Supplementary Material for: Diversity of Nontuberculous Mycobacteria in Kuwait: Rapid Identification and Differentiation of Mycobacterium Species by Multiplex PCR, INNO-LiPA Mycobacteria v2 Assay and PCR Sequencing of rDNA

Objective: Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. Materials and Methods: DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. Results: mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. Conclusions: mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.研究背景与目的：非结核分枝杆菌（Nontuberculous mycobacteria, NTM）常引发临床表现与结核病难以区分的疾病。明确病原体种类至关重要，因为感染的治疗方案会因致病分枝杆菌菌种不同而存在差异。本研究采用多重聚合酶链反应（multiplex PCR, mPCR）检测法，快速区分分枝杆菌生长指示管960系统（mycobacterial growth indicator tube 960 system, MGIT）培养物为结核分枝杆菌（Mycobacterium tuberculosis, MTB）或非结核分枝杆菌；同时结合INNO LiPA分枝杆菌v2检测法（INNO LiPA Mycobacteria v2 assay, LiPA）和/或核糖体DNA（rDNA）PCR测序技术，对科威特地区的选定结核分枝杆菌分离株与所有非结核分枝杆菌分离株进行菌种特异性鉴定。 材料与方法：从1033名疑似结核病患者的样本中获得MGIT培养物共1033份，其中664份源自肺部样本，369份源自肺外样本，本研究从上述培养物中提取DNA。采用mPCR区分结核分枝杆菌与非结核分枝杆菌。按照试剂盒说明书进行LiPA检测并解读结果。使用泛分枝杆菌引物对rDNA进行扩增与测序。 实验结果：mPCR共鉴定出979株分离株为MTB、53株为NTM，另有1株为混合培养物。经LiPA和/或PCR测序验证，979株选定分离株中的112株确认为MTB。该混合培养物包含结核分枝杆菌与偶然分枝杆菌。LiPA共得到12种检测图谱，在47株NTM分离株中鉴定出10个菌种/菌种复合体，1份培养物中同时检出堪萨斯分枝杆菌（M. kansasii）与瘰疬分枝杆菌（M. scrofulaceum），另有5株分离株仅能鉴定至属水平。PCR测序可对22株分离株实现更精准的物种/亚种水平鉴定。 研究结论：mPCR可快速区分MTB与NTM。LiPA可在物种/菌种复合体水平对52株NTM分离株中的44株进行鉴定，同时可识别2份混合培养物。PCR测序可实现更精准的物种/亚种水平鉴定。采用mPCR快速将分离株划分为MTB或NTM，再通过LiPA/PCR测序完成NTM的菌种特异性鉴定，该方案适用于科威特地区分枝杆菌感染的规范化诊疗。