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BioRxiv : the Preprint Server For... Mar 2023The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to (Pv) invasion of reticulocytes. Pv expresses a Duffy binding protein (PvDBP) on...
The Duffy antigen receptor for chemokines (DARC) expressed on erythrocytes is central to (Pv) invasion of reticulocytes. Pv expresses a Duffy binding protein (PvDBP) on merozoites, a DARC ligand, and their protein-protein interaction is central to vivax blood stage malaria. Here we compared the functional activity of humAbs derived from naturally exposed and vaccinated individuals for the first time using easily cultured (Pk) that had been genetically modified to replace its endogenous PkDBP orthologue with PvDBP to create a transgenic parasite, PkPvDBPOR. This transgenic parasite requires DARC to invade human erythrocytes but is not reticulocyte restricted. Using this model, we evaluated the invasion inhibition potential of 12 humAbs (9 naturally acquired; 3 vaccine-induced) targeting PvDBP individually and in combinations using growth inhibition assays (GIAs). The PvDBP-specific humAbs demonstrated 70-100% inhibition of PkPvDBPOR invasion with the IC values ranging from 51 to 338 μg/mL for the 9 naturally acquired (NA) humAbs and 33 to 99 μg/ml for the 3 vaccine-induced (VI) humAbs. To evaluate antagonistic, additive, or synergistic effects, six pairwise combinations were performed using select humAbs. Of these combinations tested, one NA/NA (099100/094083) combination demonstrated relatively strong additive inhibition between 10-100 μg/mL; all combinations of NA and VI humAbs showed additive inhibition at concentrations below 25 μg/mL and antagonism at higher concentrations. None of the humAb combinations showed synergy. This PkPvDBPOR model system enables efficient assessment of NA and VI humAbs individually and in combination.
PubMed: 36945444
DOI: 10.1101/2023.03.07.531647 -
The American Journal of Tropical... May 2023This study highlights the development of two lateral flow recombinase polymerase amplification assays for the diagnosis of human malaria. The lateral flow cassettes...
This study highlights the development of two lateral flow recombinase polymerase amplification assays for the diagnosis of human malaria. The lateral flow cassettes contained test lines that captured biotin-, 6-carboxyfluorescein, digoxigenin-, cyanine 5-, and dinitrophenyl-labeled amplicons. The overall process can be completed in 30 minutes. Recombinase polymerase amplification coupled with lateral flow had a detection limit of 1 copy/µL for Plasmodium knowlesi, Plasmodium vivax, and Plasmodium falciparum. No cross-reactivity was observed among nonhuman malaria parasites such as Plasmodium coatneyi, Plasmodium cynomolgi, Plasmodium brasilanium, Plasmodium inui, Plasmodium fragile, Toxoplasma gondii, Sarcocystis spp., Brugia spp., and 20 healthy donors. It is rapid, highly sensitive, robust, and easy to use. The result can be read without the need for special equipment and thus has the potential to serve as an effective alternative to polymerase chain reaction methods for the diagnosis of malaria.
Topics: Humans; Recombinases; Plasmodium; Malaria; Plasmodium knowlesi; Plasmodium falciparum; Plasmodium vivax
PubMed: 36913921
DOI: 10.4269/ajtmh.22-0657 -
BMJ Open Mar 2023malaria is a non-human simian malaria that threatens Southeast Asian rural communities. Studies indicate that non-compliant bednet usage, travelling into the forest and...
INTRODUCTION
malaria is a non-human simian malaria that threatens Southeast Asian rural communities. Studies indicate that non-compliant bednet usage, travelling into the forest and working as farmers and rubber tappers put communities at risk for infection. Despite guidelines, malaria incidence increases yearly and has become a public health concern. In addition to research gaps addressing factors that influence malaria prevention behaviour in these communities, there are no specific guidelines to facilitate strategies against the threat of malaria.
METHOD
To examine potential factors that influence malaria-prevention behaviour in communities exposed to malaria, 12 malaria experts participated in a modified Delphi study; every participant maintained their anonymity throughout the study. Three Delphi rounds were conducted via different online platforms between 15 November 2021 and 26 February 2022, and consensus was achieved when 70% of the participants agreed on a particular point with a 4-5 median. The results from the open-ended questions were then subjected to thematic analysis, and the dataset generated by this study was analysed using a deductive and inductive approach.
