Pyrazinamide susceptibility testing in Mycobacterium tuberculosis using the fast resazurin microtiter assay plate.

SETTING: Department of Clinical Analysis and Biomedicine, State University of Maringa, Maringa, PR, Brazil. OBJECTIVE: To evaluate the performance of the resazurin microtiter assay (REMA) plate at pH 5.5 in detecting Mycobacterium tuberculosis susceptibility to pyrazinamide (PZA). DESIGN: The minimal inhibitory concentration (MIC) of PZA in M. tuberculosis H37Rv and M. bovis AN5 reference strains and in 34 clinical M. tuberculosis isolates (26 PZA-susceptible and eight PZA-resistant) was determined using REMA at pH 5.5 and compared to REMA at pH 6.0. RESULTS: REMA at pH 5.5 was helpful in discriminating PZA-susceptible from resistant M. tuberculosis isolates when â©¿50 µg/ml PZA was considered as the cut-off for PZA susceptibility. Furthermore, it provided results in 8 days. However, two PZA-resistant isolates failed to grow at pH 5.5. CONCLUSION: As the REMA method is rapid, inexpensive, easy to perform and read, it would be of great usefulness in low-income countries for detecting PZA-resistant M. tuberculosis. REMA at pH 5.6-5.9 should be evaluated on an extended panel of clinical M. tuberculosis isolates with a greater range of MIC values in different laboratories for a better understanding of its utility in differentiating PZA-resistant from PZA-susceptible isolates.

Anti-tuberculosis neolignans from Piper regnellii.

The present study determined the anti-Mycobacterium tuberculosis activities of supercritical CO2 extracts, neolignans eupomatenoid-5 (1), conocarpan (4) and eupomatenoid-3 (7) and their derivatives (2, 3, 5, 6, and 8) from Piper regnellii, as well as their cytotoxicities. The supercritical CO2 extract from leaves was purified by chromatographic methods, yielding compounds (1), (4) and (7), which were identified by (1)H NMR and comparison with literature data. Anti-M. tuberculosis activity (H37Rv and clinical isolates) was evaluated using a resazurin microtiter assay plate (REMA) to determine the MIC. The cytotoxicity assay was carried out in macrophages J774G.8 by sulforhodamine B colorimetric assay. The supercritical CO2 extracts from leaves and stems, and compound (4) showed activity against M. tuberculosis (MIC 15.6 µg/ml). Compound (1) showed the best activity (MIC 1.9 µg/ml), with good SI. Compounds (7) and (8) showed low activity against M. tuberculosis H37Rv. The derivative compounds did not show increased anti-M. tuberculosis activity. This is the first report, to our knowledge, to describe neolignans from P. regnellii with activity against M. tuberculosis, and compound (1) is a potential candidate for future antituberculosis drugs.