Prediction of Cardiometabolic Health Through Changes in Plasma Proteins With Intentional Weight Loss in the DiRECT and DIADEM-I Randomized Clinical Trials of Type 2 Diabetes Remission.

OBJECTIVE: To determine the extent to which changes in plasma proteins, previously predictive of cardiometabolic outcomes, predict changes in two diabetes remission trials. RESEARCH DESIGN AND METHODS: We applied SomaSignal predictive tests (each derived from ∼5,000 plasma protein measurements using aptamer-based proteomics assay) to baseline and 1-year samples of trial intervention (Diabetes Remission Clinical Trial [DiRECT], n = 118, and Diabetes Intervention Accentuating Diet and Enhancing Metabolism [DIADEM-I], n = 66) and control (DiRECT, n = 144, DIADEM-I, n = 76) group participants. RESULTS: Mean (SD) weight loss in DiRECT (U.K.) and DIADEM-I (Qatar) was 10.2 (7.4) kg and 12.1 (9.5) kg, respectively, vs. 1.0 (3.7) kg and 4.0 (5.4) kg in control groups. Cardiometabolic SomaSignal test results showed significant improvement (Bonferroni-adjusted P < 0.05) in DiRECT and DIADEM-I (expressed as relative difference, intervention minus control) as follows, respectively: liver fat (-26.4%, -37.3%), glucose tolerance (-36.6%, -37.4%), body fat percentage (-8.6%, -8.7%), resting energy rate (-8.0%, -5.1%), visceral fat (-34.3%, -26.1%), and cardiorespiratory fitness (9.5%, 10.3%). Cardiovascular risk (measured with SomaSignal tests) also improved in intervention groups relative to control, but this was significant only in DiRECT (DiRECT, -44.2%, and DIADEM-I, -9.2%). However, weight loss >10 kg predicted significant reductions in cardiovascular risk, -19.1% (95% CI -33.4 to -4.91) in DiRECT and -33.4% (95% CI -57.3, -9.6) in DIADEM-I. DIADEM-I also demonstrated rapid emergence of metabolic improvements at 3 months. CONCLUSIONS: Intentional weight loss in recent-onset type 2 diabetes rapidly induces changes in protein-based risk models consistent with widespread cardiometabolic improvements, including cardiorespiratory fitness. Protein changes with greater (>10 kg) weight loss also predicted lower cardiovascular risk, providing a positive outlook for relevant ongoing trials.

Performance of highly sensitive and conventional rapid diagnostic tests for clinical and subclinical Plasmodium falciparum infections, and hrp2/3 deletion status in Burundi.

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.

EdU Incorporation To Assess Cell Proliferation and Drug Susceptibility in Naegleria fowleri.

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 (7-deaza-2'-deoxy-7-ethynyladenosine), 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.

Molecular analysis reveals a high diversity of Anopheles species in Karama, West Sulawesi, Indonesia.

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 km2 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 of malaria elimination.

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 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.