Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia.

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.

End-point detection of loop-mediated isothermal amplification (LAMP) on malaria by direct observation with colorimetric dyes.

In order to ascertain the results of the LAMP technique, different end-point detection methods can be employed. However, these methods require sophisticated equipment. To simplify current end-point detection methods for the diagnosis of malaria, we propose the incorporation of colorimetric dyes: malachite green (MG), phenol red (PR), and xylenol orange (XO) in the LAMP assay. To evaluate the optimum concentration of dyes, 5 different concentrations (50 µM, 75 µM, 100 µM, 125 µM, and 150 µM) were used with buffer pH 8.5 and pH 8.8, respectively. The results showed that 125 µM of MG at pH 8.8 produced the most obvious colour change. A total of 71 clinical blood samples of Plasmodium knowlesi, Plasmodium malariae, Plasmodium vivax, Plasmodium falciparum, and healthy donors were tested using MG-LAMP. It showed 100% sensitivity and specificity. The simplicity and affordability of this method make it ideal to be used as an end-point detection method for malaria diagnosis in resource limited settings.

Spatial distribution of Plasmodium knowlesi cases and their vectors in Johor, Malaysia: in light of human malaria elimination.

BACKGROUND: Plasmodium knowlesi, a simian malaria parasite infection, increases as Plasmodium falciparum and Plasmodium vivax infections decrease in Johor, Malaysia. Therefore, this study aimed to identify the distribution of vectors involved in knowlesi malaria transmission in Johor. This finding is vital in estimating hotspot areas for targeted control strategies. METHODS: Anopheles mosquitoes were collected from the location where P. knowlesi cases were reported. Cases of knowlesi malaria from 2011 to 2019 in Johor were analyzed. Internal transcribed spacers 2 (ITS2) and cytochrome c oxidase subunit I (COI) genes were used to identify the Leucosphyrus Group of Anopheles mosquitoes. In addition, spatial analysis was carried out on the knowlesi cases and vectors in Johor. RESULTS: One hundred and eighty-nine cases of P. knowlesi were reported in Johor over 10 years. Young adults between the ages of 20-39 years comprised 65% of the cases. Most infected individuals were involved in agriculture and army-related occupations (22% and 32%, respectively). Four hundred and eighteen Leucosphyrus Group Anopheles mosquitoes were captured during the study. Anopheles introlatus was the predominant species, followed by Anopheles latens. Spatial analysis by Kriging interpolation found that hotspot regions of P. knowlesi overlapped or were close to the areas where An. introlatus and An. latens were found. A significantly high number of vectors and P. knowlesi cases were found near the road within 0-5 km. CONCLUSIONS: This study describes the distribution of P. knowlesi cases and Anopheles species in malaria-endemic transmission areas in Johor. Geospatial analysis is a valuable tool for studying the relationship between vectors and P. knowlesi cases. This study further supports that the Leucosphyrus Group of mosquitoes might be involved in transmitting knowlesi malaria cases in Johor. These findings may provide initial evidence to prioritize diseases and vector surveillance.

Plasmodium knowlesi Malaria in Sabah, Malaysia, 2015-2017: Ongoing Increase in Incidence Despite Near-elimination of the Human-only Plasmodium Species.

BACKGROUND: Malaysia aims to eliminate malaria by 2020. However, while cases of Plasmodium falciparum and Plasmodium vivax have decreased substantially, the incidence of zoonotic malaria from Plasmodium knowlesi continues to increase, presenting a major challenge to regional malaria control efforts. Here we report incidence of all Plasmodium species in Sabah, including zoonotic P. knowlesi, during 2015-2017. METHODS: Microscopy-based malaria notification data and polymerase chain reaction (PCR) results were obtained from the Sabah Department of Health and State Public Health Laboratory, respectively, from January 2015 to December 2017. From January 2016 this was complemented by a statewide prospective hospital surveillance study. Databases were matched, and species was determined by PCR, or microscopy if PCR was not available. RESULTS: A total of 3867 malaria cases were recorded between 2015 and 2017, with PCR performed in 93%. Using PCR results, and microscopy if PCR was unavailable, P. knowlesi accounted for 817 (80%), 677 (88%), and 2030 (98%) malaria cases in 2015, 2016, and 2017, respectively. P. falciparum accounted for 110 (11%), 45 (6%), and 23 (1%) cases and P. vivax accounted for 61 (6%), 17 (2%), and 8 (0.4%) cases, respectively. Of those with P. knowlesi, the median age was 35 (interquartile range: 24-47) years, and 85% were male. CONCLUSIONS: Malaysia is approaching elimination of the human-only Plasmodium species. However, the ongoing increase in P. knowlesi incidence presents a major challenge to malaria control and warrants increased focus on knowlesi-specific prevention activities. Wider molecular surveillance in surrounding countries is required.

