Molecular characterization of pyrazinamide resistance in multi-drug-resistant tuberculosis

Abstract

Tuberculosis (TB) remains one of the major public health concerns with an enormous burden of disease, causing around 1.6 million deaths annually across the globe. The situation has become direr with the emergence of multidrug resistant tuberculosis (MDR-TB), which has dwarfed the effectiveness of the current anti-TB regimes and jeopardised the global effort on TB control. As such, new molecular diagnostic assays for rapid detection of MDR-TB are urgently needed. Moreover, despite the crucial role of pyrazinamide (PZA) plays in the treatment regime for both MDR-TB and drug-susceptible TB. So far, there has been little emphasis on implementing molecular assays for detecting PZA resistant TB in clinical routine service. The findings presented in this thesis aimed to bridge these research gaps.

The first part of the current study aimed to prospectively evaluate the Abbott MDR platform for direct detection of Mycobacterium tuberculosis complex (MTBC) and genetic markers for isoniazid (INH) and rifampicin (RIF) resistance using 610 clinical specimens. Against clinical and bacteriological background as the reference standard for TB diagnosis, Abbott MDR platform demonstrated an overall sensitivity and specificity of 95.2% and 99.8%, respectively. The genotypic INH and RIF resistance of 178 “MTBC detected” specimens were then analysed. Comparing to phenotypic drug susceptibility test (DST), Abbott MDR platform successfully detected genetic markers in 80% mono-RIF resistant, 66.7% mono-INH resistant and 84.6% MDR-TB specimens. Furthermore, two RIF resistant specimens harboured a novel nonsense mutation at rpoB codon position 513. Molecular model of the truncated RpoB protein demonstrated a loss of binding affinity toward rifapentine, suggesting the protein function can no longer be inhibited by the drug. The results demonstrated that Abbott MDR platform can provide rapid and reliable identification of MDR-TB in respiratory specimens.

In the last part of the study, an in house developed pncA sequencing for genotypic pyrazinamide resistance detection were first evaluated against 162 archived MTBC isolates with well-defined PZA susceptibility profiles. The results indicated 100% concordance between in house developed pncA sequencing and Mycobacterium growth indicator tube (MGIT) PZA kit among archived MTBC isolates. Meanwhile, 637 clinical specimens were prospective collected, with 158 reported as “MTBC detected” by Abbott Realtime MTB detection assay. The phenotypic and genotypic PZA susceptibility profiles of these 158 MTBC detected specimens were subsequently analysed using MGIT PZA kit and in house develop pncA sequencing. Against MGIT PZA kit, pncA sequencing reported pncA mutations in all five phenotypic PZA resistant specimens within four working days. No pncA polymorphism was observed in any PZA susceptible specimens. Combining prospective specimens with archived MTBC isolates, 27 strains were defined as phenotypic PZA resistant with pncA mutations. Of these 27 strains, six (22.2%) phenotypic PZA resistant strains harboured novel pncA mutations without additional panD and rpsA mutations. These included five with mutations situated in pncA structural gene (Del383T [1], Del 380 to 390 [1], A Ins at position 127 [1], A Ins at position 407 [1] and G Ins at position 508 [1]) and one with a mutation at the pncA promoter region at the nucleotide position -12 (T-12C). All six of these strains demonstrated no or weakened Pyrazinamidase activities. The results suggested that these novel mutations might be associated with PZA resistance. More importantly, 92.6% (25/27) of the phenotypic PZA resistant strains were later confirmed to be MDR-TB. Since PZA plays crucial roles in the MDR-TB treatment regime, implementation of direct pncA sequencing may provide rapid PZA resistance diagnosis and facilitate clinicians in the judicious use of PZA in treating PZA susceptible MDR-TB.