Publications by authors named "Sujatha Srinivasan"

49 Publications

Association between postmenopausal vulvovaginal discomfort, vaginal microbiota, and mucosal inflammation.

Am J Obstet Gynecol 2021 Mar 4. Epub 2021 Mar 4.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

Background: Half of all postmenopausal women report symptoms of vulvar, vaginal, or urinary discomfort with substantial impact on sexual function and quality of life; underlying mechanisms leading to symptoms are poorly understood.

Objective: To examine the possibility that the vaginal microbiota and/or mucosal immune response contributes to the severity of bothersome vaginal symptoms, we conducted a substudy of samples from a randomized trial of vaginal treatment for genitourinary syndrome of menopause to compare these features between women whose symptoms improved and women whose symptoms did not improve.

Study Design: This is a secondary analysis of samples collected in a 12-week randomized trial of treatment with vaginal estradiol or moisturizer vs placebo for moderate-severe postmenopausal symptoms of vaginal discomfort. We randomly selected 20 women in each arm with ≥2-point decrease in most bothersome symptom severity (responders) and 20 matched controls with ≤1-point decrease (nonresponders). At 0, 4, and 12 weeks, we characterized vaginal microbiota (16S ribosomal RNA gene sequencing), vaginal fluid metabolites (broad-based metabolomic profiling), vaginal fluid-soluble immune markers (Meso Scale Discovery), pH, and vaginal maturation index. We compared responders with nonresponders at baseline and across all visits using linear mixed models to evaluate associations with microbiota, metabolites, and immune markers, incorporating visit and participant-specific random effects while controlling for treatment arm.

Results: Here, the mean age of women was 61 years (n=120), and most women (92%) were white. At enrollment, no significant differences were observed between responders and nonresponders in age, most bothersome symptom type or severity, microbiota composition or diversity, Lactobacillus dominance, metabolome, or immune markers. There was a significant decrease in diversity of the vaginal microbiota in both responders and nonresponders (P<.001) over 12 weeks. Although this change did not differ by responder status, diversity was associated with treatment arm: more women in the estradiol arm (63%) had Lactobacillus-dominant, lower diversity bacterial communities than women in the moisturizer (35%) or dual placebo (23%) arms (P=.001) at 12 weeks. The metabolome, vaginal maturation index, and measured immune markers were not associated with responder status over the 12 weeks but varied by treatment arm.

Conclusion: Postmenopausal vaginal symptom severity was not significantly associated with vaginal microbiota or mucosal inflammatory markers in this small study. Women receiving vaginal estradiol experienced greater abundance of lactobacilli and lower vaginal pH at end of treatment.
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http://dx.doi.org/10.1016/j.ajog.2021.02.034DOI Listing
March 2021

sp. nov., sp. nov., and sp. nov.: novel bacteria isolated from the female genital tract.

Int J Syst Evol Microbiol 2019 Jun 22;71(3). Epub 2021 Feb 22.

University of Pittsburgh School of Medicine, Department of Obstetrics, Gynecology and Reproductive Sciences, Pittsburgh, PA, USA.

Six strictly anaerobic Gram-negative bacteria representing three novel species were isolated from the female reproductive tract. The proposed type strains for each species were designated UPII 199-6, KA00182 and BV3C16-1. Phylogenetic analyses based on 16S rRNA gene sequencing indicated that the bacterial isolates were members of the genus . UPII 199-6 and KA00182 had 16S rRNA gene sequence identities of 99.9 % with 16S rRNA clone sequences previously amplified from the human vagina designated as type 1 and type 2, members of the human vaginal microbiota associated with bacterial vaginosis, preterm birth and HIV acquisition. UPII 199-6 exhibited sequence identities ranging from 92.9 to 93.6 % with validly named isolates and KA00182 had 16S rRNA gene sequence identities ranging from 92.6-94.2 %. BV3C16-1 was most closely related to with a 16S rRNA gene sequence identity of 95.4 %. Cells were coccoid or diplococcoid, non-motile and did not form spores. Genital tract isolates metabolized organic acids but were asaccharolytic. The isolates also metabolized amino acids. The DNA G+C content for the genome sequences of UPII 199-6, KA00182 and BV3C16-1 were 46.4, 38.9 and 49.8 mol%, respectively. Digital DNA-DNA hybridization and average nucleotide identity between the genital tract isolates and other validly named species suggest that each isolate type represents a new species. The major fatty acid methyl esters include the following: C, C, C dimethyl acetal (DMA) and summed feature 5 (C DMA and/or C 3-OH) in UPII 199-6; C and C 9 in KA00182; C; C 3-OH; and summed feature 5 in BV3C16-1. The isolates produced butyrate, isobutyrate, and isovalerate but there were specific differences including production of formate and propionate. Together, these data indicate that UPII 199-6, KA00182 and BV3C16-1 represent novel species within the genus . We propose the following names: sp. nov. for UPII 199-6 representing the type strain of this species (=DSM 111201=ATCC TSD-205), sp. nov. for KA00182 representing the type strain of this species (=DSM 111202=ATCC TSD-206) and sp. nov. for BV3C16-1 representing the type strain of this species (=DSM 111203=ATCC TSD-207).
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http://dx.doi.org/10.1099/ijsem.0.004702DOI Listing
June 2019

Synergic effect of eugenol oleate with amphotericin B augments anti-leishmanial immune response in experimental visceral leishmaniasis in vitro and in vivo.

Int Immunopharmacol 2021 Feb 21;91:107291. Epub 2020 Dec 21.

Department of Biotechnology, Centre for Research in Infectious Diseases, School of Chemical and Biotechnology, SASTRA University, Thanjavur, India; Department of Chemistry, School of Chemical and Biotechnology, SASTRA University, Thanjavur, India. Electronic address:

Present treatment regimen on visceral leishmaniasis has multiple limitations including severe side effects, toxicity, and resistance of Leishmania strains. Amphotericin B is a well-established pharmacologically approved drug; however, mainly toxicity is a foremost issue with that drug. Recently, our group identified eugenol oleate as an anti-leishmanial immunomodulatory compound. The important objectives of this present study was to evaluate the possible synergistic effect of eugenol oleate with amphotericin B to reduce the toxicity of this approved drug. Results obtained from this study signified that combination of eugenol oleate and amphotericin B showed indifferent combinatorial effect against promastigotes with xΣFIC 1.015, while, moderate synergistic activity with xΣFIC 0.456 against amastigotes. It was also notable that eugenol oleate (2.5 μM) with low concentrations of amphotericin B (0.3125 μM) showed 96.45% parasite reduction within L. donovani-infected murine macrophages. Furthermore, eugenol oleate and amphotericin B significantly (p < 0.01) enhanced the nitrite generation, and pro-inflammatory cytokines (IL-12, IFN-γ and TNF-α) in infected macrophages in vitro and in BALB/c mice in vivo. Eugenol oleate (10 mg/Kg b. wt.) with amphotericin B (1 mg/Kg b.wt.) significantly (p < 0.01) controlled the parasite burden in liver by 96.2% and in spleen by 93.12%. Hence, this study strongly suggested the synergic potential of eugenol oleate with low concentration of amphotericin B in experimental visceral leishmaniasis through anti-leishmanial immune response.
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http://dx.doi.org/10.1016/j.intimp.2020.107291DOI Listing
February 2021

Influence of intramuscular depot-medroxyprogesterone acetate initiation on vaginal microbiota in the postpartum period.