Updates on malaria incidence and profile in Malaysia from 2013 to 2017.

BACKGROUND: To date, most of the recent publications on malaria in Malaysia were conducted in Sabah, East Malaysia focusing on the emergence of Plasmodium knowlesi. This analysis aims to describe the incidence, mortality and case fatality rate of malaria caused by all Plasmodium species between Peninsular Malaysia and East Malaysia (Sabah and Sarawak) over a 5-year period (2013-2017). METHODS: This is a secondary data review of all diagnosed and reported malaria confirmed cases notified to the Ministry of Health, Malaysia between January 2013 and December 2017. RESULTS: From 2013 to 2017, a total of 16,500 malaria cases were notified in Malaysia. The cases were mainly contributed from Sabah (7150; 43.3%) and Sarawak (5684; 34.4%). Majority of the patients were male (13,552; 82.1%). The most common age group in Peninsular Malaysia was 20 to 29 years (1286; 35.1%), while Sabah and Sarawak reported highest number of malaria cases in age group of 30 to 39 years (2776; 21.6%). The top two races with malaria in Sabah and Sarawak were Bumiputera Sabah (5613; 43.7%) and Bumiputera Sarawak (4512; 35.1%), whereas other ethnic group (1232; 33.6%) and Malays (1025; 28.0%) were the two most common races in Peninsular Malaysia. Plasmodium knowlesi was the commonest species in Sabah and Sarawak (9902; 77.1%), while there were more Plasmodium vivax cases (1548; 42.2%) in Peninsular Malaysia. The overall average incidence rate, mortality rate and case fatality rates for malaria from 2013 to 2017 in Malaysia were 0.106/1000, 0.030/100,000 and 0.27%, respectively. Sarawak reported the highest average incidence rate of 0.420/1000 population followed by Sabah (0.383/1000). Other states in Peninsular Malaysia reported below the national average incidence rate with less than 0.100/1000. CONCLUSIONS: There were different trends and characteristics of notified malaria cases in Peninsular Malaysia and Sabah and Sarawak. They provide useful information to modify current prevention and control measures so that they are customised to the peculiarities of disease patterns in the two regions in order to successfully achieve the pre-elimination of human-only species in the near future.

Comparative evaluation of two commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium knowlesi and other Plasmodium species in Sabah, Malaysia.

