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Background: Vaginal microbiota changes associated with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV), can be linked to adverse reproductive health outcomes. Evidence suggests gastrointestinal tract (GIT) infections may affect extra-GIT microbiota. Full-length 16S rRNA gene sequencing may better identify microbiota to species level. We used long-read, full-length 16S rRNA gene sequencing to characterise vulvovaginal (VVS) and anorectal microbiota and their associations with urogenital and anorectal STIs in Ecuadorian female sex workers (FSWs). Methods: VVS and anorectal DNA extracts were subselected from a wider study of FSWs from north-west Ecuador, previously tested for urogenital and anorectal CT, NG, MG and urogenital TV. Full-length 16S rRNA amplicon sequencing was performed using PacBio Hi-Fidelity (HiFi) circular consensus sequencing, reads mapped to a 16S rRNA NCBI database and analyses performed using R-based packages including phyloseq. Significance values were corrected for multiple testing. Results: A total of 124 samples were sequenced, of which 83 were VVS and 43 anorectal, including 36 VVS-anorectal pairs from the same FSWs. Following normalisation, distinct species numbers were lower in VVS compared with anorectal samples (p β = -42, p = 0.018) with no impact on anorectal Shannon and Simpson diversity. In contrast, anorectal CT or NG infection was associated with greater VVS microbiota evenness (Simpson β = 0.31, p = 0.003; Shannon β = 0.67, p = 0.01) independent of urogenital STI status, with 5-fold increased Actinobacteria abundance (p = 0.02), dominated by G. vaginalis and F. vaginae, and 43% decreased Firmicutes abundance (p = 0.05), predominantly decreased L. iners. Conclusions: Amongst vulnerable, highly sexually active Ecuadorian FSWs with high usage of vaginal products, anorectal STIs were independently associated with increased vulvovaginal microbial diversity with changes in vulvovaginal microbiota traditionally associated with poor reproductive health outcomes. [ABSTRACT FROM AUTHOR]

Background: Trichomonas vaginalis (TV) infection, which is associated with poor reproductive tract health outcomes, is the most common curable nonviral STI globally with increasing evidence of treatment failure. TV epidemiology can be highly sub-structured and linked to low socio-economic status, older age in women and female sex work. However, the large highly repetitive genome makes whole genome sequencing approaches to describe TV transmission challenging. We use nested-PCR multi-locus sequence typing (MLST) of TV directly from clinical samples to describe strain diversity among two different groups of at-risk female sexworkers (FSWs) in Ecuador. Methods: DNA was extracted from self-collected vaginal swab samples from two groups of consenting FSWs: street-based workers (SBWs) and brothelbased workers (BBWs). Behavioural and demographic data were collected. Extracts were tested for TV using real-time PCR, TV-positive extracts amplified using a previously published twostep nested PCR for seven TV genes and MLSTamplicon libraries sequenced using Illumina MiSeq. Following alignment to reference genes, strain types were assessed using MLST software and a partition model phylogenetic tree built using IQTREE. Results: Among 250 FSWs, 35/205 (17%) and 23/45 (51%) BBWs and SBWs respectively were positive for TV (p < 0.001). A subset of 35 TV-positive vaginal DNA extracts were sequenced, of which 13/35 were SBWs and 22/35 BBWs. Compared with BBWs, SBWs were older (median age 42 vs 28; p < 0.001), earnt less (median 60US$ (IQR;45-125) vs 200US$ (IQR;100-300)), had higher rates of partner turnover and were more likely to work in one location. MLST typing appeared to show considerable numbers of possible new alleles. Sequences separated into one major (A) and three minor clades (B). All SBW sequences were clade A whereas clades A and B were found among BBWs. The possible influence of mixed infections in these 'high TV exposure' FSWs is under investigation. Conclusion: This study of TV strain diversity among FSWs in north-west Ecuador suggests a high level of allelic diversity. There appeared to be complete clade homogeneity among the poorer, single location, high client-turnover SBWs, compared to BBWs. TV-typing methods may be useful for identification of population structure in TV infection. [ABSTRACT FROM AUTHOR]

Background: Mycoplasma genitalium (MG) is an important sexually transmitted infection (STI) associated with reproductive health sequelae in women, with high rates of antimicrobial resistance (AMR) to macrolides and fluoroquinolones. The small 570-590 kb genome allows for development of rapid molecular-epidemiology technologies more commonly used for viruses, such as for covid-19. We developed a multiplex multi-gene tiled-PCR scheme enabling identification and detection of resistance associated mutations (RAMs) and clade-typing. Method: A multiplex PCR panel to generate 2kb amplicons tiling across twenty-six MG genes was developed using a web-based primer design tool, optimised using co-culture positive reference and clinical MG strains. The panel was used to amplify MG-positive DNA from clinical samples previously collected from 250 female sex workers (FSWs) and 250 non sex workers (NSWs) from Ecuador, collected two years previously from vaginal, anorectal and pharyngeal sites, frozen and later transported to the U.K. Amplicons were barcoded and sequenced using Oxford Nanopore MinION. Kraken taxonomic identifier was used to confirm MG DNA presence. Phylogenetic trees were built using IQTREE and visualised using Figtree. Results: Approximately 50kb of MG genome was amplified using 29 primer pairs in three primer pools. Proportion of MG amongst all FSWs was 9.6% (24/250). DNA extracts from 13 vaginal, 12 anorectal and 1 pharyngeal sample, representing 17 FSWs, 6 of whom had 2 samples included, and 2 NSWs were sequenced. An unrooted phylogenetic tree identified three distinct clinical clades, with positive control and reference sequences confirmed as adjacent. Any two MG sequences derived from separate anatomical sites in the same individual were always of the same clade. However, there was no clear clustering by geolocation and no known clinically significant RAMs in 23S rRNA, gyrA, or parC were detected. However, 38.5% (10/26) samples harboured parC P62S, implicated in evolution of fluoroquinolone resistance. Conclusion: MgSeq is a new, potentially rapid method for long-read MG DNA sequencing, from highly fragmented, low quality clinical samples. Close clustering of sequences derived from multiple sites in a FSWs suggests anatomical contamination or mechanical sharing through sexual practices. Adaptation of this method could be utilised for other important STIs. [ABSTRACT FROM AUTHOR]

