RESULTS: A total of 36 studies met the inclusion criteria for this review. Numerous stakeholders were identified as involved in the intersectoral actions to defeat malaria amongst MMPs. Almost all studies discussed the involvement of Ministry of Health/Public Health (MOH/MOPH). The most frequently assessed intervention among the studies that were included was the coverage and utilization of insecticide-treated nets as personal protective measures (40.5%), followed by the intervention of early diagnoses and treatment of malaria (33.3%), the surveillance and response activities (13.9%) and the behaviour change communication (8.3%). There is a dearth of information on how these stakeholders shared roles and responsibilities for implementation, and about the channels of communication between-and-within the partners and with the MOH/MOPH. Despite limited details in the studies, the intermediate outcomes showed some evidence that the intersectoral collaborations contributed to improvement in knowledge about malaria, initiation and promotion of bed nets utilization, increased access to diagnosis and treatment in a surveillance context and contributed towards a reduction in malaria transmission. Overall, a high proportion of the targeted MMPs was equipped with correct knowledge about malaria transmission (70%, 95% CI 57-83%). Interventions targeting the use of bed nets utilization were two times more likely to reduce malaria incidence amongst the targeted MMPs (summary OR 2.01, 95% CI 1.43-2.6) than the non-users. The various intersectoral actions were often more vertically organized and not fully integrated in a systemic way within a given country or sub-national administrative setting.
CONCLUSION: Findings suggest that interventions supported by the multiple stakeholders had a significant impact on the reduction of malaria transmission amongst the targeted MMPs. Well-designed studies from different countries are recommended to robustly assess the role of intersectoral interventions targeted to MMPs and their impact on the reduction of transmission.
METHODS: This was a meta-analysis of diagnostic test accuracy. Relevant studies that evaluated the diagnostic performance of RDTs and microscopy for detection of asymptomatic malaria were searched in health-related electronic databases. The methodological quality of the studies included was assessed using the QUADAS-2 tool.
RESULTS: Ten studies assessing RDT and/or microscopy were identified. The diagnostic accuracies in all these studies were verified by PCR. Overall, the pooled sensitivities of RDT, as well as microscopy for detection of any malaria parasites in asymptomatic participants, were low, while their pooled specificities were almost ideal. For the detection of Plasmodium falciparum, pooled sensitivity by RDT (59%, 95%CI:16-91%) or microscopy (55%, 95%CI: 25-82%) were almost comparable. For detection of Plasmodium vivax, pooled sensitivity of RDT (51%, 95% CI:7-94%) had also the comparable accuracy of microscopy (54%, 95%CI,11-92%). Of note are the wide range of sensitivity and specificity.
CONCLUSION: The findings of this meta-analysis suggest that RDTs and microscopy have limited sensitivity and are inappropriate for the detection of asymptomatic Plasmodium infections. Other methods including a combination of PCR-based strategies, Loop-Mediated Isothermal Amplification (LAMP) technique must be considered to target these infections, in order to achieve malaria elimination. However, more data is needed for the wide acceptance and feasibility of these approaches. Studies to explore the role of asymptomatic and sub-patent infections in the transmission of malaria are of critical importance and are recommended.
METHODS: Using the recently completed genome sequences from P. malariae, P. ovale and P. knowlesi, a set of 33 candidate cell surface and secreted blood-stage antigens was selected and expressed in a recombinant form using a mammalian expression system. These proteins were added to an existing panel of antigens from P. falciparum and P. vivax and the immunoreactivity of IgG, IgM and IgA immunoglobulins from individuals diagnosed with infections to each of the five different Plasmodium species was evaluated by ELISA. Logistic regression modelling was used to quantify the ability of the responses to determine prior exposure to the different Plasmodium species.
