Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.

Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum an...

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Autores principales:	Ho Jae Lim, Eun-Rim Kang, Min Young Park, Bo Kyung Kim, Min Jin Kim, Sunkyung Jung, Kyoung Ho Roh, Nackmoon Sung, Jae-Hyun Yang, Min-Woo Lee, Sun-Hwa Lee, Yong-Jin Yang
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/aa6a9ae598f7421891b51839a0b02e77
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spelling	oai:doaj.org-article:aa6a9ae598f7421891b51839a0b02e772021-12-02T20:10:26ZDevelopment of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.1932-620310.1371/journal.pone.0253402https://doaj.org/article/aa6a9ae598f7421891b51839a0b02e772021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0253402https://doaj.org/toc/1932-6203Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.Ho Jae LimEun-Rim KangMin Young ParkBo Kyung KimMin Jin KimSunkyung JungKyoung Ho RohNackmoon SungJae-Hyun YangMin-Woo LeeSun-Hwa LeeYong-Jin YangPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 6, p e0253402 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ho Jae Lim Eun-Rim Kang Min Young Park Bo Kyung Kim Min Jin Kim Sunkyung Jung Kyoung Ho Roh Nackmoon Sung Jae-Hyun Yang Min-Woo Lee Sun-Hwa Lee Yong-Jin Yang Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
description	Classification of clinical symptoms and diagnostic microbiology are essential to effectively employ antimicrobial therapy for lower respiratory tract infections (LRTIs) in a timely manner. Empirical antibiotic treatment without microbial identification hinders the selective use of narrow-spectrum antibiotics and effective patient treatment. Thus, the development of rapid and accurate diagnostic procedures that can be readily adopted by the clinic is necessary to minimize non-essential or excessive use of antibiotics and accelerate patient recovery from LRTI-induced damage. We developed and validated a multiplex real-time polymerase chain reaction (mRT-PCR) assay with good analytical performance and high specificity to simultaneously detect four bacterial pathogens causing pneumonia: Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Moraxella catarrhalis. The analytical performance of mRT-PCR against target pathogens was evaluated by the limit of detection (LOD), specificity, and repeatability. Two hundred and ten clinical specimens from pneumonia patients were processed using an automatic nucleic acid extraction system for the "respiratory bacteria four" (RB4) mRT-PCR assay, and the results were directly compared to references from bacterial culture and/or Sanger sequencing. The RB4 mRT-PCR assay detected all target pathogens from sputum specimens with a coefficient of variation ranging from 0.29 to 1.71 and conservative LOD of DNA corresponding to 5 × 102 copies/reaction. The concordance of the assay with reference-positive specimens was 100%, and additional bacterial infections were detected from reference-negative specimens. Overall, the RB4 mRT-PCR assay showed a more rapid turnaround time and higher performance that those of reference assays. The RB4 mRT-PCR assay is a high-throughput and reliable tool that assists decision-making assessment and outperforms other standard methods. This tool supports patient management by considerably reducing the inappropriate use of antibiotics.
format	article
author	Ho Jae Lim Eun-Rim Kang Min Young Park Bo Kyung Kim Min Jin Kim Sunkyung Jung Kyoung Ho Roh Nackmoon Sung Jae-Hyun Yang Min-Woo Lee Sun-Hwa Lee Yong-Jin Yang
author_facet	Ho Jae Lim Eun-Rim Kang Min Young Park Bo Kyung Kim Min Jin Kim Sunkyung Jung Kyoung Ho Roh Nackmoon Sung Jae-Hyun Yang Min-Woo Lee Sun-Hwa Lee Yong-Jin Yang
author_sort	Ho Jae Lim
title	Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
title_short	Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
title_full	Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
title_fullStr	Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
title_full_unstemmed	Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
title_sort	development of a multiplex real-time pcr assay for the simultaneous detection of four bacterial pathogens causing pneumonia.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/aa6a9ae598f7421891b51839a0b02e77
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Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia.

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