Abstract: Drug-resistant infectious diseases pose a significant threat to public health. To overcome this challenge, drug susceptibility testing (DST) is essential for identifying the most effective drugs to treat and monitoring drug resistance in these diseases. Pyrazinamide (PZA) is a critical component of current tuberculosis (TB) treatment, shortening drug susceptible TB (DS-TB) treatment duration from 9-12 months to 6 months. PZA is also used in multidrug resistant TB (MDR-TB) treatment. Unfortunately, PZA resistance is increasing, with a pooled prevalence of PZA resistance among all TB cases estimated at 16.2%, leading to worse consequences, including death. Unlike other TB drugs, current PZA susceptibility tests are unreliable, complicated, and expensive, leading to underutilization, even in high TB burden areas. Misclassifying PZA-resistant infections as PZA-susceptible infections results in significantly worse clinical outcomes for the patients and development of further drug resistance. Conversely, considering PZA-susceptible infections as PZA-resistant infection leads to longer treatment with more expensive and toxic medicines unnecessarily. The global burden of PZA-resistant TB is estimated at 1.4 million new cases out of 10.6 million active TB cases per year, highlighting a significant challenge for current TB control. According to the CDC, treatment costs for DR-TB are significantly higher than for DS-TB, at times, costing $528,000 (2020 USD) more to treat a patient with DR-TB than to treat a patient with DS-TB. Due to the importance of PZA in many TB regimens, the emergence of PZA resistance and systematic problems in current PZA DST, reliable PZA susceptibility testing has been assigned a high priority in the target product profile by the WHO. To address this challenge, it is urgent to develop a rapid, reliable and accurate PZA DST that can be routinely performed in mycobacterial labs worldwide. This will ensure that TB patients receive the most effective and adequate treatment, prevent the further development of TB drug resistance and limit its spread. Our goal is to develop such a test, which is substantiated by our preliminary findings indicating that (1) PZA is active in liquid and agar media at pH 6.8, which is optimal for the growth of M. tuberculosis, unlike the current acidic conditions that are unstable when bacteria grow, leading to a high rate of false resistance and technical difficulties; (2) The new testing can determine the critical proportion of PZA-resistant M. tuberculosis highly correlated with treatment outcomes, with one of the important specifications of DST at 1%, compared with 10% for the current test; (3) The new PZA susceptibility testing can be conducted without the need for expensive readout equipment, which is a significant improvement from current test such as the BACTEC MGIT 960 PZA that relies completely on such equipment; (4) the turnaround time for the new test is much shorter, with results being...