RESULTS: Using sera from European travellers with diagnosed Plasmodium infections, antigens showing species-specific immunoreactivity were identified to select a panel of 22 proteins from five Plasmodium species for serological profiling. The immunoreactivity to the antigens in the panel of sera taken from travellers and individuals living in malaria-endemic regions with diagnosed infections showed moderate power to predict infections by each species, including P. ovale, P. malariae and P. knowlesi. Using a larger set of patient samples and logistic regression modelling it was shown that exposure to P. knowlesi could be accurately detected (AUC = 91%) using an antigen panel consisting of the P. knowlesi orthologues of MSP10, P12 and P38.
CONCLUSIONS: Using the recent availability of genome sequences to all human-infective Plasmodium spp. parasites and a method of expressing Plasmodium proteins in a secreted functional form, an antigen panel has been compiled that will be useful to determine exposure to these parasites.
RESULTS: Anti-ICAM-1 and CD36 monoclonal antibodies were able to inhibit and reverse P. falciparum binding of lab and recently adapted patient isolates in vitro. However, reversal of binding was incomplete and varied in its efficiency between parasite isolates.
CONCLUSIONS: The results show that, as a proof of concept, disturbing existing ligand-receptor interactions is possible and could have potential therapeutic value for severe malaria. The variation seen in the degree of reversing existing binding with different parasite isolates and the incomplete nature of reversal, despite the use of high affinity inhibitors, suggest that anti-adhesion approaches as adjunct therapies for severe malaria may not be effective, and the focus may need to be on inhibitory approaches such as vaccines.
METHODS: Mosquitoes were collected from six sites located in three different states in Sudan, Khartoum, Kassala and Sennar, using pyrethrum spray catch of indoor resting mosquitoes. Anopheline mosquitoes were identified morphologically and based on species specific nucleotide sequences in the ribosomal DNA intergenic spacers (IGS). Seven published An. gambiae microsatellite loci primers were used to amplify the DNA of An. arabiensis samples.
RESULTS: PCR confirmed that An. arabiensis was the main malaria vector found in the six localities. Of the seven microsatellite loci utilized, six were found to be highly polymorphic across populations, with high allelic richness and heterozygosity with the remaining one being monomorphic. Deviation from Hardy-Weinberg expectations were found in 21 out of 42 tests in the six populations due to heterozygote deficiency. Bayesian clustering analysis revealed two gene pools, grouping samples into two population clusters; one includes four and the other includes two populations. The clusters were not grouped according to the three states but were instead an admixture. The genetic distances between pairs of populations ranged from 0.06 to 0.24. Significant FST was observed between all pairwise analyses of An. arabiensis populations. The Kassala state population indicated high genetic differentiation (FST ranged from 0.17 to 0.24) from other populations, including one which is also located in the same state. High gene flow (Nm = 1.6-8.2) was detected among populations within respective clusters but limited between clusters particularly with respect to Kassala state. There was evidence of a bottleneck event in one of the populations (Al Haj Yousif site). No isolation by distance pattern was detected among populations.
CONCLUSIONS: This study revealed low levels of population differentiation with high gene flow among the An. arabiensis populations investigated in Sudan, with the exception of Kassala state.
METHODS: A household-based cross-sectional study was conducted in five remote villages (clusters) of Orang Asli located in the State of Kelantan, a central region of the country. Community members aged six years and above were interviewed. Demographic, socio-economic and KAP data on malaria were collected using a structured questionnaire and analysed using descriptive statistics.
RESULTS: Overall, 536 individuals from 208 households were interviewed. Household indoor residual spraying (IRS) coverage and bed net ownership were 100% and 89.2%, respectively. A majority of respondents used mosquito bed nets every night (95.1%), but only 50.2% were aware that bed nets were used to prevent malaria. Nevertheless, almost all of the respondents (97.9%) were aware that malaria is transmitted by mosquitoes. Regarding practice for managing malaria, the most common practice adopted by the respondents was seeking treatment at the health facilities (70.9%), followed by self-purchase of medication from a local shop (12.7%), seeking treatment from a traditional healer (10.5%) and self-healing (5.9%). Concerning potential zoonotic malaria, about half of the respondents (47.2%) reported seeing monkeys from their houses and 20.1% reported entering nearby forests within the last 6 months.
