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This study aimed to investigate what impact the COVID-19 pandemic and its associated restrictions had onRetrospective analysis of data collectedWhen the pandemic restrictions were installed in 2020, the number of reported chlamydia cases decreased. The decline was most pronounced in Norway 10.8% (2019:Pandemic restrictions had an impact on the number of reported chlamydia infections in all three countries, but only temporarily and did not seem to be correlated to the restriction levels. The number of reported gonorrhoea infections in Sweden and Norway significantly decreased but not in Denmark. Pandemic restrictions appear to have had a limited effect on the spread of chlamydia and gonorrhoea.

Saarentausta K, Ivarsson L, Jacobsson S, Herrmann B, Sundqvist M, Unemo M. Potential impact of the COVID-19 pandemic on the incidence, epidemiology and diagnostic testing of chlamydia and gonorrhoea in Sweden, 2020 The COVID-19 pandemic has challenged the societies and health care systems globally, and resulted in many social and physical distancing restrictions to limit the spread of SARS-CoV-2. These restrictions have also likely affected the frequency of intimate contacts and the spread of sexually transmitted infections (STIs). Compared to most other countries, Sweden especially in Spring-Autumn 2020 pursued mainly milder voluntary, i.e. not mandatory enforced by laws, recommended restrictions and the impacts of these on society and spread of STIs remain largely unknown. We describe the potential impact of the COVID-19 pandemic on the national and regional incidence, epidemiology and diagnostic testing of chlamydia and gonorrhoea in Sweden in 2020. Compared to 2019, we found a significant decrease in incidence of chlamydia (-4.5%) and gonorrhoea (-17.5%), and in diagnostic testing (-10.5% for chlamydia, -9.4% for gonorrhoea) in 2020. However, the decrease in chlamydia incidence, which has mainly been decreasing last 10 years, was not significant when compared with the average incidence in 2017-2019. The largest decrease in national incidence of both infections was observed among young and heterosexual patients, however some Swedish regions showed an increased incidence, particularly of chlamydia. Increased "internet-based self-sampling" testing approach partly compensated for a decreased attendance at STI clinics. Studies, including sexual behavior, prevention, reasons for attending STI health care, STIs in different anatomical sites, and management of STIs, are required to elucidate the impact of COVID-19-associated social and physical distancing restrictions on sexual activity and the incidence and epidemiology of chlamydia and gonorrhoea in Sweden.

Background Adolescents and young adults are at higher risk of acquiring Chlamydia trachomatis infection (chlamydia), so testing is promoted in these populations. Studies have shown that re-testing for chlamydia is common amongst them. We investigated how sexual risk behaviour profiles are associated with repeated testing for chlamydia. Methods We used baseline data from a cohort of 2814 individuals recruited at an urban STI -clinic. We applied latent class (LC) analysis using 9 manifest variables on sexual behaviour and substance use self-reported by the study participants. We fitted ordered logistic regression to investigate the association of LC membership with the outcomes repeated testing during the past 12 months and lifetime repeated testing for chlamydia. Models were fit separately for men and women. Results We identified four LCs for men and three LCs for women with increasing gradient of risky sexual behaviour. The two classes with the highest risk among men were associated with lifetime repeated testing for chlamydia: adjOR = 2.26 (95%CI: 1.50–3.40) and adjOR = 3.03 (95%CI: 1.93–4.74) as compared with the class with lowest risk. In women, the class with the highest risk was associated with increased odds of repeated lifetime testing (adjOR =1.85 (95%CI: 1.24–2.76)) and repeated testing during past 12 months (adjOR = 1.72 (95%CI: 1.16–2.54)). An association with chlamydia positive test at the time of the study and during the participant’s lifetime was only found in the male highest risk classes. Conclusion Prevention messages with regard to testing for chlamydia after unprotected sexual contact with new/casual partners seem to reach individuals in highest risk behaviour classes who are more likely to test repeatedly. Further prevention efforts should involve potentially more tailored sex-specific interventions taking into consideration risk behaviour patterns.

Background: This study aimed to investigate what impact the COVID-19 pandemic and its associated restrictions had on Chlamydia trachomatis and Neisseria gonorrhoeae infections in Sweden, Denmark and Norway, countries with very different governmental strategies for handling this pandemic. Methods: Retrospective analysis of data collected via requests to Swedish regions and to health authorities in Denmark and Norway. The data were collected for the years 2018–2020 and the data from Sweden were more detailed. Results: When the pandemic restrictions were installed in 2020, the number of reported chlamydia cases decreased. The decline was most pronounced in Norway 10.8% (2019: n = 28,446; 2020: n = 25,444) while it was only 3.1% in Denmark (2019: n = 35,688; 2020: n = 34,689) and 4.3% in Sweden (2019: n = 34,726; 2020: n = 33,339). Nucleic acid amplifications tests for chlamydia decreased in Sweden (10%) and Norway (18%) in 2020 compared to 2019, while in Denmark a 21% decrease was noted in April 2020 but thereafter increased to a higher level than 2019. The number of reported gonorrhoea cases decreased in Sweden (17%) and in Norway (39%) in 2020 compared to 2019, while a 21% increase was noted in Denmark. Conclusions: Pandemic restrictions had an impact on the number of reported chlamydia infections in all three countries, but only temporarily and did not seem to be correlated to the restriction levels. The number of reported gonorrhoea infections in Sweden and Norway significantly decreased but not in Denmark. Pandemic restrictions appear to have had a limited effect on the spread of chlamydia and gonorrhoea.

