Novel point-of-care cytokine biomarker lateral flow test for the screening for sexually transmitted infections and bacterial vaginosis: study protocol of a multicentre multidisciplinary prospective observational clinical study to evaluate the performance and feasibility of the Genital InFlammation Test (GIFT).

INTRODUCTION: A prototype lateral flow device detecting cytokine biomarkers interleukin (IL)-1α and IL-1ß has been developed as a point-of-care test-called the Genital InFlammation Test (GIFT)-for detecting genital inflammation associated with sexually transmitted infections (STIs) and/or bacterial vaginosis (BV) in women. In this paper, we describe the rationale and design for studies that will be conducted in South Africa, Zimbabwe and Madagascar to evaluate the performance of GIFT and how it could be integrated into routine care. METHODS AND ANALYSIS: We will conduct a prospective, multidisciplinary, multicentre, cross-sectional and observational clinical study comprising two distinct components: a biomedical ('diagnostic study') and a qualitative, modelling and economic ('an integration into care study') part. The diagnostic study aims to evaluate GIFT's performance in identifying asymptomatic women with discharge-causing STIs (Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG)) and BV. Study participants will be recruited from women attending research sites and family planning services. Several vaginal swabs will be collected for the evaluation of cytokine concentrations (ELISA), STIs (nucleic acid amplification tests), BV (Nugent score) and vaginal microbiome characteristics (16S rRNA gene sequencing). The first collected vaginal swab will be used for the GIFT assay which will be performed in parallel by a healthcare worker in the clinic near the participant, and by a technician in the laboratory. The integration into care study aims to explore how GIFT could be integrated into routine care. Four activities will be conducted: user experiences and/or perceptions of the GIFT device involving qualitative focus group discussions and in-depth interviews with key stakeholders; discrete choice experiments; development of a decision tree classification algorithm; and economic evaluation of defined management algorithms. ETHICS AND DISSEMINATION: Findings will be reported to participants, collaborators and local government for the three sites, presented at national and international conferences, and disseminated in peer-reviewed publications.The protocol and all study documents such as informed consent forms were reviewed and approved by the University of Cape Town Human Research Ethics Committee (HREC reference 366/2022), Medical Research Council of Zimbabwe (MRCZ/A/2966), Comité d'Ethique pour la Recherche Biomédicale de Madagascar (N° 143 MNSAP/SG/AMM/CERBM) and the London School of Hygiene and Tropical Medicine ethics committee (LSHTM reference 28046).Before the start, this study was submitted to the Clinicaltrials.gov public registry (NCT05723484). TRIAL REGISTRATION NUMBER: NCT05723484.

Prevalence and incidence of sexually transmitted infections among South African women initiating injectable and long-acting contraceptives.

BACKGROUND: South Africa is among the countries with the highest prevalence of sexually transmitted infections (STIs), including Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG). In 2017, there were an estimated 6 million new CT, 4.5 million NG and 71 000 Treponema pallidum infections among South African men and women of reproductive age. METHODS: We evaluated STI prevalence and incidence and associated risk factors in 162 women aged 18-33 years old, residing in eThekwini and Tshwane, South Africa who were part of the Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial. Women were randomised to use depot medroxyprogesterone acetate (n = 53), copper intrauterine device (n = 51), or levonorgestrel (n = 58) implant. Lateral vaginal wall swab samples were collected prior to contraceptive initiation and at months one and three following contraceptive initiation for STI testing. RESULTS: There were no significant differences in STI incidence and prevalence across contraceptive groups. At baseline, 40% had active STIs (CT, NG, Trichomonas vaginalis (TV), Mycoplasma genitalium (MG) or herpes simplex virus-2 shedding across all age groups- 18-21 years (46%), 22-25 years (42%) and 26-33 years (29%). The incidence of STIs during follow-up was exceptionally high (107.9/100 women-years [wy]), with younger women (18-21 years) more likely to acquire CT (75.9/100 wy) compared to 26-33 year olds (17.4/100 wy; p = 0.049). TV incidence was higher in the 26-33 year old group (82.7/100 wy) compared to the 18-21 year olds (8.4/100 wy; p = 0.01). CONCLUSIONS: Although the study participants received extensive counselling on the importance of condom use, this study highlights the high prevalence and incidence of STIs in South African women, especially amongst young women, emphasising the need for better STI screening and management strategies.

