Dainis, J. | bioRxiv Science

bioRxiv2025

Virulence of Naegleria fowleri isolates varies significantly in the mouse model of primary amoebic meningoencephalitis

Naegleria fowleri is a small free-living amoeba that causes an acute, fatal disease called primary amoebic meningoencephalitis (PAM). One persisting question is why few people succumb to disease when so many are potentially exposed. We tested the hypothesis that N. fowleri isolates vary in virulence and in the minimum infectious dose required to induce disease by using a mouse model of PAM and intranasally inoculating dilutions of five clinical isolates of N. fowleri. Results showed significant differences in onset of severe disease and mortality rates between isolates. Remarkably, for isolate V596, 100 amoebae produced 100% mortality within 5 days. In contrast, higher numbers of amoebae were required for other isolates and mice survived for >2 weeks. Concurrently, we developed an in vitro virulence assay by comparing feeding rates between amoebae isolates seeded onto Vero cells. We observed a positive correlation between cytopathic effects in vitro and virulence in vivo. Article Summary LineIsolates of Naegleria fowleri vary in virulence, which results in significant differences in minimal infectious concentrations of amoebae that cause primary amoebic meningoencephalitis.

microbiology↗

bioRxiv2025

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

Rapid and accurate diagnostics are needed to effectively detect and treat primary amoebic meningoencephalitis (PAM) caused by the free-living amoeba, Naegleria fowleri. Delayed diagnosis and similarities to other causes of meningitis contribute to a case mortality rate of >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. fowleri extracellular vesicles (EVs) and identified microRNAs, tRNAs and other small RNAs in N. fowleri-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. fowleri-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. fowleri 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. One Sentence SummarySmall RNAs of Naegleria fowleri can be detected in liquid biopsies and serve as early diagnostic biomarkers of primary amoebic meningoencephalitis.

microbiology↗