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: 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: 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: 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.
CASE PRESENTATION: A 23-year old splenectomized patient with beta thalassaemia major presented with fever 11 days after receiving a blood transfusion from a pre-symptomatic donor who presented with knowlesi malaria 12 days following blood donation. The infection resulted in severe disease in the recipient, with a parasite count of 84,000/µL and associated metabolic acidosis and multi-organ failure. She was treated with intravenous artesunate and made a good recovery. Sequencing of a highly diverse 649-base pair fragment of the P. knowlesi bifunctional dihydrofolate reductase-thymidylate synthase gene (pkdhfr) revealed that the recipient and donor shared the same haplotype.
CONCLUSIONS: This case demonstrates that acquisition of P. knowlesi from blood transfusion can occur, and that clinical consequences can be severe. Furthermore, this case raises the possibility that thalassaemic patients, particularly those who are splenectomized, may represent a high-risk group for TTM and severe malaria. With rising P. knowlesi incidence, further studies in Sabah are required to determine the risk of TTM in order to guide screening strategies for blood transfusion services.
METHODS: Plasmodium knowlesi, P. cynomolgi, P. coatneyi, P. inui and P. fieldi, were detected using nested PCR assays in DNA samples from 276 wild-caught long-tailed macaques. These samples had been derived from macaques captured at seven locations, two each in the Philippines (n = 68) and Indonesia (n = 70), and one each in Cambodia (n = 54), Singapore (n = 40) and Laos (n = 44). The results were compared with previous studies of malaria parasites in long-tailed macaques from other locations in Southeast Asia. Fisher exact test and Chi square test were used to examine the geographic bias of the distribution of Plasmodium species in the macaque populations.
RESULTS: Out of 276 samples tested, 177 were Plasmodium-positive, with P. cynomolgi being the most common and widely distributed among all long-tailed macaque populations (53.3 %) and occurring in all populations examined, followed by P. coatneyi (20.4 %), P. inui (12.3 %), P. fieldi (3.4 %) and P. knowlesi (0.4 %). One P. knowlesi infection was detected in a macaque from Laos, representing the first documented case of P. knowlesi in wildlife in Laos. Chi square test showed three of the five parasites (P. knowlesi, P. coatneyi, P. cynomolgi) with significant bias in prevalence towards macaques from Malaysian Borneo, Cambodia, and Southern Sumatra, respectively.
CONCLUSIONS: The prevalence of malaria parasites, including those that are transmissible to humans, varied among all sampled regional populations of long-tailed macaques in Southeast Asia. The new discovery of P. knowlesi infection in Laos, and the high prevalence of P. cynomolgi infections in wild macaques in general, indicate the strong need of public advocacy in related countries.
METHODS: Mosquito collections were carried out using human landing catches at ground and canopy levels in the Tawau Division of Sabah. Collections were conducted along an anthropogenic disturbance gradient (primary forest, lightly logged virgin jungle reserve and salvage logged forest) between 18:00 and 22:00 h.
RESULTS: Anopheles balabacensis, a vector of P. knowlesi, was the predominant species in all collection areas, accounting for 70 % of the total catch, with a peak landing time of 18:30-20:00 h. Anopheles balabacensis had a preference for landing on humans at ground level compared to the canopy (p
CASE PRESENTATION: An army officer who returned from Malaysia in October 2016 was found to be positive for Plasmodium both by microscopy and rapid diagnostic test (RDT) by the Anti Malaria Campaign Sri Lanka (AMC) during his third visit to a health care provider. Microscopy findings were suspicious of P. knowlesi infection as the smears showed parasite stages similar to both Plasmodium malariae and Plasmodium falciparum. Nested PCR at AMC confirmed Plasmodium genus, but not the species. In the absence of species confirmation, the patient was treated as a case of P. falciparum. The presence of P. knowlesi was later confirmed by a semi-nested PCR assay performed at the Environmental Health Institute, National Environmental Agency in Singapore. The parasite strain was also characterized by sequencing the circumsporozoite gene. Extensive case investigation including parasitological and entomological surveillance was carried out.
CONCLUSIONS: Plasmodium knowlesi should be suspected in patients returning from countries in the South Asian region where the parasite is prevalent and when blood smear results are inconclusive.
