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

<div><p>Background</p><p>Soil-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.</p><p>Methods</p><p>To 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 <i>Necator americanus</i>.</p><p>Findings</p><p>Diagnostic 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 <i>N</i>. <i>americanus</i> larvae were assessed and the results showed that the <i>Aac</i> polymerase used has high tolerance to inhibitors in fecal samples.</p><p>Conclusion</p><p>The <i>N</i>. <i>americanus</i> 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.</p></div

SmartAmp2 primer design for β-tubulin mutation (SNP) detection.

<p>Partial sequence of the β-tubulin isotype 1 gene carrying (<b>A</b>) SNP 167A, (<b>B</b>) SNP 198C, and (<b>C</b>) SNP 200A as well as the sequences of primers used for the SmartAmp2 assay for the three SNP positions. The locations of SNPs indicated in bold. The boost primer (BP) was used as the discrimination primer. The folding primer (FP) has a specific sequence (CCTATATATATATAGG) at the 5’ end to allow self-annealing hairpin formation.</p

Summary of multivariate analysis for risk of <i>A. lumbricoides</i>, <i>T. trichiura</i> and hookworm infection.

*<p>maximum variance inflation factor was 1.02.</p><p>Numbers within square brackets indicate the number of infected children positive for the given explanatory variable.</p

Predicted prevalence of infection with any one or more soil-transmitted helminth infection among children in the plantation sector in five districts of Sri Lanka.

<p>Predicted prevalence of infection with any one or more soil-transmitted helminth infection among children in the plantation sector in five districts of Sri Lanka.</p

Geographical location of study schools and laboratories in the districts of Kandy, Kegalle, Nuwara Eliya, Badulla and Ratnapura, together with prevalence of infection with any one or more soil-transmitted helminth infection at each school.

<p>Geographical location of study schools and laboratories in the districts of Kandy, Kegalle, Nuwara Eliya, Badulla and Ratnapura, together with prevalence of infection with any one or more soil-transmitted helminth infection at each school.</p

Genotyping results of <i>N</i>. <i>americanus</i> samples at codon 198 with SmartAmp2 and conventional sequencing.

<p><b>(A)</b> SmartAmp2 amplification of 198A/C polymorphs using wild-type (WT) and mutant-type (MT)-primer sets. Left, center, and right panels show assay results for homozygous WT (WT/WT), mixed (WT/MT), and homozygous MT (MT/MT) samples, respectively. <b>(B)</b> Conventional sequencing of β-tubulin gene from left to right, WT (A/A), mixed (A/C), and MT (C/C).</p

SmartAmp2 assay optimization for <i>N</i>. <i>americanus</i> β-tubulin SNP at codon 200T/A.

<p>(<b>A</b>) Wild-type (WT)-specific primer amplification of WT plasmid (red circle)and no amplification with mutant-type (MT) plasmid (blue triangle) or NC (no template) (green diamond). (<b>B</b>) MT-specific primer amplification of MT plasmid (blue triangle) and no amplification with WT plasmid (red circle) or NC (green diamond). Assays were run in duplicate.</p

Evaluation of SmartAmp2 SNP detection assay in pools and individual hookworm samples of eggs or larvae.

<p>(<b>A</b>) Wild-type (WT)-specific primer amplification of gDNA from egg and larval pools with complete suppression of amplification using MT-specific primers. (<b>B</b>) WT-specific primer amplification of gDNA from single eggs and larva with complete suppression of amplification using the MT-specific primers. Amplification with WT primers, larva(e) (red circle), egg(s) (green square), and amplification with MT primers (blue triangle), for egg(s) or larva(e).</p

Prevalence of soil-transmitted helminth infections in plantation sector school children by district.

<p>Prevalence of soil-transmitted helminth infections in plantation sector school children by district.</p

SmartAmp2 assay specificity of mutant-type (MT) allele detection in the presence of wild-type (WT) DNA.

<p>SmartAmp2 amplification for SNPs at codon 167 (first panel), and codons 198 or 200 (second panel), using the MT-specific primers with a mixture of MT plasmid and WT plasmid, 1:1 (50%; red circle), 1:9 (10%; blue triangle), 1:99; (1%; green diamond), and 0:100 (0%; purple square). Duplicate assays.</p