Malaria causes high global mortality and morbidity annually. Plasmodium knowlesi has been recognised as the fifth human Plasmodium sp. and its infection is widely distributed in Southeast Asia. Merozoite surface protein-119 (MSP-119) appears as a potential candidate for malaria blood stage vaccine as it could induce protective immunity. In this study, codon optimized P. knowlesi MSP-119 (pkMSP-119) was expressed and purified in yeast Pichia pastoris expression system. The purified recombinant protein was further evaluated using Western blot assay using knowlesi malaria, non-knowlesi human malaria, non-malarial parasitic infections and healthy serum samples (n = 50). The sensitivity of purified pkMSP-119 towards detection of knowlesi infection was as 28.6% (2/7). pkMSP-119 did not react with all nonmalarial parasitic infections and healthy donor sera, yet reacted with some non-knowlesi human malaria sera, therefore lead to a specificity of 86.0% (37/43).

Circumsporozoite protein (CSP) central repeat region is one of the main target regions of the RTS,S/AS01 vaccine for falciparum infection as it consists of immunodominant B cell epitopes. However, there is a lack of study for P. knowlesi CSP central repeat region. This study aims to characterise the CSP repeat motifs of P. knowlesi isolates in Peninsular Malaysia. CSP repeat motifs of 64 P. knowlesi isolates were identified using Rapid Automatic Detection and Alignment of Repeats (RADAR). Antigenicity of the repeat motifs and linear B cell epitopes were predicted using VaxiJen 2.0, BepiPred-2.0 and BCPred, respectively. A total of 35 dominant repeat motifs were identified. The repeat motif “AGQPQAQGDGANAGQPQAQGDGAN” has the highest repeat frequency (n=15) and antigenicity index of 1.7986. All the repeat regions were predicted as B cell epitopes. In silico approaches revealed that all repeat motifs were antigenic and consisted of B cell epitopes which could be designed as knowlesi malaria vaccine.

In Malaysia presently, the main cause of human malaria is by the zoonotic monkey parasite Plasmodium knowlesi. A previous study has suggested that the P. knowlesi merozoite surface protein 1 (Pkmsp-1) block IV to be a suitable multiplicity of infection (MOI) genotyping marker for knowlesimalaria. This study therefore aimed to investigate the usefulness of Pkmsp-1 block IV in assessing the MOI of P. knowlesi in clinical isolates from Malaysia. Two allele-specific PCR primer pairs targeting the two allelic families of block IV (T1 and T2) were designed, and used to genotype P. knowlesi in 200 blood samples (100 from Peninsular Malaysia and 100 from Malaysian Borneo). Results showed that the mean MOI in Malaysian Borneo was slightly higher as compared to Peninsular Malaysia (1.58 and 1.40, respectively). Almost half of the total blood samples from Malaysian Borneo (52%) had polyclonal infections (i.e., more than one allele of any family type) as compared to Peninsular Malaysia (33%) samples. The T1 allelic family was more prevalent in Peninsular Malaysia (n=75) than in Malaysian Borneo (n=60). The T2 allelic family, however, was more prevalent in the Malaysian Borneo (n=87 vs n=53 respectively). This study shows that the single locus Pkmsp-1 block IV can serve as a simple alternative genetic marker for estimating knowlesi malaria MOI in a population. Future MOI studies should focus on macaque populations as macaques are the natural host of P. knowlesi.

Normocyte binding protein Xa (NBPXa) has been implied to play a significant role in parasite invasion of human erythrocytes. Previous phylogenetic studies have reported the existence of three types of NBPXa for Plasmodium knowlesi (PkNBPXa). PkNBPXa region II (PkNBPXaII) of type 1, type 2 and type 3 were expressed on mammalian cell surface and interacted with human and macaque (Macaca fascicularis) erythrocytes. The binding activities of PkNBPXaII towards human and macaque erythrocytes were evaluated using erythrocyte-binding assay (EBA). Three parameters were evaluated to achieve the optimal protein expression of PkNBPXaII and erythrocyte binding activity in EBA: types of mammalian cells, post transfection time and erythrocyte incubation time. COS-7, HEK-293, and CHO-K1 cells showed successful expression of PkNBPXaII, despite the protein expression is weak compared to the positive control. COS-7 was used in EBA. All three types of PkNBPXaII showed rosette formation with macaque erythrocytes but not with human erythrocytes. Future studies to enhance the PkNBPXaII expression on surface of mammalian cells is indeed needed in order to elucidate the specific role of PkNBPXaII in erythrocytes invasion.

