{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:31:15Z","timestamp":1785421875128,"version":"3.56.0"},"posted":{"date-parts":[[2025,1,14]]},"group-title":"Microbiology","reference-count":33,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T00:00:00Z","timestamp":1736812800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nd\/4.0\/"}],"funder":[{"award":["R03AI141709"],"award-info":[{"award-number":["R03AI141709"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"award":["T32AI060546"],"award-info":[{"award-number":["T32AI060546"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/030689123","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,5,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Rapid and accurate diagnostics are needed to effectively detect and treat primary amoebic meningoencephalitis (PAM) caused by the free-living amoeba,\n                  <jats:italic>Naegleria fowleri<\/jats:italic>\n                  . Delayed diagnosis and similarities to other causes of meningitis contribute to a case mortality rate of &gt;97%, and current testing requires a spinal tap to obtain cerebrospinal fluid (CSF). Thus, there is an unmet medical need for a non-invasive liquid biopsy diagnostic method. We sequenced\n                  <jats:italic>N. fowleri<\/jats:italic>\n                  extracellular vesicles (EVs) and identified microRNAs, tRNAs and other small RNAs in\n                  <jats:italic>N. fowleri-<\/jats:italic>\n                  EVs. From these data we selected two prevalent small RNAs as biomarker candidates. We developed an RT-qPCR assay and both small RNAs were detected in\n                  <jats:italic>N. fowleri-<\/jats:italic>\n                  EVs and amoeba-conditioned media. In the mouse model of PAM, both small RNA biomarkers were detected in 100% of mouse plasma samples at the end-stage of infection. Notably, smallRNA-1 was detected in the urine of infected mice at timepoints as early as 24h post infection (18\/23 mice) and in the plasma as early as 60h post infection (8\/8 mice). Additionally, smallRNA-1 was detected in 100% (n=6) of CSF samples from human PAM cases, and in whole blood samples, but not in human plasma from PAM cases. In this study, we discovered small RNAs as biomarkers of\n                  <jats:italic>N. fowleri<\/jats:italic>\n                  infection\u2014one which can be detected reliably in CSF, urine, and whole blood. The RT-qPCR assay is a highly sensitive diagnostic assay that can be conducted in \u223c3h after receipt of liquid biopsy. The data suggest detection of smallRNA-1 biomarker could provide earlier diagnosis of PAM and be used to monitor biomass of amoebae during treatment.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>One Sentence Summary<\/jats:title>\n                  <jats:p>\n                    Small RNAs of\n                    <jats:italic>Naegleria fowleri<\/jats:italic>\n                    can be detected in liquid biopsies and serve as early diagnostic biomarkers of primary amoebic meningoencephalitis.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.01.11.632551","type":"posted-content","created":{"date-parts":[[2025,1,14]],"date-time":"2025-01-14T08:45:17Z","timestamp":1736844317000},"source":"Crossref","is-referenced-by-count":0,"title":["Secreted small RNAs of\n                  <i>Naegleria fowleri<\/i>\n                  are biomarkers for diagnosis of primary amoebic meningoencephalitis"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4725-1758","authenticated-orcid":false,"given":"A. Cassiopeia","family":"Russell","sequence":"first","affiliation":[{"name":"University of Georgia, Department of Infectious Diseases, Athens, GA, USA, 30602"},{"name":"University of Georgia, Center for Tropical and Emerging Global Diseases, Athens, GA, USA, 30602"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-8455-8237","authenticated-orcid":false,"given":"Joseph","family":"Dainis","sequence":"additional","affiliation":[{"name":"University of Georgia, Department of Infectious Diseases, Athens, GA, USA, 30602"},{"name":"University of Georgia, Center for Tropical and Emerging Global Diseases, Athens, GA, USA, 30602"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-0854-7122","authenticated-orcid":false,"given":"Jose","family":"Alexander","sequence":"additional","affiliation":[{"name":"AdventHealth Central Florida, Microbiology Department, Orlando, FL, USA, 32803"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9808-3227","authenticated-orcid":false,"given":"Ibne Karim M.","family":"Ali","sequence":"additional","affiliation":[{"name":"Centers for Disease Control and Prevention, Waterborne Disease Prevention Branch, DFWED\/NCEZID, Atlanta, GA, USA, 30333"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0238-965X","authenticated-orcid":false,"given":"Dennis E.","family":"Kyle","sequence":"additional","affiliation":[{"name":"University of Georgia, Department of Infectious Diseases, Athens, GA, USA, 30602"},{"name":"University of Georgia, Center for Tropical and Emerging Global Diseases, Athens, GA, USA, 30602"},{"name":"University of Georgia, Department of Cellular Biology, Athens, GA, USA, 