Improved Aedes/dengue field surveillance using Gravid Oviposition Sticky trap and dengue NS1 tests: Epidemiological, entomological outcomes and community acceptance.

The aim of this study is to investigate the feasibility and outcomes of using Gravid Oviposition Sticky (GOS) trap and dengue NS1 antigen tests for indoor and outdoor dengue/Aedes surveillance in the field. A one-year community-based study was carried out at Sungai Buloh Hospital Quarters, Selangor, Malaysia. GOS traps were first placed outdoors in three apartment blocks (Anggerik, Bunga Raya and Mawar). Beginning 29th week of the study, indoor traps were set in two apartment units on every floor in Anggerik. All female Aedes mosquitoes caught were tested for the presence of dengue NS1 antigen. Dengue seroprevalence and knowledge, attitude and practices on dengue prevention of the community and their reception to the surveillance approach were also assessed. Dengue-positive mosquitoes were detected at least 1 week before a dengue onset. More mosquitoes were caught indoors than outdoors in block Anggerik, but the total number of mosquitoes caught in all 3 blocks were similar. There was a significant difference in distribution of Ae. aegypti and Ae. albopictus between the 3 blocks. 66.1% and 3.4% of the community were positive for dengue IgG and IgM, respectively. Most respondents think that this surveillance method is Good (89%) and support its use nationwide. Dengue case ratio in the study apartment blocks decreased from year 2018 to 2019. This study demonstrated the practicality of performing proactive dengue/Aedes surveillance inside apartment units using the GOS traps. This surveillance method can be performed with immediate result output in the field.

Case report: recurrence of Plasmodium vivax malaria due to defective cytochrome P450 2D6 function in Pos Lenjang, Pahang, Malaysia.

Five children in Pos Lenjang, Pahang, Malaysia were PCR-positive for vivax malaria and were admitted to the hospital from 5 to 26 July 2019. One of the patients experienced three episodes of recurrence of vivax malaria. Microsatellite analysis showed that reinfection is unlikely. Drug resistance analysis indicated that Riamet (artemether-lumefantrine) is effective. Cytochrome P450 2D6 (CYP2D6) testing showed that this patient has defective CYP2D6 function. Primaquine failure to clear the Plasmodium vivax hypnozoites may be the cause of recurring infections in this patient. This report highlights the need for the development of liver-stage curative antimalarials that do not require metabolism by the CYP2D6 enzyme.

Natural Plasmodium infection in wild macaques of three states in peninsular Malaysia.

Zoonotic cases of Plasmodium knowlesi account for most malaria cases in Malaysia, and humans infected with P. cynomolgi, another parasite of macaques have recently been reported in Sarawak. To date the epidemiology of malaria in its natural Macaca reservoir hosts remains little investigated. In this study we surveyed the prevalence of simian malaria in wild macaques of three states in Peninsular Malaysia, namely Pahang, Perak and Johor using blood samples from 103 wild macaques (collected by the Department of Wildlife and National Parks Peninsular Malaysia) subjected to microscopic examination and nested PCR targeting the Plasmodium small subunit ribosomal RNA gene. As expected, PCR analysis yielded significantly higher prevalence (64/103) as compared to microscopic examination (27/103). No relationship between the age and/or sex of the macaques with the parasitaemia and the Plasmodium species infecting the macaques could be identified. Wild macaques in Pahang had the highest prevalence of Plasmodium parasites (89.7%), followed by those of Perak (69.2%) and Johor (28.9%). Plasmodium inui and P. cynomolgi were the two most prevalent species infecting the macaques from all three states. Half of the macaques (33/64) harboured two or more Plasmodium species. These data provide a baseline survey, which should be extended by further longitudinal investigations that should be associated with studies on the bionomics of the anopheline vectors. This information will allow an accurate evaluation of the risk of zoonotic transmission to humans, and to elaborate effective strategies to control simian malaria.

Distribution of the Duffy genotypes in Malaysian Borneo and its relation to Plasmodium knowlesi malaria susceptibility.

