Markus Klint

High-Resolution Genotyping of Chlamydia trachomatis Strains by Multilocus Sequence Analysis

ABSTRACT Genotyping of Chlamydia trachomatis is limited by the low sequence variation in the genome, and no adequate method is available for analysis of the spread of chlamydial infections in the community. We have developed a multilocus sequence typing (MLST) system based on five target regions and compared it with analysis of ompA, the single gene most extensively used for genotyping. Sequence determination of 16 reference strains, comprising all major serotypes, serotypes A to L3, showed that the number of genetic variants in the five separate target regions ranged from 8 to 16. The genetic variation in 47 clinical C. trachomatis isolates of representative serotypes (14 serotype D, 12 serotype E, 11 serotype G, and 10 serotype K strains) was analyzed; and the MLST system detected 32 variants, whereas 12 variants were detected by using ompA analysis. Specimens of the predominant serotype, serotype E, were differentiated into seven genotypes by MLST but into only two by ompA analysis. The MLST system was applied to C. trachomatis specimens from a population of men who have sex with men and was able to differentiate 10 specimens of one predominant ompA genotype G variant into four distinct MLST variants. To conclude, our MLST system can be used to discriminate C. trachomatis strains and can be applied to high-resolution molecular epidemiology.

Emergence and Spread of Chlamydia trachomatis Variant, Sweden

A variant of Chlamydia trachomatis that had escaped detection by commonly used systems was discovered in Sweden in 2006. In a nationwide study, we found that it is now prevalent across Sweden, irrespective of the detection system used. Genetic analysis by multilocus sequence typing identified a predominant variant, suggesting recent emergence.

Lymphogranuloma Venereum Prevalence in Sweden among Men Who Have Sex with Men and Characterization of Chlamydia trachomatis ompA Genotypes

ABSTRACT An outbreak of lymphogranuloma venereum (LGV) infections has recently been reported from The Netherlands and other European countries. The Swedish surveillance system has identified three LGV cases since 2004, all with clinically suspected infection in men who have sex with men (MSM). In order to assess the prevalence of LGV in a high-risk group of MSM and include clinically atypical cases, retrospective analysis of 197 Chlamydia trachomatis-infected men was performed. Sequencing of the ompA gene showed a different serotype distribution compared to recent Swedish studies in heterosexual populations. The most common types were G (45%), D (27%), and J (26%), whereas the normally predominant type E accounted for only 4% of the chlamydia cases. Furthermore, certain ompA genotype variants of the dominant serotypes were highly prevalent among MSM, and the reason for this is discussed. No additional case of LGV was detected by retrospective analysis of the high-risk MSM population. This indicates that, thus far, LGV in Sweden is only a result of sporadic import from infected MSM clusters abroad.

Typing of lymphogranuloma venereum Chlamydia trachomatis strains

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.

Chlamydophila psittaci in Fulmars, the Faroe Islands

Chlamydophila psittaci was detected in 10% of 431 fulmars examined from the Faroe Islands. Analysis of ompA showed a sequence almost identical to that of the type strain. The origin of C. psittaci outbreaks in fulmars is discussed. Despite a high level of exposure, the risk for transmission of C. psittaci to humans is low.

Characterisation of Chlamydia trachomatis by ompA sequencing and multilocus sequence typing in a Swedish county before and after identification of the new variant

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 false-negative results. The aim of this study was to characterise consecutive Chlamydia tissue culture-positive samples from 2006 in Örebro County, after identification of the nvCT, and to compare the results from samples collected in the same county in 1999–2000. 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 culture-positive samples. Results A significant (p<0.001) increase of genotype E, from 47% in 1999–2000 to 69% in 2006, was detected. All 41 nvCT isolates from 2006 displayed an identical ompA genotype E and MLST profile. Excluding the nvCT isolates, the distribution of ompA genotypes is similar to the genotyping results from 1999–2000. Among the wild-type genotype E isolates from 2006, 14 unique MLST sequence types were obtained from 26 isolates while they were identical in ompA genotyping. The discriminatory power (D) of C trachomatis strains in this material was 83.5% using the MLST system compared with 49.5% utilising ompA sequencing. Conclusion In all, MLST enables improved studies of the molecular epidemiology of C trachomatis. All nvCT isolates from 2006 displayed an identical ompA genotype E and MLST profile, which strongly indicates a clonal spread of the nvCT.

Prevalence trends in Sweden for the new variant of Chlamydia trachomatis

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.

Multilocus sequence typing of urogenital Chlamydia trachomatis from patients with different degrees of clinical symptoms

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.

Mosaic structure of intragenic repetitive elements in histone H1-like protein Hc2 varies within serovars of Chlamydia trachomatis

BackgroundThe histone-like protein Hc2 binds DNA in Chlamydia trachomatis and is known to vary in size between 165 and 237 amino acids, which is caused by different numbers of lysine-rich pentamers. A more complex structure was seen in this study when sequences from 378 specimens covering the hctB gene, which encodes Hc2, were compared.ResultsThis study shows that the size variation is due to different numbers of 36-amino acid long repetitive elements built up of five pentamers and one hexamer. Deletions and amino acid substitutions result in 14 variants of repetitive elements and these elements are combined into 22 configurations. A protein with similar structure has been described in Bordetella but was now also found in other genera, including Burkholderia, Herminiimonas, Minibacterium and Ralstonia.Sequence determination resulted in 41 hctB variants that formed four clades in phylogenetic analysis. Strains causing the eye disease trachoma and strains causing invasive lymphogranuloma venereum infections formed separate clades, while strains from urogenital infections were more heterogeneous. Three cases of recombination were identified. The size variation of Hc2 has previously been attributed to deletions of pentamers but we show that the structure is more complex with both duplication and deletions of 36-amino acid long elements.ConclusionsThe polymorphisms in Hc2 need to be further investigated in experimental studies since DNA binding is essential for the unique biphasic life cycle of the Chlamydiacae. The high sequence variation in the corresponding hctB gene enables phylogenetic analysis and provides a suitable target for the genotyping of C. trachomatis.