Unravelling Chlamydia trachomatis diversity in Amhara, Ethiopia: MLVA-ompA sequencing as a molecular typing tool for trachoma.

Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections, alongside improving facial cleanliness and environmental conditions to reduce transmission. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes could be useful. While many studies have used the Ct major outer membrane protein gene (ompA) for genotyping, it has limitations. Our study applies a typing system novel to trachoma, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing 3 variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control.

Use of real-time PCR as an alternative to conventional genotyping methods for the laboratory detection of lymphogranuloma venereum (LGV).

Lymphogranuloma venereum (LGV) can be differentiated from non-LGV chlamydial infection using Sanger sequencing or molecular assays, including those that are commercially-available internationally. Here, we describe the performance of a rapid real-time PCR (RT-PCR)-based strategy in differentiating Chlamydia trachomatis infections associated with LGV or non-LGV serovars. One hundred three rectal swabs, previously genotyped using Sanger sequencing of the ompA gene as a reference method, were tested in the RT-PCR assays. All non-LGV specimens were correctly identified, but the RT-PCR failed to detect 1 LGV specimen, resulting in a sensitivity of 87.5% for the non-LGV/LGV RT-PCR assay. Additional performance characteristics (e.g., specificity, accuracy, and reproducibility) were all between 93% and 100% with a limit of detection ≤100 copies/reaction. Thus, this rapid RT-PCR method for LGV detection in clinical specimens is comparable to the reference method.

Ongoing evolution of Chlamydia trachomatis lymphogranuloma venereum: exploring the genomic diversity of circulating strains.

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis, is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

Tryptophan Operon Diversity Reveals Evolutionary Trends among Geographically Disparate Chlamydia trachomatis Ocular and Urogenital Strains Affecting Tryptophan Repressor and Synthase Function.

The obligate intracellular pathogen Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections and blindness globally. To date, Ct urogenital strains are considered tryptophan prototrophs, utilizing indole for tryptophan synthesis within a closed-conformation tetramer comprised of two α (TrpA)- and two ß (TrpB)-subunits. In contrast, ocular strains are auxotrophs due to mutations in TrpA, relying on host tryptophan pools for survival. It has been speculated that there is strong selective pressure for urogenital strains to maintain a functional operon. Here, we performed genetic, phylogenetic, and novel functional modeling analyses of 595 geographically diverse Ct ocular, urethral, vaginal, and rectal strains with complete operon sequences. We found that ocular and urogenital, but not lymphogranuloma venereum, TrpA-coding sequences were under positive selection. However, vaginal and urethral strains exhibited greater nucleotide diversity and a higher ratio of nonsynonymous to synonymous substitutions [Pi(a)/Pi(s)] than ocular strains, suggesting a more rapid evolution of beneficial mutations. We also identified nonsynonymous amino acid changes for an ocular isolate with a urogenital backbone in the intergenic region between TrpR and TrpB at the exact binding site for YtgR-the only known iron-dependent transcription factor in Chlamydia-indicating that selective pressure has disabled the response to fluctuating iron levels. In silico effects on protein stability, ligand-binding affinity, and tryptophan repressor (TrpR) affinity for single-stranded DNA (ssDNA) measured by calculating free energy changes (ΔΔG) between Ct reference and mutant tryptophan operon proteins were also analyzed. We found that tryptophan synthase function was likely suboptimal compared to other bacterial tryptophan prototrophs and that a diversity of urogenital strain mutations rendered the synthase nonfunctional or inefficient. The novel mutations identified here affected active sites in an orthosteric manner but also hindered α- and ß-subunit allosteric interactions from distant sites, reducing efficiency of the tryptophan synthase. Importantly, strains with mutant proteins were inclined toward energy conservation by exhibiting an altered affinity for their respective ligands compared to reference strains, indicating greater fitness. This is not surprising as l-tryptophan is one of the most energetically costly amino acids to synthesize. Mutations in the tryptophan repressor gene (trpR) among urogenital strains were similarly detrimental to function. Our findings indicate that urogenital strains are evolving more rapidly than previously recognized with mutations that impact tryptophan operon function in a manner that is energetically beneficial, providing a novel host-pathogen evolutionary mechanism for intracellular survival.IMPORTANCEChlamydia trachomatis (Ct) is a major global public health concern causing sexually transmitted and ocular infections affecting over 130 million and 260 million people, respectively. Sequelae include infertility, preterm birth, ectopic pregnancy, and blindness. Ct relies on available host tryptophan pools and/or substrates to synthesize tryptophan to survive. Urogenital strains synthesize tryptophan from indole using their intact tryptophan synthase (TS). Ocular strains contain a trpA frameshift mutation that encodes a truncated TrpA with loss of TS function. We found that TS function is likely suboptimal compared to other tryptophan prototrophs and that urogenital stains contain diverse mutations that render TS nonfunctional/inefficient, evolve more rapidly than previously recognized, and impact operon function in a manner that is energetically beneficial, providing an alternative host-pathogen evolutionary mechanism for intracellular survival. Our research has broad scientific appeal since our approach can be applied to other bacteria that may explain evolution/survival in host-pathogen interactions.

