Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs
Abstract Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the s...
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doaj-7f348df1f413460ca864fce6d83becdc2021-09-26T11:33:21ZengBMCParasites & Vectors1756-33052021-09-0114111210.1186/s13071-021-04941-wImproving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggsJavier Gandasegui0Berta Grau-Pujol1María Cambra-Pelleja2Valdemiro Escola3Maria Antonietta Demontis4Anelsio Cossa5José Carlos Jamine6Rafael Balaña-Fouce7Lisette van Lieshout8José Muñoz9María Martínez-Valladares10The Stopping Transmission of Intestinal Parasites (STOP) Project ConsortiumInstituto de Ganadería de Montaña (CSIC-Universidad de León)ISGlobal, Hospital Clínic - Universitat de BarcelonaInstituto de Ganadería de Montaña (CSIC-Universidad de León)Centro de Investigação Em Saúde de Manhiça (CISM)Departement of Parasitology, Leiden University Medical CenterCentro de Investigação Em Saúde de Manhiça (CISM)Centro de Investigação Em Saúde de Manhiça (CISM)Departamento de Ciencias Biomédicas, Universidad de LeónDepartement of Parasitology, Leiden University Medical CenterISGlobal, Hospital Clínic - Universitat de BarcelonaInstituto de Ganadería de Montaña (CSIC-Universidad de León)Abstract Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstracthttps://doi.org/10.1186/s13071-021-04941-wSoil-transmitted helminthsBenzimidazolesAnthelmintic resistancePyrosequencing |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Javier Gandasegui Berta Grau-Pujol María Cambra-Pelleja Valdemiro Escola Maria Antonietta Demontis Anelsio Cossa José Carlos Jamine Rafael Balaña-Fouce Lisette van Lieshout José Muñoz María Martínez-Valladares The Stopping Transmission of Intestinal Parasites (STOP) Project Consortium |
spellingShingle |
Javier Gandasegui Berta Grau-Pujol María Cambra-Pelleja Valdemiro Escola Maria Antonietta Demontis Anelsio Cossa José Carlos Jamine Rafael Balaña-Fouce Lisette van Lieshout José Muñoz María Martínez-Valladares The Stopping Transmission of Intestinal Parasites (STOP) Project Consortium Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs Parasites & Vectors Soil-transmitted helminths Benzimidazoles Anthelmintic resistance Pyrosequencing |
author_facet |
Javier Gandasegui Berta Grau-Pujol María Cambra-Pelleja Valdemiro Escola Maria Antonietta Demontis Anelsio Cossa José Carlos Jamine Rafael Balaña-Fouce Lisette van Lieshout José Muñoz María Martínez-Valladares The Stopping Transmission of Intestinal Parasites (STOP) Project Consortium |
author_sort |
Javier Gandasegui |
title |
Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs |
title_short |
Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs |
title_full |
Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs |
title_fullStr |
Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs |
title_full_unstemmed |
Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs |
title_sort |
improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in trichuris trichiura and necator americanus pooled eggs |
publisher |
BMC |
series |
Parasites & Vectors |
issn |
1756-3305 |
publishDate |
2021-09-01 |
description |
Abstract Background There is an urgent need for an extensive evaluation of benzimidazole efficacy in humans. In veterinary science, benzimidazole resistance has been mainly associated with three single-nucleotide polymorphisms (SNPs) in the isotype-1 β-tubulin gene. In this study, we optimized the stool sample processing methodology and resistance allele frequency assessment in Trichuris trichiura and Necator americanus anthelmintic-related SNPs by pyrosequencing, and standardized it for large-scale benzimidazole efficacy screening use. Methods Three different protocols for stool sample processing were compared in 19 T. trichiura-positive samples: fresh stool, egg concentration using metallic sieves with decreasing pore size, and egg concentration followed by flotation with saturated salt solution. Yield of each protocol was assessed by estimating the load of parasite DNA by real-time PCR. Then, we sequenced a DNA fragment of the β-tubulin gene containing the putative benzimidazole resistance SNPs in T. trichiura and N. americanus. Afterwards, resistant and susceptible-type plasmids were produced and mixed at different proportions, simulating different resistance levels. These mixtures were used to compare previously described pyrosequencing assays with processes newly designed by our own group. Once the stool sample processing and the pyrosequencing methodology was defined, the utility of the protocols was assessed by measuring the frequencies of putative resistance SNPs in 15 T. trichiura- and 15 N. americanus-positive stool samples. Results The highest DNA load was provided by egg concentration using metallic sieves with decreasing pore size. Sequencing information of the β-tubulin gene in Mozambican specimens was highly similar to the sequences previously reported, for T. trichiura and N. americanus, despite the origin of the sample. When we compared pyrosequencing assays using plasmids constructs, primers designed in this study provided the most accurate SNP frequencies. When pooled egg samples were analysed, none of resistant SNPs were observed in T. trichiura, whereas 17% of the resistant SNPs at codon 198 were found in one N. americanus sample. Conclusions We optimized the sample processing methodology and standardized pyrosequencing in soil-transmitted helminth (STH) pooled eggs. These protocols could be used in STH large-scale screenings or anthelmintic efficacy trials. Graphical Abstract |
topic |
Soil-transmitted helminths Benzimidazoles Anthelmintic resistance Pyrosequencing |
url |
https://doi.org/10.1186/s13071-021-04941-w |
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