Use of the atmospheric generators for capnophilic bacteria Genbag-CO2 for the evaluation of in vitro Plasmodium falciparum susceptibility to standard anti-malarial drugs

BACKGROUND: The aim of this study was to evaluate the cultivation system in which the proper atmospheric conditions for growing Plasmodium falciparum parasites were maintained in a sealed bag. The Genbag(® )system associated with the atmospheric generators for capnophilic bacteria Genbag CO2(® )was used for in vitro susceptibility test of nine standard anti-malarial drugs and compared to standard incubator conditions. METHODS: The susceptibility of 36 pre-identified parasite strains from a wide panel of countries was assessed for nine standard anti-malarial drugs (chloroquine, quinine, mefloquine, monodesethylamodiaquine, lumefantrine, dihydroartemisinin, atovaquone and pyrimethamine) by the standard 42-hour (3)H-hypoxanthine uptake inhibition method using the Genbag CO2(® )system and compared to controlled incubator conditions (5% CO(2 )and 10% O(2)). RESULTS: The counts per minute values in the control wells in incubator atmospheric conditions (5% CO(2 )and 10% O(2)) were significantly higher than those of Genbag(® )conditions (2738 cpm vs 2282 cpm, p < 0.0001). The geometric mean IC(50 )estimated under the incubator atmospheric conditions was significantly lower for atovaquone (1.2 vs 2.1 nM, p = 0.0011) and higher for the quinolines: chloroquine (127 vs 94 nM, p < 0.0001), quinine (580 vs 439 nM, p < 0.0001), monodesethylamodiaquine (41.4 vs 31.8 nM, p < 0.0001), mefloquine (57.5 vs 49.7 nM, p = 0.0011) and lumefantrine (23.8 vs 21.2 nM, p = 0.0044). There was no significant difference of IC(50 )between the 2 conditions for dihydroartemisinin, doxycycline and pyrimethamine. To reduce this difference in term of anti-malarial susceptibility, a specific cut-off was estimated for each drug under Genbag(® )conditions by regression. The cut-off was estimated at 77 nM for chloroquine (vs 100 nM in 10% O(2)), 611 nM for quinine (vs 800 nM), 30 nM for mefloquine (vs 30 nM), 61 nM for monodesethylamodiaquine (vs 80 nM) and 1729 nM for pyrimethamine (vs 2000 nM). CONCLUSIONS: The atmospheric generators for capnophilic bacteria Genbag CO2(® )is an appropriate technology that can be transferred to the field for epidemiological surveys of drug-resistant malaria. The present data suggest the importance of the gas mixture on in vitro microtest results for anti-malarial drugs and the importance of determining the microtest conditions before comparing and analysing the data from different laboratories and concluding on malaria resistance.