Resolving the cause of recurrent Plasmodium vivax malaria probabilistically.

Relapses arising from dormant liver-stage Plasmodium vivax parasites (hypnozoites) are a major cause of vivax malaria. However, in endemic areas, a recurrent blood-stage infection following treatment can be hypnozoite-derived (relapse), a blood-stage treatment failure (recrudescence), or a newly acquired infection (reinfection). Each of these requires a different prevention strategy, but it was not previously possible to distinguish between them reliably. We show that individual vivax malaria recurrences can be characterised probabilistically by combined modelling of time-to-event and genetic data within a framework incorporating identity-by-descent. Analysis of pooled patient data on 1441 recurrent P. vivax infections in 1299 patients on the Thailand-Myanmar border observed over 1000 patient follow-up years shows that, without primaquine radical curative treatment, 3 in 4 patients relapse. In contrast, after supervised high-dose primaquine only 1 in 40 relapse. In this region of frequent relapsing P. vivax, failure rates after supervised high-dose primaquine are significantly lower (∼3%) than estimated previously.

Real time PCR detection of common CYP2D6 genetic variants and its application in a Karen population study.

BACKGROUND: Plasmodium vivax malaria is characterized by relapses arising from the hypnozoite stages in the liver. The only currently registered drug for radical treatment to prevent relapse is primaquine. Primaquine, a prodrug, requires metabolism through the liver cytochrome CYP2D6 isoenzyme to its active metabolite. Mutations in the CYP2D6 gene may thus affect primaquine efficacy. A SNPs genotyping technique was developed to characterize the CYP2D6 genetic variants and tested this in the patients with Plasmodium vivax infection collected in a Karen population on the Thailand-Myanmar border, where P. vivax malaria is endemic. METHODS: Direct sequencing of PCR-reamplified products (DSP) was used to uncover exonic CYP2D6 sequence variations. Subsequently, an allele-specific oligonucleotide probe real-time SNPs genotyping (ASO) assay was developed for rapid detection of the four clinically relevant CYP2D6 variants occurring in this population. These two in-house developed assays were used to genotype CYP2D6 mutations in blood samples obtained from 70 Karen adults. RESULTS: Results showed a high degree of concordance between the DSP and ASO methods. Six CYP2D6 point mutations were identified within the Karen population: C100T, C1039T, G1661C, G1846A, C2850T and G4180C, at frequencies of 0.43, 0.43, 0.76, 0.02, 0.32 and 0.76, respectively. The CYP2D6*2, *4, *5, *10 and *36 allelic frequencies were 0.33, 0.02, 0.03, 0.40 and 0.01, respectively. Alleles conferring an intermediate CYP2D6 metabolizer phenotype comprised 46% of the total number of alleles. CONCLUSION: The newly developed ASO assay is a reliable and rapid tool for large-scale CYP2D6 genotyping. The high frequency of the CYP2D6*10 allele in the Karen population warrants further assessment of its association with the radical curative efficacy of primaquine.