{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T17:32:22Z","timestamp":1792690342165,"version":"4.5.0"},"reference-count":82,"publisher":"American Society of Tropical Medicine and Hygiene","issue":"6","license":[{"start":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T00:00:00Z","timestamp":1780444800000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,6,3]]},"abstract":"<jats:title>ABSTRACT.<\/jats:title>\n                  <jats:p>\n                    Soil-transmitted helminth (STH) infections are prevalent worldwide, but the true burden of strongyloidiasis is unclear due to lack of sensitive and field-friendly diagnostic tools. Diagnosis is often based on serological assays that are typically not point-of-care (POC). Although polymerase chain reaction (PCR) tests are sensitive and specific, the need for expensive equipment and highly skilled personnel limits their use in resource limited areas. Isothermal amplification assays are largely instrument-free, making them simpler to implement without loss of either sensitivity or specificity. We developed two recombinase polymerase amplification (RPA) assays to detect\n                    <jats:italic>Strongyloides stercoralis<\/jats:italic>\n                    (\n                    <jats:italic>Ss<\/jats:italic>\n                    ) in human stool samples and a complementary CRISPR-Cas12a detection system with visual readouts. Primers, probes, and guide RNAs (crRNAs) for these assays were designed targeting the\n                    <jats:italic>Ss<\/jats:italic>\n                    -NIE sequence and\n                    <jats:italic>Ss<\/jats:italic>\n                    dispersed repetitive sequence (\n                    <jats:italic>Ss<\/jats:italic>\n                    -DRS). The assay\u2019s specificities and limits of detection (LOD) were assessed using gDNA from\n                    <jats:italic>Ss<\/jats:italic>\n                    L3 larvae or from other STH and filariae. The NIE RPA showed a LOD of 1 fg\/\n                    <jats:italic>\u00b5<\/jats:italic>\n                    L, whereas the LOD for the\n                    <jats:italic>Ss<\/jats:italic>\n                    -DRS RPA was 1 pg\/\n                    <jats:italic>\u00b5<\/jats:italic>\n                    L. The LOD was 500 fg\/\n                    <jats:italic>\u00b5<\/jats:italic>\n                    L for the NIE RPA CRISPR-Cas12a assay. No cross-reactivity with any filarial parasite or other STH was observed. Because the NIE assays were more sensitive than the\n                    <jats:italic>Ss<\/jats:italic>\n                    -DRS assay, six patient samples positive for\n                    <jats:italic>Ss<\/jats:italic>\n                    by real-time PCR (qPCR) were tested using the NIE assays, of which four were positive. Though assay refinement and clinical validation are needed, this study establishes fast, highly sensitive and field-applicable POC diagnostic tools for\n                    <jats:italic>Ss<\/jats:italic>\n                    detection that are ideal for use in endemic areas with limited resources.\n                  <\/jats:p>","DOI":"10.4269\/ajtmh.26-0042","type":"journal-article","created":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T18:08:54Z","timestamp":1776794934000},"page":"1157-1164","source":"Crossref","is-referenced-by-count":2,"title":["Development of Recombinase Polymerase Amplification and CRISPR-Cas12a\u2013Enhanced Isothermal Amplification Assays for Strongyloides stercoralis DNA Detection: A Pilot Study"],"prefix":"10.4269","volume":"114","author":[{"given":"Robertine","family":"Lontuo-Fogang","sequence":"first","affiliation":[{"name":"Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sasisekhar","family":"Bennuru","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas B.","family":"Nutman","sequence":"additional","affiliation":[{"name":"Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2795","reference":[{"issue":"1","key":"b1","series-title":"J Nematol","first-page":"20240019","article-title":"Introduction to Strongyloides stercoralis anatomy","volume":"56","author":"Castelletto","year":"2024"},{"issue":"10","key":"b2","series-title":"Trans R Soc Trop Med Hyg","first-page":"967","article-title":"Strongyloidiasis\u2013The