Intermittent Lactobacilli-containing Vaginal Probiotic or Metronidazole Use to Prevent Bacterial Vaginosis Recurrence: A Pilot Study Incorporating Microscopy and Sequencing

Abstract

Bacterial vaginosis (BV) is associated with HIV acquisition and adverse pregnancy outcomes. Recurrence after metronidazole treatment is high. HIV-negative, non-pregnant Rwandan BV patients were randomized to four groups (n = 17/group) after seven-day oral metronidazole treatment: behavioral counseling only (control), or counseling plus intermittent use of oral metronidazole, Ecologic Femi+ vaginal capsule (containing multiple Lactobacillus and one Bifidobacterium species), or Gynophilus LP vaginal tablet (L. rhamnosus 35) for two months. Vaginal microbiota assessments at all visits included Gram stain Nugent scoring and 16S rRNA gene qPCR and HiSeq sequencing. All interventions were safe. BV (Nugent 7-10) incidence was 10.18 per person-year at risk in the control group, and lower in the metronidazole (1.41/person-year; p = 0.004), Ecologic Femi+ (3.58/person-year; p = 0.043), and Gynophilus LP groups (5.36/person-year; p = 0.220). In mixed effects models adjusted for hormonal contraception/pregnancy, sexual risk-taking, and age, metronidazole and Ecologic Femi+ users, each compared to controls, had higher Lactobacillus and lower BV-anaerobes estimated concentrations and/or relative abundances, and were less likely to have a dysbiotic vaginal microbiota type by sequencing. Inter-individual variability was high and effects disappeared soon after intervention cessation. Lactobacilli-based vaginal probiotics warrant further evaluation because, in contrast to antibiotics, they are not expected to negatively affect gut microbiota or cause antimicrobial resistance.

Figure 1

Flow diagram of numbers of women seen, study procedures, and samples collected. Abbreviations: BV, bacterial vaginosis; D7, day 7 visit; DNA, desoxyribonucleic acid; M1/2/6, month 1/2/6 visit; qPCR, quantitative polymerase chain reaction; RU, Rinda Ubuzima; STI, sexually transmitted infection; TV, Trichomonas vaginalis; UTI, urinary tract infection. *Totals to 110 reasons among 102 women because there could be more than one reason per woman. ^†No speculum exam performed; molecular testing of self-sampled vaginal swabs. ^‡Reasons: outside of metronidazole treatment window (n = 5), enrollment target already met (n = 4), has a mental disorder (n = 1), did not complete screening procedures and was subsequently lost to follow-up (n = 1), withdrew consent during the screening visit because she thought the reimbursement was too low (n = 1). ^§Three women in each randomization group were selected for self-sampling every other day during the first month of follow-up. ^¶Reasons: moved away from Kigali (n = 2), lost interest because symptoms resolved (n = 1), was verbally harassed by partner and sister about study participation (n = 1). ^||These were all physician-collected. See Supplement 1 for information about samples that should have been but were not collected or were lost during processing.

Figure 2

Preliminary efficacy by Nugent score, alpha diversity, and bacterial group estimated concentration. Abbreviations: D7, Day 7 visit; EF+, Ecologic Femi+; Enr, enrollment visit; est conc, estimated concentration; GynLP, Gynophilus LP; ITT, intention to treat; M1/2/6, month 1/2/6 visit; Scr, screening visit; VMB, vaginal microbiota. Changes in VMB outcomes over time per randomization group. See Supplement Table S4 for 95% confidence intervals. (A) Mean Nugent scores over time, only including women (n = 51) who were BV-negative by modified Amsel and Nugent criteria (modified ITT analysis). (B) Mean alpha diversity over time. (C) Mean estimated bacterial cell concentration over time. (D) Difference in mean estimated bacterial cell concentration with enrollment, over time. (E) Mean estimated lactobacilli concentration over time. (F) Difference in mean estimated lactobacilli concentration with enrollment, over time. (G) Mean estimated BV-anaerobes concentration over time. (H) Difference in mean estimated BV-anaerobes concentration with enrollment, over time. (I) Mean estimated pathobionts concentration over time. (J) Difference in mean estimated pathobionts concentration with enrollment, over time. (K) Mean estimated other bacteria concentration over time. (L) Difference in mean estimated other bacteria concentration with enrollment, over time.

Figure 3

Detection of probiotic strains during the trial. Abbreviations: D7, Day 7 visit; EF+, Ecologic Femi+; Enr, enrollment visit; GynLP, Gynophilus LP; M1/2/6, month 1/2/6 visit; Scr, screening visit. Mean relative abundance (A) and mean estimated concentration (B) of Lactobacillus species over time in the EF+ group; mean relative abundance (C) and mean estimated concentration (D) of Lactobacillus species over time in the GynLP group. The length of bars in (A,C) depicts total relative abundance of all Lactobacillus species combined.