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ABSTRACT: Macrolide treatment failure in syphilis patients is associated with a single point mutation (either A2058G or A2059G) in both copies of the 23S rRNA gene in T. pallidum strains. The conventional method for the detection of both point mutations uses nested PCR combined with restriction enzyme digestions, which is laborious and time-consuming. We initially developed a TaqMan-based real-time duplex PCR for detecting the A2058G mutation and upon discovery of the A2059G mutation we modified the assay into a triplex format to simultaneously detecting both mutations. The point mutations detected by the real-time triplex PCR were confirmed by pyrosequencing. A total of 129 T. pallidum PCR-positive specimens obtained from an azithromycin resistance surveillance study conducted in the U.S. were analyzed. Sixty-six of 129 (51.2%) samples were identified with the A2058G mutation by both real-time PCR assays. Of the remaining 63 samples that were identified as having a macrolide-susceptible genotype by the duplex PCR assay, 17 (27%) were found to contain the A2059G mutation using the triplex PCR. The proportion of macrolide-susceptible versus -resistant genotypes harboring either the A2058G or A2059G mutation among T. pallidum strains was 35.6%, 51.2% and 13.2%, respectively. None of the T. pallidum strains examined had both point mutations. The TaqMan-based real-time triplex PCR assay offers an alternative to conventional nested PCR and restriction fragment length polymorphism (RFLP) analysis for rapidly detecting both point mutations associated with macrolide resistance in T. pallidum.
Journal of clinical microbiology 01/2013; · 4.16 Impact Factor
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ABSTRACT: Highly sensitive and specific nucleic acid amplification tests (NAATs) have emerged as the gold standard diagnostic tests for many infectious diseases. Real-time PCR has further refined the technology of nucleic acid amplification with detection in a closed system and enabled multiplexing to simultaneously detect multiple pathogens. It is a versatile, fast, and high-throughput system for pathogen detection that has reduced the risk of PCR contamination, eliminated post-PCR manipulations, and improved the cost-effectiveness of testing. In addition, real-time PCR can be applied to self-collected noninvasive specimens. Here, we describe an in-house developed TaqMan-based real-time multiplex PCR (M-PCR) assay for the diagnosis of sexually transmitted genital ulcer disease (GUD) and discuss briefly on issues associated with validation of assay performance.
Methods in molecular biology (Clifton, N.J.) 01/2012; 903:103-12.
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ABSTRACT: Formalin-fixed paraffin-embedded (FFPE) tissue blocks are routinely used for histopathological examination and are also useful for specific pathogen detection by polymerase chain reaction (PCR). FFPE tissue is stable at ambient temperature for an extended period of time and relatively easy to transport compared to fresh tissue, which has to be processed or frozen immediately. In addition, archival material is an invaluable source for retrospective molecular and clinical investigation. This chapter describes detailed procedures for nucleic acid extraction and PCR detection of Treponema pallidum using FFPE tissue.
Methods in molecular biology (Clifton, N.J.) 01/2012; 903:295-306.
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ABSTRACT: We report a case of yaws in a patient with puritic cutaneous eruption who was initially suspected of infection with monkeypox. The diagnosis was established by real-time PCR and sequencing of specific treponemal DNA sequences. This is the first report describing the use of DNA sequencing to identify Treponema pallidum subsp. pertenue-specific sequences in a patient with active yaws.
Journal of clinical microbiology 09/2011; 49(11):4013-5. · 4.16 Impact Factor
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ABSTRACT: To develop and evaluate a real-time quadriplex PCR for the diagnosis of lymphogranuloma venereum (LGV) and non-LGV chlamydial infections using rectal swab specimens.
The design of the real-time quadriplex PCR assay incorporates an LGV-specific, a non-LGV-specific target sequence, a Chlamydia trachomatis plasmid target, and the human RNase P gene as an internal control. The performance of the quadriplex PCR was compared with a previously reported real-time duplex PCR assay on which LGV diagnosis was based on exclusion.
Very good agreement (85 of 89 specimens, 95.5%) was found between the two multiplex PCR assays for the detection of C trachomatis DNA (kappa value 0.93, 95% CI 0.86 to 0.99). Both assays identified 34 LGV, 35 non-LGV C trachomatis and 16 negative specimens. Of two specimens that tested positive for non-LGV by the duplex PCR, one was found to be a mixed infection and the other was positive only for plasmid and RNase P targets by the quadriplex PCR. Two additional specimens that had equivocal results for non-LGV by the duplex PCR also tested positive only for plasmid target and human DNA by the quadriplex PCR. In addition, six specimens that tested negative by the duplex PCR assay were found to be invalid when using the quadriplex PCR.
A real-time quadriplex PCR assay has been developed that is capable of detecting LGV, non-LGV, or mixed infections simultaneously in rectal specimens. The assay also contains a supplemental amplification target for the confirmation of C trachomatis infection as well as a human DNA control for monitoring sample adequacy and PCR inhibition.
Sexually transmitted infections 03/2008; 84(4):273-6. · 2.18 Impact Factor
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ABSTRACT: The objectives of this study were to evaluate the use of a real-time multiplex polymerase chain reaction (M-PCR) assay to differentiate between trachoma and lymphogranuloma venereum (LGV) biovars of Chlamydia trachomatis and to validate its performance with the conventional genotyping method.
Swab specimens from 115 patients with anorectal symptoms or syndromes associated with LGV were tested by a real-time M-PCR assay and the results compared with the PCR-based restriction fragment length polymorphism analysis of the major outer membrane protein gene (omp1).
A high agreement of 96.5% (111 of 115 specimens) was found between the real-time M-PCR testing and the standard genotyping method for the detection of C. trachomatis DNA (kappa value, 0.945, P <0.00001). Both methods identified 53 LGV, 32 non-LGV C. trachomatis, and 26 negative specimens.
The real-time M-PCR assay simultaneously detects and differentiates LGV from non-LGV strains using swab specimens. This assay offers a relatively rapid and sensitive alternative for the diagnosis of LGV infection and is a useful tool for screening and for outbreak investigations.
Sex Transm Dis 07/2007; 34(7):451-5. · 2.87 Impact Factor
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ABSTRACT: The objective of this study was to determine predictors of Trichomonas vaginalis among women and their partners in Moshi, Tanzania.
Women (N = 1440) and their partners (N = 588) were interviewed and specimens for detection of T. vaginalis and sexually transmitted infections (STIs) were collected.
Prevalence of T. vaginalis was 10.7% in women and 6.3% in men. Having a partner with T. vaginalis was the strongest risk factor in women (adjusted odds ratio [OR], 19.44; 95% confidence interval [CI], 7.84-48.25) and men (adjusted OR, 19.01; 95% CI, 6.8-52.40). Risk of T. vaginalis infection was increased in subjects with less education. Other risk factors in women were daily alcohol consumption, being separated, reporting infertility problems, having a partner who had children with other women, and other STIs; and in men, the risk factor was having no income. T. vaginalis was not associated with HIV-1 in women and men.
Prevention of T. vaginalis and other STIs among couples is a major priority. Reduction of alcohol consumption in women is an important intervention.
Sex Transm Dis 01/2007; 33(12):712-8. · 2.87 Impact Factor
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ABSTRACT: We report on a case of gastric syphilis in a patient with chronic dyspepsia. The diagnosis was established by serology and the demonstration of spirochetes in diffusely inflammed gastric mucosa by staining with a fluorescent monoclonal antibody specific for pathogenic treponemes and by the detection of specific treponemal DNA sequences by a real-time PCR.
Journal of Clinical Microbiology 10/2006; 44(9):3452-6. · 4.15 Impact Factor
