Enhanced Ex Vivo Plasmodium vivax Intraerythrocytic Enrichment and Maturation for Rapid and Sensitive Parasite Growth Assays

Abstract

chloroquine resistance has been documented in nearly every region where this malaria-causing parasite is endemic. Unfortunately, resistance surveillance and drug discovery are challenging due to the low parasitemias of patient isolates and poor parasite survival through maturation that reduce the sensitivity and scalability of current antimalarial assays. Using cryopreserved patient isolates from Brazil and fresh patient isolates from India, we established a robust enrichment method for parasites. We next performed a medium screen for formulations that enhance survival. Finally, we optimized an isotopic metabolic labeling assay for measuring maturation and its sensitivity to antimalarials. A KCl Percoll density gradient enrichment method increased parasitemias from small-volume isolates by an average of>40-fold. The use of Iscove's modified Dulbecco's medium for culture approximately doubled the parasite survival through maturation. Coupling these with [H]hypoxanthine metabolic labeling permitted sensitive and robust measurements of parasite maturation, which was used to measure the sensitivities of Brazilian isolates to chloroquine and several novel antimalarials. These techniques can be applied to rapidly and robustly assess the isolate sensitivities to antimalarials for resistance surveillance and drug discovery.

Amplification of Duffy binding protein-encoding gene allows Plasmodium vivax to evade host anti-DBP humoral immunity

“…Cryopreserved infected RBCs were thawed and cultured in IMDM medium (Gibco) supplemented with 0.5% Albumax II (Gibco), 2.5% heat-inactivated human serum, 25 mM HEPES (Gibco), 20 μg ml −1 gentamicin (Sigma) and 0.2 mM hypoxanthine (C-C Pro) for~24 or~48 h until a majority of schizont stage parasites were observed. The schizont-infected erythrocytes were enriched using KCl-Percoll density gradient 38 , then mixed at a ratio of 1 erythrocyte to 1 reticulocyte enriched from cord blood and labeled with CellTrace Far Red dye following manufacturer's instructions. The cultures were incubated for~8 h in a final volume of 50 μL in 96 well plates or 20 μL in 384 well plates, in presence of the human monoclonal antibodies.…”

Monitoring the Efficacy of Chloroquine-Primaquine Therapy for Uncomplicated Plasmodium vivax Malaria in the Main Transmission Hot Spot of Brazil

“…increasing drug concentrations, but the use of these assays has been limited until recently by the lack of practical and standardized protocols (4,5). Moreover, results may be affected by previous or concomitant antimalarial use, patterns of parasite synchronicity in clinical samples, and time delays in processing field-collected samples (6).…”

Plasmodium vivax infection compromises reticulocyte stability.

“…Having established that the youngest CD71 + reticulocytes that are preferred by P. vivax for invasion 13,14 are the most osmotically stable of all enucleated RBCs, we next assessed the impact of P. vivax infection on reticulocyte osmotic stability. In the absence of continuous P. vivax in vitro culture, cryopreserved clinical P. vivax samples are an invaluable resource for investigating P. vivax biology [40][41][42][43] . Cognizant of the decreased stability of cryopreserved RBCs, however ( Fig.…”

“…Indian and Brazilian clinical P. vivax samples and P. falciparum 3D7 P2G12 were cultured at 100 × 10 6 cells per mL in IMDM (Gibco) containing 10% AB + heat-inactivated sera and 50 µg/mL gentamicin at 37 o C in 5% CO 2, 1% O 2 , and 94% N 2 as previously described 40 . Hemacolor-stained cytospins were prepared and 200 cells per slide (1000x magni cation) were called as either asexual stage (I-V) or gametocyte 60 .…”