Comparative studies of serum-free media and detection techniques for <i>in vitro</i> drug sensitivity assessment of <i>Plasmodium falciparum</i>

Abstract

Malaria continues to be a devastating disease. In a previous study, we formulated a chemically defined culture medium that is able to sustain the complete intraerythrocytic growth of Plasmodium falciparum. We tested the feasibility of using the medium (CDRPMI) as well as human serum-free media enriched with commercially available human-serum substitutes (GFSRPMI and ALBRPMI) to assess the drug sensitivity of P. falciparum, using chloroquine diphosphate (CQ) and dihydroartemisinin (DHART) as conventional antimalarial drugs. Growth inhibition was measured by four different methods: flow cytometry with SYBR Green I (FCM), microscopy (Giemsa method), enzymatic estimation of parasite lactate dehydrogenase (pLDH), and histidine-rich protein 2 (HRPII) determination. In drug sensitivity tests on asynchronous parasites cultured for 96 h in the presence of drugs, the dose-response curves were similar and differences in the 50% growth inhibition concentrations for the drugs, which were estimated by the four methods, were not statistically significant for the three culture media. The effect of the drugs on the growth of synchronous parasites at the ring stage was also assessed in micro-volume tests by three different methods of FCM: tracking fluorescent erythrocytes, schizont test, and merozoite test. Dose-response curves for the drugs were similar, and differences in the 50% growth inhibition concentrations were not statistically significant for CDRPMI and GFSRPMI.Thus CDRPMI as well as GFSRPMI and ALBRPMI can be similarly useful media for drug sensitivity testing of P. falciparum. The FCM, pLDH and HRPII estimations were fast and reliable detection methods, with FCM allowing schizont and merozoite tests to be performed with shorter periods of culture.

Beninese Plant Extracts with Antiplasmodial Activity Select New Allele Variants Msp1 and Msp2 in Plasmodium falciparum

“…Briefly, parasites were cultured at 4% hematocrit (O + red blood cells (RBCs)) in complete medium (RPMI-1640 containing 2 mM L-glutamine, 25 mM HEPES, 0.85 g/L sodium bicarbonate supplemented with 50 mg/L hypoxanthine (Gibco), and 0.25% ( w / v ) Albumax II (Gibco) as human AB + serum substitute [ 24 ] without gentamycin). Cell culture flasks (T-75 cm 2 Nunclon™, Denmark) were provided with 3% O 2 , 5% CO 2 , and 92% N 2 (AIR LIQUIDE France Industrie) and incubated in a CO 2 incubator settled at 37°C.…”

A new Plasmodium falciparum field isolate from Benin in continuous culture: Infectivity of fresh-produced and cryopreserved gametocytes to Anopheles gambiae

“…In vitro culturing of both NF54 and the eld isolates strains was implemented following standard protocols with minor modi cations 52,53 . Brie y, after centrifugation of 3 4 mL of blood, at 800 g for 5 minutes, plasma was removed and the pellets washed and then adjusted to 5% haematocrit with RPMI-1640 (with 2 mM L-glutamine, 25 mM HEPES, 0.85 g/L Sodium bicarbonate) supplemented with 50 mg/L Hypoxanthine (Gibco), 0.25% (w/v) Albumax II (Gibco) as human AB + serum substitute [54][55][56][57] without gentamycin. Cell culture asks (T-75 cm 2 Nunclon TM , Denmark) were used under a reduced oxygen environment of 3% O 2 , 5% CO 2 and 92% N 2 (AIR LIQUID France Industrie) and incubated at 37°C.…”