PCR-RFLP screening of polymorphisms associated with benzimidazole resistance in Necator americanus and Ascaris lumbricoides from different geographical regions in Brazil

Abstract

Ascaris lumbricoides and Necator americanus are soil-transmitted parasites with global geographic distribution, and they represent some of the most common and neglected infections in the world. Periodic treatment with mass drug administration (MDA) in endemic areas is the recommended action put forth by the World Health Organization. However, MDA can cause the selection of subpopulations that possess the genetic ability to overcome the mechanism of drug action. In fact, beta-tubulin gene mutations (codons 167, 198 and 200) are correlated with benzimidazole resistance in nematodes of veterinary importance. It is possible that these SNPs also have strong correlation with treatment resistance in the human geohelminths A. lumbricoides, Trichuris trichiura and hookworms. Here, we aimed to investigate the presence of some of these canonical molecular markers associated with parasite resistance to benzimidazole in N. americanus and A. lumbricoides collected from six Brazilian states. Nested-PCR and PCR-RFLP were used to detect mutations at codons 167 and 198 in 601 individual eggs of A. lumbricoides collected from 62 human stool samples; however, no mutations were found. Codons 198 and 200 were tested in 552 N. americanus eggs collected from 48 patients using the same methodology, which presented a relative frequency of 1.4% and 1.1%, respectively. The presence of these SNPs in N. americanus eggs is an important finding, indicating that with high benzimidazole drug pressure there is potential for benzimidazole resistance to be selected in this hookworm. However, at these low frequencies it does not indicate that there is at present any benzimidazole resistance problem. This is the first systematic study performed in South America, and the study yielded a landscape of the genetic variants in the beta-tubulin gene and anthelmintic resistance to soil-transmitted parasites detected by a simple, rapid and affordable genotyping assay of individual eggs.

Molecular evidence of hybridization between pig and human Ascaris indicates an interbred species complex infecting humans

“…The finding that A. suum and A. lumbricoides form a genetic complex has important public health implications. Reduced treatment efficacy is not currently a common issue in Ascaris infections among humans or pigs ( Levecke et al, 2018 ; Vercruysse et al, 2011 ; Zuccherato et al, 2018 ), although low efficacy of benzimidazoles is an issue for Trichuris trichiura in humans ( Diawara et al, 2009 ; Furtado et al, 2016 ; Olsen et al, 2009 ) and various intestinal nematodes of veterinary importance ( Jaeger and Carvalho-Costa, 2017 ; Kaplan and Vidyashankar, 2012 ; Wolstenholme et al, 2004 ). Extensive albendazole use in either human or pig populations could lead to resistance in both populations, if cross-species infections are common and produce fertile offspring.…”

Egg genotyping reveals the possibility of patent Ancylostoma caninum infection in human intestine

“…Sampling and DnA extraction. The stool samples were processed, and DNA extractions were performed for single hookworm eggs from patients collected in six Brazilian states exactly as described previously by Zuccherato and coworkers 53 . The initial isolation of the eggs was performed according to Ritchie 54 with modifications, and samples that were positive for hookworm were stored in 10% formaldehyde for later molecular analysis.…”

Anthelmintic drug discovery: target identification, screening methods and the role of open science

“…Unfortunately, drug resistance against benzimidazoles 1 and 2 and other anthelmintics have been detected in veterinary parasites, with mutations in the beta-tubulin gene [31,32]. To date there is limited evidence of resistance to benzimidazoles in human STHs [33], although benzimidazole resistance alleles have been found with increased frequency following anthelmintic treatment [34,35]. However, as the use of these anthelmintics has increased in the past decade, thanks to the donation of millions of doses of these drugs to enable mass drug administration, the selective drug pressure on the STHs has increased.…”