Could LASCA-imaging of GB-speckles be applied for a high discrimination and typing of pathogenic bacteria?

In this article, the method of analysis of GB-speckles (gene-based speckles) has been adapted to the problem of detecting the differences in a group of genes (usually 5-7), used in Multi Locus Sequence Typing (MLST). This method is based on s-LASCA imaging (spatial Laser Speckle Contrast Analysis) of virtual GB-speckle and on the technique of RGB coordinates for GB-speckles, processed by the s-LASCA method. A very high sensitivity and accuracy of the new method for detecting gene polymorphism as a great alternative to classical MLST has been demonstrated. The analysis of GB-speckles, obtained for the concatenated sequences of seven genes (gatA, gidA, enoA, fumC, hemN, hflX, oppA) of three different Chlamydia trachomatis strains (E/Bour, ST94; G/9301, ST95; G/11222, ST94) has been applied as the model. The high efficiency of usage of s-LASCA-imaging of GB-speckles has been shown. The data obtained represent a significant progress in digital biology as a whole and improvements in the bio-digitalization of bacterial DNA.

Lymphogranuloma venereum genovariants in men having sex with men in Italy.

OBJECTIVES: Lymphogranuloma venereum (LGV) is an STI caused by Chlamydia trachomatis serovars L1-L3. In Europe, the current epidemic is caused mainly by L2b genovariant, although increasing cases associated with other L2 variants have been reported. Here, we assessed the distribution of rectal LGV genovariants among men having sex with men (MSM) in Italy. METHODS: From 2016 to 2020, all the anorectal swabs collected from MSM attending the STI Clinic of St. Orsola-Malpighi Hospital in Bologna and positive for C. trachomatis were stored. LGV infection was confirmed by a pmpH PCR, and, subsequently, a fragment of the ompA gene was amplified and sequenced. Sequences were aligned to reference strains representing different LGV variants. RESULTS: LGV cases accounted for one-third of all chlamydial rectal infections with a total prevalence of 4.1% (76/1852). Total number of LGV cases per year remained constant. LGV was mainly found in symptomatic patients (>65%), older than 30 years, with a high burden of other STIs (63.7% HIV-positive, 35.5% with concurrent rectal gonorrhoea, 19.7% with early syphilis). A decreasing trend in HIV-LGV co-infection was noticed over time. Three main LGV genovariants were detected (L2f, 46.1%; L2b, 23.0%; L2-L2b/D-Da, 16.9%), together with other known L2b variants (mainly L2bV2 and L2bV4). Two novel L2b ompA variants with non-synonymous single-nucleotide polymorphisms were found. Over time, the percentage of L2f cases dropped gradually, with a significant increase in L2-L2b/D-Da cases (p=0.04). CONCLUSIONS: In our area, LGV is endemic among MSM with different circulating genovariants. Active surveillance and genotyping programmes are needed to reduce re-establishing of LGV infection.

Characterization of Chlamydia trachomatis ompA Genotypes Among Sexually Transmitted Disease Patients in Korea.

