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A Multinational Analysis of Mutations and Heterogeneity in PZase, RpsA, and PanD Associated with Pyrazinamide
S M Ramirez-Busby1, T C Rodwell2, L Fink1
1Biological and Medical Informatics Research Center, San Diego State University, San Diego, California, USA.
Abstract:
Pyrazinamide (PZA) is an important first-line drug in all existing and new tuberculosis (TB) treatment regimens. 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.
Insights
Molecular diagnostics for pyrazinamide (PZA) resistance in tuberculosis (TB) show promise. A pncA-based test achieved 90% sensitivity, with potential improvements from including rpsA and panD genes.
Area of Science:
- Microbiology
- Genetics
- Drug Resistance
Background:
- Pyrazinamide (PZA) is a critical first-line tuberculosis (TB) drug.
- Increasing PZA resistance, particularly in multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB, necessitates improved diagnostics.
- Current PZA drug susceptibility testing (DST) methods face challenges, driving the need for molecular alternatives.
Purpose of the Study:
- To comprehensively assess molecular diagnostic tools for PZA resistance in M/XDR TB cases.
- To investigate the role of pncA, rpsA, and panD genes in PZA resistance.
- To evaluate the diagnostic performance of molecular markers for PZA resistance.
Main Methods:
- Whole-genome sequencing of 296 M. tuberculosis isolates from four countries.
- Drug susceptibility testing (DST) for eight drugs and confirmatory Wayne's assay.
- Analysis of mutations in pncA, rpsA, and panD genes associated with PZA resistance.
Main Results:
- A pncA-based molecular test demonstrated 90% sensitivity and 65% specificity for PZA resistance.
- Inclusion of rpsA and panD genes offered a potential 2% increase in sensitivity.
- Molecular heterogeneity in pncA correlated with resistance, suggesting its utility as a diagnostic marker.
- Euro-American TB isolates showed a higher likelihood of wild-type PZase, potentially evading molecular detection.
Conclusions:
- Molecular diagnostics targeting pncA, rpsA, and panD show significant potential for detecting PZA resistance in TB.
- Genetic variations in pncA are strongly associated with PZA resistance and warrant further diagnostic evaluation.
- A notable proportion of PZA resistance (8-10%) may be mediated by mechanisms beyond the investigated genes.
- Further research, including confirmatory mutagenesis, could refine the specificity and sensitivity of molecular PZA resistance tests.