Clin Infect Dis 2020 Dec 23. Epub 2020 Dec 23.

Departments of Medicine and Global Health, University of Washington, Seattle, WA, USA.

Background: The vaginal microbiome plays a key role in women's reproductive health. Use of exogenous hormones, such as intramuscular depot-medroxyprogesterone acetate (DMPA-IM), may alter the composition of vaginal bacterial community.

Methods: Vaginal swabs were collected from postpartum Kenyan women initiating DMPA-IM or non-hormonal contraception (non-HC). Bacterial vaginosis was assessed by Nugent score (Nugent-BV) and bacterial community composition was evaluated using broad-range 16S rRNA gene PCR with high-throughput sequencing. Changes in Nugent score, alpha diversity (Shannon diversity index), and total bacterial load between contraceptive groups from enrollment to three-months post-initiation were estimated using multivariable linear mixed effects regression.

Results: Among 54 HIV-negative women, 33 choosing DMPA-IM and 21 choosing non-HC, Nugent-BV was more common among DMPA-IM users at enrollment. At follow-up, Nugent score had decreased significantly among DMPA-IM users (Δ=-1.89 (95%CI:-3.53, -0.25; p=0.02) while alpha diversity remained stable (Δ=0.03, 95%CI:-0.24, 0.30; p=0.83). Conversely, Nugent score remained relatively stable among non-HC users (Δ=-0.73, 95%CI:-2.18, 0.73; p=0.33) while alpha diversity decreased (Δ=-0.34, 95%CI:-0.67, -0.001; p=0.05). Total bacterial load decreased slightly in DMPA-IM users and increased slightly among non-HC users, resulting in a significant difference in change between the contraceptive groups (difference=-0.64 log10 gene copies/swab, 95%CI:-1.19, -0.08; p=0.02). While significant changes in Nugent score and alpha diversity were observed within contraceptive groups, changes between groups were not significantly different.

Conclusions: Postpartum vaginal bacterial diversity did not change in DMPA-IM users despite a reduction in Nugent-BV, but decreased significantly among women using non-HC. Choice of contraception may influence Lactobacillus recovery in postpartum women.
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http://dx.doi.org/10.1093/cid/ciaa1876DOI Listing
December 2020

Vaginal bacteria and risk of incident and persistent infection with high risk sub-types of human papillomavirus: a cohort study among Kenyan women.

Sex Transm Dis 2020 Dec 15;Publish Ahead of Print. Epub 2020 Dec 15.

Department of Epidemiology, University of Washington; Seattle, WA, USA Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center; Seattle, WA, USA Department of Medical Microbiology, University of Nairobi; Nairobi, Kenya University of Nairobi Institute for Tropical and Infectious Diseases, University of Nairobi; Nairobi, Kenya Department of Medicine, University of Washington; Seattle, WA, USA Department of Global Health, University of Washington; Seattle, WA, USA.

Background: Bacterial vaginosis (BV) is associated with increased risk of high-risk HPV (hrHPV), whereas Lactobacillus-dominated vaginal microbiotas are associated with reduced burden of hrHPV. Few epidemiologic studies have prospectively investigated the relationships between vaginal bacteria and hrHPV, particularly among women from countries in Africa.

Methods: We conducted a prospective cohort study nested within the Preventing Vaginal Infections trial to evaluate associations between vaginal bacteria and hrHPV incidence and persistence. Sexually active, HIV-seronegative women age 18-45 who had a vaginal infection at screening were eligible to enroll. Analyses were restricted to participants enrolled in Kenya and randomized to placebo. At enrollment and months 2, 4, 6, 8, 10, and 12, hrHPV testing, quantitative PCR (measuring taxon quantity per swab), and 16S rRNA gene amplicon sequencing of the vaginal microbiota were performed. Generalized estimating equations multinomial logistic regression models were fit to evaluate associations between vaginal bacteria and incident and persistent hrHPV.

Results: Eighty-four participants were included in this analysis. Higher concentrations of Lactobacillus crispatus were inversely associated with persistent hrHPV detection. Specifically, one tertile higher L. crispatus concentration was associated with 50% reduced odds of persistent hrHPV detection (OR = 0.50, 95% CI 0.29-0.85).

Conclusions: This study is consistent with reports that vaginal L. crispatus is associated with reduced susceptibility to hrHPV persistence. Evidence from in vitro studies provides insight into potential mechanisms by which L. crispatus may mediate hrHPV risk. Future studies should further explore in vivo mechanisms that may drive this relationship and opportunities for intervention.
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http://dx.doi.org/10.1097/OLQ.0000000000001343DOI Listing
December 2020

Genetic Variation in Toll-Like Receptor 5 and Colonization with Flagellated Bacterial Vaginosis-Associated Bacteria.

Infect Immun 2021 Feb 16;89(3). Epub 2021 Feb 16.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

Bacterial vaginosis (BV) is a vaginal dysbiotic condition linked to negative gynecological and reproductive sequelae. Flagellated bacteria have been identified in women with BV, including spp. and BV-associated bacterium-1 (BVAB1), an uncultivated, putatively flagellated species. The host response to flagellin mediated through Toll-like receptor 5 (TLR5) has not been explored in BV. Using independent discovery and validation cohorts, we examined the hypothesis that TLR5 deficiency-defined by a dominant negative stop codon polymorphism, rs5744168-is associated with an increased risk for BV and increased colonization with flagellated bacteria associated with BV (BVAB1, , and ). TLR5 deficiency was not associated with BV status, and TLR5-deficient women had decreased colonization with BVAB1 in both cohorts. We stimulated HEK-hTLR5-overexpressing NF-κB reporter cells with whole, heat-killed or and with partially purified flagellin from these species; as BVAB1 is uncultivated, we used cervicovaginal lavage (CVL) fluid supernatant from women colonized with BVAB1 for stimulation. While heat-killed and CVL fluid from women colonized with BVAB1 stimulate a TLR5-mediated response, heat-killed did not. In contrast, partially purified flagellin from both species stimulated a TLR5-mediated response We observed no correlation between vaginal interleukin 8 (IL-8) and flagellated BVAB concentrations among TLR5-sufficient women. Interspecies variation in accessibility of flagellin recognition domains may be responsible for these observations, as reflected in the potentially novel flagellin products encoded by species versus those encoded by BVAB1.
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http://dx.doi.org/10.1128/IAI.00060-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8097276PMC
February 2021

Oral administration of eugenol oleate cures experimental visceral leishmaniasis through cytokines abundance.

Cytokine 2020 Oct 28:155301. Epub 2020 Oct 28.