BACKGROUND: Chlamydia trachomatis is the leading cause of bacterial sexually transmitted diseases (STDs) world-wide. The aim of this study was to characterize the C. trachomatis genetic profiles of clinical isolates in the Korean population by sequence analysis of the ompA gene. METHODS: Endocervical specimens from patients who were confirmed as C. trachomatis infection by real-time PCR were used for ompA sequencing. The individual sequences (about 890 nucleotides) were determined by comparison with those from known C. trachomatis strains using the BLAST search tool. Sequence variations were analyzed by comparing them with sequences from prototype strains. RESULTS: Sequence analysis using BLAST similarity search of the ompA gene from the 106 clinical isolates revealed that the most prevalent genotype corresponded to E (n = 28, 26.2%), followed by F (n = 20, 18.9%), D (n = 16, 15.1%), J (n = 16, 15.1%), G (n = 9, 8.5%), H (n = 8, 7.5%), K (n = 5, 4.7%), B (n = 2, 1.9%), and I (n = 2, 1.9%). Detailed sequence analysis based on comparison with each prototype revealed that there are sequence differences in 44 specimens (41.5%). CONCLUSIONS: This study is the update on the distribution of C. trachomatis ompA genotypes in Korea in more than a decade. The genotypes have become more diversified, with genotypes K, B, and I being newly detected. C. trachomatis genotyping is crucial for regional and global epidemiological studies, as well as for vaccine development.

Chlamydia trachomatis isolated from cervicovaginal samples in Sapporo, Japan, reveals the circulation of genetically diverse strains.

BACKGROUND: This study was conducted to understand the molecular epidemiology of circulating Chlamydia trachomatis (Ct) strains in Sapporo, Japan. METHODS: A total of 713 endocervical samples collected from April 2016 to March 2019 were screened for Ct. The obtained Ct positive samples were analyzed by ompA genotyping and multilocus sequence analysis (MLSA). RESULTS: Eighty-three (11.6%) samples were positive for Ct plasmid DNA. Sequence analysis of the ompA gene from the 61 positive cases revealed eight genotypes: F (40.9%), E (19.6%), D (14.7%), G (9.8%), H (6.5%), I (3.2%), K (3.2%), and J (1.6%). The globally dominant genotype E and F strains were highly conserved with 13 ompA genetic variants being detected, whereas genotype D strains were the most diverse. Genetic characterization of D strains revealed that D1 genetic variants may be potentially specific to Sapporo. MLSA revealed 13 unique sequence types (STs) including four novel STs from 53 positive samples, with the globally dominant STs 39 and 19 being predominant. STs 39, 34, and 21 were exclusively associated with genotypes E and F indicating their global dominance. Novel ST70 and ST30 were specifically associated with genotype D. CONCLUSION: Our study has revealed the circulation of genetically diverse Ct strains in the women population of Sapporo, Japan. We suggest identifying a transmission network of those successful strains and implementing public health prevention strategies to control the spread of Ct in Sapporo.

Extragenital Mycoplasma genitalium infections among men who have sex with men.

OBJECTIVES: There are limited data on the prevalence of Mycoplasma genitalium (Mgen) coinfection with rectal chlamydia (Chlamydia trachomatis (CT)) and rectal gonorrhoea (Neisseria gonorrhoeae (NG)) infections and few studies examining the prevalence of pharyngeal Mgen in men who have sex with men (MSM). Using transcription-mediated amplification assay, this study aimed to determine the proportion of rectal CT and rectal NG infections in MSM who are coinfected with rectal Mgen, and the proportion of MSM with Mgen detected in the pharynx in order to inform clinical practice. METHODS: This was a cross-sectional study conducted at Melbourne Sexual Health Centre in Australia. Consecutively collected rectal swabs from MSM that tested positive for CT (n=212) or NG (n=212), and consecutively collected pharyngeal samples (n=480) from MSM were tested for Mgen using the Aptima Mycoplasma genitalium Assay (Hologic, San Diego). Samples were linked to demographic data and symptom status. RESULTS: Rectal Mgen was codetected in 27 of 212 rectal CT (13%, 95% CI 9 to 18) and in 29 of 212 rectal NG (14%, 95% CI 9 to 19) samples, with no difference in the proportion positive for Mgen. MSM with rectal CT/Mgen coinfection had more sexual partners than those with rectal CT monoinfection (mean 6 vs 11, p=0.06). MSM with rectal NG/Mgen coinfection were more likely to be HIV-positive than those with rectal NG monoinfection (OR=2.96, 95% CI 1.21 to 7.26, p=0.023). MSM with rectal CT/Mgen coinfection were more likely to be using pre-exposure prophylaxis than MSM with rectal NG/Mgen coinfection (OR 0.25, 95% CI 0.10 to 0.65, p=0.002). Pharyngeal Mgen was uncommon and detected in 8 of 464 samples (2%, 95% CI 1% to 3%). Pharyngeal Mgen was associated with having a rectal STI (OR=10.61, 95% CI 2.30 to 48.87, p=0.002), and there was a borderline association with being HIV-positive (p=0.079). CONCLUSION: These data indicate one in seven MSM treated for rectal CT or rectal NG will have undiagnosed Mgen that is potentially exposed to azithromycin during treatment of these STIs. Rectal Mgen coinfection was associated with specific risk factors which may inform testing practices. Pharyngeal Mgen was uncommon.

