Genetic diversity and genetic relatedness in Plasmodium falciparum parasite population in individuals with uncomplicated malaria based on microsatellite typing in Eastern and Western regions of Uganda, 2019-2020

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 Plasmodium falciparum populations in Uganda is limited. Microsatellite markers and DNA sequencing were used 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 of 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% CI14.2-38.9) 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: 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 observed in this study is consistent with the high malaria transmission intensity in these settings. Parasite 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.

Keywords: Genetic diversity; Malaria; Microsatellite markers; Multiclonal infections; Multiplicity of infection; Parasite relatedness; Pfhrp2 deletion; Rapid diagnostic tests.

Fig. 1

Geographical distribution of DNA samples used for genotyping. The red and green dots indicate the location where the sequenced and microsatellite typed samples were collected respectively. Black lines indicate district boundaries

Fig. 2

Genetic relatedness of P. falciparum populations. a and b Legend: Genetic relatedness of P. falciparum populations from Uganda differing in pfhrp2/3 genetic gene status (a) and comparison of parasite populations from Uganda and Eritrea (b). The relatedness among parasites was constructed using seven neutral microsatellite markers/loci indicated in Additional file 1: Table S1. Plots were produced using Phyloviz software at cutoff value of 3 minimum differences for 4 loci). Numbered circles indicate specific haplotypes. Circle sizes are proportional to the number of samples with a particular haplotype. The number of samples are indicated inside each circle. Pfhrp2/pfhrp3 status is indicated as: Positive/Positive (wild type), Negative/Negative (dual pfhrp2/3 deletion), Positive/Negative (pfhrp3 deletion) and Negative/Positive (Pfhrp2 deletion)

Fig. 3

Sequence length of pfhrp2 exon2. LM means light microscopy

Fig. 4

Phylogeny bootstrap consensus neighbour-join tree. D = discordant samples (RDT−/LM +), C = concordant samples (RDT+/LM +)

Fig. 5

GIS mapping for distribution of sequences across sites. The different colored dots represent the different HRP2 sequence types