Biology subjects

Rice, C. A.

Publications and source records attributed to Rice, C. A..

6 recordsLinked to original sources

bioRxiv2026

Development of a novel loop-mediated isothermal amplification (LAMP) assay for Balamuthia mandrillaris

Balamuthia mandrillaris is a free-living amoeba that can cause cutaneous skin lesions, systemic disease, and a severe life-threatening CNS infection, Balamuthia amoebic encephalitis (BAE), which has a high mortality rate (~92%). The current diagnostic methods for B. mandrillaris have many limitations including the long detection time, low sensitivity, and specific diagnostic tests only available in highly specialized centers. The early stage of diagnosis is important for the treatment because of how quickly BAE infection then develops. Therefore, we developed, optimized, and evaluated a novel loop-mediated isothermal amplification (LAMP) assay for the 18S rRNA gene of B. mandrillaris, which can be observed by naked eyes with phenol red. This novel diagnostic assay can detect ten different B. mandrillaris isolates with no cross-reactivity with the DNA of other free-living amoeba, protozoa, fungi, or bacteria tested. Compared with conventional PCR, our LAMP assay displayed ~10-100-fold higher sensitivity from extracted DNA, heat-treated samples, and non-treated samples, with the limit of detection (LOD) of 10 fg/L DNA, 10 trophozoite/100 L for non-treated cells, and 1 trophozoite/100 L for heat-treated cells, respectively. This method was further validated using two newly developed qPCR and dPCR methods to determine the LOD of approximately 1 copy/L. In addition, there is no tolerance for complicated matrices, that LAMP maintained efficacy in cerebrospinal fluid (CSF), urine, and blood-spiked samples. Due to the simplicity, robustness, and high sensitivity of the developed LAMP method, it will be useful for quick clinical detection and diagnosis of B. mandrillaris, particularly in non-specialized centers and resource-limited areas. Here we report the first LAMP diagnostic method for B. mandrillaris; publicly disclosed on Tuesday, November 4th, 2025 at the 20th (XX) International Meeting on the Biology and Pathogenicity of Free-Living Amoebae (FLAM 2025) at Puerto Morelos, Mexico.

microbiology↗

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↗

bioRxiv2024

Discovery of cyclic peptide natural product inhibitors of Balamuthia mandrillaris

Balamuthia mandrillaris is a pathogenic free-living amoeba that causes infection of central nervous system, called Balamuthia amoebic encephalitis (BAE), as well as cutaneous and systemic diseases. Patients infected with B. mandrillaris have a high mortality rate due to the lack of effective treatments. A combination of non-optimized antimicrobial drug regimen is typically recommended; however, they have poor parasite activity and can cause various severe side effects. Cyclic peptides exhibit a broad spectrum of antimicrobial activities and lower cytotoxicity. In this study, we evaluated the anti-B. mandrillaris effect of cyclic peptides. The predicted natural product-43 (pNP-43), identified from the SNaPP (Synthetic Natural Product Inspired Cyclic Peptides) library, and its derivates displayed a significant inhibition for B. mandrillaris trophozoites. Eight pNPs had IC50s <5 M. Furthermore, all hit pNPs demonstrated minimal hemolytic and cytotoxic effects on human cells. Our study first indicates the anti-B. mandrillaris effect of cyclic peptides, which provides a new direction for drug development. Further studies of the mechanism of action and in vivo effects will be elucidated to confirm the potency as a treatment for B. mandrillaris infection in the future.

pharmacology and toxicology↗

bioRxiv2020

Naegleria fowleri: protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brains frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogens high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. N. fowleri genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

molecular biology↗

bioRxiv2020

The transcriptome of Balamuthia mandrillaris trophozoites for structure-based drug design

Balamuthia mandrillaris, a pathogenic free-living amoeba (FLA), causes cutaneous skin lesions as well as the brain-eating disease: Balamuthia granulomatous amoebic encephalitis (GAE). These diseases, and diseases caused by other pathogenic FLA, Naegleria fowleri or Acanthamoeba species, are minimally studied from a drug discovery perspective; few targets have been validated or characterized at the molecular level, and little is known about the biochemical pathways necessary for parasite survival. Chemotherapies for CNS disease caused by B. mandrillaris require vast improvement. Current therapeutics are limited to a small number of drugs that were previously discovered in the last century through in vitro testing or identified after use in the small pool of surviving reports. Using our recently published methodology to identify potentially useful therapeutics, we screened a collection of 85 compounds that have previously been reported to have antiparasitic activity. We identified 59 compounds that impacted growth at concentrations below 220 M. Since there is no fully annotated genome or proteome, we used RNA-Seq to reconstruct the transcriptome of B. mandrillaris and locate the coding sequences of the specific genes potentially targeted by the compounds identified to inhibit trophozoite growth. We determined the sequence of 17 of these target genes and obtained expression clones for 15 that we validated by direct sequencing.

genomics↗

bioRxiv2020

Discovery of anti-amoebic inhibitors from screening the MMV Pandemic Response Box on Balamuthia mandrillaris, Naegleria fowleri and Acanthamoeba castellanii

Pathogenic free-living amoebae, Balamuthia mandrillaris, Naegleria fowleri and several Acanthamoeba species are the etiological agents of severe brain diseases, with case mortality rates >90%. A number of constraints including misdiagnosis and partially effective treatments lead to these high fatality rates. The unmet medical need is for rapidly acting, highly potent new drugs to reduce these alarming mortality rates. Herein, we report the discovery of new drugs as potential anti-amoebic agents. We used the CellTiter-Glo 2.0 high-throughput screening methods to screen the Medicines for Malaria Ventures (MMV) Pandemic Response Box in a search for new active chemical scaffolds. Initially we screened the library as a single-point assay at 10 and 1 {micro}M. From these data, we reconfirmed hits by conducting quantitative dose response assays and identified 12 hits against B. mandrillaris, 29 against N. fowleri and 14 against A. castellanii ranging from nanomolar to low micromolar potency. We further describe 11 novel molecules with activity against B. mandrillaris, 22 against N. fowleri and 9 against A. castellanii. These structures serve as a starting point for medicinal chemistry studies and demonstrate the utility of phenotypic screening for drug discovery to treat diseases caused by free-living amoebae.

microbiology↗