LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis
BACKGROUND: Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less access...
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| author | Eva Dueñas Ingrid Tirado Percy Huaihua Ariana Parra del Riego Luis Cabrera-Sosa Jose A. Nakamoto Maria Manuel Cruz Carlos M. Restrepo Jorge Arévalo Vanessa Adaui |
| author_browse | Ariana Parra del Riego Carlos M. Restrepo Eva Dueñas Ingrid Tirado Jorge Arévalo Jose A. Nakamoto Luis Cabrera-Sosa Maria Manuel Cruz Percy Huaihua Vanessa Adaui |
| author_facet | Eva Dueñas Ingrid Tirado Percy Huaihua Ariana Parra del Riego Luis Cabrera-Sosa Jose A. Nakamoto Maria Manuel Cruz Carlos M. Restrepo Jorge Arévalo Vanessa Adaui |
| author_sort | Eva Dueñas |
| collection | Producción científica |
| description | BACKGROUND: Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the multicopy 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP). METHODOLOGY/PRINCIPAL FINDINGS: LAMP-coupled CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the L. (Viannia) subgenus, while the 18S assay showed pan-Leishmania detection capability. There was no cross-reactivity with other protozoan (Trypanosoma cruzi and Plasmodium falciparum) and bacterial (Mycobacterium tuberculosis) pathogen DNA, nor with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% (95% CI: 80.1-97.0%) for kDNA and 72.7% (95% CI: 59.0-83.9%) for 18S rDNA, both with 100% (95% CI: 90-100%) specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load. CONCLUSIONS/SIGNIFICANCE: These new proof-of-concept LAMP-CRISPR assays, combining high sensitivity, multiple Leishmania species detection capability, and a portable lateral flow readout, hold promise as next-generation molecular tools to improve leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control. |
| format | Article |
| id | oai:cris.upeu.edu.pe:20.500.15037/886 |
| institution | UPeU |
| language | English |
| publishDate | 2026 |
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| spelling | oai:cris.upeu.edu.pe:20.500.15037/8862026-10-30T16:14:57ZLAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasisEva DueñasIngrid TiradoPercy HuaihuaAriana Parra del RiegoLuis Cabrera-SosaJose A. NakamotoMaria Manuel CruzCarlos M. RestrepoJorge ArévaloVanessa AdauiLeishmaniasisLeishmaniaDiseaseMedicineTropical diseaseCutaneous leishmaniasisVisceral leishmaniasisNeglected tropical diseasesComputational biologyMolecular diagnosticsImmunologyVirologyProtozoal diseaseBiologyZoonotic diseaseDiagnostic testLeishmaniasisLeishmaniaDiseaseTropical diseaseCutaneous leishmaniasisVisceral leishmaniasisHumansHumansHumansLeishmania geneticsLeishmania geneticsLeishmania geneticsLeishmania isolation & purificationLeishmania isolation & purificationLeishmania isolation & purificationLeishmania braziliensis geneticsLeishmania braziliensis geneticsLeishmania braziliensis geneticsLeishmania braziliensis isolation & purificationLeishmania braziliensis isolation & purificationLeishmania braziliensis isolation & purificationLeishmaniasis diagnosisLeishmaniasis diagnosisLeishmaniasis diagnosisLeishmaniasis parasitologyLeishmaniasis parasitologyLeishmaniasis parasitologyRNA, Ribosomal, 18S geneticsRNA, Ribosomal, 18S geneticsRNA, Ribosomal, 18S geneticsSensitivity and SpecificitySensitivity and SpecificitySensitivity and SpecificityDNA, Protozoan geneticsDNA, Protozoan geneticsDNA, Protozoan geneticsLeishmaniasis, Cutaneous diagnosisLeishmaniasis, Cutaneous diagnosisLeishmaniasis, Cutaneous diagnosisLeishmaniasis, Cutaneous parasitologyLeishmaniasis, Cutaneous parasitologyLeishmaniasis, Cutaneous parasitologyDNA, Kinetoplast geneticsDNA, Kinetoplast geneticsDNA, Kinetoplast geneticsNucleic Acid Amplification Techniques methodsNucleic Acid Amplification Techniques methodsNucleic Acid Amplification Techniques methodsMolecular Diagnostic Techniques methodsMolecular Diagnostic Techniques methodsMolecular Diagnostic Techniques methodsCRISPR-Cas SystemsCRISPR-Cas SystemsCRISPR-Cas SystemsHealth Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis