A quantitative adaptation of the Wayne test for pyrazinamide resistance.

BACKGROUND: Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. METHODS: We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 µg/ml and 800 µg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. RESULTS: The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 µg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 µg/ml PZA were more accurate than 800 µg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. CONCLUSIONS: The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

pncA gene expression and prediction factors on pyrazinamide resistance in Mycobacterium tuberculosis.

Mutations in the pyrazinamidase (PZAse) coding gene, pncA, have been considered as the main cause of pyrazinamide (PZA) resistance in Mycobacterium tuberculosis. However, recent studies suggest there is no single mechanism of resistance to PZA. The pyrazinoic acid (POA) efflux rate is the basis of the PZA susceptibility Wayne test, and its quantitative measurement has been found to be a highly sensitive and specific predictor of PZA resistance. Based on biological considerations, the POA efflux rate is directly determined by the PZAse activity, the level of pncA expression, and the efficiency of the POA efflux pump system. This study analyzes the individual and the adjusted contribution of PZAse activity, pncA expression and POA efflux rate on PZA resistance. Thirty M. tuberculosis strains with known microbiological PZA susceptibility or resistance were analyzed. For each strain, PZAse was recombinantly produced and its enzymatic activity measured. The level of pncA mRNA was estimated by quantitative RT-PCR, and the POA efflux rate was determined. Mutations in the pncA promoter were detected by DNA sequencing. All factors were evaluated by multiple regression analysis to determine their adjusted effects on the level of PZA resistance. Low level of pncA expression associated to mutations in the pncA promoter region was observed in pncA wild type resistant strains. POA efflux rate was the best predictor after adjusting for the other factors, followed by PZAse activity. These results suggest that tests which rely on pncA mutations or PZAse activity are likely to be less predictive of real PZA resistance than tests which measure the rate of POA efflux. This should be further analyzed in light of the development of alternate assays to determine PZA resistance.

A new approach for pyrazinamide susceptibility testing in Mycobacterium tuberculosis.

BACKGROUND: Pyrazinamide (PZA) is an important drug in the treatment of tuberculosis. Microbiological methods of PZA susceptibility testing are controversial and have low reproducibility. After conversion of PZA into pyrazinoic acid (POA) by the bacterial pyrazinamidase enzyme, the drug is expelled from the bacteria by an efflux pump. OBJECTIVE: To evaluate the rate of POA extrusion from Mycobacterium tuberculosis as a parameter to detect PZA resistance. METHODS: The rate of POA extrusion and PZA susceptibility determined by BACTEC 460 were measured for 34 strains in a previous study. PZA resistance was modeled in a logistic regression with the pyrazinoic efflux rate. RESULT: POA efflux rate predicted PZA resistance with 70.83%-92.85% sensitivity and 100% specificity compared with BACTEC 460. CONCLUSION: POA efflux rate could be a useful tool for predicting PZA resistance in M. tuberculosis. Further exploration of this approach may lead to the development of new tools for diagnosing PZA resistance, which may be of public health importance.

The effect of pyrazinamide and rifampicin on isoniazid metabolism in rats.

Hepatotoxicity is the main concern during tuberculosis chemotherapy with the first-line drugs isoniazid (INH), rifampicin (RMP) and pyrazinamide (PYR). Since these hepatotoxic events have been associated with INH metabolites, the study aimed to measure the area under curve (AUC) parameter for INH and its metabolites acetylisoniazid (AcINH), hydrazine (Hz) and acetylhydrazine (AcHz), when groups of rats were pre-treated for 21 days with INH alone or in combination with RMP and/or PYR, in the following amounts per kg body weight: INH 100 mg; INH 100 mg + RMP 100 mg; INH 100 mg + PYR 350 mg; INH 100 mg + PYR 350 mg + RMP 100 mg. It was found that co-administration of RMP, PYR and RMP + PYR caused a significant decrease in the AUC for INH. Co-administration of PYR was the only treatment that caused a significant increase in the AUC for Hz and a decrease in the AUC for its acetylated product AcHz. The AUC for AcINH was not significantly altered in any experimental group. In conclusion, the increased metabolism of INH in all the drug combinations and the significantly higher production of Hz in the group INH + PYR might be linked with exacerbated hepatotoxic effects of these drug associations.

Role of rifampicin in arthralgia induced by pyrazinamide

Arthralgia during daily treatment with chemotherapy regimens containing pyrazinamide was found to be considerably less in patients who received rifampicin concomitantly...