Keerthirathne Thilini Piushani, Magana-Arachchi Dhammika Nayoma, Madegedara Dushantha, Sooriyapathirana Suneth Sithumini
National Institute of Fundamental Studies, Kandy, Sri Lanka.
Respiratory Disease Treatment Unit &Teaching Hospital, Kandy, Sri Lanka.
BMC Infect Dis. 2016 Oct 26;16(1):607. doi: 10.1186/s12879-016-1943-y.
Mycobacteria have a spectrum of virulence and different susceptibilities to antibiotics. Distinguishing mycobacterial species is vital as patients with non-tuberculous mycobacterial (NTM) infections present clinical features that are similar to those of patients with tuberculosis. Thus, rapid differentiation of Mycobacterium tuberculosis complex from NTM is critical to administer appropriate treatment. Hence the aim of the study was to rapid identification of mycobacterial species present in bronchial washings using multiplex real time Polymerase Chain Reaction (PCR) and to determine the drug susceptibility in identified mycobacterial species.
Sputum smear negative bronchoscopy specimens (n = 150) were collected for a period of one year, from patients attending the General Hospital Kandy, Sri Lanka. The specimens were processed with modified Petroff's method and were cultured on Löwenstein- Jensen medium. DNA, extracted from the mycobacterial isolates were subjected to a SYBR green mediated real time multiplex, PCR assay with primers specific for the M. tuberculosis complex, M. avium complex, M. chelonae-M.abscessus group and M. fortuitum group. DNA sequencing was performed for the species confirmation, by targeting the 16S rRNA gene and the drug susceptibility testing was performed for the molecularly identified isolates of M. tuberculosis and NTM.
The optimized SYBR Green mediated multiplex real-time PCR assay was able to identify the presence of genus Mycobacterium in 25 out of 26 AFB positive isolates, two M. tuberculosis complex, three M. avium complex and two isolates belonging to M. chelonae-M. abscessus group. DNA sequencing confirmed the presence of M. tuberculosis, M. chelonae-M. abscessus, M. intracellulare, M. avium, Rhodococcus sp. and M. celatum. Remaining isolates were identified as Mycobacterium sp. All the NTM isolates were sensitive to amikacin and seven were resistant to ciproflaxacin. Twenty two of the NTM isolates and the isolate Rhodococcus was resistant to clarithromycin. The two isolates of M. tuberculosis were sensitive to all first line anti tuberculosis drugs.
The optimized SYBR Green mediated multiplex real time PCR assay could be an effective tool for the rapid differentiation of pathogenic M. tuberculosis complex from the opportunistic nontuberculous mycobacteria and also it confirmed the presence of NTM in 15.3 % of the study population.
分枝杆菌具有不同的毒力和对抗生素的不同敏感性。区分分枝杆菌种类至关重要，因为非结核分枝杆菌（NTM）感染患者的临床特征与结核病患者相似。因此，快速区分结核分枝杆菌复合群与NTM对于给予恰当治疗至关重要。因此，本研究的目的是使用多重实时聚合酶链反应（PCR）快速鉴定支气管灌洗物中存在的分枝杆菌种类，并确定已鉴定分枝杆菌种类的药物敏感性。
从斯里兰卡康提总医院的患者中收集了为期一年的痰涂片阴性支气管镜检查标本（n = 150）。标本采用改良的彼得罗夫方法处理，并在罗-琴培养基上培养。从分枝杆菌分离株中提取的DNA，使用针对结核分枝杆菌复合群、鸟分枝杆菌复合群、龟分枝杆菌-脓肿分枝杆菌组和偶然分枝杆菌组的引物，进行SYBR绿介导的实时多重PCR检测。通过靶向16S rRNA基因进行DNA测序以确认菌种，并对分子鉴定的结核分枝杆菌和NTM分离株进行药物敏感性测试。
优化后的SYBR绿介导的多重实时PCR检测能够在26株抗酸杆菌阳性分离株中的25株中鉴定出分枝杆菌属的存在，其中包括两株结核分枝杆菌复合群、三株鸟分枝杆菌复合群以及两株属于龟分枝杆菌-脓肿分枝杆菌组的分离株。DNA测序证实存在结核分枝杆菌、龟分枝杆菌-脓肿分枝杆菌、胞内分枝杆菌、鸟分枝杆菌、红球菌属和celatum分枝杆菌。其余分离株被鉴定为分枝杆菌属。所有NTM分离株对阿米卡星敏感，七株对环丙沙星耐药。22株NTM分离株和红球菌属分离株对克拉霉素耐药。两株结核分枝杆菌分离株对所有一线抗结核药物敏感。
优化后的SYBR绿介导的多重实时PCR检测可能是一种有效的工具，用于从机会性非结核分枝杆菌中快速区分致病性结核分枝杆菌复合群，并且它证实了在15.3%的研究人群中存在NTM。
