Displaying publications 81 - 100 of 220 in total

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  1. 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.
  2. Fulltext Giemsa-stained wet mount based method for reticulocyte quantification: a viable alternative in resource limited or malaria endemic settings
    Lee WC, Russell B, Lau YL, Fong MY, Chu C, Sriprawat K, et al.
    PLoS One, 2013;8(4):e60303.
    PMID: 23565221 DOI: 10.1371/journal.pone.0060303
    The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on 'subvital' staining. The specialist 'subvital' stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa's stain (commonly used microbiology and parasitology) in a 'subvital' manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.
  3. Fulltext Deciphering the Draft Genome of Toxoplasma gondii RH Strain
    Lau YL, Lee WC, Gudimella R, Zhang G, Ching XT, Razali R, et al.
    PLoS One, 2016;11(6):e0157901.
    PMID: 27355363 DOI: 10.1371/journal.pone.0157901
    Toxoplasmosis is a widespread parasitic infection by Toxoplasma gondii, a parasite with at least three distinct clonal lineages. This article reports the whole genome sequencing and de novo assembly of T. gondii RH (type I representative strain), as well as genome-wide comparison across major T. gondii lineages. Genomic DNA was extracted from tachyzoites of T. gondii RH strain and its identity was verified by PCR and LAMP. Subsequently, whole genome sequencing was performed, followed by sequence filtering, genome assembly, gene annotation assignments, clustering of gene orthologs and phylogenetic tree construction. Genome comparison was done with the already archived genomes of T. gondii. From this study, the genome size of T. gondii RH strain was found to be 69.35Mb, with a mean GC content of 52%. The genome shares high similarity to the archived genomes of T. gondii GT1, ME49 and VEG strains. Nevertheless, 111 genes were found to be unique to T. gondii RH strain. Importantly, unique genes annotated to functions that are potentially critical for T. gondii virulence were found, which may explain the unique phenotypes of this particular strain. This report complements the genomic archive of T. gondii. Data obtained from this study contribute to better understanding of T. gondii and serve as a reference for future studies on this parasite.
  4. Fulltext Expression and Evaluation of Recombinant Plasmodium knowlesi Merozoite Surface Protein-3 (MSP-3) for Detection of Human Malaria
    De Silva JR, Lau YL, Fong MY
    PLoS One, 2016;11(7):e0158998.
    PMID: 27391270 DOI: 10.1371/journal.pone.0158998
    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.
  5. Fulltext Draft Genomes of Anopheles cracens and Anopheles maculatus: Comparison of Simian Malaria and Human Malaria Vectors in Peninsular Malaysia
    Lau YL, Lee WC, Chen J, Zhong Z, Jian J, Amir A, et al.
    PLoS One, 2016;11(6):e0157893.
    PMID: 27347683 DOI: 10.1371/journal.pone.0157893
    Anopheles cracens has been incriminated as the vector of human knowlesi malaria in peninsular Malaysia. Besides, it is a good laboratory vector of Plasmodium falciparum and P. vivax. The distribution of An. cracens overlaps with that of An. maculatus, the human malaria vector in peninsular Malaysia that seems to be refractory to P. knowlesi infection in natural settings. Whole genome sequencing was performed on An. cracens and An. maculatus collected here. The draft genome of An. cracens was 395 Mb in size whereas the size of An. maculatus draft genome was 499 Mb. Comparison with the published Malaysian An. maculatus genome suggested the An. maculatus specimen used in this study as a different geographical race. Comparative analyses highlighted the similarities and differences between An. cracens and An. maculatus, providing new insights into their biological behavior and characteristics.
  6. Fulltext Effects of low-powered RF sweep between 0.01-20 GHz on female Aedes Aegypti mosquitoes: A collective behaviour analysis
    Poh AH, Moghavvemi M, Shafiei MM, Leong CS, Lau YL, Mahamd Adikan FR, et al.
    PLoS One, 2017;12(6):e0178766.
    PMID: 28582398 DOI: 10.1371/journal.pone.0178766
