Introduction: Malaria is still a major public health problem in Côte d'Ivoire. Both treatment and control there are hampered by the spread of resistance to common antimalarial drugs, especially in the south where multidrug-resistant malaria is highly prevalent. Recent treatment guidelines require in vitro tests and the adaptation of drug policies according to local resistance rates. In addition to performing clinical assays in the field, we sought to establish a national map of drug resistance by using in vitro tests with clinical surveys. These make it possible to detect changes in susceptibility and are expected to prevent the emergence of resistance against the most recently introduced combined therapy.
Materials and methods: Isolates of Plasmodium falciparum. Isolates of P. falciparum were collected from symptomatic adults and paediatric patients seen at Anonkoua-Kouté Hospital or at the Pasteur Institute of Côte d'Ivoire. Venous blood samples were collected in heparinized vacutainer tubes (Becton Dickinson, Rutherford, NJ, USA). Giemsa-stained thin and thick blood smears were examined for infection by P. falciparum and parasite density was determined. Only blood samples with a parasite density>4,000 parasites/microL of blood were used. When parasite density exceeded 10,000 parasites/microL, freshly washed uninfected red blood cells were added to adjust parasite density to this level. All drug susceptibility assays were performed within 48 h after blood samples were taken.
Drugs: Stock solutions of chloroquine, quinine and artesunate were prepared in methanol. The final concentration of methanol did not exceed 0.05%. The concentrations of the solutions tested ranged from 12.5 to 1,600 nM for chloroquine, 25 to 2 400 nM for quinine and 0.12 to 100 nM for artesunate. In vitro assays The in vitro drug sensitivity of the Ivorian isolates was assessed with a standard 48-h isotope test. Briefly, fresh blood samples were washed three times with RPMI 1640 medium (GibcoTM, Invitrogen Corporation, France) and centrifuged (1,500xg, 5 minutes). The parasites were then tested directly without culture adaptation. If parasitemia > 0.5%, fresh uninfected erythrocytes were added to adjust it to 0.3%. The infected erythrocytes (1.5% hematocrit, 0.1-1% parasitemia) were suspended in complete RPMI medium supplemented with 10% decomplemented human AB+ serum (Biomedia, France) and buffered with 25 mM/L HEPES and 25 mM/L NaHCO3. The mixture was distributed (200 microL per well) into 96-well test plates pre-coated with antimalarial agents. Each plate included two drug-free control wells and one control well without parasites. The culture plates were incubated for 48 h at 37 degrees C in a 5% CO2 atmosphere. [3H]Hypoxanthine (0.5 mCi/well; Amersham Biosciences, France) was used to assess parasite growth. Each isolate was tested once in duplicate in the microplates with serial drug dilutions. Drug response was quantified by monitoring [3H] hypoxanthine uptake in a Wallac MicroBeta Trilux counter (Perkin-Elmer, France).
Data analysis: The IC50 values (defined as the drug concentration that resulted in a level of 3H-hypoxanthine uptake 50% lower than that measured in the drug-free control wells) were determined by nonlinear regression analysis of the plot of the concentration logarithm against growth inhibition. Data were adapted to fit the log-probit model (Excel, Microsoft; Redmond, WA, USA). The threshold IC50 values for in vitro resistance to chloroquine, quinine and artésunate have previously been estimated to be >100 nM, >800 nM and >19.81 nM respectively.
Results: In all 23, 21 and 19 P. falciparum isolates grew satisfactorily in quinine, artésunate and chloroquine, respectively, and yielded interpretable results for these drugs. The geometric mean IC50 for quinine was 272.12 nM with values ranging from 2.08 to 660.28. For artésunate, the IC50 values ranged from 0.03 to 43.84 nM and the geometric mean was 7.49 nM. The IC50 values for chloroquine ranged from 17.71 to 359.19 nM, with a geometric mean for the 23 isolates of 93.72 nM. The proportions of resistant isolates were 26.10% for chloroquine (IC50>100 nM), 9.5% for artesunate (IC50>9.66 nM) and 0% for quinine. No multidrug-resistant isolates (resistant to more than three drugs) were found.
Conclusion: The decreased susceptibility to artesunate of isolates collected in Abidjan justifies an improved surveillance program for drug resistance to malaria in Côte d'Ivoire.