Background. Nontuberculous mycobacteria (NTM) are widely present in environments, such as soil and water, and have recently been recognized as important pathogenic bacteria. The incidence of NTM-related infections is steadily increasing. As the diagnosis and treatment of NTM infection should be distinguished from tuberculosis, and the treatment should be specific to the species of NTM acquired, accurate species identification is required. Methods. In this study, two-step multiplex PCR (mPCR) and multigene sequence-based analysis were used to accurately identify NTM species in 320 clinical isolates from Gyeongsang National University Hospital (GNUH). In particular, major mycobacterial strains with a high isolation frequency as well as coinfections with multiple species were diagnosed through two-step mPCR. Multigene sequencing was performed to accurately identify other NTM species not detected by mPCR. Variable regions of the genes 16S rRNA, rpoB, hsp65, and 16S-23S rRNA internal transcribed spacer were included in the analysis. Results. Two-step mPCR identified 234 (73.1%) cases of M. intracellulare, 26 (8.1%) cases of M. avium subsp. avium, and 13 (4.1%) cases of M. avium subsp. hominissuis infection. Additionally, 9 (2.8%) M. fortuitum, 9 (2.8%) M. massiliense, 2 (0.6%) M. abscessus, and 4 (1.2%) M. kansasii isolates were identified. Coinfection was identified in 7 (2.2%) samples. The sixteen samples not classified by two-step mPCR included 6 (1.9%) cases of M. chimaera, 4 (1.3%) M. gordonae, 1 (0.3%) M. colombiense, 1 (0.3%) M. mageritense, and 1 (0.3%) M. persicum identified by sequence analysis. Conclusions. The results of this study suggest a strategy for rapid detection and accurate identification of species using two-step mPCR and multigene sequence-based analysis. To the best of our knowledge, this study is the first to report the identification of NTM species isolated from patients in Gyeongnam/Korea.

中文翻译：

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使用多重 PCR 和基于多基因序列的分析鉴定韩国庆南地区肺部疾病患者中的非结核分枝杆菌

背景。非结核分枝杆菌 (NTM) 广泛存在于环境中，如土壤和水，最近被认为是重要的病原菌。NTM 相关感染的发生率正在稳步上升。由于 NTM 感染的诊断和治疗应与结核病相鉴别，治疗应针对获得性 NTM 的种属，因此需要进行准确的种属鉴定。方法. 在这项研究中，使用两步多重 PCR (mPCR) 和基于多基因序列的分析来准确鉴定庆尚国立大学医院 (GNUH) 的 320 个临床分离株中的 NTM 物种。特别是，通过两步 mPCR 诊断出具有高分离频率的主要分枝杆菌菌株以及与多个物种的共感染。进行多基因测序以准确识别 mPCR 未检测到的其他 NTM 物种。分析中包括基因 16S rRNA、rpoB、hsp65和 16S-23S rRNA 内部转录间隔区的可变区。结果。两步 mPCR 鉴定了 234 例（73.1%）M病例。细胞内，26 (8.1%) 例M。鸟亚种 avium和 13 (4.1%) 例M。鸟亚种 人类感染。此外，9 (2.8%) M。偶然, 9 (2.8%) M . massiliense , 2 (0.6%) M . 脓肿和 4 (1.2%) M。堪萨斯分离株被鉴定。在 7 个 (2.2%) 样本中发现了合并感染。未通过两步 mPCR 分类的 16 例样本包括 6 例（1.9%）M病例。嵌合体, 4 (1.3% ) M. gordonae , 1 (0.3%) M .哥伦比亚, 1 (0.3% ) M. mageritense和 1 (0.3%) M。通过序列分析鉴定出白蜡菌。结论。这项研究的结果提出了一种使用两步 mPCR 和基于多基因序列的分析来快速检测和准确识别物种的策略。据我们所知，这项研究是第一个报告从庆南/韩国患者中分离出的 NTM 物种的鉴定。