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

Correction to: Microbiome 8, 165 (2020) https://doi.org/10.1186/s40168-020-00932-

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-1β 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

Characterisation of the HIV inhibitory activity of vaginal lactobacilli isolates from young South African women at high risk of HIV acquisition

Bacterial vaginosis (BV) is an important predisposing factor for the acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs) in South African women. However, the microbial causes and the immunomodulatory effects of BV are not yet fully understood, and effective treatment strategies do not exist. BV is associated with upregulated inflammatory cytokine levels in the female genital tract (FGT), which in turn may increase HIV infection risk by recruiting and activating HIV target cells, reducing epithelial barrier function and directly promoting HIV replication. Lactobacillus species on the other hand are thought to protect against HIV by competitive exclusion, producing virucidal hydrogen peroxide (H2O2), maintaining an acidic pH by producing lactic acid and regulating immune responses in the FGT. This dissertation aimed to characterise the relative HIV inhibitory properties of clinical Lactobacillus isolates, to evaluate the immunoregulatory properties of lactobacilli, and determine the mechanisms underlying these relationships. Vaginal Lactobacillus isolates (n=103), including L. crispatus, L. jensenii, L. johnsonii, L. mucosae, L. plantarum, L. ruminis, L. salivarius and L. vaginalis, were isolated from young South African women who participated in the Women's Initiative in Sexual Health (WISH) study. The production of pro-inflammatory cytokines (IL-6, IL-1α, IL-1β), chemokines (IL-8, IP-10, MIP-3α, MIP-1α, MIP-1β) and regulatory IL-1RA by vaginal epithelial cells in response to lactobacilli in the presence or absence of Gardnerella vaginalis ATCC 14018 and Prevotella bivia ATCC 29303, was measured using Luminex. Growth rates, bacterial sizes, adhesion to cervical (Ca Ski) and vaginal epithelial cells (VK2), culture pH changes and D/L-lactate production by the lactobacilli were also measured in vitro. The properties of vaginal Lactobacillus isolates were also compared to those of commercial probiotics and ATCC reference strains. In order to evaluate differences between lactobacilli isolates that induced low (termed “non-inflammatory”) versus high (termed “inflammatory”) levels of inflammatory cytokine production, the proteomic profiles of 22 inflammatory and 22 non-inflammatory Lactobacillus isolates were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the underlying mechanisms leading to the different inflammatory profiles. Lastly, the influence of Lactobacillus culture supernatants (n=16) on HIV infectivity was evaluated using a Luciferase Reporter Gene Assay in TZM-BL cells. Lactobacilli isolated from women with non-optimal microbiota produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1a, IL-1b, MIP-1a and MIP-1b production significantly elevated. Proteomics analysis showed that 164 proteins were differentially abundant between inflammatory lactobacilli and non-inflammatory lactobacilli. Functional analysis revealed that isolates inducing low levels of inflammatory cytokine production had a significantly higher relative abundance of membrane-associated cellular components, metabolic biological processes and enzymatic molecular functions compared to isolates that induced higher levels of inflammation. A subset of sixteen lactobacilli significantly suppressed IL-6 (adjusted p<0.001) and IL-8 (adjusted p=0.0170) responses to G. vaginalis while L. crispatus isolates suppressed inflammatory cytokines responses to P. bivia. Culture supernatants from the same 16 isolates significantly suppressed HIV infectivity in TZM-BL cells (p=0.0078). Lactobacilli adhesion to VK2 cells correlated negatively with IL-6, IL-8, MIP-1a and IL-1RA production. Lactobacillus beneficial characteristics were highly strainspecific and vaginal isolates out-performed commercial probiotics and ATCC strains. Lactobacillus growth rates, bacterial sizes and adhesion to VK2 cells did not differ significantly between isolates from women with non-optimal microbiota versus those from women with optimal microbiota. These findings show that, while cervicovaginal lactobacilli suppressed overall inflammatory responses to G. vaginalis and P. bivia, isolates from women with non-optimal microbiota were more inflammatory, had lower relative protein abundance and produced less antimicrobial lactic acid than isolates from women with optimal microbiota. Additionally, vaginal Lactobacillus isolates performed better than existing commercial probiotics, suggesting room for improvement of current probiotic formulations available on the South African market to improve BV treatment outcomes and reduce inflammation in the FGT

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

CITATION: Alisoltani, A., et al. 2020. Microbial function and genital inflammation in young South African women at high risk of HIV infection. Microbiome, 8:165, doi:10.1186/s40168-020-00932-8.The original publication is available at https://microbiomejournal.biomedcentral.comBackground: 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.https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-020-00932-8Publisher's versio