    There are many products claiming to be an electronic solution towards repelling mosquitoes. Several reviews were published in debunking these claims. However, there is a lack of a systematic study on effects of electromagnetic (EM) or more specifically, radio frequency (RF) waves against mosquitoes due to the conclusions made in those years. Therefore, we attempt to establish a fundamental study on female Aedes Aegypti (Linnaeus) mosquitoes by quantifying the collective behavior of the mosquitoes against a continuous stream of low-powered RF signals via a broadband horn antenna using image processing methods. By examining the average lateral and vertical positions of the mosquitoes versus frequency and time, the data shows negligible consistency in the reactions of the mosquitoes toward the different frequencies ranging from 10 to 20,000.00 MHz, with a step of 10 MHz. This was done by examining 33 hours of spatiotemporal data, which was divided into three sessions. All three sessions showed totally different convolutions in the positions in arbitrary units based on the raster scan of the image processing output. Several frequencies apparently showed up to 0.2-70% shift in both lateral and vertical components along the spectrum, without repeatability for all three sessions. This study contributes to the following: A pilot study for establishing the collective effects of RF against mosquitoes, open-source use, and finally a low-cost and easily adaptable platform for the study of EM effects against any insects.
  7. Fulltext Collective behavior quantification on human odor effects against female Aedes aegypti mosquitoes-Open source development
    Poh AH, Moghavvemi M, Leong CS, Lau YL, Safdari Ghandari A, Apau A, et al.
    PLoS One, 2017;12(2):e0171555.
    PMID: 28152031 DOI: 10.1371/journal.pone.0171555
    Classifying and quantifying mosquito activity includes a plethora of categories, ranging from measuring flight speeds, repellency, feeding rates, and specific behaviors such as home entry, swooping and resting, among others. Entomologists have been progressing more toward using machine vision for efficiency for this endeavor. Digital methods have been used to study the behavior of insects in labs, for instance via three-dimensional tracking with specialized cameras to observe the reaction of mosquitoes towards human odor, heat and CO2, although virtually none was reported for several important fields, such as repellency studies which have a significant need for a proper response quantification. However, tracking mosquitoes individually is a challenge and only limited number of specimens can be studied. Although tracking large numbers of individual insects is hailed as one of the characteristics of an ideal automated image-based tracking system especially in 3D, it also is a costly method, often requiring specialized hardware and limited access to the algorithms used for mapping the specimens. The method proposed contributes towards (a) unlimited open source use, (b) a low-cost setup, (c) complete guide for any entomologist to adapt in terms of hardware and software, (d) simple to use, and (e) a lightweight data output for collective behavior analysis of mosquitoes. The setup is demonstrated by testing a simple response of mosquitoes in the presence of human odor versus control, one session with continuous human presence as a stimuli and the other with periodic presence. A group of female Aedes aegypti (Linnaeus) mosquitoes are released into a white-background chamber with a transparent acrylic panel on one side. The video feed of the mosquitoes are processed using filtered contours in a threshold-adjustable video. The mosquitoes in the chamber are mapped on the raster where the coordinates of each mosquito are recorded with the corresponding timestamp. The average distance of the blobs within the frames against time forms a spectra where behavioral patterns can be observed directly, whether any collective effect is observed. With this method, 3D tracking will not be required and a more straightforward data output can be obtained.
  8. Fulltext Detection of Naegleria species in environmental samples from Peninsular Malaysia
    Ithoi I, Ahmad AF, Nissapatorn V, Lau YL, Mahmud R, Mak JW
    PLoS One, 2011;6(9):e24327.
    PMID: 21915311 DOI: 10.1371/journal.pone.0024327
    BACKGROUND: In Malaysia, researchers and medical practitioners are unfamiliar with Naegleria infections. Thus little is known about the existence of pathogenic Naegleria fowleri, and the resultant primary amoebic meningoencephalitis (PAM) is seldom included in the differential diagnosis of central nervous system infections. This study was conducted to detect the presence of Naegleria species in various environmental samples.