RESULTS
After a systematic, iterative process, knowledge and belief, social support, cognitive and environmental factors, past experience as a malaria patient, and the affordability and feasibility of a given intervention were critical contributors to malaria-prevention behaviour.
CONCLUSION
Future research on malaria could adapt this study's findings for a more nuanced understanding of factors that influence malaria-prevention behaviour and improve malaria programmes based on the expert consensus.
Topics: Humans; Plasmodium knowlesi; Delphi Technique; Malaria; Public Health; Forests; Malaysia
PubMed: 36898744
DOI: 10.1136/bmjopen-2022-067451 -
Frontiers in Microbiology 2023The emergence of potentially life-threatening zoonotic malaria caused by nearly two decades ago has continued to challenge Malaysia healthcare. With a total of 376...
The emergence of potentially life-threatening zoonotic malaria caused by nearly two decades ago has continued to challenge Malaysia healthcare. With a total of 376 infections notified in 2008, the number increased to 2,609 cases in 2020 nationwide. Numerous studies have been conducted in Malaysian Borneo to determine the association between environmental factors and knowlesi malaria transmission. However, there is still a lack of understanding of the environmental influence on knowlesi malaria transmission in Peninsular Malaysia. Therefore, our study aimed to investigate the ecological distribution of human malaria in relation to environmental factors in Peninsular Malaysia. A total of 2,873 records of human infections in Peninsular Malaysia from 1st January 2011 to 31st December 2019 were collated from the Ministry of Health Malaysia and geolocated. Three machine learning-based models, maximum entropy (MaxEnt), extreme gradient boosting (XGBoost), and ensemble modeling approach, were applied to predict the spatial variation of disease risk. Multiple environmental parameters including climate factors, landscape characteristics, and anthropogenic factors were included as predictors in both predictive models. Subsequently, an ensemble model was developed based on the output of both MaxEnt and XGBoost. Comparison between models indicated that the XGBoost has higher performance as compared to MaxEnt and ensemble model, with AUC values of 0.933 ± 0.002 and 0.854 ± 0.007 for train and test datasets, respectively. Key environmental covariates affecting human occurrence were distance to the coastline, elevation, tree cover, annual precipitation, tree loss, and distance to the forest. Our models indicated that the disease risk areas were mainly distributed in low elevation (75-345 m above mean sea level) areas along the Titiwangsa mountain range and inland central-northern region of Peninsular Malaysia. The high-resolution risk map of human knowlesi malaria constructed in this study can be further utilized for multi-pronged interventions targeting community at-risk, macaque populations, and mosquito vectors.
PubMed: 36876062
DOI: 10.3389/fmicb.2023.1126418 -
Journal of Ayub Medical College,... 2023
Topics: Humans; Plasmodium knowlesi; Malaria; Fatal Outcome
PubMed: 36849407
DOI: 10.55519/JAMC-01-11418 -
Cellular and Molecular Life Sciences :... Feb 2023Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen...
Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a β-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1-RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1-RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.
Topics: Humans; Cell Membrane; Erythrocytes; Malaria; Membrane Proteins; Protozoan Proteins
PubMed: 36847896
DOI: 10.1007/s00018-023-04712-z -
Malaria Journal Feb 2023The incidence of zoonotic Plasmodium knowlesi infections in humans is rising in Southeast Asia, leading to clinical studies to monitor the efficacy of anti-malarial...
BACKGROUND
The incidence of zoonotic Plasmodium knowlesi infections in humans is rising in Southeast Asia, leading to clinical studies to monitor the efficacy of anti-malarial treatments for knowlesi malaria. One of the key outcomes of anti-malarial drug efficacy is parasite clearance. For Plasmodium falciparum, parasite clearance is typically estimated using a two-stage method, that involves estimating parasite clearance for individual patients followed by pooling of individual estimates to derive population estimates. An alternative approach is Bayesian hierarchical modelling which simultaneously analyses all parasite-time patient profiles to determine parasite clearance. This study compared these methods for estimating parasite clearance in P. knowlesi treatment efficacy studies, with typically fewer parasite measurements per patient due to high susceptibility to anti-malarials.
