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

Timothy G Geary,Luciana Werneck Zuccherato,Celi Da Silva Medeiros,Élida M Rabelo,Luis Fernando Furtado,Carina Da Silva Pinheiro

doi:10.1371/journal.pntd.0006766

Timothy G Geary, Luciana Werneck Zuccherato + Show 4 more

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https://doi.org/10.1371/journal.pntd.0006766

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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.

Highlights

Soil-transmitted helminth infections represent important, neglected, tropical diseases that affect approximately one-fourth of the global population
A. lumbricoides samples showed no mutations in any codons, and mutation at codons 198 and 200 were observed at low frequencies in N. americanus
The observed data posit important public health issues: the mutations are a substrate for selective pressure and a long-term mass drug administration could allow to the selection of parasites resistant to benzimidazole in Brazil

Summary

Introduction

Soil-transmitted helminth infections represent important, neglected, tropical diseases that affect approximately one-fourth of the global population. The standard approach for geohelminth control, including hookworms and A. lumbricoides, is large-scale preventive chemotherapy predominantly using benzimidazoles through mass drug administration (MDA), based on the drug’s performance in overall reductions in prevalence and reductions in the extent and severity of infection [4]. This inexpensive and highly effective strategy can potentially select subpopulations of parasites that become resistant to treatment

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