Cargando…
Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy
Mycoplasma pneumoniae is a common causative pathogen of community-acquired pneumonia. An accurate and sensitive detection method is important for evaluating disease severity and treatment efficacy. Digital droplet PCR (ddPCR) is a competent method enabling the absolute quantification of DNA copy num...
Autores principales: | , , , , , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2023
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10324665/ https://www.ncbi.nlm.nih.gov/pubmed/37426001 http://dx.doi.org/10.3389/fmicb.2023.1177273 |
_version_ | 1785069191855865856 |
---|---|
author | Zhao, Hanqing Yan, Chao Feng, Yanling Du, Bing Feng, Junxia Cui, Xiaohu Cui, Jinghua Gan, Lin Fan, Zheng Xu, Ziying Fu, Tongtong Yu, Zihui Yuan, Jing Xue, Guanhua |
author_facet | Zhao, Hanqing Yan, Chao Feng, Yanling Du, Bing Feng, Junxia Cui, Xiaohu Cui, Jinghua Gan, Lin Fan, Zheng Xu, Ziying Fu, Tongtong Yu, Zihui Yuan, Jing Xue, Guanhua |
author_sort | Zhao, Hanqing |
collection | PubMed |
description | Mycoplasma pneumoniae is a common causative pathogen of community-acquired pneumonia. An accurate and sensitive detection method is important for evaluating disease severity and treatment efficacy. Digital droplet PCR (ddPCR) is a competent method enabling the absolute quantification of DNA copy number with high precision and sensitivity. We established ddPCR for M. pneumoniae detection, using clinical specimens for validation, and this showed excellent specificity for M. pneumoniae. The limit of detection of ddPCR was 2.9 copies/reaction, while that for real-time PCR was 10.8 copies/reaction. In total, 178 clinical samples were used to evaluate the ddPCR assay, which correctly identified and differentiated 80 positive samples, whereas the real-time PCR tested 79 samples as positive. One sample that tested negative in real-time PCR was positive in ddPCR, with a bacterial load of three copies/test. For samples that tested positive in both methods, the cycle threshold of real-time PCR was highly correlated with the copy number of ddPCR. Bacterial loads in patients with severe M. pneumoniae pneumonia were significantly higher than those in patients with general M. pneumoniae pneumonia. The ddPCR showed that bacterial loads were significantly decreased after macrolide treatment, which could have reflected the treatment efficacy. The proposed ddPCR assay was sensitive and specific for the detection of M. pneumoniae. Quantitative monitoring of bacterial load in clinical samples could help clinicians to evaluate treatment efficacy. |
format | Online Article Text |
id | pubmed-10324665 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-103246652023-07-07 Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy Zhao, Hanqing Yan, Chao Feng, Yanling Du, Bing Feng, Junxia Cui, Xiaohu Cui, Jinghua Gan, Lin Fan, Zheng Xu, Ziying Fu, Tongtong Yu, Zihui Yuan, Jing Xue, Guanhua Front Microbiol Microbiology Mycoplasma pneumoniae is a common causative pathogen of community-acquired pneumonia. An accurate and sensitive detection method is important for evaluating disease severity and treatment efficacy. Digital droplet PCR (ddPCR) is a competent method enabling the absolute quantification of DNA copy number with high precision and sensitivity. We established ddPCR for M. pneumoniae detection, using clinical specimens for validation, and this showed excellent specificity for M. pneumoniae. The limit of detection of ddPCR was 2.9 copies/reaction, while that for real-time PCR was 10.8 copies/reaction. In total, 178 clinical samples were used to evaluate the ddPCR assay, which correctly identified and differentiated 80 positive samples, whereas the real-time PCR tested 79 samples as positive. One sample that tested negative in real-time PCR was positive in ddPCR, with a bacterial load of three copies/test. For samples that tested positive in both methods, the cycle threshold of real-time PCR was highly correlated with the copy number of ddPCR. Bacterial loads in patients with severe M. pneumoniae pneumonia were significantly higher than those in patients with general M. pneumoniae pneumonia. The ddPCR showed that bacterial loads were significantly decreased after macrolide treatment, which could have reflected the treatment efficacy. The proposed ddPCR assay was sensitive and specific for the detection of M. pneumoniae. Quantitative monitoring of bacterial load in clinical samples could help clinicians to evaluate treatment efficacy. Frontiers Media S.A. 2023-06-22 /pmc/articles/PMC10324665/ /pubmed/37426001 http://dx.doi.org/10.3389/fmicb.2023.1177273 Text en Copyright © 2023 Zhao, Yan, Feng, Du, Feng, Cui, Cui, Gan, Fan, Xu, Fu, Yu, Yuan and Xue. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Zhao, Hanqing Yan, Chao Feng, Yanling Du, Bing Feng, Junxia Cui, Xiaohu Cui, Jinghua Gan, Lin Fan, Zheng Xu, Ziying Fu, Tongtong Yu, Zihui Yuan, Jing Xue, Guanhua Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy |
title | Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy |
title_full | Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy |
title_fullStr | Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy |
title_full_unstemmed | Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy |
title_short | Absolute quantification of Mycoplasma pneumoniae in infected patients by droplet digital PCR to track disease severity and treatment efficacy |
title_sort | absolute quantification of mycoplasma pneumoniae in infected patients by droplet digital pcr to track disease severity and treatment efficacy |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10324665/ https://www.ncbi.nlm.nih.gov/pubmed/37426001 http://dx.doi.org/10.3389/fmicb.2023.1177273 |
work_keys_str_mv | AT zhaohanqing absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT yanchao absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT fengyanling absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT dubing absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT fengjunxia absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT cuixiaohu absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT cuijinghua absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT ganlin absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT fanzheng absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT xuziying absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT futongtong absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT yuzihui absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT yuanjing absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy AT xueguanhua absolutequantificationofmycoplasmapneumoniaeininfectedpatientsbydropletdigitalpcrtotrackdiseaseseverityandtreatmentefficacy |