Detection of Rifampicin Resistance in Mycobacterium tuberculosis by Using Middlebrook 7H9 Broth Medium with 2,3-Diphenyl-5-Thienyl-(2)-Tetrazolium Chloride / 대한임상미생물학회지

BACKGROUND: A simple and cost-effective method is needed for the detection of rifampicin resistance in Mycobacterium tuberculosis in resource-limited settings. We suggest a broth medium-based method using 2,3-diphenyl-5-thienyl-(2)-tetrazolium chloride (STC) for detection of rifampin resistance of tubercle bacilli within a reasonable time frame. METHODS: The type strain (M. tuberculosis H37Rv) and 45 cultured clinical strains of M. tuberculosis (35 rifampin-susceptible and 10 rifampin-resistant) were used. Phenotypes of rifampicin resistance were tested by the Korea Institute of Tuberculosis, and confirmed by GenoType MTBDRplus (Hain Lifescience, Germany). Susceptibility tests were performed using STC-containing OADC-enriched Middlebrook 7H9 broth (BD, USA). RESULTS: All tests were finished in 3 to 6 days. The same results were obtained with the standard and current methods for all 45 clinical isolates (100% sensitivity and specificity for resistance detection). CONCLUSION: The current method using STC is a good alternative for detecting M. tuberculosis rifampin resistance in a cost-effective and timely fashion, which is particularly important in resource-limited settings.

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis).</p><p><b>METHODS</b>12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay.</p><p><b>RESULTS</b>The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively.</p><p><b>CONCLUSION</b>Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.</p>

Establishment and application of multi-PCR-SSCP assay for rapid detection of isoniazid- and rifampin-resistance Mycobacterium tuberculosis isolates / 中华微生物学和免疫学杂志

Objective To establish multi-PCR-single strand conformational polymorphism analysis (mPCR-SSCP) for rapid detection of isoniazid (INH) and rifampin (RIF) resistance associated katG,inhA and rpoB genes of Mycobacterium tuberculosis isolates.Methods The INH-and RFP-resistance of 134 isolates was determined by using drug susceptibility test.The primers were designed for detecting INH and RFP resistance-associated katG,inhA and rpoB gene in the isolates by mPCR-SSCP.PCR-DS technique was applied to detect the mutations in katG,inhA and rpoB genes.All the results from different assays were subsequently analyzed as well as compared.Results All of the 134 tested isolates had katG,inhA and rpoB genes.Of the 134 isolates,42 (31.3％) and 45 (33.6％) strains were INH-and RFP-resistant,respectively.The results of mPCR-SSCP and PCR-DS showed that all the 92 INH-susceptible isolates had no mutation in katG and inhA genes with 100％ specificity.In the 89 RFP-susceptible isolates,2 and 1 had mutation in rpoB genes confirmed by mPCR-SSCP and PCR-DS with 97.8％ or 98.9％ specificity,respectively.Among the 42 INH-resistance isolates,33 and 36 strains had the mutations in katG and/or inhA genes due to the results of mPCR-SSCP and PCR-DS with 78.6％ or 85.7％ sensitivity,respectively.The results of mPCR-SSCP and PCR-DS also demonstrated that in the 45 RFP-resistance isolates,41 and 43 strains had the mutations in rpoB gene with 91.1％ or 95.6％ sensitivity,respectively.Conclusion The mPCR-SSCP established in this study can be used to rapidly detect INH and RFP-resistance associated mutations in katG,inhA and rpoB genes of M.tuberculosis with convenience,specificity and sensitivity,which shows a good prospect for application in clinic.

Direct Detection of Rifampin-Resistant Mycobacterium Tuberculosis by Polymerase Chain Reaction and Line Probe Assay in Sputums / 대한임상병리학회지

BACKGROUND: Recently, the control of tuberculosis may be threatened by widespread emergence of multidrug-resistant tuberculosis. Rifampin resistance is considered as a useful marker of multidrug-resistant tuberculosis and is developed by missense mutations in region of the rpoB gene encoding the beta-subunit of RNA polymerase. This study aimed to evaluate the line probe assay for direct detection of rifampin-resistant Mycobacterium tuberculosis in clinical samples. METHODS: Twenty-seven smear-positive and 13 smear-negative clinical samples, 13 M. tuberculosis strains and 2 nontuberculous mycobacteria strains were analyzed with line probe assay (Innogenetics, Belgium) and results were compared with conventional susceptibility test. I also reviewed conventional antimicrobial susceptibility testing patterns of 128 M. tuberculosis isolates from January 1996 to March 1997. RESULTS: All (100%) of total 30 patients who had rifampin-resistant strains revealed 3 or more drug resistances, but 5 (5%) out of 88 patients who had rifampin-sensitive strains revealed 2 or more drug resistances. By the line probe assay, 3 out of 13 smear-negative samples, 6 out of 27 smear-positive samples and 6 out of 13 M. tuberculosis strains were rifampin-resistant. Overall concordance of the line probe assay with conventional susceptibility test was 100%. Most common mutation site of rpoB gene was codon 531 (53.3%) followed by codon 526 (40%) and codon 516 (6.7%). CONCLUSIONS: These suggest that rifampin resistance will be a useful marker of multidrug- resistant tuberculosis and line probe assay will be a rapid and direct diagnostic tool for the detection of rifampin-resistant M. tuberculosis in clinical samples, especially AFB smear-negative samples.