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Journal of Vector Borne Diseases Apr 2024Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into...
Detection of Plasmodium knowlesi in whole blood samples with sandwich enzyme-linked immunosorbent assay (ELISA) using rhoptry-associated protein 1 specific polyclonal antibodies.
BACKGROUND OBJECTIVES
Plasmodium knowlesi, a simian malaria species, is now known to infect humans. Due to disadvantages in the current diagnosis methods, many efforts have been placed into developing new methods to diagnose the disease. This study assessed the ability of the PkRAP-1 sandwich enzyme-linked immunosorbent (ELISA) to detect P knowlesi antigens in whole blood specimens.
METHODS
Western blot assay was conducted to evaluate the ability of raised mouse and rabbit anti-PkRAP-1 polyclonal antibodies to bind to the native proteins in P. knowlesi lysate. The polyclonal antibodies were then used in sandwich ELISA to detect P. knowlesi. In the sandwich ELISA, mouse and rabbit polyclonal antibodies were used as the capture and detection antibodies, respectively. The limit of detection (LOD) of the assay was determined using P. knowlesi A1H1 culture and purified recombinant PkRAP-1.
RESULTS
Western blot results showed positive reactions towards the proteins in P. knowlesi lysate. The LOD of the assay from three technical replicates was 0.068% parasitaemia. The assay performance in detecting P. knowlesi was 83% sensitivity and 70% specificity with positive and negative predictive values of 74% and 80%, respectively. The anti-PkRAP-1 polyclonal antibodies did not cross-react with P. falciparum and healthy samples, but P. vivax by detecting all 12 samples.
INTERPRETATION CONCLUSION
PkRAP-1 has the potential as a biomarker for the development of a new diagnostic tool for P. knowlesi detection. Further studies need to be conducted to establish the full potential of the usage of anti-PkRAP-1 antibodies for P. knowlesi detection.
Topics: Plasmodium knowlesi; Enzyme-Linked Immunosorbent Assay; Animals; Malaria; Antibodies, Protozoan; Rabbits; Sensitivity and Specificity; Mice; Protozoan Proteins; Humans; Antigens, Protozoan; Blotting, Western; Limit of Detection
PubMed: 38922654
DOI: 10.4103/jvbd.jvbd_55_23 -
Acta Tropica Jun 2024Malaria continues to be a global public health problem although it has been eliminated from many countries. Sri Lanka and China are two countries that recently achieved... (Review)
Review
Malaria continues to be a global public health problem although it has been eliminated from many countries. Sri Lanka and China are two countries that recently achieved malaria elimination status, and many countries in Southeast Asia are currently in the pipeline for achieving the same goal by 2030. However, Plasmodium knowlesi, a non-human primate malaria parasite continues to pose a threat to public health in this region, infecting many humans in all countries in Southeast Asia except for Timor-Leste. Besides, other non-human primate malaria parasite such as Plasmodium cynomolgi and Plasmodium inui are infecting humans in the region. The non-human primates, the long-tailed and pig-tailed macaques which harbour these parasites are now increasingly prevalent in farms and forest fringes close by to the villages. Additionally, the Anopheles mosquitoes belonging to the Lecuosphyrus Group are also present in these areas which makes them ideal for transmitting the non-human primate malaria parasites. With changing landscape and deforestation, non-human primate malaria parasites will affect more humans in the coming years with the elimination of human malaria. Perhaps due to loss of immunity, more humans will be infected as currently being demonstrated in Malaysia. Thus, control measures need to be instituted rapidly to achieve the malaria elimination status by 2030. However, the zoonotic origin of the parasite and the changes of the vectors behaviour to early biting seems to be the stumbling block to the malaria elimination efforts in this region. In this review, we discuss the challenges faced in malaria elimination due to deforestation and the serious threat posed by non-human primate malaria parasites.
PubMed: 38908421
DOI: 10.1016/j.actatropica.2024.107280 -
Comparative Medicine Jun 2024Malaria is a parasitic disease caused by protozoan species of the genus and transmitted by female mosquitos of the genus and other Culicidae. Most of the parasites of...
