A new rapid and simple colorimetric method to detect pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide
A new rapid and simple colorimetric method to detect pyrazinamide resistance in Mycobacterium tuberculosis using nicotinamide
复制标题
DOI：
10.1093/jac/dkl231
复制
发表时间：
2006-08-01
影响因子：
5.2
通讯作者：
Portaels, Francoise
中科院分区：
文献类型：
--
作者：
Martin, Anandi;Takiff, Howard;Portaels, Francoise
目的：建立和评价一种快速检测结核分枝杆菌对吡嗪酰胺耐药性的方法。方法：采用氧化还原指示剂REMA(REMA)检测96孔烟酰胺对结核分枝杆菌的耐药性，并比较BACTEC460-TB系统与两种浓度的吡津酰胺(100和300 mg/L)的检测结果，以及韦恩法检测吡津酰胺酶活性。结果：95株结核分枝杆菌临床分离株经韦恩药敏试验、BACTEC(300 mg/L)药敏试验和瑞玛烟酰胺药敏试验，25株均为耐药菌株。以烟酰胺MIC>；250 mg/L作为抗药性的界限，只有一株菌株被错误地标记为抗药性。Rema烟酰胺试验的敏感性为100%，特异性为98%。BACTEC(100 mg/L)错误地将8个菌株归类为抗性。DNA测序检测到22株吡嗪酰胺耐药株中18/22的pncA基因发生突变。结论：烟酰胺REMA平板法检测吡嗪胺耐药性是一种简便、快速的方法，可在资源有限的国家推广应用。
Objectives: The purpose of this study was to develop and assess a rapid method for pyrazinamide resistance detection in Mycobacterium tuberculosis using nicotinamide in a colorimetric resazurin assay.Methods: We have tested M. tuberculosis isolates using nicotinamide in a 96-well format with the redox indicator resazurin (REMA) and compared results using the BACTEC 460-TB system with two concentrations of pyrazinamide (100 and 300 mg/L), as well as the Wayne method for detecting pyrazinamidase activity. Mutations in the pncA gene were detected by DNA sequencing of the pyrazinamide-resistant strains.Results: Out of 95 clinical isolates of M. tuberculosis tested, 25 were determined to be resistant by the Wayne, BACTEC (300 mg/L), and the REMA nicotinamide methods. Using a nicotinamide MIC > 250 mg/L as the cut-off for defining resistance, only one strain was falsely labelled as resistant. The REMA nicotinamide assay demonstrated a sensitivity of 100% and a specificity of 98%. The BACTEC (100 mg/L) falsely classified 8 strains as resistant. DNA sequencing detected mutations in 18/22 of the pncA genes from pyrazinamide-resistant strains.Conclusions: The REMA plate using nicotinamide to detect resistance to pyrazinamide is a simple and rapid method that could be useful in limited-resource countries.