Chlamydia trachomatis serovars in urogenital and ocular samples collected 2014-2017 from Austrian patients.

Infection of humans with Chlamydia trachomatis, a bacterial pathogen with a unique intracellular replication cycle, may cause a variety of clinical manifestations. These are linked to various serovars of the pathogen; trachoma to serovars A-C, oculogenital infections to serovars D-K, and lymphogranuloma venereum to serovars L1-L3. Nineteen serovars are known as human pathogens. The aim of the study was to determine the serovars of 401 C. trachomatis DNA positive extracts from original clinical specimens of patients in Austria including cervical and urethral swabs, urine, genital secretions and conjunctival swabs - collected from 2014 to 2017. Sequence analysis of the omp1 gene, encoding major outer-membrane protein was performed on each sample. In 50.1% of samples serovar E was identified and serovars F, D/Da and G/Ga were found in 16.2%, 9.7% and 9.0%, respectively. Remaining serovars were J (6.0%), K (4.7%), H (2.7%), B/Ba (1.0%), and I/Ia (0.5%). In 19 patients follow up samples could be tested. The majority of C. trachomatis serovars were associated with urogenital tract infections (D-K), however, one of them - serovar B/Ba - is linked to both, ocular and genital tract infection.

Bacterial Load of Chlamydia trachomatis in the Posterior Oropharynx, Tonsillar Fossae, and Saliva among Men Who Have Sex with Men with Untreated Oropharyngeal Chlamydia.

The aim of this study was to determine whether Chlamydia trachomatis could be detected in saliva and if infection is specific to an anatomical site in the oropharynx. Men who have sex with men (MSM) who were diagnosed with oropharyngeal chlamydia at the Melbourne Sexual Health Centre in 2017-2018 were invited to participate upon returning for treatment. Swabs at the tonsillar fossae and posterior oropharynx and a saliva sample were collected. Throat samples were tested for C. trachomatis by the Aptima Combo 2 assay. The bacterial loads of C. trachomatis in all samples were assessed by quantitative PCR (qPCR) detecting the ompA gene. We calculated the positivity and bacterial load of C. trachomatis for all samples. Forty-two MSM were included. The median age was 28 years (interquartile range [IQR], 24 to 33 years). Thirty-two participants (76.2%; 95% confidence interval [CI], 60.5% to 87.9%) had C. trachomatis detected by qPCR at both the tonsillar fossae and the posterior oropharynx, followed by 9.5% (n = 4; 95% CI, 2.7% to 22.6%) positive at the posterior oropharynx only and 4.8% (n = 2; 95% CI, 0.58% to 16.2%) positive at the tonsillar fossae only. Twenty-nine MSM had C. trachomatis detected in saliva (69.0%; 95% CI, 52.9% to 82.3%). The median C. trachomatis load in saliva was 446 copies/ml (IQR, 204 to 1,390 copies/ml), that in the tonsillar fossae was 893 copies/swab (IQR, 390 to 13,224 copies/ml), and that in the posterior oropharynx was 1,204 copies/swab (IQR, 330 to 16,211). There was no significant difference in C. trachomatis load between the tonsillar fossae and the posterior oropharynx (P = 0.119). Among MSM with oropharyngeal chlamydia, nearly three-quarters had chlamydia DNA detected in saliva, although the viability and implications for transmission are unknown.

