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

Nour Rashwan,Nipul Kithsiri Gunawardena,Nilanthi De Silva,Catherine Bourguinat,Roger Prichard,Kathy Keller,Cinzia Cantacessi

doi:10.1371/journal.pntd.0005113

Nour Rashwan, Nipul Kithsiri Gunawardena + Show 5 more

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https://doi.org/10.1371/journal.pntd.0005113

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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.

Highlights

Intestinal helminths cause a major burden on human health in developing countries, infecting more than 2 billion people worldwide [1]
The N. americanus SmartAmp2 single nucleotide polymorphism (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
We developed a novel genotyping assay to screen for β-tubulin polymorphisms in N. americanus, using the SmartAmp2 method

Summary

Introduction

Intestinal helminths cause a major burden on human health in developing countries, infecting more than 2 billion people worldwide [1]. Hookworms are one of the major STHs and the second most prevalent intestinal helminth of humans [2], infecting an estimated 438.9 million people in resource-constrained countries in the tropics and subtropics [3], and causing 22.1 million disability-adjusted life years [4]. A single dose of either drug shows suboptimal efficacy against hookworms [9,10,11,12,13] Treatment with these drugs is the major hookworm control strategy recommended by the World Health Organization (WHO) [14, 15] as there is no vaccine available. Previous molecular assays for putative resistance mutations have been based mainly on PCR amplification and sequencing These techniques are complicated and time consuming and not suitable for resourceconstrained situations. A simple, rapid and sensitive genotyping method is required to monitor for possible developing resistance to BZ drugs

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