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: Forty full-length pktrap sequences from clinical isolates of Malaysia along with the reference H-strain were downloaded from published databases. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. McDonald-Kreitman test was conducted using P. vivax and Plasmodium coatneyi as ortholog sequence in DnaSP 5.10 software. Population genetic differentiation index (FST) of parasite populations was determined using Arlequin v3.5. Phylogenetic relationships between trap ortholog genes were determined using MEGA 5.0 software.
RESULTS: Comparison of 40 full-length pktrap sequences along with the H-strain identified 74 SNPs (53 non-synonymous and 21 synonymous substitutions) resulting in 29 haplotypes. Analysis of the full-length gene showed that the nucleotide diversity was lower compared to its nearest ortholog pvtrap. Domain-wise analysis indicated that the proline/asparagine rich region had higher nucleotide diversity compared to the von Willebrand factor domain and the thrombospondin-type-1 domain. McDonald-Kreitman test identified that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. knowlesi was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. knowlesi and P. vivax. The von Willebrand factor domain also indicated balancing selection using MK test, however, it did not give significant results when tested with P. coatneyi as an outgroup. Phylogenetic analysis of full-length genes identified three distinct sub-clusters of P. knowlesi, one originating from Peninsular Malaysia and two originating from Malaysian Borneo. High population differentiation values was observed within samples from Peninsular Malaysia and Malaysian Borneo.
CONCLUSIONS: This study is the first to report on the genetic diversity and natural selection of full-length pktrap. Low level of genetic diversity was found across the full-length gene of pktrap. Balancing selection of the von Willebrand factor domain indicated that TRAP could be a target in inducing immune response against P. knowlesi infections. However, higher number of samples would be necessary to further confirm the findings.
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: Eleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5.
RESULTS: Sequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain.
CONCLUSIONS: This study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries.
CASE PRESENTATION: A 46-year-old man presented with fever and acute surgical abdomen with concomitant P. knowlesi malaria infection at Kapit Hospital. He was in compensated shock upon arrival to the hospital. He had generalized abdominal tenderness, maximal at the epigastric region. Bedside focused abdominal ultrasonography revealed free fluid in the abdomen. He underwent emergency exploratory laparotomy in view of haemodynamic instability and worsening peritonism. Intraoperatively, haemoperitoneum and bleeding from the spleen was noted. Splenectomy was performed. Histopathological examination findings were suggestive of splenic rupture and presence of malarial pigment. Analysis of his blood sample by nested PCR assays confirmed P. knowlesi infection. The patient completed a course of anti-malarial treatment and recovered well post-operation.
CONCLUSIONS: Spontaneous splenic rupture is a rare complication of malaria. This is the first reported case of splenic rupture in P. knowlesi malaria infection. Detection of such a complication requires high index of clinical suspicion and is extremely challenging in hospitals with limited resources.
METHODS: Thick and thin blood films were made prior to administration of anti-malarial treatment in patients who were subsequently confirmed as having single species knowlesi infections by PCR assays. Giemsa-stained blood films, prepared from 10 randomly selected patients with a parasitaemia ranging from 610 to 236,000 parasites per microl blood, were examined.
RESULTS: The P. knowlesi infection was highly synchronous in only one patient, where 97% of the parasites were at the late trophozoite stage. Early, late and mature trophozoites and schizonts were observed in films from all patients except three; where schizonts and early trophozoites were absent in two and one patient, respectively. Gametocytes were observed in four patients, comprising only between 1.2 to 2.8% of infected erythrocytes. The early trophozoites of P. knowlesi morphologically resemble those of P. falciparum. The late and mature trophozoites, schizonts and gametocytes appear very similar to those of P. malariae. Careful examinations revealed that some minor morphological differences existed between P. knowlesi and P. malariae. These include trophozoites of knowlesi with double chromatin dots and at times with two or three parasites per erythrocyte and mature schizonts of P. knowlesi having 16 merozoites, compared with 12 for P. malariae.
CONCLUSION: Plasmodium knowlesi infections in humans are not highly synchronous. The morphological resemblance of early trophozoites of P. knowlesi to P. falciparum and later erythrocytic stages to P. malariae makes it extremely difficult to identify P. knowlesi infections by microscopy alone.