Genital microbiota of women using a 90 day tenofovir or tenofovir and levonorgestrel intravaginal ring in a placebo controlled randomized safety trial in Kenya

Abstract

In a phase-IIa trial, we investigated the influence of 90 days continuous-delivery tenofovir (TFV) intravaginal rings (IVRs) with/without levonorgestrel (LNG) on the genital microbiota of Kenyan women. Eligible women (n = 27; 18-34 years; negative for HIV, sexually transmitted infections, and Amsel-bacterial vaginosis) were randomized 2:2:1 to use of IVRs containing TFV, TFV/LNG, or placebo. Using vaginal wall and IVR swabs at IVR insertion and removal, the genital microbial composition was determined using 16S rRNA gene sequencing. The presence of Candida spp. was determined using qPCR. The vaginal total bacterial burden appeared to decrease with TFV and TFV/LNG IVR use (log₁₀0.57 and log₁₀0.27 decrease respectively; p > 0.05). The TFV/LNG IVR was more 'stabilizing': 50% of the participants' microbiota community state types remained unchanged and 50% shifted towards higher Lactobacillus abundance. Specifically, TFV/LNG IVR use was accompanied by increased abundances of Lactobacillus gasseri/hominis/johnsonii/taiwanensis (16.3-fold) and L. fermentum/reuteri/vaginalis (7.0-fold; all p < 0.01). A significant shift in the overall microbial α-diversity or β-diversity was not observed for either IVR, and IVR use did not influence Candida spp. prevalence. TFV/LNG and TFV IVRs did not adversely affect the genital microbiota and are safe to use. Our findings support further studies assessing their efficacy in preventing HIV/HSV-2 and unintended pregnancies.

Trial registration: ClinicalTrials.gov NCT03762382.

Figure 1

Change in bacterial load between intravaginal ring (IVR) insertion and removal among women randomized to use tenofovir/levonorgestrel (TFV/LNG), tenofovir (TFV), and placebo IVRs, Kisumu, Kenya, 2019. Comparing (A) vaginal log₁₀ total 16S copies per swab between the two visits, (B) the degree of change in vaginal wall log₁₀ total bacterial 16S copies from baseline to the IVR removal visit among women and (C) difference in total log₁₀ 16S copies between the vaginal wall and IVR surface (log₁₀ bacterial load of vaginal wall − log₁₀ bacterial load of IVR) at the time of IVR removal. Values above and below the dotted red line indicate an increase and decrease in bacterial loads respectively. p values were determined using the Wilcoxon Signed Rank test (A) and Mann–Whitney U test (B,C).

Figure 2

Impact of length of tenofovir/levonorgestrel (TFV/LNG), tenofovir (TFV), or placebo intravaginal ring (IVR) use on total bacterial load, Kisumu, Kenya, 2019. Linear regressions showing the association between number of days of IVR use and change in bacterial load between visits. The coloured shading represents the 95% confidence interval around the slope.

Figure 3

No change in overall microbiota diversity with use of tenofovir/levonorgestrel (TFV/LNG), tenofovir (TFV), or placebo intravaginal ring (IVR), Kisumu, Kenya, 2019. (A) Comparing the change in Shannon diversity (within-participant diversity) with each IVR. The dotted red line at zero indicates no change over time. (B–D) Principal component analysis plots showing the overlap between samples at baseline and the IVR removal visit for the TFV/LNG, TFV and Placebo IVRs, based on Bray–Curtis distances (between-participant diversity). p values were determined using the (A) Mann–Whitney U test or the (B–D) Adonis/PERMANOVA test based on permutations of distance matrices. NMDS non-metric multidimensional scaling.

Figure 4

Transitions in bacterial communities within each study arm, Kisumu, Kenya, 2019. (A) Shifts in community state types (CSTs) in the genital tract from baseline to the intravaginal ring (IVR) removal visit. (B) Differences in CST between the genital tract and the IVR surface at the IVR removal visit. Each line represents one participant’s transition within the tenofovir/levonorgestrel (TFV/LNG), tenofovir (TFV), or placebo study arms.

Figure 5

Fold changes in abundance of specific bacteria with the tenofovir/levonorgestrel (TFV/LNG), tenofovir (TFV), and placebo intravaginal rings (IVRs) respectively, Kisumu, Kenya, 2019. Dots on the right hand side of the gray solid line show a fold change increase in bacterial abundance and dots on the left hand side show a fold change decrease. The red and blue dotted lines represent 0.5-fold and twofold changes in bacterial abundance. Differential abundances with an adjusted p value of ≤ 0.01 are shown.

Figure 6

Longitudinal Candida status within each study arm for (A) C. albicans, (B) C. krusei, and (C) C. glabrata, Kisumu, Kenya, 2019. Women who remained Candida-negative are shown in green. Cleared infections are shown in yellow and new infections are shown in red.

Figure 7

Vaginal tenofovir (TFV) concentrations across community state types (CSTs), Kisumu, Kenya, 2019. Comparing the difference in log₁₀ vaginal TFV concentrations at the IVR removal visit among women using the TFV and tenofovir/levonorgestrel arms categorized by their CST. The p value was determined using the Mann–Whitney U test.