Standardization and application of a modified RFLP-PCR methodology for analysis of polymorphisms linked to treatment resistance in Ancylostoma braziliense

Abstract

Background: Single nucleotide polymorphisms (SNPs) in codons 167, 198 and 200 of the beta-tubulin isotype 1 gene are associated with benzimidazoles resistance in many helminths. Codon 167 mutation has never been described in hookworms; however, polymorphisms in codons 198 and 200 have been described for Ancylostoma caninum and Necator americanus. These mutations have never been investigated in Ancylostoma braziliense; therefore, it is not known if they are present in this species and whether they are correlated with treatment resistance. The RFLP-PCR technique has been used to analyze these polymorphisms in some nematodes, but depending on the species, these alterations do not create or eliminate any restriction enzyme cleavage site, making it impossible to use this technique. Here, we describe the standardization and application of a modified RFLP-PCR technique for detecting polymorphisms in individual A. braziliense worms recovered from naturally infected dogs in two Brazilian states. Results: The molecular techniques used were sensitive, specific, and easy to apply. To our knowledge, we report for the first time the presence of a polymorphism at codon 198 of the beta-tubulin gene of A. braziliense (1/81; 95% CI: 0-3.69%). Conclusions: It is not known whether the presence of the mutation in codon 198 of the beta-tubulin gene of A. braziliense has importance for this parasite. However, based on studies of other helminths, it is possible that this polymorphism is directly related to the resistance to benzimidazoles. This may be a major concern, since this nematode has considerable relevance as a parasite of canids and felids and as one of the agents of cutaneous larva migrans in humans. Standardized methodologies will be useful for screening for polymorphisms in the beta-tubulin gene of canine hookworms in a broader population. The method could also be adapted for the analysis of other SNPs in other nematode species.