Direct detection of isoniazid resistant mycobacterium tuberculosis in respiratory specimens by real time PCR

Abstract

Tuberculosis (TB) is one of the major global health problems with high mortality rate. With emerging drug resistance, multi-drug resistant tuberculosis (MDR-TB) is becoming a major public health issue. With advanced molecular technology, rapid and accurate detection for Mycobacterium tuberculosis (MTB) and drug resistance are possible in diagnostic laboratory.

This study aimed to develop a molecular protocol for rapid and accurate detection for Isoniazid (INH)-resistant MTB in direct respiratory specimens, which can help clinician for treatment planning and better patient management.

MTB was first detected by an in-house single-tube nested real-time PCR targeting IS6110 gene, which is an insertion genetic element found in MTB complex. INH resistance of MTB positive samples were subsequently analyzed by another in-house duplex HybProbe assay targeting katG S315T (AGC-ACC) mutation and MabA-15 CT mutation. MTB culture and drug susceptibility test (DST) were used as gold standard for evaluation of the performance of the assays. AFB smear results of the cases were also included for analysis.

A total of 200 respiratory specimens, including 177 sputum, 17 bronchial aspirate and 6 bronchoalveolar lavage, were collected from Pamela Youde Nethersode Eastern Hospital during the period from June to December 2016. Among the 200 respiratory specimens, IS6110 gene of MTB was detected in 65 samples by PCR. In comparing to MTB culture, the overall specificity, sensitivity, PPV (positive predictive value) and NPV (negative predictive value) of the PCR assay were 98.5%, 96.9%, 98.5% and 96.9% respectively. In AFB smear positive cases, sensitivity and PPV were 100%, indicating the test was having excellent performance on detection of MTB in smear positive specimens. In AFB smear negative cases, the sensitivity and the specificity were 96% and 98.5%. Besides, the internal control of the assay revealed that no PCR inhibition was found during the study.

INH-resistance genotypes of the 65 MTB positive cases were further evaluated by the HybProbe assay which targeted katG S315T and MabA-15 mutations. Mutant and wildtype were differentiated by the differences in melting temperature, with mutant at 60℃ and wildtype at 68℃. Among the 65 MTB positive cases, 23 cases were failed in amplification due to low concentration of DNA input. The failure rate was 35.3%. The failed cases were INH sensitive according to DST result. For the 42 cases with successful amplification, 3 were found to have katG S315T (AGCACC) mutation, which was confirmed by DST. The remaining samples were found to be wildtype in katG S315T and MabA-15 position. Comparing the PCR result with DST result, 100% specificity, sensitivity, PPV and NPV were obtained in smear positive cases. The specificity, sensitivity, PPV and NPV were also 100% for smear negative specimens but there were 35.3% cases.

Turnaround time of the molecular protocol was compared with MTB culture and DST for identification of INH-resistant MTB. The average time required for the molecular protocol was found to be 1.5-2 days. The average time for the conventional laboratory protocol comprising MTB culture and DST required around 9 weeks. Therefore the turnaround time was a lot improved with the help of the molecular protocol in INH resistance MTB detection.

In conclusion, the finding of the study suggested that the molecular protocol was having satisfactory performance that the sensitivity and specificity were high. Also, the turnaround time of the molecular protocol was found to be greatly shorter than MTB culture and DST for the detection of INH resistant MTB. Therefore, it is believed that with this molecular protocol, rapid and accurate detection for INH-resistant MTB in direct respiratory specimens is possible. It enables clinician to make accurate treatment planning, hence improving patient management.