Introduction. Malaria is currently one of the most prevalent parasite-transmitted diseases caused by parasites of the genus Plasmodium. Misidentification of human malaria parasites especially P. knowlesi based on microscopic examination is very common. The objectives of this paper were to accurately identify the incidence of human malaria parasites in the interior division of Sabah, Malaysian Borneo, based on small subunit ribosomal RNA (ssrRNA) and to determine the misidentification rate in human malaria parasites. Methods. Nested PCR was used to detect the presence of human malaria parasites. A total of 243 blood spot samples from patients who had requested for blood film for malaria parasite (BFMP) analyses were used in this study. Results. Nested PCR findings showed that there was no P. malariae infection while the highest prevalent malaria parasite was P. knowlesi, followed by P. vivax, P. falciparum, and mixed infection. Only 69.5% of the 243 samples giving consistent nested PCR and microscopic results. Conclusion. The preliminary findings from molecular detection of malaria showed that P. knowlesi was the most prevalent Plasmodium species in the interior division of Sabah. The findings from this paper may provide a clearer picture on the actual transmission of different Plasmodium species in this region.

BackgroundThe sulphadoxine/pyrimethamine (SDX/PYR) combination had been chosen to treat uncomplicated falciparum malaria in Malaysia for more than 30 years. Non-silent mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are responsible for the resistance to pyrimethamine and sulphadoxine, respectively. This study reports the mutational analysis of pfdhfr and pfdhps in single Plasmodium falciparum infection isolates from the interior division of Sabah, Malaysian Borneo.MethodsA total of 22 P. falciparum single infection isolates collected from two districts of the interior division of Sabah from February to November 2010 were recruited for the mutational study of pfdhfr and pfdhps. Both genes were amplified by nested PCR prior to DNA sequencing and mutational analysis.ResultsA total of three pfdhfr and four pfdhps alleles were identified. The most prevalent pfdhfr allele is ANRNL (86%) involving triple mutation at position 108(S to N), 59(C to R) and 164(I to L). In pfdhps, two novel alleles, SGTGA (73%) and AAKAA (5%) were identified. Alleles involving triple mutation in both pfdhfr (ANRNL) and pfdhps (SGTGA), which were absent in Sabah in a study conducted about 15 years ago, are now prevalent.ConclusionsHigh prevalence of mutations in SDX/PYR associated drug resistance genes are reported in this study. This mutational study of pfdhps and pfdhfr indicating that SDX/PYR should be discontinued in this region.

Plasmodium knowlesi had been recognized as the fifth human malaria parasite due to its wide distribution of naturally acquired infection among the human populations in many parts of Southeast Asia. The aim of this molecular epidemiological study was to determine the incidence of human malaria parasites with special attention to P.knowlesi in four districts in the interior division of Sabah, Malaysian Borneo. Nested-PCR assays were used to identify the presence of Plasmodium species in the DNA extracted from 243 dried blood spots collected for six months in year 2010 from patients suspected to have malaria. A total of 107 (44%) of the samples were positive for Plasmodium sp. based on genus-specific nested-PCR detection. Among these Plasmodium positive samples, there were 63 (58.9%) single P.knowlesi infection and two cases of mixed-species infection with P.knowlesi (1 P.knowlesi/P.falciparum and 1 P.knowlesi/P.vivax).DNA sequencing of 20 randomly selected P.knowlesi isolates from this study showed that these isolates were similar with the sexual type of naturally acquired P.knowlesi in human. P.knowlesi was the most prevalent Plasmodium sp. in this region and it had been consistently found in all the four study sites with the highest incidence in the Tenom district which accounted for 53.8% (35 cases) of all P.knowlesi cases reported in this region. This preliminary study provides a clearer picture on the actual transmission of the fifth human malaria parasites in the interior division of Sabah. The high incidence and widespread of potentially fatal P.knowlesi infection in this region should pose an important concern where proper treatment and management is needed timely.Index Terms-Malaysian Borneo, Molecular epidemiology, Malaria, Plasmodium knowlesi.