Chlamydial infection is the most common bacterial sexually transmitted infection in Sweden and worldwide, despite extensive testing, efficient treatment and partner notification. The incidence of Lymphogranuloma venereum, currently occurring among men who have sex with men and most often in the rectum, shows no tendency to decline. This infection is usually symptomatic but still evading diagnosis due to similarities to other causes of proctitis. There is evidence that rectal chlamydia is common also in women not practicing anal sex.

In a previous study, we developed a Multi-Locus VNTRs Analysis (MLVA) typing system, called MLVA-5, for the discrimination of Chlamydia trachomatis genovar E strain. The results suggested the clonal spread of a MLVA-5 type 21 strain among men who have sex with men (MSM). We applied the MLVA-5 typing method on 157 French anorectal genovar E specimens and 19 Swedish specimens collected between 2010 and 2015. A total of 29 MLVA-5 types was obtained, with three predominant types among French samples: 78 specimens belonged to MLVA-5 type 21, two other types, 11 and 13, included 9 and 14 specimens, respectively. In 15 cases, one unique MLVA-5 type was observed for a single patient, 7 of which were new types not previously described. The distribution of MLVA-5 types according to sexual orientation showed that the 7 anorectal specimens from heterosexual patients belonged to 6 genotypes, and the 12 anorectal specimens from bisexual patients comprised eight types. The 95 anorectal specimens from MSM were distributed into 22 types, but 55 (57.9%) of them belonged to MLVA-5 type 21. Among the Swedish specimens from MSM, eight were from MLVA-type 21 (4 urines and 4 anorectal specimens). The results support the hypothesis of the spread of clonal genovar E strain among MSM.

Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI)

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.

A study on notified sexually acquired chlamydia cases and testing in Sweden found that recently decreasing chlamydia notification rates are not affected by testing volumes and most likely reflect decreasing incidence rates. Supplemental digital content is available in the text., Background We investigated the notification trends of sexually acquired chlamydia (chlamydia) and its association with testing in Sweden before (1992–2004) and after (2009–2018) the discovery of a new variant of Chlamydia trachomatis (nvCT). Methods We applied monthly time series analysis to study chlamydia trends and annual time series to study chlamydia rates adjusted for testing. We analyzed incidence nationally and by county group (based on able and unable to detect nvCT at time of discovery). Results We present data on 606,000 cases of chlamydia and 9.9 million persons tested. We found a U-shaped chlamydia trend during the period 1992–2004, with an overall increase of 83.7% from 1996 onward. The period 2009–2018 began with a stable trend at a high incidence level followed by a decrease of 19.7% during the period 2015–2018. Peaks were seen in autumn and through during winter and summer. Similar results were observed by groups of county, although with varying levels of increase and decrease in both periods. Furthermore, increased testing volume was associated with increased chlamydia rates during the first period (P = 0.019) but not the second period. Conclusions Our results showed that chlamydia trends during the period 2009–2018 were not driven by testing, as they were during the period 1992–2004. This suggests less biased notified chlamydia rates and thus possibly a true decrease in chlamydia incidence rates. It is important to adjust case rates for testing intensity, and future research should target other potential factors influencing chlamydia rates.

An investigation of unexpected positive test results for Chlamydia trachomatis at a women's health clinic in Sweden revealed that samples were contaminated by RNA in the clinic. The risk for RNA contamination at a clinic has been postulated previously. We are, however, not aware that this has actually been demonstrated in practice.

The new Abbott Alinity m STI Assay was compared with Abbott m2000 RealTime PCR. For Chlamydia trachomatis, 26 (7.5%) of 347 samples were positive in the Alinity assay and 24 (6.9%) in the m2000 assay. Corresponding figures for Neisseria gonorrhoeae were 23 (6.6%) and 17 (4.9%). For Mycoplasma genitalium, 22 (7.9%) of 279 samples were positive in the Alinity assay and 18 (6.5%) in the m2000 assay, for which DNA extraction was performed on an m2000sp instrument combined with in-house real-time PCR. The Alinity assay has at least the same sensitivity as the m2000 assay. The specificity was evaluated by discrepancy analysis.

This study aimed to determine the incidence of lymphogranuloma venereum (LGV) in Sweden since 2004 and to study in detail a consecutive number of Chlamydia trachomatis cases in men who have sex with men (MSM) during a 10 month period (September 2014 to July 2015). LGV increased from sporadic import cases in 2004 to comprise a spread within Sweden in 2016. Initially, only the L2b ompA genotype was detected, but in 2015 half of the genotyped LGV cases were L2 genotype. The changing genotype distribution in Sweden is linked to increased LGV spread in Europe. High-resolution multilocus sequence typing of 168 C . trachomatis cases from MSM in 2015 resulted in 29 sequence types, of which 3 accounted for 49 % of cases. The increased rates and different genotypes of LGV indicate that more concern for high-risk taking MSM is needed to avoid further spread of this invasive infection.

