Background: Malaysia recorded the highest number of dengue cases between 2014 and 2017. There are 13 states and three federal territories in Malaysia, and each area varies in their prevalence of dengue. Sabah is one of the states situated in Borneo, Malaysia. Although dengue has been increasing for the last several years, no study was being done to understand the burden and serotype distribution of the dengue virus (DENV) in Sabah. Therefore, the present study was carried out to understand the epidemiology of the dengue infection and the factors responsible for severe dengue in Sabah. Methods: Data on dengue infection were extracted from the dengue database of the state of Sabah from 2013 through 2018. DENV NS-1-positive serum samples from multiple sites throughout Sabah were sent to the state public health laboratory, Kota Kinabalu Public Health Laboratory, for serotype determination. The analysis of factors associated with severe dengue was determined from the data of 2018 only. Results: In 2013, there were 724 dengue cases; however, from 2014, dengue cases increased exponentially and resulted in 3423 cases in 2018. Increasing dengue cases also led to increased dengue mortality. The number of dengue deaths in 2013 was only five which then gradually increased, and in 2018, 29 patients died. This is an increase of 580% from 2013 to 2018. Deaths were considerably more in the districts of the east coast of Sabah compared with districts in the west coast. During the study period, all DENV serotypes could be identified as serotypes circulating in Sabah. In 2018, the predominant serotype was DENV-3. The monthly peak of dengue infection varied in different years. In the logistic regression analysis, it was identified that children were 6.5 times, patients infected with mixed serotype of DENV were 13 times, and cases from the districts of the east coast were 5.2 times more likely to develop severe dengue. Conclusions: An increasing trend of dengue infection has been observed in Sabah. The burden of dengue, severe dengue, and mortality was noted especially in the districts of the east coast of Sabah. Severe dengue was most likely developed in children, cases from the east coast, and patients infected with mixed serotype of DENV.

The incidence of zoonotic malaria, Plasmodium knowlesi, infection is increasing and now is the major cause of malaria in Malaysia. Here, we describe a WarmStart colorimetric loop-mediated isothermal amplification (LAMP) assay for the detection of Plasmodium spp. The detection limit for this assay was 10 copies/μL for P knowlesi and Plasmodium ovale and 1 copy/μL for Plasmodium falciparum, Plasmodium vivax, and Plasmodium malariae. To test clinical sensitivity and specificity, 100 microscopy-positive and 20 malaria-negative samples were used. The WarmStart colorimetric LAMP was 98% sensitive and 100% specific. Amplification products were visible for direct observation, thereby eliminating the need for post-amplification processing steps. Therefore, WarmStart colorimetric LAMP is suitable for use in resourcelimited settings.

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Dengue fever is an arthropod-borne viral disease caused by the Dengue virus (genus Falvivirus, family Flaviviridae). It has rapidly spread all over the world affecting approximately 400 million people annually. Human dengue infection is caused by four types of closely related viruses (also called serotypes) namely DENV-1, DENV-2, DENV-3, and DENV-4, all of which can be all found in Sabah, Malaysia. Each serotype can then be divided into unique groups based on its genotypes. In Malaysia, dengue has been reported as the most prevalent disease of the country with a ratio of 328.3 cases per 100,000 populations. Exacerbating this further, it was also recently reported in 2017 of the emergence of a newly identified Asian lineage dengue virus i.e. type 3 genotype II (D3GII) in Malaysia. We have aimed, through this study, to examine the serotypes and the genotypes of dengue virus circulating in Sabah. This study was conducted for a period of 8 months i.e. from January to August 2017. A total of 52 NS1 (50.9% were males and 49.1% were females) positive dengue patient serum samples were genotyped. Viral RNA was extracted from serum using QIAamp viral RNA mini kit and DNA sequencing was done on Applied Biosystems 3730xl DNA analyzer. The results showed that serotype DENV-3 was the most predominant dengue circulating virus in Sabah with 23 cases detected. These were further grouped under three genotypes namely D3GI (1 case), D3GII (14 cases) and D3GIII (8 cases). Serotype DENV-1 was the second most common circulating virus in Sabah with 17 cases and grouped under two genotypes, D1Gia (15 cases) and D1Gic (2 cases), respectively. On the other hand, only one genotype (D4GII) was detected for DENV-4 (9 cases), and two genotypes (D2 Cosmopolitan Clade I and D2 Cosmopolitan Clade Ib) for DENV-2, each with one case per genotype, respectively. Understanding of genotype diversity will be useful in designing strategies for dengue management in epidemiological surveillance and vaccine design.

The Plasmodium knowlesi secreted protein with an altered thrombospondin repeat (PkSPATR) is an important protein that helps in the parasite's invasion into the host cell. This protein has been regarded as one of the potential vaccine candidates against P. knowlesi infection. This study investigates the genetic diversity and natural selection of PkSPATR gene of P. knowlesi clinical isolates from Malaysia. PCR amplification of the full length PkSPATR gene was performed on 60 blood samples of infected P. knowlesi patients from Peninsular Malaysia and Malaysian Borneo. The amplified PCR products were cloned and sequenced. Sequence analysis of PkSPATR from Malaysia showed higher nucleotide diversity (CDS p: 0.01462) than previously reported Plasmodium vivax PvSPATR (p = 0.0003). PkSPATR from Peninsular Malaysia was observed to have slightly higher diversity (CDS p: 0.01307) than those from Malaysian Borneo (CDS p: 0.01212). Natural selection analysis on PkSPATR indicated significant purifying selection. Multiple amino acid sequence alignment revealed 69 polymorphic sites. The phylogenetic tree and haplotype network did not show any distinct clustering of PkSPATR. The low genetic diversity level, natural selection and absence of clustering implied functional constrains of the PkSPATR protein.

Plasmodium knowlesi is a simian malaria parasite that causes significant zoonotic infections in Southeast Asia, particularly in Malaysia. The Plasmodium thrombospondin-related apical merozoite protein (TRAMP) plays an essential role in the invasion of the parasite into its host erythrocyte. The present study investigated the genetic polymorphism and natural selection of the full length PkTRAMP from P. knowlesi clinical isolates from Malaysia. Blood samples (n = 40) were collected from P. knowlesi malaria patients from Peninsular Malaysia and Malaysian Borneo. The PkTRAMP gene was amplified using PCR, followed by cloning into a plasmid vector and sequenced. Results showed that the nucleotide diversity of PkTRAMP was low (π: 0.009). Z-test results indicated negative (purifying) selection of PkTRAMP. The alignment of the deduced amino acid sequences of PkTRAMP of Peninsular Malaysia and Malaysian Borneo revealed 38 dimorphic sites. A total of 27 haplotypes were identified from the amino acid sequence alignment. Haplotype analysis revealed that there was no clustering of PkTRAMP from Peninsular Malaysia and Malaysian Borneo.

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