METHODS: A hydrolysis probe for a real-time PCR assay was designed to recognize a specific DNA sequence within the P. knowlesi small subunit ribosomal RNA gene. The sensitivity, linearity and specificity of the assay were determined using plasmids containing P. knowlesi DNA and genomic DNA of P. falciparum, P. knowlesi, P. malariae, P. ovale and P. vivax isolated from clinical samples. DNA samples of the simian malaria parasites Plasmodium cynomolgi and Plasmodium inui that can infect humans under experimental conditions were also examined together with human DNA samples.

RESULTS: Analytical sensitivity of the P. knowlesi-specific assay was 10 copies/μL and quantitation was linear over a range of 10-106 copies. The sensitivity of the assay is equivalent to nested PCR and P. knowlesi DNA was detected from all 40 clinical P. knowlesi specimens, including one from a patient with a parasitaemia of three parasites/μL of blood. No cross-reactivity was observed with 67 Plasmodium DNA samples (31 P. falciparum, 23 P. vivax, six P. ovale, three P. malariae, one P. malariae/P. ovale, one P. falciparum/P. malariae, one P. inui and one P. cynomolgi) and four samples of human DNA.

METHODS: Using CRISPR/Cas9 gene editing two chimeric P. falciparum parasites, were generated, where the pfcsp gene has been replaced by either one of the two major pvcsp alleles, VK210 or VK247.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.

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.

METHODS: A household-based cross-sectional study was conducted in March 2024 in six Semai sub-ethnic indigenous villages in Pos Lenjang, Kuala Lipis, Pahang. A structured questionnaire was administered to randomly selected individuals (≥ 12 years old) to collect data on sociodemographic characteristics and KAP. Data were analysed using descriptive statistics and predictors of KAP were determined using logistic regression. A p-value less than 0.05 was considered statistically significant.

METHODS: Twenty-five P. knowlesi samples collected in 2018-2023 were sequenced for the 42-kDa region of pkmsp1 and compared with 24 retrieved sequences in 2000-2009, focusing on nucleotide diversity, natural selection, recombination rate, and population differentiation.

METHODS: A total of 71 malaria microscopy positive blood samples collected in blood spots were obtained from the Sarawak State Health Department. Using 18s rRNA as the target gene, nested PCR and SYBR green I LAMP assay were performed following the DNA extraction. The colour changes of LAMP end products were observed by naked eyes.

RESULTS: LAMP assay demonstrated a detection limit of 10 copies/µL in comparison with 100 copies/µL nested PCR. Of 71 P. knowlesi blood samples collected, LAMP detected 69 microscopy-positive samples. LAMP exhibited higher sensitivity than nested PCR assay. The SYBR green I LAMP assay was 97.1% sensitive (95% CI 90.2-99.7%) and 100% specific (95% CI 83.2-100%). Without opening the cap, incorporation of SYBR green I into the inner cap of the tube enabled the direct visualization of results upon completion of amplification. The positives instantaneously turned green while the negatives remained orange.

METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.

RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.

METHODS: A total of 189 whole blood samples were collected from Telupid Health Clinic, Sabah, Malaysia, from 2008 to 2011. All patients who participated in the study were microscopically malaria positive before recruitment. Complete demographic details and haematological profiles were obtained from 85 patients (13 females and 72 males). Identification of Plasmodium species was conducted using PlasmoNex™ targeting the 18S ssu rRNA gene.

RESULTS: A total of 178 samples were positive for Plasmodium species by using PlasmoNex™. Plasmodium falciparum was identified in 68 samples (38.2%) followed by 64 cases (36.0%) of Plasmodium vivax, 42 (23.6%) cases of P. knowlesi, two (1.1%) cases of Plasmodium malariae and two (1.1%) mixed-species infections (i e, P. vivax/P. falciparum). Thirty-five PlasmoNex™ positive P. knowlesi samples were misdiagnosed as P. malariae by microscopy. Plasmodium knowlesi was detected in all four districts of Sandakan division with the highest incidence in the Kinabatangan district. Thrombocytopaenia and anaemia showed to be the most frequent malaria-associated haematological complications in this study.

METHODS: The prevalence of G6PD deficiency among 408 Thai participants diagnosed with malaria by microscopy (71), and malaria-negative controls (337), was assessed using a phenotypic test based on water-soluble tetrazolium salts. High-resolution melting (HRM) curve analysis was developed from a previous study to enable the detection of 15 common missense, synonymous and intronic G6PD mutations in Asian populations. The identified mutations were subjected to biochemical and structural characterisation to understand the molecular mechanisms underlying enzyme deficiency.

RESULTS: Based on phenotypic testing, the prevalence of G6PD deficiency (T) and intronic (c.1365-13T>C and c.486-34delT) mutations was detected with intermediate to normal enzyme activity. The double missense mutations were less catalytically active than their corresponding single missense mutations, resulting in severe enzyme deficiency. While the mutations had a minor effect on binding affinity, structural instability was a key contributor to the enzyme deficiency observed in G6PD-deficient individuals.

METHODS: A descriptive cross-sectional study with a semi-structured questionnaire was carried out among 100 and 123 households from forest-aboriginal and rural areas, respectively.

RESULTS: Knowledge about malaria and its transmission is significantly higher among the rural participants than the aborigines (86.2% vs 76%, p < 0.01). However, use of medicinal plants and beliefs in witchcraft and sorcery in treating febrile diseases were significantly higher among the aboriginal population (p < 0.01). There were no significant differences between the two communities in terms of the knowledge about malaria symptoms, attitudes towards its severity and practices in preventive measures against malaria by using mosquito bed nets. However, the knowledge and practice of different preventive measures to combat malaria, such as insecticide and the elimination of breeding areas, was significantly higher among the rural population than the aborigines (p < 0.001).

METHODS: This study conducted the PRISMA-ScR scoping review and formulated a set of research questions to identify current trends in vector-borne diseases in Borneo. These questions aim to identify which diseases have been studied, what geographical areas have been covered by the research, how the One Health approach-encompassing human, animal and environmental factors-is integrated, and what gaps and challenges exist in addressing these diseases.

RESULTS: A total of 2241 references were screened for eligibility and 117 articles were selected for review. The majority of the materials focused on mosquitoes and malaria, and the One Health elements focused mainly on humans.

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.