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Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries

Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to de...

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Autores principales: Chakraborty, Subhra, Connor, Sean, Velagic, Mirza
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8827434/
https://www.ncbi.nlm.nih.gov/pubmed/35089927
http://dx.doi.org/10.1371/journal.pntd.0010180
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author Chakraborty, Subhra
Connor, Sean
Velagic, Mirza
author_facet Chakraborty, Subhra
Connor, Sean
Velagic, Mirza
author_sort Chakraborty, Subhra
collection PubMed
description Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay–"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user’s bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user’s need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~10(5) CFU/gm of stool for LT, STh, and STp and ~10(4) CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella.
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spelling pubmed-88274342022-02-10 Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries Chakraborty, Subhra Connor, Sean Velagic, Mirza PLoS Negl Trop Dis Research Article Enterotoxigenic E. coli (ETEC) and Shigella spp (Shigella) are complex pathogens. The diagnostic assays currently used to detect these pathogens are elaborate or complicated, which make them difficult to apply in resource poor settings where these diseases are endemic. The culture methods used to detect Shigella are not sensitive, and the methods used to detect ETEC are only available in a few research labs. To address this gap, we developed a rapid and simple diagnostic assay–"Rapid LAMP based Diagnostic Test (RLDT)." The six minutes sample preparation method directly from the fecal samples with lyophilized reaction strips and using established Loop-mediated Isothermal Amplification (LAMP) platform, ETEC [heat labile toxin (LT) and heat stable toxins (STh, and STp) genes] and Shigella (ipaH gene) detection was made simple, rapid (<50 minutes), and inexpensive. This assay is cold chain and electricity free. Moreover, RLDT requires minimal equipment. To avoid any end user’s bias, a battery-operated, handheld reader was used to read the RLDT results. The results can be read as positive/negative or as real time amplification depending on the end user’s need. The performance specifications of the RLDT assay, including analytical sensitivity and specificity, were evaluated using fecal samples spiked with ETEC and Shigella strains. The limit of detection was ~10(5) CFU/gm of stool for LT, STh, and STp and ~10(4) CFU/gm of stool for the ipaH gene, which corresponds to about 23 CFU and 1 CFU respectively for ETEC and Shigella per 25uL reaction within 40 minutes. The RLDT assay from stool collection to result is simple, and rapid and at the same time sufficiently sensitive. RLDT has the potential to be applied in resource poor endemic settings for the rapid diagnosis of ETEC and Shigella. Public Library of Science 2022-01-28 /pmc/articles/PMC8827434/ /pubmed/35089927 http://dx.doi.org/10.1371/journal.pntd.0010180 Text en © 2022 Chakraborty 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
Chakraborty, Subhra
Connor, Sean
Velagic, Mirza
Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries
title Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries
title_full Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries
title_fullStr Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries
title_full_unstemmed Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries
title_short Development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic E. coli and Shigella spp applicable to endemic countries
title_sort development of a simple, rapid, and sensitive diagnostic assay for enterotoxigenic e. coli and shigella spp applicable to endemic countries
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8827434/
https://www.ncbi.nlm.nih.gov/pubmed/35089927
http://dx.doi.org/10.1371/journal.pntd.0010180
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