ObjectivesIn Sweden, an increasing number of tests for sexually transmitted infections are conducted. Self-sampling services are provided free of charge at the national eHealth website. Our aim was to obtain a deeper understanding of users’ beliefs and experiences of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) self-sampling services.MethodsThis qualitative study is part of the national project ‘Internet-based chlamydia and gonorrhoea self-sampling test’, conducted in Sweden. Individuals ordering a CT/NG self-sampling test at home from the eHealth website were invited to participate. Of the 114 individuals who agreed, a purposeful sample including 20 women and men aged 18–49 years (mean, 30.8 years) participated in a telephone interview in 2019.ResultsThe test service for CT/NG was highly appreciated by men and women of different ages. Round-the-clock accessibility, avoiding clinical visits, ease of use, confidentiality and a rapid test result were reasons for this appreciation. Language, uncertainty about the correct sampling procedure, unreliable postal services and concerns about handling of personal data were mentioned as barriers. Reasons for testing were checking after unprotected sex, symptoms, checking a partner’s fidelity or a regular routine—‘to be on the safe side’. Knowledge about the infections and their consequences was limited; some considered them severe, especially if they could threaten fertility, and others were less concerned. Disclosing an infection was described as emotionally stressful. Participants had high self-efficacy in relation to the test and would not hesitate to use the service again, even if it involved a cost.ConclusionsInternet-based CT/NG self-sampling at home was highly appreciated and was used for individual health reasons, but also out of concern for others’ health and for society as a whole. The benefits seem to outweigh the barriers, and the service may therefore continue to be widely offered.

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.

The gram-negative, obligate intracellular human pathogen, Chlamydia trachomatis has a bi-phasic developmental cycle. The histone H1-like C. trachomatis DNA binding protein, Hc2, is produced late during the developmental cycle when the dividing reticulate body transforms into the smaller, metabolically inactive elementary body. Together with Hc1, the two proteins compact the chlamydial chromosome and arrest replication and transcription. Hc2 is heterogeneous in length due to variation in the number of lysine rich pentamers. Six pentamers and one hexamer constitute a 36 amino acid long repetitive unit that, in spite of variations, is unique for Chlamydiaceae. Using synthetic peptides, the DNA-binding capacity of the 36 amino acid peptide and that of a randomized peptide was analyzed. Both peptides bound and compacted plasmid DNA, however, electron microscopy of peptide/DNA complexes showed major differences in the resulting aggregated structures. Fluorescence spectroscopy was used to analyze the binding. After complexing plasmid DNA with each of three different intercalating dyes, increasing amounts of peptides were added and fluorescence spectroscopy performed. The major groove binder, methyl green, was displaced by both peptides at low concentrations, while the minor groove binder, Hoechts, and the intercalating dye, Ethidium Bromide, were displaced only at high concentrations of peptides.

Background Chlamydia trachomatis (CT) testing in Sweden is free of charge and now exceeds 600,000 annual tests in a population of 10 million. These tests include internet-based self-sampling tests, ...

ObjectivesA new variant ofChlamydia trachomatis(nvCT) was discovered in Sweden in 2006. The nvCT has a plasmid deletion, which escaped detection by two nucleic acid amplification tests (Abbott/Roche, AR), which were used in 14 of 21 Swedish counties. The objectives of this study were to assess when and where nvCT emerged in Sweden, the proportion of nvCT in each county, and the role of a potential fitness difference between nvCT and co-circulating wild-type strains (wtCT).MethodsWe used a compartmental mathematical model describing the spatial and temporal spread of nvCT and wtCT. We parameterised the model using sexual behaviour data and Swedish spatial and demographic data. We used Bayesian inference to fit the model to surveillance data about reported diagnoses of chlamydia infection in each county and data from four counties that assessed the proportion of nvCT in multiple years.ResultsModel results indicated that nvCT emerged in central Sweden (Dalarna, Gävleborg, Västernorrland), reaching a proportion of 1% of prevalent CT infections in late 2002 or early 2003. The diagnostic selective advantage enabled rapid spread of nvCT in the presence of high treatment rates. After detection, the proportion of nvCT decreased from 30-70% in AR counties and 5-20% in counties that Becton Dickinson tests, to around 5% in 2015 in all counties. The decrease in nvCT was consistent with an estimated fitness cost of around 5% in transmissibility or 17% reduction in infectious duration.ConclusionsWe reconstructed the course of a natural experiment in which a mutant strain ofC. trachomatisspread across Sweden. Our modelling study provides support, for the first time, of a reduced transmissibility or infectious duration of nvCT. This mathematical model improved our understanding of the first nvCT epidemic in Sweden and can be adapted to investigate the impact of future diagnostic escape mutants.Key messagesThe dynamics of a new variant ofChlamydia trachomatis(nvCT) that escaped testing and treatment in Sweden can be reconstructed using a mathematical transmission model.Our study for the first time provides support of a reduced transmissibility or infectious duration of the nvCT in Sweden.This mathematical model improved our understanding of the nvCT epidemic in Sweden and can be adapted to investigate the impact of future diagnostic escape mutants.

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.

Introduction Lymphogranuloma venereum (LGV) has become prevalent among men who have sex with men (MSM) in Europe since an outbreak in The Netherlands in 2003. The aim of this study was to describe the development of LGV in relation to HIV since 2004, and genotypes of LGV and other Chlamydia trachomatis (Ct) genovars in a MSM population in 2014/15. Methods All testing for LGV in Sweden is referred to Uppsala University Hospital. LGV-specific pmpH-gene PCR was used for detection. High-resolution genotyping based on ompA gene and multilocus sequence typing (MLST) was performed on all Ct-positive cases from an STI clinic for MSM in Stockholm between 1.9. 2014 and 1.7.2015. Results The annual numbers of detected LGV cases in Sweden were 2 in 2004 to 2006, between 5 and 20 in 2007 to 2012, and between 23 and 38 in 2013 to 2016. The number of LGV-tests increased from 68 in 2008 to 268 in 2016. During the study in 2014/15 31 LGV cases were identified in 309 patients with successful PCR-results. In 39% (12/31) LGV was unexpected and had not been detected without extended screening. The HIV-prevalence among LGV-positive patients decreased from 88% 2006–2013% to 68% 2014–2015. Of ompA genotyped LGV cases 69% were L2% and 31% were L2b type. This contrasts to earlier Swedish and European data from 2004–2009 when only L2b was found. Complete genotyping, including ompA and MLST, was obtained for 151 patients with non-LGV Ct and resulted in genovar D, 27%; E, 14%; G, 30% and J 21%. MLST resulted in 27 STs of which 3 predominated and accounted for 51%. Eight STs were new when compared to the database http://mlstdb.bmc.uu.se comprising 540 STs from >3300 specimens. Conclusion In Sweden LGV has gone from sporadic import cases to a probable endemic spread among HIV-negative MSM, which emphasises the need for LGV-testing This emphasises the need for LGV-testing within this high risk group. LGV has developed from being of clonal nature to occur as different strains among MSM.

