Genetic diversity and genetic relatedness in P. falciparum parasite population in individuals with uncomplicated malaria based on Microsatellite typing in Eastern and Western regions of Uganda, 2019-2020.
Authors: Bosco Agaba B. , Anderson Karen , Gresty Karryn , Prosser Christiane , Smith David , Nankabirwa Joaniter , Nsobya Sam , Yeka Adoke , Namubiru Rhoda , Arinaitwe Emmanuel , Mbaka Paul , Kissa John , Lim Chae Seung , Karamagi Charles , Kalyango Joan N. , Kamya Moses R. , Cheng Qin 
Published: March 2021

Abstract
Background: Genetic diversity and parasite relatedness are essential parameters for assessing impact of interventions and understanding transmission dynamics of malaria parasites however data on its status in P. falciparum populations in Uganda is limited. We used microsatellite markers and DNA sequencing to determine diversity and molecular characterization of P. falciparum parasite populations in Uganda. Methods: A total of 147 P. falciparum genomic DNA samples collected from cross-sectional surveys in symptomatic individuals 2-10 years were characterized by genotyping of seven highly polymorphic neutral microsatellite markers (n=85) and genetic sequencing of the Histidine Rich Protein 2 (pfhrp2) gene (n=62). ArcGIS was used to map the geographical distribution of isolates while statistical testing was done using Student's t-test or Wilcoxon's rank-sum test and Fisher’s exact test as appropriate at P ≤ 0.05. Results: Overall, 75.5% (95% CI: 61.1 - 85.8) and 24.5 % (95% CI:14.2 - 38.9) of parasites examined were of multiclonal (mixed genotype) and single clone infections respectively. Multiclonal infections occurred more frequently in the Eastern region 73.7% (95% CI: 48.8 - 89.1), P<0.05. Overall, multiplicity of infection (MOI) was 1.9 (95% CI: 1.7 - 2.1), P=0.01 that was similar between age groups (1.8 vs 1.9), P=0.60 and regions (1.9 vs 1.8), P=0.43 for the <5 and ≥5 years and Eastern and Western regions respectively. Genomic sequencing of the pfhrp2 exon2 revealed a high level of genetic diversity reflected in 96.8% (60/62) unique sequence types. Repeat type AHHAAAHHATD and HRP2 sequence Type C were more frequent in RDT-/PCR+ samples (1.9% vs 1.5%) and (13% vs 8%), P<0.05 respectively. Genetic relatedness analysis revealed small clusters of gene deleted parasites in Uganda, but no clustering with Eritrean parasites. Conclusion: We observed a high level of genetic diversity of P. falciparum parasites reflected in the frequency of multiclonal infections, multiplicity of infection and variability of the pfhrp2 gene in these samples. These findings are consistent with the high malaria transmission intensity and endemicity in these settings despite the scaling up of malaria interventions. Findings highlight the need for selection of appropriate molecular tools for detection of drug resistance and pfhrp2 gene deletions in multiclonal infections. Genetic analysis suggested spontaneous emergence and clonal expansion of pfhrp2 deleted parasites that require close monitoring to inform national malaria diagnosis and case management policies. We recommend future molecular epidemiological surveys on parasite genomics that are more representative with wider coverage.