METHODS
Using parasite clearance data from 714 patients with knowlesi malaria and enrolled in three trials, the Worldwide Antimalarial Resistance Network (WWARN) Parasite Clearance Estimator (PCE) standard two-stage approach and Bayesian hierarchical modelling were compared. Both methods estimate the parasite clearance rate from a model that incorporates a lag phase, slope, and tail phase for the parasitaemia profiles.
RESULTS
The standard two-stage approach successfully estimated the parasite clearance rate for 678 patients, with 36 (5%) patients excluded due to an insufficient number of available parasitaemia measurements. The Bayesian hierarchical estimation method was applied to the parasitaemia data of all 714 patients. Overall, the Bayesian method estimated a faster population mean parasite clearance (0.36/h, 95% credible interval [0.18, 0.65]) compared to the standard two-stage method (0.26/h, 95% confidence interval [0.11, 0.46]), with better model fits (compared visually). Artemisinin-based combination therapy (ACT) is more effective in treating P. knowlesi than chloroquine, as confirmed by both methods, with a mean estimated parasite clearance half-life of 2.5 and 3.6 h, respectively using the standard two-stage method, and 1.8 and 2.9 h using the Bayesian method.
CONCLUSION
For clinical studies of P. knowlesi with frequent parasite measurements, the standard two-stage approach (WWARN's PCE) is recommended as this method is straightforward to implement. For studies with fewer parasite measurements per patient, the Bayesian approach should be considered. Regardless of method used, ACT is more efficacious than chloroquine, confirming the findings of the original trials.
Topics: Animals; Humans; Antimalarials; Plasmodium knowlesi; Parasites; Bayes Theorem; Artemisinins; Malaria; Chloroquine; Plasmodium falciparum; Zoonoses; Parasitemia
PubMed: 36782162
DOI: 10.1186/s12936-023-04483-9 -
Frontiers in Veterinary Science 2023The imminent risk of zoonoses of non-human malaria parasites is not far from reality in India, as has been observed in the case of Plasmodium knowlesi (Pk), and so is...
INTRODUCTION
The imminent risk of zoonoses of non-human malaria parasites is not far from reality in India, as has been observed in the case of Plasmodium knowlesi (Pk), and so is possible with (Pc), already reported from South East Asian countries. Therefore, a novel multiplex qPCR assay was developed and evaluated for detection of non-human malaria parasites- Pk and Pc in populations at risk.
METHODS
The qPCR primers were designed in-house with fluorescence labeled probes (HEX for Pk and FAM for Pc). DNA samples of Pk and Pc were used as templates and further the qPCR assay was evaluated in 250 symptomatic and asymptomatic suspected human blood samples from malaria endemic areas of North Eastern states of India.
RESULTS
The qPCR assay successfully amplified the target 18S rRNA gene segment from Pk and Pc and was highly specific for Pk and Pc parasites only, as no cross reactivity was observed with (Pf), (Pv), (Pm), and (Po). Standard curves were generated to estimate the limit of detection (LOD) of Pk and Pc parasites DNA (0.00275 & 0.075 ng/μl, respectively). Due to COVID-19 pandemic situation during 2020-21, the sample accessibility was difficult, however, we managed to collect 250 samples. The samples were tested for Pf and Pv using conventional PCR- 14 Pf and 11 Pv infections were observed, but no Pk and Pc infections were detected. For Pk infections, previously reported conventional PCR was also performed, but no Pk infection was detected.
DISCUSSION
The multiplex qPCR assay was observed to be robust, quick, cost-effective and highly sensitive as compared to the currently available conventional PCR methods. Further validation of the multiplex qPCR assay in field setting is desirable, especially from the high-risk populations. We anticipate that the multiplex qPCR assay would prove to be a useful tool in mass screening and surveillance programs for detection of non-human malaria parasites toward the control and elimination of malaria from India by 2030.
PubMed: 36777671
DOI: 10.3389/fvets.2023.1127273 -
Scientific Reports Feb 2023The zoonotic Plasmodium knowlesi parasite is a growing public health concern in Southeast Asia, especially in Malaysia, where elimination of P. falciparum and P. vivax...
