Deciphering the sequential events during in vivo acquisition of drug resistance in Mycobacterium tuberculosis

Tuberculosis [TB] is caused by Mycobacterium tuberculosis [MTB] and the disease has remained a major health problem in most of the developing countries, particularly after the emergence of multidrug-resistant TB [MDR-TB]. The MDR-TB is an intriguing subject and very little is known about the in vivo processes which take place during the acquisition of MDR. This study describes a unique case of pulmonary TB [PTB] from which four sequential isolates of MTB could be isolated while the patient was on anti-tubercular treatment. The first baseline isolate was sensitive to all drugs, but the subsequent three isolates acquired resistance to multiple drugs and finally the patient died after 27 months post-diagnosis when his fourth isolate became resistant to isoniazid, rifampicin, ethambutol and kanamycin. All sequential cultures were identified as MTB using conventional and molecular methods, including 16s RNA sequencing and the spoligotyping. Spoligotyping followed by comparison with SITVITWEB database revealed that all the isolates belonged to the family of the Central Asian Strain Delhi [CAS1_Delhi, ST26] genotype, and no cross or mixed infections were observed. The drug resistance was further characterized at the molecular level by sequencing the target genes [katG, inhA, rpoB, embB, eis promoter region and rrs]. The results revealed mutated alleles associated with resistance to the respective drugs. This unique case indicates that it is possible to isolate MTB during treatment if the strain is acquiring resistance. The data presented from four sequential isolates provides an insight into what sequential genetic and proteomic changes occur in the bacteria during the in vivo acquisition of MDR.

Comparison of an in-house multiplex PCR with two commercial immuno-chromatographic tests for rapid identification and differentiation of MTB from NTM isolates

Species specific diagnosis of mycobacterial infection is crucial because treatment of infections caused by Mycobacterium tuberculosis [MTB] differs from that of non-tuberculous mycobacterial [NTM] species. The species identification used to be cumbersome and non-reproducible a decade ago. Recently, some commercial tests have been made available to differentiate the MTB and NTM growths in culture media. Sensitivity and specificity of these tests was evaluated. In this double blind study 572 clinical samples were cultured in an automated BACTEC-MGIT-960 system. A total of 147 [25.7%] samples were MGIT culture positive. These cultures were subjected to an in-house m-PCR [which amplifies hsp-65, esat-6 and ITS region for MAC], two commercial immune-chromatographic tests [ICTs] and phenotypic tests. Of the 147 MGIT positive cultures, m-PCR was able to correctly identify MTB in 123 cultures and NTM in 24 which included 3 MAC isolates. m-PCR showed 100% agreement with two gold standard methods-the nitrate reductase assay and PNB tests-in correctly identifying MTB. Commercial strips were able to correctly identify MTB in 120 [97.5%] of 123 cultures, while 3 [2.5%] isolates were falsely identified as NTM. However, none of the growth negative spent medium gave false positive results in any of the tests. None of the commercial strips misidentified any of the 24 NTM as MTB; hence, specificity of these strips was 100%. Of the 2 IC test systems, both SD Bioline and BD TBc strip tests missed 2.5% of MTB isolates and misidentified these as NTM. The in-house m-PCR was found to be the most accurate and efficient tool for identifying the MTB, MAC and other NTMs