scholarly journals Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Javier Gandasegui ◽  
Berta Grau-Pujol ◽  
María Cambra-Pelleja ◽  
Valdemiro Escola ◽  
Maria Antonietta Demontis ◽  
...  
Keyword(s):  
Pore Size ◽  
Stool Sample ◽  
Large Scale ◽  
Trichuris Trichiura ◽  
Sample Processing ◽  
Tubulin Gene ◽  
Necator Americanus ◽  
Benzimidazole Resistance ◽  
Extensive Evaluation ◽  
Β Tubulin

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

Genotypic profile of benzimidazole resistance associated with SNP F167Y in the beta-tubulin gene of Necator americanus helminths obtained from Brazilian populations

2020 ◽  
Vol 86 ◽  
pp. 104594
Author(s):  
Luis Fernando Viana Furtado ◽  
Talita Rodrigues dos Santos ◽  
Valéria Nayara Gomes Mendes de Oliveira ◽  
Élida Mara Leite Rabelo
Keyword(s):  
Tubulin Gene ◽  
Beta Tubulin ◽  
Necator Americanus ◽  
Benzimidazole Resistance ◽  
Genotypic Profile ◽  
Beta Tubulin Gene ◽  
Brazilian Populations

Sequence variation in the Trichuris trichiura β-tubulin locus: implications for the development of benzimidazole resistance

2002 ◽  
Vol 32 (12) ◽  
pp. 1519-1528 ◽  
Author(s):  
A.B. Bennett ◽  
T.J.C. Anderson ◽  
G.C. Barker ◽  
E. Michael ◽  
D.A.P. Bundy
Keyword(s):  
Sequence Variation ◽  
Trichuris Trichiura ◽  
Benzimidazole Resistance ◽  
Β Tubulin

First identification of the F200Y SNP in the β-tubulin gene linked to benzimidazole resistance in Ancylostoma caninum

2014 ◽  
Vol 206 (3-4) ◽  
pp. 313-316 ◽  
Author(s):  
Luis Fernando Viana Furtado ◽  
Ana Cristina Passos de Paiva Bello ◽  
Hudson Andrade dos Santos ◽  
Maria Raquel Santos Carvalho ◽  
Élida Mara Leite Rabelo
Keyword(s):  
Tubulin Gene ◽  
Ancylostoma Caninum ◽  
Benzimidazole Resistance ◽  
Β Tubulin

scholarly journals Teladorsagia Circumcincta Beta Tubulin: The Presence of the E198L Polymorphism on Its Own is Associated with Benzimidazole Resistance

2020 ◽  
Author(s):  
María Martínez Valladares ◽  
Elora Valderas-García ◽  
Javier Gandasegui ◽  
Philip Skuce ◽  
Alison Morrison ◽  
...  
Keyword(s):  
Phenotypic Variability ◽  
Linear Regression Analysis ◽  
Significant Variable ◽  
Multivariate Linear Regression Analysis ◽  
Tubulin Gene ◽  
Phenotypic Resistance ◽  
Pyrosequencing Assay ◽  
Teladorsagia Circumcincta ◽  
Benzimidazole Resistance ◽  
Β Tubulin

Abstract Background: Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta.Methods: The benzimidazole phenotypic resistance was measured by the Faecal Egg Count Reduction Test (FECRT) and the Egg Hatch Test (EHT) using a discriminating dose in 39 sheep flocks. Around 1,000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta . The resistance alleles frequencies were measured for F200Y and E198A. A 371 bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results: The percentage of resistant flocks was 32% by FECRT or 26% by EHT, however F200Y and E198A SNPs were absent in T. circumcincta . The amplification a 371 bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929; p = 0.000) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4 and 80.7% before treatment, and from 82.3 to 92.8% after treatment. Very high and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720; p = 0.000), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; p = 0.000). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions: The E198L polymorphism can confer BZ resistance on its own in T. circumcincta .

scholarly journals Teladorsagia circumcincta beta tubulin: the presence of the E198L polymorphism on its own is associated with benzimidazole resistance

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
María Martínez-Valladares ◽  
Elora Valderas-García ◽  
Javier Gandasegui ◽  
Philip Skuce ◽  
Alison Morrison ◽  
...  
Keyword(s):  
Phenotypic Variability ◽  
Linear Regression Analysis ◽  
Significant Variable ◽  
Multivariate Linear Regression Analysis ◽  
Tubulin Gene ◽  
Phenotypic Resistance ◽  
Pyrosequencing Assay ◽  
Teladorsagia Circumcincta ◽  
Benzimidazole Resistance ◽  
Β Tubulin

Abstract Background Benzimidazole resistance is associated with isotype-1 β-tubulin gene F200Y, E198A and F167Y SNPs. In this study, the recently described polymorphism E198L was reported and analysed in Teladorsagia circumcincta. Methods The benzimidazole phenotypic resistance was measured by the faecal egg count reduction test (FECRT) and the egg hatch test (EHT) using a discriminating dose (DD) in 39 sheep flocks. Around 1000 larvae collected before and after treatment were used for DNA extraction. The resistant species identified in all flocks was T. circumcincta. The resistance alleles frequencies were measured for F200Y and E198A. A 371-bp fragment of the isotype-1 β-tubulin gene was analysed, including the three codons of interest, and a new pyrosequencing assay was designed for testing E198L. Results The percentage of resistant flocks was 35% by FECRT or 26% by EHT; however, F200Y and E198A SNPs were absent in T. circumcincta. The amplification of a 371-bp fragment confirmed the absence of F167Y and F200Y in 6 resistant flocks. Regarding codon 198, all samples after treatment carried a leucine (CTA). A pyrosequencing assay analysed the allele frequencies for the first two bases at codon 198 independently, G/C and A/T. The correlation between C and T frequencies was almost 1 (r = 0.929, P < 0.0001) and the mean value of both was calculated to measure the leucine frequency; this value ranged between 10.4–80.7% before treatment, and 82.3–92.8% after treatment. High and similar correlations were reported between the genotypic variables (C frequency, T frequency or mean of both frequencies) and phenotypic resistance (r > 0.720, P < 0.0001), although negatively associated with the FECRT and positively with the EHT. According to multivariate linear regression analysis, the T frequency was the most significant variable influencing the phenotypic resistance (FECRT or EHT; P < 0.0001). In the EHT, 67.1% of the phenotypic variability is associated with the T frequency but in the FECRT only 33.4%; therefore, the EHT using a DD seems to detect the genotypic resistance more accurately than the FECRT. Conclusions The E198L polymorphism can confer BZ resistance on its own in T. circumcincta.

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