Comparison of PZA-LPA2 and WGS for determining PZA resistance of baseline Mycobacterium tuberculosis isolates collected from patients enrolled in the STREAM I clinical trial.
Background: Pyrazinamide (PZA) remains an important component of anti-tuberculosis regimens. The Nipro Genoscholar PZA TB II line probe assay (PZA-LPA2) indirectly detects mutations in pncA by targeting the wild-type sequence. We compared results obtained by PZA-LPA2 with whole genome sequencing (WGS) as a reference standard to assess PZA resistance in Mycobacterium tuberculosis (Mtb) isolates.  Design/Methods: PZA-LPA2 and WGS were performed on multidrug-resistant Mtb isolates from the STREAM stage I clinical trial. DNA extracts were subjected to PZA-LPA2 and Illumina WGS. Probe profiles were interpreted by two operators for PZA-LPA2 and WGS data were analyzed using TB-Profiler. We compared overall genotypic agreement on PZA resistance calling. Non-synonymous pncA mutations and insertions/deletions identified by WGS were interpreted as resistant (R) and the wildtype sequence as susceptible (S). For LPA an absent probe was interpreted as R and the presence of a probe as S. Sanger sequencing and MGIT PZA phenotypic drug susceptibility testing were done on selected gDNA extracts and/or stored isolates to resolve discrepancies between discordant WGS-LPA, MGIT PZA results.  Results: Of 323 isolates with PZA-LPA2 and WGS results, 54 (16.7%, 95%CI 12.6-20.8) were invalid and 4 negative by PZA-LPA2 (Table 1). Interpretable results by both methods were obtained for 265 isolates, of which 242 were concordant (91.3%, 95%CI 87.9-94.7). For 21 isolates with PZA-LPA2-S/WGS-R results the discordance may be explained partly by undetected heteroresistance (n=7) and undetected mutations in the pncA promotor region (n=4) by PZA-LPA2. For two isolates PZA-LPA2-R was reported while WGS showed pncA wild-type (WGS-S). Of the 242 isolates with concordant resistance calling, the mutation position (n=142) or wildtype sequence (n=88) detected in 230 (95.0%, 95% CI 92.3-97.8) isolates corresponded to the absent or present PZA-LPA2 probe, respectively.  Conclusions: The concordance between PZA-LPA2 and WGS to detect PZA resistance was high (>90%). Most discordances were observed in the PZA-LPA2-S/WGS-R group; the current design of the PZA-LPA2 does not allow heteroresistance detection and hinders the detection of pncA promotor mutations upstream of the pncA gene.  Summary: Detecting pyrazinamide resistance is paramount. Nipro Genoscholar PZA-TB II LPA was compared to whole genome sequencing to assess PZA-R in Mycobacterium tuberculosis isolates. Concordance was obtained in 91.3% of all interpretable results. The majority of missed resistance detection by LPA was due to heteroresistance and undetected pncA promotor mutations.