Polimorfismos envolvidos no processos de resistência aos benzimidazóis em ancilostomídeos

Abstract

Mass administration of benzimidazoles corresponds to the main method of control of hookworms, however, this strategy may select strains of parasites resistant to the drug. Single nucleotide polymorphism (SNP) at codons 167, 198 and 200 of the β-tubulin isotype 1 gene are associated with resistance to benzimidazoles in some nematodes. The mutation at codon 167 has never been described for hookworms, whereas mutations at codons 198 and 200 have already been described for these helminths, at low frequency in nature. The objective of this work was to investigate the participation of polymorphisms in the β-tubulin isotype 1 gene in the resistance process to benzimidazoles in hookworms. A screening of the canonical SNPs related to resistance in different species of hookworms was carried out. Adult worms of Ancylostoma braziliense (81) and Ancylostoma caninum (327) were collected from naturally infected dogs from two and six states of Brazil, respectively, and Necator americanus eggs (552) from faecal samples from six states of Brazil. Codons 167 and 198 of A. caninum were evaluated by ARMS-PCR and Tetraprimer ARMS-PCR, respectively (codon 200 of this species was analyzed in a previous work). All codons of A. braziliense and N. americanus were analyzed by RFLP-PCR. Mutations were observed only to the codon 198 of A. braziliense and N. americanus, with a frequency of 1.2% and 1.4%, respectively, and in the codon 200 of N. americanus, with a frequency of 1.1%. Considering the low frequency of these SNPs in nature and the possibility of other mechanisms being involved in the benzimidazole resistance, the selection of a resistant strain of Ancylostoma ceylanicum in hamsters was performed by selective drug pressure. Since the canonical SNPs in the β-tubulin gene of this strain were not observed, a β-tubulin isotype 1 cDNA mini library was constructed to the resistant worms. Sequence analyzes of the clones showed three independent SNPs not reproducible among the samples. Considering that changes in the parasite biology could occur, a comparison between the sensitive and resistant strain was made, based on parasitological and pathogenic parameters. A lower recovery of worms was observed in the resistant strain, with a lower amount of eggs released in the faeces, indicating a lower adaptation of this strain to the infection. It is suggested that other mechanisms, independent of the drug binding target, are related to resistance to benzimidazoles in hookworms.