most neglected of the neglected tropical diseases?","volume":"103","author":"Olsen","year":"2009"},{"issue":"7","key":"b3","series-title":"PLoS Negl Trop Dis","first-page":"e2288","article-title":"Strongyloides stercoralis: Global distribution and risk factors","volume":"7","author":"Sch\u00e4r","year":"2013"},{"issue":"4","key":"b4","series-title":"Trans R Soc Trop Med Hyg","first-page":"314","article-title":"Strongyloides hyperinfection syndrome: An emerging global infectious disease","volume":"102","author":"Marcos","year":"2008"},{"issue":"6","key":"b5","series-title":"Pathogens","first-page":"468","article-title":"The global prevalence of Strongyloides stercoralis infection","volume":"9","author":"Buonfrate","year":"2020"},{"issue":"1","key":"b6","series-title":"Eur J Clin Microbiol Infect Dis","first-page":"27","article-title":"Direct detection of Strongyloides infection via molecular and antigen detection methods","volume":"40","author":"Balachandra","year":"2021"},{"issue":"1","key":"b7","series-title":"Nat Rev Dis Primers","first-page":"6","article-title":"Strongyloidiasis","volume":"10","author":"Gordon","year":"2024"},{"issue":"3","key":"b8","series-title":"Am J Trop Med Hyg","first-page":"248","article-title":"Efficacy of stool examination for detection of Strongyloides infection","volume":"53","author":"Sato","year":"1995"},{"issue":"9","key":"b9","series-title":"Diagnostics (Basel)","first-page":"615","article-title":"Effectiveness of Strongyloides recombinant IgG immunoreactive antigen in detecting IgG and IgG4 subclass antibodies for diagnosis of human strongyloidiasis using rapid immunochromatographic tests","volume":"10","author":"Boonroumkaew","year":"2020"},{"issue":"4","key":"b10","series-title":"PLoS Negl Trop Dis","first-page":"e0013018","article-title":"Recombinant antigen-based lateral flow tests for the detection of Strongyloides stercoralis infection","volume":"19","author":"Lontuo-Fogang","year":"2025"},{"key":"b11","series-title":"Eur J Clin Microbiol Infect Dis","first-page":"465","article-title":"Development of immunochromatographic device as a point-of-care tool for serodiagnosis of human strongyloidiasis cases","volume":"39","author":"Sadaow","year":"2020"},{"issue":"1","key":"b12","series-title":"Parasit Vectors","first-page":"535","article-title":"Evaluation of novel recombinant antigen-based (NIE\/SsIR) immunochromatographic rapid tests for Strongyloides stercoralis: An accuracy study","volume":"17","author":"Scarso","year":"2024"},{"issue":"1","key":"b13","series-title":"PLoS Negl Trop Dis","first-page":"e0010126","article-title":"Strongy detect: Preliminary validation of a prototype recombinant Ss-NIE\/Ss-IR based ELISA to detect Strongyloides stercoralis infection","volume":"16","author":"Sears","year":"2022"},{"issue":"2","key":"b14","series-title":"Am J Trop Med Hyg","first-page":"432","article-title":"Lateral flow dipstick test for serodiagnosis of strongyloidiasis","volume":"101","author":"Yunus","year":"2019"},{"issue":"1","key":"b15","series-title":"PLoS Negl Trop Dis","first-page":"e2640","article-title":"Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection","volume":"8","author":"Bisoffi","year":"2014"},{"issue":"2","key":"b16","series-title":"PLoS Negl Trop Dis","first-page":"e0006229","article-title":"Accuracy of molecular biology techniques for the diagnosis of Strongyloides stercoralis infection\u2013A systematic review and meta-analysis","volume":"12","author":"Buonfrate","year":"2018"},{"key":"b17","series-title":"Parasit Vectors","first-page":"38","article-title":"Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: Field-based inferences on the impact of mass deworming","volume":"9","author":"Easton","year":"2016"},{"issue":"3","key":"b18","series-title":"PLoS Negl Trop Dis","first-page":"e0004578","article-title":"Improved PCR-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design","volume":"10","author":"Pilotte","year":"2016"},{"issue":"7","key":"b19","series-title":"Clin Infect Dis","first-page":"e1594","article-title":"AcanR3990 qPCR: A novel, highly sensitive, bioinformatically-informed assay to detect Angiostrongylus cantonensis infections","volume":"73","author":"Sears","year":"2021"},{"issue":"4","key":"b20","series-title":"Trans