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Fulltext Pathogenesis of Plasmodium berghei ANKA infection in the gerbil (Meriones unguiculatus) as an experimental model for severe malaria

Junaid QO, Khaw LT, Mahmud R, Ong KC, Lau YL, Borade PU, et al.

Parasite, 2017;24:38.
PMID: 29034874 DOI: 10.1051/parasite/2017040

BACKGROUND: As the quest to eradicate malaria continues, there remains a need to gain further understanding of the disease, particularly with regard to pathogenesis. This is facilitated, apart from in vitro and clinical studies, mainly via in vivo mouse model studies. However, there are few studies that have used gerbils (Meriones unguiculatus) as animal models. Thus, this study is aimed at characterizing the effects of Plasmodium berghei ANKA (PbA) infection in gerbils, as well as the underlying pathogenesis.
METHODS: Gerbils, 5-7 weeks old were infected by PbA via intraperitoneal injection of 1 × 106 (0.2 mL) infected red blood cells. Parasitemia, weight gain/loss, hemoglobin concentration, red blood cell count and body temperature changes in both control and infected groups were monitored over a duration of 13 days. RNA was extracted from the brain, spleen and whole blood to assess the immune response to PbA infection. Organs including the brain, spleen, heart, liver, kidneys and lungs were removed aseptically for histopathology.
RESULTS: Gerbils were susceptible to PbA infection, showing significant decreases in the hemoglobin concentration, RBC counts, body weights and body temperature, over the course of the infection. There were no neurological signs observed. Both pro-inflammatory (IFNγ and TNF) and anti-inflammatory (IL-10) cytokines were significantly elevated. Splenomegaly and hepatomegaly were also observed. PbA parasitized RBCs were observed in the organs, using routine light microscopy and in situ hybridization.
CONCLUSION: Gerbils may serve as a good model for severe malaria to further understand its pathogenesis.
Fulltext Diagnostic tools in childhood malaria

Amir A, Cheong FW, De Silva JR, Lau YL

Parasit Vectors, 2018 01 23;11(1):53.
PMID: 29361963 DOI: 10.1186/s13071-018-2617-y

Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR, qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.
Fulltext Reduced red blood cell deformability in Plasmodium knowlesi malaria

Barber BE, Russell B, Grigg MJ, Zhang R, William T, Amir A, et al.

Blood Adv, 2018 02 27;2(4):433-443.
PMID: 29487058 DOI: 10.1182/bloodadvances.2017013730

The simian parasite Plasmodium knowlesi can cause severe and fatal human malaria. However, little is known about the pathogenesis of this disease. In falciparum malaria, reduced red blood cell deformability (RBC-D) contributes to microvascular obstruction and impaired organ perfusion. In P knowlesi infection, impaired microcirculatory flow has been observed in Macaca mulatta (rhesus macaques), unnatural hosts who develop severe and fatal disease. However, RBC-D has not been measured in human infection or in the natural host M fascicularis (long-tailed macaques). Using ektacytometry, we measured RBC-D in adults with severe and non-severe knowlesi and falciparum malaria and in healthy controls. In addition, we used micropipette aspiration to determine the relative stiffness of infected RBCs (iRBCs) and uninfected RBCs (uRBCs) in P knowlesi-infected humans and M fascicularis Ektacytometry demonstrated that RBC-D overall was reduced in human knowlesi malaria in proportion to disease severity, and in severe knowlesi malaria, it was comparable to that of severe falciparum malaria. RBC-D correlated inversely with parasitemia and lactate in knowlesi malaria and HRP2 in falciparum malaria, and it correlated with hemoglobin nadir in knowlesi malaria. Micropipette aspiration confirmed that in humans, P knowlesi infection increased stiffness of both iRBCs and uRBCs, with the latter mostly the result of echinocytosis. In contrast, in the natural host M fascicularis, echinocyte formation was not observed, and the RBC-D of uRBCs was unaffected. In unnatural primate hosts of P knowlesi, including humans, reduced deformability of iRBCs and uRBCs may represent a key pathogenic mechanism leading to microvascular accumulation, impaired organ perfusion, and anemia.
Fulltext Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