30602"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026041121552301000_2025.01.11.632551v2.1","doi-asserted-by":"publisher","DOI":"10.1093\/jpids\/piu103"},{"key":"2026041121552301000_2025.01.11.632551v2.2","doi-asserted-by":"publisher","DOI":"10.3201\/eid2401.170545"},{"key":"2026041121552301000_2025.01.11.632551v2.3","doi-asserted-by":"publisher","DOI":"10.1097\/QCO.0000000000000923"},{"key":"2026041121552301000_2025.01.11.632551v2.4","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/cir961"},{"key":"2026041121552301000_2025.01.11.632551v2.5","doi-asserted-by":"publisher","DOI":"10.1016\/S1473-3099(19)30675-9"},{"key":"2026041121552301000_2025.01.11.632551v2.6","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(12)61730-9"},{"key":"2026041121552301000_2025.01.11.632551v2.7","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-695X.2007.00232.x"},{"key":"2026041121552301000_2025.01.11.632551v2.8","doi-asserted-by":"publisher","DOI":"10.1016\/j.pt.2021.10.004"},{"key":"2026041121552301000_2025.01.11.632551v2.9","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciy813"},{"key":"2026041121552301000_2025.01.11.632551v2.10","doi-asserted-by":"publisher","DOI":"10.1093\/ofid\/ofac682"},{"key":"2026041121552301000_2025.01.11.632551v2.11","doi-asserted-by":"publisher","DOI":"10.1155\/2022\/2359883"},{"key":"2026041121552301000_2025.01.11.632551v2.12","doi-asserted-by":"publisher","DOI":"10.3390\/pathogens9080638"},{"key":"2026041121552301000_2025.01.11.632551v2.13","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0008353"},{"key":"2026041121552301000_2025.01.11.632551v2.14","doi-asserted-by":"publisher","DOI":"10.1007\/s00203-023-03518-8"},{"key":"2026041121552301000_2025.01.11.632551v2.15","doi-asserted-by":"publisher","DOI":"10.1093\/ajcp\/aqz103"},{"key":"2026041121552301000_2025.01.11.632551v2.16","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.00875-06"},{"key":"2026041121552301000_2025.01.11.632551v2.17","doi-asserted-by":"publisher","DOI":"10.5858\/2004-128-e33-PQCHIA"},{"key":"2026041121552301000_2025.01.11.632551v2.18","doi-asserted-by":"crossref","first-page":"291","DOI":"10.3347\/kjp.2019.57.3.291","article-title":"Primary Amebic Meningoencephalitis: A Case Report","volume":"57","year":"2019","journal-title":"Korean J Parasitol"},{"key":"2026041121552301000_2025.01.11.632551v2.19","doi-asserted-by":"publisher","DOI":"10.1186\/s12879-017-2638-8"},{"key":"2026041121552301000_2025.01.11.632551v2.20","doi-asserted-by":"publisher","DOI":"10.1001\/archpedi.1979.02130100049010"},{"key":"2026041121552301000_2025.01.11.632551v2.21","doi-asserted-by":"publisher","DOI":"10.15585\/mmwr.mm6443a5"},{"key":"2026041121552301000_2025.01.11.632551v2.22","doi-asserted-by":"crossref","unstructured":"A. C. Russell , P. Bush , G. Grigorean , D. E. Kyle , Characterization of the extracellular vesicles, ultrastructural morphology, and intercellular interactions of multiple clinical isolates of the brain-eating amoeba, Naegleria fowleri . Frontiers in Microbiology 14, (2023).","DOI":"10.3389\/fmicb.2023.1264348"},{"key":"2026041121552301000_2025.01.11.632551v2.23","doi-asserted-by":"crossref","unstructured":"S. Lertjuthaporn et al., Extracellular Vesicles from Naegleria fowleri Induce IL-8 Response in THP-1 Macrophage. Pathogens 11, (2022).","DOI":"10.3390\/pathogens11060632"},{"key":"2026041121552301000_2025.01.11.632551v2.24","doi-asserted-by":"crossref","unstructured":"L. Retana Moreira et al., Characterization of Extracellular Vesicles Secreted by a Clinical Isolate of Naegleria fowleri and Identification of Immunogenic Components within Their Protein Cargo. Biology (Basel) 11, (2022).","DOI":"10.3390\/biology11070983"},{"key":"2026041121552301000_2025.01.11.632551v2.25","doi-asserted-by":"crossref","unstructured":"H. G. Le , J. M. Kang , T. C. Vo , W. G. Yoo , B. K. Na , Naegleria fowleri Extracellular Vesicles Induce Proinflammatory Immune Responses in BV-2 Microglial Cells. Int J Mol Sci 24, (2023).","DOI":"10.3390\/ijms241713623"},{"key":"2026041121552301000_2025.01.11.632551v2.26","doi-asserted-by":"publisher","DOI":"10.1186\/s12879-021-06932-9"},{"key":"2026041121552301000_2025.01.11.632551v2.27","doi-asserted-by":"crossref","unstructured":"M. Pavletic et al., Fast, Reliable, and Simple Point-of-Care-like Adaptation of RT-qPCR for the Detection of SARS-CoV-2 for Use in Hospital Emergency Departments. Viruses 13, (2021).","DOI":"10.3390\/v13122413"},{"key":"2026041121552301000_2025.01.11.632551v2.28","doi-asserted-by":"crossref","first-page":"e0189921","DOI":"10.1128\/spectrum.01899-21","article-title":"Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri","volume":"10","year":"2022","journal-title":"Microbiol Spectr"},{"key":"2026041121552301000_2025.01.11.632551v2.29","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.81.8.2406"},{"key":"2026041121552301000_2025.01.11.632551v2.30","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0198827"},{"key":"2026041121552301000_2025.01.11.632551v2.31","doi-asserted-by":"publisher","DOI":"10.1261\/rna.035279.112"},{"key":"2026041121552301000_2025.01.11.632551v2.32","doi-asserted-by":"crossref","unstructured":"M. F. Kramer , Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol Chapter 15, Unit 15 10 (2011).","DOI":"10.1002\/0471142727.mb1510s95"},{"key":"2026041121552301000_2025.01.11.632551v2.33","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkn188"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.01.11.632551","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T04:55:29Z","timestamp":1775969729000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.01.11.632551"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,14]]},"references-count":33,"URL":"https:\/\/doi.org\/10.1101\/2025.01.11.632551","relation":{"has-version":[{"id-type":"doi","id":"10.7554\/eLife.105994.1","asserted-by":"object"},{"id-type":"doi","id":"10.7554\/eLife.105994.2","asserted-by":"object"}]},"subject":[],"published":{"date-parts":[[2025,1,14]]},"subtype":"preprint"}}