Advances in mass spectrometry technologies to characterize cervicovaginal microbiome functions that impact spontaneous preterm birth.

Preterm birth (PTB) is a leading cause of morbidity and mortality in young children. Infection is a major cause of this adverse outcome, particularly in PTBs characterised by spontaneous rupture of membranes, referred to as spontaneous (s)PTB. However, the aetiology of sPTB is not well defined and specific bacteria associated with sPTB differ between studies and at the individual level. This may be due to many factors including a lack of understanding of strain-level differences in bacteria that influence how they function and interact with each other and the host. Metaproteomics and metabolomics are mass spectrometry-based methods that enable the collection of detailed microbial and host functional information. Technological advances in this field have dramatically increased the resolution of these approaches, enabling the simultaneous detection of thousands of proteins or metabolites. These data can be used for taxonomic analysis of vaginal bacteria and other microbes, to understand microbiome-host interactions, and identify diagnostic biomarkers or therapeutic targets. Although these methods have been used to assess host proteins and metabolites, few have characterized the microbial compartment in the context of pregnancy. The utilisation of metaproteomic and metabolomic-based approaches has the potential to vastly improve our understanding of the mechanisms leading to sPTB.

Cost-effectiveness analysis of different screening and diagnostic strategies for sexually transmitted infections and bacterial vaginosis in women attending primary health care facilities in Cape Town.

Background: Genital inflammation associated with sexually transmitted infections (STIs) and bacterial vaginosis (BV) is considered a key driver in the HIV epidemic. A new rapid point-of-care test (POC) that detects genital inflammation in women-Genital InFlammation Test (GIFT)-was recently developed by researchers at the University of Cape Town. The objective of this study was to establish the cost-effectiveness of this novel intervention relative to other relevant screening and diagnostic strategies for the management of STIs and BV in women seeking care in the public health sector in South Africa. Methods: A decision analysis model was developed for five different screening and diagnostic strategies for women incorporating syndromic management, screening with GIFT and using etiological diagnosis. A decision tree was constructed using Microsoft Excel Office 365, and cost and effectiveness parameters were obtained from published literature and market prices. The model incorporated all clinic-level and treatment costs associated with diagnosing and treating a single episode of disease. The effectiveness of each approach was proxied by its sensitivity. One-way and threshold sensitivity analyses were conducted to test key uncertainties and assumptions in the model. Results: Screening with GIFT, and following with antibiotic treatment according to syndromic management guidelines for GIFT-positive cases, was the most cost-effective strategy with an incremental cost-effectiveness ratio (ICER) of USD 11.08 per women diagnosed with an STI(s) and/or BV and provided treatment. This strategy resulted in lower rates of overtreatment compared to syndromic management, but higher rates compared to etiological diagnosis using nucleic acid amplification tests and microscopy. However, following a GIFT positive test with etiological diagnosis prior to treatment did not increase the effectiveness, but dramatically increased the cost. Conclusion: Screening with GIFT and treating positive cases according to syndromic management guidelines is the most cost-effective strategy for the management of STIs and BV. GIFT has a potential to significantly improve the management of STIs and BV in women by identifying asymptomatic women and reducing their risk of HIV infection. This analysis presents a first step in establishing the cost-effectiveness of these interventions and paves the way for further research to develop optimal context-specific implementation strategies.

Shifting the power: scale-up of access to point-of-care and self-testing for sexually transmitted infections in low-income and middle-income settings.

