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Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples.

Wang Y, Skilton RJ, Cutcliffe LT, Andrews E, Clarke IN, Marsh P - PLoS ONE (2011)

Bottom Line: Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture.Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology and Infection, School of Medicine, University of Southampton, Southampton, United Kingdom.

ABSTRACT

Background: Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV) and the genitourinary genotypes D-K. Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.

Principal findings: We evaluated variable number tandem repeat (VNTR) and ompA sequencing (now called multi-locus VNTR analysis and ompA or "MLVA-ompA") to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM) clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche) PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59%) were fully genotyped by MLVA-ompA. Only one mixed infection (E & D) in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.

Conclusions: Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

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Related in: MedlinePlus

Distribution of different VNTR types according to ompA genotype (where loci appeared mixed in individual samples, both numerals are given separated by a ‘/’).
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pone-0016971-g002: Distribution of different VNTR types according to ompA genotype (where loci appeared mixed in individual samples, both numerals are given separated by a ‘/’).

Mentions: There were different MLVA types within each genotype, including the two genotype D variants (Figure 2). Overall, the combination of the ompA and MLVA as a genotyping panel gave, for this set of results, a Simspon's discriminatory index [15] of 0.96. Of the eight samples which could only be genotyped following culture (Table S3), five (MLVA-ompA types: 3.2.4-K; 3.6a.3-D/UW-3; 12.5a.2-G; 3.4a.2-J; and 8.8.2-F) were unique genotypes which had no replicates that were also detectable directly from the original DNA samples. Some MLVA signatures were common to different ompA types, for example 8.5.2 was found in ompA types D (both sub-types), E, F and J; whilst 8.6.2 was found in D/IC-CAL8, E, and F. In most cases however, MLVA signatures were unique to ompA types. As well as defining some new VNTR sequences for the three loci (Table 3), we further noted that in some cases there were differences in the flanking regions between two samples where the VNTR locus sequence itself was identical. For example, VNTR CT1299 variant 4 (Table 3) has a tandem repeat of ten cytosine residues (10C). In our survey, we found three versions of CT1299 variant 4 with 10C, with three different sequences between the conserved flanking regions, namely 10C (variant 4), 10C-T3C (variant 4a) and CT-10C-T3C (variant 4b). We therefore extended the signature numbering system of Pedersen et al [10] to enable differentiation by inclusion of the eight-base flanking regions. Two samples which were genotypes E and J, had a new CT1299 variant (which we have designated as variant 9; Table 3). This variant is common in LGV strains, which may indicate a closer linkage of particular genital tract strains to LGV than others.


Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples.

Wang Y, Skilton RJ, Cutcliffe LT, Andrews E, Clarke IN, Marsh P - PLoS ONE (2011)

Distribution of different VNTR types according to ompA genotype (where loci appeared mixed in individual samples, both numerals are given separated by a ‘/’).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037941&req=5

pone-0016971-g002: Distribution of different VNTR types according to ompA genotype (where loci appeared mixed in individual samples, both numerals are given separated by a ‘/’).
Mentions: There were different MLVA types within each genotype, including the two genotype D variants (Figure 2). Overall, the combination of the ompA and MLVA as a genotyping panel gave, for this set of results, a Simspon's discriminatory index [15] of 0.96. Of the eight samples which could only be genotyped following culture (Table S3), five (MLVA-ompA types: 3.2.4-K; 3.6a.3-D/UW-3; 12.5a.2-G; 3.4a.2-J; and 8.8.2-F) were unique genotypes which had no replicates that were also detectable directly from the original DNA samples. Some MLVA signatures were common to different ompA types, for example 8.5.2 was found in ompA types D (both sub-types), E, F and J; whilst 8.6.2 was found in D/IC-CAL8, E, and F. In most cases however, MLVA signatures were unique to ompA types. As well as defining some new VNTR sequences for the three loci (Table 3), we further noted that in some cases there were differences in the flanking regions between two samples where the VNTR locus sequence itself was identical. For example, VNTR CT1299 variant 4 (Table 3) has a tandem repeat of ten cytosine residues (10C). In our survey, we found three versions of CT1299 variant 4 with 10C, with three different sequences between the conserved flanking regions, namely 10C (variant 4), 10C-T3C (variant 4a) and CT-10C-T3C (variant 4b). We therefore extended the signature numbering system of Pedersen et al [10] to enable differentiation by inclusion of the eight-base flanking regions. Two samples which were genotypes E and J, had a new CT1299 variant (which we have designated as variant 9; Table 3). This variant is common in LGV strains, which may indicate a closer linkage of particular genital tract strains to LGV than others.

Bottom Line: Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture.Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology and Infection, School of Medicine, University of Southampton, Southampton, United Kingdom.

ABSTRACT

Background: Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV) and the genitourinary genotypes D-K. Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.

Principal findings: We evaluated variable number tandem repeat (VNTR) and ompA sequencing (now called multi-locus VNTR analysis and ompA or "MLVA-ompA") to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM) clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche) PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59%) were fully genotyped by MLVA-ompA. Only one mixed infection (E & D) in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.

Conclusions: Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.

Show MeSH
Related in: MedlinePlus