Wong YP, Othman S, Lau YL, Radu S, Chee HY

J Appl Microbiol, 2018 Mar;124(3):626-643.
PMID: 29165905 DOI: 10.1111/jam.13647

Loop-mediated isothermal amplification (LAMP) amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions by using a DNA polymerase with high displacement strand activity and a set of specifically designed primers to amplify targeted DNA strands. Following its first discovery by Notomi et al. ( Nucleic Acids Res 28: E63), LAMP was further developed over the years which involved the combination of this technique with other molecular approaches, such as reverse transcription and multiplex amplification for the detection of infectious diseases caused by micro-organisms in humans, livestock and plants. In this review, available types of LAMP techniques will be discussed together with their applications in detection of various micro-organisms. Up to date, there are varieties of LAMP detection methods available including colorimetric and fluorescent detection, real-time monitoring using turbidity metre and detection using lateral flow device which will also be highlighted in this review. Apart from that, commercialization of LAMP technique had also been reported such as lyophilized form of LAMP reagents kit and LAMP primer sets for detection of pathogenic micro-organisms. On top of that, advantages and limitations of this molecular detection method are also described together with its future potential as a diagnostic method for infectious disease.
Fulltext Recombinase Polymerase Amplification Combined with a Lateral Flow Strip for the Detection of Plasmodium knowlesi

Lai MY, Ooi CH, Lau YL

Am J Trop Med Hyg, 2018 03;98(3):700-703.
PMID: 29260656 DOI: 10.4269/ajtmh.17-0738

The aim of this study was to develop a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method for specific diagnosis of Plasmodium knowlesi. With incubation at 37°C, the 18S rRNA gene of P. knowlesi was successfully amplified within 12 minutes. By adding a specifically designed probe to the reaction solution, the amplified RPA product can be visualized on a LF strip. The RPA assay exhibited high sensitivity with limits of detection down to 10 parasites/μL of P. knowlesi. Nonetheless, it was demonstrated that all P. knowlesi (N = 41) and other Plasmodium sp. (N = 25) were positive while negative samples (N = 8) were negative. Therefore, a combination of RPA and LF strip detection is a highly promising approach with the potential to be suitable for use in resource-limited settings.
Fulltext Cross-species analysis of apical asparagine-rich protein of Plasmodium vivax and Plasmodium knowlesi

Muh F, Ahmed MA, Han JH, Nyunt MH, Lee SK, Lau YL, et al.

Sci Rep, 2018 04 10;8(1):5781.
PMID: 29636493 DOI: 10.1038/s41598-018-23728-1

The Plasmodium falciparum apical asparagine (Asn)-rich protein (AARP) is one of malarial proteins, and it has been studied as a candidate of malaria subunit vaccine. Basic characterization of PvAARP has been performed with a focus on its immunogenicity and localization. In this study, we further analyzed the immunogenicity of PvAARP, focusing on the longevity of the antibody response, cross-species immunity and invasion inhibitory activity by using the primate malaria parasite Plasmodium knowlesi. We found that vivax malaria patient sera retained anti-PvAARP antibodies for at least one year without re-infection. Recombinant PvAARP protein was strongly recognized by knowlesi malaria patients. Antibody raised against the P. vivax and P. knowlesi AARP N-termini reacted with the apical side of the P. knowlesi merozoites and inhibited erythrocyte invasion by P. knowlesi in a concentration-dependent manner, thereby suggesting a cross-species nature of anti-PvAARP antibody against PkAARP. These results can be explained by B cell epitopes predicted in conserved surface-exposed regions of the AARP N-terminus in both species. The long-lived anti-PvAARP antibody response, cross-reactivity, and invasion inhibitory activity of anti-PvAARP support a critical role of AARP during the erythrocyte invasion and suggest that PvAARP induces long-lived cross-species protective immunity against P. vivax and P. knowlesi.
Fulltext Importance of Proactive Malaria Case Surveillance and Management in Malaysia

Liew JWK, Mahpot RB, Dzul S, Abdul Razak HAB, Ahmad Shah Azizi NAB, Kamarudin MB, et al.

Am J Trop Med Hyg, 2018 06;98(6):1709-1713.
PMID: 29877176 DOI: 10.4269/ajtmh.17-1010

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.
Fulltext In vitro invasion inhibition assay using antibodies against Plasmodium knowlesi Duffy binding protein alpha and apical membrane antigen protein 1 in human erythrocyte-adapted P. knowlesi A1-H.1 strain

Muh F, Lee SK, Hoque MR, Han JH, Park JH, Firdaus ER, et al.

