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.

Background Helminth and protozoan infections affect more than 1 billion children globally. Improving water quality, sanitation, handwashing, and nutrition could be more sustainable control strategies for parasite infections than mass drug administration, while providing other quality of life benefits. Methods and findings We enrolled geographic clusters of pregnant women in rural western Kenya into a cluster-randomized controlled trial (ClinicalTrials.gov NCT01704105) that tested 6 interventions: water treatment, improved sanitation, handwashing with soap, combined water treatment, sanitation, and handwashing (WSH), improved nutrition, and combined WSH and nutrition (WSHN). We assessed intervention effects on parasite infections by measuring Ascaris lumbricoides , Trichuris trichiura , hookworm, and Giardia duodenalis among children born to the enrolled pregnant women (index children) and their older siblings. After 2 years of intervention exposure, we collected stool specimens from 9,077 total children aged 2 to 15 years in 622 clusters, including 2,346 children in an active control group (received household visits but no interventions), 1,117 in the water treatment arm, 1,160 in the sanitation arm, 1,141 in the handwashing arm, 1,064 in the WSH arm, 1,072 in the nutrition arm, and 1,177 in the WSHN arm. In the control group, 23% of children were infected with A . lumbricoides , 1% with T . trichiura , 2% with hookworm, and 39% with G . duodenalis . The analysis included 4,928 index children (median age in years: 2) and 4,149 older siblings (median age in years: 5); study households had an average of 5 people, <10% had electricity access, and >90% had dirt floors. Compared to the control group, Ascaris infection prevalence was lower in the water treatment arm (prevalence ratio [PR]: 0.82 [95% CI 0.67, 1.00], p = 0.056), the WSH arm (PR: 0.78 [95% CI 0.63, 0.96], p = 0.021), and the WSHN arm (PR: 0.78 [95% CI 0.64, 0.96], p = 0.017). We did not observe differences in Ascaris infection prevalence between the control group and the arms with the individual interventions sanitation (PR: 0.89 [95% CI 0.73, 1.08], p = 0.228), handwashing (PR: 0.89 [95% CI 0.73, 1.09], p = 0.277), or nutrition (PR: 86 [95% CI 0.71, 1.05], p = 0.148). Integrating nutrition with WSH did not provide additional benefit. Trichuris and hookworm were rarely detected, resulting in imprecise effect estimates. No intervention reduced Giardia . Reanalysis of stool samples by quantitative polymera...

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BackgroundProper collection and storage of fecal samples is necessary to guarantee the subsequent reliability of DNA-based soil-transmitted helminth diagnostic procedures. Previous research has examined various methods to preserve fecal samples for subsequent microscopic analysis or for subsequent determination of overall DNA yields obtained following DNA extraction. However, only limited research has focused on the preservation of soil-transmitted helminth DNA in stool samples stored at ambient temperature or maintained in a cold chain for extended periods of time.MethodologyQuantitative real-time PCR was used in this study as a measure of the effectiveness of seven commercially available products to preserve hookworm DNA over time and at different temperatures. Results were compared against “no preservative” controls and the “gold standard” of rapidly freezing samples at -20°C. The preservation methods were compared at both 4°C and at simulated tropical ambient temperature (32°C) over a period of 60 days. Evaluation of the effectiveness of each preservative was based on quantitative real-time PCR detection of target hookworm DNA.ConclusionsAt 4°C there were no significant differences in DNA amplification efficiency (as measured by Cq values) regardless of the preservation method utilized over the 60-day period. At 32°C, preservation with FTA cards, potassium dichromate, and a silica bead two-step desiccation process proved most advantageous for minimizing Cq value increases, while RNA later, 95% ethanol and Paxgene also demonstrate some protective effect. These results suggest that fecal samples spiked with known concentrations of hookworm-derived egg material can remain at 4°C for 60 days in the absence of preservative, without significant degradation of the DNA target. Likewise, a variety of preservation methods can provide a measure of protection in the absence of a cold chain. As a result, other factors, such as preservative toxicity, inhibitor resistance, preservative cost, shipping requirements, sample infectivity, and labor costs should be considered when deciding upon an appropriate method for the storage of fecal specimens for subsequent PCR analysis. Balancing logistical factors and the need to preserve the target DNA, we believe that under most circumstances 95% ethanol provides the most pragmatic choice for preserving stool samples in the field.