Abstract
BackgroundThe benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trichiura to BZ treatment. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established.MethodsThe objective of this study was to develop new genotyping assays to screen for the presence of β-tubulin SNPs in T. trichiura and Ascaris lumbricoides. Rapid, simple and accurate genotyping assays were developed based on the SmartAmp2 method. Primer sets were optimized and selected to distinguish the SNP-variant genotypes. After initial optimization on control plasmids, the feasibility of the assay was assessed in field samples from Haiti and Panama. Finally, spiked fecal samples were assessed to determine the tolerance of Aac polymerase to fecal inhibitors.FindingsRapid SNP genotyping assays were developed to target β-tubulin polymorphisms in T. trichiura and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to PCR inhibitors in fecal samples.ConclusionThese assays proved to be robust and efficient with the potential to be used as field tools for monitoring SNPs that could be associated with BZ resistance. However, further work is needed to validate the assays on large numbers of field samples before and after treatment.
Highlights
Soil-transmitted helminths (STHs) are a major cause of morbidity in developing countries
The assays showed high sensitivity and specificity in field samples and demonstrated high tolerance to PCR inhibitors in fecal samples. These assays proved to be robust and efficient with the potential to be used as field tools for monitoring single nucleotide polymorphism (SNP) that could be associated with BZ resistance
We developed novel genotyping assays to screen for β-tubulin polymorphisms in T. trichiura and A. lumbricoides based on the SmartAmp2 method
Summary
Soil-transmitted helminths (STHs) are a major cause of morbidity in developing countries. The current control strategy against STHs is the regular administration of a single-dose of ABZ (400 mg) or MBZ (500 mg) as preventive chemotherapy in large-scale mass drug administration (MDA) programs [7] with the ultimate goal of elimination of STHs as a public health problem by 2020 [3] These programs have been greatly expanded in recent years by massive donations of these drugs. Intensive and prolonged reliance on two drugs of the same anthelmintic class with the same mode of action and suboptimal efficacy greatly increases the probability that BZ resistance may develop [12,13,14] This would raise serious complications for control of STHs [4]. The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs) Their intensive use increases the possibility that BZ resistance may develop. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established
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