Plasmodium falciparum Population Structure Inferred by msp1 Amplicon Sequencing of Parasites Collected from Areas of Differing Transmission in Kenya

Abstract

An important measure of Plasmodium falciparum parasite diversity is multiplicity of in-fection (MOI), usually derived from the highly polymorphic genes such as msp1, msp2, glurp and microsatellites. MOI is used to distinguish recrudescence and new infecting clones to inform malaria control interventions. Conventional methods of deriving MOI lack fine resolution needed to discriminate minor clones. The aim of the current study was to infer P. falciparum population structure by msp1 amplicon sequencing of para-sites collected from areas of differing transmissions in Kenya. A total of 264 P. falcipa-rum positive blood samples collected from patients with acute febrile illnesses were re-trieved from frozen sample repository. Samples were collected over a 10-year period from 2010 to 2019 and originated from areas of varying malaria endemicities. Pfmsp1 gene was amplified from extracted total DNA, amplicon libraries prepared and se-quenced on an Illumina MiSeq platform. Children <5 years had higher parasitaemia (mean=23.5±5 SD, p = 0.03) than those ≥5-14 (mean=25.3±5 SD), and those ≥15 (mean=25.1±6 SD). MOIs and haplotype dynamics were derived and stratified by spa-tial and temporal measures. Of the 1014 alleles detected, 553 (54.5%) were K1, 250 (24.7%) were MAD20 and 211 (20.8%) RO33, that clustered into 19 K1 allelic families (108-270 bp), 14 MAD20 (108-216 bp) and one RO33(153 bp). By amplicon sequenc-ing, the mean MOI was 4.8(±0.78, 95% CI) for the malaria endemic Lake Victoria re-gion Alupe, Kombewa and Kisumu, 4.4(±1.03, 95% CI) for the epidemic prone Kisii highland (Kisii and Nyamira) and 3.4(±0.62, 95% CI) for the seasonal malaria semi-arid regions of Marigat, Isiolo, Lodwar, Iftin and Gilgil. High levels of clonal diversity were identified throughout the different transmission settings (He=0.98). This thesis describes additional 176 distinct allelic sequences to this database highlighting the added ad-vantages that a highly sensitive tool such as AmpliSeq brings in malaria surveillance. The key findings of this study provide information that informs malaria control interventions such as msp1-based vaccine candidates.