Malaria is a parasitic disease caused by protozoan species of the genus and transmitted by female mosquitos of the genus and other Culicidae. Most of the parasites of the genus are highly species specific with more than 200 species described affecting different species of mammals, birds, and reptiles. species strictly affecting humans are , , , and More recently, and other nonhuman primate plasmodia were found to naturally infect humans. Currently, malaria occurs mostly in poor tropical and subtropical areas of the world, and in many of these countries it is the leading cause of illness and death. For more than 100 y, animal models, have played a major role in our understanding of malaria biology. Avian species were the first to be used as models to study human malaria. Malaria parasite biology and immunity were first studied using mainly and . Rodent malarias, particularly and , have been used extensively as models to study malaria in mammals. Several species of from nonhuman primates have been used as surrogate models to study human malaria immunology, pathogenesis, candidate vaccines, and treatments. , , and are important models for studying malaria produced by and , while is used as a model for studying severe malaria. Other nonhuman primate malarias used in research are , , , , and . Very few nonhuman primate species can develop an infection with human malarias. Macaques in general are resistant to infection with , , , and . Only apes and a few species of New World monkeys can support infection with human malarias. Herein we review the most common, and some less common, avian, reptile, and mammal plasmodia species used as models to study human malaria.
PubMed: 38902006
DOI: 10.30802/AALAS-CM-24-000019 -
International Journal of Molecular... May 2024is the only that causes zoonotic disease among the that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate...
is the only that causes zoonotic disease among the that cause infection in humans. It is fatal due to its short asexual growth cycle within 24 h. Lactate dehydrogenase (LDH), an enzyme that catalyzes the final step of glycolysis, is a biomarker for diagnosing infection by spp. parasite. Therefore, this study aimed to efficiently produce the soluble form of LDH (PkLDH) using a bacterial expression system for studying malaria caused by . Recombinant pET-21a(+)- plasmid was constructed by inserting the gene into a pET-21a(+) expression vector. Subsequently, the recombinant plasmid was inserted into the protein-expressing Rosetta(DE3) strain, and the optimal conditions for overexpression of the PkLDH protein were established using this strain. We obtained a yield of 52.0 mg/L PkLDH from the Rosetta(DE3) strain and confirmed an activity of 483.9 U/mg through experiments. This methodology for high-efficiency PkLDH production can be utilized for the development of diagnostic methods and drug candidates for distinguishing malaria caused by .
Topics: Plasmodium knowlesi; L-Lactate Dehydrogenase; Cloning, Molecular; Malaria; Recombinant Proteins; Escherichia coli; Animals; Humans; Gene Expression; Protozoan Proteins
PubMed: 38891805
DOI: 10.3390/ijms25115615 -
Proteomes of plasmodium knowlesi early and late ring-stage parasites and infected host erythrocytes.Journal of Proteomics Jun 2024The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo...
The emerging malaria parasite Plasmodium knowlesi threatens the goal of worldwide malaria elimination due to its zoonotic spread in Southeast Asia. After brief ex-vivo culture we used 2D LC/MS/MS to examine the early and late ring stages of infected Macaca mulatta red blood cells harboring P. knowlesi. The M. mulatta clathrin heavy chain and T-cell and macrophage inhibitor ERMAP were overexpressed in the early ring stage; glutaredoxin 3 was overexpressed in the late ring stage; GO term differential enrichments included response to oxidative stress and the cortical cytoskeleton in the early ring stage. P. knowlesi clathrin heavy chain and 60S acidic ribosomal protein P2 were overexpressed in the late ring stage; GO term differential enrichments included vacuoles in the early ring stage, ribosomes and translation in the late ring stage, and Golgi- and COPI-coated vesicles, proteasomes, nucleosomes, vacuoles, ion-, peptide-, protein-, nucleocytoplasmic- and RNA-transport, antioxidant activity and glycolysis in both stages. SIGNIFICANCE: Due to its zoonotic spread, cases of the emerging human pathogen Plasmodium knowlesi in southeast Asia, and particularly in Malaysia, threaten regional and worldwide goals for malaria elimination. Infection by this parasite can be fatal to humans, and can be associated with significant morbidity. Due to zoonotic transmission from large macaque reservoirs that are untreatable by drugs, and outdoor biting mosquito vectors that negate use of preventive measures such as bed nets, its containment remains a challenge. Its biology remains incompletely understood. Thus we examine the expressed proteome