PURPOSE OF REVIEW: Point-of-care (POC) testing for sexually transmitted infections (STIs) can provide complementary coverage to existing HIV testing services in LMICs. This review summarizes current and emerging technologies for detecting STIs in LMICs, with an emphasis on women, discharge-causing infections (chlamydia, gonorrhoea, trichomoniasis, and syphilis), true POC, self-testing, ethics, and economic considerations related to equitable access. RECENT FINDINGS: The WHO have recently adapted guidelines for treatment of STIs in women that advise the use of true-POC or near-POC tests to improve case finding. The number of rapid, sensitive, and specific POC diagnostics for STIs has increased significantly over the past 10 years, although adoption of these in low-income and middle-income countries (LMICs) remains limited. Barriers to POC adoption by patients include the cost of tests, the inconvenience of lengthy clinic visits, low perceived risk, stigma, lack of partner notification, and lack of trust in healthcare providers. Lowering the cost of true POC lateral flow devices, interfacing these with digital or eHealth technologies, and enabling self-testing/self-sampling will overcome some of these barriers in LMICs. Ensuring linkage of diagnostic tests to subsequent care remains one of the major concerns about self-testing, irrespective of geography, although available evidence from HIV self-testing suggests that linkage to care is similar to that for facility-based testing. SUMMARY: Increasing access to sensitive STI true POC tests will strengthen reproductive healthcare in LMICs. Although HIV self-testing is demonstrably useful in LMICs, there is an urgent need for randomized trials evaluating the utility and cost-effectiveness of similar tests for other sexually transmitted infections.

The Impact of Over-The-Counter Lactic Acid Containing Vaginal Gels on the Integrity and Inflammatory State of the Vaginal Epithelium in vitro.

The vaginal microbiome influences a wide range of health outcomes in women, where a microbiome dominated by Lactobacillus spp. is considered optimal and associated with reduced risk of pre-term birth and acquisition of sexually transmitted infections including HIV. Conversely, replacement of lactobacilli by non-optimal bacteria leads to the development of bacterial vaginosis, which is associated with increased risk of these outcomes. Lactobacilli produce the metabolite lactic acid (LA) which is a potent antibacterial and antiviral agent. The potential therapeutic benefits of LA have prompted the development of numerous over-the-counter LA-containing gels for use in the vagina, although a comprehensive analysis of the impact of these formulations on the cervicovaginal epithelium and pro-inflammatory cytokine/chemokine responses, has not been assessed. Here, we evaluated the properties of 11 over-the-counter gels, including 9 containing LA, marketed for use in the vagina. Ten of the 11 gels had an osmolality greater than vaginal fluid from women with Lactobacillus-dominated microbiota (370 ± 40 mOsmol/kg in women with Nugent score 0-3), with six gels that were hyperosmolal >2,000 mOsmol/kg. Using a reconstructed primary cell model of the vaginal epithelium, we found hyperosmolal gels had a detrimental impact on epithelial barrier integrity, resulting in substantial cellular toxicity (<10% viability as compared to untreated cells) and reduced epithelial barrier integrity [≈30% of untreated cells, assessed by transepithelial electrical resistance (TEER)]. Treatment of vaginal tissues with most of the gels elicited the production of pro-inflammatory factors including IL-1α (8 of 11) and IL-1ß (10 of 11) which are associated with heightened risk of HIV acquisition in vivo. The majority of the OTC gels elicited moderate tissue damage as determined by histology. The detrimental effects of these gels on the human vaginal epithelium in vitro may predict compromised epithelial barrier integrity and genital inflammation in vivo, which has implications for sexual and reproductive health. This study highlights the importance of evaluating the impact of intravaginal products on the integrity and inflammatory status of the mucosal epithelium to avoid unfavorable off target effects.

Lactic acid from vaginal microbiota enhances cervicovaginal epithelial barrier integrity by promoting tight junction protein expression.

BACKGROUND: Women with a cervicovaginal microbiota dominated by Lactobacillus spp. are at reduced risk of acquiring sexually transmitted infections including HIV, but the biological mechanisms involved remain poorly defined. Here, we performed metaproteomics on vaginal swab samples from young South African women (n = 113) and transcriptomics analysis of cervicovaginal epithelial cell cultures to examine the ability of lactic acid, a metabolite produced by cervicovaginal lactobacilli, to modulate genital epithelial barrier function. RESULTS: Compared to women with Lactobacillus-depleted microbiota, women dominated by vaginal lactobacilli exhibit higher abundance of bacterial lactate dehydrogenase, a key enzyme responsible for lactic acid production, which is independently associated with an increased abundance of epithelial barrier proteins. Physiological concentrations of lactic acid enhance epithelial cell culture barrier integrity and increase intercellular junctional molecule expression. CONCLUSIONS: These findings reveal a novel ability of vaginal lactic acid to enhance genital epithelial barrier integrity that may help prevent invasion by sexually transmitted pathogens. Video abstract.