Department of Biotechnology, Centre for Research in Infectious Diseases, School of Chemical and Biotechnology, SASTRA Deemed To Be University, Thanjavur, India. Electronic address:

Visceral leishmaniasis (VL) is an endemic fatal infectious disease in tropical and subtropical nations. The limited treatment options, long treatment regimens, invasive mode of administration of drugs, and lack of effective vaccination are the main reasons for the search of new alternative therapeutics against VL. On this quest, from a series of eugenol derivatives, we had demonstrated eugenol oleate as a lead immunomodulatory anti-VL molecule earlier. In this report, the oral efficacy and mechanism of eugenol oleate in inducing immunomodulatory anti-VL activity has been studied in BALB/c mice model. The plasma pharmacokinetic and acute toxicity studies suggested that the eugenol oleate is safe with an appreciable pharmacokinetic profile. Eugenol oleate (30 mg/kg B.W.) showed 86.5% of hepatic and 84.1% of splenic parasite clearance. The increased Th1 cytokine profile and decreased Th2 cytokine profile observed from ELISA and qRTPCR suggested that the eugenol oleate induced the parasite clearance through the activation of the host immune system. Subsequently, the mechanistic insights behind the anti-leishmanial activity of eugenol oleate were studied in peritoneal macrophages in vitro by inhibitor response study and immunoblotting. The results inferred that eugenol oleate activated the PKC-βII-p38 MAPK and produced IL-12 and IFN-γ which intern activated the iNOS2 to produce NO free radicals that cleared the intracellular parasite.
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http://dx.doi.org/10.1016/j.cyto.2020.155301DOI Listing
October 2020

Bacterial communities associated with abnormal Nugent score are different in pre- versus postmenopausal women.

J Infect Dis 2020 Oct 27. Epub 2020 Oct 27.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

Nugent score is the gold standard for bacterial vaginosis (BV) diagnosis but has not been validated in postmenopausal women. We compared relative abundances from 16S rRNA gene sequencing of vaginal microbiota with Nugent score in cohorts of premenopausal (n = 220) and postmenopausal (n = 144) women. In premenopausal women 33 taxa were significantly correlated with Nugent score, including the classic BV-associated taxa Gardnerella, Atopobium, Sneathia, Megasphaera and Prevotella. In postmenopausal women, 11 taxa were significantly associated with Nugent score, including Prevotella but no other BV-associated genera. High Nugent score should not be used to infer BV in postmenopausal women.
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http://dx.doi.org/10.1093/infdis/jiaa675DOI Listing
October 2020

Urethral microbiota in men: Association of Haemophilus influenzae and Mycoplasma penetrans with nongonococcal urethritis.

Clin Infect Dis 2020 Aug 5. Epub 2020 Aug 5.

Department of Epidemiology, University of Washington, Seattle, WA, USA.

Background: Nongonococcal urethritis (NGU) is a common syndrome with no known etiology in up to 50% of cases. We estimated associations between urethral bacteria and NGU in men who have sex with men (MSM) and men who have sex with women (MSW).

Methods: Urine was collected from NGU cases (129 MSM; 121 MSW) and controls (70 MSM; 114 MSW) attending a Seattle STD clinic. Cases had ≥5 polymorphonuclear leukocytes on Gram stain plus symptoms or discharge; controls had <5 PMNs, no symptoms, no discharge. NGU was considered idiopathic when Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Trichomonas vaginalis, adenovirus, and herpes simplex virus were absent. The urethral microbiota was characterized using 16S rRNA gene sequencing. Compositional lasso analysis was conducted to identify associations between bacterial taxa and NGU, and to select bacteria for targeted quantitative PCR (qPCR).

Results: Among NGU cases, 45.2% were idiopathic. Based on compositional lasso analysis, we selected Haemophilus influenzae (HI) and Mycoplasma penetrans (MP) for targeted qPCR. Compared to 182 men without NGU, the 249 men with NGU were more likely to have HI (14% vs. 2%) and MP (21% vs. 1%) (both p≤0.001). In stratified analyses, detection of HI was associated with NGU among MSM (12% vs. 3%, p=0.036) and MSW (17% vs. 1%, p<0.001), but MP was associated with NGU only among MSM (13% vs. 1%, p=0.004). Associations were stronger in men with idiopathic NGU.

Conclusions: HI and MP are potential causes of male urethritis. MP was more often detected among MSM than MSW with urethritis.
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http://dx.doi.org/10.1093/cid/ciaa1123DOI Listing
August 2020

Association Between Vaginal Bacterial Microbiota and Vaginal Yeast Colonization.

J Infect Dis 2021 Mar;223(5):914-923

University of Washington, Department of Medicine, Seattle, Washington, USA.

Background: Vaginal yeast is frequently found with Lactobacillus-dominant microbiota. The relationship between vaginal yeast and other bacteria has not been well characterized.

Methods: These analyses utilized data from the Preventing Vaginal Infections trial. Relative abundance of vaginal bacteria from 16S ribosomal ribonucleic acid gene amplicon sequencing and quantities of 10 vaginal bacteria using taxon-directed polymerase chain reaction assays were compared at visits with and without detection of yeast on microscopy, culture, or both.

Results: Higher relative abundances of Megasphaera species type 1 (risk ratio [RR], 0.70; 95% confidence interval [CI], 0.52-0.95), Megasphaera species type 2 (RR, 0.81; 95% CI, 0.67-0.98), and Mageeibacillus indolicus (RR, 0.46; 95% CI, 0.25-0.83) were associated with lower risk of detecting yeast. In contrast, higher relative abundances of Bifidobacterium bifidum, Aerococcus christensenii, Lactobacillus mucosae, Streptococcus equinus/infantarius/lutentiensis, Prevotella bivia, Dialister propionicifaciens, and Lactobacillus crispatus/helveticus were associated with yeast detection. Taxon-directed assays confirmed that increasing quantities of both Megasphaera species and M indolicus were associated with lower risk of detecting yeast, whereas increasing quantities of L crispatus were associated with higher risk of detecting yeast.

Conclusions: Despite an analysis that examined associations between multiple vaginal bacteria and the presence of yeast, only a small number of vaginal bacteria were strongly and significantly associated with the presence or absence of yeast.
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http://dx.doi.org/10.1093/infdis/jiaa459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938175PMC
March 2021

Roll-over shape of a prosthetic foot: a finite element evaluation and experimental validation.

Med Biol Eng Comput 2020 Oct 18;58(10):2259-2270. Epub 2020 Jul 18.

TTK Center for Rehabilitation Research and Device Development (R2D2), Department of Mechanical Engineering, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 600036, India.

Prosthetic feet have generally been designed experimentally by adopting a trial-and-error technique. The objective of this research is to introduce a novel numerical approach for the a priori evaluation of the roll-over shape (ROS) of a prosthetic foot for application in its systematic design and development. The ROS was achieved numerically by employing a non-linear finite element model incorporating the augmented Lagrangian and multi-point constraint contact formulations, a hyperelastic material model and a higher-order strain definition. The Ottobock Solid Ankle Cushion Heel (SACH) foot was chosen to experimentally validate the numerical model. The geometry of the foot was evaluated from optical scans, and the material properties were obtained from uniaxial tensile, shear and volumetric compression tests. A new setup was designed for an improved experimental determination of the ROS, with the inclusion of an extended moment arm and variable loading. Error analysis of the radius of curvature of the ROS between the numerical and experimental results showed the percentage error to be 7.52%, thereby establishing the validity of the model. A numerical design model of this kind can be utilised to vary the input design parameters to arrive at a prosthetic foot with specified performance characteristics effectively and economically. Graphical abstract.
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http://dx.doi.org/10.1007/s11517-020-02214-9DOI Listing
October 2020

Extra-vaginal Bacterial Colonization and Risk for Incident Bacterial Vaginosis in a Population of Women who Have Sex with Men.

