Although genetically distinct malaria parasites have been shown to simultaneously infect an individual, the total number of unique parasites has not been systematically studied. We examined multiple clones (8-38) from individual blood samples collected from Papua New Guinean children for polymorphisms in the Plasmodium vivax Duffy binding protein (dbpII) and the merozoite surface protein 3alpha (msp3alpha). We found a median of 4 (range = 2-6) and 12 (range = 2-23) unique genotypes based on dbpII and msp3alpha, respectively, per person at one time point and at least 12-33 unique genotypes per person over a four-month period. Control polymerase chain reactions (PCRs) detected 0-31% of clones with haplotypes that arose from PCR artifacts, indicating that caution must be taken when using PCR-based analysis to examine complex infections. To reduce artifacts from clones, analysis was based on haplotypes unlikely to have been generated by PCR artifacts or had been previously identified. Plasmodium vivax infections can be highly complex in disease-endemic areas, suggesting continual genetic mixing that could have significant implications for the use of antimalarial drugs and malaria vaccines.
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