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Introduction: Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by Chlamydia trachomatis genotypes L1-L3. A combination of techniques with high discriminatory capacity such as multilocus sequence typing (MLST) and the analysis of the ompA gene may be useful to determine the greater penetration of certain strains in transmission networks and their relationship with certain tropisms., Aim: The aim of this study was to investigate the molecular epidemiology of LGV isolates from different regions of Spain., Methods: Genetic characterisation of LGV isolates detected in six hospitals from Spain between 2018 and 2019 was performed. MLST (five variable regions: hctB , CT058, CT144, CT172 and pbpB ) and ompA sequence determination were used to study the LGV strains., Results: Most of the 161 LGV isolates (93.8%) were detected in men who have sex with men (MSM). At least 43.5% of the patients presented with HIV coinfection and 53.4% were symptomatic, with proctitis being the most prevalent symptom (73.3%). Most isolates were detected in Barcelona (n=129).The distribution of ompA genovariants was as follows: 56.1% belonged to L2, 24.3% to L2b, 5.4% to L2bV1, 4.7% to L2bV4, 4.1% to L1, 2.7% to L2b/D-Da, 2.0% to L2bV2 and 0.7% to L2bV7. MLST was successfully performed in 81 samples and 9 different sequence types (STs) were detected. The ompA and MLST combination obtained 17 different genetic profiles, with L2-ST53 and L2-ST58 being the most prevalent (29.5% and 14.1%, respectively). L1 genotype strains belonged to ST23 (n=3) and ST2 (n=3)., Conclusion: LGV infections were mainly found in MSM living with HIV and with proctitis. The joint analysis of ompA and MLST genetic characterisation techniques showed a high discriminatory capacity. Our findings suggest a cocirculation of L2 and L2b ompA genotypes, and with the inclusion of MLST characterisation, the most prevalent profiles were ompA genotype L2-MLST ST53 and L2-MLST ST58., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: The objective of this study lies in identifying the dominant genotype of C. trachomatis isolated in Republic of Korea (ROK) between 2017 and 2018., Methods: A total of 504 clinical cervicovaginal swabs from patients were collected and inoculated on McCoy cell monolayers to isolate Chlamydial agents. C. trachomatis isolates were analyzed by sequencing of its ompA gene., Results: A total of 54 C. trachomatis isolates were obtained. The constructed phylogenetic tree revealed the genotypes of isolates are D/Ep6 (48, 88.89%), D/Ep6-like (5, 9.26%), and Ja (1, 1.85%). The C. trachomatis D/Ep6-like have one single nucleotide polymorphism (SNP) compared to C. trachomatis D/Ep6, leading to E870K amino acid change., Conclusions: Phylogenetic analysis demonstrated globally rare C. trachomatis D/Ep6 was dominant in the ROK from 2017 to 2018. Findings in this study act as a keystone, bridging past and present molecular epidemiology of C. trachomatis in context of ROK.

Background: Chlamydia trachomatis is the causative agent of the most prevalent bacterial sexually transmitted infections globally. Whole genome sequencing is essential for molecular Chlamydia surveillance; however, its application is hampered by the pathogen's low abundance in clinical specimens and the expensive labor-intensive nature of existing enrichment methodologies for Chlamydia., Methods: We developed a targeted whole genome amplification tool termed SWITCH by integrating phi29 DNA polymerase-mediated amplification with meticulously designed primer sets to enrich the C trachomatis genome, followed by whole genome sequencing. This method underwent evaluation through testing synthetic and clinical specimens., Results: SWITCH demonstrated robust ability to achieve up to 98.3% genomic coverage of C trachomatis from as few as 26.4 genomic copies present in synthetic specimens, and it exhibited excellent performance across diverse C trachomatis serovars. Utilizing SWITCH, we directly generated 21 Chlamydia genomes from 26 clinical samples, enabling us to gain insights into the genetic relationships and phylogeny of current Chlamydia strains circulating in the country. Remarkably, this study marked the first instance of generating Chinese Chlamydia genomes directly from clinical samples., Conclusions: SWITCH represents a practical cost-efficient approach to enrich the Chlamydia genome directly from clinical specimens, offering an efficient avenue for molecular surveillance of Chlamydia., Competing Interests: Potential conflicts of interest. All authors: No reported conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Several Chlamydia trachomatis lineages identified through outer membrane protein A genotyping or multilocus sequence typing have been circulating worldwide among men who have sex with men. In a study in Tokyo, Japan, we demonstrate that such lineages commonly belong to a specific polymorphic membrane protein E clade across genotypes.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Harte et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of competing interest The authors report no conflicts of interest relevant to this article., (Published by Elsevier Inc.)

