Nitrate reductase assay for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide
Nitrate reductase assay for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide
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DOI：
10.1093/jac/dkm418
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发表时间：
2008-01-01
影响因子：
5.2
通讯作者：
Palomino, Juan Carlos
中科院分区：
文献类型：
--
作者：
Martin, Anandi;Cubillos-Ruiz, Andres;Palomino, Juan Carlos
目的：本研究的目的是开发硝酸还原酶测定法（NRA），用于快速检测结核分枝杆菌吡嗪酰胺耐药性，使用烟酰胺耐药性作为吡嗪酰胺耐药性的标志物，在Lowenstein-Jensen（LJ）培养基中，在中性pH.Methods：我们测试68 M。在LJ培养基中通过NRA使用三种不同浓度（1000、500和250 mg/L）的烟酰胺对结核分离株进行分离，并将结果与使用BACTEC 460-TB或BACTEC MGIT 960作为参考标准方法获得的结果进行比较。结果：在34株耐吡嗪酰胺的M.结核病吡嗪酰胺耐药菌株中，31株对1000和500 mg/L烟酰胺耐药，敏感性和特异性分别为91%和94%。当烟酰胺浓度为250 mg/L时，敏感性和特异性分别降至91%和71%。平均10天就能得到结果。根据这些结果，确定烟酰胺的暂定折点浓度为500 mg/L。pncA基因的DNA测序在34只M中的26只中检测到突变。结论：使用烟酰胺在LJ培养基中检测对吡嗪酰胺的耐药性的NRA是一种快速准确的方法，可用于BACTEC 460-TB或BACTEC MGIT 960系统不可用的资源有限的国家。
Objectives: The purpose of this study was to develop the nitrate reductase assay (NRA) for the rapid detection of pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide resistance as a marker of pyrazinamide resistance in Lowenstein-Jensen (LJ) medium at neutral pH.Methods: We tested 68 M. tuberculosis isolates using nicotinamide at three different concentrations (1000, 500 and 250 mg/L) by the NRA in LJ medium and compared the results with those obtained with the BACTEC 460-TB or the BACTEC MGIT 960 as reference standard methods. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant isolates.Results: Out of 34 M. tuberculosis pyrazinamide-resistant isolates, 31 were found to be resistant to 1000 and 500 mg/L nicotinamide giving sensitivity and specificity of 91% and 94%, respectively. At 250 mg/L nicotinamide, the sensitivity and specificity decreased to 91% and 71%, respectively. Results were obtained in an average of 10 days. Based on these results, a tentative breakpoint concentration of 500 mg/L nicotinamide was defined. DNA sequencing of the pncA gene detected mutations in 26 out of 34 M. tuberculosis isolates resistant to pyrazinamide.Conclusions: The NRA using nicotinamide to detect resistance to pyrazinamide in LJ medium is a rapid and accurate method that could be useful in limited-resource countries where the BACTEC 460-TB or the BACTEC MGIT 960 system is not available.