Malar J, 2018 Jul 27;17(1):272.
PMID: 30049277 DOI: 10.1186/s12936-018-2420-4

BACKGROUND: The rapid process of malaria erythrocyte invasion involves ligand-receptor interactions. Inducing antibodies against specific ligands or receptors that abrogate the invasion process is a key challenge for blood stage vaccine development. However, few candidates were reported and remain to be validated for the discovery of new vaccine candidates in Plasmodium knowlesi.
METHODS: In order to investigate the efficacy of pre-clinical vaccine candidates in P. knowlesi-infected human cases, this study describes an in vitro invasion inhibition assay, using a P. knowlesi strain adapted to in vitro growth in human erythrocytes, PkA1-H.1. Recombinant proteins of P. knowlesi Duffy binding protein alpha (PkDBPα) and apical membrane antigen 1 (PkAMA1) were produced in Escherichia coli system and rabbit antibodies were generated from immune animals.
RESULTS: PkDBPα and PkAMA1 recombinant proteins were expressed as insoluble and produced as a functional refolded form for this study. Antibodies against PkDBPα and PkAMA1 specifically recognized recombinant proteins and native parasite proteins in schizont-stage parasites on the merozoite organelles. Single and combination of anti-PkDBPα and anti-PkAMA1 antibodies elicited strong growth inhibitory effects on the parasite in concentration-dependent manner. Meanwhile, IgG prevalence of PkDBPα and PkAMA1 were observed in 13.0 and 46.7% in human clinical patients, respectively.
CONCLUSION: These data provide support for the validation of in vitro growth inhibition assay using antibodies of DBPα and AMA1 in human-adapted P. knowlesi parasite PkA1-H.1 strain.
Fulltext Diversity and natural selection on the thrombospondin-related adhesive protein (TRAP) gene of Plasmodium knowlesi in Malaysia

Ahmed MA, Lau YL, Quan FS

Malar J, 2018 Jul 27;17(1):274.
PMID: 30053885 DOI: 10.1186/s12936-018-2423-1

BACKGROUND: Plasmodium knowlesi a parasite of the macaques is currently the most common cause of human malaria in Malaysia. The thrombospondin-related adhesive protein (TRAP) gene is pre-erythrocytic stage antigen. It is a well-characterized vaccine candidate in Plasmodium vivax and Plasmodium falciparum, however, no study has been done in the orthologous gene of P. knowlesi. This study investigates nucleotide diversity, haplotypes, natural selection and population differentiation of full-length pktrap genes in clinical samples from Malaysia.
METHODS: Forty full-length pktrap sequences from clinical isolates of Malaysia along with the reference H-strain were downloaded from published databases. Genetic diversity, polymorphism, haplotype and natural selection were determined using DnaSP 5.10 software. McDonald-Kreitman test was conducted using P. vivax and Plasmodium coatneyi as ortholog sequence in DnaSP 5.10 software. Population genetic differentiation index (FST) of parasite populations was determined using Arlequin v3.5. Phylogenetic relationships between trap ortholog genes were determined using MEGA 5.0 software.
RESULTS: Comparison of 40 full-length pktrap sequences along with the H-strain identified 74 SNPs (53 non-synonymous and 21 synonymous substitutions) resulting in 29 haplotypes. Analysis of the full-length gene showed that the nucleotide diversity was lower compared to its nearest ortholog pvtrap. Domain-wise analysis indicated that the proline/asparagine rich region had higher nucleotide diversity compared to the von Willebrand factor domain and the thrombospondin-type-1 domain. McDonald-Kreitman test identified that the ratio of the number of nonsynonymous to synonymous polymorphic sites within P. knowlesi was significantly higher than that of the number of nonsynonymous to synonymous fixed sites between P. knowlesi and P. vivax. The von Willebrand factor domain also indicated balancing selection using MK test, however, it did not give significant results when tested with P. coatneyi as an outgroup. Phylogenetic analysis of full-length genes identified three distinct sub-clusters of P. knowlesi, one originating from Peninsular Malaysia and two originating from Malaysian Borneo. High population differentiation values was observed within samples from Peninsular Malaysia and Malaysian Borneo.
CONCLUSIONS: This study is the first to report on the genetic diversity and natural selection of full-length pktrap. Low level of genetic diversity was found across the full-length gene of pktrap. Balancing selection of the von Willebrand factor domain indicated that TRAP could be a target in inducing immune response against P. knowlesi infections. However, higher number of samples would be necessary to further confirm the findings.
Aedes aegypti(Linnaeus) larvae from dengue outbreak areas in Selangor showing resistance to pyrethroids but susceptible to organophosphates