Whole-genome sequencing of ocular Chlamydia trachomatis isolates from Gadarif State, Sudan.

BACKGROUND: Trachoma, caused by ocular Chlamydia trachomatis, is the leading infectious cause of blindness worldwide. Sudan first reported trachoma in the 1930s and has since been consistently endemic. Ocular C. trachomatis previously isolated from trachoma patients in Sudan in 1963 was antigenically identical to an isolate from Saudi Arabia (A/SA1). No contemporary ocular C. trachomatis whole genome sequences have been reported from Sudan. METHODS: This study sequenced twenty ocular C. trachomatis isolates to improve understanding of pathogen diversity in North-East Africa and examine for genomic variation specific to Sudan, possibly related to the persistence of trachoma in surveyed communities. High quality, whole genome sequences were obtained from 12/20 isolates. RESULTS: All isolates were serovar A and had tarP and trpA sequences typical of classical, ocular C. trachomatis isolates. The Sudanese isolates formed a closely related subclade within the T2-trachoma clade of C. trachomatis phylogeny distinct from geographically disparate ocular isolates, with little intra-population diversity. We found 333 SNPs that were conserved in Sudanese ocular isolates but rare compared to other ocular C. trachomatis populations, which were focused in two genomic loci (CTA0172-CTA0173 and CTA0482). CONCLUSIONS: Limited intra-population diversity and geographical clustering of ocular C. trachomatis suggests minimal transmission between and slow diversification within trachoma-endemic communities. However, diversity may have been higher pre-treatment in these communities. Over-representation of Sudan-specific SNPs in three genes suggests they may have an impact on C. trachomatis growth and transmission in this population.

Rectal Microbiota Associated With Chlamydia trachomatis and Neisseria gonorrhoeae Infections in Men Having Sex With Other Men.

Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) represent the most common agents of sexually transmitted rectal infections among men having sex with other men (MSM). In this study, we assessed the bacterial composition of the rectal microbiota associated with CT and/or NG infections in a cohort of men reporting unsafe rectal intercourse. A total of 125 rectal swabs were collected and four groups were compared: non-infected subjects (n = 53), patients with CT (n = 37), or NG rectal infection (n = 17) and patients with contemporary positivity for CT/NG (n = 18). CT and NG infections were detected by a real-time commercial test and the rectal microbiota composition was analyzed from rectal swabs through sequencing of the hypervariable V3-V4 regions of the 16S rRNA gene. The rectal microbiota of all subgroups was dominated by Prevotellaceae, Enterobacteriaceae, and Ruminococcaceae families. Irrespective of the analyzed subgroup, we found that the rectal environment of all the enrolled MSM was rich in Prevotella and Escherichia genera. Moreover, a shift in the bacterial composition between patients with sexually transmitted rectal infections and controls was noticed: infected patients were characterized by a depletion of Escherichia species, associated with an increase of anaerobic genera, including Peptoniphilus, Peptostreptococcus, and Parvimonas. Overall, the presence of rectal symptoms did not significantly modify the rectal microbiota profiles among the four groups of analyzed patients. We confirmed that HIV-positive patients are characterized by a lower bacterial richness than HIV-negative subjects. However, we found that the presence of HIV has a different impact on bacterial rectal communities compared to CT and NG infections, modifying the relative abundance of several genera, including Gardnerella, Lactobacillus, Corynebacterium, and Sutterella. Information about the rectal microbiota composition in CT and NG infections could shed light on the pathogenesis of these conditions and could contribute to the onset of new strategies for their control.

Impact of a single round of mass drug administration with azithromycin on active trachoma and ocular Chlamydia trachomatis prevalence and circulating strains in The Gambia and Senegal.

