Quantitative MODS-Wayne Assay for Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis from Sputum Samples

BackgroundTuberculosis (TB) remains a significant global health challenge, particularly with the rise of drug-resistant strains, such as Pyrazinamide (PZA)-resistant TB. This resistance hampers treatment effectiveness. Currently, theres a lack of affordable and precise quantitative tests for detecting PZA resistance, highlighting the need for accessible diagnostic tools. Our study introduces a direct, accurate, and accessible susceptibility test for PZA resistance by quantifying pirazinoic acid (POA). MethodsWe analyzed 264 TB-positive samples (MP and TBN) to assess PZA susceptibility by measuring POA production. This was done using the MODS-Wayne qualitative assay and our developed quantitative approach (MODS-WQ). For MODS-WQ, TBN samples were processed in 7H9 media (MODS-WQ 7H9) and MP samples in citrate buffer (MODS-WQ CB). We measured POA levels using spectrophotometry against a calibration curve, with PZA susceptibility determined by a composite standard. We also investigated the correlation between POA levels and pyrazinamidase mutations. Multidrug-resistant samples were assessed using the MODS test, and statistical analyses were conducted. ResultsApproximately 23.48% of the samples showed PZA resistance, which included 62.8% of the multidrug-resistant (MDR) samples. The MODS-Wayne assay demonstrated an 87.8% sensitivity and 95.9% specificity for MP samples, and 62.5% sensitivity with 93.7% specificity for TBN samples. The MODS-WQ identified specific cut-off points--123.25 M for MODS-WQ 7H9 and 664.7 M for MODS-WQ CB--with corresponding sensitivities of 81.25% and 92.31%, and specificities of 77.22% and 95.93%. Significantly lower POA levels were observed in samples with mutations in the metal binding site compared to mutations on the enzymes periphery. ConclusionsThe citrate-buffered MODS-WQ demonstrates high sensitivity and specificity in quantifying POA. It offers a significant improvement over qualitative methods, effectively overcoming their subjectivity limitations in PZA resistance detection.