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Acta Tropica Dec 2023The 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of...
BACKGROUND
The 2022 malaria WHO reported around 4000 P. knowlesi infections in the South-East Asia region. In the same period, 72 positive cases were reported by the Department of Disease Control in Thailand, suggesting a persistent infection. Little is known about dihydrofolate reductase (pkdhfr) and dihydropteroate synthase (pkdhps), putative antimalarial resistance markers for P. knowlesi. The relevant amplification and sequencing protocol are presently unavailable. In this study, we developed a protocol for amplifying and evaluating pkdhps mutations. The haplotype pattern of pkdhfr-pkdhps in Thai isolates was analyzed, and the effects of these pkdhps mutations were predicted by using a computer program.
METHODS
Pkdhps were amplified and sequenced from 28 P. knowlesi samples collected in 2008 and 2020 from nine provinces across Thailand. Combining pkdhfr sequencing data from previous work with pkdhps data to analyze polymorphisms of pkdhfr and pkdhps haplotype. Protein modeling and molecular docking were constructed using two inhibitors, sulfadoxine and sulfamethoxazole, and further details were obtained through analyses of protein-ligand interactions by using the Genetic Optimisation for Ligand Docking program. A phylogenetic tree cluster analysis was reconstructed to compare the P. knowlesi Malaysia isolates.
RESULTS
Five nonsynonymous mutations in the pkdhps were detected outside the equivalence of the binding pocket sites to sulfadoxine and sulfamethoxazole, which are at N391S, E421G, I425R, A449S, and N517S. Based on the modeling and molecular docking analyses, the N391S and N517S mutations located close to the enzyme-binding pocket demonstrated a different docking score and protein-ligand interaction in loop 2 of the enzyme. These findings indicated that it was less likely to induce drug resistance. Of the four haplotypes of pkdhfr-pkdhps, the most common one is the R34L pkdhfr mutation and the pkdhps quadruple mutation (GRSS) at E421G, I425R, A449S, and N517S, which were observed in P. knowlesi in southern Thailand (53.57%). Based on the results of neighbor-joining analysis for pkdhfr and pkdhps, the samples isolated from eastern Thailand displayed a close relationship with Cambodia isolates, while southern Thailand isolates showed a long branch separated from the Malaysian isolates.
CONCLUSIONS
A new PCR protocol amplification and evaluation of dihydropteroate synthase mutations in Knowlesi (pkdhps) has been developed. The most prevalent pkdhfr-pkdhps haplotypes (53.57%) in southern Thailand are R34L pkdhfr mutation and pkdhps quadruple mutation. Further investigation requires additional phenotypic data from clinical isolates, transgenic lines expressing mutant alleles, or recombinant proteins.
Topics: Sulfadoxine; Pyrimethamine; Tetrahydrofolate Dehydrogenase; Dihydropteroate Synthase; Plasmodium knowlesi; Thailand; Molecular Docking Simulation; Ligands; Phylogeny; Antimalarials; Drug Resistance; Sulfamethoxazole; Plasmodium falciparum
PubMed: 37683820
DOI: 10.1016/j.actatropica.2023.107016 -
Tropical Medicine and Infectious Disease Jul 2023The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in...
The initial and vital stage in the diagnosis of malaria involves extracting DNA. The efficiency of malaria testing is restricted by the multiple steps involved in commercial DNA extraction kits. We attempted to improve an existing loop-mediated isothermal amplification (LAMP) for the detection of by using a simple DNA extraction approach, making it a feasible option for mass screening. We utilized a simple nucleic acid extraction method directly from whole blood for the detection of , taking only 5 min to complete. The extracted DNA was evaluated by two fluorescent-based LAMP and one colorimetric-based LAMP assay. The detection limit for both SYTO-LAMP and SYBR green-LAMP was 0.00001% and 0.0001% parasitemia, respectively. Meanwhile, neutral red-LAMP had a detection limit of 0.01% parasitemia. Combining this simple and inexpensive DNA extraction method, SYTO-LAMP could serve as an alternative molecular diagnosis for the detection of and other human spp.