of the early and late ex-vivo cultured ring stages, the first intraerythrocyte developmental stages after infection of host rhesus macaque erythrocytes. We used GO term enrichment strategies and differential protein expression to compare early and late ring stages. The early ring stage is characterized by the enrichment of P. knowlesi vacuoles, and overexpression of the M. mulatta clathrin heavy chain, important for clathrin-coated pits and vesicles, and clathrin-mediated endocytosis. The M. mulatta protein ERMAP was also overexpressed in the early ring stage, suggesting a potential role in early ring stage inhibition of T-cells and macrophages responding to P. knowlesi infection of reticulocytes. This could allow expansion of the host P. knowlesi cellular niche, allowing parasite adaptation to invasion of a wider age range of RBCs than the preferred young RBCs or reticulocytes, resulting in proliferation and increased pathogenesis in infected humans. Other GO terms differentially enriched in the early ring stage include the M. mulatta cortical cytoskeleton and response to oxidative stress. The late ring stage is characterized by overexpression of the P. knowlesi clathrin heavy chain. Combined with late ring stage GO term enrichment of Golgi-associated and coated vesicles, and enrichment of COPI-coated vesicles in both stages, this suggests the importance to P. knowlesi biology of clathrin-mediated endocytosis. P. knowlesi ribosomes and translation were also differentially enriched in the late ring stage. With expression of a variety of heat shock proteins, these results suggest production of folded parasite proteins is increasing by the late ring stage. M. mulatta endocytosis was differentially enriched in the late ring stage, as were clathrin-coated vesicles and endocytic vesicles. This suggests that M. mulatta clathrin-based endocytosis, perhaps in infected reticulocytes rather than mature RBC, may be an important process in the late ring stage. Additional ring stage biology from enriched GO terms includes M. mulatta proteasomes, protein folding and the chaperonin-containing T complex, actin and cortical actin cytoskeletons. P knowlesi biology also includes proteasomes, as well as nucleosomes, antioxidant activity, a variety of transport processes, glycolysis, vacuoles and protein folding. Mature RBCs have lost internal organelles, suggesting infection here may involve immature reticulocytes still retaining organelles. P. knowlesi parasite proteasomes and translational machinery may be ring stage drug targets for known selective inhibitors of these processes in other Plasmodium species. To our knowledge this is the first examination of more than one timepoint within the ring stage. Our results expand knowledge of both host and parasite proteins, pathways and organelles underlying P. knowlesi ring stage biology.
Topics: Plasmodium knowlesi; Macaca mulatta; Animals; Erythrocytes; Proteome; Protozoan Proteins; Malaria; Humans; Host-Parasite Interactions
PubMed: 38759952
DOI: 10.1016/j.jprot.2024.105197 -
ELife May 2024Zoonotic disease dynamics in wildlife hosts are rarely quantified at macroecological scales due to the lack of systematic surveys. Non-human primates (NHPs) host a... (Meta-Analysis)
Meta-Analysis
Zoonotic disease dynamics in wildlife hosts are rarely quantified at macroecological scales due to the lack of systematic surveys. Non-human primates (NHPs) host a zoonotic malaria of public health concern and the main barrier to malaria elimination in Southeast Asia. Understanding of regional infection dynamics in wildlife is limited. Here, we systematically assemble reports of NHP and investigate geographic determinants of prevalence in reservoir species. Meta-analysis of 6322 NHPs from 148 sites reveals that prevalence is heterogeneous across Southeast Asia, with low overall prevalence and high estimates for Malaysian Borneo. We find that regions exhibiting higher prevalence in NHPs overlap with human infection hotspots. In wildlife and humans, parasite transmission is linked to land conversion and fragmentation. By assembling remote sensing data and fitting statistical models to prevalence at multiple spatial scales, we identify novel relationships between in NHPs and forest fragmentation. This suggests that higher prevalence may be contingent on habitat complexity, which would begin to explain observed geographic variation in parasite burden. These findings address critical gaps in understanding regional epidemiology and indicate that prevalence in simian reservoirs may be a key spatial driver of human spillover risk.
Topics: Animals; Humans; Asia, Southeastern; Ecosystem; Malaria; Plasmodium knowlesi; Prevalence; Primate Diseases; Primates; Zoonoses
PubMed: 38753426
DOI: 10.7554/eLife.88616 -
MedRxiv : the Preprint Server For... May 2024The emergence of the zoonotic monkey parasite as the dominant cause of malaria in Malaysia has disrupted current national WHO elimination goals. Malaysia has free...
