Plasmodium falciparum population structure inferred by msp1 amplicon sequencing of parasites collected from febrile patients in Kenya

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Abstract Background Multiplicity of infection (MOI) is an important measure of Plasmodium falciparum diversity, usually derived from the highly polymorphic genes such as msp1, msp2 and glurp as well as microsatellites. Conventional methods of deriving MOI lack fine resolution needed to discriminate minor clones. This study used amplicon sequencing (AmpliSeq) of Pfmsp1 to measure spatial and temporal genetic diversity of P. falciparum. Methods 264 P. falciparum positive blood samples collected from areas of differing malaria endemicities between 2010 to 2019 were used. Pfmsp1 gene was amplified and amplicon libraries sequenced on Illumina MiSeq. Sequences were aligned against a reference sequence (NC_004330.2) and clustered to detect fragment length polymorphism and amino acid variations. Results Children <5 years had higher parasitemia (median = 23.5+5 SD, p = 0.03) than the >5-14 (= 25.3+5 SD), and those >15 (=25.1+6 SD). Of the alleles detected, 553 (54.5%) were K1, 250 (24.7%) MAD20 and 211 (20.8%) RO33 that grouped into 19 K1 allelic families (108-270 bp), 14 MAD20 (108-216 bp) and one RO33 (153 bp). AmpliSeq revealed nucleotide polymorphisms in alleles that had similar sizes, thus increasing the K1 to 104, 58 for MAD20 and 14 for RO33. By AmpliSeq, the mean MOI was 2.9 (+0.73, 95% CI) for the malaria endemic Lake Victoria region, 2.7 (+ 0.97, 95% CI) for the epidemic prone Kisii Highland and 1.4 (+ 0.19, 95% CI) for the seasonal malaria Semi-Arid region. MOI decreased with age: 2.5 (+ 0.68, 95% CI) for children <5 years, compared to 2.3 (+ 0.65, 95% CI) for ages 5 to 14 and 1.8 (+ 0.71, 95% CI) for those >15. Females had a higher MOI of 2.5 (+ 0.61, 95% CI), compared to males (1.9 (+ 0.49, 95% CI), though not statistically significant. In all regions, the number of alleles increased from the 2014-2015 period, more so in the Lake Victoria and the seasonal transmission arid regions. Conclusion Our findings highlight the added advantages of AmpliSeq in allele discrimination. MOI was influenced by age, gender and transmission settings, highlighting an additional level of complexity of P. falciparum population structure.

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