Development and Evaluation of a Multiplex Quantitative Real-Time Polymerase Chain Reaction for Hookworm Species in Human Stool

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 (R ² ≥ 0.9004) and naturally egg-infected individuals (R ² = 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.

Figure 1.

Singleplex and multiplex qPCR efficiencies. Assay optimization to compare the efficiencies of single vs. multiplex PCR assays using gBlock gene targets for (A) Necator americanus, (B) Ancylostoma ceylanicum, and (C) Ancylostoma duodenale fragments (IDT^® Technologies) as standard curve controls. Ct = cycle threshold. This figure appears in color at www.ajtmh.org.

Figure 2.

Cycle threshold (Ct) values plotted against known quantities of purified seeded eggs of (A) Necator americanus, (B) Ancylostoma caninum, and (C) Ancylostoma ceylanicum. An excellent correlation was displayed between seeded average egg counts and average Ct measured for N. americanus, Ancylostoma duodenale/A. caninum, and A. ceylanicum by the multiplex hookworm quantitative PCR. This figure appears in color at www.ajtmh.org.

Figure 3.

Hookworm prevalence by multiplex hookworm quantitative PCR (qPCR), multiplex soil-transmitted helminth (STH) qPCR conventional PCR, and sodium nitrate flotation (microscopy). Data presented combined for all 192 study participants, showing higher recorded percentage prevalence across all genera and species of hookworms, including mixed species infections, by multiplex hookworm qPCR.

Figure 4.

Relationship between cycle threshold (Ct) value for multiplex hookworm qPCR and fecal flotation derived egg counts (EPG) for Necator americanus. Microscopy negative Ct values not displayed. A moderate-to-good correlation was displayed between average N. americanus egg counts measured by sodium nitrate flotation and average Ct measured by the multiplex hookworm qPCR. EPG = eggs per gram. This figure appears in color at www.ajtmh.org.