PubMed: 37624327
DOI: 10.3390/tropicalmed8080389 -
Tropical Medicine and Infectious Disease Jul 2023The cell-traversal protein for ookinetes and sporozoites (CelTOS), expressed on the surface of ookinetes and sporozoitesin , is a promising malaria vaccine candidate....
The cell-traversal protein for ookinetes and sporozoites (CelTOS), expressed on the surface of ookinetes and sporozoitesin , is a promising malaria vaccine candidate. CelTOS is essential for parasite invasion into mosquito midgut and human hepatocytes, thereby contributing to malaria transmission and disease pathogenesis. This study explores the genetic diversity, polymorphisms, haplotypes, natural selection, phylogenetic analysis, and epitope prediction in the full-length gene in clinical samples from Sarawak, Malaysian Borneo, and long-term laboratory strains from Peninsular Malaysia and the Philippines. Our analysis revealed a high level of genetic variation in the gene, with a nucleotide diversity of π ~ 0.021, which was skewed towards the 3' end of the gene. This level of diversity is double that observed in and 20 times that observed in from worldwide clinical samples. Tests of natural selection revealed evidence for positive selection within clinical samples. Phylogenetic analysis of the amino acid sequence of revealed the presence of two distinct groups, although no geographical clustering was observed. Epitope prediction analysis identified two potential epitopes (96AQLKATA102 and 124TIKPPRIKED133) using the IEDB server and one epitope (125IKPPRIKED133) by Bcepred server on the C' terminal region of protein. Both the servers predicted a common epitope region of nine amino acid length (IKPPRIKED) peptide, which can be studied in the future as a potential candidate for vaccine development. These findings shed light on the genetic diversity, polymorphism, haplotypes, and natural selection within in clinical samples and provide insights about its future prospects as a potential candidate for malaria vaccine development.
PubMed: 37624318
DOI: 10.3390/tropicalmed8080380 -
MedRxiv : the Preprint Server For... Aug 2023is a zoonotic parasite that causes malaria in humans. The pathogen has a natural host reservoir in certain macaque species and is transmitted to humans via mosquitoes...
BACKGROUND
is a zoonotic parasite that causes malaria in humans. The pathogen has a natural host reservoir in certain macaque species and is transmitted to humans via mosquitoes of the Leucosphyrus Group. The risk of human infection varies across Southeast Asia and is dependent upon environmental factors. Understanding this geographic variation in risk is important both for enabling appropriate diagnosis and treatment of the disease and for improving the planning and evaluation of malaria elimination. However, the data available on occurrence are biased towards regions with greater surveillance and sampling effort. Predicting the spatial variation in risk of malaria requires methods that can both incorporate environmental risk factors and account for spatial bias in detection.
METHODS & RESULTS
We extend and apply an environmental niche modelling framework as implemented by a previous mapping study of transmission risk which included data up to 2015. We reviewed the literature from October 2015 through to March 2020 and identified 264 new records of , with a total of 524 occurrences included in the current study following consolidation with the 2015 study. The modelling framework used in the 2015 study was extended, with changes including the addition of new covariates to capture the effect of deforestation and urbanisation on transmission.
DISCUSSION
Our map of relative transmission suitability estimates that the risk posed by the pathogen is highest in Malaysia and Indonesia, with localised areas of high risk also predicted in the Greater Mekong Subregion, The Philippines and Northeast India. These results highlight areas of priority for surveillance and prospective sampling to address the challenge the disease poses to malaria elimination planning.
PubMed: 37609228
DOI: 10.1101/2023.08.04.23293633 -
Infection, Genetics and Evolution :... Oct 2023Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic...
Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic characterization on P. knowlesi SERA3 Ag2 (PkSERA3 Ag2) is lacking. In the present study, nucleotide diversity, natural selection, and haplotypes of PkSERA3 Ag2 in clinical samples from Peninsular Malaysia and Malaysian Borneo were investigated. A total of 50 P. knowlesi clinical samples were collected from Peninsular Malaysia and Malaysian Borneo. The PkSERA3 Ag2 gene was amplified using PCR, and subsequently cloned and sequenced. Genetic diversity, haplotype, natural selection as well as genetic structure and differentiation of PkSERA3 Ag2 were analysed. In addition, in silico analyses were performed to identify repeat motifs, B-cell epitopes, and antigenicity indices of the protein. Analysis of 114 PkSERA3 Ag2 sequences revealed high nucleotide diversity of the gene in Malaysia. A codon-based Z-test indicated that the gene underwent purifying selection. Haplotype and population structure analyses identified two distinct PkSERA3 Ag2 clusters (K = 2, ΔK = 721.14) but no clear genetic distinction between PkSERA3 Ag2 from Peninsular Malaysia and Malaysian Borneo. F index indicated moderate differentiation of the gene. In silico analyses revealed unique repeat motifs among PkSERA3 Ag2 isolates. Moreover, the amino acid sequence of PkSERA3 Ag2 exhibited potential B-cell epitopes and possessed high antigenicity indices. These findings enhance the understanding of PkSERA3 Ag2 gene as well as its antigenic properties. Further validation is necessary to ascertain the utility of PkSERA3 Ag2 as a serological marker for P. knowlesi infection.
Topics: Genetic Variation; Protozoan Proteins; Plasmodium knowlesi; Malaysia; Epitopes, B-Lymphocyte; Nucleotides
PubMed: 37595939
DOI: 10.1016/j.meegid.2023.105490 -
Parasites & Vectors Aug 2023Indonesia is home to many species of non-human primates (NHPs). Deforestation, which is still ongoing in Indonesia, has substantially reduced the habitat of NHPs in the...
BACKGROUND
Indonesia is home to many species of non-human primates (NHPs). Deforestation, which is still ongoing in Indonesia, has substantially reduced the habitat of NHPs in the republic. This has led to an intensification of interactions between NHPs and humans, which opens up the possibility of pathogen spillover. The aim of the present study was to determine the prevalence of malarial parasite infections in NHPs in five provinces of Indonesia in 2022. Species of the genus Anopheles that can potentially transmit malarial pathogens to humans were also investigated.
METHODS
An epidemiological survey was conducted by capturing NHPs in traps installed in several localities in the five provinces, including in the surroundings of a wildlife sanctuary. Blood samples were drawn aseptically after the NHPs had been anesthetized; the animals were released after examination. Blood smears were prepared on glass slides, and dried blood spot tests on filter paper. Infections with Plasmodium spp. were determined morphologically from the blood smears, which were stained with Giemsa solution, and molecularly through polymerase chain reaction and DNA sequencing using rplU oligonucleotides. The NHPs were identified to species level by using the mitochondrial cytochrome c oxidase subunit I gene and the internal transcribed spacer 2 gene as barcoding DNA markers. Mosquito surveillance included the collection of larvae from breeding sites and that of adults through the human landing catch (HLC) method together with light traps.
RESULTS
Analysis of the DNA extracted from the dried blood spot tests of the 110 captured NHPs revealed that 50% were positive for Plasmodium, namely Plasmodium cynomolgi, Plasmodium coatneyi, Plasmodium inui, Plasmodium knowlesi and Plasmodium sp. Prevalence determined by microscopic examination of the blood smears was 42%. Species of the primate genus Macaca and family Hylobatidae were identified by molecular analysis. The most common mosquito breeding sites were ditches, puddles and natural ponds. Some of the Anopheles letifer captured through HLC carried sporozoites of malaria parasites that can cause the disease in primates.
CONCLUSIONS
The prevalence of malaria in the NHPs was high. Anopheles letifer, a potential vector of zoonotic malaria, was identified following its collection in Central Kalimantan by the HLC method. In sum, the potential for the transmission of zoonotic malaria in several regions of Indonesia is immense.