Leong CS, Vythilingam I, Wong ML, Wan Sulaiman WY, Lau YL

Acta Trop, 2018 Sep;185:115-126.
PMID: 29758171 DOI: 10.1016/j.actatropica.2018.05.008

The resistance status of Selangor Aedes aegypti (Linnaeus) larvae against four major groups of insecticides (i.e., organochlorines, carbamates, organophosphates and pyrethroids) was investigated. Aedes aegypti were susceptible against temephos (organophosphate), although resistance (RR50 = 0.21-2.64) may be developing. The insecticides susceptibility status of Ae. aegypti larvae were found heterogeneous among the different study sites. Results showed that Ae. aegypti larvae from Klang, Sabak Bernam and Sepang were susceptible against all insecticides tested. However, other study sites exhibited low to high resistance against all pyrethroids (RR50 = 1.19-32.16). Overall, the application of synergists ethacrynic acid, S.S.S.- tributylphosphorotrithioate and piperonyl butoxide increased the toxicity of insecticides investigated. However, the application failed to increase the mortality to susceptible level (>97%) for certain populations, therefore there are chances of alteration of target site resistance involved. Biochemical assays revealed that α-esterase, (Gombak, Kuala Langat, Kuala Selangor and Sabak Bernam strains) β-esterase (Klang and Sabak Bernam strains), acetylcholinesterase (Kuala Selangor and Sabak Bernam strains), glutathione-S-transferase (Kuala Selangor and Sabak Bernam strains) and mono-oxygenases (Gombak, Hulu Langat, Hulu Selangor and Kuala Langat strains) were elevated. Spearman rank-order correlation indicated a significant correlation between resistance ratios of: DDT and deltamethrin (r = 0.683, P = 0.042), cyfluthrin and deltamethrin (r = 0.867, P =0.002), cyflyuthrin and lambdacyhalothrin (r = 0.800, P =0.010), cyfluthrin and permethrin (r = 0.770, P =0.015) deltamethrin and permethrin (r = 0.803, P =0.088), propoxur and malathion (r = 0.867, P = 0.002), malathion and temephos (r = 0.800, P = 0.010), etofenprox and MFO enzyme (r = 0.667, P =0.050). The current study provides baseline information for vector control programs conducted by local authorities. The susceptibility status of Ae. aegypti should be monitored sporadically to ensure the effectiveness of current vector control strategy in Selangor.
Fulltext Development of Loop-Mediated Isothermal Amplification-Based Lateral Flow Device Method for the Detection of Malaria

Mallepaddi PC, Lai MY, Podha S, Ooi CH, Liew JW, Polavarapu R, et al.

Am J Trop Med Hyg, 2018 09;99(3):704-708.
PMID: 29943720 DOI: 10.4269/ajtmh.18-0177

The present study aims to develop a method for rapid diagnosis of malaria using loop-mediated isothermal amplification (LAMP) combined with a lateral flow device (LFD). By adding the biotin-labeled and fluorescein amidite-labeled loop primers to the LAMP reaction solution, the end product can be visualized on a LFD. The entire procedure takes approximately 42 minutes to complete, LAMP assay exhibited high sensitivity, as the detection limit was 0.01 pg/μL for all five Plasmodium species. It was demonstrated that all Plasmodium knowlesi (N = 90) and Plasmodium vivax (N = 56) were positively amplified by LAMP-LFD assay, whereas healthy donor samples (N = 8) were negative. However, not all mixed infections were positive, and other infected nonmalaria samples were negative. Loop-mediated isothermal amplification-LFD represents a robust approach with potential suitability for use in resource-constrained laboratories. We believe that LAMP-LFD has a potential to be developed as point-of-care diagnostic tool in future.
Fulltext Erythrocyte-binding assays reveal higher binding of Plasmodium knowlesi Duffy binding protein to human Fya+/b+ erythrocytes than to Fya+/b- erythrocytes

