{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T05:07:18Z","timestamp":1787029638835,"version":"build-2736575974"},"reference-count":45,"publisher":"MDPI AG","issue":"24","license":[{"start":{"date-parts":[[2024,12,12]],"date-time":"2024-12-12T00:00:00Z","timestamp":1733961600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Aase og Ejnar Danielsen Fond"},{"name":"VPCIR biosciences"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Sensors"],"abstract":"<jats:p>Malaria poses a serious global health problem, with half the world population being at risk. Regular screening is crucial for breaking the transmission cycle and combatting the disease spreading. However, current diagnostic tools relying on blood samples face challenges in many malaria-epidemic areas. In the present study, we demonstrate the detection of the malaria-causing Plasmodium parasite in non-invasive saliva samples (N = 61) from infected individuals by combining a DNA-based Rolling-circle-Enhanced-Enzyme-Activity-Detection (REEAD) sensor system with a chemiluminescence readout that could be detected with an in-house-developed affordable and battery-powered portable reader. We successfully transferred the technology to sub-Saharan Africa, where the malaria burden is high, and demonstrated a proof of concept in a small study (N = 40) showing significant differences (p &lt; 0.00001) between malaria-positive individuals (N = 33) and presumed asymptomatic negative individuals (N = 7) all collected in Gabon. This is the first successful application of the REEAD sensor system for the detection of malaria in saliva in a high-epidemic area and holds promise for the potential future use of REEAD for malaria diagnosis or surveillance based on non-invasive specimens in sub-Saharan Africa.<\/jats:p>","DOI":"10.3390\/s24247947","type":"journal-article","created":{"date-parts":[[2024,12,12]],"date-time":"2024-12-12T06:29:30Z","timestamp":1733984970000},"page":"7947","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Non-Invasive Malaria Detection in Sub-Saharan Africa Using a DNA-Based Sensor System"],"prefix":"10.3390","volume":"24","author":[{"given":"Trine","family":"Juul-Kristensen","sequence":"first","affiliation":[{"name":"Department of Molecular Biology and Genetics, Universitetsbyen 81, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-2236-9521","authenticated-orcid":false,"given":"Celine","family":"Thiesen","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Universitetsbyen 81, Aarhus University, 8000 Aarhus, Denmark"},{"name":"Department of Biomedicine, H\u00f8egh-Guldbergs Gade 10, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-5499-3230","authenticated-orcid":false,"given":"Line","family":"Wulff Haurum","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Universitetsbyen 81, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Josephine Geertsen","family":"Keller","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Universitetsbyen 81, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Romeo Wenceslas","family":"Lendamba","sequence":"additional","affiliation":[{"name":"Centre de Recherches M\u00e9dicales de Lambar\u00e9n\u00e9 (CERMEL), Lambar\u00e9n\u00e9 BP 242, Gabon"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rella","family":"Zoleko Manego","sequence":"additional","affiliation":[{"name":"Centre de Recherches M\u00e9dicales de Lambar\u00e9n\u00e9 (CERMEL), Lambar\u00e9n\u00e9 BP 242, Gabon"},{"name":"Department of Implementation Research, Bernhard Nocht Institute for Tropical Medicine, & I. Dep. of Medicine, University Medical Centre Hamburg-Eppendorf, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4206-1333","authenticated-orcid":false,"given":"Madeleine Eunice","family":"Betouke Ongwe","sequence":"additional","affiliation":[{"name":"Centre de Recherches M\u00e9dicales de Lambar\u00e9n\u00e9 (CERMEL), Lambar\u00e9n\u00e9 BP 242, Gabon"},{"name":"Institut de Recherche en Ecologie Tropicale, CENAREST, Libreville BP 13354, Gabon"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Birgitta Ruth","family":"Knudsen","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Universitetsbyen 81, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduardo","family":"Pareja","sequence":"additional","affiliation":[{"name":"Nisonide SL, 18008 Granada, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eduardo","family":"Pareja-Tobes","sequence":"additional","affiliation":[{"name":"Nisonide SL, 18008 Granada, Spain"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rodrigo","family":"Labouriau","sequence":"additional","affiliation":[{"name":"Department of