The Duffy blood group plays a key role in Plasmodium knowlesi and Plasmodium vivax invasion into human erythrocytes. The geographical distribution of the Duffy alleles differs between regions with the FY*A allele having high frequencies in many Asian populations, the FY*B allele is found predominately in European populations and the FY*Bes allele found predominantly in African regions. A previous study in Peninsular Malaysia indicated high homogeneity of the dominant FY*A/FY*A genotype. However, the distribution of the Duffy genotypes in Malaysian Borneo is currently unknown. In the present study, the distribution of Duffy blood group genotypes and allelic frequencies among P. knowlesi infected patients as well as healthy individuals in Malaysian Borneo were determined. A total of 79 P. knowlesi patient blood samples and 76 healthy donor samples were genotyped using allele specific polymerase chain reaction (ASP-PCR). Subsequently a P. knowlesi invasion assay was carried out on FY*AB/ FY*A and FY*A/ FY*A Duffy genotype blood to investigate if either genotype conferred increased susceptibility to P. knowlesi invasion. Our results show almost equal distribution between the homozygous FY*A/FY*A and heterozygous FY*A/FY*B genotypes. This is in stark contrast to the Duffy distribution in Peninsular Malaysia and the surrounding Southeast Asian region which is dominantly FY*A/FY*A. The mean percent invasion of FY*A/FY*A and FY*A/FY*B blood was not significantly different indicating that neither blood group confers increased susceptibility to P. knowlesi invasion.

Whole genome sequencing of amplified Plasmodium knowlesi DNA from unprocessed blood reveals genetic exchange events between Malaysian Peninsular and Borneo subpopulations.

The zoonotic Plasmodium knowlesi parasite is the most common cause of human malaria in Malaysia. Genetic analysis has shown that the parasites are divided into three subpopulations according to their geographic origin (Peninsular or Borneo) and, in Borneo, their macaque host (Macaca fascicularis or M. nemestrina). Whilst evidence suggests that genetic exchange events have occurred between the two Borneo subpopulations, the picture is unclear in less studied Peninsular strains. One difficulty is that P. knowlesi infected individuals tend to present with low parasitaemia leading to samples with insufficient DNA for whole genome sequencing. Here, using a parasite selective whole genome amplification approach on unprocessed blood samples, we were able to analyse recent genomes sourced from both Peninsular Malaysia and Borneo. The analysis provides evidence that recombination events are present in the Peninsular Malaysia parasite subpopulation, which have acquired fragments of the M. nemestrina associated subpopulation genotype, including the DBPß and NBPXa erythrocyte invasion genes. The NBPXb invasion gene has also been exchanged within the macaque host-associated subpopulations of Malaysian Borneo. Our work provides strong evidence that exchange events are far more ubiquitous than expected and should be taken into consideration when studying the highly complex P. knowlesi population structure.

Plasmodium knowlesi malaria: current research perspectives.

Originally known to cause simian malaria, Plasmodium knowlesi is now known as the fifth human malaria species. Since the publishing of a report that largely focused on human knowlesi cases in Sarawak in 2004, many more human cases have been reported in nearly all of the countries in Southeast Asia and in travelers returning from these countries. The zoonotic nature of this infection hinders malaria elimination efforts. In order to grasp the current perspective of knowlesi malaria, this literature review explores the different aspects of the disease including risk factors, diagnosis, treatment, and molecular and functional studies. Current studies do not provide sufficient data for an effective control program. Therefore, future direction for knowlesi research is highlighted here with a final aim of controlling, if not eliminating, the parasite.

Importance of Proactive Malaria Case Surveillance and Management in Malaysia.

Although Plasmodium vivax infections in Malaysia are usually imported, a significant autochthonous outbreak of vivax malaria was detected in a remote indigenous (Orang Asli) settlement located in northern peninsular Malaysia. Between November 2016 and April 2017, 164 cases of P. vivax infection were detected. Although 83.5% of the vivax cases were identified through passive case detection and contact screening during the first 7 weeks, subsequent mass blood screening (combination of rapid diagnostic tests, blood films, and polymerase chain reaction [PCR]) of the entire settlement (N = 3,757) revealed another 27 P. vivax infections, 19 of which were asymptomatic. The mapped data from this active case detection program was used to direct control efforts resulting in the successful control of the outbreak in this region. This report highlights the importance of proactive case surveillance and timely management of malaria control in Malaysia as it nears malaria elimination.

