Abstract
Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis However, no drug susceptibility test (DST) is considered sufficiently reliable for routine application. Although molecular tests are endorsed, their application is limited to known PZA resistance associated mutations. Microbiological DSTs for PZA have been restricted by technical limitations, especially the necessity for an acidic pH. Here, for the first time, MODS culture at neutral pH was evaluated using high PZA concentrations (400 and 800 μg/ml) to determine PZA susceptibility directly from sputum samples. Sputum samples were cultured with PZA for up to 21 days at 37°C. Plate reading was performed at two time points: R1 (mean, 10 days) and R2 (mean, 13 days) for each PZA concentration. A consensus reference test, composed of MGIT-PZA, pncA sequencing, and the classic Wayne test, was used. A total of 182 samples were evaluated. The sensitivity and specificity for 400 μg/ml ranged from 76.9 to 89.7 and from 93.0 to 97.9%, respectively, and for 800 μg/ml ranged from 71.8 to 82.1 and from 95.8 to 98.6%, respectively. Compared to MGIT-PZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZA-sensitive and -resistant isolates, respectively). In conclusion, MODS-PZA is presented as a fast, simple, and low-cost DST that could complement the MODS assay to evaluate resistance to the principal first-line antituberculosis drugs. Further optimization of test conditions would be useful in order to increase its performance.
Highlights
Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis
For the first time, we evaluated the use of microscopic observation drug susceptibility (MODS) to determine PZA resistance directly from sputum samples
Unlike other studies that have evaluated the application of MODS for PZA resistance determination in M. tuberculosis strains [46,47,48], our findings have demonstrated the possibility of directly evaluating sputum samples with high PZA concentration (400 and 800 g/ml) without the need for additional processing except for a decontamination step
Summary
Pyrazinamide (PZA) is considered the pivot drug in all tuberculosis treatment regimens due to its particular action on the persistent forms of Mycobacterium tuberculosis. The development of molecular diagnostic tests to detect PZA resistance have been limited due to the large number of mutations along the pncA gene [14], the lack of a mutation hot spot, and the fact that not all mutations cause PZAse impairment or have been associated with a specific PZA susceptibility phenotype [15, 22,23,24,25]. As a matter of fact, the correlation between pncA sequencing and detection of mutations with PZA resistance varies among countries low and high endemicities, with reported estimates of 41% in Taiwan, 67% in South Africa, 72 to 84% in Brazil, 91% in China, and 97% in China and Japan [7, 18]
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