Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China
Background: Diseases caused by M. tuberculosis (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. Methods: To develop and assess a mul...
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Elsevier
2025-01-01
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2405844024174154 |
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| author | Tingting Fang Lijun Peng Tingting Yang Qingshan Cai Huanyu Li Hao Li Long Cai |
| author_facet | Tingting Fang Lijun Peng Tingting Yang Qingshan Cai Huanyu Li Hao Li Long Cai |
| author_sort | Tingting Fang |
| collection | DOAJ |
| description | Background: Diseases caused by M. tuberculosis (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. Methods: To develop and assess a multiplex real-time fluorescence PCR (Multiplex PCR) method for rapid identification of MTB, M. avium complex (MAC), M. chelonae-M. abscessus group (MCAG), and M. kansasii 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. Results: 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 (p > 0.05) and higher than Culture, especially in extrapulmonary samples (P < 0.0001). For NTM detection in pulmonary samples, the sensitivity was slightly lower than Culture (P > 0.05) but higher than CapitalBio RT-PCR (P < 0.05). Overall, the Multiplex PCR showed significantly higher sensitivity for mycobacterial diseases compared to the other three methods (P < 0.01), with a specificity of 96 %. Conclusions: 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. |
| format | Article |
| id | doaj-art-21b7b5ab2655493c86622049cb8053ea |
| institution | Kabale University |
| issn | 2405-8440 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Heliyon |
| spelling | doaj-art-21b7b5ab2655493c86622049cb8053ea2025-01-17T04:51:06ZengElsevierHeliyon2405-84402025-01-01111e41384Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern ChinaTingting Fang0Lijun Peng1Tingting Yang2Qingshan Cai3Huanyu Li4Hao Li5Long Cai6Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaClinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaClinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaClinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaClinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaClinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaCorresponding author.; Clinical Laboratory Center, Hangzhou Red Cross Hospital, Hangzhou, Zhejiang, 310003, ChinaBackground: Diseases caused by M. tuberculosis (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. Methods: To develop and assess a multiplex real-time fluorescence PCR (Multiplex PCR) method for rapid identification of MTB, M. avium complex (MAC), M. chelonae-M. abscessus group (MCAG), and M. kansasii 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. Results: 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 (p > 0.05) and higher than Culture, especially in extrapulmonary samples (P < 0.0001). For NTM detection in pulmonary samples, the sensitivity was slightly lower than Culture (P > 0.05) but higher than CapitalBio RT-PCR (P < 0.05). Overall, the Multiplex PCR showed significantly higher sensitivity for mycobacterial diseases compared to the other three methods (P < 0.01), with a specificity of 96 %. Conclusions: 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.http://www.sciencedirect.com/science/article/pii/S2405844024174154Mycobacterium tuberculosis complexNon-tuberculous mycobacteriaMultiplex real-time fluorescence PCRRapid identification |
| spellingShingle | Tingting Fang Lijun Peng Tingting Yang Qingshan Cai Huanyu Li Hao Li Long Cai Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China Heliyon Mycobacterium tuberculosis complex Non-tuberculous mycobacteria Multiplex real-time fluorescence PCR Rapid identification |
| title | Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China |
| title_full | Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China |
| title_fullStr | Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China |
| title_full_unstemmed | Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China |
| title_short | Development and evaluation of multiplex real-time PCR for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern China |
| title_sort | development and evaluation of multiplex real time pcr for rapid identifying major pathogenic mycobacteria from pulmonary and extrapulmonary clinical samples in eastern china |
| topic | Mycobacterium tuberculosis complex Non-tuberculous mycobacteria Multiplex real-time fluorescence PCR Rapid identification |
| url | http://www.sciencedirect.com/science/article/pii/S2405844024174154 |
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