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 µM. 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.

Background Understanding local Anopheles species compositions and bionomic traits are vital for an effective malaria vector intervention strategy. Though eight malaria vectors, including species complexes, have been documented across the island of Sulawesi, Indonesia, a comprehensive survey linking morphological and molecular species identification has not been conducted in this global hotspot of biodiversity. Results Eighteen distinct species of Anopheles were molecularly identified in a 1 km 2 area in Karama village, West Mamuju Province, Sulawesi. Known species included An. aconitus , An. karwari , An. peditaeniatus , An. vagus , An. barbirostris , An. tessellatus , An. nigerrimus , An. crawfordi , An. maculatus, An. flavirostris and An. kochi . Of the 18 distinct sequence groups identified through both ribosomal DNA internal transcribed spacer region 2, and mitochondrial DNA cytochrome c oxidase subunit 1 loci, 8 could not be identified to species through comparison to published sequences. The comparison of morphological and molecular identities determined that interpretations of local species compositions for primary and expected species in Karama ( An. barbirostris and An. vagus ) had the highest rate of accuracy (92.1% and 87.6%, respectively) when compared to molecular analysis. However, the remaining distinct sequences molecularly identified to species were identified correctly by morphological methods less frequently, from 0 to 83%. Conclusions Karama, Indonesia has a high diversity of Anopheles spp. The unexpected high number of Anopheles species in a small area points to possible complex transmission dynamics and limitations with vector control based on possible varying behaviors and interactions with both humans and interventions. Morphological identification of Anopheles spp. in this study was more accurate for primary and expected species than secondary or unexpected species. Finally, the inability to identify seven sequence groups to species with consensus sequences implies that future studies employing sequencing are required to clarify species compositions in the Nigerrimus Subgroup, among others, as well as their distribution and vector status. Use of molecular methods in conjunction with morphological investigations for analysis of species composition, population dynamics and bionomic characteristics is directly implicated in understanding drivers of malaria transmission, intervention effectiveness, and the pursuit...

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 µM. 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.

Rapid diagnostic tests (RDTs) are a key tool for the diagnosis of malaria infections among clinical and subclinical individuals. Low-density infections, and deletions of the P. falciparum hrp2/3 genes (encoding the HRP2 and HRP3 proteins detected by many RDTs) present challenges for RDT-based diagnosis. The novel Rapigen Biocredit three-band Plasmodium falciparum HRP2/LDH RDT was evaluated among 444 clinical and 468 subclinical individuals in a high transmission setting in Burundi. Results were compared to the AccessBio CareStart HRP2 RDT, and qPCR with a sensitivity of <0.3 parasites/μL blood. Sensitivity compared to qPCR among clinical patients for the Biocredit RDT was 79.9% (250/313, either of HRP2/LDH positive), compared to 73.2% (229/313) for CareStart (P = 0.048). Specificity of the Biocredit was 82.4% compared to 96.2% for CareStart. Among subclinical infections, sensitivity was 72.3% (162/224) compared to 58.5% (131/224) for CareStart (P = 0.003), and reached 88.3% (53/60) in children <15 years. Specificity was 84.4% for the Biocredit and 93.4% for the CareStart RDT. No (0/362) hrp2 and 2/366 hrp3 deletions were observed. In conclusion, the novel RDT showed improved sensitivity for the diagnosis of P. falciparum.

Naegleria fowleri is a pathogenic free-living amoeba that is commonly found in warm, freshwater and can cause a rapidly fulminant disease known as primary amoebic meningoencephalitis (PAM). New drugs are urgently needed to treat PAM, as the fatality rate is >97%. Until recently, few advances have been made in the discovery of new drugs for N. fowleri and one drawback is the lack of validated tools and methods to enhance drug discovery and diagnostics research. In this study we aimed to validate alternative methods to assess cell proliferation that are commonly used for other cell types and develop a novel drug screening assay to evaluate drug efficacy on N. fowleri replication. EdU (5-ethynyl-2’-deoxyuridine) is a pyrimidine analog of thymidine that can be used as a quantitative endpoint for cell proliferation. EdU incorporation is detected via a copper catalyzed click reaction with an Alexa Fluor linked azide. EdU incorporation in replicating N. fowleri was validated using fluorescence microscopy and quantitative methods for assessing EdU incorporation were developed by using an imaging flow cytometer. Currently used PAM therapeutics inhibited N. fowleri replication and EdU incorporation in vitro. EdA (5’ethynyl-2’-deoxyadenosine), an adenine analog, also was incorporated by N. fowleri, but was more cytotoxic than EdU. In summary, EdU incorporation could be used as a complimentary method for drug discovery for these neglected pathogens.

