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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 ompA types discriminated according to whether they were genotyped directly from clinical samples, whether any of these were subsequently cultured, and those that could only be genotyped following culture (n = 93).
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pone-0016971-g001: Distribution of ompA types discriminated according to whether they were genotyped directly from clinical samples, whether any of these were subsequently cultured, and those that could only be genotyped following culture (n = 93).

Mentions: Prior to this study we tested a collection of Taq DNA polymerase types, and due to its high fidelity concluded that we should use Phusion Taq DNA polymerase (New England Biolabs UK, Hitchin, UK) for all PCR reactions. This is a high fidelity enzyme which we employed for several reasons, including to eliminate the possibility of PCR error in amplification of tandem repeats of monothymidines of greater than eleven base-pairs [13]. Direct genotyping from the DNA extracts used for the NAAT test that deployed the three VNTR loci and ompA was possible for 85 of the samples. The ompA sequences were aligned with 18 known reference ompA sequences from the GenBank sequence database [14]. The genotypes which most regularly produced a positive genotype were; D, E and F, comprising 20.4%, 40.9% and 16.1% of the genotyped positives respectively (Figure 1). Genotype D could be sub-divided into two divergent types by comparing the differing sequences available on the NCBI database. Therefore the proportion that were genotype D (20.4%) comprised 11.8% of the total which aligned with D/UW-3/CX, and 8.6% of the total which aligned with D/IC-CAL3. No genotype H was found.


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 ompA types discriminated according to whether they were genotyped directly from clinical samples, whether any of these were subsequently cultured, and those that could only be genotyped following culture (n = 93).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0016971-g001: Distribution of ompA types discriminated according to whether they were genotyped directly from clinical samples, whether any of these were subsequently cultured, and those that could only be genotyped following culture (n = 93).
Mentions: Prior to this study we tested a collection of Taq DNA polymerase types, and due to its high fidelity concluded that we should use Phusion Taq DNA polymerase (New England Biolabs UK, Hitchin, UK) for all PCR reactions. This is a high fidelity enzyme which we employed for several reasons, including to eliminate the possibility of PCR error in amplification of tandem repeats of monothymidines of greater than eleven base-pairs [13]. Direct genotyping from the DNA extracts used for the NAAT test that deployed the three VNTR loci and ompA was possible for 85 of the samples. The ompA sequences were aligned with 18 known reference ompA sequences from the GenBank sequence database [14]. The genotypes which most regularly produced a positive genotype were; D, E and F, comprising 20.4%, 40.9% and 16.1% of the genotyped positives respectively (Figure 1). Genotype D could be sub-divided into two divergent types by comparing the differing sequences available on the NCBI database. Therefore the proportion that were genotype D (20.4%) comprised 11.8% of the total which aligned with D/UW-3/CX, and 8.6% of the total which aligned with D/IC-CAL3. No genotype H was found.

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