BACKGROUND
The emergence of the zoonotic monkey parasite as the dominant cause of malaria in Malaysia has disrupted current national WHO elimination goals. Malaysia has free universal access to malaria care; however, out-of-pocket costs are unknown. This study estimated household costs of illness attributable to malaria due to against other non-zoonotic species infections in Sabah, Malaysia.
METHODOLOGY/PRINCIPAL FINDINGS
Household costs were estimated from patient-level surveys collected from four hospitals between 2013 and 2016. Direct costs including medical and associated travel costs, and indirect costs due to lost productivity were included. One hundred and fifty-two malaria cases were enrolled: (n=108), (n=22), (n=16), and (n=6). Costs were inflated to 2022 Malaysian Ringgits and reported in United States dollars (US$). Across all cases, the mean total costs were US$138 (SD=108), with productivity losses accounting for 58% of costs (US$80; SD=73). had the highest mean total household cost at US$210, followed by (US$127), (US$126), and (US$105). Most patients (80%) experienced direct health costs above 10% of monthly income, with 58 (38%) patients experiencing health spending over 25% of monthly income, consistent with catastrophic health expenditure.
CONCLUSIONS/SIGNIFICANCE
Despite Malaysia's free health-system care for malaria, patients and families face other related medical, travel, and indirect costs. Household out-of-pocket costs were driven by productivity losses; primarily attributed to infections in working-aged males in rural agricultural-based occupations. Costs for were comparable to and lower than The higher costs related to direct health facility costs for repeat monitoring visits given the liver-stage treatment required.
AUTHOR SUMMARY
Knowlesi malaria is due to infection with a parasite transmitted by mosquitos from monkeys to humans. Most people who are infected work or live near the forest. It is now the major type of malaria affecting humans in Malaysia. The recent increase of knowlesi malaria cases in humans has impacted individuals, families, and health systems in Southeast Asia. Although the region has made substantial progress towards eliminating human-only malaria species, knowlesi malaria threatens elimination targets as traditional control measures do not address the parasite reservoir in monkeys. The economic burden of illness due to knowlesi malaria has not previously been estimated or subsequently compared with other malaria species. We collected data on the cost of illness to households in Sabah, Malaysia, to estimate their related total economic burden. Medical costs and time off work and usual activities were substantial in patients with the four species of malaria diagnosed during the time of this study. This research highlights the financial burden which households face when seeking care for malaria in Malaysia, despite the free treatment provided by the government.
PubMed: 38746350
DOI: 10.1101/2024.05.02.24306734 -
Acta Tropica Jul 2024Natural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic...
BACKGROUND
Natural human infections by Plasmodium cynomolgi and P. inui have been reported recently and gain the substantial attention from Southeast Asian countries. Zoonotic transmission of non-human malaria parasites to humans from macaque monkeys occurred through the bites of the infected mosquitoes. The objective of this study is to establish real-time fluorescence loop-mediated isothermal amplification (LAMP) assays for the detection of zoonotic malaria parasites by combining real-time fluorescent technology with the isothermal amplification technique.
METHODS
By using 18S rRNA as the target gene, the primers for P. cynomolgi, P. coatneyi and P. inui were newly designed in the present study. Four novel real-time fluorescence LAMP assays were developed for the detection of P. cynomolgi, P. coatneyi, P. inui and P. knowlesi. The entire amplification process was completed in 60 min, with the assays performed at 65 °C. By using SYTO-9 as the nucleic acid intercalating dye, the reaction was monitored via real-time fluorescence signal.
RESULTS
There was no observed cross-reactivity among the primers from different species. All 70 field-collected monkey samples were successfully amplified by real-time fluorescence LAMP assays. The detection limit for P. cynomolgi, P. coatneyi and P. knowlesi was 5 × 10 copies/µL. Meanwhile, the detection limit of P. inui was 5 × 10 copies/µL.
CONCLUSION
This is the first report of the detection of four zoonotic malaria parasites by real-time fluorescence LAMP approaches. It is an effective, rapid and simple-to-use technique. This presented platform exhibits considerable potential as an alternative detection for zoonotic malaria parasites.