This study compared conventional ompA genotyping of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus typing (MLT) DNA microarray. DNA extracts of 104 C. trachomatis positive specimens were analyzed by ompA sequencing and MLST and of these 76 by MLT array. Obtained MLST sequence types (STs) were compared to sequences in the database http://mlstdb.uu.se. The resolution obtained for MLST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 1.3 higher than MLT array (27 MLT groups). Among the 104 samples the predominant genotype E could be divided into 5 ompA variants and 23 STs of which 16 had not been reported in previous studies. The most common STs, ST3 and ST56, were identified as founders and are common in several countries on a global scale. The MLST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing.

High-resolution genotyping of Chlamydia trachomatis improves the characterization of strains infecting different patient groups and sexual networks. In this study, multilocus sequence typing (MLST) and ompA sequence determination were used for an analysis of C. trachomatis strains from 203 men who have sex with men (MSM) from Sweden, the Netherlands, and the United States. The results obtained were compared with data from 153 heterosexual women from Sweden and the Netherlands. The overlap in MLST/ ompA profiles between MSM from Sweden and the Netherlands was 68%, while the overlap between heterosexual populations from these countries was only 18%. The distribution of genotypes in MSM from the United States was less similar to that in MSM from the European countries, with 45% and 46% overlaps for MSM in Sweden and the Netherlands, respectively. Minimum-spanning-tree analysis of MLST/ ompA sequence types identified two large clusters that contained almost exclusively samples from MSM and comprised 74% of all MSM samples. Three other clusters were predominated by samples from women but also contained MSM specimens. Of 19 detected variants of the MLST target CT144, three variants were highly associated with MSM. Our study supports the hypotheses of both tissue tropism as well as epidemiological network structures as explanations for the linkage between specific genetic variants and sexual orientation.

A nucleic acid amplification test (NAAT) may enable the laboratory to detect Chlamydia trachomatis and Neisseria gonorrhoeae with high sensitivity and specificity in traditional urogenital swabs and in different types of samples obtained noninvasively by patients at home or in other settings. This chapter provides guidance in selecting the most appropriate NAATs for C. trachomatis and N. gonorrhoeae among available commercial and in-house assays. The selection of targets for detection of C. trachomatis and N. gonorrhoeae is a major point in determining which assay to use for routine diagnostics. Sequence variation in the target region may lead to false-negative results, whereas the presence of the target gene in other species may lead to false-positive results. Routine diagnostics of C. trachomatis infections is predominantly performed with commercial NAAT high-volume test systems, but there are still applications where in-house-developed methods are useful. The number of samples in a pool depends on the prevalence, and it is calculated from the number of samples, which needs to be tested individually from positive pools. Samples from a negative pool should be reported as negative. The rapid spread of a mutant variant of C. trachomatis in Sweden escaping detection by some NAATs has been a warning that vigilance for drug-resistant mutants should be enforced.

Background: In the past, contradictory results have been obtained linking Chlamydia trachomatis serovars (ompA gene) to different clinical courses of infection. Methods: A high resolution multilocus sequence typing (MLST) system was used to genotype 6 genetic regions, including ompA, in 70 Dutch urogenital C. trachomatis strains from patients with different degrees of defined clinical symptoms (asymptomatic, symptomatic, and lower abdominal pain), to determine if MLST genotypes correlated with clinical manifestations of infection. Results and conclusions: We identified 46 MLST types, with only a small overlap to Swedish MLST types. This study found no correlation between MLST profiles and symptomatology. To understand the clinical course of infection, future studies should not only consider bacterial factors but also look on the immunogenetics of the host

We aimed to compare conventional ompA typing of Chlamydia trachomatis with multilocus sequence typing (MLST) and multilocus variable-number tandem-repeat (VNTR) analysis (MLVA). Previously used MLST and MLVA systems were compared to modified versions that used shorter target regions and nested PCR. Heterosexual couples were selected from among persons with urogenital C. trachomatis infections visiting the sexually transmitted infection outpatient clinic in Amsterdam, The Netherlands. We identified 30 couples with a total of 65 C. trachomatis -positive samples on which MLST and MLVA for eight target regions were performed. All regions were successfully sequenced in 52 samples, resulting in a complete profile for 18 couples and 12 individuals. Nine ompA genovars from D to K, with two variants of genovar G, were found. The numbers of sequence type and MLVA type profiles were 20 for MLST and 21 for MLVA, and a combination of MLST and MLVA yielded 28 profiles, with discriminatory indexes ( D ) ranging from 0.95 to 0.99. Partners in 17 couples shared identical profiles, while partners in 1 couple had completely different profiles. Three persons had infections at multiple anatomical locations, and within each of these three individuals, all profiles were identical. The discriminatory capacity of all MLST and MLVA methods is much higher than that of ompA genotyping ( D = 0.78). No genotype variation was found within the samples of the same person or from heterosexual couples with a putative single transmission. This shows that the chlamydial genome in clinical specimens has an appropriate polymorphism to enable epidemiological cluster analysis using MLST and MLVA.