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

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 β (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 (ΔΔ G ) 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 β-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 ( trp R) 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. IMPORTANCE Chlamydia 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 trp A 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., (Copyright © 2021 Bommana et al.)

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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., Competing Interests: Competing interests: The Melbourne Sexual Health Centre receives funding from SpeeDx (Australia) for research projects on Mycoplasma genitalium; however, no funding from SpeeDx was received or used to support this project., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

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.

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., (Copyright © 2019 American Society for Microbiology.)

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.

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-1β, 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., (Copyright © 2019 Faris, Andersen, McCullough, Gourronc, Klingelhutz and Weber.)

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.

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.

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., (Copyright © 2019 Ceccarani, Marangoni, Severgnini, Camboni, Laghi, Gaspari, D'Antuono, Foschi, Re and Consolandi.)

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%); p trend =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%); p trend =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., Competing Interests: Competing interests: None declared, (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

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; I 2 =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; I 2 =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; I 2 =80.1%), PR=32.2 (95% CI 25.6 to 40.7; I 2 =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; I 2 =89.9%), PR=8.8 (95% CI 6.8 to 11.5; I 2 =58.1%), respectively). Anal intercourse was associated with anorectal NG (PR=4.3; 95% CI 2.2 to 8.6; I 2 =0.0%) but not with anorectal CT (PR=1.0; 95% CI 0.7 to 1.4; I 2 =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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

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 β subunits that encode TrpA and TrpB, respectively, and form a functional αββα 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 αββα 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. IMPORTANCE Chlamydia 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., (Copyright © 2019 Somboonna et al.)

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., (Copyright © 2018 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Background: Chlamydia trachomatis may coinfect with human papillomavirus (HPV) and complicate the cervical pathogenesis. This study aimed to evaluate the prevalence, risk factors, and clinical outcomes of HPV/C. trachomatis coinfection in women from Inner Mongolia, China., Methods: We performed a polymerase chain reaction (PCR)-based HPV/C. trachomatis screening and cervical samples were analyzed by thinprep cytologic test. Statistical analysis was used to assess the association between demographic factors and coinfection., Results: Among the 2345 women recruited, the prevalences of HPV, C. trachomatis, and HPV/C. trachomatis coinfection were 36.0%, 14.3%, and 4.8%, respectively. The rate of multiple HPV genotypes was higher in coinfected women. HPV66 was the most frequently identified genotype in coinfected participants. The HPV DNA load was significantly higher in HPV monoinfected cases. In contrast, the DNA load of C. trachomatis was significantly higher in the coinfection group. Risk factors, including single women (odds ratio [OR] = 6.0, 95% confidence interval [CI], 4.044-8.782) and women with multiple sex partners (OR = 1.9, 95% CI, 1.324-2.824), were associated with coinfection. Importantly, coinfection was associated with increased risk for high-grade squamous intraepithelial lesions., Conclusions: HPV and C. trachomatis coinfection is an important risk factor for the progression of cervical lesions.

The transmission of the urogenital serovars of Chlamydia trachomatis can be significantly influenced by vaginal gels. Hydroxyethyl cellulose is a commonly used gelling agent that can be found in vaginal gels. Hydroxyethyl cellulose showed a concentration-dependent growth-enhancing effect on C. trachomatis serovars D and E, with a 26.1-fold maximal increase in vitro and a 2.57-fold increase in vivo ., (Copyright © 2018 American Society for Microbiology.)

