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 sequenc
ing (AmpliSeq) of P. falciparum msp1 (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 and 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 poly
morphism and amino acid variations.
Results Children < 5 years had higher parasitaemia (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 4.8 (± 0.78, 95% CI) for the malaria endemic Lake Victoria region,
4.4 (± 1.03, 95% CI) for the epidemic prone Kisii Highland and 3.4 (± 0.62, 95% CI) for the seasonal malaria Semi-Arid
region. MOI decreased with age: 4.5 (± 0.76, 95% CI) for children < 5 years, compared to 3.9 (± 0.70, 95% CI) for ages
5 to 14 and 2.7 (± 0.90, 95% CI) for those > 15. Females’ MOI (4.2 ± 0.66, 95% CI) was not different from males 4.0
(± 0.61, 95% CI). In all regions, the number of alleles were high in the 2014–2015 period, more so in the Lake Victoria
and the seasonal transmission arid regions.
Conclusion These findings highlight the added advantages of AmpliSeq in haplotype discrimination and the associ
ated improvement in unravelling complexity of P. falciparum population structure.
Keywords Malaria, Multiplicity of infection, P. falciparum, P. falciparum msp1, Deep sequencing, Genetic diversity