    METHODS/FINDINGS: A total of 41 Naegleria-like isolates were isolated from water and dust samples. All these isolates were subjected to PCR using two primer sets designed from the ITS1-ITS2 regions. The N. fowleri species-specific primer set failed to produce the expected amplicon. The Naegleria genus-specific primers produced amplicons of 408 bp (35), 450 bp (2), 457 bp (2) or 381 bp (2) from all 41 isolates isolated from aquatic (33) and dust (8) samples. Analysis of the sequences from 10 representative isolates revealed that amplicons with fragments 408, 450 and 457 bp showed homology with non-pathogenic Naegleria species, and 381 bp showed homology with Vahlkampfia species. These results concurred with the morphological observation that all 39 isolates which exhibited flagella were Naegleria, while 2 isolates (AC7, JN034055 and AC8, JN034056) that did not exhibit flagella were Vahlkampfia species.

    CONCLUSION: To date, pathogenic species of N. fowleri have not been isolated from Malaysia. All 39 isolates that produced amplicons (408, 450 and 457 bp) from the genus-specific primers were identified as being similar to nonpathogenic Naegleria. Amplicon 408 bp from 5 representative isolates showed 100% and 99.7% identity to Naegleria philippinensis isolate RJTM (AM167890) and is thus believed to be the most common species in our environment. Amplicons 450 bp and 457 bp were respectively believed to be from 2 new species of Naegleria, since representative isolates showed lower homology and had a longer base pair length when compared to the reference species in the Genbank, Naegleria schusteri (AJ566626) and Naegleria laresi (AJ566630), respectively.

  9. Fulltext Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)
    Lau YL, Ismail IB, Mustapa NIB, Lai MY, Tuan Soh TS, Haji Hassan A, et al.
    PLoS One, 2021;16(1):e0245164.
    PMID: 33406112 DOI: 10.1371/journal.pone.0245164
    Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.
  10. Fulltext Genotyping of the Duffy blood group among Plasmodium knowlesi-infected patients in Malaysia
    De Silva JR, Lau YL, Fong MY
    PLoS One, 2014;9(9):e108951.
    PMID: 25268233 DOI: 10.1371/journal.pone.0108951
    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.
  11. Fulltext Metabolite profiling of endophytic Streptomyces spp. and its antiplasmodial potential
    Ahmad SJ, Mohamad Zin N, Mazlan NW, Baharum SN, Baba MS, Lau YL
    PeerJ, 2021;9:e10816.
    PMID: 33777509 DOI: 10.7717/peerj.10816
    Background: Antiplasmodial drug discovery is significant especially from natural sources such as plant bacteria. This research aimed to determine antiplasmodial metabolites of Streptomyces spp. against Plasmodium falciparum 3D7 by using a metabolomics approach.

    Methods: Streptomyces strains' growth curves, namely SUK 12 and SUK 48, were measured and P. falciparum 3D7 IC50 values were calculated. Metabolomics analysis was conducted on both strains' mid-exponential and stationary phase extracts.

    Results: The most successful antiplasmodial activity of SUK 12 and SUK 48 extracts shown to be at the stationary phase with IC50 values of 0.8168 ng/mL and 0.1963 ng/mL, respectively. In contrast, the IC50 value of chloroquine diphosphate (CQ) for antiplasmodial activity was 0.2812 ng/mL. The univariate analysis revealed that 854 metabolites and 14, 44 and three metabolites showed significant differences in terms of strain, fermentation phase, and their interactions. Orthogonal partial least square-discriminant analysis and S-loading plot putatively identified pavettine, aurantioclavine, and 4-butyldiphenylmethane as significant outliers from the stationary phase of SUK 48. For potential isolation, metabolomics approach may be used as a preliminary approach to rapidly track and identify the presence of antimalarial metabolites before any isolation and purification can be done.

