Meinzen Christopher, Proaño Alvaro, Gilman Robert H, Caviedes Luz, Coronel Jorge, Zimic Mirko, Sheen Patricia
Asociación Benéfica PRISMA, Peru.
Faculty of Medicine 'Alberto Hurtado', Universidad Peruana Cayetano Heredia, Peru.
Tuberculosis (Edinb). 2016 Jul;99:41-46. doi: 10.1016/j.tube.2016.03.011. Epub 2016 Apr 6.
Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important.
We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 μg/ml and 800 μg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth.
The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 μg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 μg/ml PZA were more accurate than 800 μg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results.
The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.
吡嗪酰胺（PZA）是治疗潜伏性结核病（TB）最重要的药物，用于一线和二线治疗方案。耐多药结核病的持续增加以及PZA耐药性的流行使得开发用于快速鉴定PZA耐药性的替代检测方法变得更加重要。
我们对韦恩检测法（QW）的定量变体进行了标准化和评估，以确定结核分枝杆菌菌株对PZA的耐药性。该检测法使用可见分光光度法定量检测结核分枝杆菌对PZA的代谢及吡嗪酸（POA）的产生。我们在3天、5天和7天的孵育期使用400μg/ml和800μg/ml的PZA浓度评估了该方法。还使用标准韦恩检测法、四氮唑微量板检测法（TEMA）、Bactec 460TB和pncA测序对68份痰标本中的结核分枝杆菌菌株进行了检测。我们使用一致的Bactec 460TB和pncA测序，将QW和标准韦恩检测法与二分法参考分类进行比较。其次，我们确定了QW值与TEMA针对Bactec 460TB百分比生长的最低抑菌浓度（MIC）之间的定量相关性。
标准韦恩检测法的灵敏度为88%，特异性为97.5%，与参考检测相比，约登指数（YI）为0.855。QW在第7天、PZA浓度为400μg/ml时显示出最大约登指数0.934，灵敏度为96%，特异性为97.4%。400μg/ml PZA的吸光度OD值比800μg/ml PZA更准确。虽然QW对PZA敏感性显示出较高的准确性，但它与Bactec百分比生长没有定量相关性。TEMA检测不可靠，且与Bactec结果不相关。
所提出的QW检测法是一种廉价的方法，能够实现比色法PZA耐药性检测的标准化和自动化，其鉴别能力优于标准韦恩检测法。