Genital inflammatory status and the innate immune response to contraceptive initiation.

PROBLEM: Data on the effects of contraceptives on female genital tract (FGT) immune mediators are inconsistent, possibly in part due to pre-existing conditions that influence immune mediator changes in response to contraceptive initiation. METHODS: This study included 161 South African women randomised to injectable depot medroxyprogesterone acetate (DMPA-IM), copper intrauterine device (IUD), or levonorgestrel (LNG) implant in the Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial. We measured thirteen cytokines and antimicrobial peptides previously associated with HIV acquisition in vaginal swabs using Luminex and ELISA, before, and at 1 and 3 months after contraceptive initiation. Women were grouped according to an overall baseline inflammatory profile. We evaluated modification of the relationships between contraceptives and immune mediators by baseline inflammation, demographic, and clinical factors. RESULTS: Overall, LNG implant and copper IUD initiation were associated with increases in inflammatory cytokines, while no changes were observed following DMPA-IM initiation. However, when stratifying by baseline inflammatory profile, women with low baseline inflammation in all groups experienced significant increases in inflammatory cytokines, while those with a high baseline inflammatory profile experienced no change or decreases in inflammatory cytokines. CONCLUSION: We conclude that pre-contraceptive initiation immune profile modifies the effect of contraceptives on the FGT innate immune response.

Rapid Point-of-Care Testing for Genital Tract Inflammatory Cytokine Biomarkers to Diagnose Asymptomatic Sexually Transmitted Infections and Bacterial Vaginosis in Women: Cost Estimation and Budget Impact Analysis.

BACKGROUND: Screening for genital inflammation can reveal asymptomatic cases of sexually transmitted infections (STIs) and bacterial vaginosis (BV), useful in settings where only syndromic management is available. This study aimed to estimate the incremental cost of screening using a new cytokine biomarker rapid test and determine the budget impact of providing this service in primary health facilities in South Africa. METHODS: Costs of adding genital inflammation screening to existing family planning services were estimated for women (15-49 years) attending 3 different family planning clinics in US $2016. The predicted unit cost per patient screened from a provider's perspective was calculated using bottom-up and top-down approaches and was used to analyze the budget impact of scaling up and providing this service in primary health facilities countrywide. Univariate sensitivity analyses tested the robustness of the findings. RESULTS: The incremental cost per woman screened for genital inflammation ranged between US $3.19 and US $4.79. The scaled-up costs ranged between US $7,245,775 and US $22,212,636 countrywide, annually. This was based on the number of women of reproductive age currently seeking contraceptive care at all primary health care facilities, as a proxy for those most susceptible to asymptomatic STIs/BV. The cost estimates were sensitive to changes in personnel costs, utilization rate, and population coverage rates. CONCLUSIONS: This screening tool is likely to increase case detection, contributing to better STI/BV management and control, in addition to reducing women's risk of HIV acquisition. The incremental cost estimates could make implementation affordable.

The Effect of Exogenous Sex Steroids on the Vaginal Microbiota: A Systematic Review.

