Abstract
BackgroundDiagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies. Molecular diagnostics are powerful tools to identify closely related species, but the requirement for costly equipment makes their implementation difficult in low-resource or field settings. Rapid, sensitive and cost-effective diagnostic tools are crucial for accurate estimation of STH infection intensity in MDA programmes in which the goal is to reduce morbidity following repeated rounds of chemotherapy.ResultsIn this study, colourimetric isothermal assays were developed using SmartAmp2 primer sets and reagents in loop-mediated amplification (LAMP) assays. Species-specific primer sets, designed on a specific target sequence in the β-tubulin gene, were used to identify Necator americanus, Trichuris trichiura and Ascaris lumbricoides. After initial optimization on control plasmids and genomic DNA from adult worms, assays were evaluated on field samples. Assays showed high sensitivity and demonstrated high tolerance to inhibitors in spiked faecal samples. Rapid and sensitive colourimetric assays were successfully developed to identify the STHs in field samples using hydroxy napthol blue (HNB) dye.ConclusionsRapid and simple colourimetric diagnostic assays, using the SmartAmp2 method, were developed, with the potential to be applied in the field for detection of STH infections and the estimation of response to treatment. However, further validation on large numbers of field samples is needed.
Highlights
Diagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies
Selection of SmartAmp2 primers Several primer sets were designed to amplify a target DNA sequence within the β-tubulin gene. Screening of these primer combinations and assay conditions identified an optimal primer set that completed the amplification within 20–30 min from the target genomic DNA (10 ng)
Primer sets were assessed based on speed, yield of amplification and efficiency to differentiate a target sequence from a highly related alternative target
Summary
Diagnosis of soil-transmitted helminths (STHs) has traditionally relied on stool microscopy, which has a number of critical deficiencies. Mass drug administration (MDA) programmes are the major control strategy and target the most important STH, namely A. lumbricoides, hookworms and T. trichiura, and involve a single oral dose of albendazole (ALB; 400 mg) or mebendazole (MEB; 500 mg) administered periodically [5, 6]. Both of these front-line anthelmintics are benzimidazoles and concern has been expressed as to the possible selection for resistance to both benzimidazoles [7]. With expansion of MDA programmes for STHs, there is a need to monitor infection levels in the programme areas
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