Detection and Identification of Mycobacteria in Clinical Specimens

通过常规方法检测和鉴定分枝杆菌属的抗酸杆菌需要从患者标本中培养生物体，然后测试分离株的各种表型特征。该实验室先前报告了一种聚合酶链反应（PCR）和核酸测序检测方法的开发，靶向SecA1管家基因，可用于在种属水平上识别大多数分枝杆菌。此外，初步研究表明，该测试可直接用于患者样本，以检测和鉴定临床标本中的分枝杆菌，从而避免了传统培养和鉴定方法所涉及的漫长延迟。 共76个呼吸道标本（8支气管肺泡灌洗液，68支气管）收集和冷冻在NIH分枝杆菌实验室进行了测试，我们的PCR SecA1检测，与抗酸涂片和培养的结果进行了比较。76份标本中共有69份为分枝杆菌阳性，SecA1检测试剂盒的检测灵敏度为87%，特异性为100%。对镜检抗酸杆菌阳性标本的敏感性为100%，对涂片阴性、培养阳性标本的敏感性为72%。最近，开始了第二项研究，样本来自乌干达结核病诊所的患者。从大约140名患者身上采集标本，然后冷冻运往美国。NIH分枝杆菌实验室通过涂片和培养处理所有标本，我们对标本进行PCR分析。大约65%的患者的涂片和分支杆菌培养呈阳性。对PCR数据的初步分析显示，检测灵敏度为91%，特异性为84%。正在进行额外的分析，以确定不一致的结果是否与该患者人群的既往抗分枝杆菌治疗有关。

Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. This laboratory previously reported the development of a polymerase chain reaction (PCR) and nucleic acid sequencing assay, targeting SecA1 housekeeping gene, that can be used to identify most mycobacteria at the species level. Additionally, preliminary studies indicated this test could be used directly with patient samples to detect and identify mycobacteria in clinical specimens, thus avoiding the lengthy delays involved with the traditional culture and identification methods. A total of 76 respiratory specimens (8 bronchial alveolar lavages, 68 sputa) collected and frozen at the NIH Mycobacteriology Laboratory were tested by our PCR SecA1 assay, with the results compared with acid-fast smears and cultures. A total of 69 of 76 specimens were positive for mycobacteria, and the SecA1 assay had a test sensitivity of 87% and specificity of 100%. The sensitivity of the assay was 100% for specimens with many acid-fast baciili seen by microscopy and 72% for the smear-negative, culture-positive specimens. Recently, a second study was initiated with specimens collected from patients in Uganda who presented to a Tuberculosis Clinic. Specimens were collected from approximately 140 patients and then shipped frozen to the United States. The NIH Mycobacteriology Lab processed all specimens by smear and culture, and we performed PCR analysis on the specimens. Approximately 65% of the patients had positive smears and cultures for mycobacteria. The initial analysis of the PCR data revealed a test sensitivity of 91% and specificityof 84%. Additional analysis is underway to determine if the discrepant results are related to prior antimycobacterial therapy in this patient population.