Background: Exogenous sex steroids within hormonal contraception and menopausal hormone therapy (MHT) have been used for family planning and management of menopausal symptoms, without consideration of their effects on the vaginal microbiota. This is largely because their use predates our understanding of the importance of the vaginal microbiome on human health. We conducted a systematic review (PROSPERO: CRD42018107730) to determine the influence of exogenous sex steroids, stratified by oestrogen-containing or progestin-only types of contraception, and MHT on the vaginal microbiome, as measured by molecular methods. Methods: Embase, PubMed and Medline were searched for relevant literature published through to December 1st 2020. Eligible studies reported on the effect of specific exogenous sex steroids on the vaginal microbiome using a molecular method. Data regarding the 'positive', 'negative' or 'neutral' effect of each type of contraceptive or MHT on the vaginal microbiome was extracted and summarised. A positive effect reflected sex steroid exposure that was associated with increased abundance of lactobacilli, a change to, or maintenance of, an optimal vaginal microbiota composition, or a decrease in bacterial diversity (specifically reflecting a low-diversity optimal microbiota state), relative to the control group. An exogenous sex steroid was designated as having a negative effect on the vaginal microbiome if it resulted in opposing effects (i.e. loss of lactobacilli, a non-optimal microbiota state). When no significant change was found, this was considered neutral/inconclusive. Results: We identified 29 manuscripts reporting on the effect of exogenous sex steroids on the vaginal microbiome; 25 investigating hormonal contraceptives, and 4 investigating MHT. Oestrogen-containing contraception, particularly reflecting the combined oestrogen and progestin-containing contraceptive pill, had a positive effect on the composition of the vaginal microbiota. Progestin-only contraception, particularly reflecting depo-medroxyprogesterone acetate, had mixed effects on the microbiota. Among post-menopausal women using MHT, exogenous oestrogen applied topically was associated with increased prevalence of lactobacilli. Conclusion: Our findings suggest that oestrogen-containing compounds may promote an optimal vaginal microbiota, which could have clinical applications. The impact of progestin-only contraceptives on the vaginal microbiota is less clear; more data is needed to determine how progestin-only contraceptives contribute to adverse reproductive and sexual health outcomes.

Recent Advances and New Challenges in Cisgender Women's Gynecologic and Obstetric Health in the Context of HIV.

Although rates of human immunodeficiency virus (HIV) have declined globally over the past 10 years, United Nations Programme on HIV/AIDS estimates 1.7 million new infections occurred in 2019, with cisgender women (cis women) and girls accounting for 48%. Acquired immune deficiency syndrome-related illnesses are the leading global cause of mortality in cis women aged 15 to 49, and in many sub-Saharan Africa countries, young women face substantially higher HIV risk than their male counterparts. Drivers of this increased risk include sexual and reproductive health characteristics unique to cis women. This review discusses the role of sexually transmitted infections, contraception and pregnancy in HIV risk, and biomedical HIV prevention technologies available and in development.

Il4ra-independent vaginal eosinophil accumulation following helminth infection exacerbates epithelial ulcerative pathology of HSV-2 infection.

How helminths influence the pathogenesis of sexually transmitted viral infections is not comprehensively understood. Here, we show that an acute helminth infection (Nippostrongylus brasiliensis [Nb]) induced a type 2 immune profile in the female genital tract (FGT). This leads to heightened epithelial ulceration and pathology in subsequent herpes simplex virus (HSV)-2 infection. This was IL-5-dependent but IL-4 receptor alpha (Il4ra) independent, associated with increased FGT eosinophils, raised vaginal IL-33, and enhanced epithelial necrosis. Vaginal eosinophil accumulation was promoted by IL-33 induction following targeted vaginal epithelium damage from a papain challenge. Inhibition of IL-33 protected against Nb-exacerbated HSV-2 pathology. Eosinophil depletion reduced IL-33 release and HSV-2 ulceration in Nb-infected mice. These findings demonstrate that Nb-initiated FGT eosinophil recruitment promotes an eosinophil, IL-33, and IL-5 inflammatory circuit that enhances vaginal epithelial necrosis and pathology following HSV-2 infection. These findings identify a mechanistic framework as to how helminth infections can exacerbate viral-induced vaginal pathology.

Impact of point-of-care testing and treatment of sexually transmitted infections and bacterial vaginosis on genital tract inflammatory cytokines in a cohort of young South African women.