BACKGROUND: Mass drug administration (MDA) with azithromycin is a cornerstone of the trachoma elimination strategy. Although the global prevalence of active trachoma has declined considerably, prevalence persists or even increases in some communities and districts. To increase understanding of MDA impact, we investigated the prevalence of active trachoma and ocular C. trachomatis prevalence, organism load, and circulating strains at baseline and one-year post-MDA in The Gambia and Senegal. METHODS: Pre- and one-year post-MDA, children aged 0-9 years were examined for clinical signs of trachoma in six Gambian and 12 Senegalese villages. Ocular swabs from each child's right conjunctiva were tested for evidence of ocular C. trachomatis infection and organism load (ompA copy number), and ompA and multi-locus sequence typing (MLST) was performed. RESULTS: A total of 1171 children were examined at baseline and follow-up in The Gambia. Active trachoma prevalence decreased from 23.9% to 17.7%, whereas ocular C. trachomatis prevalence increased from 3.0% to 3.8%. In Senegal, 1613 and 1771 children were examined at baseline and follow-up, respectively. Active trachoma prevalence decreased from 14.9% to 8.0%, whereas ocular C. trachomatis prevalence increased from 1.8% to 3.6%. Higher organism load was associated with having active trachoma and severe inflammation. Sequence typing demonstrated that all Senegalese samples were genovar A, whereas Gambian samples were a mix of genovars A and B. MLST provided evidence of clustering at village and household levels and demonstrated differences of strain variant frequencies in Senegal, indicative of an "outbreak". MLST, including partial ompA typing, provided greater discriminatory power than complete ompA typing. CONCLUSIONS: We found that one round of MDA led to an overall decline in active trachoma prevalence but no impact on ocular C. trachomatis infection, with heterogeneity observed between villages studied. This could not be explained by MDA coverage or number of different circulating strains pre- and post-MDA. The poor correlation between active trachoma and infection prevalence supports the need for further work on alternative indicators to clinical signs for diagnosing ocular C. trachomatis infection. MLST typing has potential molecular epidemiology utility, including better understanding of transmission dynamics, although relationship to whole-genome sequence variability requires further exploration.

Clinical Persistence of Chlamydia trachomatis Sexually Transmitted Strains Involves Novel Mutations in the Functional αßßα Tetramer of the Tryptophan Synthase Operon.

Clinical persistence of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) is a major public health concern. In vitro persistence is known to develop through interferon gamma (IFN-γ) induction of indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, an essential amino acid for Ct replication. The organism can recover from persistence by synthesizing tryptophan from indole, a substrate for the enzyme tryptophan synthase. The majority of Ct strains, except for reference strain B/TW-5/OT, contain an operon comprised of α and ß subunits that encode TrpA and TrpB, respectively, and form a functional αßßα tetramer. However, trpA mutations in ocular Ct strains, which are responsible for the blinding eye disease known as trachoma, abrogate tryptophan synthesis from indole. We examined serial urogenital samples from a woman who had recurrent Ct infections over 4 years despite antibiotic treatment. The Ct isolates from each infection episode were genome sequenced and analyzed for phenotypic, structural, and functional characteristics. All isolates contained identical mutations in trpA and developed aberrant bodies within intracellular inclusions, visualized by transmission electron microscopy, even when supplemented with indole following IFN-γ treatment. Each isolate displayed an altered αßßα structure, could not synthesize tryptophan from indole, and had significantly lower trpBA expression but higher intracellular tryptophan levels compared with those of reference Ct strain F/IC-Cal3. Our data indicate that emergent mutations in the tryptophan operon, which were previously thought to be restricted only to ocular Ct strains, likely resulted in in vivo persistence in the described patient and represents a novel host-pathogen adaptive strategy for survival.IMPORTANCEChlamydia trachomatis (Ct) is the most common sexually transmitted bacterium with more than 131 million cases occurring annually worldwide. Ct infections are often asymptomatic, persisting for many years despite treatment. In vitro recovery from persistence occurs when indole is utilized by the organism's tryptophan synthase to synthesize tryptophan, an essential amino acid for replication. Ocular but not urogenital Ct strains contain mutations in the synthase that abrogate tryptophan synthesis. Here, we discovered that the genomes of serial isolates from a woman with recurrent, treated Ct STIs over many years were identical with a novel synthase mutation. This likely allowed long-term in vivo persistence where active infection resumed only when tryptophan became available. Our findings indicate an emerging adaptive host-pathogen evolutionary strategy for survival in the urogenital tract that will prompt the field to further explore chlamydial persistence, evaluate the genetics of mutant Ct strains and fitness within the host, and their implications for disease pathogenesis.

