Rodent Malaria Erythrocyte Preference Assessment by an Ex Vivo Tropism Assay.

Circulating red blood cells consist of young erythrocytes (early and late reticulocytes) and mature erythrocytes (normocytes). The human malaria parasites, Plasmodium falciparum and P. vivax, have a preference to invade reticulocytes during blood-stage infection. Rodent malaria parasites that also prefer reticulocytes could be useful tools to study human malaria reticulocyte invasion. However, previous tropism studies of rodent malaria are inconsistent from one another, making it difficult to compare cell preference of different parasite species and strains. In vivo measurements of cell tropism are also subjected to many confounding factors. Here we developed an ex vivo tropism assay for rodent malaria with highly purified fractions of murine reticulocytes and normocytes. We measured invasion into the different erythrocyte populations using flow cytometry and evaluated the tropism index of the parasite strains. We found that P. berghei ANKA displayed the strongest reticulocyte preference, followed by P. yoelii 17X1.1, whereas P. chabaudi AS and P. vinckei S67 showed mixed tropism. These preferences are intrinsic and were maintained at different reticulocyte and normocyte availabilities. Our study shed light on the true erythrocyte preference of the parasites and paves the way for future investigations on the receptor-ligand interactions mediating erythrocyte tropism.

Recent Molecular Assessment of Plasmodium vivax and Plasmodium falciparum Asymptomatic Infections in Botswana.

In 2016, we reported the presence of Plasmodium vivax in Botswana through active case detection. A real-time PCR was used during a similar study in 10 districts to assess changes in the P. vivax prevalence. We assessed 1,614 children (2-13 years of age) for hemoglobin (Hb; g/dL) and Plasmodium parasites. The median age of all participants was 5.0 years (25th percentile, 3 years; 75th percentile, 8 years). The median Hb (g/dL) level was 12.1, but 18.3% of the participants had anemia (Hb < 11.0 g/dL); these participants were clustered in the younger than 5 years age group in all districts (P < 0.001). The risk of anemia decreased with age 5 years or older (odds ratio [OR], 0.26; 95% confidence interval [CI], 0.197-0.34; P < 0.001). The prevalence rates of Plasmodium parasites were as follows: P. vivax, 12.7%; P. falciparum, 12.7%; P. malariae, 0.74%; and P. ovale (P. ovale curtisi), 0.68%. Mixed infection rates were as follows: P. falciparum and P. vivax, 2.35%; P. falciparum and P. ovale curtisi, 0.56%; P. vivax and P. malariae, 0.06%; and P. falciparum and P. malariae, 0.68%. The infections were largely asymptomatic (99.6%). Using logistic regression, the risk of infection with P. vivax was highest in Kweneng East (OR, 6.2; 95% CI, 2.9-13.1), followed by South East (OR, 5.6; 95% CI, 2.5-12.3) and Ngami (OR, 5.1; 95% CI, 2.2-12.0). Compared to the risk of infection for children younger than 5 years, the risk of infection decreased for children 5 years or older in regions with high rates of P. vivax and P. falciparum infections. P. vivax and P. falciparum have expanded within the asymptomatic population in Botswana; therefore, careful attention is required for their elimination.

Unambiguous determination of Plasmodium vivax reticulocyte invasion by flow cytometry.

The invasion of CD71+ reticulocytes by Plasmodium vivax is a crucial yet poorly characterised event. The application of flow cytometry to ex vivo invasion assays promises to facilitate the quantitative analysis of P. vivax reticulocyte invasion. However, current protocols suffer from a low level of sensitivity due to the absence of a particular design for P. vivax cell tropism. Importantly, merozoite invasion into contaminating red blood cells from the schizont inoculum (auto-invasion) may confound the analysis. Here we present a stable two-color flow cytometry assay for the accurate quantification of P. vivax merozoite invasion into intracellularly labelled CD71+ reticulocytes. Various enzymatic treatments, antibodies and invasion inhibitory molecules were used to successfully demonstrate the utility of this method. Fluorescent labelling of red blood cells did not affect the invasion and early intra-erythrocytic development of P. vivax. Importantly, this portable field assay allows for the economic usage of limited biological material (parasites and reticulocytes) and the intracellular labeling of the target cells reduces the need for highly purified schizont inoculums. This assay will facilitate the study of P. vivax merozoite biology and the testing of vaccine candidates against vivax malaria.