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Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens

Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to...

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Autores principales: Nourrisson, Céline, Brunet, Julie, Flori, Pierre, Moniot, Maxime, Bonnin, Virginie, Delbac, Frédéric, Poirier, Philippe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696706/
https://www.ncbi.nlm.nih.gov/pubmed/33187097
http://dx.doi.org/10.3390/microorganisms8111768
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author Nourrisson, Céline
Brunet, Julie
Flori, Pierre
Moniot, Maxime
Bonnin, Virginie
Delbac, Frédéric
Poirier, Philippe
author_facet Nourrisson, Céline
Brunet, Julie
Flori, Pierre
Moniot, Maxime
Bonnin, Virginie
Delbac, Frédéric
Poirier, Philippe
author_sort Nourrisson, Céline
collection PubMed
description Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and compare the performance of (i) two DNA extraction methods (manual versus automated), and (ii) four qPCR assays (three “in-house” and one commercialized), for detection of Blastocystis sp. in human stools. One hundred and forty stools were included, among which 76 were confirmed to be positive for Blastocystis. The manual DNA extraction method allowed for the identification of significantly more positive specimens than the automated method (p < 0.05). In particular, specimens with a low parasite load were negative when DNA was extracted with the automated process. The four qPCR assays also had variable performances, with the commercialized assay being the most sensitive (84%) but the least specific (82%). Overall, for all qPCR assays, the specificity decreased when the sensitivity increased. Blastocystis’ subtype, notably the subtype 4, influenced these performances. Our results indicate that the positivity rate for the detection of Blastocystis in stools could be variable according to the DNA extraction method and the qPCR assay used. These pitfalls need to be considered for the selection of method and interpretation of results, particularly considering the search of this intestinal parasite in a donor before fecal microbiota transplantation.
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spelling pubmed-76967062020-11-29 Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens Nourrisson, Céline Brunet, Julie Flori, Pierre Moniot, Maxime Bonnin, Virginie Delbac, Frédéric Poirier, Philippe Microorganisms Article Diagnosis of Blastocystis in stool may be challenging, as microscopic examination and culture-based methods have demonstrated low sensitivity. Molecular detection assays are now available for this enteric parasite, based on “in-house” or commercial-developed techniques. The aim of this study was to assess and compare the performance of (i) two DNA extraction methods (manual versus automated), and (ii) four qPCR assays (three “in-house” and one commercialized), for detection of Blastocystis sp. in human stools. One hundred and forty stools were included, among which 76 were confirmed to be positive for Blastocystis. The manual DNA extraction method allowed for the identification of significantly more positive specimens than the automated method (p < 0.05). In particular, specimens with a low parasite load were negative when DNA was extracted with the automated process. The four qPCR assays also had variable performances, with the commercialized assay being the most sensitive (84%) but the least specific (82%). Overall, for all qPCR assays, the specificity decreased when the sensitivity increased. Blastocystis’ subtype, notably the subtype 4, influenced these performances. Our results indicate that the positivity rate for the detection of Blastocystis in stools could be variable according to the DNA extraction method and the qPCR assay used. These pitfalls need to be considered for the selection of method and interpretation of results, particularly considering the search of this intestinal parasite in a donor before fecal microbiota transplantation. MDPI 2020-11-11 /pmc/articles/PMC7696706/ /pubmed/33187097 http://dx.doi.org/10.3390/microorganisms8111768 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Nourrisson, Céline
Brunet, Julie
Flori, Pierre
Moniot, Maxime
Bonnin, Virginie
Delbac, Frédéric
Poirier, Philippe
Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
title Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
title_full Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
title_fullStr Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
title_full_unstemmed Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
title_short Comparison of DNA Extraction Methods and Real-Time PCR Assays for the Detection of Blastocystis sp. in Stool Specimens
title_sort comparison of dna extraction methods and real-time pcr assays for the detection of blastocystis sp. in stool specimens
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7696706/
https://www.ncbi.nlm.nih.gov/pubmed/33187097
http://dx.doi.org/10.3390/microorganisms8111768
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