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We analyzed by multilocus sequence typing 77 lymphogranuloma venereum Chlamydia trachomatis strains from men who have sex with men in Europe and the United States. Specimens from an outbreak in 2003 in Europe were monoclonal. In contrast, several strains were in the United States in the 1980s, including a variant from Europe.

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 MIST and of these 76 by MLT array. Obtained MIST sequence types (STs) were compared to sequences in the database http://mIstdb.uu.se. The resolution obtained for MIST (35 STs) was 2.1 higher than for ompA sequencing (17 variants) and 13 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 MIST and the MLT array provided similar strain discrimination capacity and showed considerably higher resolution than conventional ompA sequencing.

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 t, Manuscript title: High-resolution Multilocus Sequence Typing of Chlamydia trachomatis reveals multiple new genotypes in North and Central Norway

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.

Chlamydia psittaci in birds may be transmitted to humans and cause respiratory infections, sometimes as severe disease. Our study investigated the C. psittaci prevalence in migratory birds in Sweden by real-time PCR. Fecal specimens or cloacal swabs were collected from 497 birds from 22 different species, mainly mallards (Anas platyrhynchos), at two bird observatories in Sweden. DNA from C. psittaci was found in six (1.2%) birds from three different species. Five of the positive specimens were infected with four novel strains of C. psittaci, based on sequencing of partial 16S rRNA gene and ompA gene, and the sixth was indentified as a recently described Chlamydiaceae-like bacterium. Considering exposure to humans it is concluded that the risk of zoonotic infection is low.

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 genot

BACKGROUND: Chlamydia psittaci is an intracellular bacterium primarily causing respiratory diseases in birds but may also be transmitted to other animals, including humans. The prevalence of the pathogen in wild birds in Sweden is largely unknown. METHODS: DNA was extracted from cloacae swabs and screened for C. psittaci by using a 23S rRNA gene PCR assay. Partial 16S rRNA and ompA gene fragments were sequence determined and phylogenies were analysed by the neighbour-joining method. RESULTS AND CONCLUSION: The C. psittaci prevalence was 1.3% in 319 Peregrine Falcons and White-tailed Sea Eagles, vulnerable top-predators in Sweden. 16S rRNA and ompA gene analysis showed that novel Chlamydia species, as well as novel C. psittaci strains, are to be found among wild birds.

In 2006, a new variant of Chlamydia trachomatis (nvCT) was discovered in Sweden that was not detectable with Abbott m2000 (Abbott) and Amplicor/COBAS Amplicor/TaqMan48 (Roche). The proportion of nvCT was 20-64% of the detected Chlamydia cases in counties using Abbott/Roche test systems. Although the ProbeTec system from Becton Dickinson (BD) could detect nvCT, the proportion of nvCT in counties using BD was 7-19%. The objective of the current study was to follow the nvCT proportions from 2007 to 2009 in two counties that used Roche and had introduced test systems able to detect nvCT in late 2006. The nvCT was also followed in two counties that used BD, and in all four counties the effect of nvCT on the serotype distribution of C. trachomatis wild-type strains was analysed. A total of 2576 specimens positive for C. trachomatis were collected in the four counties at three time points, and analysed for nvCT and serotype E. The proportion of nvCT declined significantly in the two counties using Roche, from 65% and 48% in 2007 to 24% for both counties in 2009 (p < 0.001). The nvCT proportion increased in Norrbotten county, which used BD, from 9% in 2007 to 19% in 2009 (p 0.03). In Uppsala county, which also used BD but was surrounded by counties using detection systems from Roche, the proportion of nvCT declined from 24% in 2007 to 18% in 2009 (p < 0.03). No major difference in the level of serotype E was seen. The proportion of nvCT seems to rapidly converge in the Swedish counties after the selective diagnostic advantage for nvCT has been lost in the Abbott/Roche counties.

Typing of Chlamydia trachomatis is important to understandingits epidemiology. Currently used methods such as DNA sequencingof the ompA gene and multilocus sequence typing (MLST) eitheroffer limited epidemiological resolution or are laborious andexpensive, or both. DNA microarray technology using the ArrayStripformat is an affordable alternative for genotyping. In thisstudy, we developed a new multilocus typing (MLT) DNA microarray,based on the target regions of a high-resolution MLST systemas well as software for easy analysis. Validation of the arraywas done by typing 80 previously MLST-typed clinical specimensfrom unselected adolescents in school. The MLT array showed100% specificity and provided 2.4-times-higher resolution thanompA sequencing, separating the commonly predominating ompAE/Bour genotype into 7 MLT array genotypes. The MLT array reproducedepidemiological findings revealed by the MLST system and showedsufficient sensitivity to work with clinical specimens. Comparedto MLST analysis, the expenses needed for testing a sample withthe MLT array are considerably lower. Moreover, testing canbe completed within 1 working day rather than 3 or 4 days, withdata analysis not requiring highly specialized personnel. Thepresent MLT array represents a powerful alternative in C. trachomatisgenotyping.

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 degrees 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.