5.1 Proyectos de Innovación y transferencia tecnológica

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https://hdl.handle.net/20.500.12390/380

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Examinando 5.1 Proyectos de Innovación y transferencia tecnológica por Autor "rp00525"
  • Publicación
    Direct determination of pyrazinamide (PZA) susceptibility by sputum microscopic observation drug susceptibility (MODS) culture at neutral pH: The MODS-PZA assay
    (American Society for Microbiology, 2020)
    Alcántara R.
    ;
    Fuentes P.
    ;
    Marin L.
    ;
    Kirwan D.E.
    ;
    Gilman R.H.
    ;
    Zimic M.
    ;
    Sheen P.
    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 _g/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 MGITPZA, our test showed a similar turnaround time (medians of 10 and 12 days for PZAsensitive 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. Copyright © 2020 Alcántara et al.
  • Publicación
    MODS-Wayne, a colorimetric adaptation of the Microscopic-Observation Drug Susceptibility (MODS) assay for detection of mycobacterium tuberculosis pyrazinamide resistance from sputum samples
    (American Society for Microbiology, 2019)
    Alcántara R.
    ;
    Fuentes P.
    ;
    Antiparra R.
    ;
    Santos M.
    ;
    Gilman R.H.
    ;
    Kirwan D.E.
    ;
    Zimic M.
    ;
    Sheen P.
    Although pyrazinamide (PZA) is a key component of first- and second-line tuberculosis treatment regimens, there is no gold standard to determine PZA resistance. Approximately 50% of multidrug-resistant tuberculosis (MDR-TB) and over 90% of extensively drug-resistant tuberculosis (XDR-TB) strains are also PZA resistant. pncA sequencing is the endorsed test to evaluate PZA susceptibility. However, molecular methods have limitations for their wide application. In this study, we standardized and evaluated a new method, MODS-Wayne, to determine PZA resistance. MODS-Wayne is based on the detection of pyrazinoic acid, the hydrolysis product of PZA, directly in the supernatant of sputum cultures by detecting a color change following the addition of 10% ferrous ammonium sulfate. Using a PZA concentration of 800 μg/ml, sensitivity and specificity were evaluated at three different periods of incubation (reading 1, reading 2, and reading 3) using a composite reference standard (MGIT-PZA, pncA sequencing, and the classic Wayne test). MODS-Wayne was able to detect PZA resistance, with a sensitivity and specificity of 92.7% and 99.3%, respectively, at reading 3. MODS-Wayne had an agreement of 93.8% and a kappa index of 0.79 compared to the classic Wayne test, an agreement of 95.3% and kappa index of 0.86 compared to MGIT-PZA, and an agreement of 96.9% and kappa index of 0.90 compared to pncA sequencing. In conclusion, MODS-Wayne is a simple, fast, accurate, and inexpensive approach to detect PZA resistance, making this an attractive assay especially for low-resource countries, where TB is a major public health problem. Copyright © 2019 Alcántara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.