A Novel Ex Vivo Drug Assay for Assessing the Transmission-Blocking Activity of Compounds on Field-Isolated Plasmodium falciparum Gametocytes.

The discovery and development of transmission-blocking therapies challenge malaria elimination and necessitate standard and reproducible bioassays to measure the blocking properties of antimalarial drugs and candidate compounds. Most of the current bioassays evaluating the transmission-blocking activity of compounds rely on laboratory-adapted Plasmodium strains. Transmission-blocking data from clinical gametocyte isolates could help select novel transmission-blocking candidates for further development. Using freshly collected Plasmodium falciparum gametocytes from asymptomatic individuals, we first optimized ex vivo culture conditions to improve gametocyte viability and infectiousness by testing several culture parameters. We next pre-exposed ex vivo field-isolated gametocytes to chloroquine, dihydroartemisinin, primaquine, KDU691, GNF179, and oryzalin for 48 h prior to direct membrane feeding. We measured the activity of the drug on the ability of gametocytes to resume the sexual life cycle in Anopheles after drug exposure. Using 57 blood samples collected from Malian volunteers aged 6 to 15 years, we demonstrate that the infectivity of freshly collected field gametocytes can be preserved and improved ex vivo in a culture medium supplemented with 10% horse serum at 4% hematocrit for 48 h. Moreover, our optimized drug assay displays the weak transmission-blocking activity of chloroquine and dihydroartemisinin, while primaquine and oryzalin exhibited a transmission-blocking activity of ~50% at 1 µM. KDU691 and GNF179 both interrupted Plasmodium transmission at 1 µM and 5 nM, respectively. This new approach, if implemented, has the potential to accelerate the screening of compounds with transmission-blocking activity.

Ex Vivo Plasmodium malariae Culture Method for Antimalarial Drugs Screen in the Field.

In vitro and ex vivo cultivation of Plasmodium (P) falciparum has facilitated active research into the malaria parasite toward the quest for basic knowledge and the discovery of effective drug treatments. Such a drug discovery program is currently difficult for P. malariae simply because of the absence of in vitro and ex vivo cultivation system for its asexual blood stages supporting antimalarial evaluation. Despite availability of artemisinin combination therapies effective on P. falciparum, P. malariae is being increasingly detected in malaria endemic countries. P. malariae is responsible for chronic infections and is associated with a high burden of anemia and morbidity. Here, we optimized and adapted ex vivo conditions under which P. malariae can be cultured and used for screening antimalarial drugs. Subsequently, this enabled us to test compounds such as artemether, chloroquine, lumefantrine, and quinine for ex vivo antimalarial activity against P. malariae.

Plasmodium malariae and Plasmodium falciparum comparative susceptibility to antimalarial drugs in Mali.

OBJECTIVES: To evaluate Plasmodium malariae susceptibility to current and lead candidate antimalarial drugs. METHODS: We conducted cross-sectional screening and detection of all Plasmodium species malaria cases, which were nested within a longitudinal prospective study, and an ex vivo assessment of efficacy of a panel of antimalarials against P. malariae and Plasmodium falciparum, both PCR-confirmed mono-infections. Reference compounds tested included chloroquine, lumefantrine, artemether and piperaquine, while candidate antimalarials included the imidazolopiperazine GNF179, a close analogue of KAF156, and the Plasmodium phosphatidylinositol-4-OH kinase (PI4K)-specific inhibitor KDU691. RESULTS: We report a high frequency (3%-15%) of P. malariae infections with a significant reduction in ex vivo susceptibility to chloroquine, lumefantrine and artemether, which are the current frontline drugs against P. malariae infections. Unlike these compounds, potent inhibition of P. malariae and P. falciparum was observed with piperaquine exposure. Furthermore, we evaluated advanced lead antimalarial compounds. In this regard, we identified strong inhibition of P. malariae using GNF179, a close analogue of KAF156 imidazolopiperazines, which is a novel class of antimalarial drug currently in clinical Phase IIb testing. Finally, in addition to GNF179, we demonstrated that the Plasmodium PI4K-specific inhibitor KDU691 is highly inhibitory against P. malariae and P. falciparum. CONCLUSIONS: Our data indicated that chloroquine, lumefantrine and artemether may not be suitable for the treatment of P. malariae infections and the potential of piperaquine, as well as new antimalarials imidazolopiperazines and PI4K-specific inhibitor, for P. malariae cure.

Uptake of Plasmodium falciparum Gametocytes During Mosquito Bloodmeal by Direct and Membrane Feeding.

Plasmodium falciparum remains one of the leading causes of child mortality, and nearly half of the world's population is at risk of contracting malaria. While pathogenesis results from replication of asexual forms in human red blood cells, it is the sexually differentiated forms, gametocytes, which are responsible for the spread of the disease. For transmission to succeed, both mature male and female gametocytes must be taken up by a female Anopheles mosquito during its blood meal for subsequent differentiation into gametes and mating inside the mosquito gut. Observed circulating numbers of gametocytes in the human host are often surprisingly low. A pre-fertilization behavior, such as skin sequestration, has been hypothesized to explain the efficiency of human-to-mosquito transmission but has not been sufficiently tested due to a lack of appropriate tools. In this study, we describe the optimization of a qPCR tool that enables the relative quantification of gametocytes within very small input samples. Such a tool allows for the quantification of gametocytes in different compartments of the host and the vector that could potentially unravel mechanisms that enable highly efficient malaria transmission. We demonstrate the use of our gametocyte quantification method in mosquito blood meals from both direct skin feeding on Plasmodium gametocyte carriers and standard membrane feeding assay. Relative gametocyte abundance was not different between mosquitoes fed through a membrane or directly on the skin suggesting that there is no systematic enrichment of gametocytes picked up in the skin.

Effect of artemether-lumefantrine (Coartem) on cytomegalovirus urine viral load during and following treatment for malaria in children.

BACKGROUND: Artemisinins, commonly used to treat malaria, have shown activity against cytomegalovirus (CMV) in vitro, in an animal model, and in case reports; however, the in vivo anti-CMV activity has not been well investigated. OBJECTIVES: To evaluate whether artemisinins affect CMV shedding among subjects co-infected with CMV and malaria. STUDY DESIGN: A prospective observational study of children in Mali (6 month-10 year) presenting with fever. Urine samples were collected at day 0, 3, and 14 from children treated with artemether-lumefantrine (Coartem(®)) for malaria and those who had other illnesses not treated with Coartem. CMV DNA was quantified using a real-time PCR. Resulting urine viral loads were compared between the groups at three time points. RESULTS: 164 malaria cases and 143 non-malaria comparisons were enrolled. Eighty-one (49%) cases and 88 (62%) comparisons shed CMV at day 0. Day 0 and day 3 viral loads were similar, but at day 14 the median viral load of cases was lower than that of comparisons (360 vs 720 copies/mL or 2.56 vs 2.86 log10), p=0.059. A stratified analysis of day 0 high viral shedders (defined as >1000 copies/mL) showed significantly lower median viral load at day 14 among cases (490 copies/mL, 2.69 log10) vs comparisons (1200 copies/mL, 3.08 log10), p=0.045. CONCLUSION: A high rate of urinary CMV shedding was found in a malaria-endemic area. Among high virus shedders artemether-lumefantrine decreased urine viral load, but the effect was not observed when analysis of both high and low shedders was performed. These results support additional studies of artemisinin dosing and duration in CMV infection.