The zoonotic Plasmodium knowlesi parasite is a growing public health concern in Southeast Asia, especially in Malaysia, where elimination of P. falciparum and P. vivax malaria has been the focus of control efforts. Understanding of the genetic diversity of P. knowlesi parasites can provide insights into its evolution, population structure, diagnostics, transmission dynamics, and the emergence of drug resistance. Previous work has revealed that P. knowlesi fall into three main sub-populations distinguished by a combination of geographical location and macaque host (Macaca fascicularis and M. nemestrina). It has been shown that Malaysian Borneo groups display profound heterogeneity with long regions of high or low divergence resulting in mosaic patterns between sub-populations, with some evidence of chromosomal-segment exchanges. However, the genetic structure of non-Borneo sub-populations is less clear. By gathering one of the largest collections of P. knowlesi whole-genome sequencing data, we studied structural genomic changes across sub-populations, with the analysis revealing differences in Borneo clusters linked to mosquito-related stages of the parasite cycle, in contrast to differences in host-related stages for the Peninsular group. Our work identifies new genetic exchange events, including introgressions between Malaysian Peninsular and M. nemestrina-associated clusters on various chromosomes, including in parasite invasion genes (DBP[Formula: see text], NBPX[Formula: see text] and NBPX[Formula: see text]), and important proteins expressed in the vertebrate parasite stages. Recombination events appear to have occurred between the Peninsular and M. fascicularis-associated groups, including in the DBP[Formula: see text] and DBP[Formula: see text] invasion associated genes. Overall, our work finds that genetic exchange events have occurred among the recognised contemporary groups of P. knowlesi parasites during their evolutionary history, leading to apparent mosaicism between these sub-populations. These findings generate new hypotheses relevant to parasite evolutionary biology and P. knowlesi epidemiology, which can inform malaria control approaches to containing the impact of zoonotic malaria on human communities.
Topics: Animals; Humans; Genetic Variation; Plasmodium knowlesi; Macaca fascicularis; Malaria; Malaysia; Malaria, Vivax; Malaria, Falciparum; Genetics, Population; Selection, Genetic
PubMed: 36750737
DOI: 10.1038/s41598-023-29368-4 -
Microbial Biotechnology Apr 2023Currently, malaria is still one of the major public health problems commonly caused by the four Plasmodium species. The similar symptoms of malaria and the COVID-19...
Currently, malaria is still one of the major public health problems commonly caused by the four Plasmodium species. The similar symptoms of malaria and the COVID-19 epidemic of fever or fatigue lead to frequent misdiagnosis. The disadvantages of existing detection methods, such as time-consuming, costly, complicated operation, need for experienced technicians, and indistinguishable typing, lead to difficulties in meeting the clinical requirements of rapid, easy, and accurate typing of common Plasmodium species. In this study, we developed and optimized a universal two-dimensional labelled probe-mediated melting curve analysis (UP-MCA) assay based on multiplex and asymmetric PCR for rapid and accurate typing of five Plasmodium species, including novel human Plasmodium, Plasmodium knowlesi (Pk), in a single closed tube following genome extraction. The assay showed a limit of detection (LOD) of 10 copies per reaction and could accurately distinguish Plasmodium species from intra-plasmodium and other pathogens. Additionally, we proposed and validated different methods of fluorescence quenching and tag design for probes that are suitable for UP-MCA assays. Moreover, the clinical performance of the Plasmodium UP-MCA assay using a base-quenched universal probe was evaluated using 226 samples and showed a sensitivity of 100% (164/164) and specificity of 100% (62/62) at a 99% confidence interval, with the microscopy method as the gold standard. In summary, the UP-MCA assay showed excellent sensitivity, specificity, and accuracy for genotyping Plasmodium species spp. Additionally, it facilitates convenient and rapid Plasmodium detection in routine clinical practice and has great potential for clinical translation.
Topics: Humans; Multiplex Polymerase Chain Reaction; Sensitivity and Specificity; DNA, Protozoan; COVID-19; Plasmodium; Malaria; COVID-19 Testing
PubMed: 36745663
DOI: 10.1111/1751-7915.14232