Genetic Diversity of Plasmodium falciparum Populations in Malaria Declining Areas of Sabah, East Malaysia

Abstract

Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751-800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651-700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0.532). The genetic data from the present study highlighted the limited diversity and contrasting genetic pattern of P. falciparum populations in the malaria declining areas of Sabah.

Fig 1. Malaria study areas in Sabah, Malaysia.

In the present study, samples were collected from individuals living in two Sabah divisions from Kalabakan district in the Tawau Division and Kota Marudu district in the Kudat Division. This map was generated using SimpleMappr online software [18].

Fig 2. The allele frequencies of 10 microsatellite loci in the Kalabakan and Kota Marudu P. falciparum isolates.

Allele sizes, determined by fragment analysis, are shown on the x-axis and their frequencies on the y-axis. Vertical bars for allele frequencies of each microsatellite locus were determined using GENALEX 6.0. Only 1 allele (96 bp) was detected on PFG377 locus for both Kalabakan and Kota Marudu P. falciparum isolates (S1 Table).

Fig 3. Dendogram showing the clustering of 43 P. falciparum isolates from Kalabakan (KB) and Kota Marudu (KM).

The genetic relatedness between microsatellite haplotypes was examined by generating a similarity matrix based on number of tri-nucleotide repeats of each allele from 10 microsatellite loci to construct an unweighted pair group method with arithmetic mean (UPGMA) dendogram. The color coded haplotypes show the distribution of different clonal P. falciparum isolates within each cluster. The major haplotypes (A, B and C) persisted in every year of sampling (2008 and 2009 for Kalabakan; 2011 and 2012 for Kota Marudu). ND, the haplotype was not determined due to missing genotypes or more than 2 alleles of any microsatellite locus.