Objective: The development of an accurate, sensitive, specific, rapid, reproducible, stable-at-room-temperature and cost-effective diagnostic kit, and a low-cost portable fluorescence detector to fulfil the requirements of diagnostic facilities in developing countries., Methods: We developed the 'Chlamy and Ness CT/NG kit' based on molecular beacons for the detection of Chlamydia trachomatis (CT) and Neisseriagonorrhoeae (NG). Multi-centric evaluation of the CT/NG kit was performed using the commercially available nucleic acid amplification test (NAAT)-based FTD Urethritis basic kit for comparison from December 2014 to November 2016. The stability of the kit reagents at 4 and 37 ˚C and the inter-day reproducibility of results were also analysed., Results: The sensitivity and specificity of the kit were found to be 95.83 and 100.00 % for the detection of C. trachomatis and 93.24 and 99.75 % for N. gonorrhoeae, respectively, when tested against the commercial kit. The positive predictive value (PPV) was 100.00 and 98.57 %, whereas the negative predictive value (NPV) was 99.54 and 98.79 % for C. trachomatis and N. gonorrhoeae, respectively. Analysis of the kappa statistics enhanced the 'inter-rater' κ=0.976 for Chlamydia and κ=0.943 for Neisseria., Conclusion: Our kit was found to be as sensitive and specific as commercially available kits. Its low cost and ease of use will make it suitable for the routine diagnosis of C. trachomatis and N. gonorrhoeae in the resource-limited settings of developing countries.

Objectives: Chlamydia trachomatis ompA genotypes A and B, primarily associated with trachoma, were unexpectedly detected in urogenital samples of patients in Spain, a trachoma-free country. In this study, we aimed to explain this finding using analysis of organotropism-related genes and a multilocus sequence typing (MLST) technique., Methods: C. trachomatis genotypes A or B were detected in 8/930 (0.9%) infection episodes between 2006 and 2012. In these strains, organotropism-related genes (polymorphic membrane protein gene H, tryptophan synthase gene A, CTA0934, and cytotoxin) were studied. Further, the strains were analysed by MLST, using a polymerase chain reaction that amplifies five highly variable genomic loci (hctB, CT058, CT144, CT172, and pbpB). Amplicons were sequenced and phylogenetic analysis was conducted., Results: Seven strains were detected in the eight infection episodes (in one patient, an identical strain being found in two episodes). Analysis of organotropism-related genes showed that these strains shared genetic features characteristic of genitotropic genotypes but not of trachoma strains. Three strains of genotype A showed a unique and new MLST-sequence type (ST551, allele profile 8-8-2-27-69). The four strains of genotype B belonged to ST138., Conclusions: C. trachomatis ompA genotypes A and B associated with trachoma, but detected sporadically in urogenital samples in trachoma-free countries, may be the result of recombination between strains adapted to trachoma and strains adapted to sexual transmission., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We present AAI-profiler, a web server for exploratory analysis and quality control in comparative genomics. AAI-profiler summarizes proteome-wide sequence search results to identify novel species, assess the need for taxonomic reclassification and detect multi-isolate and contaminated samples. AAI-profiler visualises results using a scatterplot that shows the Average Amino-acid Identity (AAI) from the query proteome to all similar species in the sequence database. Taxonomic groups are indicated by colour and marker styles, making outliers easy to spot. AAI-profiler uses SANSparallel to perform high-performance homology searches, making proteome-wide analysis possible. We demonstrate the efficacy of AAI-profiler in the discovery of a close relationship between two bacterial symbionts of an omnivorous pirate bug (Orius) and a thrip (Frankliniella occidentalis), an important pest in agriculture. The symbionts represent novel species within the genus Rosenbergiella so far described only in floral nectar. AAI-profiler is easy to use, the analysis presented only required two mouse clicks and was completed in a few minutes. AAI-profiler is available at http://ekhidna2.biocenter.helsinki.fi/AAI.