CONCLUSION: This study found that most populations living in the villages have an acceptable level of knowledge and awareness about malaria. However, positive attitudes and practices concerning managing malaria require marked improvement.
METHODS: In order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.
RESULTS: PkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.
CONCLUSION: These data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.
METHODS: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey.
RESULTS: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC50) values of blood stages measured by NanoLuc activity proved comparable to IC50 values measured by the standard SYBR Green method.
CONCLUSION: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.
METHODS: Using CRISPR/Cas9 gene editing two chimeric P. falciparum parasites, were generated, where the pfcsp gene has been replaced by either one of the two major pvcsp alleles, VK210 or VK247. In addition, a P. falciparum parasite line that lacks CSP expression was also generated. These parasite lines have been analysed for sporozoite production in An. stephensi mosquitoes.
RESULTS: The two chimeric Pf-PvCSP lines exhibit normal asexual and sexual blood stage development in vitro and produce sporozoite-containing oocysts in An. stephensi mosquitoes. Expression of the corresponding PvCSP was confirmed in oocyst-derived Pf-PvCSP sporozoites. However, most oocysts degenerate before sporozoite formation and sporozoites were not found in either the mosquito haemocoel or salivary glands. Unlike the chimeric Pf-PvCSP parasites, oocysts of P. falciparum parasites lacking CSP expression do not produce sporozoites.
CONCLUSIONS: Chimeric P. falciparum parasites expressing P. vivax circumsporozoite protein fail to produce salivary gland sporozoites. Combined, these studies show that while PvCSP can partially complement the function of PfCSP, species-specific features of CSP govern full sporozoite maturation and development in the two human malaria parasites.
METHODS: The protocol of this systematic review was registered in the PROSPERO International Prospective Register of Systematic Reviews (ID = CRD42020204770). Studies reporting the misidentification of P. knowlesi as P. malariae by microscopy and confirmation of this by molecular methods in MEDLINE, Web of Science and Scopus were reviewed. The risk of bias in the included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS). The pooled prevalence and 95% confidence interval (CI) of the misidentification of P. knowlesi as P. malariae by microscopy were estimated using a random effects model. Subgroup analysis of the study sites was performed to demonstrate any differences in the misidentification rates in different areas. Heterogeneity across the included studies was assessed and quantified using Cochran's Q and I2 statistics, respectively. Publication bias in the included studies was assessed using the funnel plot, Egger's test and contour-enhanced funnel plot.
RESULTS: Among 375 reviewed studies, 11 studies with a total of 1569 confirmed P. knowlesi cases in humans were included. Overall, the pooled prevalence of the misidentification of P. knowlesi as P. malariae by microscopy was estimated at 57% (95% CI 37-77%, I2: 99.3%). Subgroup analysis demonstrated the highest rate of misidentification in Sawarak, Malaysia (87%, 95% CI 83-90%, I2: 95%), followed by Sabah, Malaysia (85%, 95% CI 79-92%, I2: 85.1%), Indonesia (16%, 95% CI 6-38%), and then Thailand (4%, 95% CI 2-9%, I2: 95%).
CONCLUSION: Although the World Health Organization (WHO) recommends that all P. malariae-positive diagnoses made by microscopy in P. knowlesi endemic areas be reported as P. malariae/P. knowlesi malaria, the possibility of microscopists misidentifying P. knowlesi as P. malariae is a diagnostic challenge. The use of molecular techniques in cases with malariae-like Plasmodium with high parasite density as determined by microscopy could help identify human P. knowlesi cases in non-endemic countries.
METHODS: The concordance rate, sensitivity and specificity of the duplex ddPCR assay were determined and compared to nested PCR and duplex qPCR.
RESULTS: The duplex ddPCR assay had higher analytical sensitivity (P. vivax = 10 copies/µL and P. knowlesi = 0.01 copies/µL) compared to qPCR (P. vivax = 100 copies/µL and P. knowlesi = 10 copies/µL). Moreover, the ddPCR assay had acceptable clinical sensitivity (P. vivax = 80% and P. knowlesi = 90%) and clinical specificity (P. vivax = 87.84% and P. knowlesi = 81.08%) when compared to nested PCR. Both ddPCR and qPCR detected more double infections in the samples.
