Isothermal Diagnostic Assays for Monitoring Single Nucleotide Polymorphisms in Necator americanus Associated with Benzimidazole Drug Resistance

Abstract

BackgroundSoil-transmitted helminths (STHs) are the most prevalent intestinal helminths of humans, and a major cause of morbidity in tropical and subtropical countries. The benzimidazole (BZ) drugs albendazole (ABZ) and mebendazole (MBZ) are used for treatment of human STH infections and this use is increasing dramatically with massive drug donations. Frequent and prolonged use of these drugs could lead to the emergence of anthelmintic resistance as has occurred in nematodes of livestock. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing. However, these techniques are complicated and time consuming and not suitable for resource-constrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs.MethodsTo address this problem, single nucleotide polymorphism (SNP) detection assays were developed based on the Smart amplification method (SmartAmp2) to target codons 167, 198, and 200 in the β-tubulin isotype 1 gene for the hookworm Necator americanus.FindingsDiagnostic assays were developed and applied to analyze hookworm samples by both SmartAmp2 and conventional sequencing methods and the results showed high concordance. Additionally, fecal samples spiked with N. americanus larvae were assessed and the results showed that the Aac polymerase used has high tolerance to inhibitors in fecal samples.ConclusionThe N. americanus SmartAmp2 SNP detection assay is a new genotyping tool that is rapid, sensitive, highly specific and efficient with the potential to be used as a field tool for monitoring SNPs associated with BZ resistance. However, further validation on large numbers of field samples is required.

Prospects for elimination of soil-transmitted helminths

“…Anthelmintic resistance, including multidrug resistance [85], is a well documented issue in animal populations [86] and some mutations associated with benzimidazole resistance have been identified in eggs from human stool [87]. Although any largescale MDA with benzimidazoles may risk producing resistance [54], in animal husbandry, strategies to combat resistance include the exclusion of a subset of the population from deworming to maintain refugia [88,89].…”

Gold Nanoparticle‐Based Paper Sensor for Simultaneous Detection of 11 Benzimidazoles by One Monoclonal Antibody

“…As shown in Figure 3, 2-(methoxycarbonylamino)-3H-benzimidazole-5-carboxylic acid (Hapten) not only has the general chemical structures of carbamate BZs, but it also has a carboxyl, which can conjugate with carrier protein directly. Compared with other haptens used in previous research, 1H-benzo[d] [1,2,3]triazol-1-yl6-((tert-butoxycarbonyl) amino) hexanoate [12a] and ABZNH 2 SO 2 [12c] had relatively large structure difference with BZs. Thus, it is more likely to get one antibody that can recognize different kinds of BZs simultaneously by using this hapten.…”

“…BZs have advantages of being broad spectrum and have high effectiveness and low toxicity. However, the abusing the use of BZs can lead to the emergence of drugresistant anthelmintics, [3] and can generate residues in foods of animal origin, such as milk and pork. [4] In addition, animal experiments showed that BZs had a certain embryo toxicity, polyploidy, and teratogenicity.…”

Rapid Genotyping of β-tubulin Polymorphisms in Trichuris trichiura and Ascaris lumbricoides

“…This technique has been previously used to develop assays for the hookworm, N . americanus [30]. The SmartAmp2 is a DNA amplification method for detection of DNA mutations, deletions or SNPs under isothermal conditions and in a single step.…”