Streamlining the Development of Targeted Treatments for Neglected Amoebic Diseases

Free-living amoebae are commonly found in freshwater lakes and ponds, as well as in soils. While most species are not harmful to humans, there are a few species that can cause severe eye conditions and fatal brain diseases. Treatment options remain limited, and central nervous system infections are associated with extremely high mortality rates, with only a small number of survivors documented worldwide despite aggressive clinical intervention.

This is largely due to rapid disease progression, which leaves little time for suspicion, definitive diagnostic tests, and the initiation of treatment, as well as inadequate and severe side effects from the current treatment options. For instance, Acanthamoeba keratitis (AK) is the most common disease caused by Acanthamoeba spp. and mainly appears when contact lenses become contaminated by tap water containing the pathogen. It is a debilitating infection of the cornea that can cause sight loss, trigeminal neuralgia, and intense light sensitivity.

The first-line treatment for AK is typically polyhexamethylene biguanide (PHMB), administered as eye drops several times daily for weeks to months, and sometimes longer in refractory cases. Treatment is painful, inconvenient, and slow acting, and there is a chance of disease recurrence, with severe cases taking several years to fully resolve. Unfortunately, PHMB is cytotoxic, and patients have reported thinning of the cornea and higher intraocular pressure, with many eventually requiring corneal transplantation, or even enucleation of the eye in rare cases.

The high mortality rates associated with free-living amoeba diseases of the central nervous system are partly caused by the poor detection methods available for these species. Many cases are consequently misdiagnosed, leading to inappropriate treatment.

For instance, primary amoebic meningoencephalitis (PAM), caused by Naegleria fowleri, and granulomatous amoebic encephalitis (GAE), caused by Acanthamoeba spp., are often incorrectly diagnosed due to the delayed onset of non-specific symptoms that overlap with more common conditions, such as herpes simplex virus, fungal infection, or bacterial meningitis. The difficulty in obtaining a definitive diagnosis indicates the urgent need for faster, more accurate diagnostic tools to support earlier treatment onset and higher survival rates. 

Ramping up progress in drug discovery

Research into new treatment methods for amoebic diseases has historically been limited, with only a few groups currently actively working on drug discovery for free-living amoebae worldwide, despite their high morbidity and mortality.

This can be attributed in part to a lack of awareness surrounding these pathogens, as well as to their relatively low global incidence. In addition, novel drugs exhibit high failure rates during clinical trials, possibly due to genetics and host-pathogen interactions, though these effects also require further study.

Though still assumed to be rare, the prevalence of these amoebic diseases is on the rise, further highlighting the pressing need for new treatments. The Rice Research Group within the Department of Comparative Pathobiology at Purdue University is dedicated to addressing this unmet medical need by identifying safer, more selective, and more effective therapies for infections caused by free-living amoebae.

The group’s work focuses on the development of rapid and easy-to-use diagnostics, and on elucidating the mechanism of action of select amoeba inhibitors. This will enable the team to discover more targeted therapeutics that will reduce host toxicity and improve disease outcomes, as well as to repurpose existing drugs, which is an ideal strategy for accelerating new treatments for orphan diseases.

For example, miltefosine—a breast cancer chemotherapeutic agent and anti-leishmanial drug—has already been repurposed for the treatment of pathogenic free-living amoebae, albeit with inconsistent outcomes. Additionally, nitroxoline, an FDA-approved drug for bacterial urinary tract infections, has also been successfully repurposed for the treatment of GAE caused by Balamuthia mandrillaris.

The lab studies three types of amoebic pathogens: Acanthamoeba spp., which cause both AK and GAE; Balamuthia mandrillaris, which leads to Balamuthia amoebic encephalitis (BAE); and N. fowleri, the species responsible for PAM.

Working concurrently on multiple amoebae is what makes the Rice Research Group unique, as most institutions have traditionally studied one species at a time. This means the group can screen numerous drug candidates against all three parasites simultaneously, speeding up the process of identifying a broad-spectrum anti-amoebic or genus-targeted drug. This kind of pan-amoeba inhibitor could be delivered empirically to patients with an amoebic disease without needing to do extensive and time-consuming tests to identify the specific species present. 

Adopting automation for consistency and comparability

Before the establishment of his research group in 2022, principal investigator Dr. Rice had personally screened close to half a million drug compounds over the course of a decade. Dr. Rice recognized that batch-to-batch differences, purity, and manual handling can all influence drug screening results.

His team has therefore developed a high-throughput screening assay for all three amoebae, using laboratory automation to create a highly standardized drug discovery pipeline that allows direct comparison of how each species, genotype, and strain responds to different compounds and therapeutic strategies. This enabled them to screen a further half a million compounds in just two years, drastically speeding up progress towards novel treatments for multiple deadly amoebic diseases. October 2025 marked a significant milestone for the team; the lab screened its one millionth compound.

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The lab uses acoustic nanodrop dispensing to prepare large batches of identical 384-well assay plates, each containing panels of around 20 candidate drug compounds at fixed concentrations. These plates are then loaded onto an automated stacker (INTEGRA Biosciences) located in a BSL 2 hood, which allows walk-away assay set-up for up to 50 plates at a time, while also providing consistent reagent dispensing in both 96- and 384-well formats.

The lab also uses a portable 96-channel electronic pipette (INTEGRA Biosciences) to rapidly perform plate-to-plate transfers, clone drug plates, and distribute parasites into assay plates. Together, these intuitive and compact systems support a flexible, scalable workflow that reduces time and manual pipetting, and minimizes the risk of batch-to-batch variability in results.

Screening assays are run in triplicate with an injection-based dispenser (INTEGRA Biosciences), using the same drug batches, concentrations, and protocols across experiments to reduce inter-assay variability and generate directly comparable datasets. Automating liquid transfers and plate set-ups with a 96-well pipette, dispenser, and stacker allows the group to normalize variables and speed up data acquisition and analysis. Applying the same automated liquid handling tools to all screening workflows also enables the lab to distinguish genuine biological differences between strains from experimental variability.

The lab’s streamlined screening approach has enabled it to establish a centralized drug activity repository—known as the RADAR project—which is designed to track susceptibility patterns and put amoebae “on people’s radar”. The repository includes parasite strains from both clinical cases and the environment, and early findings suggest that some strains are already displaying higher drug-resistant phenotypes than other biobanked samples.

Associating a specific genotype with a particular drug susceptibility could allow the development of personalized care plans specific to each patient and parasite, potentially leading to more effective treatments and faster disease resolution. The repository also holds promise for helping to predict future treatment challenges associated with drug resistance.   

Bringing effective, low-toxicity treatments within reach

Standardized, high-throughput workflows using automation have traditionally only been available to industrialized drug discovery. However, thanks to the development of compact, cost-effective tools, smaller institutions are increasingly able to adopt this type of highly efficient set-up, supporting vital research into fatal diseases such as AK, GAE, BAE, and PAM.

Bringing high-throughput into an academic setting is already accelerating the development of safer, more selective drugs for these orphan diseases, and enhancing knowledge of circulating and emerging resistance patterns. These discoveries will no doubt contribute to the improvement of disease outcomes and provide hope that, soon, patients will finally have a real chance of survival.