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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: Lim, Ho Jae, Kang, Eun-Rim, Park, Min Young, Kim, Bo Kyung, Kim, Min Jin, Jung, Sunkyung, Roh, Kyoung Ho, Sung, Nackmoon, Yang, Jae-Hyun, Lee, Min-Woo, Lee, Sun-Hwa, Yang, Yong-Jin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211157/
https://www.ncbi.nlm.nih.gov/pubmed/34138947
http://dx.doi.org/10.1371/journal.pone.0253402
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author Lim, Ho Jae
Kang, Eun-Rim
Park, Min Young
Kim, Bo Kyung
Kim, Min Jin
Jung, Sunkyung
Roh, Kyoung Ho
Sung, Nackmoon
Yang, Jae-Hyun
Lee, Min-Woo
Lee, Sun-Hwa
Yang, Yong-Jin
author_facet Lim, Ho Jae
Kang, Eun-Rim
Park, Min Young
Kim, Bo Kyung
Kim, Min Jin
Jung, Sunkyung
Roh, Kyoung Ho
Sung, Nackmoon
Yang, Jae-Hyun
Lee, Min-Woo
Lee, Sun-Hwa
Yang, Yong-Jin
author_sort Lim, Ho Jae
collection PubMed
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 × 10(2) 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.
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spelling pubmed-82111572021-06-29 Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia Lim, Ho Jae Kang, Eun-Rim Park, Min Young Kim, Bo Kyung Kim, Min Jin Jung, Sunkyung Roh, Kyoung Ho Sung, Nackmoon Yang, Jae-Hyun Lee, Min-Woo Lee, Sun-Hwa Yang, Yong-Jin PLoS One Research Article 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 × 10(2) 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. Public Library of Science 2021-06-17 /pmc/articles/PMC8211157/ /pubmed/34138947 http://dx.doi.org/10.1371/journal.pone.0253402 Text en © 2021 Lim et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Lim, Ho Jae
Kang, Eun-Rim
Park, Min Young
Kim, Bo Kyung
Kim, Min Jin
Jung, Sunkyung
Roh, Kyoung Ho
Sung, Nackmoon
Yang, Jae-Hyun
Lee, Min-Woo
Lee, Sun-Hwa
Yang, Yong-Jin
Development of a multiplex real-time PCR assay for the simultaneous detection of four bacterial pathogens causing pneumonia
title 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_short 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
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8211157/
https://www.ncbi.nlm.nih.gov/pubmed/34138947
http://dx.doi.org/10.1371/journal.pone.0253402
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