Abstract
.Hookworm disease caused by Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum affects half a billion people worldwide. The prevalence and intensity of infection of individual hookworm species are vital for assessing morbidity and generating targeted intervention programs for their control. The present study aims to evaluate a multiplex real-time quantitative PCR (qPCR) assay to determine the prevalence and egg intensity of all three hookworm species and compare this with standard microscopy and published genus-based conventional and real-time multiplex qPCRs. Performance of the diagnostic assays was evaluated using DNA extracted from 192 fecal samples collected as part of a soil-transmitted helminth (STH) survey in northern Cambodia. The prevalence of hookworms as detected by the multiplex hookworm qPCR of 84/192 (43.8%) was significantly higher than that using microscopy of 49/192 (25.5%). The hookworm multiplex qPCR showed very good agreement for the detection of both N. americanus (Kappa 0.943) and Ancylostoma spp. (Kappa 0.936) with a multiplex STH qPCR. A strong and moderate quantitative correlation between cycle threshold and eggs per gram (EPG) feces was obtained for the hookworm qPCR for seeded DNA egg extracts (R2 ≥ 0.9004) and naturally egg-infected individuals (R2 = 0.6848), respectively. The newly developed hookworm quantitative multiplex qPCR has the potential for application in anthelmintic efficacy trials and for monitoring the success of mass deworming programs targeting individual species of anthroponotic and zoonotic hookworms.
Highlights
Hookworm disease, caused by blood-feeding worms residing in the small intestine, affects nearly half a billion people worldwide.[1]
No cross-reaction in the quantitative PCR (qPCR) assays was observed between hookworm species when singleplex and multiplex N. americanus, A. ceylanicum, and A. duodenale assays were assessed using gBlock gene fragments (IDT® Technologies, Skokie, Illinois, USA) consisting of individual hookworm species targets, or with genomic DNA templates extracted from individual hookworm species
The newly developed multiplex hookworm qPCR assay was developed and validated against a well-described set of 192 field samples previously tested for hookworm infection using microscopy and both conventional and a multiplex soil-transmitted helminth (STH) qPCR
Summary
Hookworm disease, caused by blood-feeding worms residing in the small intestine, affects nearly half a billion people worldwide.[1]. All three species have similar life cycles but have important differences in pathogenicity and host specificity. These differences may have important public health implications, such as assessing the success of intervention programs in a community. The presence of A. duodenale in a population causes higher morbidity through increased blood loss[5,6] and results in significantly different seasonal and age-related epidemiology to the other species, owing to the ability of larvae to undergo tissue hypobiosis.[7,8] hookworm species may vary in their response to chemotherapeutic intervention[9]; current tools do not allow us to accurately assess this as part of chemotherapeutic efficacy trials
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