J Infect Dis 2020 May 7. Epub 2020 May 7.

Fred Hutchinson Cancer Research Center, Seattle, WA.

Bacterial vaginosis (BV) is a common cause of vaginal discharge and associated with vaginal acquisition of BV-associated bacteria (BVAB). We used quantitative PCR assays to determine if presence or concentrations of BVAB in the mouth, anus, vagina, or labia prior to BV predict risk of incident BV in 72 women who have sex with men. Baseline vaginal and extra-vaginal colonization with Gardnerella spp., Megasphaera spp., Sneathia spp., BVAB-2, Dialister sp. type 2. and other BVAB was more common among subjects with incident BV. Prior colonization with BVAB is a consistent risk for BV.
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http://dx.doi.org/10.1093/infdis/jiaa233DOI Listing
May 2020

Complementing 16S rRNA Gene Amplicon Sequencing with Total Bacterial Load To Infer Absolute Species Concentrations in the Vaginal Microbiome.

mSystems 2020 Apr 7;5(2). Epub 2020 Apr 7.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Whereas 16S rRNA gene amplicon sequencing quantifies relative abundances of bacterial taxa, variation in total bacterial load between samples restricts its ability to reflect absolute concentrations of individual bacterial species. Quantitative PCR (qPCR) can quantify individual species, but it is not practical to develop a suite of qPCR assays for every bacterium present in a diverse sample. We sought to determine the accuracy of an inferred measure of bacterial concentration using total bacterial load and relative abundance. We analyzed 1,320 samples from 20 women with a history of frequent bacterial vaginosis who self-collected vaginal swabs daily over 60 days. We inferred bacterial concentrations by taking the product of species relative abundance (assessed by 16S rRNA gene amplicon sequencing) and bacterial load (measured by broad-range 16S rRNA gene qPCR). Log-converted inferred concentrations correlated with targeted qPCR ( = 0. 935, < 2.2e-16) for seven key bacterial species. The mean inferred concentration error varied across bacteria, with rarer bacteria associated with larger errors. A total of 92% of the >0.5-log errors occurred when the relative abundance was <10%. Many errors occurred during early bacterial expansion from or late contraction to low abundance. When the relative abundance of a species is >10%, inferred concentrations are reliable proxies for targeted qPCR in the vaginal microbiome. However, targeted qPCR is required to capture bacteria at low relative abundance and is preferable for characterizing growth and decay kinetics of single species. Microbiome studies primarily use 16S rRNA gene amplicon sequencing to assess the relative abundance of bacterial taxa in a community. However, these measurements do not accurately reflect absolute taxon concentrations. We sought to determine whether the product of species' relative abundance and total bacterial load measured by broad-range qPCR is an accurate proxy for individual species' concentrations, as measured by taxon-specific qPCR assays. Overall, the inferred bacterial concentrations were a reasonable proxy of species-specific qPCR values, particularly when bacteria are present at a higher relative abundance. This approach offers an opportunity to assess the concentrations of bacterial species and how they change in a community over time without developing individual qPCR assays for each taxon.
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http://dx.doi.org/10.1128/mSystems.00777-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7141891PMC
April 2020

Presence and Concentrations of Select Bacterial Vaginosis-Associated Bacteria Are Associated With Increased Risk of Pelvic Inflammatory Disease.

Sex Transm Dis 2020 05;47(5):344-346

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA.

In a vaginal 16S ribosomal RNA gene quantitative PCR study of 17 pelvic inflammatory disease (PID) cases and 17 controls who tested positive for Chlamydia trachomatis, women who additionally tested positive for Atopobium vaginae, Sneathia spp., Megasphaera spp., Eggerthella-like bacterium or Prevotella amnii were more likely to develop PID.
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http://dx.doi.org/10.1097/OLQ.0000000000001164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167351PMC
May 2020

Changes in key vaginal bacteria among postpartum African women initiating intramuscular depot-medroxyprogesterone acetate.

PLoS One 2020 5;15(3):e0229586. Epub 2020 Mar 5.

Department of Global Health, University of Washington, Seattle, WA, United States of America.

Background: The ECHO trial has relieved apprehension about intramuscular depot medroxyprogesterone acetate (DMPA-IM), however it is still important to understand how DMPA-IM affects the vaginal environment. We sought to describe how DMPA-IM initiation influences vaginal bacteria associated with HIV acquisition in postpartum women.

Methods: Vaginal swabs were collected for Nugent score determination and taxon-specific quantitative PCR of eight bacteria. Enrollment occurred at contraceptive initiation (DMPA-IM or non-hormonal contraception (non-HC)) and repeat vaginal swabs were collected after three months. Generalized estimating equations were used to estimate changes in Nugent score, total bacterial load, and taxa concentrations among contraceptive groups.

Results: Women who chose DMPA-IM (n = 33) were more likely to be married (97%vs.67%) and have resumed intercourse since delivery (52%vs.29%) compared to women who chose non-HC (n = 21). After three months, significant decreases in the concentrations of Sneathia species, Mycoplasma hominis, and Parvimonas species Type 1 were seen among non-HC users, however concentrations remained stable among DMPA-IM users; contraceptive method was associated with significantly different changes in M. hominis concentration between groups (p = 0.010).

Conclusions: Our findings suggest that postpartum use of DMPA-IM and non-HC may have differential impacts on the vaginal concentrations of some bacteria that have previously been associated with HIV acquisition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0229586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7058341PMC
June 2020

Impact of preconception vaginal microbiota on women's risk of spontaneous preterm birth: protocol for a prospective case-cohort study.

BMJ Open 2020 02 25;10(2):e035186. Epub 2020 Feb 25.

Department of Epidemiology, University of Washington, Seattle, Washington, USA

Introduction: Bacterial vaginosis (BV) and vaginal microbiota disruption during pregnancy are associated with increased risk of spontaneous preterm birth (SPTB), but clinical trials of BV treatment during pregnancy have shown little or no benefit. An alternative hypothesis is that vaginal bacteria present around conception may lead to SPTB by compromising the protective effects of cervical mucus, colonising the endometrial surface before fetal membrane development, and causing low-level inflammation in the decidua, placenta and fetal membranes. This protocol describes a prospective case-cohort study addressing this hypothesis.

Methods And Analysis: HIV-seronegative Kenyan women with fertility intent are followed from preconception through pregnancy, delivery and early postpartum. Participants provide monthly vaginal specimens during the preconception period for vaginal microbiota assessment. Estimated date of delivery is determined by last menstrual period and first trimester obstetrical ultrasound. After delivery, a swab is collected from between the fetal membranes. Placenta and umbilical cord samples are collected for histopathology. Broad-range 16S rRNA gene PCR and deep sequencing of preconception vaginal specimens will assess species richness and diversity in women with SPTB versus term delivery. Concentrations of key bacterial species will be compared using quantitative PCR (qPCR). Taxon-directed qPCR will also be used to quantify bacteria from fetal membrane samples and evaluate the association between bacterial concentrations and histopathological evidence of inflammation in the fetal membranes, placenta and umbilical cord.