Background: This is the first report to characterize the prevalence and genovar distribution of genital chlamydial infections among random heterosexual patients in the multi-ethnic Saratov Region, located in Southeast Russia., Methods: Sixty-one clinical samples (cervical or urethral swabs) collected from a random cohort of 856 patients (7.1%) were C. trachomatis (CT) positive in commercial nucleic acid amplification tests (NAATs) and duplex TaqMan PCRs., Results: Sequence analysis of the VDII region of the ompA gene revealed seven genovars of C. trachomatis in PCR-positive patients. The overall genovars were distributed as E (41.9%), G (21.6%), F (13.5%), K (9.5%), D (6.8%), J (4.1%), and H (2.7%). CT-positive samples were from males (n = 12, 19.7%), females (n = 42, 68.8%), and anonymous (n = 7, 11.5%) patients, with an age range of 19 to 45 years (average 26.4), including 12 different ethnic groups representative of this region. Most patients were infected with a single genovar (82%), while 18% were co-infected with either two or three genovars. The 1156 bp-fragment of the ompA gene was sequenced in 46 samples to determine single nucleotide polymorphisms (SNP) among isolates. SNP-based subtyping and phylogenetic reconstruction revealed the presence of 13 variants of the ompA gene, such as E (E1, E2, E6), G (G1, G2, G3, G5), F1, K, D (D1, Da2), J1, and H2. Differing genovar distribution was identified among urban (E>G>F) and rural (E>K) populations, and in Slavic (E>G>D) and non-Slavic (E>G>K) ethnic groups. Multilocus sequence typing (MLST) determined five sequences types (STs), such as ST4 (56%, 95% confidence interval, CI, 70.0 to 41.3), ST6 (10%, 95% CI 21.8 to 3.3), ST9 (22%, 95% CI 35.9 to 11.5), ST10 (2%, 95% CI 10.7 to 0.05) and ST38 (10%, 95% CI 21.8 to 3.3). Thus, the most common STs were ST4 and ST9., Conclusion: C. trachomatis is a significant cause of morbidity among random heterosexual patients with genital chlamydial infections in the Saratov Region. Further studies should extend this investigation by describing trends in a larger population, both inside and outside of the Saratov Region to clarify some aspects for the actual application of C. trachomatis genotype analysis for disease control.

Two techniques based on ompA amplification of Chlamydia trachomatis were compared, being reverse hybridization (RHM) and ompA sequencing (OSA), to investigate the concordance between them and to study the epidemiological relevance of each method. In addition, phylogenetic analysis was performed on the ompA sequences. One hundred and seven C. trachomatis positive samples from Tunisian patients and female sex workers were analyzed using both the RHM and ompA sequencing. The overall genovar distribution obtained with both techniques was very similar. The RHM identified nine genovars, being B, D, E, F, G, H, I, J and K, where B, I, J, and K were only found in mixed infections versus 7 types for the OSA being D, E, F, G, H, I, and K. The agreement between both typing techniques was 87.8%. Both methods showed that genovar E was the most predominant type. In 24.3% of the analyzed samples, mixed infections were detected. In 96.1% of these, the genovar identified by OSA was also detected using the RHM. OmpA sequencing allowed determination of six genovar types that could not be typed using RHM. The analyses of ompA nucleotide variation in the 107 clinical specimens detected ompA genovar variants with distinct ompA mutational patterns for types D2, G1, G2, and H1. In conclusion, RHM and OSA showed a high agreement in C. trachomatis genotyping results with each having their specific benefits., (© 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Studies have rarely assessed the genotype distribution of Chlamydia trachomatis (CT) in urine among men attending sexually transmitted disease clinics (MSCs) in China. This study was aimed at investigating the prevalence and molecular epidemiology of CT infection by examining urine samples among MSCs from different geographic areas of Guangdong Province, China. A cross-sectional study was conducted among MSCs from 10 human immunodeficiency virus sentinel surveillance sites in Guangdong Province. CT DNA was extracted from male urine samples and analyzed using a Roche cobas 4800 CT/NG. The ompA genes were amplified by nested PCR and sequenced. The leukocyte esterase test was performed by routine urine analysis at local clinics. Of the 1,903 samples, 163 (8.6%, 95% confidence interval [CI] 3.8-16.3%) tested positive for CT. The highest prevalence (10.5%) of CT infection was observed among participants aged between 21 and 30 years. A total of 130 CT-positive samples (79.8%, 130/163) were successfully genotyped by nested PCR, resulting in 8 genotypes. The most prevalent genotypes were D, E, F, and J, with proportions of 20.8%, 20.0%, 17.7%, and 16.9%, respectively. There were no significant correlations between the geographical areas, leukocyte esterase test results and genotype distribution. Promotion of detection and molecular epidemiology research is needed for effective and comprehensive prevention and control programs.

