Journal Article

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

Belizario, Vicente Y., Stringer, Oliver W., de Hostos, Eugenio L., Cantera, Jason L., Forrest, Matthew S., White, Heather N., de los Santos, Tala, Storey, Helen L.

2021

Overview

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.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Amplification / Assaying / Biology and Life Sciences / Cognitive ability / Control / Control programs / Cross-reactivity / Decision making / Deoxyribonucleic acid / Diagnosis / DNA / DNA probes / DNA sequencing / DNA, Helminth - genetics / Engineering and Technology / Enzymes / Feces - parasitology / Gene amplification / Gene sequencing / Helminthiasis / Helminthiasis - diagnosis / Helminthiasis - parasitology / Helminthiasis - transmission / Helminths / Humans / Infections / Instrumentation / Intestine / Intestines / Luminous intensity / Medicine and Health Sciences / Methods / Microscopy / Molecular diagnostic techniques / Morbidity / Nematodes / Nucleic Acid Amplification Techniques - standards / Nucleic acids / Nucleotide sequence / Nucleotide sequencing / Ova / Pathogens / PCR / Polymerase chain reaction / Primers / Quantitation / Real time / Recombinase / Recombinases - chemistry / Recombinases - metabolism / Reproducibility of Results / Research and Analysis Methods / Samples / Sensitivity and Specificity / Soil / Soil - parasitology / Soils / Target detection / Tropical diseases