The study provides an optimised protocol for sample processing and a standardised pyrosequencing methodology which could be implemented in large-scale studies
A new publication by STOP researchers provides an optimised protocol that allows large-scale screening of human stool samples for the detection of benzimidazole-resistant alleles in whipworm (Trichuris trichiura) and hookworm (Necator americanus).
Soil-transmitted helminths affect an estimated 1.5 billion people worldwide. The current strategy recommended by the WHO to control these intestinal infections is to conduct massive administration campaigns (MDA) of albendazole or mebendazole to all schoolchildren, women of child-bearing age, and certain adults, once or twice a year.
However, the widespread use of these drugs can result in the selection of drug-resistant helminths, leading to a decrease in the efficacy of MDA campaigns. In some countries, the efficacy against Trichuris trichiura has decreased from 72.6% to 43% over the last twenty years. A decrease in efficacy has also been observed with hookworm. “Therefore, there is an urgent need to extensively evaluate benzimidazole resistance in soil-transmitted helminths,” says Javier Gandasegui, first author of the study.
“We know that worms infecting cows and other ruminants can develop resistance to benzimidazole,” explains María Martinez-Valladares, from the University of León and senior author of the study. This resistance in veterinary practice has been associated with specific changes in the DNA of a gene (three single nucleotide polymorphisms or SNPs in the isotype-1 b-tubulin gene).
The aim of this study was to develop a methodology that allows large-scale screening of human stool samples for the detection of benzimidazole-resistant alleles in whipworm (Trichuris trichiura) and hookworm (Necator americanus). The STOP team first optimised the processing of stool samples that were obtained in the Manhiça district (Mozambique) in order to obtain a high yield and quality of eggs. They then determined the best pyrosequencing protocol (a rapid method of DNA sequencing that relies on light detection as DNA is synthesised), capable of measuring different levels of resistant alleles in the sample.
Using this technology on some of the stool samples from Manhiça, the team detected only one sample with a drug-resistant genotype. “However, the proportion of samples analysed does not allow to infer the real prevalence of SNPs in the community. Also, further work must evaluate the association of these genetic markers of resistance with treatment response to benzimidazoles”, says Gandasegui. The authors also point to the need of identifying new genetic markers of drug-resistance in these worms by using other approaches, such as genome-wide approaches.
Regardless of the resistance marker analysed, this study provides an optimised protocol for sample processing and a standardised pyrosequencing methodology which could be implemented in the large-scale monitoring of responses to anthelmintic drugs or in drug efficacy trials.
Gandasegui J, Grau-Pujol B, Cambra-Pelleja M, et al. Improving stool sample processing and pyrosequencing for quantifying benzimidazole resistance alleles in Trichuris trichiura and Necator americanus pooled eggs. Parasites Vectors 14, 490 (2021). https://doi.org/10.1186/s13071-021-04941-w