METHODS: Malaria disease incidence rates by active case detection in cohorts of children, and indicators of insecticide resistance in local vectors were monitored in each of approximately 300 separate locations (clusters) with high coverage of malaria vector control over multiple malaria seasons. Phenotypic and genotypic resistance was assessed annually. In two countries, Sudan and India, clusters were randomly assigned to receive universal coverage of ITNs only, or universal coverage of ITNs combined with high coverage of IRS. Association between malaria incidence and insecticide resistance, and protective effectiveness of vector control methods and insecticide resistance were estimated, respectively.

RESULTS: Cohorts have been set up in all five countries, and phenotypic resistance data have been collected in all clusters. In Sudan, Kenya, Cameroon and Benin data collection is due to be completed in 2015. In India data collection will be completed in 2016.

METHODS: This study investigated blood samples from malaria centres in Southern Thailand. Genetic loci associated with drug resistance were amplified and sequenced. Drug resistance associated genes Pvmdr1, Pvcrt-o, Pvdhfr, and Pvdhps were characterized for 145 cases of P. vivax malaria, as well as the artemisinin resistance-associated Pfkelch13 gene from 91 cases of P. falciparum malaria.

RESULTS: Plasmodium vivax samples from Southern Thai provinces showed numerous chloroquine and antifolate resistance-associated mutations, including SNP and Pvcrt-o K10-insertion combinations suggestive of chloroquine resistant P. vivax phenotypes. A high proportion of the C580Y coding mutation (conferring artemisinin resistance) was detected in P. falciparum samples originating from Ranong and Yala (where the mutation was previously unreported).

METHODS: A total of 95 blood samples from long-tailed macaques in the Philippines were collected from three locations; 30 were from captive macaques at the National Wildlife Rescue and Rehabilitation Center (NWRRC) in Luzon, 25 were from captive macaques at the Palawan Wildlife Rescue and Conservation Center (PWRCC) in Palawan and 40 were from wild macaques from Puerto Princesa Subterranean River National Park (PPSRNP) in Palawan. The Plasmodium spp. infecting the macaques were identified using nested PCR assays on DNA extracted from these blood samples.

RESULTS: All 40 of the wild macaques from PPSRNP in Palawan and 5 of 25 captive macaques from PWRCC in Palawan were Plasmodium-positive; while none of the 30 captive macaques from the NWRRC in Luzon had any malaria parasites. Overall, P. inui was the most prevalent malaria parasite (44.2%), followed by P. fieldi (41.1%), P. cynomolgi (23.2%), P. coatneyi (21.1%), and P. knowlesi (19%). Mixed species infections were also observed in 39 of the 45 Plasmodium-positive macaques. There was a significant difference in the prevalence of P. knowlesi among the troops of wild macaques from PPSRNP.

METHODS: Thirty-six full-length pkmsp7D gene sequences (along with the reference H-strain: PKNH_1266000) obtained from clinical isolates of Malaysia, which were orthologous to pvmsp7H (PVX_082680) were downloaded from public databases. Population genetic, evolutionary and phylogenetic analyses were performed to determine the level of genetic diversity, polymorphism, recombination and natural selection.

RESULTS: Analysis of 36 full-length pkmsp7D sequences identified 147 SNPs (91 non-synonymous and 56 synonymous substitutions). Nucleotide diversity across the full-length gene was higher than its ortholog in Plasmodium vivax (msp7H). Region-wise analysis of the gene indicated that the nucleotide diversity at the central region was very high (π = 0.14) compared to the 5' and 3' regions. Most hyper-variable SNPs were detected at the central domain. Multiple test for natural selection indicated the central region was under strong positive natural selection however, the 5' and 3' regions were under negative/purifying selection. Evidence of intragenic recombination were detected at the central region of the gene. Phylogenetic analysis using full-length msp7D genes indicated there was no geographical clustering of parasite population.

METHODS: A total of 93 blood samples from Macaca fascicularis, Macaca leonina and Macaca arctoides were collected from four locations in Thailand: 32 were captive M. fascicularis from Chachoengsao Province (CHA), 4 were wild M. fascicularis from Ranong Province (RAN), 32 were wild M. arctoides from Prachuap Kiri Khan Province (PRA), and 25 were wild M. leonina from Nakornratchasima Province (NAK). DNA was extracted from these samples and analysed by nested PCR assays to detect Plasmodium, and subsequently to detect P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.

