Secreted small RNAs of Naegleria fowleri are biomarkers for diagnosis of primary amoebic meningoencephalitis

Rapid and accurate diagnostics are needed to effectively detect and treat primary amoebic meningoencephalitis (PAM) caused by Naegleria fowleri ( Nf ). Delayed diagnosis and similarities to other causes of meningitis contribute to a case mortality rate of >97%. Thus, there is an unmet medical need for a non-invasive liquid biopsy diagnostic method. We sequenced Nf extracellular vesicles (EVs) and identified microRNAs, tRNAs and other small RNAs in Nf- 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 Nf- 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 Nf infection, one 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 ∼3h 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.