OBJECTIVES: STIs cause inflammation that is detrimental for both HIV risk and reproductive health. We assessed the impact of point-of-care (POC) STI testing, immediate treatment and expedited partner therapy (EPT) on genital tract cytokines among a cohort of young South African women. METHODS: HIV-negative women underwent POC testing for Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and Trichomonas vaginalis (TV) by Xpert CT/NG and OSOM TV, and for bacterial vaginosis (BV) by microscopy. Women with STIs and/or BV received immediate treatment, EPT for STIs and retested after 6 and 12 weeks. Concentrations of 48 cytokines were measured in cervicovaginal fluid at each visit using multiplex ELISA technology. The impact of STI treatment on cytokine concentrations was assessed by multivariable linear mixed models and principal component analysis. RESULTS: The study enrolled 251 women with median age of 23 years (IQR 21-27). The prevalence of CT, NG and TV were 14.3%, 4.4% and 4.0%, and 34.3% had BV. Women with STIs or BV at baseline (n=94) had significantly higher concentrations of pro-inflammatory cytokines (interleukin (IL)-1α, IL-1ß, IL-6, tumour necrosis factor (TNF)-α, TNF-ß, IL-18 and macrophage inflammatory factor (MIF)) and chemokines (IL-8, IL-16, macrophage inflammatory protein (MIP)-1α, IFN-α2, monokine induced by gamma interferon (MIG), monocyte chemoattractant protein (MCP)-3, regulated on activation normal T cell expressed and secreted and eotaxin) compared with women without (n=157). STI treatment was strongly associated with reduced concentrations of pro-inflammatory cytokines IL-6 (p=0.004), IL-1ß (p=0.013), TNF-α (p=0.018) and chemokines MIG (p=0.008) and growth-related oncogene (GRO)-α (p=0.025). A lower Nugent score was associated with a reduction in pro-inflammatory cytokines IL-1α (p=0.003), TNF-related apoptosis-inducing ligand (p=0.004), MIF (p=0.010) and IL-18 (p<0.001), but an increase in chemokines MIG (p=0.020), GRO-α (p<0.001), IP-10 (p<0.001), MIP-1ß (p=0.008) and MCP-1 (p=0.005). Principal component analysis showed differences in STI and BV-related inflammatory profiles, but that resolution restored a profile consistent with vaginal health. CONCLUSIONS: A comprehensive STI intervention effectively reduced genital inflammation among young women, thereby improving vaginal health and potentially reducing HIV risk.

Lactic acid-containing products for bacterial vaginosis and their impact on the vaginal microbiota: A systematic review.

OBJECTIVE: The vaginal microbiota in bacterial vaginosis (BV) typically has low abundance of lactic acid producing lactobacilli. Lactic acid has properties that may make it effective for treating BV and/or restoring an optimal lactobacillus-dominated vaginal microbiota. We conducted a systematic review to describe the effect of intravaginal lactic acid-containing products on BV cure, and their impact on vaginal microbiota composition (PROSPERO registration: CRD42018115982). METHODS: PubMed, Embase and OVID were searched from inception to November 2019 to identify eligible studies. Included studies evaluated an intravaginal lactic acid-containing product and reported BV cure using established diagnostic methods, and/or vaginal microbiota composition using molecular methods. Studies were independently screened and assessed, and the proportion of women cured post-treatment was calculated. Study results were described in a qualitative manner. RESULTS: We identified 1,883 articles and assessed 57 full-texts for eligibility. Seven different lactic acid-containing products were evaluated and differed with respect to excipients, lactic acid concentration and pH. Most studies had medium or high risk of bias. Three trials compared the efficacy of a lactic acid-containing product to metronidazole for BV cure. One study found lactic acid to be equivalent to metronidazole and two studies found lactic acid to be significantly inferior to metronidazole. Two studies included a control group receiving a placebo or no treatment. One reported lactic acid to be superior than no treatment and the other reported lactic acid to be equivalent to placebo. Lactic acid-containing products did not significantly impact the vaginal microbiota composition. CONCLUSION: There is a lack of high-quality evidence to support the use of lactic acid-containing products for BV cure or vaginal microbiota modulation. However, adequately powered and rigorous randomised trials with accompanying vaginal microbiota data are needed to evaluate the efficacy of lactic acid as a BV treatment strategy.