Ethics And Dissemination: This study was approved by ethics committees at Kenyatta National Hospital and the University of Washington. Results will be disseminated to clinicians at study sites and partner institutions, presented at conferences and published in peer-reviewed journals. The findings of this study could shift the paradigm for thinking about the mechanisms linking vaginal microbiota and prematurity by focusing attention on the preconception vaginal microbiota as a mediator of SPTB.
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http://dx.doi.org/10.1136/bmjopen-2019-035186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7045118PMC
February 2020

Vaginal Microbiota and Mucosal Immune Markers in Women With Vulvovaginal Discomfort.

Sex Transm Dis 2020 04;47(4):269-274

Vaccine and Infectious Diseases Division, Fred Hutchinson Research Center, Seattle, WA.

Background: Up to 30% of women with vaginal symptoms are not assigned a diagnosis after standard diagnostic assessment.

Methods: We compared premenopausal women with idiopathic vaginitis (IV) or vulvodynia (VVD) to healthy controls. Microbiota were characterized using rRNA sequencing. Cytokines/chemokines (IL-10, IL-1α, IL-1β, IL-6, IL-8, IL-2, IL-18, IL-4, IL-9, and IL-13) were measured in vaginal lavage fluid using the Meso Scale Discovery platform or ELISA (IL-1ra). Immunoglobulins were measured in vaginal lavage fluid using a bead-based immunoassay (Millipore). Cases and controls were compared using Kruskal-Wallis, analysis of variance, and linear regression or (for microbiome composition) the Bray-Curtis dissimilarity statistic.

Results: We compared 20 women with IV, 30 with VVD, and 52 controls. Most (80%) had greater than 90% 16S rRNA gene sequences from Lactobacillus crispatus, L. jensenii, L. gasseri, or L. iners. In analyses adjusted for age and hormonal contraception (HC), Gardnerella vaginalis was less prevalent and abundant in women with VVD (2/30, 7%) versus controls (16/52, 31%) or IV (5/20, 25%) (P = 0.030). Bray-Curtis dissimilarity was not significantly different between IV and controls or VVD. Fungal sequences were only detected in 5 participants: 2 control, 1 IV, 2 VVD. In univariate analysis, cytokines were not associated with diagnosis. Median vaginal concentration of IgE (but not other immunoglobulins) was lower in women with VVD (P = 0.006).

Conclusions: Minimal differences in vaginal microbiota and inflammatory markers between women with IV, VVD or controls suggest no striking association between vaginal bacteria, fungi or inflammation and diagnosis in these women.
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http://dx.doi.org/10.1097/OLQ.0000000000001143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071966PMC
April 2020

Human gut microbiota is associated with HIV-reactive immunoglobulin at baseline and following HIV vaccination.

PLoS One 2019 23;14(12):e0225622. Epub 2019 Dec 23.

HIV Vaccine Trials Network, Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States of America.

Antibodies that recognize commensal microbial antigens may be cross reactive with a part of the human immunodeficiency virus (HIV) envelope glycoprotein gp41. To improve understanding of the role of the microbiota in modulating the immune response to HIV vaccines, we studied the associations of the gut microbiota composition of participants in the HIV Vaccine Trials Network 096 clinical trial with their HIV-specific immune responses in response to vaccination with a DNA-prime, pox virus boost strategy designed to recapitulate the only efficacious HIV-vaccine trial (RV144). We observed that both levels of IgG antibodies to gp41 at baseline and post-vaccination levels of IgG antibodies to the Con.6.gp120.B, ZM96.gp140 and gp70 B.CaseA V1-V2 antigens were associated with three co-occurring clusters of family level microbial taxa. One cluster contained several families positively associated with gp41-specific IgG and negatively associated with vaccine-matched gp120, gp140 and V1-V2-specific IgG responses. A second cluster contained families that negatively associated with gp41 and positively associated with gp120, gp140 and V1-V2-specific IgG responses. A third cluster contained microbial groups that did not correlate with any immune responses. Baseline and post-vaccination levels of gp41 IgG were not significantly correlated, suggesting that factors beyond the microbiome that contribute to immune response heterogeneity. Sequence variant richness was positively associated with gp41, p24, pg140 and V1-V2 specific IgG responses, gp41 and p24 IgA responses, and CD4+ T cell responses to HIV-1 proteins. Our findings provide preliminary evidence that the gut microbiota may be an important predictor of vaccine response.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0225622PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927600PMC
April 2020

Measuring open defecation in India using survey questions: evidence from a randomised survey experiment.

BMJ Open 2019 09 26;9(9):e030152. Epub 2019 Sep 26.

r.i.c.e, India.

Objectives: To investigate differences in reported open defecation between a question about latrine use or open defecation for every household member and a household-level question.

Setting: Rural India is home to most of the world's open defecation. India's Demographic and Health Survey (DHS) 2015-2016 estimates that 54% of households in rural India defecate in the open. This measure is based on a question asking about the behaviour of all household members in one question. Yet, studies in rural India find substantial open defecation among individuals living in households with latrines, suggesting that household-level questions underestimate true open defecation.

Participants: In 2018, we randomly assigned latrine-owning households in rural parts of four Indian states to receive one of two survey modules measuring sanitation behaviour. 1215 households were asked about latrine use or open defecation individually for every household member. 1216 households were asked the household-level question used in India's DHS: what type of facility do members of the household usually use?

Results: We compare reported open defecation between households asked the individual-level questions and those asked the household-level question. Using two methods for comparing open defecation by question type, the individual-level question found 20-21 (95% CI 16 to 25 for both estimates) percentage points more open defecation than the household-level question, among all households, and 28-29 (95% CI 22 to 35 for both estimates) percentage points more open defecation among households that received assistance to construct their latrines.

Conclusions: We provide the first evidence that individual-level questions find more open defecation than household-level questions. Because reducing open defecation in India is essential to meeting the Sustainable Development Goals, and exposure to open defecation has consequences for child mortality and development, it is essential to accurately monitor its progress.

Trial Registration Number: Registry for International Development Impact Evaluations (5b55458ca54d1).
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http://dx.doi.org/10.1136/bmjopen-2019-030152DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773353PMC
September 2019

Optimizing bacterial DNA extraction in urine.

PLoS One 2019 24;14(9):e0222962. Epub 2019 Sep 24.

Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United states of America.

Urine is an acceptable, non-invasive sample for investigating the human urogenital microbiota and for the diagnosis of sexually transmitted infections. However, low quantities of bacterial DNA and PCR inhibitors in urine may prevent efficient PCR amplification for molecular detection of bacteria. Furthermore, cold temperatures used to preserve DNA and bacteria in urine can promote precipitation of crystals that interfere with DNA extraction. Saline, Dulbecco's Phosphate Buffered Saline, or Tris-EDTA buffer were added to urine from adult men to determine if crystal precipitation could be reversed without heating samples beyond ambient temperature. Total bacterial DNA concentrations and PCR inhibition were measured using quantitative PCR assays to compare DNA yields with and without buffer addition. Dissolution of crystals with Tris-EDTA prior to urine centrifugation was most effective in increasing bacterial DNA recovery and reducing PCR inhibition. DNA recovery using Tris-EDTA was further tested by spiking urine with DNA from bacterial isolates and median concentrations of Lactobacillus jensenii and Escherichia coli 16S rRNA gene copies were found to be higher in urine processed with Tris-EDTA. Maximizing bacterial DNA yield from urine may facilitate more accurate assessment of bacterial populations and increase detection of specific bacteria in the genital tract.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222962PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6759279PMC
April 2020

Tenofovir disoproxil fumarate intravaginal ring for HIV pre-exposure prophylaxis in sexually active women: a phase 1, single-blind, randomised, controlled trial.

