A multitarget real-time PCR (MRT-PCR) for detection of

We have developed and validated a nested real-time PCR (NRT-PCR) for the genotyping of Chlamydia trachomatis and used it specifically for the typing of either eight genovars from D to K or three genovars of lymphogranuloma venereum (LGV). The 11 probes used in the NRT-PCR correctly identified the DNA from D to K and LGV reference strains and did not cross-react with the DNA from 26 strains representing the bacterial pathogens and commensals of the oropharynx, genital tract, and rectum. The NRT-PCR had a 95% probability of detection at four genome copies (confidence interval, three to six copies) of C. trachomatis per reaction. One hundred cervical and urethral swab specimens containing C. trachomatis DNA from 63 women and 37 men were used to validate the method. The results from the NRT-PCR and the DNA sequencing of amplicons generated from the omp1 gene showed 100% correlation for these samples. The assay also identified the LGV-II genotype in 24 of 48 rectal swab specimens containing C. trachomatis DNA that were obtained from men having sex with men. The Sexually Transmitted Bacteria Reference Laboratory, London, independently confirmed these results using group-specific LGV real-time PCR and restriction fragment length polymorphism analysis. Compared with the NRT-PCR, non-NRT-PCR was found to be less sensitive: it typed C. trachomatis DNA in only 80% of the genital samples and 90% of the rectal swab samples. This is the first successful demonstration of the use of real-time PCR for the genotype-specific typing of C. trachomatis strains that cause sexually transmitted diseases.

Background/objectives: The polymer conjugate enhanced enzyme immunoassay (IDEIA) and Cobas Amplicor polymerase chain reaction Chlamydia trachomatis (CT) (Amplicor PCR) are two commonly used assays for the diagnosis of CT infection. The performance of these assays was compared for the diagnosis of genital CT infection among 1000 consecutive patients attending a genitourinary medicine (GUM) clinic. Confirmation of positive results and the clinical significance of the absence of cryptic plasmid in chlamydia on the diagnosis of infection by Amplicor PCR were also investigated. Methods: IDEIA, Amplicor PCR, and two nested in-house PCR assays targeting cryptic plasmid and omp1 gene were performed on all samples. DNA from Amplicor PCR negative samples was pooled for in-house PCR assays. Each pool contained DNA from seven Amplicor PCR negative samples. Results: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and efficiency of IDEIA in the diagnosis of genital CT infection were 80%, 97%, 80%, 97%, and 95%, respectively. Sensitivity, specificity, PPV, NPV and efficiency of Amplicor PCR were 99%, 98%, 89%, 100%, and 98%, respectively. 16 (11%) of 144 Amplicor PCR positive results were identified as false positive by in-house PCR assays. No isolate of plasmid free CT was detected among the study population. Conclusions: IDEIA should not be used for the diagnosis of CT infection because of its poor sensitivity. Although the analytic specificity of Amplicor PCR was 98%, because of the adverse medical, social, and psychological impact of false positive results for patients, confirmation of Amplicor PCR positive results by a different assay with comparable sensitivity is essential. Amplification assays targeting cryptic plasmid are appropriate for the diagnosis of genital CT infections.

High-risk subtypes of human papillomavirus (HPV) are the main causative agents of cervical cancer, for which Chlamydia trachomatis (CT) may sometimes be a co-factor. Vaccines have been developed against some subtypes of human papillomavirus and a vaccine against CT is in development. The objective of this study was to determine the prevalence of the subtypes of HPV and CT in genitourinary (GU) medicine clinic attenders. In total, 1000 consecutive patients attending the GU clinic participated in this anonymized point-prevalence study. Urethral swabs from 437 men and urethral plus cervical swabs as a single specimen from 563 women were tested for the subtypes of both organisms. Nested major outer membrane protein (MOMP) polymerase chain reaction detected CT chromosomal DNA in 44/437 (10%) of the men and 73/563 (13%) of the women. Genotypes E, F, and D were the most common. In all, 55/437 (13%) of men and 244/563 (43%) of women were infected with at least one high-risk HPV type. In conclusion, the new HPV vaccines, Gardasil and Cervarix, would have protected against 58% and 45%, respectively, of the high-risk subtypes found in women in this population. The rate of high-risk HPV infection (43%) found in women in this study raises concern.

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