BACKGROUND: The monkey parasite Plasmodium knowlesi is an emerging public health issue in Southeast Asia. In Sabah, Malaysia, P. knowlesi is now the dominant cause of human malaria. Molecular detection methods for P. knowlesi are essential for accurate diagnosis and in monitoring progress towards malaria elimination of other Plasmodium species. However, recent commercially available PCR malaria kits have unpublished P. knowlesi gene targets or have not been evaluated against clinical samples. METHODS: Two real-time PCR methods currently used in Sabah for confirmatory malaria diagnosis and surveillance reporting were evaluated: the QuantiFast™ Multiplex PCR kit (Qiagen, Germany) targeting the P. knowlesi 18S SSU rRNA; and the abTES™ Malaria 5 qPCR II kit (AITbiotech, Singapore), with an undisclosed P. knowlesi gene target. Diagnostic accuracy was evaluated using 52 P. knowlesi, 25 Plasmodium vivax, 21 Plasmodium falciparum, and 10 Plasmodium malariae clinical isolates, and 26 malaria negative controls, and compared against a validated reference nested PCR assay. The limit of detection (LOD) for each PCR method and Plasmodium species was also evaluated. RESULTS: The sensitivity of the QuantiFast™ and abTES™ assays for detecting P. knowlesi was comparable at 98.1% (95% CI 89.7-100) and 100% (95% CI 93.2-100), respectively. Specificity of the QuantiFast™ and abTES™ for P. knowlesi was high at 98.8% (95% CI 93.4-100) for both assays. The QuantiFast™ assay demonstrated falsely-positive mixed Plasmodium species at low parasitaemias in both the primary and LOD analysis. Diagnostic accuracy of both PCR kits for detecting P. vivax, P. falciparum, and P. malariae was comparable to P. knowlesi. The abTES™ assay demonstrated a lower LOD for P. knowlesi of ≤ 0.125 parasites/µL compared to QuantiFast™ with a LOD of 20 parasites/µL. Hospital microscopy demonstrated a sensitivity of 78.8% (95% CI 65.3-88.9) and specificity of 80.4% (95% CI 67.6-89.8) compared to reference PCR for detecting P. knowlesi. CONCLUSION: The QuantiFast™ and abTES™ commercial PCR kits performed well for the accurate detection of P. knowlesi infections. Although the QuantiFast™ kit is cheaper, the abTES™ kit demonstrated a lower LOD, supporting its use as a second-line referral-laboratory diagnostic tool in Sabah, Malaysia.

Plasmodium falciparum artemisinin resistance monitoring in Sabah, Malaysia: in vivo therapeutic efficacy and kelch13 molecular marker surveillance.

BACKGROUND: Spreading Plasmodium falciparum artemisinin drug resistance threatens global malaria public health gains. Limited data exist to define the extent of P. falciparum artemisinin resistance southeast of the Greater Mekong region in Malaysia. METHODS: A clinical efficacy study of oral artesunate (total target dose 12 mg/kg) daily for 3 days was conducted in patients with uncomplicated falciparum malaria and a parasite count < 100,000/µL admitted to 3 adjacent district hospitals in Sabah, East Malaysia. On day 3 and 4 all patients were administered split dose mefloquine (total dose 25 mg/kg) and followed for 28 days. Twenty-one kelch13 polymorphisms associated with P. falciparum artemisinin resistance were also evaluated in P. falciparum isolates collected from patients presenting to health facilities predominantly within the tertiary referral area of western Sabah between 2012 and 2016. RESULTS: In total, 49 patients were enrolled and treated with oral artesunate. 90% (44/49) of patients had cleared their parasitaemia by 48 h and 100% (49/49) within 72 h. The geometric mean parasite count at presentation was 9463/µL (95% CI 6757-13,254), with a median time to 50% parasite clearance of 4.3 h (IQR 2.0-8.4). There were 3/45 (7%) patients with a parasite clearance slope half-life of ≥ 5 h. All 278 P. falciparum isolates evaluated were wild-type for kelch13 markers. CONCLUSION: There is no suspected or confirmed evidence of endemic artemisinin-resistant P. falciparum in this pre-elimination setting in Sabah, Malaysia. Current guidelines recommending first-line treatment with ACT remain appropriate for uncomplicated malaria in Sabah, Malaysia. Ongoing surveillance is needed southeast of the Greater Mekong sub-region.

Phylogeographic Evidence for 2 Genetically Distinct Zoonotic Plasmodium knowlesi Parasites, Malaysia.

Infections of humans with the zoonotic simian malaria parasite Plasmodium knowlesi occur throughout Southeast Asia, although most cases have occurred in Malaysia, where P. knowlesi is now the dominant malaria species. This apparently skewed distribution prompted an investigation of the phylogeography of this parasite in 2 geographically separated regions of Malaysia, Peninsular Malaysia and Malaysian Borneo. We investigated samples collected from humans and macaques in these regions. Haplotype network analyses of sequences from 2 P. knowlesi genes, type A small subunit ribosomal 18S RNA and cytochrome c oxidase subunit I, showed 2 genetically distinct divergent clusters, 1 from each of the 2 regions of Malaysia. We propose that these parasites represent 2 distinct P. knowlesi types that independently became zoonotic. These types would have evolved after the sea-level rise at the end of the last ice age, which separated Malaysian Borneo from Peninsular Malaysia.

