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Objectives: Buruli ulcer (BU) is an infected cutaneous lesion, the etiological agent of which is Mycobacterium ulcerans. Diagnosis is confirmed by the identification of acid-fast bacilli and culture. In clinically suspicious forms with negative bacteriological or Ziehl-Neelsen (ZN) findings, molecular tests are used. This study compared the concordance of nested polymerase chain reaction (PCR) (targeting IS2404) and PCR (targeting IS2606) in different clinical situations., Methods: A total of 57 samples were sourced from 39 BU patients. Control samples (n = 43) were obtained from non-BU ulcers in 38 patients. Samples were divided into two pieces and submitted to, respectively, histological examination and ZN staining, and PCR. Subsamples submitted to PCR were divided and submitted to nested PCR IS2404 and PCR IS2606, respectively., Results: Of the 57 BU biopsies, positive results were obtained by nested PCR in 18 (31.6%) and by IS2606 PCR in 37 (64.9%) cases. Sequencing of the positive samples confirmed the specificity of amplicons in all nested PCR samples and in 26 of 37 (70.2%) samples positive to IS2606. Hence, nested PCR was more specific (100% vs. 93%) and less sensitive (32% vs. 46%) than IS2606 PCR. In the BU samples, nested PCR was negative in 15 instances, and IS2606 PCR was negative in 11 instances in which ZN histology had been positive (false negatives). Both PCRs were positive in six ZN-negative smears., Conclusions: We considered 57 samples from 39 BU patients in various clinical stages and at different times after the beginning of therapy. These provided positive results in 18 cases with IS2404 nested PCR and in 37 cases with PCR IS2606; only 26 of the latter remained positive subsequent to sequencing. Hence, even if IS2404 PCR is considered more specific, in subjects who appear to fail to respond to therapy, it is advisable to also carry out IS2606 PCR. A possible interpretation of the discordance between the two techniques due to unavoidable technical errors as well as to different sensitivity of the two tests at M. ulcerans DNA low concentration (i.e. in recent infection and in well-treated cases) is discussed., (© 2013 The International Society of Dermatology.)

An evaluation of the utility of rep PCR typing compared to the 15 loci discriminatory set of MIRU-VNTR was undertaken. Twenty-nine isolates of Mycobacterium tuberculosis from patients were examined. Genomic DNA was extracted from the isolates by standard method. The number of copies of tandem repeats of the 15 MIRU-VNTR loci was determined by PCR amplification and agarose gel electrophoresis of the amplicons. M. tuberculosis outbreak-related strains were distinguished from other isolates. MIRU-VNTR typing identified 4 major clusters of strains. The same isolates clustered together after RFLP typing, but rep-PCR identified only 3 of them. The concordance between RFLP and MIRU-VNTR typing was complete, with the exception of two isolates with identical RFLP patterns that differed in the number of tandem repeat copies at two MIRU-VNTR alleles. A further isolate, even sharing the same RFLP pattern, differed by one repeat from the rest of its cluster. We also tested the use of an automated rep-PCR for clinical laboratory applications but it failed to identify the link between two pairs of epidemiologically related strains clustered by the other 2 techniques. For superior discrimination, ease of comparison of results and lower cost, MIRU-VNTR typing should be the favored PCR-based typing tool.

We have developed a Real-Time PCR assay to detect M. tuberculosis using the iCycler iQ detection system by TaqMan assay directly on the clinical specimen. A total of 513 clinical samples were taken from patients with suspected tuberculosis and other patients that had an active mycobacterial infection, as well as patients with diagnosed tuberculosis who were receiving antitubercular therapy. The sensitivity and specificity of this assay, 10% and 100%, respectively, were compared to those of conventional microbiological methods.

Objectives: Tuberculosis (TB) and drug-resistant TB (DR-TB) are national health burdens in Vietnam. In this study, we investigated the prevalence of rifampicin (RIF) and/or isoniazid (isonicotinic acid hydrazide, INH) resistance in patients with suspected TB, and applied appropriate techniques to help rapidly target DR-TB. Methods: In total, 1,547 clinical specimens were collected and cultured using the BACTEC MGIT system (Becton Dickinson and Co.). A resazurin microtiter assay (REMA) was used to determine the proportions of RIF and/or INH resistance. A real-time polymerase chain reaction panel with TaqMan probes was employed to identify the mutations of rpoB and katG associated with DR-TB in clinical isolates. Genotyping of the identified mutations was also performed. Results: A total of 468 Mycobacterium tuberculosis isolates were identified using the REMA. Of these isolates, 106 (22.6%) were found to be resistant to 1 or both antibiotics. Of the resistant isolates, 74 isolates (69.8%) were resistant to isoniazid (INH) only, while 1 isolate (0.94%) was resistant to RIF only. Notably, 31 isolates (29.24%) were resistant to both antibiotics. Of the 41 phenotypically INH-resistant isolates, 19 (46.3%) had the Ser315Thr mutation. There were 8 different rpoB mutations in 22 (68.8%) of the RIF-resistant isolates. The most frequently detected mutations were at codons 531 (37.5%), 526 (18.8%), and 516 (6.3%). Conclusion: To help prevent new cases of DR-TB in Vietnam, it is crucial to gain a comprehensive understanding of the genotypic DR-TB isolates. [ABSTRACT FROM AUTHOR]