StudiesHealth Sciences Medicine Epidemiology Trypanosoma species research and implicationsLife Sciences Immunology and Microbiology Parasitology Parasites and Host InteractionsBACKGROUND: Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the multicopy 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP). METHODOLOGY/PRINCIPAL FINDINGS: LAMP-coupled CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the L. (Viannia) subgenus, while the 18S assay showed pan-Leishmania detection capability. There was no cross-reactivity with other protozoan (Trypanosoma cruzi and Plasmodium falciparum) and bacterial (Mycobacterium tuberculosis) pathogen DNA, nor with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% (95% CI: 80.1-97.0%) for kDNA and 72.7% (95% CI: 59.0-83.9%) for 18S rDNA, both with 100% (95% CI: 90-100%) specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load. CONCLUSIONS/SIGNIFICANCE: These new proof-of-concept LAMP-CRISPR assays, combining high sensitivity, multiple Leishmania species detection capability, and a portable lateral flow readout, hold promise as next-generation molecular tools to improve leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control.Articlehttps://pubmed.ncbi.nlm.nih.gov/41686849https://doi.org/10.1371/journal.pntd.001345610.1371/journal.pntd.0013456https://hdl.handle.net/20.500.15037/886W7128800596enPLoS neglected tropical diseases1935-27271935-27352026-02-132026-06-13T09:39:36Z2026-06-13T09:39:36Z |
| spellingShingle | Leishmaniasis Leishmania Disease Medicine Tropical disease Cutaneous leishmaniasis Visceral leishmaniasis Neglected tropical diseases Computational biology Molecular diagnostics Immunology Virology Protozoal disease Biology Zoonotic disease Diagnostic test Leishmaniasis Leishmania Disease Tropical disease Cutaneous leishmaniasis Visceral leishmaniasis Humans Humans Humans Leishmania genetics Leishmania genetics Leishmania genetics Leishmania isolation & purification Leishmania isolation & purification Leishmania isolation & purification Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis isolation & purification Leishmania braziliensis isolation & purification Leishmania braziliensis isolation & purification Leishmaniasis diagnosis Leishmaniasis diagnosis Leishmaniasis diagnosis Leishmaniasis parasitology Leishmaniasis parasitology Leishmaniasis parasitology RNA, Ribosomal, 18S genetics RNA, Ribosomal, 18S genetics RNA, Ribosomal, 18S genetics Sensitivity and Specificity Sensitivity and Specificity Sensitivity and Specificity DNA, Protozoan genetics DNA, Protozoan genetics DNA, Protozoan genetics Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous parasitology Leishmaniasis, Cutaneous parasitology Leishmaniasis, Cutaneous parasitology DNA, Kinetoplast genetics DNA, Kinetoplast genetics DNA, Kinetoplast genetics Nucleic Acid Amplification Techniques methods Nucleic Acid Amplification Techniques methods Nucleic Acid Amplification Techniques methods Molecular Diagnostic Techniques methods Molecular Diagnostic Techniques methods Molecular Diagnostic Techniques methods CRISPR-Cas Systems CRISPR-Cas Systems CRISPR-Cas Systems Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies Health Sciences Medicine Epidemiology Trypanosoma species research and implications Life Sciences Immunology and Microbiology Parasitology Parasites and Host Interactions Eva Dueñas Ingrid Tirado Percy Huaihua Ariana Parra del Riego Luis Cabrera-Sosa Jose A. Nakamoto Maria Manuel Cruz Carlos M. Restrepo Jorge Arévalo Vanessa Adaui LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis |
| title | LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis |
| title_full | LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis |
| title_fullStr | LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis |
| title_full_unstemmed | LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis |
| title_short | LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis |
| title_sort | lamp coupled crispr cas12a assays a promising new tool for molecular diagnosis of leishmaniasis |