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

Infection with Chlamydia trachomatis drives severe mucosal immunopathology; however, the immune responses that are required for mediating pathology vs. protection are not well understood. Here, we employed a mouse model to identify immune responses required for C. trachomatis -induced upper genital tract pathology and to determine whether these responses are also required for bacterial clearance. In mice as in humans, immunopathology was characterized by extravasation of leukocytes into the upper genital tract that occluded luminal spaces in the uterus and ovaries. Flow cytometry identified these cells as neutrophils at early time points and CD4 + and CD8 + T cells at later time points. To determine what draws these cells to C. trachomatis -infected tissue, we measured the expression of 700 inflammation-related genes in the upper genital tract and found an up-regulation of many chemokines, including a node of interaction between CXCL9/10/11 and their common receptor CXCR3. Either depleting neutrophils or reducing T-cell numbers by CXCR3 blockade was sufficient to significantly ameliorate immunopathology but had no effect on bacterial burden, demonstrating that these responses are necessary for mucosal pathology but dispensable for C. trachomatis clearance. Therapies that specifically target these host responses may therefore prove useful in ameliorating C. trachomatis -induced pathology without exacerbating infection or transmission., Competing Interests: The authors declare no conflict of interest.

Background: Chlamydia trachomatis (Ct) plasmid has been shown to encode genes essential for infection. We evaluated the population structure of Ct using whole-genome sequence data (WGS). In particular, the relationship between the Ct genome, plasmid and disease was investigated., Results: WGS data from 157 Ct isolates deposited in the Chlamydiales pubMLST database ( http://pubMLST.org/chlamydiales/ ) were annotated with 902 genes including the core and accessory genome. Plasmid associated genes were annotated and a plasmid MLST scheme was defined allowing plasmid sequence types to be determined. Plasmid allelic variation was investigated. Phylogenetic relationships were examined using the Genome Comparator tool available in pubMLST. Phylogenetic analyses identified four distinct Ct core genome clusters and six plasmid clusters, with a strong association between the chromosomal genotype and plasmid. This in turn was linked to ompA genovars and disease phenotype. Horizontal genetic transfer of plasmids was observed for three urogenital-associated isolates, which possessed plasmids more commonly found in isolates resulting from ocular infections. The pgp3 gene was identified as the most polymorphic plasmid gene and pgp4 was the most conserved., Conclusion: A strong association between chromosomal genome, plasmid type and disease was observed, consistent with previous studies. This suggests co-evolution of the Ct chromosome and their plasmids, but we confirmed that plasmid transfer can occur between isolates. These data provide a better understanding of the genetic diversity occurring across the Ct genome in association with the plasmid content.