Factors associated with anorectal Chlamydia trachomatis or Neisseria gonorrhoeae test positivity in women: a systematic review and meta-analysis.

OBJECTIVES: There has been considerable discussion about anorectal Chlamydia trachomatis (CT) in women, with some calling for anorectal CT screening, but little about anorectal Neisseria gonorrhoeae (NG). Given that urogenital NG is more strongly associated with pelvic inflammatory disease, this is an evidence gap. This systematic review and meta-analysis investigates the associations between anorectal CT in women and CT positivity at other sites (urogenital/oropharyngeal) and with anal intercourse, and compares these with anorectal NG within the same study populations. METHODS: Electronic databases were searched for English-language studies published to October 2018 using the following terms: ("Chlamydia" OR "Chlamydia trachomatis") AND (("anal" OR "rect*" OR "anorect*") OR ("extra?genital" OR "multi?site")). Studies were included if anorectal NG data were available. Random-effects meta-analyses calculated pooled estimates; heterogeneity was investigated using meta-regression. RESULTS: 25 studies were eligible. Anorectal CT positivity ranged from 0% to 17.5%, with a summary estimate of 8.0% (95% CI 7.0 to 9.1; I2=88.5%). Anorectal NG positivity ranged from 0% to 17.0%, with a summary estimate of 2.1% (95% CI 1.6 to 2.8; I2=92.7%). The association between urogenital and anorectal positivity was stronger for NG than CT (summary prevalence ratio (PR)=89.3 (95% CI 53.1 to 150.3; I2=80.1%), PR=32.2 (95% CI 25.6 to 40.7; I2=70.3%), respectively), and between oropharyngeal and anorectal positivity it was stronger for NG than CT (PR=34.8 (95% CI 10.2 to 118.2; I2=89.9%), PR=8.8 (95% CI 6.8 to 11.5; I2=58.1%), respectively). Anal intercourse was associated with anorectal NG (PR=4.3; 95% CI 2.2 to 8.6; I2=0.0%) but not with anorectal CT (PR=1.0; 95% CI 0.7 to 1.4; I2=0.0%). CONCLUSIONS: Anorectal CT is more common than anorectal NG, but anorectal NG is more strongly associated with anal intercourse, urogenital and oropharyngeal NG, suggesting that ongoing discussion about anorectal CT should also include NG. Longitudinal data are required to further understanding of the aetiology of anorectal STIs and assess whether anorectal screening is needed in women. TRIAL REGISTRATION NUMBER: CRD42df017080188.

Prevalence of genital and oropharyngeal chlamydia and gonorrhoea among female sex workers in Melbourne, Australia, 2015-2017: need for oropharyngeal testing.

OBJECTIVE: The Victorian legislation requires sex workers to have quarterly screening for genital chlamydia and gonorrhoea, but screening for oropharyngeal infection is not mandatory in Victoria, Australia. In 2017, oropharyngeal screening for gonorrhoea and chlamydia was added as part of the routine quarterly screening for sex workers attending the Melbourne Sexual Health Centre (MSHC). The aim of this study was to examine the prevalence of oropharyngeal gonorrhoea and chlamydia among female sex workers (FSW). METHODS: We included females who (1) self-identified as sex workers or were attended MSHC for a sex work certificate and (2) had tested for any STI or HIV, between March 2015 and December 2017. The prevalence of HIV, syphilis, chlamydia and gonorrhoea was calculated. RESULTS: There were 8538 FSW consultations among 2780 individuals during the study period. There was a twofold increase in genital gonorrhoea (from 0.5% (95% CI 0.3% to 0.9%) to 1.1% (95% CI 0.8% to 1.5%); ptrend=0.047) and a 1.5-fold increase in genital chlamydia (from 2.2% (95% CI 1.6% to 2.8%) to 3.2% (95% CI 2.6% to 3.8%); ptrend=0.031) during the period. Overall, the prevalence of HIV (0.2% (95% CI 0.1% to 0.3%)) and syphilis (0.1% (95% CI 0.0% to 0.2%)) remained low and did not change over time. In 2017, the prevalence of oropharyngeal gonorrhoea was 2.0% (95% CI 1.6% to 2.6%) and oropharyngeal chlamydia was 2.1% (95% CI 1.6% to 2.7%). Among FSW who were tested positive for gonorrhoea and chlamydia, 55% (n=41) and 34% (n=45) only tested positive in the oropharynx but not genital for gonorrhoea and chlamydia, respectively. CONCLUSION: The prevalence of oropharyngeal gonorrhoea and chlamydia is similar to the prevalence at genital sites and is often independent of genital infection. It is important to test the oropharynx and genital site for chlamydia and gonorrhoea among FSW.

