Fang Tingting, Peng Lijun, Yang Tingting, Cai Qingshan, Li Huanyu, Li Hao, Cai Long
Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, China.
Heliyon. 2024 Dec 27;11(1):e41384. doi: 10.1016/j.heliyon.2024.e41384. eCollection 2025 Jan 15.
Diseases caused by (MTB) and non-tuberculous mycobacteria (NTM) have similar clinical symptoms but require different treatments. Rapid and accurate identification of MTB and NTM is essential for proper patient management and treatment.
To develop and assess a multiplex real-time fluorescence PCR (Multiplex PCR) method for rapid identification of MTB, complex (MAC), M. chelonae-M. abscessus group (MCAG), and in clinical samples. The specificity and limit of detection (LOD) were tested using standard strains and clinical isolates. The accuracy of the Multiplex PCR was validated with DNA from 228 known clinical samples confirmed by Targeted next-generation sequencing (tNGS). Additionally, 901 consecutive clinical samples were assessed to evaluate the Multiplex PCR's diagnostic performance in detecting mycobacteria in pulmonary and extrapulmonary samples.
LOD of the four mycobacteria ranged from 11.7 to 360.0 CFU/mL in water, 43.8-922.0 CFU/mL in sputum, and 53.2-859.3 CFU/mL in sputum mixed infection, with 98.7 % sample detection accuracy and 100 % strains identification accuracy. Based on the composite reference standard, the sensitivity of the Multiplex PCR for detecting tuberculosis in pulmonary and extrapulmonary samples was comparable to Xpert ( > 0.05) and higher than Culture, especially in extrapulmonary samples ( < 0.0001). For NTM detection in pulmonary samples, the sensitivity was slightly lower than Culture ( > 0.05) but higher than CapitalBio RT-PCR ( < 0.05). Overall, the Multiplex PCR showed significantly higher sensitivity for mycobacterial diseases compared to the other three methods ( < 0.01), with a specificity of 96 %.
The Multiplex PCR demonstrated excellent diagnostic performance in both pulmonary and extrapulmonary samples, offering a low-cost, rapid identifying tool for major pathogenic mycobacteria in eastern China.
结核分枝杆菌（MTB）和非结核分枝杆菌（NTM）引起的疾病具有相似的临床症状，但需要不同的治疗方法。快速准确地鉴定MTB和NTM对于患者的合理管理和治疗至关重要。
开发并评估一种多重实时荧光PCR（多重PCR）方法，用于快速鉴定临床样本中的MTB、鸟分枝杆菌复合群（MAC）、龟分枝杆菌-脓肿分枝杆菌组（MCAG）和胞内分枝杆菌。使用标准菌株和临床分离株测试其特异性和检测限（LOD）。通过靶向二代测序（tNGS）确认的228份已知临床样本的DNA验证多重PCR的准确性。此外，评估了901份连续的临床样本，以评估多重PCR在检测肺内和肺外样本中分枝杆菌的诊断性能。
四种分枝杆菌在水中的LOD范围为11.7至360.0 CFU/mL，在痰中为43.8 - 922.0 CFU/mL，在痰混合感染中为53.2 - 859.3 CFU/mL，样本检测准确率为98.7%，菌株鉴定准确率为100%。基于综合参考标准，多重PCR检测肺内和肺外样本中结核病的敏感性与Xpert相当（P>0.05），高于培养法，尤其是在肺外样本中（P<0.0001）。对于肺内样本中NTM的检测，敏感性略低于培养法（P>0.05）但高于博奥生物RT-PCR（P<0.05）。总体而言，与其他三种方法相比，多重PCR对分枝杆菌病的敏感性显著更高（P<0.01），特异性为96%。
多重PCR在肺内和肺外样本中均表现出优异的诊断性能，为中国东部主要致病性分枝杆菌提供了一种低成本、快速的鉴定工具。
