Detection of Benzimidazole Resistance-Associated Single-Nucleotide Polymorphisms in the Beta-Tubulin Gene in Trichuris trichiura from Brazilian Populations

Abstract

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.

Figure 1.

Scheme of the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) methodology adopted for the polymorphism analysis in codons (A) 167, (B) 198, and (C) 200 of the Trichuris trichiura β-tubulin gene. The PCR primers TtTubF and TtTubR are represented by blue arrows, which produce a 445-base pair fragment. The specific primer RrTt167 that only anneals in the presence of the mutation (TAT) is identified by the orange arrow and in combination with TtTubF produces a 173-base pair fragment. The specific primer RrTt198 that only anneals in the presence of the mutation (GCA) is identified by the yellow arrow and in combination with TtTubF produces a 317-base pair fragment. The specific primer Rr200Tt that only anneals in the presence of the mutation (TAC) is identified by the green arrow and in combination with TtTubF produces a 319-base pair fragment. This figure appears in color at www.ajtmh.org.

Figure 2.

Schematic representation of the distribution of the mutated samples, with their respective genotypes, for codon 198 among the five Brazilian regions. Twenty single-nucleotide polymorphisms (SNPs) were found in feces from patients from four locations, which corresponds to 4.76% of the total eggs analyzed. This figure appears in color at www.ajtmh.org.

Figure 3.

Representative amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) results from the screening of the beta-tubulin gene at codons (A) 167, (B) 198 , and (C) 200 in Trichuris trichiura. Genomic DNA samples from individual eggs were subjected to ARMS-PCR amplification with the two sets of primers. The lanes indicated by odd numbers correspond to ARMS-PCR products obtained using the primer combination to detect the fragment without the mutation (codon 167: 315 base pair (bp); codon 198: 172 bp; codon 200: 167 bp) and the even-numbered lanes correspond to ARMS-PCR products obtained using the primer combination to detect the fragment with the mutation (codon 167: 173 bp; codon 198: 317 bp; codon 200: 319 bp). In lanes 1–6, synthesized controls were used (lanes 1 and 2: wild-type plasmid; lanes 3 and 4: mutated plasmid; lanes 5 and 6: wild-type and mutated plasmid mix. For each sample, the two reactions were analyzed side-by-side. Products marked in red represent mutated samples at codon 198. The image shows an agarose gel (2.0%) that was stained with GelRed^™ (Biotium, USA). MW: 100 bp molecular weight. This figure appears in color at www.ajtmh.org.