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 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

Single-Nucleotide Polymorphisms in the Beta-Tubulin Gene and Its Relationship with Treatment Response to Albendazole in Human Soil-Transmitted Helminths in Southern Mozambique

“…The amplified products were then sent to the Instituto Aragonés de Ciencias de la Salud (IACS) where the pyrosequencing technique was performed following the protocol by Gandasegui et al. 21 …”

“…Egg concentration for pyrosequencing. Soil-transmitted helminth eggs from pretreatment and posttreatment samples were concentrated for later pyrosequencing analysis following the protocol described by Gandasegui et al 21 (Supplemental Figure 1). Briefly, the fresh stool was homogenized after shaking with water and glass beads in a 50-mL bottle for 1-2 minutes.…”

“…Pyrosequencing assay. The pyrosequencing assay to determine the frequency of the resistance alleles related to benzimidazole resistance for each species (T. trichiura and N. americanus) was performed following the protocol previously described by Gandasegui et al 21 Briefly, a first PCR was conducted at the University of Le on to amplify a fragment of the beta-tubulin gene including the three resistance-associated codons (167, 198, and 200), for each species, in all pre-and posttreatment samples. In all PCRs, plasmid constructions for each species including the susceptible genotype for the three SNPs, the resistant genotype, and a mixture of both as an intermediate genotype (1:1) were included as internal controls for the pyrosequencing assay.…”

“…In all PCRs, plasmid constructions for each species including the susceptible genotype for the three SNPs, the resistant genotype, and a mixture of both as an intermediate genotype (1:1) were included as internal controls for the pyrosequencing assay. The amplified products were then sent to the Instituto Aragon es de Ciencias de la Salud (IACS) where the pyrosequencing technique was performed following the protocol by Gandasegui et al 21 Data analysis. All statistical analysis was carried out in R Statistical Software Version 3.5.3 26 (The R Foundation for Statistical Computing, Vienna, Austria) and STATA version 16 (StataCorp., College Station, TX).…”

Anthelmintic resistance in soil-transmitted helminths: One-Health considerations

“…F167Y, E198A, E198L, F200Y Deep amplicon sequencing A. lumbricoides , A. suum x Ethiopia, Tanzania, Belgium (Roose et al 2021 ) 8. F167Y, F200Y, E198A Pyrosequencing T. trichiura , N. americanus x Low Mozambique (Gandasegui et al 2021 ) 9. F167Y, F200Y, E198A Semi-nested PCR, sequencing A. lumbricoides , T. trichiura x Honduras (Matamoros et al 2019 ) x means SNP was not detected …”