Development and validation of a multiplexed-tandem qPCR tool for diagnostics of human soil-transmitted helminth infections

Abstract

Soil-transmitted helminths (STH) are a major cause of morbidity in tropical developing countries with a global infection prevalence of more than one billion people and disease burden of around 3.4 million disability adjusted life years. Infection prevalence directly correlates to inadequate sanitation, impoverished conditions and limited access to public health systems. Underestimation of infection prevalence using traditional microscopy-based diagnostic techniques is common, specifically in populations with access to benzimidazole mass treatment programs and a predominance of low intensity infections. In this study, we developed a multiplexed-tandem qPCR (MT-PCR) tool to identify and quantify STH eggs in stool samples. We have assessed this assay by measuring infection prevalence and intensity in field samples of two cohorts of participants from Timor-Leste and Cambodia, which were collected as part of earlier epidemiological studies. MT-PCR diagnostic parameters were compared to a previously published multiplexed qPCR for STH detection. The MT-PCR assay agreed strongly with qPCR data and showed a diagnostic specificity of 99.60-100.00% (sensitivity of 83.33-100.00%) compared to qPCR and kappa agreement exceeding 0.85 in all tests. In addition, the MT-PCR has the added advantage of distinguishing Ancylostoma spp. species, namely Ancylostoma duodenale and Ancylostoma ceylanicum. This semi-automated platform uses a standardized, manufactured reagent kit, shows excellent run-to-run consistency/repeatability and supports high-throughput detection and quantitation at a moderate cost.

Fig 1

Overall infection prevalence of the major STH species comparing multiplexed qPCR and MT-PCR diagnostic approaches in cohorts from Timor-Leste (B) Cambodia (C) and combined (A). (A) Representation of combined infection prevalence for Timor-Leste and Cambodian cohort in percent with total numbers of positive infections as seen on individual bars. (B) Estimation of infection prevalence in Timor-Leste cohort of 462 stool samples with percentage infection prevalence on y-axis and STH species by diagnostic method on x-axis. (C) Estimation of infection prevalence in Cambodian cohort of 302 stool samples with percentage infection prevalence on y-axis and STH species by diagnostic method on x-axis. Estimation of infection by qPCR has been conducted for hookworm infections only. Field study setting is known to show very little to no A. lumbricoides or T. trichuris infections as to why these infections have not been included in estimation of infection prevalence by qPCR with confirmation of zero positive infections by MT-PCR within the scope of this study (*).

Fig 2. Cycle threshold (Ct) value scatterplot for all investigated STH species showing agreement of multiplexed qPCR and MT-PCR for all samples tested infection positive by either none, one or both molecular diagnostic methods.

Ascaris lumbricoides (A) Trichuris trichiura (B) Necator americanus (C) Ancylostoma spp. combined values for A. duodenale and A. ceylanicum (D). Highlighted samples were removed for analysis of coefficient of determination which determines the closeness of data to a fitted linear regression line using only samples deemed infection positive by both molecular diagnostic methods (*).