The family Chlamydiaceae contains several bacterial pathogens of important human and veterinary medical concern, such as Chlamydia trachomatis and Chlamydophila psittaci. Within the order Chlamydia ...

Background: Genital chlamydia infection (chlamydia) is the most commonly reported sexually transmitted infection (STI) in Sweden. To guide prevention needs, we aimed to investigate factors associated with chlamydia. Methods: A cohort of visitors aged 20-40 years at an urban STI clinic in Sweden was recruited. Behavioural data were collected using a self-administered questionnaire. Self-sampled specimens were tested for chlamydia by a DNA amplification assay. Statistically significant (p

Pharmacodynamic knowledge about Chlamydia trachomatis exposed to antibiotics is hampered due to methodological limitations. We have developed a quantitative real-time PCR method (qRT-PCR) for determination of viable C. trachomatis. The method measures specific RNA transcripts of omp2 (omcB) as an expression of viable C. trachomatis. Two clinical isolates (one strain derived from a patient with recurrent symptoms despite doxycycline treatment) were cultured in McCoy cells and exposed to doxycycline at concentrations of 0.0078-64 mg/L. MIC values were evaluated microscopically by immunofluorescence (IF) and by qRT-PCR performed on cDNA prepared from the total RNA. The MIC for two C. trachomatis strains were determined to 0.016 and 0.031 mg/L by both qRT-PCR and IF. The qRT-PCR assay enabled MIC determinations without subjective evaluation, which is a problem when visually evaluating inclusions. The presented qRT-PCR is a suitable method for MIC determination of C. trachomatis. It has the advantage of giving quantitative measurements of chlamydial RNA levels and the method is useful in pharmacodynamic studies of C. trachomatis.

The vast majority of genital Chlamydia trachomatis infections are non-symptomatic or mildly symptomatic. Efficient contact tracing and screening programmes are the most effective means to reveal these infections. We conducted a prospective study on the effects of contact tracing performed by specially trained midwives in a centralized municipality-based organization during one year. In order to improve the efficiency of the contact tracing, all C. trachomatis-positive isolates in the county during one year were typed by sequencing the ompA gene. Contact tracing in two municipalities the year prior to the study revealed 1.6 partners for each index case ( n = 110). The corresponding figure for the whole county during the study period was 2.1 partners per index case ( n = 2065). Genotyping added valuable information in mapping individual sexual networks, but was not essential for achieving satisfactory contact tracing results.

A new variant of Chlamydia trachomatis (nvCT) was discovered in Sweden in 2006, and it could not be detected by diagnostic systems from Abbott and Roche, whereas the third system used, from Becton Dickinson (BD), detects nvCT. We analyzed 3648 samples from 2 counties that used Roche and 2 counties that used BD methods from 2007 to 2011. After implementation of a Roche method that detects nvCT, its proportion has decreased and converged in the 4 counties but are still at different levels in Roche and BD counties. Future studies are needed to see if nvCT will decline further.

BACKGROUND: Accurate information about the prevalence of Chlamydia trachomatis is needed to assess national prevention and control measures. METHODS: We systematically reviewed population-based cross-sectional studies that estimated chlamydia prevalence in European Union/European Economic Area (EU/EEA) Member States and non-European high income countries from January 1990 to August 2012. We examined results in forest plots, explored heterogeneity using the I² statistic, and conducted random effects meta-analysis if appropriate. Meta-regression was used to examine the relationship between study characteristics and chlamydia prevalence estimates. RESULTS: We included 25 population-based studies from 11 EU/EEA countries and 14 studies from five other high income countries. Four EU/EEA Member States reported on nationally representative surveys of sexually experienced adults aged 18-26 years (response rates 52-71%). In women, chlamydia point prevalence estimates ranged from 3.0-5.3%; the pooled average of these estimates was 3.6% (95% CI 2.4, 4.8, I² 0%). In men, estimates ranged from 2.4-7.3% (pooled average 3.5%; 95% CI 1.9, 5.2, I² 27%). Estimates in EU/EEA Member States were statistically consistent with those in other high income countries (I² 0% for women, 6% for men). There was statistical evidence of an association between survey response rate and estimated chlamydia prevalence; estimates were higher in surveys with lower response rates, (p = 0.003 in women, 0.018 in men). CONCLUSIONS: Population-based surveys that estimate chlamydia prevalence are at risk of participation bias owing to low response rates. Estimates obtained in nationally representative samples of the general population of EU/EEA Member States are similar to estimates from other high income countries Background: Accurate information about the prevalence of Chlamydia trachomatis is needed to assess national prevention and control measures.Methods: We systematically reviewed population-based cross-sectional studies that estimated chlamydia prevalence in European Union/European Economic Area (EU/EEA) Member States and non-European high income countries from January 1990 to August 2012. We examined results in forest plots, explored heterogeneity using the I2 statistic, and conducted random effects meta-analysis if appropriate. Meta-regression was used to examine the relationship between study characteristics and chlamydia prevalence estimates.Results: We included 25 population-based studies from 11 EU/EEA countries and 14 studies from five other high income countries. Four EU/EEA Member States reported on nationally representative surveys of sexually experienced adults aged 18–26 years (response rates 52–71%). In women, chlamydia point prevalence estimates ranged from 3.0–5.3%; the pooled average of these estimates was 3.6% (95% CI 2.4, 4.8, I2 0%). In men, estimates ranged from 2.4–7.3% (pooled average 3.5%; 95% CI 1.9, 5.2, I2 27%). Estimates in EU/EEA Member States were statistically consistent with those in other high income countries (I2 0% for women, 6% for men). There was statistical evidence of an association between survey response rate and estimated chlamydia prevalence; estimates were higher in surveys with lower response rates, (p = 0.003 in women, 0.018 in men).