The continued absence of an effective and safe tetravalent dengue vaccine and the lack of specific anti-viral treatment have made mosquito vector control using chemical insecticides as the mainstream for dengue prevention and control. However, the long-term use of chemical insecticides may induce resistance. Hence detection of insecticide resistance in dengue vectors is crucially important in ensuring the insecticide-based intervention in dengue control program is still effective and reliable. In this study, the susceptibility status of Aedes aegypti from five selected dengue hotspots in Klang Valley, Malaysia against pyrethroids was determined by employing the World Health Organization (WHO) protocol of adult bioassay. Four types of pyrethroids were tested against adult female Aedes aegypti to determine the knockdown rate, post 24-h adult mortality and resistance ratio. All field-collected Aedes aegypti strains were resistant to the four pyrethroids tested, except for the Taman Sungai Jelok (TSJ) strain. Permethrin exhibited the lowest knockdown rate against Aedes aegypti, followed by deltamethrin, cyfluthrin and lambda-cyhalothrin. This present study indicated the widespread of pyrethroid resistance in Aedes aegypti in Klang Valley, indicating the needs of implementing alternative measures in vector control program. The data in this study can be utilised as an input for insecticide resistance management of Aedes aegypti in Malaysia.

Recent reports of natural human infection by Plasmodium cynomolgi indicate the increased risk of zoonotic transmission by this simian parasite. The P. cynomolgi Duffy binding protein 2 (PcDBP2) has a potential role in the invasion pathway of host erythrocytes, and it is a possible vaccine candidate against cynomolgi malaria. This study investigates the genetic diversity, haplotypes, and natural selection of PcDBP2 region II from isolates collected from wild macaques in Peninsular Malaysia. Blood samples from 50 P. cynomolgi-infected wild macaques were used in the study. Genomic DNA extracted from the blood samples was used as template for PCR amplification of the PcDBP2 region II. The amplicons were cloned into a plasmid vector and sequenced. MEGA X and DnaSP ver.6.12.03 programmes were used to analyse the DNA sequences. A genealogical relationship of PcDBP2 region II were determined using haplotype network tree on NETWORK ver.10.2. Result showed high genetic diversity (ð = 0.017 ± 0.002; Hd = 1.000 ± 0.001) of the PcDBP2 region II. The Z-test indicates a purifying selection, with population expansion as shown in Tajima’s D analysis. A total of 146 haplotypes of PcDBP2 region II were observed. Phylogenetic tree analysis showed that these haplotypes were grouped into three allelic types (136 for Strain B type, 9 for Berok type, and 1 recombinant type). In the haplotype network, PcDBP2 region II revealed no geographical groupings but was divided into two distinct clusters.

Diseases such as malaria, dengue, Zika and chikungunya remain endemic in many countries. Setting and deploying traps to capture the host/vector species are fundamental to understand their density and distributions. Human effort to manage the trap data accurately and timely is an exhaustive endeavour when the study area expands and period prolongs. One stop mobile app to manage and monitor the process of targeted species trapping, from field to laboratory level is still scarce. Toward this end, we developed a new mobile app named “PesTrapp” to acquire the vector density index based on the mobile updates of ovitraps and species information in field and laboratory. This study aimed to highlight the mobile app’s development and design, elucidate the practical user experiences of using the app and evaluate the preliminary user assessment of the mobile app. The mobile app was developed using mobile framework and database. User evaluation of the mobile app was based on the adjusted Mobile App Rating Scale and Standardized User Experience Percentile Rank Questionnaire. The process flows of system design and detailed screen layouts were described. The user experiences with and without the app in a project to study Aedes surveillance in six study sites in Selangor, Malaysia were elucidated. The overall mean user evaluation score of the mobile app was 4.0 out of 5 (SD=0.6), reflects its acceptability of the users. The PesTrapp, a one-stop solution, is anticipated to improve the entomological surveillance work processes. This new mobile app can contribute as a tool in the vector control countermeasure strategies.