Microbial function and genital inflammation in young South African women at high risk of HIV infection.

BACKGROUND: Female genital tract (FGT) inflammation is an important risk factor for HIV acquisition. The FGT microbiome is closely associated with inflammatory profile; however, the relative importance of microbial activities has not been established. Since proteins are key elements representing actual microbial functions, this study utilized metaproteomics to evaluate the relationship between FGT microbial function and inflammation in 113 young and adolescent South African women at high risk of HIV infection. Women were grouped as having low, medium, or high FGT inflammation by K-means clustering according to pro-inflammatory cytokine concentrations. RESULTS: A total of 3186 microbial and human proteins were identified in lateral vaginal wall swabs using liquid chromatography-tandem mass spectrometry, while 94 microbial taxa were included in the taxonomic analysis. Both metaproteomics and 16S rRNA gene sequencing analyses showed increased non-optimal bacteria and decreased lactobacilli in women with FGT inflammatory profiles. However, differences in the predicted relative abundance of most bacteria were observed between 16S rRNA gene sequencing and metaproteomics analyses. Bacterial protein functional annotations (gene ontology) predicted inflammatory cytokine profiles more accurately than bacterial relative abundance determined by 16S rRNA gene sequence analysis, as well as functional predictions based on 16S rRNA gene sequence data (p < 0.0001). The majority of microbial biological processes were underrepresented in women with high inflammation compared to those with low inflammation, including a Lactobacillus-associated signature of reduced cell wall organization and peptidoglycan biosynthesis. This signature remained associated with high FGT inflammation in a subset of 74 women 9 weeks later, was upheld after adjusting for Lactobacillus relative abundance, and was associated with in vitro inflammatory cytokine responses to Lactobacillus isolates from the same women. Reduced cell wall organization and peptidoglycan biosynthesis were also associated with high FGT inflammation in an independent sample of ten women. CONCLUSIONS: Both the presence of specific microbial taxa in the FGT and their properties and activities are critical determinants of FGT inflammation. Our findings support those of previous studies suggesting that peptidoglycan is directly immunosuppressive, and identify a possible avenue for biotherapeutic development to reduce inflammation in the FGT. To facilitate further investigations of microbial activities, we have developed the FGT-DB application that is available at http://fgtdb.org/ . Video Abstract.

HIV-1 subtype C transmitted founders modulate dendritic cell inflammatory responses.

BACKGROUND: Heterosexual transmission remains the main route of HIV-1 transmission and female genital tract (FGT) inflammation increases the risk of infection. However, the mechanism(s) by which inflammation facilitates infection is not fully understood. In rhesus macaques challenged with simian immunodeficiency virus, dendritic cell (DC) mediated recruitment of CD4+ T cells to the FGT was critical for infection. The aim of this study was to delineate the mechanisms underlying DC-mediated HIV infection by comparing chemokine and pro-inflammatory cytokine production in response to transmitted founder (TF) and chronic infection (CI) Envelope (Env) pseudotyped viruses (PSV). RESULTS: Monocyte-derived DCs (MDDCs) were stimulated with PSV and recombinant gp140 representing matched TF and CI pairs of four individuals and cytokine secretion measured by multiplex immuno-assay. We found that 4/9 Env induced robust MDDC inflammatory responses and of those, three were cloned from TFs. Overall, TF Env induced MDDCs from healthy donors to secrete higher concentrations of inflammatory cytokines and chemokines than those from CI, suggesting TF Env were better inducers of inflammation. Assessing the signalling pathway associated with inflammatory cytokines, we found that PSV of matched TF and CI variants and a gp140 clone activated ERK and JNK to similar levels. Recombinant soluble DC-SIGN inhibited cytokine release and activation of ERK by PSV, suggesting that Env-DC-SIGN binding was partly involved in MDDC stimulation. Therefore, Env clones might differentially stimulate MDDC immune responses via alternative, yet unidentified signalling pathways. CONCLUSION: Overall, this could suggest that the genetics of the virus itself influences inflammatory responses during HIV infection. In the absence of pre-existing infections, induction of greater inflammatory response by TFs might favour virus survival within the healthy FGT by driving an influx of target cells to sites of infection while suppressing immune responses via IL-10.