R Soc Trop Med Hyg","first-page":"342","article-title":"Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR","volume":"103","author":"Verweij","year":"2009"},{"issue":"2","key":"b21","series-title":"Am J Trop Med Hyg","first-page":"306","article-title":"A loop-mediated isothermal amplification (LAMP) assay for 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malaria","volume":"117","author":"Lee","year":"2020"},{"issue":"5","key":"b25","series-title":"ACS Synth Biol","first-page":"1772","article-title":"RPA\/CRISPR\/Cas12a-Based On-Site and rapid nucleic acid detection of Toxoplasma gondii in the environment","volume":"11","author":"Lei","year":"2022"},{"issue":"4","key":"b26","series-title":"Bioinformatics","first-page":"682","article-title":"PrimedRPA: Primer design for recombinase polymerase amplification assays","volume":"35","author":"Higgins","year":"2019"},{"issue":"6","key":"b27","series-title":"Can J Microbiol","first-page":"383","article-title":"Recombinase polymerase amplification in the molecular diagnosis of microbiological targets and its applications","volume":"68","author":"Mota","year":"2022"},{"key":"b28","series-title":"Front Bioeng Biotechnol","first-page":"1330047","article-title":"Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing","volume":"11","author":"Xiao","year":"2023"},{"issue":"7","key":"b29","series-title":"Expert Rev Mol Diagn","first-page":"725","article-title":"Critical insight into recombinase polymerase amplification technology","volume":"22","author":"Munawar","year":"2022"},{"issue":"9","key":"b30","series-title":"J Clin Microbiol","first-page":"e0118521","article-title":"RPAcan3990: An ultrasensitive recombinase polymerase assay to detect Angiostrongylus cantonensis DNA","volume":"59","author":"Sears","year":"2021"},{"key":"b31","series-title":"Parasite","first-page":"60","article-title":"Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography","volume":"30","author":"Boonroumkaew","year":"2023"},{"issue":"6","key":"b32","series-title":"Clin Microbiol Infect","first-page":"543","article-title":"Novel approaches to the diagnosis of Strongyloides stercoralis infection","volume":"21","author":"Buonfrate","year":"2015"},{"issue":"8","key":"b33","series-title":"PLoS Negl Trop Dis","first-page":"e3002","article-title":"Development of a rapid serological assay for the diagnosis of strongyloidiasis using a novel diffraction-based biosensor technology","volume":"8","author":"Pak","year":"2014"},{"issue":"1","key":"b34","series-title":"Parasit Vectors","first-page":"412","article-title":"Diagnostic accuracy of a novel enzyme-linked immunoassay for the detection of IgG and IgG4 against Strongyloides stercoralis based on the recombinant antigens NIE\/SsIR","volume":"14","author":"Tamarozzi","year":"2021"},{"key":"b35","series-title":"Trends Food Sci Technol","first-page":"211","article-title":"CRISPR\/Cas12a-based technology: A powerful tool for biosensing in food safety","volume":"122","author":"Mao","year":"2022"},{"issue":"6","key":"b36","series-title":"Am J Trop Med Hyg","first-page":"1041","article-title":"A novel, multi-parallel, real-time polymerase chain reaction approach for eight gastrointestinal parasites provides improved diagnostic capabilities to resource-limited at-risk populations","volume":"88","author":"Mejia","year":"2013"},{"issue":"1","key":"b37","series-title":"Sci Rep","first-page":"4216","article-title":"Chronic Strongyloides stercoralis infection increases presence of the Ruminococcus torques group in the gut and alters the microbial proteome","volume":"13","author":"Tran","year":"2023"},{"issue":"4","key":"b38","series-title":"Clin Chem","first-page":"363","article-title":"Tandem repeat generation and novel isothermal amplification based on nonspecific tailing and replication slippage","volume":"69","author":"Luo","year":"2023"},{"issue":"2","key":"b39","series-title":"J Infect Dis","first-page":"e471","article-title":"From skin to sequence: Pan-species detection and speciation of Leishmania.","volume":"233","author":"Duncan","year":"2026"},{"issue":"1","key":"b40","series-title":"Biomed J","first-page":"8","article-title":"CRISPR-Cas12a: Functional overview and applications","volume":"43","author":"Paul","year":"2020"},{"issue":"10","key":"b41","series-title":"Microbiol