Quantitative 1H Nuclear Magnetic Resonance Assay for the Rapid Detection of Pyrazinamide Resistance in Mycobacterium tuberculosis from Sputum Samples.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the 10 leading killer diseases in the world. At least one-quarter of the population has been infected, and there are 1.3 million deaths annually. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains challenges TB treatments. One of the drugs widely used in first- and second-line regimens is pyrazinamide (PZA). Statistically, 50% of MDR and 90% of XDR clinical strains are resistant to PZA, and recent studies have shown that its use in patients with PZA-resistant strains is associated with higher mortality rates. Therefore, the is an urgent need for the development of an accurate and efficient PZA susceptibility assay. PZA crosses the M. tuberculosis membrane and is hydrolyzed to its active form, pyrazinoic acid (POA), by a nicotinamidase encoded by the pncA gene. Up to 99% of clinical PZA-resistant strains have mutations in this gene, suggesting that this is the most likely mechanism of resistance. However, not all pncA mutations confer PZA resistance, only the ones that lead to limited POA production. Therefore, susceptibility to PZA may be addressed simply by its ability to form, or not, POA. Here, we present a nuclear magnetic resonance method to accurately quantify POA directly in the supernatant of sputum cultures collected from TB patients. The ability of the clinical sputum culture to hydrolyze PZA was determined, and the results were correlated with the results of other biochemical and molecular PZA drug susceptibility assays. The excellent sensitivity and specificity values attained suggest that this method could become the new gold standard for the determination of PZA susceptibility.

Evaluation of three alternatives cost-effective culture media for Mycobacterium tuberculosis detection and drug susceptibility determination using the microscopic observation drug susceptibility (MODS) assay.

Tuberculosis phenotypic detection assays are commonly used in low-resource countries. Therefore, reliable detection methods are crucial for early diagnosis and treatment. The microscopic observation drug susceptibility (MODS) assay is a culture-based test to detect Mycobacterium tuberculosis and characterize drug resistance in 7-10 days directly from sputum. The use of MODS is limited by the availability of supplies necessary for preparing the enriched culture. In this study, we evaluated three dry culture media that are easier to produce and cheaper than the standard one used in MODS [1]: an unsterilized powder-based mixed (Boldú et al., 2007) [2], a sterile-lyophilized medium, and (Sengstake et al., 2017) [3] an irradiated powder-based mixed. Mycobacterial growth and drug susceptibility were evaluated for rifampin, isoniazid, and pyrazinamide (PZA). The alternative cultures were evaluated using 282 sputum samples with positive acid-fast smears. No significant differences were observed in the positivity test rates. The positivity time showed high correlations (Rho) of 0.925, 0.889, and 0.866 between each of the three alternative media and the standard. Susceptibility testing for MDR and PZA showed an excellent concordance of 1 compared to the reference test. These results demonstrate that dry culture media are appropriate and advantageous for use in MODS in low-resource settings.

Alternative cost-effective media to facilitate MODS culture for diagnostics of tuberculosis.

Most culture-based methods for tuberculosis diagnosis remain low-cost options for low- and mid-income countries. The MODS culture is a rapid and low-cost assay to diagnose tuberculosis and determine drug susceptibility. However, its implementation is limited due to the low accessibility to supplies required for the enriched medium. In this study, we evaluate two alternative culture media: A powder-based mixed (PM) and a lyophilized media (LM). Catalase, PANTA, and gamma irradiation were evaluated as additions to PM and LM. The culture performance of the alternative media was compared with the standard MODS medium (MM) using Mycobacterium tuberculosis isolates and positive acid-fast smear sputum samples. Overall, no significant difference was observed in the bacterial growth between PM and LM with MM. However, PANTA and gamma irradiation combined reduced bacterial growth significantly in all media variants. A median positivity day of 6 ± 5 days was observed for sputum samples, regardless of the culture medium. The preliminary results show that the two variants culture media have a similar performance to the standard MODS medium. The powder-based media with PANTA (PM_P) showed a time-to-positivity and sensitivity similar to the standard MODS medium. It is the simplest to prepare and does not require any sterilization process.