RESULTS: Twenty-seven of the 93 (29%) samples were Plasmodium-positive by nested PCR assays. Among wild macaques, all 4 M. fascicularis at RAN were infected with malaria parasites followed by 50% of 32 M. arctoides at PRA and 20% of 25 M. leonina at NAK. Only 2 (6.3%) of the 32 captive M. fascicularis at CHA were malaria-positive. All 5 species of Plasmodium were detected and 16 (59.3%) of the 27 macaques had single infections, 9 had double and 2 had triple infections. The composition of Plasmodium species in macaques at each sampling site was different. Macaca arctoides from PRA were infected with P. knowlesi, P. coatneyi, P. cynomolgi, P. inui and P. fieldi.

METHODS: This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis for Diagnostic Test Accuracy (PRISMA-DTA) guideline. Relevant studies in the health-related electronic databases were searched. According to the criteria set for this study, eligible studies were identified. The quality of included studies was evaluated with the use of a quality assessment checklist. A summary performance estimates such as pooled sensitivity and specificity were stratified by type of LAMP. Bivariate model for data analyses was applied. Summary receiver operating characteristics plots were created to display the results of individual studies in a receiver operating characteristics space. Meta-regression analysis was performed to investigate the sources of heterogeneity among individual studies.

RESULTS: Twenty-seven studies across 17 endemic countries were identified. The vast majority of studies were with unclear risk of bias in the selection of index test. Overall, the pooled test performances were high for Pan LAMP (sensitivity: 0.95, 95% CI 0.91 to 0.97; specificity: 0.98, 95% CI 0.95 to 0.99), Plasmodium falciparum (Pf) LAMP (sensitivity: 0.96, 95% CI 0.94 to 0.98; specificity: 0.99, 95% CI 0.96 to 1.00) or for Plasmodium vivax (Pv) LAMP from 6 studies (sensitivity: 0.98, 95% CI 0.92 to 0.99; specificity: 0.99, 95% CI 0.72 to 1.00). The area under the curve for Pan LAMP (0.99, 95% CI 0.98-1.00), Pf LAMP (0.99, 95% CI 0.97-0.99) and Pv LAMP was (1.00, 95% CI 0.98-1.00) indicated that the diagnostic performance of these tests were within the excellent accuracy range. Meta-regression analysis showed that sample size had the greatest impact on test performance, among other factors.

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.

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.

METHODS: Study participants included 73 uncomplicated malaria patients with PCR species confirmation: 50 P. knowlesi, 20 P. falciparum and 3 P. vivax. Nineteen malaria-negative, non-endemic area controls were also included. The sensitivity of the Eiken Loopamp™ MALARIA Pan Detection kit (Pan LAMP) for detecting each Plasmodium species was evaluated. Sensitivity and specificity of the Eiken Loopamp™ MALARIA Pf Detection kit (Pf LAMP) for P. falciparum were also determined. The limit of detection for each LAMP assay was evaluated, with results compared to PCR. All P. knowlesi patients were also tested by CareStart™ (Pf/VOM) and OptiMAL-IT™ (Pan/Pf) RDTs.

RESULTS: The sensitivity of the Pan LAMP assay was 100% for P. knowlesi (95% CI 92.9-100), P. falciparum (95% CI 83.2-100), and P. vivax (95% CI 29.2-100). The Pf LAMP was 100% sensitive and specific for P. falciparum detection, with all P. knowlesi samples having a negative reaction. LAMP sensitivity was superior to both RDTs, with only 10 and 28% of P. knowlesi samples testing positive to CareStart™ and OptiMAL-IT™, respectively. Limit of detection using the Pan LAMP for both P. knowlesi and P. vivax was 2 parasites/μL, comparable to PCR. For P. falciparum both the Pan LAMP and Pf LAMP demonstrated a limit of detection of 20 parasites/μL.

