Sensitive and semiquantitative detection of soil-transmitted helminth infection in stool using a recombinase polymerase amplification-based assay

Abstract

Soil-transmitted helminths (STHs) are parasitic nematodes that inhabit the human intestine. They affect more than 1.5 billion people worldwide, causing physical and cognitive impairment in children. The global strategy to control STH infection includes periodic mass drug administration (MDA) based on the results of diagnostic testing among populations at risk, but the current microscopy method for detecting infection has diminished sensitivity as the intensity of infection decreases. Thus, improved diagnostic tools are needed to support decision-making for STH control programs. We developed a nucleic acid amplification test based on recombinase polymerase amplification (RPA) technology to detect STH in stool. We designed primers and probes for each of the four STH species, optimized the assay, and then verified its performance using clinical stool samples. Each RPA assay was as sensitive as a real-time polymerase chain reaction (PCR) assay in detecting copies of cloned target DNA sequences. The RPA assay amplified the target in DNA extracted from human stool samples that were positive for STH based on the Kato-Katz method, with no cross-reactivity of the non-target genomic DNA. When tested with clinical stool samples from patients with infections of light, moderate, and heavy intensity, the RPA assays demonstrated performance comparable to that of real-time PCR, with better results than Kato-Katz. This new rapid, sensitive and field-deployable method for detecting STH infections can help STH control programs achieve their goals. Semi-quantitation of target by RPA assay is possible and is comparable to real-time PCR. With proper instrumentation, RPA assays can provide robust, semi-quantification of STH DNA targets as an alternative field-deployable indicator to counts of helminth eggs for assessing infection intensity. More than 1.5 billion people are infected with parasitic intestinal worms called soil-transmitted helminths. Infection is transmitted by helminth eggs in human feces, which contaminate soil in areas with poor sanitation. Adverse health effects include physical and cognitive impairment in children. A key strategy to control infection is periodic mass drug administration for populations with a high prevalence of disease based on the results of diagnostic testing. The current microscopy method for detecting infection, however, has limited ability to detect disease as the intensity of infection decreases with repeated mass drug administration. To address limitations of current diagnostic methods, we developed a novel technique to diagnose infections, including those at very low levels of intensity, by detecting helminth DNA in stool samples. Our initial studies suggest that the new diagnostic technique reliably detects the presence of intestinal worms, even at low intensities of infection, and may be more useful than currently available diagnostic tools for guiding the use of periodic mass drug administration to eliminate disease in low-resource settings.