Malaria evolution in South Asia: knowledge for control and elimination.

The study of malaria parasites on the Indian subcontinent should help us understand unexpected disease outbreaks and unpredictable disease presentations from Plasmodium falciparum and Plasmodium vivax infections. The Malaria Evolution in South Asia (MESA) research program is one of ten International Centers of Excellence for Malaria Research (ICEMR) sponsored by the US National Institutes of Health. In this second of two reviews, we describe why population structures of Plasmodia in India will be characterized and how we will determine their consequences on disease presentation, outcome and patterns. Specific projects will determine if genetic diversity, possibly driven by parasites with higher genetic plasticity, plays a role in changing epidemiology, pathogenesis, vector competence of parasite populations and whether innate human genetic traits protect Indians from malaria today. Deep local clinical knowledge of malaria in India will be supplemented by basic scientists who bring new research tools. Such tools will include whole genome sequencing and analysis methods; in vitro assays to measure genome plasticity, RBC cytoadhesion, invasion, and deformability; mosquito infectivity assays to evaluate changing parasite-vector compatibilities; and host genetics to understand protective traits in Indian populations. The MESA-ICEMR study sites span diagonally across India and include a mixture of very urban and rural hospitals, each with very different disease patterns and patient populations. Research partnerships include government-associated research institutes, private medical schools, city and state government hospitals, and hospitals with industry ties. Between 2012 and 2017, in addition to developing clinical research and basic science infrastructure at new clinical sites, our training workshops will engage new scientists and clinicians throughout South Asia in the malaria research field.

Improving malaria control in West Africa: interruption of transmission as a paradigm shift.

With the paradigm shift from the reduction of morbidity and mortality to the interruption of transmission, the focus of malaria control broadens from symptomatic infections in children ≤5 years of age to include asymptomatic infections in older children and adults. In addition, as control efforts intensify and the number of interventions increases, there will be decreases in prevalence, incidence and transmission with additional decreases in morbidity and mortality. Expected secondary consequences of these changes include upward shifts in the peak ages for infection (parasitemia) and disease, increases in the ages for acquisition of antiparasite humoral and cellular immune responses and increases in false-negative blood smears and rapid diagnostic tests. Strategies to monitor these changes must include: (1) studies of the entire population (that are not restricted to children ≤5 or ≤10 years of age), (2) study sites in both cities and rural areas (because of increasing urbanization across sub-Saharan Africa) and (3) innovative strategies for surveillance as the prevalence of infection decreases and the frequency of false-negative smears and rapid diagnostic tests increases.

Sahel, savana, riverine and urban malaria in West Africa: Similar control policies with different outcomes.

The study sites for the West African ICEMR are in three countries (The Gambia, Senegal, Mali) and are located within 750 km of each other. In addition, the National Malaria Control Programmes of these countries have virtually identical policies: (1) Artemisinin Combination Therapies (ACTs) for the treatment of symptomatic Plasmodium falciparum infection, (2) Long-Lasting Insecticide-treated bed Nets (LLINs) to reduce the Entomololgic Inoculation Rate (EIR), and (3) sulfadoxine-pyrimethamine for the Intermittent Preventive Treatment of malaria during pregnancy (IPTp). However, the prevalence of P. falciparum malaria and the status of malaria control vary markedly across the four sites with differences in the duration of the transmission season (from 4-5 to 10-11 months), the intensity of transmission (with EIRs from unmeasurably low to 4-5 per person per month), multiplicity of infection (from a mean of 1.0 to means of 2-5) and the status of malaria control (from areas which have virtually no control to areas that are at the threshold of malaria elimination). The most important priority is the need to obtain comparable data on the population-based prevalence, incidence and transmission of malaria before new candidate interventions or combinations of interventions are introduced for malaria control.

Novel inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with anti-malarial activity in the mouse model.

Plasmodium falciparum, the causative agent of the most deadly form of human malaria, is unable to salvage pyrimidines and must rely on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in the pyrimidine biosynthetic pathway and represents a potential target for anti-malarial therapy. A high throughput screen and subsequent medicinal chemistry program identified a series of N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides with low nanomolar in vitro potency against DHODH from P. falciparum, P. vivax, and P. berghei. The compounds were selective for the parasite enzymes over human DHODH, and x-ray structural data on the analog Genz-667348, demonstrated that species selectivity could be attributed to amino acid differences in the inhibitor-binding site. Compounds from this series demonstrated in vitro potency against the 3D7 and Dd2 strains of P. falciparum, good tolerability and oral exposure in the mouse, and ED(50) values in the 4-day murine P. berghei efficacy model of 13-21 mg/kg/day with oral twice-daily dosing. In particular, treatment with Genz-667348 at 100 mg/kg/day resulted in sterile cure. Two recent analogs of Genz-667348 are currently undergoing pilot toxicity testing to determine suitability as clinical development candidates.

Identification and characterization of small molecule inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase.

Plasmodium falciparum causes the most deadly form of malaria and accounts for over one million deaths annually. The malaria parasite is unable to salvage pyrimidines and relies on de novo biosynthesis for survival. Dihydroorotate dehydrogenase (DHOD), a mitochondrially localized flavoenzyme, catalyzes the rate-limiting step of this pathway and is therefore an attractive antimalarial chemotherapeutic target. Using a target-based high throughput screen, we have identified a series of potent, species-specific inhibitors of P. falciparum DHOD (pfDHOD) that are also efficacious against three cultured strains (3D7, HB3, and Dd2) of P. falciparum. The primary antimalarial mechanism of action of these compounds was confirmed to be inhibition of pfDHOD through a secondary assay with transgenic malaria parasites, and the structural basis for enzyme inhibition was explored through in silico structure-based docking and site-directed mutagenesis. Compound-mediated cytotoxicity was not observed with human dermal fibroblasts or renal epithelial cells. These data validate pfDHOD as an antimalarial drug target and provide chemical scaffolds with which to begin medicinal chemistry efforts.

Analysis of pfcrt, pfmdr1, dhfr, and dhps mutations and drug sensitivities in Plasmodium falciparum isolates from patients in Vietnam before and after treatment with artemisinin.

We have analyzed artemisinin sensitivity in Plasmodium falciparum isolates obtained from patients in South Vietnam and show that artemisinin sensitivity does not differ before and after drug treatment. There was an increase in the level of mefloquine resistance in the isolates after drug treatment that was concomitant with a decrease in chloroquine resistance, suggesting that treatment with artemisinin has selected for increased mefloquine resistance. Mutations in the pfmdr1 gene, previously shown to be associated with sensitivity to mefloquine, were selected against. All isolates resistant to chloroquine encoded Thr-76 in the pfcrt gene consistent with an essential role in the mechanism of chloroquine resistance. Mutations in pfmdr1 also were linked to chloroquine resistance. High levels of mutation in dhfr and dhps genes, which have previously been associated with Fansidar resistance, also were found, suggesting that this drug would not be useful for malaria control in this part of Vietnam.