| topic | Leishmaniasis Leishmania Disease Medicine Tropical disease Cutaneous leishmaniasis Visceral leishmaniasis Neglected tropical diseases Computational biology Molecular diagnostics Immunology Virology Protozoal disease Biology Zoonotic disease Diagnostic test Leishmaniasis Leishmania Disease Tropical disease Cutaneous leishmaniasis Visceral leishmaniasis Humans Humans Humans Leishmania genetics Leishmania genetics Leishmania genetics Leishmania isolation & purification Leishmania isolation & purification Leishmania isolation & purification Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis isolation & purification Leishmania braziliensis isolation & purification Leishmania braziliensis isolation & purification Leishmaniasis diagnosis Leishmaniasis diagnosis Leishmaniasis diagnosis Leishmaniasis parasitology Leishmaniasis parasitology Leishmaniasis parasitology RNA, Ribosomal, 18S genetics RNA, Ribosomal, 18S genetics RNA, Ribosomal, 18S genetics Sensitivity and Specificity Sensitivity and Specificity Sensitivity and Specificity DNA, Protozoan genetics DNA, Protozoan genetics DNA, Protozoan genetics Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous parasitology Leishmaniasis, Cutaneous parasitology Leishmaniasis, Cutaneous parasitology DNA, Kinetoplast genetics DNA, Kinetoplast genetics DNA, Kinetoplast genetics Nucleic Acid Amplification Techniques methods Nucleic Acid Amplification Techniques methods Nucleic Acid Amplification Techniques methods Molecular Diagnostic Techniques methods Molecular Diagnostic Techniques methods Molecular Diagnostic Techniques methods CRISPR-Cas Systems CRISPR-Cas Systems CRISPR-Cas Systems Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies Health Sciences Medicine Epidemiology Trypanosoma species research and implications Life Sciences Immunology and Microbiology Parasitology Parasites and Host Interactions |
| topic_facet | Leishmaniasis Leishmania Disease Medicine Tropical disease Cutaneous leishmaniasis Visceral leishmaniasis Neglected tropical diseases Computational biology Molecular diagnostics Immunology Virology Protozoal disease Biology Zoonotic disease Diagnostic test Leishmaniasis Leishmania Disease Tropical disease Cutaneous leishmaniasis Visceral leishmaniasis Humans Humans Humans Leishmania genetics Leishmania genetics Leishmania genetics Leishmania isolation & purification Leishmania isolation & purification Leishmania isolation & purification Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis genetics Leishmania braziliensis isolation & purification Leishmania braziliensis isolation & purification Leishmania braziliensis isolation & purification Leishmaniasis diagnosis Leishmaniasis diagnosis Leishmaniasis diagnosis Leishmaniasis parasitology Leishmaniasis parasitology Leishmaniasis parasitology RNA, Ribosomal, 18S genetics RNA, Ribosomal, 18S genetics RNA, Ribosomal, 18S genetics Sensitivity and Specificity Sensitivity and Specificity Sensitivity and Specificity DNA, Protozoan genetics DNA, Protozoan genetics DNA, Protozoan genetics Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous diagnosis Leishmaniasis, Cutaneous parasitology Leishmaniasis, Cutaneous parasitology Leishmaniasis, Cutaneous parasitology DNA, Kinetoplast genetics DNA, Kinetoplast genetics DNA, Kinetoplast genetics Nucleic Acid Amplification Techniques methods Nucleic Acid Amplification Techniques methods Nucleic Acid Amplification Techniques methods Molecular Diagnostic Techniques methods Molecular Diagnostic Techniques methods Molecular Diagnostic Techniques methods CRISPR-Cas Systems CRISPR-Cas Systems CRISPR-Cas Systems Health Sciences Medicine Public Health, Environmental and Occupational Health Research on Leishmaniasis Studies Health Sciences Medicine Epidemiology Trypanosoma species research and implications Life Sciences Immunology and Microbiology Parasitology Parasites and Host Interactions |
| url | https://pubmed.ncbi.nlm.nih.gov/41686849 https://doi.org/10.1371/journal.pntd.0013456 https://hdl.handle.net/20.500.15037/886 |
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