Chest
Original ResearchLung InfectionClinical Efficacy of Direct DNA Sequencing Analysis on Sputum Specimens for Early Detection of Drug-Resistant Mycobacterium tuberculosis in a Clinical Setting
Section snippets
Study Subjects
In total, 111 patients were enrolled from Hallym University Medical Center, Korea, between October 2000 and December 2006, and their medical records were retrospectively reviewed.
A total of 113 sputum specimens from the 111 patients, who were suspected of drug-resistant TB by experienced pulmonologists and showed positive response to PCR for M tuberculosis, were used for direct DNA sequencing analysis for early detection of drug-resistant TB in the beginning of treatment regardless of acid-fast
Clinical Characteristics of the Study Subjects
The mean age of the study subjects was 49.3 ± 18.8 (mean ± SD) years with a range of 13 to 87 years; 72 (66.1%) of the subjects were men. Of 113 sputum specimens, 93 (82.3%) were smear positive, 18 (15.9%) were smear negative, and 2 (1.8%) were missed. The results of drug susceptibility tests were available from 93 culture-positive sputum specimens, except one missing value for INH resistance. A total of 41 clinical sputa (44.6%) showed resistance to one of the four drugs; 33 sputa (35.9%) were
Discussion
Most efforts to develop rapid detection of drug-resistant TB in the past few years have focused on the limited number of mutations reported in the literature as the most frequently mutated loci: rpoB516, rpoB526, rpoB531, katG315, and embB306.10, 11, 12, 13, 14 More than 90% of RIF-resistant M tuberculosis have sequence alterations in an 81-bp core region (codon 507-533) of the rpoB gene, which encodes RNA polymerase β-subunit.5, 6,14 Although a majority of the rpoB mutations in our study
Conclusion
Our study demonstrated that direct DNA sequencing analysis is a rapid and useful molecular genetic method for detecting drug resistance to INH, RIF, EMB, and PZA. The method can be used directly with sputum specimens from patients and gives interpretable results within 1 week. Furthermore, we showed good clinical application of the direct DNA sequencing results in a clinical setting. Further study with a larger cohort in a prospective manner is needed to elucidate the clinical efficacy of the
Acknowledgments
Author contributions: All of the authors provided final approval of the version to be published.
Dr Choi: contributed to analyzing and interpreting clinical data and drafting and revising the manuscript.
Dr K. W. Lee: contributed to performing molecular assays, analyzing and interpreting the results of molecular assays, and revising the manuscript critically for intellectual content.
Dr Kang: contributed to collecting, analyzing, and interpreting clinical data and revising the manuscript for
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2014, Journal of Infection and Public HealthCitation Excerpt :PCR amplification of target regions, followed by sequencing or pyrosequencing of amplicons, has also been used for the detection of MDR-TB and XDR-TB strains in culture isolates and clinical specimens. Direct DNA sequencing analysis in sputum (including smear-negative, culture-positive) samples detected 64%, 96%, 69%, 100%, and 50% of M. tuberculosis strains resistant to INH, RIF, EMB, PZA, and INH + RIF (MDR-TB), respectively [44]. Although complete genome sequencing is not yet practical with clinical specimens, it may become routine in the near future, enabling the detection of resistance-conferring mutations in multiple target genes of MDR-TB and XDR-TB strains [45].
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2012, European Journal of Medicinal ChemistryCitation Excerpt :The results of antimicrobial study indicated that the presence of electron withdrawing groups on the benzoic acid moiety improved antimicrobial activity [25,26]. Isoniazid (INH), rifampin (RIF), ethambutol (EMB) and pyrazinamide (PZA) are well known antituberculosis drugs and direct DNA sequencing analysis of their genes i.e., katG, rpoB, embB and pncA, respectively, has been reported as a rapid and useful molecular genetic method for the detection and treatment of drug-resistant Mycobacterium tuberculosis [27]. In present studies, an attempt is made to investigate the interaction of isonicotinic acid hydrazide (INH) and its two analogs; pyrazine carboxylic acid hydrazide (PCH) and 2,4-dihydroxy benzoic acid hydrazide (2,4-DHBAH) (Scheme 1) with chicken blood double stranded (ds)-DNA (ck.DNA) using UV–Vis spectroscopic (UV) and cyclic voltammetric (CV) techniques at stomach (4.7) and blood (7.4) pH under body temperature (37 °C).
Molecular characterization of drug-resistant and -susceptible Mycobacterium tuberculosis isolated from patients with tuberculosis in Korea
2012, Diagnostic Microbiology and Infectious DiseaseCitation Excerpt :Park et al. (2005) reported that, among 231 MDR strains, 45.5% (n = 105), 32.5% (n = 75), and 10.8% (n = 25) possessed the point mutations in codon 531, 526, and 516, respectively. Also, a recent study with direct DNA sequencing analysis revealed that 39 clinical sputa had 1 or more single-point mutations in the rpoB gene and the most prevalent mutation was Ser531Leu (66.7%) (Choi et al., 2010). Between these results and our result, there was little difference in the relative frequency of the mutations, presumably due to the differences in sample collection periods and regions.
Rapid molecular detection of tuberculosis and rifampicin drug resistance: Retrospective analysis of a national UK molecular service over the last decade
2012, ThoraxCitation Excerpt :Routinely, specimens received at the NMRL are first cultured and then identified as MTBC using GenoType-Series molecular assay or DNA sequencing. The advantage of ‘Fastrack’ is that TB and MDRTB can be diagnosed within a day (permitting appropriate clinical, infection control and public health action, and improving patient outcomes2–4) and the specimens are subjected to rapid automated culture. Specimens known to have MDRTB produce cultures that can then be analysed for all first-line and reserve drugs simultaneously.
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