Abstract
ObjectivesThe development of rapid molecular diagnostic assays for pyrazinamide (PZA) resistance is considered technically challenging as mutations are highly diverse, scattered along the full length of the pncA gene and not all are associated with PZA resistance. We evaluated the performance of the novel Genoscholar PZA-TB II line probe assay (PZA-LPA2; NIPRO Corporation, Japan). MethodsTo evaluate the applicability of the PZA-LPA2 in clinical settings, we compared the performance of the PZA-LPA2 to a composite reference standard pncA Sanger and Illumina sequencing plus phenotypic susceptibility testing on a panel of 87 Mycobacterium tuberculosis isolates from World Health Organization (WHO) drug resistance surveys, harbouring mutations previously classified as associated or not associated with resistance according to data from peer-reviewed literature. In addition, the PZA-LPA2 was challenged against a selection of isolates with lineage-specific and non-resistance-associated mutations, for which the frequency among clinical isolates is unknown, and tested directly on 59 sputum extracts. ResultsFor the survey isolates, the PZA-LPA2 reached an overall agreement with the composite reference of 97.6% (80/82) or 94.3% (82/87) excluding or including heteroresistance, respectively. The PZA-LPA2 failed on 8.5% (5/59) of clinical samples; among valid results, 100% (14/14) sensitivity and 100% (7/7) specificity was reached relative to pncA Sanger sequencing. ConclusionsThe PZA-LPA2 represents a valid and rapid alternative for indirect PZA susceptibility testing. Preliminary findings on clinical samples show promise for direct testing. Further studies are needed to assess the clinical risk of missing heteroresistance and falsely detecting lineage-specific, silent and nonassociated mutations.
Highlights
BCCM/ITM isolates and isolates from South Africa The five isolates from South Africa with point mutations not associated with resistancedincluding the synonymous mutation Lys96Lysdand previously identified as MGIT 960 PZA susceptible yielded a false-resistant PZA-LPA2 result, while the high confidence isolate with large deletion, showing an absence of wild-type bands 31e48, was found resistant by MGIT 960 and the PZA-LPA2
The two M. bovis isolates with a His57Asp high confidence mutation and the two M. canettii isolates with Ala46Ala silent mutations in pncA were found to be resistant by both MGIT 960 PZA and PZALPA2
The intrinsic PZA resistance of M. canettii might be due to mutations in rpsA or panD [17], i.e. not included in the PZA-LPA2
Summary
M. Driesen et al / Clinical Microbiology and Infection 24 (2018) 60e64 short (9 month) regimens for patients with multidrug-resistant TB [1,2]. PZA is a prodrug that is converted to its active form, pyrazinoic acid, by pyrazinamidase [3,4]. The main mechanism of PZA resistance in Mycobacterium tuberculosis (MTB) is the inactivation of pyrazinamidase as a result of mutations affecting the pncA gene encoding this enzyme. About 72e99% of PZA-resistant MTB strains have a mutation in pncA [5,6]. The nonessential nature of the pncA gene allows the development of multiple mutations dispersed along the gene and promotor region without affecting bacterial fitness [7]
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