Molecular Characterization of Mutations in Isoniazid- and Rifampicin-Resistant Mycobacterium tuberculosis Isolated in Thailand.

The control of drug-resistant tuberculosis (TB) is a major challenge. The frequency and mutation characteristics indicate the efficiency of molecular tests for the rapid detection of TB drug resistance. This study examined the existence of katG and inhA mutations for isoniazid (INH) resistance and rpoB mutations for rifampicin (RFP) resistance. In total, 178 drug-resistant Mycobacterium tuberculosis (MTB) isolates were analyzed. Mutations in katG encoding and inhA regulatory regions were detected in 136/168 (81.0%) and 29/168 (17.3%), respectively, with the most prominent mutation of Ser315Thr substitution in katG in 126/168 (75.0%), and -15 C to T substitution in the regulatory region of the inhA (26/168; 15.5%). Two distinct katG mutations (Tyr337Cys, 1003InsG) were identified. Of 125 RFP-resistant isolates, 118 (94.4%) carried mutations affecting the 81-bp RFP resistance-determining region, with the most commonly affected codons 450, 445, and 435 identified in 74 (59.2%), 26 (20.8%), and 12 (9.6%) isolates, respectively. Genetic mutations were highly associated with phenotypic INH and RFP resistance, and the majority shared similarities with those reported in previous studies in Thailand and other Asian countries. These data are useful for guiding the use and improvement of molecular tests for TB drug resistance.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand.

Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.

Evaluation of an in-house loop-mediated isothermal amplification for Mycobacterium tuberculosis detection in a remote reference laboratory, Thailand

ABSTRACT Loop-mediated isothermal amplification (LAMP) is a simple and efficient nucleic acid amplification method for the rapid diagnosis of infectious diseases. This study assessed the performance of an in-house LAMP for tuberculosis (TB) diagnosis at a remote reference laboratory in the endemic setting of Thailand. As part of the routine service, 1,882 sputum samples were processed for mycobacterial culture in Lowenstein-Jensen and MGIT media. The DNA was extracted from the remaining decontaminated samples after the culture procedure for real-time polymerase chain reaction (PCR) analysis using Anyplex plus MTB/NTM detection kit. 785 (40.28%) were positive by mycobacterial culture. Of these, 222 DNA remnants were available and subjected to LAMP analysis. Based on culture as reference (Mycobacterium tuberculosis; MTB= 209/ non-tuberculous mycobacteria; NTM= 13), the overall sensitivity of LAMP and Anyplex plus assays for MTB detection were 89.95% (188/209; 95% confidential interval [CI]: 85.05-93.67%) and 96.65% (202/209; 95% CI: 93.22-98.64%), and the accuracy values were 88.74% (197/222; 95% CI: 83.83-92.58) and 96.40% (214/222; 93.02-98.43%), respectively. The sensitivity and accuracy of the in-house LAMP and the Anyplex plus real-time PCR assay were high in comparison to culture results. The high sensitivity and accuracy suggested that this in-house LAMP was promising and might be useful for early TB diagnosis.