Quantitative MODS-Wayne assay for rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis from sputum samples.

Tuberculosis (TB) remains a significant global health challenge, exacerbated by the emergence of drug-resistant strains, such as those resistant to pyrazinamide (PZA). The current scarcity of affordable and precise quantitative diagnostic tests for PZA resistance underscores the urgent need for more accessible diagnostic tools. We evaluated PZA susceptibility in 264 TB-positive samples by quantifying pyrazinoic acid (POA) production, using both the MODS-Wayne qualitative assay and our newly developed quantitative approach (MODS-WQ). The MODS-WQ was assessed in 7H9 medium (MODS-WQ_7H9) or citrate buffer (MODS-WQ_CB), with POA levels measured via spectrophotometry against a calibration curve. PZA susceptibility determinations were based on a composite reference standard. Associations between POA levels and pyrazinamidase mutations were explored. The composite standard detected PZA resistance in 23.5% of the samples, which accounts for 62.8% of the multidrug-resistant (MDR) samples. The MODS-WQ established specific POA cutoffs of 123.25 µM for MODS-WQ_7H9 and 664.7 µM for MODS-WQ_CB, with sensitivities of 81.3% and 92.3% and specificities of 77.2% and 95.9%, respectively. Notably, samples with mutations in the pyrazinamidase metal-binding site exhibited significantly lower POA levels compared with mutations in the enzyme periphery. Furthermore, a significant correlation was found between POA production and PZA resistance, Bactec Growth Index, and minimum inhibitory concentration (MIC) values. This study presents a novel, direct, and accessible susceptibility test for PZA resistance that quantifies POA, enhancing the detection capabilities for this condition. The citrate-buffered MODS-WQ assay demonstrated high sensitivity and specificity for quantifying POA, confirming that POA production is a reliable indicator of PZA resistance.

Importance: PZA susceptibility testing continues to be a challenge, particularly in countries with high TB incidence. In response to this pressing need, we have developed a quantitative MODS-Wayne (MODS-WQ) assay. This approach offers a direct and cost-effective solution representing a significant advancement in TB diagnostics, particularly benefiting resource-limited laboratories, primarily in developing regions. The MODS-WQ assay stands out for its ability to quantify pyrazinoic acid (POA) production, as a reliable indicator of PZA resistance. Unlike traditional qualitative assays, MODS-WQ eliminates the inherent subjectivity in interpretation, providing more accurate and actionable results. Moreover, the MODS-WQ approach accounts for critical factors influencing PZA resistance, including enzymatic efficiency and efflux pump activity. By integrating these factors into the detection process, our methodology offers a comprehensive understanding of PZA resistance levels, enabling tailored treatment strategies for patients.