Inflammatory and antimicrobial properties differ between vaginal Lactobacillus isolates from South African women with non-optimal versus optimal microbiota.

Female genital tract (FGT) inflammation increases HIV infection susceptibility. Non-optimal cervicovaginal microbiota, characterized by depletion of Lactobacillus species and increased bacterial diversity, is associated with increased FGT cytokine production. Lactobacillus species may protect against HIV partly by reducing FGT inflammation. We isolated 80 lactobacilli from South African women with non-optimal (Nugent 4-10; n = 18) and optimal microbiota (Nugent 0-3; n = 14). Cytokine production by vaginal epithelial cells in response to lactobacilli in the presence and absence of Gardnerella vaginalis was measured using Luminex. Adhesion to vaginal epithelial cells, pH, D/L-lactate production and lactate dehydrogenase relative abundance were assessed. Lactobacilli from women with non-optimal produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1α, IL-1ß and MIP-1α/ß production significantly elevated. Overall, lactobacilli suppressed IL-6 (adjusted p < 0.001) and IL-8 (adjusted p = 0.0170) responses to G. vaginalis. Cytokine responses to the lactobacilli were inversely associated with lactobacilli adhesion to epithelial cells and D-lactate dehydrogenase relative abundance. Thus, while cervicovaginal lactobacilli reduced the production of the majority of inflammatory cytokines in response to G. vaginalis, isolates from women with non-optimal microbiota were more inflammatory and produced less lactic acid than isolates from women with optimal microbiota.

Impact of Hormonal Contraceptives on Cervical T-helper 17 Phenotype and Function in Adolescents: Results from a Randomized, Crossover Study Comparing Long-acting Injectable Norethisterone Oenanthate (NET-EN), Combined Oral Contraceptive Pills, and Combined Contraceptive Vaginal Rings.

BACKGROUND: Adolescents in sub-Saharan Africa are at risk for human immunodeficiency virus (HIV) infection and unintended pregnancies. Observational studies suggest that injectable hormonal contraceptives (HCs) increase the HIV risk, although their effects on genital inflammation, particularly HIV-susceptible T-helper 17 (Th17) cells, are unknown. In a randomized crossover study, the effect of injectable norethisterone oenanthate (NET-EN), combined contraceptive vaginal rings (CCVR; NuvaRing), and combined oral contraceptive pills (COCPs) on cervical Th17 cells and cytokines were compared. METHODS: Adolescents (n = 130; 15-19 years) were randomly assigned 1:1:1 to NET-EN, CCVR, or COCPs for 16 weeks, then subsequently crossed over to another HC for 16 weeks. Estrogen, follicular stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured. Chemokine receptor 5 (CCR5), human leukocyte antigen (HLA) DR isotope, and cluster of differentiation 38 (CD38) expression by cervical cytobrush-derived CD4+ T cells was assessed by fluorescence-activated cell sorting. Th17 cells were defined as CCR6+ and CCR10-. Cervicovaginal Th17-related cytokines were measured by Luminex. RESULTS: CCVR use for the first 16 weeks was associated with reduced Th17 frequencies and lower FSH and LH concentrations, as compared to NET-EN and COCPs, with FSH concentrations and Th17 frequencies correlating significantly. However, Th17-related cytokine concentrations (interleukin [IL]-21, IL-1ß, tumor necrosis factor-α, interferon-γ) and CCR5, HLA-DR, CD38, and Th17 frequencies were significantly higher in CCVR than NET-EN and COCP. At crossover, CCVR users changing to COCPs or NET-EN did not resolve activation or cytokines, although switching from COCP to CCVRs increased cytokine concentrations. CONCLUSIONS: CCVR use altered endogenous hormone levels and associated cervical Th17 cell frequencies to a greater extent than use of NET-EN or COCPs, although Th17 cells were more activated and Th17-related cytokine concentrations were elevated. While CCVRs may impact the HIV risk by regulating Th17 numbers, increased activation and inflammation may balance any risk gains.