For this study, we reasoned that persistently high concentrations of BV-associated bacteria could prevent colonization with exogenous Lactobacillus. Vaginal colonization with L. crispatus CTV-05 was achieved in 61% of women at either day 10 and/or day 28; while 44% were colonized with CTV-05 at day 28. A comparison with other studies on probiotics for BV treatment is difficult because most used different strains, tested colonization in healthy women, used a vaginal or oral capsules, and/or they did not measure specific colonization but assessed BV recurrence using Nugent’s criteria or clinical cure.
Antonio et al
studied L. crispatus
CTV-05 in 90 healthy women without BV using gelatin vaginal capsules and reported a colonization efficiency of 69% at one or more follow-up visits and of 59% at day 28. Mastromarino et al
, reported treatment success after 21 days of follow-up (Nugent score <7) in 61% of Lactobacillus
-treated patients compared to 19% in the placebo-treated group and reached a general Lactobacillus
species colonization rate of 74% at day 21. Martinez et al
, using vaginal capsules of L. rhamnosus
GR-1 and L. reuteri
RC-14 over 4 weeks following a single 2g dose of tinidazole, reported a cure rate of 87.5% in the Lactobacillus
group compared to 50% in the placebo group. However, often the reasons for failure to colonize or for BV recurrence are not explored in detail.
Our qPCR data on the vaginal concentrations of fastidious BV-associated bacteria prior to treatment suggest an association between these levels and subsequent colonization with exogenous L. crispatus CTV-05 and also suggest that high concentrations may be a more important influence on CTV-05 colonization than the mere presence of the bacteria. Although the median concentrations of BV-associated bacteria were fairly similar at screening and enrollment (after MetroGel® treatment) for both groups, the change from these baseline values to day 28 (after LACTIN-V treatment) was more pronounced in participants who colonized with CTV-05, especially for Atopobium spp. (p=0.04). Higher median levels of BV-associated bacteria at the screening, enrollment and day 28 follow-up visits were generally associated with a decreased likelihood of colonization with L. crispatus CTV-05 at the final (day 28) follow-up visit.
Swidsinski et al
followed 18 patients with BV after a 7-day treatment with oral metronidazole and reported consistently observing the resurgence of a dense and active bacterial bio-film on the vaginal mucosa, primarily consisting of G. vaginalis
and A. vaginae
. It is possible that these metronidazole resistant biofilms were present in those of our study participants with sustained high concentration of specific BV-associated bacteria and that these bio-films prevented the exogenous L. crispatus
CTV-05 from adhering to the vaginal epithelial cells. Consequently, it may be crucial for future probiotic studies to break down these biofilms before treatment with Lactobacillus
probiotics using higher doses of oral and/or intravaginal antibiotics and/or longer treatment courses. Additionally, longer periods of probiotic treatment could optimize vaginal colonization with high numbers of H2
and lactic acid producing lactobacilli.
Vaginal Gram stains from healthy women without BV typically show lactobacilli, but geographic and racial variations regarding the predominant Lactobacillus
have been recorded. Studies in China33
have reported the predominance of L. crispatus
in normal women including pregnant women. In contrast, Anukam et al
reported that L. iners
is the most abundant vaginal Lactobacillus
species in premenopausal Nigerian women, many of them with BV. Matu et al
reported a higher diversity of lactobacilli in Kenyan women with normal flora compared to women with BV, with L. jensenii
as the predominant species in addition to L. iners
. Fredricks et al
, using a combination of broad-range PCR assays of 16S rRNA genes and fluorescence in situ hybridization (FISH) performed directly on vaginal fluid, found L. crispatus
to be the predominant species in BV-negative women and L. iners
to be the predominant lactobacillus species in BV-positive women. Our study also found high levels of L. iners
, maintained throughout the study, in participants who colonized with CTV-05 as well as in those who did not. This suggests that the vaginal presence of L. iners
neither hinders nor aids CTV-05 colonization and that L. iners
may be more resistant to replacement by BV-associated bacteria.
Vaginal presence of endogenous L. crispatus
at baseline was found to be associated with a reduced odds of colonization with the L. crispatus
CTV-05 strain, similar to findings of Antonio et al
who studied L. crispatus
CTV-05 colonization in women without BV. While 15 of 18 participants receiving LACTIN-V (83%) had qPCR detectable L. crispatus
species at day 28, only eight (53%) of these participants had the exogenous L. crispatus
CTV-05 strain detectable by rep-PCR, suggesting that endogenous strains of L. crispatus
could have prevented CTV-05 colonization. Based these findings, future study designs should generally include either rep-PCR probes or qPCR probes to directly detect the administered strain (e.g. L. crispatus
CTV-05) and to differentiate it from endogenous lactobacilli.
Limited information is available about the influence of sexual intercourse on levels of vaginal bacteria or its effect on vaginal colonization with exogenous Lactobacillus
. In participants with a history of douching, sex within the past week was associated with increased likelihood of BV.39
Schellenberg et al
found that longer self-reported time since last sexual intercourse was independently associated with increased counts of bacterial cell-units (BCU) per gram of vaginal fluid. High BCU were associated with normal Hay–Ison score41
suggesting the presence and higher quantities of Lactobacillus
in these women with longer periods of sexual abstinence. In the present study, we found that vaginal intercourse during the trial significantly decreased the likelihood of successful CTV-05 colonization. A similar observation was previously also reported by Antonio et al
The high pH of seminal fluid or one of its components may affect the adherence of CTV-05 to vaginal epithelial cells and/or its survival in the vaginal vault.
We recognize that our study has several limitations. First, the number of time points for assessment was limited and our sample size was small, having been drawn from the treatment arm of a Phase 2A trial designed to investigate safety and colonization efficiency of L. crispatus
CTV-05, and excluding the placebo arm unexposed to L. crispatus
However, we still found significant association between the vaginal concentration of some BV-associated bacteria DNA and the likelihood of colonization with L. crispatus
CTV-05. Second, we performed qPCR assays targeting selected fastidious bacteria recently associated with BV using molecular methods. The qPCR platform could be used to assay other vaginal bacteria that may play a role in the pathogenesis of BV. Future research should seek to measure how vaginal levels of these bacteria correlate with the BV status and how they influence colonization with endogenous and exogenous lactobacilli. Third, our detection threshold for each assay was 375 to 750 copies per swab. The use of a larger fraction of vaginal fluid DNA for each assay would reduce the detection thresholds but would also compromise one’s ability to run multiple assays. Finally, the results of this study may not be generalizable to women not initially treated with metronidazole before probiotic treatment. One of the strengths of this study is the extensive use of PCR controls to monitor for false-positive and false-negative results, thereby increasing the reliability of the bacterial qPCR data reported.