Mathematics, Aarhus University, Ny Munkegade 118, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2658-8706","authenticated-orcid":false,"given":"Ghyslain","family":"Mombo-Ngoma","sequence":"additional","affiliation":[{"name":"Centre de Recherches M\u00e9dicales de Lambar\u00e9n\u00e9 (CERMEL), Lambar\u00e9n\u00e9 BP 242, Gabon"},{"name":"Department of Implementation Research, Bernhard Nocht Institute for Tropical Medicine, & I. Dep. of Medicine, University Medical Centre Hamburg-Eppendorf, Bernhard-Nocht-Strasse 74, 20359 Hamburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Cinzia","family":"Tesauro","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology and Genetics, Universitetsbyen 81, Aarhus University, 8000 Aarhus, Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2024,12,12]]},"reference":[{"key":"ref_1","unstructured":"WHO (2023). World Malaria Report 2023, WHO."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Wanjala, C.L., and Kweka, E.J. (2016). Impact of Highland Topography Changes on Exposure to Malaria Vectors and Immunity in Western Kenya. Front. Public Health, 4.","DOI":"10.3389\/fpubh.2016.00227"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1555","DOI":"10.1038\/s41467-021-21815-y","article-title":"Malaria trends in Ethiopian highlands track the 2000 \u2018slowdown\u2019 in global warming","volume":"12","author":"Martinez","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Carlson, C.J., Bannon, E., Mendenhall, E., Newfield, T., and Bansal, S. (2023). Rapid range shifts in African Anopheles mosquitoes over the last century. Biol. Lett., 19.","DOI":"10.1098\/rsbl.2022.0365"},{"key":"ref_5","unstructured":"Gasimov, E. (2024). Update on Malaria Elimination, Including Zoonotic Malaria in Malaria Policy Advisory Group Meeting, World Health Organization."},{"key":"ref_6","unstructured":"WHO (2024, July 10). Countries and Territories Certified Malaria-Free by WHO. Available online: https:\/\/www.who.int\/teams\/global-malaria-programme\/elimination\/countries-and-territories-certified-malaria-free-by-who."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/S0065-308X(01)50029-9","article-title":"The malaria-infected red blood cell: Structural and functional changes","volume":"50","author":"Cooke","year":"2001","journal-title":"Adv. Parasitol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.ijpara.2008.09.007","article-title":"Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites","volume":"39","author":"Gilson","year":"2009","journal-title":"Int. J. Parasitol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"533","DOI":"10.1136\/jcp.49.7.533","article-title":"ACP Broadsheet no 148. July 1996. Laboratory diagnosis of malaria","volume":"49","author":"Warhurst","year":"1996","journal-title":"J. Clin. Pathol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"93","DOI":"10.3347\/kjp.2009.47.2.93","article-title":"Malaria diagnosis: A brief review","volume":"47","author":"Tangpukdee","year":"2009","journal-title":"Korean J. Parasitol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1128\/CMR.00035-07","article-title":"Update on rapid diagnostic testing for malaria","volume":"21","author":"Murray","year":"2008","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Gitta, B., and Kilian, N. (2020). Diagnosis of Malaria Parasites Plasmodium spp. in Endemic Areas: Current Strategies for an Ancient Disease. Bioessays, 42.","DOI":"10.1002\/bies.201900138"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"3138","DOI":"10.1021\/ja056957p","article-title":"An electronic, aptamer-based small-molecule sensor for the rapid, label-free detection of cocaine in adulterated samples and biological fluids","volume":"128","author":"Baker","year":"2006","journal-title":"J. Am. Chem. Soc."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"10008","DOI":"10.1039\/C5NR01158B","article-title":"Design of ultrasensitive DNA-based fluorescent pH sensitive nanodevices","volume":"7","author":"Halder","year":"2015","journal-title":"Nanoscale"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1424","DOI":"10.1021\/ac3023982","article-title":"Graphene signal amplification for sensitive and real-time fluorescence anisotropy detection of small molecules","volume":"85","author":"Liu","year":"2013","journal-title":"Anal. Chem."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"13666","DOI":"10.1021\/ja0651456","article-title":"Label-free and reagentless aptamer-based sensors for small molecules","volume":"128","author":"Zayats","year":"2006","journal-title":"J. Am. Chem. Soc."