Topics: Animals; Nucleic Acid Amplification Techniques; Malaria; RNA, Ribosomal, 18S; Molecular Diagnostic Techniques; Plasmodium; Sensitivity and Specificity; Zoonoses; Humans; DNA Primers; Fluorescence; Macaca; Monkey Diseases
PubMed: 38740319
DOI: 10.1016/j.actatropica.2024.107249 -
MedRxiv : the Preprint Server For... Apr 2024Malaria remains a major public health concern with substantial morbidity and mortality worldwide. In Malaysia, the emergence of has led to a surge in zoonotic malaria...
INTRODUCTION
Malaria remains a major public health concern with substantial morbidity and mortality worldwide. In Malaysia, the emergence of has led to a surge in zoonotic malaria cases and deaths in recent years. Signs of cerebral involvement have been observed in a non-comatose, fatal case of severe knowlesi infection, but the potential impact of this malaria species on the brain remains underexplored. To address this gap, we investigated circulating levels of brain injury, inflammation, and vascular biomarkers in a cohort of knowlesi-infected patients and controls.
METHODS
Archived plasma samples from 19 patients with confirmed symptomatic knowlesi infection and 19 healthy, age-matched controls from Peninsular Malaysia were analysed. A total of 52 plasma biomarkers of brain injury, inflammation, and vascular activation were measured using Luminex and SIMOA assays. Wilcoxon tests were used to examine group differences, and biomarker profiles were explored through hierarchical clustering heatmap analysis.
RESULTS
Bonferroni-corrected analyses revealed significantly elevated brain injury biomarker levels in knowlesi-infected patients, including S100B (p<0.0001), Tau (p=0.0007), UCH-L1 (p<0.0001), αSyn (p<0.0001), Park7 (p=0.0006), NRGN (p=0.0022), and TDP-43 (p=0.005). Compared to controls, levels were lower in the infected group for BDNF (p<0.0001), CaBD (p<0.0001), CNTN1 (p<0.0001), NCAM-1 (p<0.0001), GFAP (p=0.0013), and KLK6 (p=0.0126). Hierarchical clustering revealed distinct group profiles for circulating levels of brain injury and vascular activation biomarkers.
CONCLUSIONS
Our findings highlight for the first time the impact of infection on the brain, with distinct alterations in cerebral injury and endothelial activation biomarker profiles compared to healthy controls. Further studies are warranted to investigate the pathophysiology and clinical significance of these altered surrogate markers, through both neuroimaging and long-term neurocognitive assessments.
PubMed: 38712121
DOI: 10.1101/2024.04.25.24306382 -
Vaccine Jun 2024Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development....
Recent data indicate increasing disease burden and importance of Plasmodium vivax (Pv) malaria. A robust assay will be essential for blood-stage Pv vaccine development. Results of the in vitro growth inhibition assay (GIA) with transgenic P. knowlesi (Pk) parasites expressing the Pv Duffy-binding protein region II (PvDBPII) correlate with in vivo protection in the first PvDBPII controlled human malaria infection (CHMI) trials, making the PkGIA an ideal selection tool once the precision of the assay is defined. To determine the precision in percentage of inhibition in GIA (%GIA) and in GIA (antibody concentration that gave 50 %GIA), ten GIAs with transgenic Pk parasites were conducted with four different anti-PvDBPII human monoclonal antibodies (mAbs) at concentrations of 0.016 to 2 mg/mL, and three GIAs with eighty anti-PvDBPII human polyclonal antibodies (pAbs) at 10 mg/mL. A significant assay-to-assay variation was observed, and the analysis revealed a standard deviation (SD) of 13.1 in the mAb and 5.94 in the pAb dataset for %GIA, with a LogGIA SD of 0.299 (for mAbs). Moreover, the ninety-five percent confidence interval (95 %CI) for %GIA or GIA in repeat assays was calculated in this investigation. The error range determined in this study will help researchers to compare PkGIA results from different assays and studies appropriately, thus supporting the development of future blood-stage malaria vaccine candidates, specifically second-generation PvDBPII-based formulations.
Topics: Malaria Vaccines; Plasmodium knowlesi; Protozoan Proteins; Plasmodium vivax; Antigens, Protozoan; Humans; Receptors, Cell Surface; Antibodies, Protozoan; Malaria, Vivax; Antibodies, Monoclonal; Vaccine Development; Animals
PubMed: 38704253
DOI: 10.1016/j.vaccine.2024.04.073