Genetic clustering and polymorphism of the merozoite surface protein-3 of Plasmodium knowlesi clinical isolates from Peninsular Malaysia.

BACKGROUND: The simian malaria parasite Plasmodium knowlesi has been reported to cause significant numbers of human infection in South East Asia. Its merozoite surface protein-3 (MSP3) is a protein that belongs to a multi-gene family of proteins first found in Plasmodium falciparum. Several studies have evaluated the potential of P. falciparum MSP3 as a potential vaccine candidate. However, to date no detailed studies have been carried out on P. knowlesi MSP3 gene (pkmsp3). The present study investigates the genetic diversity, and haplotypes groups of pkmsp3 in P. knowlesi clinical samples from Peninsular Malaysia. METHODS: Blood samples were collected from P. knowlesi malaria patients within a period of 4 years (2008-2012). The pkmsp3 gene of the isolates was amplified via PCR, and subsequently cloned and sequenced. The full length pkmsp3 sequence was divided into Domain A and Domain B. Natural selection, genetic diversity, and haplotypes of pkmsp3 were analysed using MEGA6 and DnaSP ver. 5.10.00 programmes. RESULTS: From 23 samples, 48 pkmsp3 sequences were successfully obtained. At the nucleotide level, 101 synonymous and 238 non-synonymous mutations were observed. Tests of neutrality were not significant for the full length, Domain A or Domain B sequences. However, the dN/dS ratio of Domain B indicates purifying selection for this domain. Analysis of the deduced amino acid sequences revealed 42 different haplotypes. Neighbour Joining phylogenetic tree and haplotype network analyses revealed that the haplotypes clustered into two distinct groups. CONCLUSIONS: A moderate level of genetic diversity was observed in the pkmsp3 and only the C-terminal region (Domain B) appeared to be under purifying selection. The separation of the pkmsp3 into two haplotype groups provides further evidence of the existence of two distinct P. knowlesi types or lineages. Future studies should investigate the diversity of pkmsp3 among P. knowlesi isolates in North Borneo, where large numbers of human knowlesi malaria infection still occur.

Expression and Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-3 (MSP-3) for Detection of Human Malaria.

Malaria remains a major health threat in many parts of the globe and causes high mortality and morbidity with 214 million cases of malaria occurring globally in 2015. Recent studies have outlined potential diagnostic markers and vaccine candidates one of which is the merozoite surface protein (MSP)-3. In this study, novel recombinant Plasmodium knowlesi MSP-3 was cloned, expressed and purified in an Escherichia coli system. Subsequently, the recombinant protein was evaluated for its sensitivity and specificity. The recombinant pkMSP-3 protein reacted with sera from patients with P. knowlesi infection in both Western blot (61%) and ELISA (100%). Specificity-wise, pkMSP-3 did not react with healthy donor sera in either assay and only reacted with a few non-malarial parasitic patient sera in the ELISA assay (3 of 49). In conclusion, sensitivity and specificity of pkMSP-3 was found to be high in the ELISA and Western Blot assay and thus utilising both assays in tandem would provide the best sero-diagnostic result for P. knowlesi infection.

Genotyping of the Duffy blood group among Plasmodium knowlesi-infected patients in Malaysia.

The Duffy blood group is of major interest in clinical medicine as it plays an important role in Plasmodium knowlesi and Plasmodium vivax infection. In the present study, the distribution of Duffy blood group genotypes and allelic frequencies among P. knowlesi infected patients as well as healthy individuals in Peninsular Malaysia were determined. The blood group of 60 healthy blood donors and 51 P. knowlesi malaria patients were genotyped using allele specific polymerase chain reaction (ASP-PCR). The data was analyzed using Fisher's exact test in order to assess the significance of the variables. Our results show a high proportion of the FY*A/FY*A genotype (>85% for both groups) and a high frequency of the FY*A allele (>90% for both groups). The FY*A/FY*A genotype was the most predominant genotype in both infected and healthy blood samples. The genotype frequency did not differ significantly between the donor blood and the malaria patient groups. Also, there was no significant correlation between susceptibility to P. knowlesi infection with any Duffy blood genotype.