The Uppsala University Chlamydia trachomatis multilocus sequence type (MLST) database ( http://mlstdb.bmc.uu.se ) is based on five target regions (non-housekeeping genes) and the ompA gene. Each target has various numbers of alleles— hctB , 89; CT058, 51; CT144, 30; CT172, 38; and pbpB , 35—derived from 13 studies. Our aims were to perform an overall analysis of all C. trachomatis MLST sequence types (STs) in the database, examine STs with global spread, and evaluate the phylogenetic capability by using the five targets. A total of 415 STs were recognized from 2,089 specimens. The addition of 49 ompA gene variants created 459 profiles. ST variation and their geographical distribution were characterized using eBURST and minimum spanning tree analyses. There were 609 samples from men having sex with men (MSM), with 4 predominating STs detected in this group, comprising 63% of MSM cases. Four other STs predominated among 1,383 heterosexual cases comprising, 31% of this group. The diversity index in ocular trachoma cases was significantly lower than in sexually transmitted chlamydia infections. Predominating STs were identified in 12 available C. trachomatis whole genomes which were compared to 22 C. trachomatis full genomes without predominating STs. No specific gene in the 12 genomes with predominating STs could be linked to successful spread of certain STs. Phylogenetic analysis showed that MLST targets provide a tree similar to trees based on whole-genome analysis. The presented MLST scheme identified C. trachomatis strains with global spread. It provides a tool for epidemiological investigations and is useful for phylogenetic analyses.

Objectives: To analyse trends in rates of genital chlamydial infection and ectopic pregnancy between 1985 and 1995 in a county in Sweden. Setting: Uppsala county where screening for chlamydial infection, treatment, and contact tracing has been widespread and where ectopic pregnancies are recorded. Methods: Rates of chlamydial infections and ectopic pregnancy between 1985 and 1995 were calculated for women aged 20-39 years. Poisson and linear regression were used to examine the association between the risk of ectopic pregnancy and the current rates of chlamydial infection and rates of chlamydial infection from up to 5 years earlier. Main outcome measures: Rates of chlamydial infection per 100 examinations, rates of ectopic pregnancy per 1000 pregnancies, rate ratios and 95% confidence intervals for an increase in chlamydial infections of 5 new cases per 100 examinations. Results: 103 870 cervical samples from women aged 15-39 years were analysed; 5648 (5.4%) were positive for chlamydia. 51 630 pregnancies were analysed; 930 (1.8%) were ectopic. Both rates declined over time. For women aged 20-24 years there was a strong correlation between the rate of ectopic pregnancy and the rate of chlamydial infection in the same year (r=0.93, P Conclusions: Declining rates of genital chlamydial infections have probably led to a fall in the rate of ectopic pregnancies. The timing of the decline in the rate of ectopic pregnancies varies with age. Among young women falling rates of chlamydial infection have been accompanied by an immediate reduction in the rate of ectopic pregnancy. Key messages The effect of measures to diagnose and treat genital chlamydial infections on the risk of ectopic pregnancy was examined in a county in Sweden Rates of chlamydial infection and ectopic pregnancy both declined between 1985 and 1995 Chlamydial infections seem to be the main cause of ectopic pregnancy in young women; the risk of ectopic pregnancy decreased concurrently with the decline in chlamydial infections In older women the associations were less pronounced

A patient with proctitis and inguinal buboes diagnosed with lymphogranuloma venereum (LGV) was treated with doxycycline 21 days, azithromycin 20 days and moxifloxacin for a further 12 days because of progressive worsening of inguinal symptoms. Despite extensive antibiotic treatment, the inguinal LGV lesions persisted; however, the patient recovered spontaneously after three months.

The amplification of DNA from Chlamydia trachomatis by PCR with degenerated primers yielded a 345-bp fragment of the putative RNase P RNA gene. From the deduced DNA sequence of this gene in C. trachomatis, a modified primer pair was designed. The primer pair was subsequently used to obtain the corresponding gene products from Chlamydia pneumoniae and Chlamydia psittaci. Sequence comparisons revealed similarities of 76.6% between C. trachomatis and C. pneumoniae, 79.5% between C. trachomatis and C. psittaci, and 84.7% between C. pneumoniae and C. psittaci. Furthermore, the three species were differentiated by fragment length polymorphism analysis after restriction enzyme cleavage of the PCR products. Sequence variations among 14 serotypes of C. trachomatis were confined to one purine base substitution in the putative RNase P RNA gene of lymphogranuloma venereum strains L1 to L3. Complete sequence similarity was found for nine strains of C. pneumoniae of different geographic origins. Taken together, our results indicate a possibility of the general application of this method in clinical bacteriology. Analysis of the secondary structures of the putative RNase P RNA genes from the different Chlamydia species suggested that a novel structural element in the domain of RNase P RNA is involved in base pairing with the 3'-terminal CCA motif of a tRNA precursor. This structure has not previously been found among RNase P RNAs of members of the division Bacteria.