Topics: Animals; Humans; Indonesia; Mosquito Vectors; Malaria; Plasmodium knowlesi; Primates; Macaca; Anopheles
PubMed: 37550692
DOI: 10.1186/s13071-023-05880-4 -
Malaria Journal Aug 2023The recent deforestation for agricultural, mining, and human re-settlement has significantly reduced the habitat of many non-human primates (NHPs) in Indonesia and...
BACKGROUND
The recent deforestation for agricultural, mining, and human re-settlement has significantly reduced the habitat of many non-human primates (NHPs) in Indonesia and intensifies interaction between the NHPs and humans and thus opening the possibility of pathogen spill-over. The emergence of zoonotic malaria, such as Plasmodium knowlesi, poses an immense threat to the current malaria control and elimination that aims for the global elimination of malaria by 2030. As malaria in humans and NHPs is transmitted by the female Anopheles mosquito, malaria vector control is very important to mitigate the spill-over of the malaria parasite to humans. The present study aims to explore the Anopheles species diversity in human settlements adjacent to the wildlife sanctuary forest in Buton Utara Regency, Southeast Sulawesi, Indonesia, and identify the species that potentially transmit the pathogen from monkey to human in the area.
METHODS
Mosquito surveillance was conducted using larval and adult collection, and the collected mosquitoes were identified morphologically and molecularly using the barcoding markers, cytochrome oxidase subunit I (COI), and internal transcribed species 2 (ITS2) genes. Plasmodium sporozoite carriage was conducted on mosquitoes collected through human landing catch (HLC) and human-baited double net trap (HDNT).
RESULTS
The results revealed several Anopheles species, such as Anopheles flavirostris (16.6%), Anopheles sulawesi (3.3%), Anopheles maculatus (3.3%), Anopheles koliensis (1.2%), and Anopheles vagus (0.4%). Molecular analysis of the sporozoite carriage using the primate-specific malaria primers identified An. sulawesi, a member of the Leucosphyrus group, carrying Plasmodium inui sporozoite.
CONCLUSIONS
This study indicates that the transmission of zoonotic malaria in the area is possible and alerts to the need for mitigation efforts through a locally-tailored vector control intervention and NHPs habitat conservation.
Topics: Animals; Adult; Humans; Female; Malaria; Animals, Wild; Anopheles; Indonesia; Mosquito Vectors; Plasmodium knowlesi; Haplorhini
PubMed: 37528368
DOI: 10.1186/s12936-023-04647-7 -
Nature Communications Aug 2023Invasion of red blood cells (RBCs) by Plasmodium merozoites is critical to their continued survival within the host. Two major protein families, the Duffy binding-like...
Invasion of red blood cells (RBCs) by Plasmodium merozoites is critical to their continued survival within the host. Two major protein families, the Duffy binding-like proteins (DBPs/EBAs) and the reticulocyte binding like proteins (RBLs/RHs) have been studied extensively in P. falciparum and are hypothesized to have overlapping, but critical roles just prior to host cell entry. The zoonotic malaria parasite, P. knowlesi, has larger invasive merozoites and contains a smaller, less redundant, DBP and RBL repertoire than P. falciparum. One DBP (DBPα) and one RBL, normocyte binding protein Xa (NBPXa) are essential for invasion of human RBCs. Taking advantage of the unique biological features of P. knowlesi and iterative CRISPR-Cas9 genome editing, we determine the precise order of key invasion milestones and demonstrate distinct roles for each family. These distinct roles support a mechanism for phased commitment to invasion and can be targeted synergistically with invasion inhibitory antibodies.
Topics: Animals; Humans; Carrier Proteins; Parasites; Malaria; Plasmodium knowlesi; Protozoan Proteins; Erythrocytes; Merozoites; Plasmodium falciparum
PubMed: 37528099
DOI: 10.1038/s41467-023-40357-z