Lancet HIV 2019 08 15;6(8):e498-e508. Epub 2019 Jul 15.

Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY, USA. Electronic address:

Background: An intravaginal ring that releases the tenofovir prodrug, tenofovir disoproxil fumarate, provided 100% protection in macaques against simian HIV and was safe in a 14-day clinical trial in sexually abstinent women. We aimed to assess the safety and pharmacokinetics of this intravaginal ring over 90 days in sexually active women.

Methods: We did a phase 1, single-blind, randomised, placebo-controlled trial to assess safety, pharmacokinetics, and acceptability of a tenofovir disoproxil fumarate intravaginal ring used continuously with monthly ring changes for 3 months. Sexually active women who were HIV negative were randomly assigned (3:1) to a tenofovir disoproxil fumarate ring or placebo ring. Primary safety endpoint was the proportion of women who had grade 2 or higher genitourinary adverse events judged related to study product and any grade 2 or higher adverse event as defined by the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events. We quantified tenofovir disoproxil fumarate and tenofovir concentrations in cervicovaginal fluid, tenofovir in plasma, and tenofovir diphosphate, the active metabolite, in cervical tissue and dried blood spots 1 month after each ring insertion. We compared changes over time in cervicovaginal fluid cytokine and chemokine concentrations and vaginal microbiota. The study was electively stopped early and is registered with ClinicalTrials.gov, number NCT02762617.

Findings: Between Feb 24 and July 20, 2017, 17 women were enrolled before study termination. 12 were assigned to receive the tenofovir disoproxil fumarate ring and five were assigned to receive the placebo ring. Two participants in the tenofovir disoproxil fumarate ring group completed 3 months of continuous ring use; eight were asked to discontinue ring use early because of ulcerations (grade 1) near the ring; in the remaining two women, rings were electively removed by study staff on day 20 and day 23. Ulcers were detected a mean of 32 days after ring use (range 23-56). Four of eight participants with ulcers were symptomatic with vaginal discharge; four had ulcers identified when examined; three had two ulcers; all ulcers resolved after ring removal. No participants in the placebo group developed ulcers. No grade 2 product-related adverse events were reported in either group and four non-product-related grade 2 adverse events were reported in the tenofovir disoproxil fumarate ring group. Cervicovaginal fluid tenofovir concentrations did not differ at day 14 (p=0·14) comparing the eight patients who did (median 1·0 × 10 ng/mL [IQR 9·1 × 10-1·1 × 10]) with the four who did not (6·0 × 10 ng/mL [5·6 × 10-1·1 × 10]) develop ulcers. No significant changes in vaginal microbiota were detected in either group. Concentrations of multiple inflammatory cytokines and chemokines were significantly higher at days 14 and 28 compared with baseline in the tenofovir disoproxil fumarate ring group but not the placebo group.

Interpretation: Future studies are needed to establish whether the unanticipated finding of ulcerations is specific to this tenofovir disoproxil fumarate ring or generalisable to other sustained topical release formulations of tenofovir or its prodrugs.

Funding: National Institutes of Health.
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http://dx.doi.org/10.1016/S2352-3018(19)30145-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6719300PMC
August 2019

Specific Vaginal Bacteria Are Associated With an Increased Risk of Trichomonas vaginalis Acquisition in Women.

J Infect Dis 2019 09;220(9):1503-1510

Department of Epidemiology, University of Washington, Seattle, Washington.

Background: While bacterial vaginosis has been associated with an increased risk of Trichomonas vaginalis (TV) acquisition, it is unknown whether other characteristics of the vaginal microbiota, including the presence of key bacterial species, influence a woman's risk of TV acquisition.

Methods: The vaginal microbiota before 25 unique episodes of TV infection involving 18 women was compared to that of 50 controls who remained uninfected. TV was detected by transcription-mediated amplification. Vaginal microbiota were quantified using broad-range polymerase chain reaction analysis and taxon-specific quantitative PCR of the 16S ribosomal RNA gene.

Results: TV acquisition was significantly associated with the presence of Prevotella amnii (risk ratio [RR], 2.21; 95% confidence interval [CI], 1.12-4.38; P = .02) and Sneathia sanguinegens (RR, 2.58; 95% CI, 1.00-6.62; P = .049). When adjusted for menstrual phase, the association between P. amnii and TV acquisition remained similar (adjusted RR, 2.11; 95% CI, 1.03-4.33; P = .04), but the association between S. sanguinegens and TV acquisition was attenuated (adjusted RR, 2.31; 95% CI, .86-6.23; P = .10).

Conclusions: Key vaginal bacterial species may contribute to the susceptibility to TV acquisition. Understanding how these bacterial species increase a woman's risk of TV acquisition could help to guide the development of novel strategies to reduce women's risk of TV infection.
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http://dx.doi.org/10.1093/infdis/jiz354DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761949PMC
September 2019

Associations between vaginal bacteria implicated in HIV acquisition risk and proinflammatory cytokines and chemokines.

Sex Transm Infect 2020 02 13;96(1):3-9. Epub 2019 Jun 13.

Department of Internal Medicine, University of Washington, Seattle, Washington, USA.

Objectives: Recent studies have identified vaginal bacterial taxa associated with increased HIV risk. A possible mechanism to explain these results is that individual taxa differentially promote cervicovaginal inflammation. This study aimed to explore relationships between concentrations of bacteria previously linked to HIV acquisition and vaginal concentrations of proinflammatory cytokines and chemokines.

Methods: In this cross-sectional analysis, concentrations of 17 bacterial taxa and four proinflammatory cytokines (interleukin (IL)-1β, IL-6, IL-10 and tumour necrosis factor alpha (TNFα)) and two proinflammatory chemokines (IL-8 and interferon gamma-induced protein 10) were measured in vaginal swabs collected from 80 HIV-uninfected women. Cytokine and chemokine concentrations were compared between women with bacterial concentrations above or below the lower limit of detection as determined by quantitative PCR for each taxon. Principal component analysis was used to create a summary score for closely correlated bacteria, and linear regression analysis was used to evaluate associations between this score and increasing concentrations of TNFα and IL-1β.

Results: Detection of (p=0.01), sp type 1 (p=0.05) or (p=0.03) was associated with higher TNFα concentrations, and detection of (p<0.01) sp type 1 (p=0.04) (p=0.02) or sp type 2 (p=0.05) was associated with significantly higher IL-1β concentrations. Seven bacterial taxa (, sp type 1, sp, sp, and sp type 2) were found to be highly correlated by principal component analysis (eigenvalue 5.24, explaining 74.92% of variability). Linear regression analysis demonstrated associations between this principal component and concentrations of TNFα (β=0.55, 95% CI 0.01 to 1.08; p=0.048) and IL-1β (β=0.96, 95% CI 0.19 to 1.74; p=0.016).