Fong MY, Cheong FW, Lau YL

Parasit Vectors, 2018 Sep 26;11(1):527.
PMID: 30257710 DOI: 10.1186/s13071-018-3118-8

BACKGROUND: The merozoite of the zoonotic Plasmodium knowlesi invades human erythrocytes via the binding of its Duffy binding protein (PkDBPαII) to the Duffy antigen on the eythrocytes. The Duffy antigen has two immunologically distinct forms, Fya and Fyb. In this study, the erythrocyte-binding assay was used to quantitatively determine and compare the binding level of PkDBPαII to Fya+/b+ and Fya+/b- human erythrocytes.
RESULTS: In the erythrocyte-binding assay, binding level was determined by scoring the number of rosettes that were formed by erythrocytes surrounding transfected mammalian COS-7 cells which expressed PkDBPαII. The assay result revealed a significant difference in the binding level. The number of rosettes scored for Fya+/b+ was 1.64-fold higher than that of Fya+/b- (155.50 ± 34.32 and 94.75 ± 23.16 rosettes, respectively; t(6) = -2.935, P = 0.026).
CONCLUSIONS: The erythrocyte-binding assay provided a simple approach to quantitatively determine the binding level of PkDBPαII to the erythrocyte Duffy antigen. Using this assay, PkDBPαII was found to display higher binding to Fya+/b+ erythrocytes than to Fya+/b- erythrocytes.
Seroprevalence of sarcocystosis in the local communities of Pangkor and Tioman Islands using recombinant surface antigens 3 (rSAG3) of Sarcocystis falcatula

Tengku-Idris TIN, Fong MY, Lau YL

Trop Med Int Health, 2018 12;23(12):1374-1383.
PMID: 30286271 DOI: 10.1111/tmi.13160

OBJECTIVE: To investigate the seroprevalence of Sarcocystosis in the local communities of Pangkor and Tioman islands, Malaysia, by using antigenic recombinant surface antigens 2 and 3 from Sarcocystis falcatula (rSfSAG2 and rSfSAG3) as the target proteins via Western blot and ELISA assays.
METHODS: SfSAG2 and SfSAG3 genes were isolated from S. falcatula and expressed in Escherichia coli expression system. A total of 348 serum samples [volunteers from both islands (n = 100), non-Sarcocystis parasitic infections patients (n = 50) and healthy donors (n = 100)] were collected and tested with purified SfSAGs in Western blot and ELISA assays to measure the seroprevalence of human sarcocystosis.
RESULTS: None of the sera in this study reacted with rSfSAG2 by Western blot and ELISA. For rSfSAG3, relatively high prevalence of sarcocystosis was observed in Tioman Island (75.5%) than in Pangkor Island (34%) by Western blot. In ELISA, the different prevalence rate was observed between Tioman Island (43.8%) and Pangkor Island (37%). The prevalence rate in other parasitic infections (amoebiasis, cysticercosis, filariasis, malaria, toxocariasis and toxoplasmosis) was 30% by Western blot and 26% by ELISA. Only 8% (by Western blot) and 10% (by ELISA) of healthy donors showed reactivity towards rSfSAG3.
CONCLUSION: This is the first study reporting a seroprevalence of sarcocystosis in Pangkor and Tioman Islands, Malaysia. The combination of Western blot and ELISA is suitable to be used for serodiagnosis of sarcocystosis. With further evaluations, SfSAG3 can potentially be used to confirm infection, asymptomatic screening, surveillance and epidemiological studies.
Fulltext Plasmodium knowlesi malaria: current research perspectives

Amir A, Cheong FW, de Silva JR, Liew JWK, Lau YL

Infect Drug Resist, 2018;11:1145-1155.
PMID: 30127631 DOI: 10.2147/IDR.S148664

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.
Fulltext Plasmodium knowlesi exhibits distinct in vitro drug susceptibility profiles from those of Plasmodium falciparum

van Schalkwyk DA, Blasco B, Davina Nuñez R, Liew JWK, Amir A, Lau YL, et al.

