazole in Asymptomatic Bacterial Vaginosis to Prevent the Acquisition of Sexually Transmitted Diseases

ABSTRACT Bacterial vaginosis (BV)—the most common form of vaginitis throughout the world—has been associated with adverse outcomes including HIV infection and other sexually transmitted diseases (STDs). One reason for this may be the absence of hydrogen peroxide-producing lactobacilli that inhibit bacterial growth. At present, treatment of asymptomatic BV is not routinely recommended. This goal of this prospective study was to determine whether treating BV lessens the risk of STDs. Participants were 107 African American women with asymptomatic BV whose mean age was 25 years. Fifty-three of them were assigned randomly to apply intravaginal metronidazole gel at bedtime for 5 days and subsequently twice a week for 6 months. These women and the 54 who were only observed were followed up for a total of 12 months. Significantly longer times preceded the development of STD in women using metronidazole gel. Six-month rates of STD were 2.29 per person-year in the observed group and 1.58 per person-year in the actively treated women, a statistically significant difference. No such difference was apparent in the last 6 months of the study after treatment ended. Overall rates of STD for the entire 12-month period were 1.64 and 1.31, respectively, in the observation and treatment groups. The only significant difference in the distribution of individual pathogens was a greater frequency of chlamydia infection in the observed group. This is the first study to demonstrate that the prevention and treatment of asymptomatic BV lowers the risk of chlamydia infection. While it remains necessary to confirm these findings, especially in more diverse populations, the present findings suggest that routine treatment be considered for women with BV even if there are no symptoms.

Estrogens and Urinary‐Tract Infection

The effects of estrogen on the risk of urinary-tract infection (UTI) in women has been the subject of many studies in both humans and experimental animals. Often, these studies have come to seemingly contradictory results: some have suggested an increased risk attributable to estrogen and others that estrogens may be preventative. In part, this confusion arises because the physiological effects of estrogen on different anatomic parts of the urinary tract differ depending on the specific effect and the outcome measured. For example, in the absence of estrogen, the periurethral and vaginal microflora, which is usually predominated by hydrogen peroxide-producing lactobacilli and few Escherichia coli, changes dramatically to a flora with few or no lactobacilli but many E. coli [1]. This change in flora is associated with a markedly increased risk of recurrent E. colibladder infections. In a randomized, placebo-controlled trial of topical intravaginal estrogen cream in such women, both the reestablishment of the normal lactobacillus-dominant vaginal flora and reduced rates of UTI were demonstrated in estro-

The Vaginal Flora of Healthy Women Is Not Always Dominated byLactobacillusSpecies

Marrazzos interesting editorial commentary about the enigmatic ecological mystery of bacterial vaginosis (BV) fails to take into account the recent findings of several investigations that call into question some long-held assumptions about this disorder. Her essay begins with the pronouncement that we have known for decades that the normal vagina is dominated by hydrogen peroxide-producing lactobacilli and then follows this with the declaration that decreases in the numbers of lactobacilli have been associated with a host of problems including BV gonorrhea HIV infection and cervicitis. However several investigations conducted using cultivation-independent methods have shown that a significant proportion (~30%) of healthy women lack appreciable numbers of Lactobacillus species. (excerpt)

Reply to: ‘Is vaginal washing associated with increased risk of HIV-1 acquisition?’

In their letter, van de Wijgert and colleagues [1] report that they found no association between vaginal washing and HIV-1 acquisition among women attending family planning clinics in Zimbabwe and Uganda. These results differ from our study in a cohort of female sex workers in Kenya [2], among whom vaginal washing was associated with a significantly increased risk of HIV-1. We agree that differences in the populations and their background HIV-1 risk may partly explain the variation between the two studies. An additional factor that may help to explain the difference in study results is the frequency and timing of vaginal washing. Although we did not collect this information when the Mombasa cohort was initiated in 1993, we have collected detailed data about vaginal washing practices since 2002 from cohort participants enrolling in a trial of periodic presumptive treatment for vaginal infections. Of the 310 women enrolled, 300 (97%) reported that they perform vaginal washing, with a median frequency of three (interquartile range 2–3) times per day. Vaginal washing was performed after sex in 299 (100%), when bathing in 294 (98%), after urinating in 83 (28%), and before sex in only 16 (5%) women. The association between the frequency of vaginal washing and HIV-1 risk should be a topic for future investigation. Although methodological differences between the Kenya and Zimbabwe/Uganda studies have been noted, we feel that these are unlikely to explain the differences in results. We used the baseline report of vaginal washing as our measure of exposure because we felt that this better captured the women's usual behavior over time. More importantly, we felt that this measure was less subject to bias as a result of washing in response to genital symptoms (including symptomatic genital tract infections) or a perception of partners' risk. Nonetheless, we also evaluated the association between vaginal washing as a time-dependent covariate and HIV-1 seroconversion. In this analysis, vaginal washing with water [hazard ratio (HR) 3.63, 95% confidence interval (CI) 1.43–9.20, P = 0.007] and with soap (HR 3.82, 95% CI 1.56–9.33, P = 0.003) were significantly associated with an increased risk of HIV-1 acquisition. Differences in laboratory testing were noted between our study and the study reported by van de Wijgert and colleagues [1]. We did not include serological testing for herpes simplex virus (HSV) 2 or testing for Chlamydia trachomatis. However, we did adjust our analyses for the presence of genital ulcerations, mucopurulent cervical discharge, and microscopic evidence of cervicitis, the likely mechanisms by which herpes and Chlamydia increase the risk of HIV-1. It is important to note that neither HSV-2 serostatus nor incident chlamydial infection would confound our results unless these exposures were also associated with baseline vaginal washing. Although women may wash in response to episodes of HSV-2 or Chlamydia, it seems unlikely that practices reported at enrollment would be closely related to subsequent genital infections. We thus feel that confounding by HSV-2 and Chlamydia does not explain the increased risk of HIV-1 acquisition associated with vaginal washing. We recently evaluated the association between vaginal washing and Lactobacillus colonization (by culture). Compared with women who reported no vaginal washing, there was a step-wise decrease in the detection of vaginal Lactobacillus during follow-up among women who washed with water (HR 0.58, 95% CI 0.41–0.82, P = 0.002) and with soap (HR 0.46, 95% CI 0.36–0.61, P < 0.001). Similar findings were observed for the detection of hydrogen peroxide producing lactobacilli (water only, HR 0.53, 95% CI 0.30–0.95, P = 0.03; soap HR 0.39, 95% CI 0.27–0.55, P < 0.001). Vaginal colonization with Lactobacillus, and particularly with hydrogen peroxide-producing Lactobacillus species, has been associated with a significantly lower risk of HIV-1 acquisition [3], supporting the hypothesis that vaginal washing may increase HIV-1 susceptibility by eliminating vaginal lactobacilli. In summary, our recent study demonstrates that vaginal washing may substantially increase women's risk of acquiring HIV-1. This adds to evidence that vaginal washing, which has no confirmed medical value, increases the risk of pelvic inflammatory disease, other genital tract infections, and adverse obstetrical outcomes [4]. We feel that intervention strategies aimed at modifying intravaginal practices should be evaluated as a possible female-controlled HIV-1 prevention strategy. This should not be interpreted as indicating that vaginal washing should be addressed to the exclusion of other strategies. We agree that vaginal washing interventions could be most effective as part of a comprehensive programme including the treatment and prevention of genital tract infections, barrier methods, and other HIV-1 risk-reduction strategies. Combined interventions of this type are a focus of our current research.