Background: Since PGAP (pan-genome analysis pipeline) was published in 2012, it has been widely employed in bacterial genomics research. Though PGAP has integrated several modules for pan-genomics analysis, how to properly and effectively interpret and visualize the results data is still a challenge., Result: To well present bacterial genomic characteristics, a novel cross-platform software was developed, named PGAP-X. Four kinds of data analysis modules were developed and integrated: whole genome sequences alignment, orthologous genes clustering, pan-genome profile analysis, and genetic variants analysis. The results from these analyses can be directly visualized in PGAP-X. The modules for data visualization in PGAP-X include: comparison of genome structure, gene distribution by conservation, pan-genome profile curve and variation on genic and genomic region. Meanwhile, result data produced by other programs with similar function can be imported to be further analyzed and visualized in PGAP-X. To test the performance of PGAP-X, we comprehensively analyzed 14 Streptococcus pneumonia strains and 14 Chlamydia trachomatis. The results show that, S. pneumonia strains have higher diversity on genome structure and gene contents than C. trachomatis strains. In addition, S. pneumonia strains might have suffered many evolutionary events, such genomic rearrangements, frequent horizontal gene transfer, homologous recombination, and other evolutionary process., Conclusion: Briefly, PGAP-X directly presents the characteristics of bacterial genomic diversity with different visualization methods, which could help us to intuitively understand dynamics and evolution in bacterial genomes. The source code and the pre-complied executable programs are freely available from http://pgapx.ybzhao.com .

We are reporting the first case of lymphogranuloma venereum in women in East-Central Europe. A 22-year-old heterosexual woman attended our department of venereology. She complained about a burning sensation in the urethra and vaginal discharge. Many tests were performed, and lymphogranuloma venereum, syphilis, gonorrhea, chlamydial urethritis and cervicitis, genital herpes, genital warts, and hepatitis C were diagnosed. Lymphogranuloma venereum was originally endemic in tropical and subtropical areas, but since 2003, outbreaks of this infection have been reported in North America, Europe, and Australia in men who have sex with men (MSM) community. To date, all cases of lymphogranuloma venereum in the Czech Republic appeared in men, predominantly in HIV-positive MSM. There are not many evidences about lymphogranuloma venereum (LGV) in women in developed countries. This report underlines the need for awareness of lymphogranuloma venereum in women among gynecologists, venereologists, and other physicians not only in Western Europe, but across all European countries.

Chlamydia trachomatis (Ctr) is a bacterial pathogen that causes ocular, urogenital and lymph system infections in humans. It is highly abundant and among its serovars, E, F and D are most prevalent in sexually transmitted disease. However, the number of publicly available genome sequences of the serovars E and F, and thereby our knowledge about the molecular architecture of these serovars, is low. Here we sequenced the genomes of six E and F clinical isolates and one E lab strain, in order to study the genetic variance in these serovars. As observed before, the genomic variation inside the Ctr genomes is very low and the phylogenetic placement in comparison to publicly available genomes is as expected by ompA gene serotyping. However, we observed a large InDel carrying four to five open reading frames in one clinical E sample and in the E lab strain. We have also observed substantial variation on nucleotide and amino acid levels, especially in membrane proteins and secreted proteins. Furthermore, these two groups of proteins are also target for recombination events. One clinical F isolate was genetically heterogeneous and revealed the highest differences on nucleotide level in the pmpE gene., (© FEMS 2017.)

We detected Chlamydia trachomatis biovar L2 in vaginal swab specimens of 7 women with vaginal discharge in South Africa. Whole-genome sequencing directly from clinical specimens identified a closely related cluster of strains. The clinical role of this infection in the context of syndromic management should be clarified.

