CYP2C8 polymorphism frequencies among malaria patients in Zanzibar.

OBJECTIVE: The determination of the prevalence of the CYP2C8 main alleles in a typical set of malaria patients in Zanzibar, as these patients represent a typical population exposed to amodiaquine, an antimalarial mainly metabolized by CYP2C8. Also, to determine for the first time the frequencies of CYP2C8 alleles in native African populations. METHODS: Polymerase chain reaction-restriction fragment polymorphism for the identification of CYP2C8*1, CYP2C8*2, CYP2C8*3 and CYP2C8*4 on a random population of 165 unrelated malaria patients. RESULTS: The allele frequencies found were: CYP2C8*1 (wild type, 83.4%), CYP2C8*2 (13.9%), CYP2C8*3 (2.1%) and CYP2C8*4 (0.6%). In terms of genotypes, 70.4% of the patients showed the CYP2C8*1/ CYP2C8*1 genotypes, while heterozygous between the wild type and other minor alleles were seen in 26.0%. Finally, 3.6% of the patients were homozygous for slow metabolizer alleles. The frequencies observed are equivalent to those documented for African-Americans. CONCLUSIONS: CYP2C8 non-wild type alleles have a significant prevalence in the East African population studied. The consequent frequency of 3.6% of patients homozygous for slow metabolizer alleles represent a significant fraction of the population potentially in higher risk of adverse effects due to a less efficient metabolism of amodiaquine. As approximately 10(6) first-line treatments are currently performed in Zanzibar per year, this represents a non-negligible absolute number of amodiaquine exposures. This information constitutes a background for the pharmacovigilance programs presently being employed in Zanzibar.

Detection of atovaquone and Malarone resistance conferring mutations in Plasmodium falciparum cytochrome b gene (cytb).

Clinical treatment failures of the hydroxynaphthoquinone atovaquone or its combination with proguanil (Malarone) in Plasmodium falciparum malaria has been recently documented. These events have been associated to single nucleotide polymorphisms (SNPs) in the parasite cytochrome b gene (cytb). In this report we describe a set of nest PCR-RFLP methods developed for the fast detection of all known cytb mutations associated to resistance to these drugs. The methods were successfully applied for the analysis of phenol-chloroform extracted DNA samples from patients not cured by Malarone, and from an established parasite clone. Further, the protocol for the detection of the A803C mutation was applied to 164 DNA field samples extracted through crude methanol-based protocols, originated from several malaria settings. The PCR-RFLP methods here presented can be used as a valuable for the clinical detection and study of Malarone and atovaquone P. falciparum resistance.