Improved PCR-Based Detection of Soil Transmitted Helminth Infections Using a Next-Generation Sequencing Approach to Assay Design

Abstract

BackgroundThe soil transmitted helminths are a group of parasitic worms responsible for extensive morbidity in many of the world’s most economically depressed locations. With growing emphasis on disease mapping and eradication, the availability of accurate and cost-effective diagnostic measures is of paramount importance to global control and elimination efforts. While real-time PCR-based molecular detection assays have shown great promise, to date, these assays have utilized sub-optimal targets. By performing next-generation sequencing-based repeat analyses, we have identified high copy-number, non-coding DNA sequences from a series of soil transmitted pathogens. We have used these repetitive DNA elements as targets in the development of novel, multi-parallel, PCR-based diagnostic assays.Methodology/Principal FindingsUtilizing next-generation sequencing and the Galaxy-based RepeatExplorer web server, we performed repeat DNA analysis on five species of soil transmitted helminths (Necator americanus, Ancylostoma duodenale, Trichuris trichiura, Ascaris lumbricoides, and Strongyloides stercoralis). Employing high copy-number, non-coding repeat DNA sequences as targets, novel real-time PCR assays were designed, and assays were tested against established molecular detection methods. Each assay provided consistent detection of genomic DNA at quantities of 2 fg or less, demonstrated species-specificity, and showed an improved limit of detection over the existing, proven PCR-based assay.Conclusions/SignificanceThe utilization of next-generation sequencing-based repeat DNA analysis methodologies for the identification of molecular diagnostic targets has the ability to improve assay species-specificity and limits of detection. By exploiting such high copy-number repeat sequences, the assays described here will facilitate soil transmitted helminth diagnostic efforts. We recommend similar analyses when designing PCR-based diagnostic tests for the detection of other eukaryotic pathogens.

A review of soil transmitted helminthiasis in Nigeria

“…Similarly, a relatively high prevalence of STH across some endemic countries was confirmed by using PCR-restriction fragment length polymorphisms and Trichuris vulpi was identified in some of the children [10]. The prospect and potential of PCR-based detection methods using next-generation sequencing approach is seriously gaining attention globally [11,12].…”

The Global State of Helminth Control and Elimination in Children

“…28,29 By using multiparallel quantitative real-time polymerase chain reaction with a stool bead beating DNA extraction method to reduce the cost, the assay can be used in a capacity-building setting in resource-limited countries. 28–30 Newer techniques continue to be developed allowing for more rapid, sensitive diagnostics particularly in settings of polyparasitism and low worm burden. 28 …”

Detecting and enumerating soil-transmitted helminth eggs in soil: New method development and results from field testing in Kenya and Bangladesh

“…Molecular methods are under development for detection of STH in stool and biosolids [23,24]. Additionally, two recent studies used molecular methods for detection of hookworm species and Ascaris lumbricoides in soil [25,26].…”