Tuberculosis is one of the most serious public health problems over the world. The increasing number of multi-drug resistant strains and the HIV pandemic are threatening any control effort. Pyrazinamide (PZA) is one of the most important antituberculos drugs but the less known. Microbiological assays for PZA resistance in M. tuberculosis (MTB) are still not reliable, and the mechanism of PZA resistance is not completely understood yet. It is necessary to develop alternative methods to determine PZA resistance and to have a better understanding of the mechanism of resistance. ? ? Hypotheses. The PZA resistance is mainly caused by a null or low PZase activity, which is mainly associated to deleterious or point mutations that compromise the active site as well as the cofactor coordination site. We hypothesize that PZA-resistant strains with mutations in the cofactor coordination site, will revert its PZase activity, turning to a PZA-sensitive phenotype, under high concentrations of zinc ions, the probable cofactor. Similarly we hypothesize that PZA-resistant strains with mutations in the active site, will not recover its PZase activity under high concentrations of zinc ions. ? ? Aims. We want to evaluate an optimized Single Strand Conformation Polymorphism (SSCP), a DNA-based test, to determine PZA susceptibility of MTB directly from sputum samples of patients. We want to determine any association between mutations in the PZase coding gene (pncA), and the PZase activity, looking for the most likely 'hot spots' in the pncA of PZA resistant strains. We also plan to determine the effect of zinc and iron ions on PZase activity in both PZA resistant and PZA sensitive strains with characterized pncA mutations. ? ? Background. Microbiological PZA-susceptibility testing is performed only in reference laboratories, at high costs normally unaffordable in developing countries and taking 8 to 12 weeks. Alternate methods based on DNA, like SSCP, has been proved world wide, but unfortunately with not enough power due to a very low sample size. Not any group has ever performed an SSCP test directly on sputum samples, which we believe will dramatically reduce the diagnosis time. An ion as zinc and iron have proved to enhance PZase activity in PZA-sensitive strains, but never before was proved to recover the PZase activity in a PZA-resistant strains. ? ? Preliminary data. To overcome the PZA-susceptibility we have developed an optimized SSCP test to detect PZA-resistant in MTB clinical isolates with sensitivity around 90%. Preliminary data indicates that zinc recovers the PZase activity in PZA-resistant strains with pncA mutations associated to the cofactor coordination site. ? ? Methods. Our optimized SSCP will be tested directly in sputum samples to determine PZA susceptibility. To understand the effect of pncA mutations and ions on PZase activity we will determine PZase activity with a quantitative Wayne test on MTB total protein and recombinant PZase. ? ? Conclusion. This study will permit an appropriate and rapid tuberculosis treatment avoiding MDR- TB and MTB transmission, as well as to achieve a better understanding of the major resistance mechanism of PZA with a potential impact on therapeutics. ? ? Relevance to Public Health. PZA is an important part of TB treatment and is one of the drugs included in DOTS. However mechanisms of resistance are not well understood nor are microbiological assays for resistance well standardized or rapid. This application will advance these two areas by providing both a rapid clinical assay as well as an explanation for the mechanism of PZA resistance. ? ?

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Small Research Grants (R03)
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Special Emphasis Panel (ZRG1-DDR (01))
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Sizemore, Christine F
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Universidad Peruana Cayetano Heredia
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LIMA -31
Zimic, Mirko; Sheen, Patricia; Quiliano, Miguel et al. (2010) Peruvian and globally reported amino acid substitutions on the Mycobacterium tuberculosis pyrazinamidase suggest a conserved pattern of mutations associated to pyrazinamide resistance. Infect Genet Evol 10:346-9
Sheen, Patricia; Ferrer, Patricia; Gilman, Robert H et al. (2009) Effect of pyrazinamidase activity on pyrazinamide resistance in Mycobacterium tuberculosis. Tuberculosis (Edinb) 89:109-13
Sheen, Patricia; Mendez, Melissa; Gilman, Robert H et al. (2009) Sputum PCR-single-strand conformational polymorphism test for same-day detection of pyrazinamide resistance in tuberculosis patients. J Clin Microbiol 47:2937-43