Rapid diagnostic tests (RDTs) are a key tool for the diagnosis of malaria infections among febrile and subclinical individuals. Low-density infections, and deletions of the P. falciparum hrp2/3 genes (encoding for the HRP2 and HRP3 proteins detected by many RDTs) present challenges for RDT-based diagnosis. The novel Rapigen Biocredit three-band Plasmodium falciparum HRP2/LDH RDT was evaluated among 444 febrile and 468 subclinical individuals in a high transmission setting in Burundi. Results were compared to the AccessBio CareStart HRP2 RDT, and qPCR with a sensitivity of <0.1 parasites/µL blood. Sensitivity among clinical patients was 80.0% (250/313, either of HRP2/LDH positive), compared to 73.2% (229/313) for CareStart (P=0.048). Among subclinical infections, sensitivity was 72.3% (162/224) compared to 58.5% (131/224) for CareStart (P=0.003), and reached 88.3% (53/60) in children <15 years. No (0/362) hrp2 and 2/366 hrp3 deletions were observed. In conclusion, the novel RDT showed improved sensitivity for the diagnosis of P. falciparum.

Cardiovascular disease (CVD) is the most common non-cancer cause of death in cancer survivors and there is an unmet clinical need for easy, accurate, and safe CVD prognostic risk-stratification in adult cancer survivors. This study investigated whether a previously validated 27-plasma protein prognostic model for four-year cardiovascular (CV) events could have such a utility. We used the 27-plasma protein model to predict the four-year risk of a CV event (myocardial infarction, stroke, transient ischemic attack, heart failure hospitalization, death) in 906 participants with a prior history or active malignancy of any type of cancer and compared predictive results to follow-up CV outcome data. The participants were from the BASEL VIII or ARIC (visit 3) studies with a medically adjudicated prior diagnosis of cancer. BASEL VIII is an observational cohort study in patients with suspected coronary artery disease. ARIC is a multi-site cohort study funded by the NHLBI, NCI, and NPCR investigating risk factors for CV health. A subset analysis was conducted to assess model performance in participants with no prior history of CVD and those with stable CVD. The 27-plasma protein model accurately stratified participants into 4 distinct and non-overlapping (95% CI) risk bins. The median time to event for all cancer survivors who had an event in this study was 1.3 years. Observed 4-year event rates across the 4 risk bins (low, medium-low, medium-high, and high) were 11.0%, 17.3%, 31.2% and 60.2%, respectively, which were higher than stratified event rates from our previously published metacohort analyses (5.6%, 11.2%, 20.0% and 43.4%, respectively) in participants with elevated CVD risk factors (e.g., prior events, diabetes, kidney disease and suspected coronary artery disease). The plasma protein model accurately predicted 4-year CVD risk with a C-index of 0.71 (0.68, 0.74) and 4-year AUC of 0.74 (0.69, 0.79). Performance of the protein model was comparable between participants with no prior history of CVD (C-Index: 0.69; AUC: 0.71) and stable CVD (C-Index: 0.69; AUC: 0.72), demonstrating the model accurately predicts CV event risk in cancer survivors regardless of cardiovascular history. Cancer survivors in this cohort can be distinguished with 4-year CV event rates as high as 60.2%, underscoring the urgent need for an easy and accurate risk stratification tool for this population. Prognostic protein testing may provide a novel tool for CVD risk assessment in adult cancer survivors. Citation Format: Emma V. Troth, Matthew Ayala, Jessica Chadwick, Erin Hales, Michael Hinterberg, Jessica N. Kuzma, Clare Paterson, Rachel Ostroff, Joan E. Walter, Christian Mueller, Josef Coresh. The plasma proteome as a cardiovascular disease risk assessment tool in cancer survivors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4361.

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