The main control strategy for Ascaris lumbricoides is mass drug administration (especially with benzimidazoles), which can select strains of parasites resistant to treatment. Mutations in the beta-tubulin isotype-1 gene at codons 167, 198 and 200 have been linked to benzimidazole resistance in several nematodes. The mutation in codon 200 is the most frequent in different species of parasites, as previously observed in Necator americanus and Trichuris trichiura; however, this mutation has never been found in populations of A. lumbricoides. This study aimed to screen for single nucleotide polymorphisms (SNPs) in the beta-tubulin isotype-1 gene at codon 200 in A. lumbricoides. We developed a technique based on an amplification refractory mutation system (ARMS-PCR) for the analysis of 854 single A. lumbricoides eggs collected from 68 human stool samples from seven Brazilian states. We detected the mutation in codon 200 at a frequency of 0.5% (4/854). This is the first report of this mutation in A. lumbricoides. Although the observed frequency is low, its presence indicates that these parasite populations have the potential to develop high levels of resistance in the future. The methodology proposed here provides a powerful tool to screen for the emergence of anthelmintic resistance mutations in parasitic nematode populations.

Hookworms are intestinal parasites that cause major public health problems, especially in developing countries. To differentiate eggs from different hookworm species, it is necessary to use molecular methodologies, since the eggs are morphologically similar. Here, we performed the molecular identification of single hookworm eggs from six Brazilian states. Of the 634 eggs individually analyzed, 98.1% (622/634) represented Necator americanus, and surprisingly, 1.9% (12/634 eggs from the same patient) represented Ancylostoma caninum. DnA analysis of the A. caninum-positive stool sample revealed no contamination with animal feces. This is the first report of the presence of A. caninum eggs in human feces, which may have a direct implication for the epidemiology of hookworm infection caused by this species. This suggests the need for special attention regarding prophylaxis, as different reservoirs, previously not described, may have great relevance for the spread of A. caninum.

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Preventive chemotherapy is recommended by the WHO as the main strategy for controlling infections caused by nematodes in humans, aiming to eliminate the morbidity associated with these infections. This strategy consists of routine periodic administration of benzimidazoles, among other drugs. Although these drugs decrease the intensity of infections, they have the potential to exert selection pressure for genotypes bearing mutations associated with drug resistance, which may result in the establishment of resistant worm populations. There is evidence in the literature of resistance to these drugs in nematodes that infect humans, including in the species Trichuris trichiura. Single-nucleotide polymorphisms (SNPs) in the beta-tubulin gene located at codons 167, 198, and 200 are associated with the mechanism of resistance to benzimidazoles in nematodes. Here, we standardized a molecular technique based on an amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to analyze codons 167, 198, and 200 of T. trichiura. The ARMS-PCR methodology was successfully established to evaluate the codons of interest. A total of 420 samples of individual eggs were analyzed from populations obtained from five Brazilian states. A mutation in codon 198 was observed at a frequency of 4.8% (20/420), while for the other two codons, no polymorphism was observed. This is the first report of the presence of this mutation in populations of T. trichiura in Brazil. This fact and the emergence of the problem already observed in other species reinforces the need for regular monitoring of SNPs related to benzimidazole resistance using techniques that are highly sensitive and specific.

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