Conclusions: This study provides evidence that several highly correlated vaginal bacterial taxa may influence vaginal cytokine and chemokine concentrations. These results suggest a mechanism where the presence of specific bacterial taxa could influence HIV susceptibility by increasing vaginal inflammation.
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http://dx.doi.org/10.1136/sextrans-2018-053949DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6920574PMC
February 2020

Association between vaginal washing and detection of by culture and quantitative PCR in HIV-seronegative Kenyan women: a cross-sectional analysis.

Sex Transm Infect 2019 09 29;95(6):455-461. Epub 2019 Jan 29.

Department of Epidemiology, University of Washington, Seattle, Washington, USA.

Objectives: Vaginal washing has been associated with reductions in cultivable and an increased risk of both bacterial vaginosis (BV) and HIV infection. The effect of vaginal washing on the quantity of individual species is not well characterised. This analysis tested the hypothesis that vaginal washing would be associated with a lower likelihood of spp. detected by both culture and quantitative PCR (qPCR).

Methods: We conducted a cross-sectional study of 272 HIV-seronegative women enrolled in an open-cohort study in Mombasa, Kenya. Vaginal washing and sexual risk behaviours were assessed using face-to-face interviews. Vaginal spp. were detected using cultivation and PCR methods, with , concentrations measured using qPCR assays targeting the 16S rRNA gene. Poisson regression with robust SEs was used to assess associations between vaginal washing and detection by culture and qPCR.

Results: Eighty percent (n=217) of participants reported vaginal washing in the prior week. One-fifth (n=58) of participants had BV by Nugent score. In unadjusted analysis, vaginal washing was associated with a 45% decreased likelihood of spp. detection by culture (prevalence ratio (PR): 0.55, 95% CI 0.37 to 0.82). Adjusting for age and condomless sex in the prior week did not change the magnitude of the association (adjusted PR (aPR): 0.56, 95% CI (0.37 to 0.85). Vaginal washing was associated with approximately a 40% reduction in detection (aPR: 0.57, 95% CI 0.36 to 0.92), but was not significantly associated with (aPR: 0.68, 95% CI 0.42 to 1.09) or detection (aPR: 1.03, 95% CI 0.92 to 1.15).

Conclusions: Vaginal washing in the prior week was associated with a significantly reduced likelihood of detecting cultivable and by qPCR. Given associations between detection and improved reproductive health outcomes, these results provide motivation for additional study of vaginal washing cessation interventions to improve vaginal health.
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http://dx.doi.org/10.1136/sextrans-2018-053769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7053826PMC
September 2019

Association between vaginal washing and vaginal bacterial concentrations.

PLoS One 2019 24;14(1):e0210825. Epub 2019 Jan 24.

Department of Medicine, University of Washington, Seattle, WA, United States of America.

Vaginal washing is a common practice associated with adverse outcomes including bacterial vaginosis (BV) and HIV infection. Prior studies have not examined the associations between vaginal washing and individual vaginal bacteria, or whether these associations are independent of the effect of vaginal washing on BV. The purpose of this study was to characterize the association between vaginal washing and the presence and concentrations of vaginal bacteria associated with optimal and sub-optimal vaginal states. The analysis utilized data from participants in the placebo arm of the Preventing Vaginal Infections trial, which enrolled HIV-uninfected women from the United States and Kenya. Detection of bacterial taxa associated with BV was compared between visits with versus without reported vaginal washing. The effect of vaginal washing on a number of vaginal bacteria differed substantially (p<0.05) between the US and Kenya, so results were stratified by country. In US women, vaginal washing was associated with a significantly higher likelihood of detection of BV associated bacterium 1 (BVAB1) (relative risk [RR] 1.55, 95% confidence interval [CI] 1.15-2.09, p = 0.004), BVAB2 (RR 1.99, 95%CI 1.46-2.71, p<0.001), Mageeibacillus indolicus (RR 2.08, 95%CI 1.46-2.96, p<0.001), Atopobium vaginae (RR 1.34, 95%CI 1.13-1.59, p = 0.001), Leptotrichia/Sneathia species (RR 1.66, 95% CI 1.33-2.09, p<0.001), Megasphaera species (RR 1.78, 95%CI 1.34-2.37, p<0.001) and Gardnerella vaginalis (RR 1.08, 95%CI 1.01-1.16, p = 0.02). No significant association between vaginal washing and bacterial detection was found in Kenyan women. Adjustment for bacterial vaginosis diagnosed by Gram stain did not alter these results. This study provides evidence that the association between vaginal washing and detection of individual bacterial taxa can vary regionally. For some vaginal bacteria, the association with vaginal washing may be independent of the effect on Gram stain detection of BV. Larger prospective studies in diverse geographic settings should explore whether eliminating vaginal washing impacts the presence and concentrations of key vaginal bacteria.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0210825PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345501PMC
October 2019

Primary Syphilis in the Male Urethra: A Case Report.

Clin Infect Dis 2019 03;68(7):1231-1234

Department of Epidemiology, University of Washington.

We documented urethral Treponema pallidum infection in a man with nongonococcal urethritis and a negative syphilis serology using broad-range bacterial polymerase chain reaction (PCR) and sequencing, targeted PCR, and immunofluorescence microscopy. He subsequently seroconverted for syphilis. Early syphilis may present as urethritis. Urethral T. pallidum shedding can occur before seroconversion.
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http://dx.doi.org/10.1093/cid/ciy771DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6424071PMC
March 2019

Vaginal microbiome modulates topical antiretroviral drug pharmacokinetics.

JCI Insight 2018 07 12;3(13). Epub 2018 Jul 12.

Department of Microbiology & Immunology.

Tenofovir gel and dapivirine ring provided variable HIV protection in clinical trials, reflecting poor adherence and possibly biological factors. We hypothesized that vaginal microbiota modulates pharmacokinetics and tested the effects of pH, individual bacteria, and vaginal swabs from women on pharmacokinetics and antiviral activity. Tenofovir, but not dapivirine, uptake by human cells was reduced as pH increased. Lactobacillus crispatus actively transported tenofovir leading to a loss in drug bioavailability and culture supernatants from Gardnerella vaginalis, but not Atopobium vaginae, blocked tenofovir endocytosis. The inhibition of endocytosis mapped to adenine. Adenine increased from 65.5 μM in broth to 246 μM in Gardnerella, but decreased to 9.5 μM in Atopobium supernatants. This translated into a decrease in anti-HIV activity when Gardnerella supernatants or adenine were added to cultures. Dapivirine was also impacted by microbiota, as drug bound irreversibly to bacteria, resulting in decreased antiviral activity. When drugs were incubated with vaginal swabs, 30.7% ± 5.7% of dapivirine and 63.9% ± 8.8% of tenofovir were recovered in supernatants after centrifugation of the bacterial cell pellet. In contrast, no impact of microbiota on the pharmacokinetics of the prodrugs, tenofovir disoproxil fumarate or tenofovir alafenamide, was observed. Together, these results demonstrate that microbiota may impact pharmacokinetics and contribute to inconsistent efficacy.
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http://dx.doi.org/10.1172/jci.insight.99545DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6124523PMC
July 2018

Evaluation of the association between the concentrations of key vaginal bacteria and the increased risk of HIV acquisition in African women from five cohorts: a nested case-control study.

Lancet Infect Dis 2018 05 26;18(5):554-564. Epub 2018 Jan 26.