CONCLUSIONS: Overall, the ddPCR assay demonstrated acceptable efficiency in detection of P. knowlesi and P. vivax, and was more sensitive than nested PCR in detecting mixed infections. However, the duplex ddPCR assay still needs optimization to improve the assay's clinical sensitivity and specificity.
METHODS: A total of 151 P. falciparum isolates were collected between April 2018 and March 2019 from 12 of the governorates in Jazan region. Genomic DNA was extracted from dried blood spots and amplified using nested PCR. Polymorphisms in the propeller domain of the P. falciparum k13 (pfkelch13) gene and point mutations in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by sequencing.
RESULTS: No mutations in the pfkelch13 propeller domain were found in any of the 151 isolates. However, point mutations in the pfdhfr and pfdhps genes were detected in 90.7% (137/151) of the isolates. The pfdhfr double mutations N51I + S108N (i.e. ACICNI haplotype) and triple mutations N51I + C59R + S108N (i.e. ACIRNI haplotype) were detected in 47% and 37.8% of the isolates, respectively. Moreover, the pfdhps single mutation at codon A437G and double mutations A437G + K540E (i.e. SGEAAI haplotype) were observed in 4.6% and 51.7% of the isolates, respectively. Interestingly, 23.8%, 25.1 and 12.6% of the isolates had quintuple, quadruple and triple mutated combined pfdhfr-pfdhps genotypes, respectively. Furthermore, significant associations were found between the prevalence of mutant haplotypes and the age, gender and nationality of the patients (P
METHODS: Plasma Flt3L concentration and blood CD141+ DC, CD1c+ DC and plasmacytoid DC (pDC) numbers were assessed in (i) volunteers experimentally infected with P. falciparum and in Malaysian patients with uncomplicated (ii) P. falciparum or (iii) P. knowlesi malaria.
RESULTS: Plasmodium knowlesi caused a decline in all circulating DC subsets in adults with malaria. Plasma Flt3L was elevated in acute P. falciparum and P. knowlesi malaria with no increase in a subclinical experimental infection. Circulating CD141+ DCs, CD1c+ DCs and pDCs declined in all adults tested, for the first time extending the finding of DC subset decline in acute malaria to the zoonotic parasite P. knowlesi.
CONCLUSIONS: In adults, submicroscopic Plasmodium infection causes no change in plasma Flt3L but does reduce circulating DCs. Plasma Flt3L concentrations increase in acute malaria, yet this increase is insufficient to restore or expand circulating CD141+ DCs, CD1c+ DCs or pDCs. These data imply that haematopoietic factors, yet to be identified and not Flt3L, involved in the sensing/maintenance of circulating DC are impacted by malaria and a submicroscopic infection. The zoonotic P. knowlesi is similar to other Plasmodium spp in compromising DC in adult malaria.
METHODS: The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes.
RESULTS: Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P
METHODS: A series of qualitative interviews were conducted with policy makers and healthcare providers in four vivax-endemic countries. Routine G6PD testing is not part of current policy in Bangladesh, Cambodia or China, but it is in Malaysia. The interviews were analysed with regard to respondents perceptions of vivax malaria, -primaquine based treatment for malaria and the complexities of G6PD deficiency.
RESULTS: Three barriers to the roll-out of routine G6PD testing were identified in all sites: (a) a perceived low risk of drug-induced haemolysis; (b) the perception that vivax malaria was benign and accordingly treatment with primaquine was not regarded as a priority; and, (c) the additional costs of introducing routine testing. In Malaysia, respondents considered the current test and treat algorithm suitable and the need for an alternative approach was only considered relevant in highly mobile and hard to reach populations.
CONCLUSIONS: Greater efforts are needed to increase awareness of the benefits of the radical cure of Plasmodium vivax and this should be supported by economic analyses exploring the cost effectiveness of routine G6PD testing.