Molecular studies of clonality, transmission and severe disease in malaria

To distinguish among malaria parasites producing human infections, PCR-based amplification of the polymorphic Block 2 region of merozoite surface protein-1 (MSP-1) was used to estimate the incidence and clearance of Plasmodium falciparum infection in areas of Mali with seasonal transmission. Molecular estimates were compared prospectively with slide-based estimates in a cohort of 80 children from 6 months to 9 years of age. The molecular method detects 1.45 to 6 fold more incidence and clearance events than microscopy. However, both methods demonstrate similar variation with peaks at the end of the rainy season and troughs at the end of the dry season. The principal molecular marker used in this study allowed us to detect novel hybrid (MAD20/RO33) sequences in Block 2 of MSP-1 from recombination across allotypes between MAD20 parasites (containing 9 nucleotide repeats) and RO33 parasites (with no repeats). Feeding experiments with F1 Anopheles gambiae demonstrated that formation of hybrid parasites occurs during the meiotic reduction division in anopheline mosquito vector, which converts the diploid zygote to an oocyst midgut. The clinical significance of parasites with hybrid sequences is that they are associated with severe disease. Parasites with hybrid (MAD20/RO33) sequences in Block 2 of MSP-1 were found in 58% (14 of 24) children with cerebral malaria, versus 8% (6 of 73) children with asymptomatic infections, χ2 = 20.86, p < 0.001.