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"9447","DOI":"10.1021\/ac1021198","article-title":"Triggered polycatenated DNA scaffolds for DNA sensors and aptasensors by a combination of rolling circle amplification and DNAzyme amplification","volume":"82","author":"Bi","year":"2010","journal-title":"Anal. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"11166","DOI":"10.1021\/acsnano.5b05709","article-title":"Real-Time Label-Free Direct Electronic Monitoring of Topoisomerase Enzyme Binding Kinetics on Graphene","volume":"9","author":"Zuccaro","year":"2015","journal-title":"ACS Nano"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"4043","DOI":"10.1021\/nn9012912","article-title":"Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level","volume":"3","author":"Andersen","year":"2009","journal-title":"ACS Nano"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"5729","DOI":"10.1093\/nar\/gkr109","article-title":"Direct measurement of DNA bending by type IIA topoisomerases: Implications for non-equilibrium topology simplification","volume":"39","author":"Hardin","year":"2011","journal-title":"Nucleic AcidsRes."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1541001","DOI":"10.1142\/S1793984415410019","article-title":"Combining a Nanosensor and ELISA for Measurement of Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Activity and Protein Amount in Cell and Tissue Extract","volume":"5","author":"Jakobsen","year":"2015","journal-title":"Nano Life"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"230","DOI":"10.1016\/j.bios.2013.04.019","article-title":"Real-time detection of TDP1 activity using a fluorophore-quencher coupled DNA-biosensor","volume":"48","author":"Jensen","year":"2013","journal-title":"Biosens. Bioelectron."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"358","DOI":"10.1039\/C5NR06326D","article-title":"Specific detection of the cleavage activity of mycobacterial enzymes using a quantum dot based DNA nanosensor","volume":"8","author":"Jepsen","year":"2016","journal-title":"Nanoscale"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"9825","DOI":"10.1039\/C5NR01474C","article-title":"Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: A fast method for drug screening and determination of active enzyme concentrations","volume":"7","author":"Kristoffersen","year":"2015","journal-title":"Nanoscale"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"982","DOI":"10.1039\/c1ib00049g","article-title":"Strategies for highly sensitive biomarker detection by Rolling Circle Amplification of signals from nucleic acid composed sensors","volume":"3","author":"Stougaard","year":"2011","journal-title":"Integr. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Tesauro, C., Juul, S., Arn\u00f2, B., Nielsen, C.J., Fiorani, P., Fr\u00f8hlich, R.F., Andersen, F.F., Desideri, A., Stougaard, M., and Petersen, E. (September, January 28). Specific detection of topoisomerase I from the malaria causing P. falciparum parasite using isothermal rolling circle amplification. Proceedings of the 2012 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, San Diego, CA, USA.","DOI":"10.1109\/EMBC.2012.6346451"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"15071","DOI":"10.1021\/ja051725b","article-title":"A versatile toolbox for variable DNA functionalization at high density","volume":"127","author":"Rasched","year":"2005","journal-title":"J. Am. Chem. Soc."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"8131","DOI":"10.1021\/ac101409t","article-title":"Aptamer-based electrochemical biosensor for interferon gamma detection","volume":"82","author":"Liu","year":"2010","journal-title":"Anal. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"8935","DOI":"10.1016\/S0021-9258(18)81883-X","article-title":"Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication","volume":"264","author":"Blanco","year":"1989","journal-title":"J. Biol. Chem."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7137","DOI":"10.1021\/acs.analchem.6b01192","article-title":"Novel One-Tube-One-Step Real-Time Methodology for Rapid Transcriptomic Biomarker Detection: Signal Amplification by Ternary Initiation Complexes","volume":"88","author":"Fujita","year":"2016","journal-title":"Anal. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Wei, H., Tang, S., Hu, T., Zhao, G., and Guan, Y. (2016). Production of dumbbell probe through hairpin cleavage-ligation and increasing RCA sensitivity and specificity by circle to circle amplification. Sci. Rep., 6.","DOI":"10.1038\/srep29229"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"587","DOI":"10.1039\/C8NR07850E","article-title":"A new DNA sensor system for specific and quantitative detection of mycobacteria","volume":"11","author":"Franch","year":"2019","journal-title":"Nanoscale"},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Hede, M.S., Fjelstrup, S., L\u00f6tsch, F., Zoleko, R.M., Klicpera, A., Groger, M., Mischlinger, J., Endame, L., Veletzky, L., and Neher, R. (2018). Detection of the Malaria causing Plasmodium Parasite in Saliva from Infected Patients using Topoisomerase I Activity as a Biomarker. Sci. Rep., 8.","DOI":"10.1038\/s41598-018-22378-7"},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Keller, J.G., Petersen, K.V., Mizielinski, K., Thiesen, C., Bjergb\u00e6k, L., Reguera, R.M., P\u00e9rez-Pertejo, Y., Bala\u00f1a-Fouce, R., Trejo, A., and Masdeu, C. (2023). Gel-Free Tools for Quick and Simple Screening of Anti-Topoisomerase 1 Compounds. Pharmaceuticals, 16.","DOI":"10.3390\/ph16050657"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1039\/C6NR07428F","article-title":"Novel DNA sensor system for highly sensitive and quantitative retrovirus detection using virus encoded integrase as a biomarker","volume":"9","author":"Wang","year":"2017","journal-title":"Nanoscale"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Singh, P., Rani, K., Gotmare, A., and Bhattacharyya, S. (2022). A tale of topoisomerases and the knotty genetic material in the backdrop of Plasmodium biology. Biosci. Rep., 42.","DOI":"10.1042\/BSR20212847"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1006\/expr.1998.4362","article-title":"Plasmodium falciparum: Stage-related expression of topoisomerase I","volume":"91","author":"Tosh","year":"1999","journal-title":"Exp. Parasitol."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Keller, J.G., Mizielinski, K., Petersen, K.V., Stougaard, M., Knudsen, B.R., and Tesauro, C. (2022). Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples. J. Vis. Exp., e64484.","DOI":"10.3791\/64484-v"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"10676","DOI":"10.1021\/nn3038594","article-title":"Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement","volume":"6","author":"Juul","year":"2012","journal-title":"ACS Nano"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"500","DOI":"10.1007\/s00508-020-01794-8","article-title":"Development of sustainable research excellence with a global perspective on infectious diseases: Centre de Recherches M\u00e9dicales de Lambar\u00e9n\u00e9 (CERMEL), Gabon","volume":"133","author":"Ramharter","year":"2021","journal-title":"Wien. Klin. Wochenschr."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"599","DOI":"10.4269\/ajtmh.2001.65.599","article-title":"Comparison of methods for the rapid laboratory assessment of children with malaria","volume":"65","author":"Planche","year":"2001","journal-title":"Am. J. Trop. Med. Hyg."},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Juul-Kristensen, T., Keller, J.G., Borg, K.N., Hansen, N.Y., Foldager, A., Ladegaard, R., Ho, Y.P., Loeschcke, V., and Knudsen, B.R. (2023). Topoisomerase 1 Activity Is Reduced in Response to Thermal Stress in Fruit Flies and in Human HeLa Cells. Biosensors, 13.","DOI":"10.3390\/bios13110950"},{"key":"ref_43","unstructured":"Keun-Yeong, J. (2022). Simple and Fast DNA-Based Tool to Investigate Topoisomerase 1 Activity, a Biomarker for Drug Susceptibility in Colorectal Cancer. Recent Understanding of Colorectal Cancer Treatment, IntechOpen. Chapter 3."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1484","DOI":"10.1111\/j.1469-0691.2011.03507.x","article-title":"Improved performance with saliva and urine as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays","volume":"17","author":"Putaporntip","year":"2011","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1624","DOI":"10.1111\/j.1469-0691.2011.03639.x","article-title":"Malaria rapid diagnostic tests in elimination settings\u2014Can they find the last parasite?","volume":"17","author":"McMorrow","year":"2011","journal-title":"Clin. Microbiol. Infect."}],"container-title":["Sensors"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1424-8220\/24\/24\/7947\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:53:30Z","timestamp":1760115210000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1424-8220\/24\/24\/7947"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2024,12,12]]},"references-count":45,"journal-issue":{"issue":"24","published-online":{"date-parts":[[2024,12]]}},"alternative-id":["s24247947"],"URL":"https:\/\/doi.org\/10.3390\/s24247947","relation":{},"ISSN":["1424-8220"],"issn-type":[{"value":"1424-8220","type":"electronic"}],"subject":[],"published":{"date-parts":[[2024,12,12]]}}}