Desenvolvimento e aplicação de uma metodologia para varredura de polimorfismo relacionado à resistência aos Benzimidazóis em Ancylostoma caninum

Abstract

Ancylostoma caninum is considered an important parasite of canids, and presents great relevance as zoonotic agent of eosinophilic enteritis and skin infections in humans. The mass drug administration, especially with benzimidazoles, corresponds to the main method of control of the disease caused by members of the hookworms family. However, this strategy is considered vulnerable as it can lead to selection of drug resistant strains. Mutations in codons 167, 198 and 200 of isotype 1 -tubulin gene have been associated with benzimidazoles resistance in some nematodes. The mutation at codon 200 has been detected in humans hookworm, but so far has not being reported in A. caninum. The aim of this study was to perform a scan of polymorphisms at codons 198 and 200 of isotype 1 -tubulin gene in A. caninum collected from two different regions of Brazil. Were collected 162 worms from dogs originated from Centers for Zoonosis Control from the city of Belo Horizonte, Minas Gerais, and 149 of Teresina, Piauí. For analyze the codon 200, a molecular tool based on ARMS-PCR has been developed. For that controls for the absence of mutation, using conventional PCR, and for the presence of mutation using Megaprimer-PCR technique, were developed. Although at low frequency (0.8%), the mutation at codon 200 was observed only in worms collected in Minas Gerais, while samples from Piauí showed no mutations. A total of 75 samples were subjected to sequencing to validate the technique developed and also to scan the region comprising the codon 198. The sequencing validated the technique developed and showed no correlation between the mutations. We conclude that there is a low frequency of mutation at codon 200 in the population of A. caninum analyzed. This is the first report that determines the presence of the mutation at codon 200 in this specie population. These data reinforce the need for monitoring for benzimidazole resistant hookworms strains, given the emergence of the problem in other species.