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.

Fig 1. Schematic representation of the PCR-RFLP methodology for single cell screening of beta-tubulin polymorphisms of A) codons 167 and 198 (A. lumbricoides) and B) codons 198 and 200 (N. americanus).

PCRs were performed using the DNA isolated from a single egg as a template, followed by a nested (for A. lumbricoides) or semi-nested PCR (N. americanus). PCR-RFLP was carried out using the selected restrictions enzymes to differ between the mutated and wild-type alleles, as observed by the different sizes of fragments detected in the gel electrophoresis. The gray tails of primers Al167F, Al198R and Fb198/200Na represents the M13 universal primers added to the sequences. The numbers in the gels represent the size of the fragments in base pairs observed after the PCR-RFLP digestion.

Fig 2. Spectrum of mutations of codons 198 and 200 in 552 N. americanus samples from six Brazilian regions.

Three localities had SNPs at codon 198, representing a 1.4% (8/552) positivity: 1) Ceará with one homozygous and two heterozygous eggs from a same patient (3.0% positivity); 2) Minas Gerais with two heterozygous and one homozygous egg from two patients (3.75% positivity); and 3) Bahia with one homozygous and one heterozygous egg from one patient (2.0% positivity). The overall percentage of egg positivity for the mutation at codon 198 was 1.4% (8/552). For codon 200, the total percentage of egg positivity was 1.1% (6/552), detected in Maranhão (four homozygous eggs from three patients, positivity of 3.6%) and Bahia (two heterozygous eggs from one patient 2.0% of positivity).