Department of Medicine, University of Washington, Seattle, WA, USA; Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Background: Disruptions of vaginal microbiota might increase women's susceptibility to HIV infection. Advances in molecular microbiology have enabled detailed examination of associations between vaginal bacteria and HIV acquisition. Therefore, this study aimed to evaluate the association between the concentrations of specific vaginal bacteria and increased risk of HIV acquisition in African women.

Methods: We did a nested case-control study of participants from eastern and southern Africa. Data from five cohorts of African women (female sex workers, pregnant and post-partum women, and women in serodiscordant relationships) were used to form a nested case-control analysis between women who acquired HIV infection versus those who remained seronegative. Deep sequence analysis of broad-range 16S rRNA gene PCR products was applied to a subset of 55 cases and 55 controls. From these data, 20 taxa were selected for bacterium-specific real-time PCR assays, which were examined in the full cohort as a four-category exposure (undetectable, first tertile, second tertile, and third tertile of concentrations). Conditional logistic regression was used to generate odds ratios (ORs) and 95% CIs. Regression models were stratified by cohort, and adjusted ORs (aORs) were generated from a multivariable model controlling for confounding variables. The Shannon Diversity Index was used to measure bacterial diversity. The primary analyses were the associations between bacterial concentrations and risk of HIV acquisition.

Findings: Between November, 2004, and August, 2014, we identified 87 women who acquired HIV infection (cases) and 262 controls who did not acquire HIV infection. Vaginal bacterial community diversity was higher in women who acquired HIV infection (median 1·3, IQR 0·4-2·3) than in seronegative controls (0·7, 0·1-1·5; p=0·03). Seven of the 20 taxa showed significant concentration-dependent associations with increased odds of HIV acquisition: Parvimonas species type 1 (first tertile aOR 1·67, 95% CI 0·61-4·57; second tertile 3·01, 1·13-7·99; third tertile 4·64, 1·73-12·46; p=0·005) and type 2 (first tertile 3·52, 1·63-7·61; second tertile 0·85, 0·36-2·02; third tertile 2·18, 1·01-4·72; p=0·004), Gemella asaccharolytica (first tertile 2·09, 1·01-4·36; second tertile 2·02, 0·98-4·17; third tertile 3·03, 1·46-6·30; p=0·010), Mycoplasma hominis (first tertile 1·46, 0·69-3·11; second tertile 1·40, 0·66-2·98; third tertile 2·76, 1·36-5·63; p=0·048), Leptotrichia/Sneathia (first tertile 2·04, 1·02-4·10; second tertile 1·45, 0·70-3·00; third tertile 2·59, 1·26-5·34; p=0·046), Eggerthella species type 1 (first tertile 1·79, 0·88-3·64; second tertile 2·62, 1·31-5·22; third tertile 1·53, 0·72-3·28; p=0·041), and vaginal Megasphaera species (first tertile 3·15, 1·45-6·81; second tertile 1·43, 0·65-3·14; third tertile 1·32, 0·57-3·05; p=0·038).

Interpretation: Differences in the vaginal microbial diversity and concentrations of key bacteria were associated with greater risk of HIV acquisition in women. Defining vaginal bacterial taxa associated with HIV risk could point to mechanisms that influence HIV susceptibility and provide important targets for future prevention research.

Funding: National Institute of Child Health and Human Development.
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http://dx.doi.org/10.1016/S1473-3099(18)30058-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445552PMC
May 2018

Associations between improvement in genitourinary symptoms of menopause and changes in the vaginal ecosystem.

Menopause 2018 05;25(5):500-507

Fred Hutchinson Cancer Research Center, Seattle, WA.

Objective: The aim of the study was to identify associations between improvement in genitourinary symptoms of menopause (GSM) and vaginal microbiota, vaginal glycogen, and serum estrogen.

Methods: Thirty postmenopausal women enrolled in a hot flash treatment trial (oral estradiol vs venlafaxine vs placebo) who reported GSM and provided vaginal swabs at 0, 4, and 8 weeks were studied. Bacterial communities were characterized using deep sequencing targeting the 16S rRNA gene V3-V4 region. Participants selected a most bothersome genitourinary symptom (dryness, discharge, pain, itch/burn, or inability to have sex) and rated severity on a 10-point scale at baseline and 8 weeks. Vaginal glycogen and serum estradiol and estrone were measured at enrollment and 8 weeks. Comparisons according to improvement in most bothersome symptom (MBS) were made using χ, Wilcoxon signed-rank test, or Hotelling's t test.

Results: Of 30 participants, 21 (70%) had improvement in MBS over the 8-week study and 9 (30%) had no improvement or worsening of MBS. A higher proportion of women receiving estradiol or venlafaxine reported improvement in MBS (88%, 78%) compared with placebo (54%; P = 0.28). MBS improvement was associated with Lactobacillus-dominant vaginal microbiota at enrollment (57% vs 22%, P = 0.08). Vaginal glycogen, serum estradiol, and estrone significantly increased in women whose MBS improved.

Conclusions: A larger proportion of women whose MBS improved had a Lactobacillus dominant microbiota at enrollment than those who had no improvement during the trial, though this difference was not statistically significant. Larger trials are needed to determine whether vaginal microbiota modify or mediate treatment responses in women with GSM.
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http://dx.doi.org/10.1097/GME.0000000000001037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5898977PMC
May 2018

Graft-Derived Reconstitution of Mucosal-Associated Invariant T Cells after Allogeneic Hematopoietic Cell Transplantation.

Biol Blood Marrow Transplant 2018 02 9;24(2):242-251. Epub 2017 Oct 9.

Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, Washington; Department of Medicine, University of Washington, Seattle, Washington.

Mucosal-associated invariant T (MAIT) cells express a semi-invariant Vα7.2 T cell receptor (TCR) that recognizes ligands from distinct bacterial and fungal species. In neonates, MAIT cells proliferate coincident with gastrointestinal (GI) bacterial colonization. In contrast, under noninflammatory conditions adult MAIT cells remain quiescent because of acquired regulation of TCR signaling. Effects of inflammation and the altered GI microbiota after allogeneic hematopoietic cell transplantation (HCT) on MAIT cell reconstitution have not been described. We conducted an observational study of MAIT cell reconstitution in myeloablative (n = 41) and nonmyeloablative (n = 66) allogeneic HCT recipients and found that despite a rapid and early increase to a plateau at day 30 after HCT, MAIT cell numbers failed to normalize for at least 1 year. Cord blood transplant recipients and those who received post-HCT cyclophosphamide for graft versus host disease (GVHD) prophylaxis had profoundly impaired MAIT cell reconstitution. Sharing of TCRβ gene sequences between MAIT cells isolated from HCT grafts and blood of recipients after HCT showed early MAIT cell reconstitution was due at least in part to proliferation of MAIT cells transferred in the HCT graft. Inflammatory cytokines were required for TCR-dependent MAIT cell proliferation, suggesting that bacterial Vα7.2 TCR ligands might promote MAIT cell reconstitution after HCT. Robust MAIT cell reconstitution was associated with an increased GI abundance of Blautia spp. MAIT cells suppressed proliferation of conventional T cells consistent with a possible regulatory role. Our data identify modifiable factors impacting MAIT cell reconstitution that could influence the risk of GVHD after HCT.
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http://dx.doi.org/10.1016/j.bbmt.2017.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806215PMC
February 2018