Plasmodium falciparum Population Structure in Children with Malaria in Kisumu- Western Kenya

Abstract

Plasmodium Jalciparum population structure in malaria endemic areas is characterized by extensive genotype diversity, and wide spectrum of clinical presentation; from mild, uncomplicated malaria to severe malaria and anemia. In addition individuals may harbor parasites without any clinical presentation. These observations suggest that some parasite strains are more, virulent than others. In this study, we evaluated whether severity of P. falciparum malaria can be-attributed to a single clone that outgrows others or is due to multiple clone infections and determine the prevalent clone in the study population. Blood samples (cryopreserved at -80°C) were obtained from a case control study (2006) that enrolled 60 children «5years) with severe malarial anaemia (SMA) (defined as having blood stage asexual malaria parasites and hemoglobin (Hb) :S;6g/dL). Each case of SMA was matched by age (±2 months) and gender to a control with uncomplicated malaria (UM) (defined as having blood stage asexual malaria parasites and Hb>6 g/dL). DNA was extracted and amplified in replicate PCR reactions that targeted msp-l (Kl, MAD20 and R033) and msp-2 (FC27 and IC3D7) allelic families. Allele discrimination was achieved by use of high resolution capillary electrophoresis (CE). Genotype signatures (allele size and allele number) and multiplicity of infection (MOl) between the two groups was compared. Finally, relative abundance of parasite clones was estimated from peak height and peak area. Paired parametric t-test was used to test for statistical significance. In replicate assays, the allele numbers and sizes (up to 1 nucleotide difference) were reproducible in every case illustrating excellent assay reproducibility. MOl was not statistically different (P=0.54) between the two groups (UM=2.17, N=35 and SMA=2:0, N=35). Overall, Kl was the most prevalent allele (37%) as compared to MAD20=16%, R033=16%, FC27=22%, IC3D7=10% (P<O.OOOl). For the three alleles (Kl, MAD20 and FC27) evaluated, peak height and peak area equally predicted average allele abundance but the difference between the two groups was not significant (P>0.05). This study has demonstrated that (i) the number of alleles and their sizes obtained from a high resolution CE are reproducible up to 1 nucleotide, (ii) for relative quantification of allele abundance, either peak area or peak height can be used to define relative abundance, (iii) severity of P. falciparum malaria is not attributable to dominance of individual clones. Lastly, Mal is not predictive of disease severity.