Spectr","first-page":"e0099825","article-title":"Development of a rapid and sensitive RPA-CRISPR\/Cas12a-based assay for the detection of Brucella melitensis.","volume":"13","author":"Shen","year":"2025"},{"issue":"1","key":"b1","series-title":"J Nematol","first-page":"20240019","article-title":"Introduction to Strongyloides stercoralis anatomy","volume":"56","author":"Castelletto","year":"2024"},{"issue":"10","key":"b2","series-title":"Trans R Soc Trop Med Hyg","first-page":"967","article-title":"Strongyloidiasis\u2013The most neglected of the neglected tropical diseases?","volume":"103","author":"Olsen","year":"2009"},{"issue":"7","key":"b3","series-title":"PLoS Negl Trop Dis","first-page":"e2288","article-title":"Strongyloides stercoralis: Global distribution and risk factors","volume":"7","author":"Sch\u00e4r","year":"2013"},{"issue":"4","key":"b4","series-title":"Trans R Soc Trop Med Hyg","first-page":"314","article-title":"Strongyloides hyperinfection syndrome: An emerging global infectious disease","volume":"102","author":"Marcos","year":"2008"},{"issue":"6","key":"b5","series-title":"Pathogens","first-page":"468","article-title":"The global prevalence of Strongyloides stercoralis infection","volume":"9","author":"Buonfrate","year":"2020"},{"issue":"1","key":"b6","series-title":"Eur J Clin Microbiol Infect Dis","first-page":"27","article-title":"Direct detection of Strongyloides infection via molecular and antigen detection methods","volume":"40","author":"Balachandra","year":"2021"},{"issue":"1","key":"b7","series-title":"Nat Rev Dis Primers","first-page":"6","article-title":"Strongyloidiasis","volume":"10","author":"Gordon","year":"2024"},{"issue":"3","key":"b8","series-title":"Am J Trop Med Hyg","first-page":"248","article-title":"Efficacy of stool examination for detection of Strongyloides infection","volume":"53","author":"Sato","year":"1995"},{"issue":"9","key":"b9","series-title":"Diagnostics (Basel)","first-page":"615","article-title":"Effectiveness of Strongyloides recombinant IgG immunoreactive antigen in detecting IgG and IgG4 subclass antibodies for diagnosis of human strongyloidiasis using rapid immunochromatographic tests","volume":"10","author":"Boonroumkaew","year":"2020"},{"issue":"4","key":"b10","series-title":"PLoS Negl Trop Dis","first-page":"e0013018","article-title":"Recombinant antigen-based lateral flow tests for the detection of Strongyloides stercoralis infection","volume":"19","author":"Lontuo-Fogang","year":"2025"},{"key":"b11","series-title":"Eur J Clin Microbiol Infect Dis","first-page":"465","article-title":"Development of immunochromatographic device as a point-of-care tool for serodiagnosis of human strongyloidiasis cases","volume":"39","author":"Sadaow","year":"2020"},{"issue":"1","key":"b12","series-title":"Parasit Vectors","first-page":"535","article-title":"Evaluation of novel recombinant antigen-based (NIE\/SsIR) immunochromatographic rapid tests for Strongyloides stercoralis: An accuracy study","volume":"17","author":"Scarso","year":"2024"},{"issue":"1","key":"b13","series-title":"PLoS Negl Trop Dis","first-page":"e0010126","article-title":"Strongy detect: Preliminary validation of a prototype recombinant Ss-NIE\/Ss-IR based ELISA to detect Strongyloides stercoralis infection","volume":"16","author":"Sears","year":"2022"},{"issue":"2","key":"b14","series-title":"Am J Trop Med Hyg","first-page":"432","article-title":"Lateral flow dipstick test for serodiagnosis of strongyloidiasis","volume":"101","author":"Yunus","year":"2019"},{"issue":"1","key":"b15","series-title":"PLoS Negl Trop Dis","first-page":"e2640","article-title":"Diagnostic accuracy of five serologic tests for Strongyloides stercoralis infection","volume":"8","author":"Bisoffi","year":"2014"},{"issue":"2","key":"b16","series-title":"PLoS Negl Trop Dis","first-page":"e0006229","article-title":"Accuracy of molecular biology techniques for the diagnosis of Strongyloides stercoralis infection\u2013A systematic review and meta-analysis","volume":"12","author":"Buonfrate","year":"2018"},{"key":"b17","series-title":"Parasit Vectors","first-page":"38","article-title":"Multi-parallel qPCR provides increased sensitivity and diagnostic breadth for gastrointestinal