Impact of doxycycline pre-exposure prophylaxis (doxyPrEP) for sexually transmitted infections on the microbiome of men who have sex with men on HIV PrEP

Abstract

Doxycycline pre-exposure prophylaxis (doxyPrEP) has shown potential in preventing bacterial sexually transmitted infections, but the impact on the microbiome is unknown. This study assessed rectal microbiome changes over 48 weeks in 41 participants on HIV PrEP (tenofovir disoproxil fumarate/emtricitabine) enrolled in an open-label, randomized pilot trial comparing immediate (100 mg PO daily started immediately and continued to week 48) versus deferred doxyPrEP (100 mg PO daily starting at week 24, continued to week 48) in HIV-negative gay and bisexual men (Clinical Trial #: NCT02844634). Primary study outcomes included feasibility, adherence, and tolerability of the dual PrEP regimen, while exploratory outcomes included rectal microbiome changes. We performed 16S rRNA sequencing from participants that collected baseline, week 24, and week 48 samples. Microbial composition did not significantly change over time in either study arm as measured by individual taxa levels, or alpha and beta diversity at the genus level. A slight decrease ( < 10%) in alpha diversity was observed at the phylum level in the immediate arm, but not the deferred arm. This study shows doxyPrEP use results in minimal compositional changes in the microbiome over 12 months. Further research is needed to explore the impact of doxycycline for STI prevention on microbiome function and antimicrobial resistance.

Fig. 1. Study design and 16S rRNA sequencing analysis of DuDHS study participants.

TDF/FTC tenofovir disoproxil fumarate/emtricitabine.

Fig. 2. Rectal microbiome composition of study participants in the DuDHS study.

The composition of the rectal microbiome was determined using 16S rRNA sequencing. A Microbial composition of rectal samples of each study participant in each study arm (immediate (n = 22) and deferred (n = 19)) at their baseline visit (BL), week 24, and week 48. Different colors represent the predominant microbial taxa. Average plots of each study arm are shown on the right. B Beta-diversity of baseline microbiome profiles of study participants in each study arm (Bray–Curtis distance metric). Two-sided PERMANOVA p value is shown along with an R² value to reflect the effect size. C Differential abundance analysis of individual taxa in each study arm at baseline. Blue dots represent those P < 0.05 although none passed FDR. Each dot represents the average abundance value from two technical replicates from a single participant. Differences between groups were determined by Mann–Whitney U test (two-sided) adjusting for multiple comparisons. D Alpha diversity (Shannon’s H) of each study arm at baseline visit. Each data point represents the average of two technical replicates from each study participant. Differences between groups were determined by Mann–Whitney U test (two-sided) adjusting for multiple comparisons. Median and interquartile (IQR) ranges are shown. E Study participant covariates and effect size they have on beta diversity (R² values shown as determined by PERMANOVA).

Fig. 3. Effect of doxycycline on the rectal microbiome alpha diversity in DuDHS study participants over 24 and 48 weeks.

Rectal microbiome composition was evaluated from rectal swab samples collected at baseline, 24 and 48 weeks after randomization to either the immediate (n = 22) or deferred (n = 19) doxyPrEP study arms. A comparison of alpha diversity measures (Shannon’s H) over time in each study arm is shown at the genus (A), family (B), order (C), class (D), and phylum level (E). Box plots show the median, upper and lower quartiles. Differences between follow-up timepoints were determined using Wilcoxon signed rank tests (two-sided) in paired comparisons, and across all three timepoints using Kruskal–Wallis test.

Fig. 4. Effect of doxyPrEP on the rectal microbiome beta diversity in DuDHS study participants over 24 and 48 weeks.

Rectal microbiome composition at baseline, 24 and 48 weeks after randomization to either the immediate or deferred doxycycline study arms. A A heatmap showing top 20 most abundant taxa in all study participant samples. Study arm, study timepoint, sex behaviors, age, and concomitant antibiotic usage (non-doxycycline) are overlaid on the top. Clustering is largely driven by predominant taxa. A comparison of beta diversity measures (Bray–Curtis) over time within each study arm (B–E) and between study arms (F–G) are shown. The distance between triangles and circles reflects the relative change in microbiome composition, and degrees of significance represented by two-sided PERMANOVA p values. An R² value is provided to show the effect size. BL Baseline.

Fig. 5. Changes in abundance of individual microbial taxa over time in rectal mucosa of study participants.

Differential abundance analysis of microbial taxa for each participant was performed between timepoints in each study arm using Wilcoxon signed-rank tests, adjusted for multiple comparisons. Volcano plots show relative abundance of individual microbial taxa differences (genus) along the x axis, with the p value statistic along the y axis. Shown are analysis for A Deferred arm, baseline versus week 24; B deferred arm, baseline versus week 48; C immediate arm, baseline versus week 24; D Immediate arm, baseline versus week 48. Dots in blue are those that were below a p value significance threshold of 0.05, although none passed a 5% FDR. BL Baseline.