Int J Parasitol Drugs Drug Resist, 2019 04;9:93-99.
PMID: 30831468 DOI: 10.1016/j.ijpddr.2019.02.004

New antimalarial agents are identified and developed after extensive testing on Plasmodium falciparum parasites that can be grown in vitro. These susceptibility studies are important to inform lead optimisation and support further drug development. Until recently, little was known about the susceptibility of non-falciparum species as these had not been adapted to in vitro culture. The recent culture adaptation of P. knowlesi has therefore offered an opportunity to routinely define the drug susceptibility of this species, which is phylogenetically closer to all other human malarias than is P. falciparum. We compared the in vitro susceptibility of P. knowlesi and P. falciparum to a range of established and novel antimalarial agents under identical assay conditions. We demonstrated that P. knowlesi is significantly less susceptible than P. falciparum to six of the compounds tested; and notably these include three ATP4 inhibitors currently under development as novel antimalarial agents, and one investigational antimalarial, AN13762, which is 67 fold less effective against P. knowlesi. For the other compounds there was a less than two-fold difference in susceptibility between species. We then compared the susceptibility of a recent P. knowlesi isolate, UM01, to that of the well-established, older A1-H.1 clone. This recent isolate showed similar in vitro drug susceptibility to the A1-H.1 clone, supporting the ongoing use of the better characterised clone to further study drug susceptibility. Lastly, we used isobologram analysis to explore the interaction of a selection of drug combinations and showed similar drug interactions across species. The species differences in drug susceptibility reported by us here and previously, support adding in vitro drug screens against P. knowlesi to those using P. falciparum strains to inform new drug discovery and lead optimisation.
Fulltext Enzymatic and molecular characterization of insecticide resistance mechanisms in field populations of Aedes aegypti from Selangor, Malaysia

Leong CS, Vythilingam I, Liew JW, Wong ML, Wan-Yusoff WS, Lau YL

Parasit Vectors, 2019 May 16;12(1):236.
PMID: 31097010 DOI: 10.1186/s13071-019-3472-1

BACKGROUND: Dengue is a serious public health problem worldwide, including in Selangor, Malaysia. Being an important vector of dengue virus, Aedes aegypti are subjected to control measures which rely heavily on the usage of insecticides. Evidently, insecticide resistance in Ae. aegypti, which arise from several different point mutations within the voltage-gated sodium channel genes, has been documented in many countries. Thus, this robust study was conducted in all nine districts of Selangor to understand the mechanisms of resistance to various insecticides in Ae. aegypti. Mosquitoes were collected from dengue epidemic and non-dengue outbreak areas in Selangor.
METHODS: Using the Center for Disease Control and Prevention (CDC) bottle assays, the insecticide resistance status of nine different Ae. aegypti strains from Selangor was accessed. Synergism tests and biochemical assays were conducted to further understand the metabolic mechanisms of insecticide resistance. Polymerase chain reaction (PCR) amplification and sequencing of the IIP-IIS6 as well as IIIS4-IIIS6 regions of the sodium channel gene were performed to enable comparisons between susceptible and resistant mosquito strains. Additionally, genomic DNA was used for allele-specific PCR (AS-PCR) genotyping of the gene to detect the presence of F1534C, V1016G and S989P mutations.
RESULTS: Adult female Ae. aegypti from various locations were susceptible to malathion and propoxur. However, they exhibited different levels of resistance against dichlorodiphenyltrichloroethane (DDT) and pyrethroids. The results of synergism tests and biochemical assays indicated that the mixed functions of oxidases and glutathione S-transferases contributed to the DDT and pyrethroid resistance observed in the present study. Besides detecting three single kdr mutations, namely F1534C, V1016G and S989P, co-occurrence of homozygous V1016G/S989P (double allele) and F1534C/V1016G/S989P (triple allele) mutations were also found in Ae. aegypti. As per the results, the three kdr mutations had positive correlations with the expressions of resistance to DDT and pyrethroids.
CONCLUSIONS: In view of the above outcomes, it is important to seek new tools for vector management instead of merely relying on insecticides. If the latter must be used, regular monitoring of insecticide resistance should also be carried out at all dengue epidemic areas. Since the eggs of Ae. aegypti can be easily transferred from one location to another, it is probable that insecticide-resistant Ae. aegypti can be found at non-dengue outbreak sites as well.
Genetic characterisation of the erythrocyte-binding protein (PkβII) of Plasmodium knowlesi isolates from Malaysia

