Deep-amplicon sequencing of the complete beta-tubulin gene in Trichuris trichiura before and after albendazole treatment

Abstract

Concerns about the emergence of benzimidazole resistance in soil-transmitted helminths (STH) infections, particularly against Trichuris trichiura, have arisen. Previous studies of veterinary nematodes have linked benzimidazole resistance to single-nucleotide polymorphisms (SNPs) at three specific codons in the beta-tubulin gene, but similar associations in STH have not been consistently observed. In this work, we screened the complete beta-tubulin gene previously linked to benzimidazole resistance in T. trichiura by deep-amplicon sequencing to identify genetic variants and associate levels of diversity with drug response to albendazole. We used 99 DNA samples extracted from T. trichiura pooled eggs, previously semi-purified from human stool samples collected in Manhiça district, Mozambique. We obtained a set of 39 amplicons of the complete gene by subjecting the pooled eggs to long-read PCR and subsequently sequencing them. Of those amplicons, 22 and 17 were obtained from stool samples collected before, and 21 days after albendazole treatment, respectively. We observed genetic variation across the whole gene sequence, in both exons and introns; however, none were associated with the previously proposed resistance-associated SNPs, and none were predicted to significantly affect protein function. No significant differences in genetic diversity were observed between pre- and post-treatment samples. Using publicly available genome-wide data, we also analysed a second beta-tubulin isotype in the T. trichiura genome. We focused on detecting the canonical SNPs and assessing for signatures of genetic selection around this second isotype gene. This analysis did not reveal evidence supporting this second isotype's role in anthelmintic resistance. Despite the limitations of our study, such as a small sample size, particularly paired pre- and post-treatment samples (n = 6), or a restricted geographical area, we found no evidence linking either of the two beta-tubulin genes to benzimidazole resistance in T. trichiura, suggesting that genetic markers of drug resistance likely exist outside the beta-tubulin genes.