Background: Curable sexually transmitted infections (STIs) are a major global cause of female reproductive health morbidity. In Ecuador, there is little or no information on prevalence and risk factors for genital, anorectal and oropharyngeal STIs in atrisk women such as female sex workers (FSWs). Methods: A cross-sectional study was conducted among 250 FSWs and 250 non sex workers (NWSs) in north-west Ecuador between November 2018 and April 2019. Participants provided vulvo-vaginal (VVS), anorectal (only FSWs) and pharyngeal swabs and socio-demographic data. PCR was used to detect Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Mycoplasma genitalium (MG) from all swabs and Trichomonas vaginalis (TV) from VVS. Blood was collected for syphilis serology. Results: Overall prevalence of STIs in FSWs, at any anatomical site, vaginally, anorectally or in the oropharynx was, 39.7% (95% CI 33.4-45.4), 32.0% (95% CI 26.5-38.0), 19.7% (95%CI 15.1-25.2) and 3.2% (95%CI 1.6-6.2), respectively (See table 1). Isolated anorectal infection, that is without coinfection at other anatomical sites, of CT, NG or MG was 7.5% (95%CI: 4.8-11.6). Prevalence of positive syphilis serology (RPR/FTA ABS) was 2.4% (95% CI 1.1-5.1). In NSWs overall prevalence of having one or more STIs at any site was 5.6% (95%CI 3.4-9.2). Proportion of NSWs with at least one of CT, NG or MG in the vagina or oropharynx was 5.6% (95%CI 3.4-9.2) and 0.8% (95%CI 0.2-2.9), respectively and there were no cases of syphilis. Reported inconsistent condom use in FSWs with a stable partner and clients was 85.6% (95% CI 80.4-90.7) and 9.6% (95% CI 6.0-13.3), respectively. In NSWs, inconsistent condom use with a stable partner was 95.1% (95% CI 92.3-97.9). A high proportion of FSWs reported use of vaginal self-medication 87.2% (95% CI: 82.5-90.8). Conclusion: Overall prevalence of STIs in FSWs was high compared to NSWs, particularly for vaginal TV infection. FSWs also had a high proportion of isolated anorectal CT, NG, and MG, which calls for more research into extragenital infections, including a focus on barriers to condom negotiation. If replicated globally, findings may inform on STI prevention interventions. [ABSTRACT FROM AUTHOR]

Objectives: Anorectal sexually transmitted infections (STIs) such as Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG), present treatment challenges, potentially increase antibiotic resistance selection and if undetected may facilitate onward transmission. However, there are limited global prevalence data for anorectal STIs. We conducted a cross-sectional study to assess the prevalence and risk factors of non-viral genital and extragenital STIs in female sex workers (FSW) and female non-sex workers (NSW) in Ecuador., Methods: 250 adult street and brothel FSWs and 250 NSWs, recruited from settlements in north-west Ecuador provided oropharyngeal and vulvo-vaginal swabs (VVS) as well as socio-demographic data. FSWs also provided anorectal swabs. PCR was used to detect CT, NG, Mycoplasma genitalium (MG) from all swabs and additionally Trichomonas vaginalis (TV) from VVS. Risk factors were analysed using logistic regression., Results: Prevalence of FSW vaginal, anorectal and oropharyngeal infection was 32.0% (95% CI 26.5% to 38.0%), 19.7% (95% CI 15.1% to 25.2%) and 3.2% (95% CI 1.6% to 6.2%), respectively, with most vaginal infections being TV (23.4%; 95% CI 18.5% to 29.2%). Overall FSW STI prevalence, at any anatomical site was 39.7% (95% CI 33.8% to 46.1%), with 12.1% (95% CI 8.5% to 16.9%) of infections detected only at the anorectum. Of all the CT and/or NG infections, 64.4% (95% CI 50.4% to 78.4%) were detected only at the anorectum. STI prevalence in NSWs in the vagina and oropharynx were 5.6% (95% CI 3.4% to 9.2%) and 0.8% (95% CI 0.2% to 2.9%), respectively, with most vaginal infections being MG (3.2%; 95% CI 1.6% to 6.2%). In multivariable analysis, risk factors among brothel-based FSWs for having an anorectal STI were vaginal CT, NG or MG (p<0.001), vaginal TV (p=0.029) and being 'in a relationship' (p=0.038)., Conclusions: High prevalence of CT and NG detected only at the anorectum in these FSWs indicate the possibility of missing significant infections if providing only genital testing and calls for greater research into the potential impact on global STI estimates if extragenital infections among at-risk women are not identified., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY. Published by BMJ.)