Project Lead(s): Patricia Sheen
Lack of treatment adherence in DOTS (Directly Observed Treatment, Short-Courses) is a threat to tuberculosis (TB) control efforts, causing treatment failure and the emergence of multi-drug-resistant TB (MDR-TB).
Pyrazinamide (PZA) kills latent TB but there is growing resistance in Peru and one third of MDR-TB cases are also PZA-resistant. PZA can also produce serious hepatic side effects.
It is important to determine PZA susceptibility in the early stage of treatment, to provide a safe regimen and improve adherence. However PZA-susceptibility assays are far from adequate.
This project was a study to determine pyrazinamide (PZA)-resistance in Mycobacterium tuberculosis samples taken directly from sputum, with a microscopic observation drug susceptibility assay for pyrazinamide (MODS-PZA) and the pyrazinoic acid biomarker (MODS-Wayne).
The detection of PZA susceptibility in MODS-PZA is by microscopy evaluation of M. tuberculosis growth. The absence of growth indicates that a strain is sensitive to PZA.
MODS-Wayne was based on the team’s previous results that demonstrated that the efflux rate of pyrazinoic acid in M. tuberculosis differentiates PZA-resistant strains from PZA-sensitive strains, as PZA-sensitive strains can produce and have a higher efflux rate of pyrazinoic acid than PZA-resistant strains.
For MODS-Wayne, the detection of PZA susceptibility is by visualization of a red-brown colour after the reaction between iron (II) ammonium sulphate and pyrazinoic acid.
The assays do not require expensive equipment and reagents.
Two PZA concentrations were evaluated: 400 and 800 ug/mL.
Each methodology was compared to one composed standard that was obtained performing three methods: MGIT 960PZA, Wayne Assay and pncA mutations. The assays were evaluated in a small, controlled setting of194 samples.
The results showed that MODS-PZA and MODS-Wayne are promising methods for determining PZA susceptibility on a large scale and in a relatively short time (within 21 days), with high sensitivity (80.95% and 90.91%, respectively) and specificity (97.33% and 98.01%, respectively).
These assays showed a high correlation with the standard assays.
There is need for further validation studies using a larger sample size.
The team has applied for funding from the National Council of Sciences and Technology (CONCYTEC) and the National Program of Innovation to Competitively and Productivity (INNOVATE-Production Ministry) of Peru.