Determination of diagnostic cycle threshold cutoffs for qPCR-based prevalence surveys of soil-transmitted helminth...

World Health Organization guidelines for control of soil-transmitted helminths (STHs) rely on coproscopic methods to assess population prevalence. In low-prevalence and light-intensity STH settings, quantitative PCR (qPCR) has higher sensitivity and specificity for detection. For qPCR to accurately identify transmissible infections of public health significance, it is essential to interpret qPCR cycle threshold (Ct) results. As part of the DeWorm3 community-based cluster-randomized trial on interrupting STH transmission, we conducted population-based surveys using high-throughput qPCR and aimed to establish appropriate Ct cutoffs to detect transmissible infections. Experimental approaches, including egg and genome-equivalent spiking experiments, were hindered by inefficient fecal DNA extraction despite optimization efforts. The Ct results for 29,980 samples (pre-intervention, cross-sectional surveys) revealed a bimodal distribution for two of the four species tested, Necator americanus and Ascaris lumbricoides. The first peak was assumed to represent transmissible infections, and the second peak to represent indeterminate or non-transmissible infections. Using a finite mixture model, we defined true qPCR positivity as any Ct result with a ≥5% chance of belonging to the first peak. This approach yielded Ct cutoffs of 34.4398 for N. americanus and 28.57587 for A. lumbricoides. For hookworms, the sensitivity of qPCR was 96.7%, compared to 73.2% for Kato-Katz, and moderate- to heavy-intensity infections (median Ct, 19.1; interquartile range [IQR], 17.9-19.8) were differentiated from both light-intensity infections and Kato-Katz-negative samples (25.3; IQR, 22.5-27.9). Our findings demonstrate the feasibility and utility of evidence-based Ct cutoffs to identify transmissible STH infections in large-scale surveys and categorize infection intensity as programmatically relevant.IMPORTANCECurrently, appropriate cycle threshold (Ct) cutoff values for soil-transmitted helminth quantitative PCR (qPCR) testing do not exist. As a result, it is not possible to differentiate a qPCR "positive" that represents a transmission risk from a signal resulting from non-infectious material such as fragmented eggs or sloughed cells. Leveraging samples collected and tested as part of the DeWorm3 cluster-randomized controlled trials, we used a data-driven statistical approach to establish a qPCR Ct cutoff that distinguishes epidemiologically relevant transmissible infections from non-transmissible DNA. This study highlights the utility of evidence-based qPCR Ct cutoffs to identify soil-transmitted helminth infections when evaluating and informing deworming programs.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT03014167.