In Uppsala County, between 1985 and 1993, examinations for Chlamydia trachomatis were being performed in a central laboratory. A change in national sexually transmitted disease legislation in 1988 encouraged screening.To analyze trends in detection rates of genital chlamydial infections by age, sex, and clinic type, and to assess the influence of the legislation change.This was an analysis of 119,892 tests, representing 95.4% of all specimens sampled. Eighty-six percent of the samples were cultured, 14% were examined by enzyme immunoassay.Seventy-nine percent of specimens came from women. 7,989 positive samples were identified. Detection rates declined from 107.2 per 1,000 in 1985 to 32.3 in 1993 in women and from 183.3 to 70.7 in men. Positivity rates were highest in sexually transmitted disease and youth clinics and lowest in private practices. Among female youths, rates leveled off in later years, but the rates increased in male youths. The legislation change reduced the probability of a positive test in men but not in women.Genital chlamydial infection generally declined. However, among youths, an increase has occurred in recent years. Continued screening and the introduction of noninvasive diagnostic methods for males is warranted, particularly in youth clinics. Selective screening may be more cost-effective in other age groups.

Chlamydia is one of the most common sexually transmitted infections worldwide and can cause ectopic pregnancies and infertility. It is therefore important to have adequate genotyping tools for investigating the spread of Chlamydia trachomatis among the population. Here, we describe a high-resolution multilocus sequence typing (MLST) system able to differentiate closely related clinical strains, which makes it ideal for short-term epidemiology and outbreak investigations. It is based on five highly variable but stable target regions which are PCR amplified and DNA sequenced.

We aimed to evaluate the stability of the Chlamydia trachomatis multi locus VNTR analysis (MLVA-ompA) and multi sequence typing (MST) systems through multiple passages in tissue culture. Firstly, we analyzed the stability of these markers through adaptation of C. trachomatis to tissue culture and secondly, we examined the stability of a four-locus MLVA-ompA and a five-locus MST system after multiple passages in tissue culture. Marker sequences were monitored through successive chlamydial developmental cycles to evaluate the stability of the individual DNA markers through many bacterial divisions and this, in turn, informed us of the usefulness of using such typing systems for short and long-term molecular epidemiology. Southampton genitourinary medicine (GUM) clinic isolates from endocervical swabs collected from C. trachomatis positive women were passaged through tissue culture. MLVA-ompA typing was applied to primary swab samples and to the same samples after C. trachomatis had been passaged through cell culture (eight passages). Sequence data from time-zero and passage-eight isolates were aligned with reference sequences to determine the stability of the markers. The Swedish new variant (nvCT) underwent 72 passages in cell culture and the markers of the two schemes were similarly analyzed. Analysis of genetic markers of the MLVA-ompA typing system before and after the isolates were introduced to tissue culture showed no change in the dominant sequence. The nvCT that had been passaged 72 times over the duration of a year also showed no variation in the dominant sequence for both the genotyping schemes. MLVA-ompA and MST markers are stable upon adaptation of C. trachomatis to tissue culture following isolation of strains from primary endocervical swab samples. These markers remain stable throughout multiple rounds of cell-division in tissue culture, concomitant with the incubation period and appearance of symptoms normally associated with host-infection. Both genotyping schemes are, therefore, suitable for epidemiology of C. trachomatis.

This study describes a new multilocus variable number tandem-repeat (VNTR) analysis (MLVA) typing system for the discrimination of Chlamydia trachomatis genovar D to K isolates or specimens. We focused our MLVA scheme on genovar E which predominates in most populations worldwide. This system does not require culture and therefore can be performed directly on DNA extracted from positive clinical specimens. Our method was based on GeneScan analysis of five VNTR loci labelled with fluorescent dyes by multiplex PCR and capillary electrophoresis. This MLVA, called MLVA-5, was applied to a collection of 220 genovar E and 94 non-E genovar C. trachomatis isolates and specimens obtained from 251 patients and resulted in 38 MLVA-5 types. The genetic stability of the MLVA-5 scheme was assessed for results obtained both in vitro by serial passage culturing and in vivo using concomitant and sequential isolates and specimens. All anorectal genovar E isolates from men who have sex with men exhibited the same MLVA-5 type, suggesting clonal spread. In the same way, we confirmed the clonal origin of the Swedish new variant of C. trachomatis. The MLVA-5 assay was compared to three other molecular typing methods, ompA gene sequencing, multilocus sequence typing (MLST) and a previous MLVA method called MLVA-3, on 43 genovar E isolates. The discriminatory index was 0.913 for MLVA-5, 0.860 for MLST and 0.622 for MLVA-3. Among all of these genotyping methods, MLVA-5 displayed the highest discriminatory power and does not require a time-consuming sequencing step. The results indicate that MLVA-5 enables high-resolution molecular epidemiological characterisation of C. trachomatis genovars D to K infections directly from specimens.

Background: The Chlamydia trachomatis incidence rate in Finnmark, the most northern and sparsely populated county in Norway, has been twice the national average. This population based cross-sectional study among Finnmark high school students had the following aims: i) to examine distribution of multilocus sequence types (STs) of C. trachomatis in a previously unmapped area, ii) to compare chlamydia genetic diversity in Finnmark with that of two urban regions, and iii) to compare discriminatory capacity of multilocus sequence typing (MLST) with conventional ompA sequencing in a large number of chlamydia specimens. Methodology: ompA sequencing and a high-resolution MLST system based on PCR amplification and DNA sequencing of five highly variable genetic regions were used. Eighty chlamydia specimens from adolescents aged 15-20 years in Finnmark were collected in five high schools (n = 60) and from routine clinical samples in the laboratory (n = 20). These were compared to routine clinical samples from adolescents in Tromso (n = 80) and Trondheim (n = 88), capitals of North and Central Norway, respectively. Principal Findings: ompA sequencing detected 11 genotypes in 248 specimens from all three areas. MLST displayed 50 STs providing a five-fold higher resolution. Two-thirds of all STs were novel. The common ompA E/Bour genotype comprised 46% and resolved into 24 different STs. MLST identified the Swedish new variant of C. trachomatis not discriminated by ompA sequencing. Simpson's discriminatory index (D) was 0.93 for MLST, while a corrected D-c was 0.97. There were no statistically significant differences in ST genetic diversity between geographic areas. Finnmark had an atypical genovar distribution with G being predominant. This was mainly due to expansion of specific STs of which the novel ST161 was unique for Finnmark. Conclusions/Significance: MLST revealed multiple new STs and a larger genetic diversity in comparison to ompA sequencing and proved to be a useful tool in molecular epidemiology of chlamydia infections. Manuscript title: High-resolution Multilocus Sequence Typing of Chlamydia trachomatis reveals multiple new genotypes in North and Central Norway

