METHODS: Thirty-six full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) obtained from clinical isolates of Malaysia, which were orthologous to pvmsp7H (PVX_082680) were downloaded from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection.
RESULTS: Analysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in Plasmodium vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5' and 3' regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5' and 3' regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population.
CONCLUSIONS: High genetic diversity with hyper-variable SNPs and strong evidence of positive natural selection at the central region of MSP7D indicated exposure of the region to host immune pressure. Negative selection at the 5' and the 3' regions of MSP7D might be because of functional constraints at the unexposed regions during the merozoite invasion process of P. knowlesi. No evidence of geographical clustering among the clinical isolates from Malaysia indicated uniform selection pressure in all populations. These findings highlight the further evaluation of the regions and functional characterization of the protein as a potential blood stage vaccine candidate for P. knowlesi.
METHODS: Eleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5.
RESULTS: Sequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain.
CONCLUSIONS: This study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries.
METHODS: A total of 70 blood samples were collected from Macaca fascicularis dwelling in the forest of Hulu Selangor by the Department of Wildlife and National Parks Peninsular Malaysia, Kuala Lumpur, Malaysia. DNA was extracted using PureLink™ Genomic DNA Kits. Conventional and nested PCR were used to detect the genus and species of Plasmodium parasites respectively. In addition, phylogenetic analysis was carried out to confirm the species of Plasmodium parasites.
RESULTS: Thirty-five (50 %) of the 70 samples were positive for Plasmodium using genus-specific primers. These positive samples were then subjected to nested PCR targeting the 18S ribosomal RNA genes to detect all five simian malaria parasites: namely, P. knowlesi, Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi, and Plasmodium coatneyi. All five species of simian malaria parasites were detected. Of these, P. inui was the predominant (65.7 %), followed by P. knowlesi (60 %), P. cynomolgi (51.4 %) P. coatneyi (45.7 %) and P. fieldi (2.9 %). A total of nine macaques had mono-infection with P. knowlesi (four), P. cynomolgi (two), P. coatneyi (two) and P. fieldi (one). Eleven of the macaques had dual infections while 12 had triple infections. Three macaques were infected with four species of Plasmodium. Molecular and phylogenetic analysis confirmed the five species of Plasmodium parasites.
CONCLUSION: This study has provided evidence to elucidate the presence of transmission of malaria parasites among the local macaques in Hulu Selangor. Since malaria is a zoonosis, it is important to determine the new control strategies for the control of malaria.
METHODS: Mosquitoes found landing on humans and resting on leaves over a 5-day period at two sites in the Lawas District of northern Sarawak were collected and identified. DNA samples extracted from salivary glands of Anopheles mosquitoes were subjected to nested PCR malaria-detection assays. The small subunit ribosomal RNA (SSU rRNA) gene of Plasmodium was sequenced, and the internal transcribed spacer 2 (ITS2) and mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the mosquitoes were sequenced from the Plasmodium-positive samples for phylogenetic analysis.
RESULTS: Totals of 65 anophelines and 127 culicines were collected. By PCR, 6 An. balabacensis and 5 An. donaldi were found to have single P. knowlesi infections while 3 other An. balabacensis had either single, double or triple infections with P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Phylogenetic analysis of the Plasmodium SSU rRNA gene confirmed 3 An. donaldi and 3 An. balabacensis with single P. knowlesi infections, while 3 other An. balabacensis had two or more Plasmodium species of P. inui, P. knowlesi, P. cynomolgi and some species of Plasmodium that could not be conclusively identified. Phylogenies inferred from the ITS2 and/or cox1 sequences of An. balabacensis and An. donaldi indicate that they are genetically indistinguishable from An. balabacensis and An. donaldi, respectively, found in Sabah, Malaysian Borneo.
CONCLUSIONS: Previously An. latens was identified as the vector for P. knowlesi in Kapit, central Sarawak, Malaysian Borneo, and now An. balabacensis and An. donaldi have been incriminated as vectors for zoonotic malaria in Lawas, northern Sarawak.