Objectives: Sexually transmitted infections are frequently related to outbreaks in high-risk populations due to the dense sexual networks. We wanted to determine the dissemination of a Chlamydia trachomatis variant characterized by the pmpH-recombinant gene between L and G genotypes, which was previously described in a high-risk population., Methods: A total of 449 samples were analysed in two periods ranging from 2009 to 2015 for detection of the pmpH-recombinant gene. For those samples yielding positive amplification, a sampling was selected for phylogenetic reconstructions based on sequencing of five chromosomal genes., Results: Globally this variant was found in 113 of the 449 samples (25%). During the first years (2009-13), this variant was found almost exclusively in rectal samples (30/112 samples) of men who have sex with men and in only one non-rectal sample (1/63). In 2014, this variant was also found in urethral and pharyngeal samples (1/24 and 1/7, respectively). However, in 2015, an epidemiological change was observed as the proportion of this variant had increased in rectal samples (20/51; 39%) and non-rectal samples, including cervical samples (51/142; 36.4%). The molecular characterization revealed the replacement of the ompA gene belonging to subtype G in samples recovered from 2009 to 2013 by the ompA gene belonging to subtype J after 2013., Conclusions: Our data would support the evidence that subtype J could be a 'subtype bridge' between different sexual networks, as subtype J has been found in men who have sex with men and heterosexual populations in similar proportions. This work reveals the necessity of implementing molecular surveillance in extra-rectal samples to help us understand the gaps in transmission., (Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The objective of the present study was to determine the frequency and age distribution of different Chlamydia trachomatis (CT) genotypes causing ophthalmia neonatorum (ON) in Hungary. Using CT specific PCR, we tested 76 conjunctival samples from symptomatic infants up to 3 months old in the National Centre for Epidemiology, Budapest between 2008 and 2016. CT tested positive in 30 of 76 conjunctival samples (39.5 %). The sequencing of the positive samples was successful in every case but one, and resulted in 48 % dominance for genotype E (14/29), followed by 24 % for genotype G (7/29), 10 % for J (3/29), 6.9 % for K and F (2/29), and 3.4 % for H (1/29). CT must still be regarded as a common pathogen causing ON in Hungary. Routine screening and treatment of pregnant women can be recommended to prevent these conditions. Chronic ON cases can be reduced by early diagnosis. Further research is needed to explain the dominance of genotypes E and G.

High-resolution screening methodologies which enable the differentiation of Chlamydia trachomatis at the strain level, directly from clinical samples, can provide the detailed information required for epidemiological questions such as the dynamics of treatment failure. In addition, they give a detailed snapshot of circulating C. trachomatis genetic variation, data which are currently lacking for the Australian population. In the context of two Australian clinical trials, we assessed the genetic diversity of C. trachomatis and compared these to strains circulating globally. We used high-resolution multilocus sequence typing (MLST) of five highly variable genetic regions of C. trachomatis to examine variation in Australia. Samples with established genovars were drawn from a pool of 880 C. trachomatis-positive samples from two clinical studies, whereby 76 sample pairs which remained C. trachomatis-positive for the same genovar after treatment underwent MLST analysis to distinguish between treatment failure and reinfection. MLST analysis revealed a total of 25 sequence types (STs), six new allele variants and seven new STs not described anywhere else in the world, when compared to those in the international C. trachomatis MLST database. Of the eight most common global STs, seven were found in Australia (four derived from men who have sex with men (MSM) and three from heterosexuals). Newly identified STs were predominantly found in samples from the MSM population. In conclusion, MLST provided a diverse C. trachomatis strain profile, with novel circulating STs, and could be used to identify local sexual networks to focus on interventions such as testing and partner notification to prevent reinfection.

