Biçmen Can, Coşkun Meral, Gündüz Ayriz T, Senol Güneş, Cirak A Kadri, Tibet Gültekin
Dr. Suat Seren Göğüs Hastaliklarl ve Cerrahisi Eğitim ve Araştirma Hastanesi, Mikrobiyoloji Laboratuvari, Izmir.
Mikrobiyol Bul. 2007 Oct;41(4):503-10.
The aim of this study was the identification of atypical mycobacteria isolated from various samples of patients prediagnosed as tuberculosis between November 2004 and June 2006 by a commercial polymerase chain reaction (PCR) based reverse hybridization kit (INNO-LiPA Mycobacteria v2, Innogenetics NV, Belgium). A total of 21,060 samples obtained from 9660 patients were included to the study. After decontamination and homogenization processes, the samples were cultivated in automated MGIT Bactec 960 system and the diagnosis of atypical mycobacteria was performed in 4532 (21.5%) culture positive samples with NAP test by using Bactec 460 TB system. After DNA isolation, PCR method was performed by using the primers specific for mycobacterial 16S-23S spacer region. PCR products were then hybridized with the probes specific for Mycobacterium species on nitrocellulose strips according to the recommendations of the manufacturer and evaluated. Additionally, two different versions of another commercial Line Probe Assay (LiPA) kit [GenoType Mycobacterium and GenoType Mycobacterium AS (Additional Species), Hain Lifescience, Germany] were used for the detection of unidentified mycobacterial strains. In our study period, 10 different Mycobacterium species were identified from 44 (1%) respiratory tract samples (sputum, bronchial aspiration fluid, bronchoalveolar lavage) belonging to 30 patients. While repeated atypical mycobacterial growth was found in 13 patients on different days, 17 patients showed atypical mycobacterial growth only in one sample or in separate multiple samples taken within the same day. The species distribution among patients were as follows; M. fortuitum-M. peregrinum complex (n=5), M. intracellulare (n=4), M. avium complex (n=4), M. chelonae complex (n=4), M. gordonae (n=4), M. kansasii (n=3), M. szulgailintermedium (n=2), M. simiae (n=1) and M. scrofulaceum (n=1). Two of four samples which were unidentified by INNO-LiPA and GenoType MTBC were identified as M. szulgai/intermedium by GenoType Mycobacterium AS and the other two were found as unidentified atypical mycobacteria (Mycobacterium spp.). As a result, the frequency of atypical mycobacteria isolated in our hospital was thought to be low, however, species-level identification might be useful for the planning of therapy in such patients. In addition, after NAP test, INNO-LiPA and GenoType Mycobacterium were useful tests in microbiological identification of atypical mycobacteria, and GenoType Mycobacterium AS test could be applied in mycobacterial strains which were not identified by the former assays.
本研究的目的是通过基于商业聚合酶链反应（PCR）的反向杂交试剂盒（INNO-LiPA Mycobacteria v2，比利时Innogenetics NV公司），鉴定2004年11月至2006年6月期间从预先诊断为结核病的患者的各种样本中分离出的非结核分枝杆菌。本研究共纳入了9660例患者的21060份样本。经过去污和匀浆处理后，将样本接种于自动MGIT Bactec 960系统中培养，并使用Bactec 460 TB系统通过NAP试验对4532份（21.5%）培养阳性样本进行非结核分枝杆菌诊断。DNA提取后，使用针对分枝杆菌16S-23S间隔区的特异性引物进行PCR方法检测。然后，根据制造商的建议，将PCR产物与硝酸纤维素膜条上针对分枝杆菌菌种的探针进行杂交并评估。此外，还使用了另一种商业线性探针分析（LiPA）试剂盒的两个不同版本[GenoType Mycobacterium和GenoType Mycobacterium AS（其他菌种），德国Hain Lifescience公司]来检测未鉴定的分枝杆菌菌株。在我们的研究期间，从30例患者的44份（1%）呼吸道样本（痰液、支气管吸出液、支气管肺泡灌洗）中鉴定出10种不同的分枝杆菌菌种。13例患者在不同日期发现反复出现非结核分枝杆菌生长，17例患者仅在一个样本中或在同一天采集的多个不同样本中出现非结核分枝杆菌生长。患者中的菌种分布如下：偶然分枝杆菌-龟分枝杆菌复合群（n=5）、胞内分枝杆菌（n=4）、鸟分枝杆菌复合群（n=4）、龟分枝杆菌复合群（n=4）、戈登分枝杆菌（n=4）、堪萨斯分枝杆菌（n=3）、苏尔加分枝杆菌-中间分枝杆菌（n=2）、猿分枝杆菌（n=1）和瘰疬分枝杆菌（n=1）。INNO-LiPA和GenoType MTBC未鉴定的4份样本中的2份被GenoType Mycobacterium AS鉴定为苏尔加分枝杆菌/中间分枝杆菌，另外2份被发现为未鉴定的非结核分枝杆菌（分枝杆菌属）。结果表明，我院分离出的非结核分枝杆菌频率较低，然而菌种水平的鉴定可能有助于此类患者的治疗规划。此外，经过NAP试验后，INNO-LiPA和GenoType Mycobacterium在非结核分枝杆菌的微生物鉴定中是有用的检测方法，GenoType Mycobacterium AS试验可应用于前一种检测方法未鉴定的分枝杆菌菌株。