The apical membrane antigen-1 (AMA-1) of Plasmodium spp. is a merozoite surface antigen that is essential for the recognition and invasion of erythrocytes. Polymorphisms occurring in this surface antigen will cause major obstacles in developing effective malaria vaccines based on AMA-1. The objective of this study was to characterize ama1 gene in Plasmodium knowlesi isolates from Sabah. DNA was extracted from blood samples collected from Keningau, Kota Kinabalu and Kudat. The Pkama1 gene was amplified using nested PCR and subjected to bidirectional sequencing. Analysis of DNA sequence revealed that most of the nucleotide polymorphisms were synonymous and concentrated in domain I of PkAMA-1. Forteen haplotypes were identified based on amino acid variations and haplotype K5 was the most common haplotype. d/d ratios implied that purifying selection was prevalent in Pkama1 gene. Fu and Li's D and F values further provided evidence of negative selection acting on domain II of Pkama1. Lownucleotide diversitywas also detected for the Pkama1 sequences,which is similar to reports on Pkama1 from Peninsular Malaysia and Sarawak. The presence of purifying selection and low nucleotide diversity indicated that domain II of Pkama1 can be used as a target for vaccine development.

Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979.

Background Understanding the genetic diversity of candidate genes for malaria vaccines such as circumsporozoite protein (csp) may enhance the development of vaccines for treating Plasmodium knowlesi. Hence, the aim of this study is to investigate the genetic diversity of non-repeat regions of csp in P. knowlesi from Malaysian Borneo and Peninsular Malaysia. Methods A total of 46 csp genes were subjected to polymerase chain reaction amplification. The genes were obtained from P. knowlesi isolates collected from different divisions of Sabah, Malaysian Borneo, and Peninsular Malaysia. The targeted gene fragments were cloned into a commercial vector and sequenced, and a phylogenetic tree was constructed while incorporating 168 csp sequences retrieved from the GenBank database. The genetic diversity and natural evolution of the csp sequences were analysed using MEGA6 and DnaSP ver. 5.10.01. A genealogical network of the csp haplotypes was generated using NETWORK ver. 4.6.1.3. Results The phylogenetic analysis revealed indistinguishable clusters of P. knowlesi isolates across different geographic regions, including Malaysian Borneo and Peninsular Malaysia. Nucleotide analysis showed that the csp non-repeat regions of zoonotic P. knowlesi isolates obtained in this study underwent purifying selection with population expansion, which was supported by extensive haplotype sharing observed between humans and macaques. Novel variations were observed in the C-terminal non-repeat region of csp. Conclusions The csp non-repeat regions are relatively conserved and there is no distinct cluster of P. knowlesi isolates from Malaysian Borneo and Peninsular Malaysia. Distinctive variation data obtained in the C-terminal non-repeat region of csp could be beneficial for the design and development of vaccines to treat P. knowlesi.

No abstract

Background: Understanding the genetic diversity of candidate genes for malaria vaccines such as circumsporozoite protein (csp) may enhance the development of vaccines for treating Plasmodium knowlesi. Hence, the aim of this study is to investigate the genetic diversity of non-repeat regions of csp in P. knowlesi from Malaysian Borneo and Peninsular Malaysia. Methods: A total of 46 csp genes were subjected to polymerase chain reaction amplification. The genes were obtained from P. knowlesi isolates collected from different divisions of Sabah, Malaysian Borneo, and Peninsular Malaysia. The targeted gene fragments were cloned into a commercial vector and sequenced, and a phylogenetic tree was constructed while incorporating 168 csp sequences retrieved from the GenBank database. The genetic diversity and natural evolution of the csp sequences were analysed using MEGA6 and DnaSP ver. 5.10.01. A genealogical network of the csp haplotypes was generated using NETWORK ver. 4.6.1.3. Results: The phylogenetic analysis revealed indistinguishable clusters of P. knowlesi isolates across different geographic regions, including Malaysian Borneo and Peninsular Malaysia. Nucleotide analysis showed that the csp non-repeat regions of zoonotic P. knowlesi isolates obtained in this study underwent purifying selection with population expansion, which was supported by extensive haplotype sharing observed between humans and macaques. Novel variations were observed in the C-terminal non-repeat region of csp. Conclusions: The csp non-repeat regions are relatively conserved and there is no distinct cluster of P. knowlesi isolates from Malaysian Borneo and Peninsular Malaysia. Distinctive variation data obtained in the C-terminal non-repeat region of csp could be beneficial for the design and development of vaccines to treat P. knowlesi.