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Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing

Traditional qPCR assays for pneumococcal detection and serotype characterization require large sample volume, is expensive and labor intensive. We aimed to develop a quantitative nanofluidic Fluidigm assay to overcome some of these shortcomings. A quantitative Fluidigm assay was established to detec...

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Autores principales: Olwagen, Courtney P., Adrian, Peter V., Madhi, Shabir A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482308/
https://www.ncbi.nlm.nih.gov/pubmed/31019272
http://dx.doi.org/10.1038/s41598-019-42846-y
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author Olwagen, Courtney P.
Adrian, Peter V.
Madhi, Shabir A.
author_facet Olwagen, Courtney P.
Adrian, Peter V.
Madhi, Shabir A.
author_sort Olwagen, Courtney P.
collection PubMed
description Traditional qPCR assays for pneumococcal detection and serotype characterization require large sample volume, is expensive and labor intensive. We aimed to develop a quantitative nanofluidic Fluidigm assay to overcome some of these shortcomings. A quantitative Fluidigm assay was established to detect 11 bacterial pathogens, 55 pneumococcal serotypes and 6 serotypes of H. influenzae. The Fluidigm assay results were compared to conventional qPCR and culture. All reactions in the Fluidigm assay effectively amplified their respective targets with high sensitivity and specificity compared to qPCR. There was excellent concordance between qPCR and Fluidigm for detection of carriage prevalence (kappa > 0.75) and density (Rho > 0.95). Fluidigm identified an additional 7 (4.2%) serotypes over those detected by qPCR. There was a modest concordance between culture and Fluidigm for the majority of reactions detecting S. pneumoniae serotypes/serogroups (kappa > 0.6), with Fluidigm identifying an additional 113 (39.1%) serotypes. Discordant results between the three methods were associated with a low carriage density. The Fluidigm assay was able to detect common pneumococcal serotypes, H. influenzae serotypes, and other common nasopharyngeal bacterial organisms simultaneously. Deployment of this assay in epidemiological studies could provide better insight into the effect of PCV immunization on the nasopharyngeal microbiota in the community.
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spelling pubmed-64823082019-05-07 Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing Olwagen, Courtney P. Adrian, Peter V. Madhi, Shabir A. Sci Rep Article Traditional qPCR assays for pneumococcal detection and serotype characterization require large sample volume, is expensive and labor intensive. We aimed to develop a quantitative nanofluidic Fluidigm assay to overcome some of these shortcomings. A quantitative Fluidigm assay was established to detect 11 bacterial pathogens, 55 pneumococcal serotypes and 6 serotypes of H. influenzae. The Fluidigm assay results were compared to conventional qPCR and culture. All reactions in the Fluidigm assay effectively amplified their respective targets with high sensitivity and specificity compared to qPCR. There was excellent concordance between qPCR and Fluidigm for detection of carriage prevalence (kappa > 0.75) and density (Rho > 0.95). Fluidigm identified an additional 7 (4.2%) serotypes over those detected by qPCR. There was a modest concordance between culture and Fluidigm for the majority of reactions detecting S. pneumoniae serotypes/serogroups (kappa > 0.6), with Fluidigm identifying an additional 113 (39.1%) serotypes. Discordant results between the three methods were associated with a low carriage density. The Fluidigm assay was able to detect common pneumococcal serotypes, H. influenzae serotypes, and other common nasopharyngeal bacterial organisms simultaneously. Deployment of this assay in epidemiological studies could provide better insight into the effect of PCV immunization on the nasopharyngeal microbiota in the community. Nature Publishing Group UK 2019-04-24 /pmc/articles/PMC6482308/ /pubmed/31019272 http://dx.doi.org/10.1038/s41598-019-42846-y Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Olwagen, Courtney P.
Adrian, Peter V.
Madhi, Shabir A.
Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
title Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
title_full Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
title_fullStr Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
title_full_unstemmed Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
title_short Performance of the Biomark HD real-time qPCR System (Fluidigm) for the detection of nasopharyngeal bacterial pathogens and Streptococcus pneumoniae typing
title_sort performance of the biomark hd real-time qpcr system (fluidigm) for the detection of nasopharyngeal bacterial pathogens and streptococcus pneumoniae typing
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482308/
https://www.ncbi.nlm.nih.gov/pubmed/31019272
http://dx.doi.org/10.1038/s41598-019-42846-y
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