Mutations of pvdhfr and pvdhps genes in vivax endemic-malaria areas in Kota Marudu and Kalabakan, Sabah.

BACKGROUND: Malaria cases persist in some remote areas in Sabah and Sarawak despite the ongoing and largely successful malaria control programme conducted by the Vector Borne Disease Control Programme, Ministry Of Health, Malaysia. Point mutations in the genes that encode the two enzymes involved in the folate biosynthesis pathway, dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes confer resistance to pyrimethamine and sulfadoxine respectively, in both Plasmodium falciparum and P. vivax. The aim of the current study was to determine the mutation on both pvdhfr at codon 13, 33, 57, 58, 61, 117, and 173 and pvdhps genes at codon 383 and 553, which are potentially associated with resistance to pyrimethamine and sulfadoxine in P. vivax samples in Sabah. METHODS: Every individual was screened for presence of malaria infection using a commercial rapid dipstick assay, ParaMax-3™ (Zephyr Biomedical, India). Individuals tested positive for P. vivax had blood collected and parasite DNA extracted. The pvdhfr and pvdhps genes were amplified by nested-PCR. Restriction fragment length polymorphism (RFLP) was carried out for detection of specific mutations in pvdhfr at codons 13Leu, 33Leu, 57Ile/Leu, 58Arg, 61Met, 117Asn/Thr, and 173Leu and pvdhps at codons 383Gly and 553Gly. The PCR-RFLP products were analysed using the Agilent 2100 Bioanalyzer (Agilent Technology, AS). RESULTS: A total of 619 and 2119 individuals from Kalabakan and Kota Marudu, respectively participated in the study. In Kalabakan and Kota Marudu, 9.37 and 2.45 % were tested positive for malaria and the positivity for P. vivax infection was 4.2 and 0.52 %, respectively. No mutation was observed at codon 13, 33 and 173 on pvdhfr and at codon 553 on pvdhps gene on samples from Kalabakan and Kota Marudu. One-hundred per cent mutations on pvdhfr were at 57Leu and 117Thr. Mutation at 58Arg and 61Met was observed to be higher in Kota Marudu 72.73 %. Mutation at 383Gly on pvdhps was highest in Kalabakan with 80.77 %. There are four distinct haplotypes of pvdhfr/pvdhps combination. CONCLUSIONS: The presence of triple and quintuple mutation combination suggest that the P. vivax isolates exhibit a high degree of resistant to sulfadoxine, pyrimethamine and sulfadoxine-pyrimethamine combination therapy.

Plasmodium knowlesi malaria during pregnancy.

BACKGROUND: Plasmodium knowlesi is the commonest cause of malaria in Malaysia, but little is known regarding infection during pregnancy. METHODS: To investigate comparative risk and consequences of knowlesi malaria during pregnancy, we reviewed (1) Sabah Health Department malaria-notification records created during 2012-2013, (2) prospectively collected data from all females with polymerase chain reaction (PCR)-confirmed malaria who were admitted to a Sabah tertiary care referral hospital during 2011-2014, and (3) malaria microscopy and clinical data recorded at a Sabah tertiary care women and children's hospital during 2010-2014. RESULTS: During 2012-2013, 774 females with microscopy-diagnosed malaria were notified, including 252 (33%), 172 (20%), 333 (43%), and 17 (2%) with Plasmodium falciparum infection, Plasmodium vivax infection, Plasmodium malariae/Plasmodium knowlesi infection, and mixed infection, respectively. Among females aged 15-45 years, pregnancy was reported in 18 of 124 (14.5%), 9 of 93 (9.7%), and 4 of 151 (2.6%) P. falciparum, P. vivax, and P. malariae/P. knowlesi notifications respectively (P = .002). Three females with knowlesi malaria were confirmed as pregnant: 2 had moderate anemia, and 1 delivered a preterm low-birth-weight infant. There were 17, 7, and 0 pregnant women with falciparum, vivax, and knowlesi malaria, respectively, identified from the 2 referral hospitals. CONCLUSIONS: Although P. knowlesi is the commonest malaria species among females in Sabah, P. knowlesi infection is relatively rare during pregnancy. It may however be associated with adverse maternal and pregnancy outcomes.