Chlamydia is one of the most common sexually transmitted infections worldwide and can cause ectopic pregnancies and infertility. It is therefore important to have adequate genotyping tools for investigating the spread of Chlamydia trachomatis among the population. Here, we describe a high-resolution multilocus sequence typing (MLST) system able to differentiate closely related clinical strains, which makes it ideal for short-term epidemiology and outbreak investigations. It is based on five highly variable but stable target regions which are PCR amplified and DNA sequenced.

Typing of Chlamydia trachomatis is important to understanding its epidemiology. Currently used methods such as DNA sequencing of the ompA gene and multilocus sequence typing (MLST) either offer limited epidemiological resolution or are laborious and expensive, or both. DNA microarray technology using the ArrayStrip format is an affordable alternative for genotyping. In this study, we developed a new multilocus typing (MLT) DNA microarray, based on the target regions of a high-resolution MLST system as well as software for easy analysis. Validation of the array was done by typing 80 previously MLST-typed clinical specimens from unselected adolescents in school. The MLT array showed 100% specificity and provided 2.4-times-higher resolution than ompA sequencing, separating the commonly predominating ompA E/Bour genotype into 7 MLT array genotypes. The MLT array reproduced epidemiological findings revealed by the MLST system and showed sufficient sensitivity to work with clinical specimens. Compared to MLST analysis, the expenses needed for testing a sample with the MLT array are considerably lower. Moreover, testing can be completed within 1 working day rather than 3 or 4 days, with data analysis not requiring highly specialized personnel. The present MLT array represents a powerful alternative in C. trachomatis genotyping.

Current typing methods of Chlamydia (C) trachomatis are mainly based on the diversity of the ompA gene, which is coding for the major outer membrane protein A. The present study aimed at facilitating genotyping of strains of this obligate intracellular human pathogen by developing a DNA microarray assay using the ArrayTube (TM) format for individual samples and the ArrayStrip (TM) format for higher throughput. The new test is exploiting multiple discriminatory sites by involving a total of 61 oligonucleotide probes representing genotype-specific polymorphisms in variable domains 1, 2 and 4 of the ompA gene. After multiplex amplification of these domains using biotinylated primers, the sample is hybridized in the microarray vessel under highly stringent conditions. The resulting binding pattern is genotype specific, thus allowing direct identification. We were able to show that DNA from each of the currently accepted genotypes (serovars) yielded a unique, theoretically expected and distinct hybridization pattern. The assay was also shown to be highly sensitive as a dilution containing the equivalent of 1 inclusion-forming unit was still correctly genotyped. In addition, when 62 clinical samples were examined and compared to PCR-RFLP typing results, the genotype was correctly identified by the DNA microarray in all cases. The present test is easy to handle and economically affordable, and it allows genotyping of C. trachomatis to be accomplished within a working day, thus lending itself for epidemiological studies and routine diagnosis. (C) 2010 Elsevier Ltd. All rights reserved

Objectives In 2006 a new variant of Chlamydia trachomatis (nvCT), with a deletion in the cryptic plasmid, was reported in Sweden. This deletion included the targets for the genetic diagnostic systems used in many clinical laboratories and resulted in thousands of falsenegative results. The aim of this study was to characterise consecutive Chlamydia tissue culturepositive samples from 2006 in Orebro County, after identification of the nvCT, and to compare the results from samples collected in the same county in 1999e2000. The study also aimed to evaluate the discriminatory capacity of multilocus sequence typing (MLST) compared with ompA sequencing. Methods ompA sequencing and MLST was used to characterise 100 consecutive Chlamydia tissue culturepositive samples. Results A significant (p

Udgivelsesdato: 2009-Mar A historical review is provided of the various methods used for half a century to differentiate and type Chlamydia trachomatis strains. Typing of C. trachomatis is an important tool for revealing transmission patterns in sexual networks, and enabling association with clinical manifestations and pathogenicity. Serotyping using the major outer membrane protein (MOMP) has been the mainstay of epidemiological work for several decades. However, the development of nucleic acid amplification techniques (NAAT) and easy access to sequencing have shifted the focus from MOMP serotypes to omp1 genotypes. However, insufficient epidemiological resolution is achieved by characterization of both MOMP and omp1. This calls for new high-resolution genotyping methods applying for example a multilocus variable number tandem repeat assay (MLVA) or multilocus sequence typing (MLST). The futuristic nanotechnology already seems at hand to further simplify and automate the high-resolution genotyping method based on NAAT and sequencing of various targets in the C. trachomatis genome. Thereby, a high throughput can be achieved and more epidemiological information can be obtained. However, it is important to realize that culture of C. trachomatis may still be needed to detect and characterize new variants of C. trachomatis.