A Multinational Analysis of Mutations and Heterogeneity in PZase, RpsA, and PanD Associated with Pyrazinamide Resistance in M/XDR Mycobacterium tuberculosis

Abstract

Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatmentregimens. PZA-resistance in M. tuberculosis is increasing, especially among M/XDR cases. Noted issues with PZA Drug Susceptibility Testing (DST) have driven the search for alternative tests. This study provides a comprehensive assessment of PZA molecular diagnostics in M/XDR TB cases. A set of 296, mostly XDR, clinical M. tuberculosis isolates from four countries were subjected to DST for eight drugs, confirmatory Wayne's assay, and whole-genome sequencing. Three genes implicated in PZA resistance, pncA, rpsA, and panD were investigated. Assuming all non-synonymous mutations cause resistance, we report 90% sensitivity and 65% specificity for a pncA-based molecular test. The addition of rpsA and panD potentially provides 2% increase in sensitivity. Molecular heterogeneity in pncA was associated with resistance and should be evaluated as a diagnostic tool. Mutations near the N-terminus and C-terminus of PZase were associated with East-Asian and Euro-American lineages, respectively. Finally, Euro-American isolates are most likely to have a wild-type PZase and escape molecular detection. Overall, the 8-10% resistance without markers may point to alternative mechanisms of resistance. Confirmatory mutagenesis may improve the disconcertingly low specificity but reduce sensitivity since not all mutations may cause resistance. Pyrazinamide (PZA) is an important first-line drug recommended by the World Health Organization (WHO) for treatment of tuberculosis (TB)1 . Although most patients diagnosed with TB are successfully treated with first-line drugs, rifampicin (RIF), isoniazid (INH), ethambutol, and PZA, the incidence of multidrug resistant TB (MDR-TB) (resistant to first-line drugs) is increasing every year 1 . Along with this rate, the incidence of PZA resistance (PZA R ) is also increasing since over 60% of MDR-TB cases globally are also PZA R (rates vary regionally) 2 . In this manuscript we denote isolates that are resistant or susceptible to PZA by growth-based drug susceptibility testing (DST) as PZA R or PZA S , respectively. PZA is unique among anti-TB drugs in that, while it demonstrates powerful in vivo sterilizing activity, it exhibits no activity against actively growing Mycobacterium tuberculosis bacilli under normal culture conditions at neutral pH 3 . The drug appears to preferentially act against non-replicating persisters with low metabolic activity at acid pH in vitro or in vivo 4 . Importantly, most new drug regimens proposed for treatment of drug-resistant TB (DR-TB) show improved outcomes when combined with PZA 5-9 . Although growth-based PZA susceptibility testing is recommended by the WHO 10 , the method yields inconsistent results in some cases and suffers from a relatively high false resistance rate 11 .

Phenotypic and genotypic characterization of pyrazinamide resistance among multidrug-resistant Mycobacterium tuberculosis clinical isolates in Hangzhou, China

“…When all three genes were combined and examined via our sequencing method, the sensitivity and specificity was 98.1% and 92.3%, respectively. This sensitivity was consistent with studies from China, Asia and Belgium [15,21,29,30], and were slightly higher than the other studies [20,26,31,32]. Considering the unreliable and inconsistent results of phenotypic susceptibility tests [8e10], the effectiveness of the sequencing method to detect all three PZA resistance genes may be most useful in clinical diagnosis.…”

Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA

“…However, rpsA polymorphisms are seemingly found in some PZA-susceptible clinical strains from closely related geographical regions. Additionally, there are some low-level PZA-resistant clinical strains (minimal inhibitory concentration (MIC) = 200-300 µg/mL, pH 6.0) without pncA and rpsA mutations [8,[10][11][12][13][14][15]. Subsequently, mutations in panD encoding aspartate decarboxylase were suggested to be an additional mechanism of PZA resistance, and the PanD protein involved in β-alanine synthesis was also reported as a novel target of POA/PZA [16][17][18].…”

Prediction of pyrazinamide resistance inMycobacterium tuberculosisusing structure-based machine learning approaches

“…The model also does not take into account the introduction of protease cleavage sites or other processing abnormalities. Finally, while most pyrazinamide resistance is caused by mutations in pncA, recent studies have also implicated other genes, notably rpsA, panD, and the putative efflux pumps Rv0191, Rv3756c, Rv3008, and Rv1667c in pyrazinamide resistance 4,[25][26][27][28][29][30][31][32] . Further research is needed to determine if mutations in these genes can be reliably inferred to confer pyrazinamide resistance.…”