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 "rp00526"
  • 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
    Metallochaperones are needed for mycobacterium tuberculosis and Escherichia coli nicotinamidase-pyrazinamidase activity
    (American Society for Microbiology, 2020)
    Sheen P.
    ;
    Monsalve A.
    ;
    Campos J.
    ;
    Huerta R.
    ;
    Antiparra R.
    ;
    Arteaga H.
    ;
    Duran P.
    ;
    Bueno C.
    ;
    Kirwan D.E.
    ;
    Gilman R.H.
    ;
    Zimic M.
    Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT–Apo and PZAse-EC–Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 M) achieved 65% PZAse-EC–Apo reactivation. Rv2059 (1 M) and ZnuA (1 M) achieved 69% and 34.3% PZAse-MT–Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT–Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro. Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis. Further studies are needed for confirmation. IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide’s mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient’s individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat. Copyright © 2020 Sheen 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.