Molecular Epidemiology of Malaria in Cameroon. Xxiii. Experimental Studies on Serum Substitutes and Alternative Culture Media for in Vitro Drug Sensitivity Assays Using Clinical Isolates of Plasmodium Falciparum

Abstract

Correlation studies on the in vitro drug response of field isolates of Plasmodium falciparum and molecular markers for drug resistance are becoming important as many malaria control programs abandon monotherapies and resort to combination therapies. The standardization and optimization of the in vitro drug sensitivity assay are one of the prerequisites for validating molecular markers in the field. The present study was designed to assess and compare the growth of freshly obtained isolates for at least the first erythrocytic cycle in various culture media and determine the in vitro response to chloroquine in alternative media. Parasite growth was consistently higher in Dulbecco's modified Eagle's medium (DME)-human serum, Iscove's modified Dulbecco's medium (IMDM)-human serum, RPMI 1640 medium-goat serum, and a serum-free medium containing 1:1 (v/v) mixture of IMDM and F-12 supplemented with an ammonium sulfate fraction of adult bovine serum than in RPMI 1640 medium-human serum mixture. The level of chloroquine response determined in human serum-supplemented DME, IMDM, and RPMI 1640 media did not differ significantly (P > 0.05) from the control (RPMI 1640-human serum). This study suggests that alternative media may be used to optimize parasite growth during the critical initial phase of transition from in vivo to in vitro conditions. The capacity of these media to support long-term cultivation of P. falciparum requires further investigation.

Influence of oxygen on asexual blood cycle and susceptibility of Plasmodium falciparum to chloroquine: requirement of a standardized in vitro assay

“…Several factors influencing the results of the chemosusceptibility tests (the initial parasitaemia, the haematocrit, the time of incubation, the time point when [ 3 H]-hypoxanthine is added, the use of serum substitutes) have already been investigated by Basco [15,32]. The present data suggest the importance of the gas mixture on isotopic microtest results for chloroquine.…”

Analysis of an ordinal outcome in a multicentric randomized controlled trial: application to a 3- arm anti- malarial drug trial in Cameroon

“…In contrast, the baseline temperature was significantly associated with the outcome, showing that patients with a low temperature at inclusion were more likely to have a good treatment response. For the SP treatment, the decrease could be related to a gene mutation, responsible for a resistance towards SP [16-18]. The SP resistance was also confirmed in a recent study in South Africa [19].…”

Molecular Epidemiology of Malaria in Cameroon. XXVI. Twelve-Year In Vitro and Molecular Surveillance of Pyrimethamine Resistance and Experimental Studies to Modulate Pyrimethamine Resistance

“…Furthermore, some of the collected isolates were only used for experimental chloroquine assays. 21 Of 137 isolates, 28 did not yield an interpretable assay for pyrimethamine (chloroquine assays performed with the standard RPMI-10% human serum were successful with these 28 isolates) due to poor growth (n ‫ס‬ 22 tested with 2 batches of fetal bovine serum) or insufficient growth inhibition even at the highest drug concentration (n ‫ס‬ 6 tested with the same batch of human serum as for chloroquine).…”