Background: The biology of recurrent or long-term infections of humans by Chlamydia trachomatis is poorly understood. Because repeated or persistent infections are correlated with serious complications in humans, understanding these processes may improve clinical management and public health disease control., Methods: We conducted whole-genome sequence analysis on C. trachomatis isolates collected from a previously described patient set in which individuals were shown to be infected with a single serovar over a lengthy period., Results: Data from 5 of 7 patients showed compelling evidence for the ability of these patients to harbor the same strain for 3-5 years. Mutations in these strains were cumulative, very uncommon, and not linked to any single protein or pathway. Serovar J strains isolated from 1 patient 3 years apart did not accumulate a single base change across the genome. In contrast, the sequence results of 2 patients, each infected only with serovar Ia strains, revealed multiple same-serovar infections over 1-5 years., Conclusions: These data demonstrate examples of long-term persistence in patients in the face of repeated antibiotic therapy and show that pathogen mutational strategies are not important in persistence of this pathogen in patients., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Nucleic acid amplification tests (NAATs) are recommended by the CDC for detection of Chlamydia trachomatis (Ct) urogenital infections. Current commercial NAATs require technical expertise and sophisticated laboratory infrastructure, are time-consuming and expensive, and do not differentiate the lymphogranuloma venereum (LGV) strains that require a longer duration of treatment than non-LGV strains. The multiplexed microfluidic PCR-based assay presented in this work simultaneously interrogates 13 loci to detect Ct and identify LGV and non-LGV strain-types. Based on amplified fragment length polymorphisms, the assay differentiates LGV, ocular, urogenital, and proctocolitis clades, and also serovars L1, L2, and L3 within the LGV group. The assay was evaluated in a blinded fashion using 95 clinical swabs, with 76 previously reported as urogenital Ct-positive samples and typed by ompA genotyping and/or Multi-Locus Sequence Typing. Results of the 13-plex assay showed that 51 samples fell within urogenital clade 2 or 4, 24 samples showed both clade 2 and 4 signatures, indicating possible mixed infection, gene rearrangement, or inter-clade recombination, and one sample was a noninvasive trachoma biovar (either a clade 3 or 4). The remaining 19 blinded samples were correctly identified as LGV clade 1 (3), ocular clade 3 (4), or as negatives (12). To date, no NAAT assay can provide a point-of-care applicable turnaround time for Ct detection while identifying clinically significant Ct strain types to inform appropriate treatment. Coupled with rapid DNA processing of clinical swabs (approximately 60 minutes from swab-in to result-out), the assay has significant potential as a rapid POC diagnostic for Ct infections.

In eastern Europe, few countries have so far reported laboratory-confirmed cases of lymphogranuloma venereum (LGV). Here we describe 22 LGV cases in men who have sex with men (MSM) detected in Hungary from November 2012 to July 2016. Sequence analyses show that 16 of these 22 cases were affected by the L2c genovariant, with from 2012 to 2014, one LGV L2c case detected per year, followed by seven cases in 2015 and six up to July 2016. Of the 16 total L2c LGV cases, 10 had severe haemorrhagic proctitis. These findings are concerning as cases with this new genovariant among MSM have not been frequently reported in Europe to date. More research is needed to assess the spread of the L2c genovariant and its potential association with virulence and severe clinical manifestation., (This article is copyright of The Authors, 2017.)

Trachoma, the leading infectious cause of blindness worldwide, is an ancient human disease. Its existence in China can be traced back to as early as the twenty-seventh century BC. In modern China, the overall prevalence of trachoma has dramatically reduced, but trachoma is still endemic in many areas of the country. Here, we report that 26 (8%) of 322 students from two rural boarding schools of Qinghai province, west China, were identified as having ocular C. trachomatis infection; and 15 ocular C. trachomatis strains were isolated from these trachoma patients. Chlamydiae in 37 clinical samples were genotyped as type B based on ompA gene analyses. Three ompA variants with one or two in-between SNP differences in the second or fourth variable domain were found. C. trachomatis strains QH111L and QH111R were from the same patient's left and right conjunctival swabs, respectively, but their ompA genes have a non-synonymous base difference in the second variable domain. Moreover, this SNP only exists in this single sample, suggesting QH111L is a newly emerged ompA variant. Interestingly, chromosomal phylogeny analysis found QH111L clusters between a branch of two type B strains and a branch of both A and C strains, but is significantly divergent from both branches. Comparative chromosome analysis found that compared to sequences of reference B/TZ1A828/OT strain, 12 of 22 QH111L's chromosomal genes exhibiting more than nine SNPs have the best homology with reciprocal genes of UGT strains while 9 of 22 genes are closest to those of type C strains. Consistent with findings of UGT-type genetic features in the chromosome, the QH111L plasmid appears to be intermediate between UGT and classical ocular plasmids due to the existence of UGT-type SNPs in the QH111L plasmid. Moreover, the QH111L strain has a unique evolutionarily older cytotoxin region compared to cytotoxin regions of other C. trachomatis strains. The genome analyses suggest that the QH111L strain is derived from recombinations between UGT and classical ocular ancestors. This is the first study of culture and characterization of ocular C. trachomatis in Qinghai Tibetan areas.