Rectal Lymphogranuloma Venereum, Buenos Aires, Argentina.

Among 34 men with proctitis in Buenos Aires, Argentina, 16 (47%) had Chlamydia trachomatis infection, 11 (68.8%) of which were biovar lymphogranuloma venereum. The outbreak was probably local, as in Europe. In Argentina, lymphogranuloma venereum should be a suspected cause of proctitis in HIV-infected men who have had unprotected anal sex with men.

Identification of a new variant of Chlamydia trachomatis in Mexico.

INTRODUCTION: Chlamydia trachomatis is one of the main etiological agents of sexually transmitted infections worldwide. In 2006, a Swedish variant of C. trachomatis (Swedish-nvCT), which has a deletion of 377bp in the plasmid, was reported. In Latin America, Swedish-nvCT infections have not been reported. We investigated the presence of Swedish-nvCT in women with infertility in Mexico. METHODS: Swedish-nvCT was searched in 69C. trachomatis positive samples from 2339 endocervical specimens. We designed PCR primers to identify the deletion in the plasmid in the ORF1, and the presence of a repeated 44bp in the ORF3. The sample with the deletion was genotyped with the genes of the major outer membrane protein A (ompA) and the polymorphic membrane protein (pmpH). RESULTS: The deletion was detected in one of the 69 samples positive C. trachomatis of 2339 endocervical exudates. The nucleotide sequence analysis of the ompA shows a high degree of similarity with the Swedish nvCT (98%), however the variant found belongs to serovar D. The nucleotide sequence of the pmpH gene associates to the variant found in the genitourinary pathotype of the Swedish-nvCT but in different clusters. CONCLUSIONS: Our results revealed the presence of a new variant of C. trachomatis in Mexican patients. This variant found in Mexico belongs to serovar D based on the in silico analysis of the ompA and pmpH genes and differs to the Swedish-nvCT (serovars E). For these variants of C. trachomatis that have been found it is necessary to carry out a more detailed analysis, although the role of this mutation has not been demonstrated in the pathogenesis.

Chlamydia trachomatis Serovars Drive Differential Production of Proinflammatory Cytokines and Chemokines Depending on the Type of Cell Infected.

Chlamydia trachomatis serovars A-C infect conjunctival epithelial cells and untreated infection can lead to blindness. D-K infect genital tract epithelial cells resulting in pelvic inflammatory disease, ectopic pregnancy, and sterility while L1-L3 infect epithelial cells and macrophages, causing an invasive infection. Despite some strains of Chlamydia sharing high nucleotide sequence similarity, the bacterial and host factors that govern tissue and cellular tropism remain largely unknown. Following introduction of C. trachomatis via intercourse, epithelial cells of the vagina, foreskin, and ectocervix are exposed to large numbers of the pathogen, yet their response to infection and the dynamics of chlamydial growth in these cells has not been well-characterized compared to growth in more permissive cell types that harbor C. trachomatis. We compared intracellular replication and inclusion development of representative C. trachomatis serovars in immortalized human conjunctival epithelial, urogenital epithelial, PMA stimulated THP-1 (macrophages), and HeLa cells. We demonstrate that urogenital epithelial cells of the vagina, ectocervix, and foreskin restrict replication of serovar A while promoting robust replication and inclusion development of serovar D and L2. Macrophages restrict serovars D and A while L2 proliferates in these cells. Furthermore, we show that GM-CSF, RANTES, GROα, IL-1α, IL-1ß, IP-10, IL-8, and IL-18 are produced in a cell-type and serovar-specific manner. Collectively we have established a series of human cell lines that represent some of the first cell types to encounter C. trachomatis following exposure and show that differential production of key cytokines early during infection could regulate serovar-host cell specificity.