Detection and Identification of Mycobacteria in Clinical Specimens

通过常规程序检测和鉴定分枝杆菌种类的抗酸杆菌需要从患者样本中培养微生物，然后测试分离株的各种表型特征。该实验室此前曾报道过针对SecA1看家基因的聚合酶链式反应(PCR)和核酸测序分析的发展，可用于在物种水平上鉴定大多数分枝杆菌。此外，初步研究表明，这项测试可以直接用于患者样本中的分支杆菌检测和鉴定，从而避免了传统培养和鉴定方法涉及的漫长延迟。 对NIH分枝杆菌实验室收集和冷冻的76份呼吸道标本(8份支气管肺泡灌洗液，68份痰)进行了PCRSecA1检测，并与抗酸涂片和培养结果进行了比较。76份标本中有69份分枝杆菌阳性，SecA1试验的敏感性为87%，特异性为100%。该方法对镜检抗酸杆菌较多的标本的敏感性为100%，对涂片阴性、培养阳性的标本的敏感性为72%。最近，从提交给结核病诊所的乌干达患者身上收集的标本开始了第二项研究。从大约140名患者身上收集了样本，然后将其冷冻运往美国。NIH分枝杆菌实验室对所有标本进行涂片和培养，并对标本进行聚合酶链式反应分析。大约65%的患者涂片和培养出分枝杆菌阳性。对PCR数据的初步分析显示，检测的敏感性为91%，特异性为84%。进一步的分析正在进行中，以确定这些不一致的结果是否与该患者群体中先前的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.