Rapid detection of Mycobacterium tuberculosis DNA and genetic markers for Isoniazid resistance in Ziehl-Neelsen stained slides

BACKGROUND Early diagnosis of tuberculosis (TB) and identification of strains of Mycobacterium tuberculosis resistant to anti-TB drugs are considered the main factors for disease control. OBJECTIVES To standardise a real-time polymerase chain reaction (qPCR) assay technique and apply it to identify mutations involved in M. tuberculosis resistance to Isoniazid (INH) directly in Ziehl-Neelsen (ZN) stained slides. METHODS Were analysed 55 independent DNA samples extracted from clinical isolates of M. tuberculosis by sequencing. For application in TB diagnosis resistance, 59 ZN-stained slides were used. The sensitivity, specificity and Kappa index, with a 95% confidence interval (CI95%), were determined. FINDINGS The agreement between the tests was, for the katG target, the Kappa index of 0.89 (CI95%: 0.7-1.0). The sensitivity and specificity were 97.6% (CI95%: 87.7-99.9) and 91.7% (CI95%: 61.5-99.5), respectively. For inhA, the Kappa index was 0.92 (CI95%: 0.8-1.0), the sensitivity and specificity were 94.4% (CI95%: 72.7-99.8) and 97.3% (CI95%: 85.8-99.9), respectively. The use of ZN-stained slides for drug-resistant TB detection showed significant results when compared to other standard tests for drug resistance. MAIN CONCLUSIONS qPCR genotyping proved to be an efficient method to detect genes that confer M. tuberculosis resistance to INH. Thus, qPCR genotyping may be an alternative instead of sequencing.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis from Korea / 대한임상병리학회지

BACKGROUND: The genetic basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has been attributed to at least four genes. Mutations of the katG gene encoding catalase-eroxidase have been shown to cause resistance to INH. Among these mutations, the point mutation of codons 315 and 463 are frequently found. To determine whether simple screening method could potentially be useful for the detection of INH-esistant strains, we investigated the presence of mutation of codons 315 and 463 of katG gene in INH-esistant M. tuberculosis from Korea. METHODS: We used the polymerase chain reaction (PCR) and direct sequencing analysis to detect the point mutation of codons 315 and 463 of katG gene in 48 strains of INH-esistant and 10 strains of INH-usceptible M. tuberculosis. RESULTS: No amplification product was generated from 2 of 48 INH-esistant strains. Among the remaining 46 isolates, point mutations at codons 315 and 463 were identified in 19 isolates (41.3%) and 40 isolates (87.0%), respectively. Among the 10 INH-usceptible strains, the codon 463 point mutation was identified in 7 isolates, however, no point mutation of the codon 315 was found. CONCLUSIONS: These results suggest that the point mutation of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance. Although the mutation of codon 315 of katG gene was limited to 41.3% of INH-esistant isolates, further investigation of the codon 315 of katG gene should lead to increased understanding of resistance genes and the deveolpment of rapid molecular detection methods.

Molecular analysis of katG gene mutations in strains of Mycobacterium tuberculosis from Korea / 대한임상병리학회지

BACKGROUND: The genetic basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has been attributed to at least four genes. Mutations of the katG gene encoding catalase-eroxidase have been shown to cause resistance to INH. Among these mutations, the point mutation of codons 315 and 463 are frequently found. To determine whether simple screening method could potentially be useful for the detection of INH-esistant strains, we investigated the presence of mutation of codons 315 and 463 of katG gene in INH-esistant M. tuberculosis from Korea. METHODS: We used the polymerase chain reaction (PCR) and direct sequencing analysis to detect the point mutation of codons 315 and 463 of katG gene in 48 strains of INH-esistant and 10 strains of INH-usceptible M. tuberculosis. RESULTS: No amplification product was generated from 2 of 48 INH-esistant strains. Among the remaining 46 isolates, point mutations at codons 315 and 463 were identified in 19 isolates (41.3%) and 40 isolates (87.0%), respectively. Among the 10 INH-usceptible strains, the codon 463 point mutation was identified in 7 isolates, however, no point mutation of the codon 315 was found. CONCLUSIONS: These results suggest that the point mutation of codon 463 of katG gene of M. tuberculosis may be a polymorphism not related with INH resistance. Although the mutation of codon 315 of katG gene was limited to 41.3% of INH-esistant isolates, further investigation of the codon 315 of katG gene should lead to increased understanding of resistance genes and the deveolpment of rapid molecular detection methods.