parasites of humans: Field-based inferences on the impact of mass deworming","volume":"9","author":"Easton","year":"2016"},{"issue":"3","key":"b18","series-title":"PLoS Negl Trop Dis","first-page":"e0004578","article-title":"Improved PCR-based detection of soil transmitted helminth infections using a next-generation sequencing approach to assay design","volume":"10","author":"Pilotte","year":"2016"},{"issue":"7","key":"b19","series-title":"Clin Infect Dis","first-page":"e1594","article-title":"AcanR3990 qPCR: A novel, highly sensitive, bioinformatically-informed assay to detect Angiostrongylus cantonensis infections","volume":"73","author":"Sears","year":"2021"},{"issue":"4","key":"b20","series-title":"Trans R Soc Trop Med Hyg","first-page":"342","article-title":"Molecular diagnosis of Strongyloides stercoralis in faecal samples using real-time PCR","volume":"103","author":"Verweij","year":"2009"},{"issue":"2","key":"b21","series-title":"Am J Trop Med Hyg","first-page":"306","article-title":"A loop-mediated isothermal amplification (LAMP) assay for Strongyloides stercoralis in stool that uses a visual detection method with SYTO-82 fluorescent dye","volume":"90","author":"Watts","year":"2014"},{"issue":"4","key":"b22","series-title":"J Clin Microbiol","first-page":"e01173-18","article-title":"Comparison of loop-mediated isothermal amplification and real-time PCR assays for detection of Strongyloides larvae in different specimen matrices","volume":"57","author":"Watts","year":"2019"},{"issue":"10","key":"b23","series-title":"Pathogens","first-page":"869","article-title":"The recombinase polymerase amplification test for Strongyloides stercoralis is more sensitive than microscopy and real-time PCR in high-risk communities of Cusco, Peru","volume":"13","author":"Malaga","year":"2024"},{"issue":"41","key":"b24","series-title":"Proc Natl Acad Sci USA","first-page":"25722","article-title":"Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria","volume":"117","author":"Lee","year":"2020"},{"issue":"5","key":"b25","series-title":"ACS Synth Biol","first-page":"1772","article-title":"RPA\/CRISPR\/Cas12a-Based On-Site and rapid nucleic acid detection of Toxoplasma gondii in the environment","volume":"11","author":"Lei","year":"2022"},{"issue":"4","key":"b26","series-title":"Bioinformatics","first-page":"682","article-title":"PrimedRPA: Primer design for recombinase polymerase amplification assays","volume":"35","author":"Higgins","year":"2019"},{"issue":"6","key":"b27","series-title":"Can J Microbiol","first-page":"383","article-title":"Recombinase polymerase amplification in the molecular diagnosis of microbiological targets and its applications","volume":"68","author":"Mota","year":"2022"},{"key":"b28","series-title":"Front Bioeng Biotechnol","first-page":"1330047","article-title":"Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing","volume":"11","author":"Xiao","year":"2023"},{"issue":"7","key":"b29","series-title":"Expert Rev Mol Diagn","first-page":"725","article-title":"Critical insight into recombinase polymerase amplification technology","volume":"22","author":"Munawar","year":"2022"},{"issue":"9","key":"b30","series-title":"J Clin Microbiol","first-page":"e0118521","article-title":"RPAcan3990: An ultrasensitive recombinase polymerase assay to detect Angiostrongylus cantonensis DNA","volume":"59","author":"Sears","year":"2021"},{"key":"b31","series-title":"Parasite","first-page":"60","article-title":"Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography","volume":"30","author":"Boonroumkaew","year":"2023"},{"issue":"6","key":"b32","series-title":"Clin Microbiol Infect","first-page":"543","article-title":"Novel approaches to the diagnosis of Strongyloides stercoralis infection","volume":"21","author":"Buonfrate","year":"2015"},{"issue":"8","key":"b33","series-title":"PLoS Negl Trop Dis","first-page":"e3002","article-title":"Development of a rapid serological assay for the diagnosis of strongyloidiasis using a novel diffraction-based biosensor technology","volume":"8","author":"Pak","year":"2014"},{"issue":"1","key":"b34","series-title":"Parasit Vectors","first-page":"412","article-title":"Diagnostic accuracy of a novel enzyme-linked immunoassay for 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