Fong MY, Lau YL, Jelip J, Ooi CH, Cheong FW

J Genet, 2019 Sep;98.
PMID: 31544794

Plasmodium knowlesi contributes to the majority of human malaria incidences in Malaysia. Its uncontrollable passage among the natural monkey hosts can potentially lead to zoonotic outbreaks. The merozoite of this parasite invades host erythrocytes through interaction between its erythrocyte-binding proteins (EBPs) and their respective receptor on the erythrocytes. The regionII of P. knowlesi EBP, P. knowlesi beta (PkβII) protein is found to be mediating merozoite invasion into monkey erythrocytes by interacting with sialic acid receptors. Hence, the objective of this study was to investigate the genetic diversity, natural selection and haplotype grouping of PkβII of P. knowlesi isolates in Malaysia. Polymerase chain reaction amplifications of PkβII were performed on archived blood samples from Malaysia and 64 PkβII sequences were obtained. Sequence analysis revealed length polymorphism, and its amino acids at critical residues indicate the ability of PkβII to mediate P. knowlesi invasion into monkey erythrocytes. Low genetic diversity (π = 0.007) was observed in the PkβII of Malaysia Borneo compared to Peninsular Malaysia (π = 0.015). The PkβII was found to be under strong purifying selection to retain infectivity in monkeys and it plays a limited role in the zoonotic potential of P. knowlesi. Its haplotypes could be clustered into Peninsular Malaysia and Malaysia Borneo groups, indicating the existence of two distinct P. knowlesi parasites in Malaysia as reported in an earlier study.
Identification of Host Proteins Interacting with Toxoplasma gondii SAG1 by Yeast Two-Hybrid Assay

Lai MY, Abdul-Majid N, Lau YL

Acta Parasitol, 2019 Sep;64(3):575-581.
PMID: 31165984 DOI: 10.2478/s11686-019-00066-4

Toxoplasma gondii is one of the most successful human pathogens. To eliminate the infection, identification of receptors or binding partners from humans is indeed urgent. T. gondii surface antigen is the ultimate component involved during the attachment of parasite into host cell. However, mechanism of invasion between SAG and host-cell membrane remains unclear. Yeast two-hybrid experiment was used to identify the binding partners from cDNA human library by using T. gondii SAG1 as bait. Mated yeast cells were plated on DDO/X plates to confirm only prey plasmid that expressing interacting protein was selected. We detected 39 clones interacted with SAG1 based on a series of the selection procedures. After colony PCR, only 29 clones were positive and subsequently sent for sequencing. The yeast plasmids for true positive clones were rescued by transformation into E. coli TOP 10F' cells. Twenty-two clones were further examined by small-scale Y2H experiment. The results indicated that a strong interaction existed between Homo sapiens lysine-rich coil-coiled and SAG1 protein, which could activate the expressions of the reporter genes in diploid yeast. Co-immunoprecipitation experiment result indicated the binding between this prey and SAG1 protein was significant (Mann-Whitney U test, Z = - 1.964, P = 0.05). H. sapiens lysine-rich coil-coiled protein was found to be interacted with SAG1. This prey protein may serve as the potential drug target in vaccination study.
Fulltext Case Report: Two Cases of Recurring Ovale Malaria in Sarawak, Malaysia, after Successful Treatment of Imported Plasmodium falciparum Infection

Liew JWK, Ooi CH, Snounou G, Lau YL

Am J Trop Med Hyg, 2019 12;101(6):1402-1404.
PMID: 31595863 DOI: 10.4269/ajtmh.19-0305

Here are two cases of recurring ovale malaria in Sarawak, Malaysia, that are likely relapses that occurred 1-2 months after successful treatment of the initial imported falciparum malaria with artemisinin-based combined therapy. The patients have no history or recollection of previous malaria episodes. These cases add to the limited evidence on the relapsing nature of Plasmodium ovale, after a febrile episode. In regions where P. ovale is not known to be autochthonous, active follow-up of treated imported malaria patients is highly recommended following their return, particularly to areas nearing or having achieved elimination.
Fulltext Distribution of the Duffy genotypes in Malaysian Borneo and its relation to Plasmodium knowlesi malaria susceptibility

de Silva JR, Amir A, Lau YL, Ooi CH, Fong MY

PLoS One, 2019;14(9):e0222681.
PMID: 31536563 DOI: 10.1371/journal.pone.0222681

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.

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