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...

Descripción completa

Detalles Bibliográficos
Autores principales: 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
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