Method: Thirty-five full-length pk41 sequences from clinical isolates of Malaysia along with four laboratory lines (along with H-strain) were downloaded from public databases. For comparative analysis between species, orthologous P41 genes from P. falciparum, P. vivax, P. coatneyi and P. cynomolgi were also downloaded. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. Phylogenetic relationships between Pk41 genes were determined using MEGA 5.0 software.
Results: Analysis of 39 full-length pk41 sequences along with the H-strain identified 36 SNPs (20 non-synonymous and 16 synonymous substitutions) resulting in 31 haplotypes. Nucleotide diversity across the full-length gene was low and was similar to its ortholog in P. vivax; pv41. Domain-wise amino acid analysis of the two s48/45 domains indicated low level of polymorphisms for both the domains, and the glutamic acid rich region had extensive size variations. In the central domain, upstream to the glutamate rich region, a unique two to six (K-E)n repeat region was identified within the clinical isolates. Overall, the pk41 genes were indicative of negative/purifying selection due to functional constraints. Domain-wise analysis of the s48/45 domains also indicated purifying selection. However, analysis of Tajima's D across the genes identified non-synonymous SNPs in the s48/45 domain II with high positive values indicating possible epitope binding regions. All the 6-cysteine residues within the s48/45 domains were conserved within the clinical isolates indicating functional conservation of these regions. Phylogenetic analysis of full-length pk41 genes indicated geographical clustering and identified three subpopulations of P. knowlesi; one originating in the laboratory lines and two originating from Sarawak, Malaysian Borneo.
Conclusion: This is the first study to report on the polymorphism and natural selection of pk41 genes from clinical isolates of Malaysia. The results reveal that there is low level of polymorphism in both s48/45 domains, indicating that this antigen could be a potential vaccine target. However, genetic and molecular immunology studies involving higher number of samples from various parts of Malaysia would be necessary to validate this antigen's candidacy as a vaccine target for P. knowlesi.
METHODS: Blood samples from 511 febrile patients were collected and a partial region of the 18 s ribosomal RNA (18 s rRNA) gene was amplified using nested PCR. From the 86 positive blood samples, 13 Plasmodium falciparum and 4 Plasmodium vivax were selected and underwent cloning and, subsequently, sequencing and the sequences were subjected to phylogenetic analysis using the neighbor-joining and maximum parsimony methods.
RESULTS: Malaria was detected by PCR in 86 samples (16.8%). The majority of the single infections were caused by P. falciparum (80.3%), followed by P. vivax (5.8%). Mixed infection rates of P. falciparum + P. vivax and P. falciparum + P. malariae were 11.6% and 2.3%, respectively. All P. falciparum isolates were grouped with the strain 3D7, while P. vivax isolates were grouped with the strain Salvador1. Phylogenetic trees based on 18 s rRNA placed the P. falciparum isolates into three sub-clusters and P. vivax into one cluster. Sequence alignment analysis showed 5-14.8% SNP in the partial sequences of the 18 s rRNA of P. falciparum.
CONCLUSIONS: Although P. falciparum is predominant, P. vivax, P. malariae and mixed infections are more prevalent than has been revealed by microscopy. This overlooked distribution should be considered by malaria control strategy makers. The genetic polymorphisms warrant further investigation.