MAIN TEXT: Plasmodium knowlesi is the most common cause of malaria in Malaysia, and cases have also been reported in nearly all countries of Southeast Asia. Outside of Malaysia, P. knowlesi is frequently misdiagnosed by microscopy as Plasmodium falciparum or Plasmodium vivax. Thus, P. knowlesi may be underdiagnosed in affected regions and its true incidence underestimated. Acknowledgement in the World Malaria Report of the regional importance of P. knowlesi will facilitate efforts to improve surveillance of this emerging parasite. Furthermore, increased recognition will likely lead to improved delivery of effective treatment for this potentially fatal infection, as has occurred in Malaysia where P. knowlesi case-fatality rates have fallen despite rising incidence. In a number of knowlesi-endemic countries, substantial progress has been made towards the elimination of P. vivax and P. falciparum. However, efforts to eliminate these human-only species should not preclude efforts to reduce human malaria from P. knowlesi. The regional importance of knowlesi malaria was recognized by the WHO with its recent Evidence Review Group meeting on knowlesi malaria to address strategies for prevention and mitigation.

METHODS: The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated.

RESULTS: CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM.

METHODS: Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 Plasmodium vivax, 21 Plasmodium falciparum, and 10 Plasmodium malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated.

RESULTS: The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95% CI 89.7-100) and 100% (95% CI 93.2-100), respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95% CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi. The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤ 0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95% CI 65.3-88.9) and specificity of 80.4% (95% CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi.

METHODS: This is a secondary data review of all diagnosed and reported malaria confirmed cases notified to the Ministry of Health, Malaysia between January 2013 and December 2017.

RESULTS: From 2013 to 2017, a total of 16,500 malaria cases were notified in Malaysia. The cases were mainly contributed from Sabah (7150; 43.3%) and Sarawak (5684; 34.4%). Majority of the patients were male (13,552; 82.1%). The most common age group in Peninsular Malaysia was 20 to 29 years (1286; 35.1%), while Sabah and Sarawak reported highest number of malaria cases in age group of 30 to 39 years (2776; 21.6%). The top two races with malaria in Sabah and Sarawak were Bumiputera Sabah (5613; 43.7%) and Bumiputera Sarawak (4512; 35.1%), whereas other ethnic group (1232; 33.6%) and Malays (1025; 28.0%) were the two most common races in Peninsular Malaysia. Plasmodium knowlesi was the commonest species in Sabah and Sarawak (9902; 77.1%), while there were more Plasmodium vivax cases (1548; 42.2%) in Peninsular Malaysia. The overall average incidence rate, mortality rate and case fatality rates for malaria from 2013 to 2017 in Malaysia were 0.106/1000, 0.030/100,000 and 0.27%, respectively. Sarawak reported the highest average incidence rate of 0.420/1000 population followed by Sabah (0.383/1000). Other states in Peninsular Malaysia reported below the national average incidence rate with less than 0.100/1000.

METHODS: Relevant case-control studies that assessed the association between TLR 4/9 and malaria either in susceptibility or progression were searched in health-related electronic databases. Quality of included studies was evaluated with Newcastle-Ottawa scale. Pooled analyses for specific genetic polymorphisms were done under five genetic models. Stratified analysis was done by age and geographical region (Asian countries vs non-Asian countries).

RESULTS: Eleven studies (2716 cases and 2376 controls) from nine endemic countries were identified. Five studies (45.4%) obtained high score in quality assessment. Overall, a significant association between TLR9 (T1486C) and severity of malaria is observed in allele model (OR: 1.26, 95% CI: 1.08-1.48, I2 = 0%) or homozygous model (OR: 1.55, 95% CI: 1.08-2.28, I2 = 0%). For TLR9 (T1237C), a significant association with severity of malaria is observed in in heterozygous model (OR:1.89, 95% CI: 1.11-3.22, I2 = 75%). On stratifications, TLR9 (T1486C) is only significantly associated with a subgroup of children of non-Asian countries under allele model (OR: 1.25, 95% CI: 1.02-1.38), while 1237 is with a subgroup of adults from Asian countries under heterozygous model (OR: 2.0, 95% CI: 1.09-3.64, I2 = 39%). Regarding the susceptibility to malaria, TLR9 (T1237C) is significantly associated only with the children group under recessive model (OR: 2.21, 95% CI: 1.06-4.57, I2=85%) and homozygous model (OR: 1.49, 95% CI: 1.09-2.0, I2 = 0%). For TLR4 (D299G, T399I), none is significantly associated with either severity of malaria or susceptibility to malaria under any genetic models.

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%).

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.

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.