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The influence of several factors on parasite growth and 50% inhibitory concentration (IC50) for chloroquine was assessed. Most isolates stored at 4°C up to 72 hours grew when they were subsequently cultivated. However, parasite viability sharply decreased from 24 hours, and the mean chloroquine IC50 decreased significantly (P < 0.05). There was no evidence for selection of pre-culture populations due to storage alone. The time point when 3H-hypoxanthine was added (0 versus 18 hours) had no effect on the IC50 during the 42-hour incubation, but was associated with a lower IC50 when 3H-hypoxanthine was added after the initial 42-hour incubation during the 72-hour incubation. An increase in 3H-hypoxanthine incorporation and chloroquine IC50 was observed as the hematocrit was increased from 1.0% to 2.5%. For the same isolates, chloroquine IC50 values were generally similar when the initial parasitemia was between 0.1% and 0.5% but increased at higher (>0.75%) parasitemias. Based on these results, we recommend immediate cultivation after blood collection, a 42-hour incubation period with the addition of 3H-hypoxanthine at the beginning of incubation, a 1.5% hematocrit, and an initial parasitemia 0.1-0.5%. Further studies on serum substitutes, gas mixture, and comparison of isotopic and non-isotopic assays are needed to establish a standardized in vitro assay protocol.
Received July 13, 2003. Accepted for publication January 8, 2004.
Acknowledgments: I thank Sister Marie-Solange Oko, the personnel of Nlongkak Catholic missionary dispensary, and Delphine Ngo Ndombol for their aid in recruiting patients.
Financial support: This study was supported by the French Ministry of Research (Program PAL+).
Author’s address: Leonardo K. Basco, Unité de Recherche Paludologie Afro-Tropicale, Institut de Recherche pour le Développement, Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte contre les Endémies en Afrique Centrale, BP 288, Yaounde, Cameroon, Telephone: 237-2-232-232 Fax: 237-2-230-061, E-mails: lkbasco{at}yahoo.fr and Leonardo.Basco{at}ibaic.u-psud.fr.
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