  12. Different patterns of pfcrt and pfmdr1 polymorphism in Plasmodium falciparum isolates from Tehama region, Yemen
    Atroosh WM, Al-Mekhlafi HM, Al-Jasari A, Sady H, Dawaki SS, Elyana FN, et al.
    PeerJ, 2016;4:e2191.
    PMID: 27478699 DOI: 10.7717/peerj.2191
    Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes. Method. A molecular investigation of 10 codons of pfcrt (72-76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay. Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72-76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected in 9.3% of the isolates from the Bajil district of Hodeidah. Mutations at Y184F of pfmdr1 were found in all isolates (100%) from all districts. The mutation for codons 1034C and 86Y were found only in the isolates from the AdDahi and Khamis Bani Saad districts. Overall, the AdDahi and Khamis Bani Saad districts were similar in terms of carrying most of the mutations in the pfcrt and pfmdr1 genes, while there was a lower prevalence of mutation in the isolates from the Bajil district. Conclusion. The high prevalence of mutations in pfcrt 5 years after the official cessation of CQ use against P. falciparum suggests that there is sustained CQ pressure on P. falciparum isolates in the study area. Moreover, the low prevalence of mutations in the pfmdr1 gene could be a good indicator of the high susceptibility of P. falciparum isolates to antimalarials other than CQ. A new strategy to ensure the complete nationwide withdrawal of CQ from the private drug market is recommended.
  13. Fulltext Quantitative real-time PCR analysis of Anopheles dirus TEP1 and NOS during Plasmodium berghei infection, using three reference genes
    Liew JWK, Fong MY, Lau YL
    PeerJ, 2017;5:e3577.
    PMID: 28761783 DOI: 10.7717/peerj.3577
    Quantitative reverse transcription PCR (qRT-PCR) has been an integral part of characterizing the immunity of Anopheles mosquitoes towards Plasmodium invasion. Two anti-Plasmodium factors of Anopheles, thioester-containing protein 1 (TEP1) and nitric oxide synthase (NOS), play a role in the refractoriness of Anopheles towards Plasmodium infection and are generally expressed during infection. However, these are less studied in Anopheles dirus, a dominant malaria vector in Southeast Asia. Furthermore, most studies used a single reference gene for normalization during gene expression analysis without proper validation. This may lead to erroneous quantification of expression levels. Therefore, the present study characterized and investigated the expression profiles of TEP1 and NOS of Anopheles dirus during P. berghei infection. Prior to that, the elongation factor 1-alpha (EF1), actin 1 (Act) and ribosomal protein S7 (S7) genes were validated for their suitability as a set of reference genes. TEP1 and NOS expressions in An. dirus were found to be significantly induced after P. berghei infection.
  14. Fulltext Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of SARS-CoV-2
    Lau YL, Ismail I, Mustapa NI, Lai MY, Tuan Soh TS, Hassan A, et al.
    PeerJ, 2020;8:e9278.
    PMID: 32547882 DOI: 10.7717/peerj.9278
    BACKGROUND: Highly sensitive real-time reverse transcription polymerase chain reaction (RT-qPCR) methods have been developed for the detection of SARS-CoV-2. However, they are costly. Loop-mediated isothermal amplification (LAMP) assay has emerged as a novel alternative isothermal amplification method for the detection of nucleic acid.

    METHODS: A rapid, sensitive and specific real-time reverse transcription LAMP (RT-LAMP) assay was developed for SARS-CoV-2 detection.

    RESULTS: This assay detected one copy/reaction of SARS-CoV-2 RNA in 30 min. Both the clinical sensitivity and specificity of this assay were 100%. The RT-LAMP showed comparable performance with RT-qPCR. Combining simplicity and cost-effectiveness, this assay is therefore recommended for use in resource resource-limited settings.

  15. Immunogenic characterization of the chimeric surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii expressed in the yeast Pichia pastoris
    Lau YL, Thiruvengadam G, Lee WW, Fong MY
    Parasitol Res, 2011 Sep;109(3):871-8.
    PMID: 21455621 DOI: 10.1007/s00436-011-2315-6
    In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-γ, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P 
  16. Evaluation of Toxoplasma gondii-recombinant dense granular protein (GRA2) for serodiagnosis by western blot
    Ching XT, Lau YL, Fong MY, Nissapatorn V
    Parasitol Res, 2013 Mar;112(3):1229-36.
    PMID: 23274488 DOI: 10.1007/s00436-012-3255-5
    Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the food industry. Commonly used serological tests involve preparation of whole Toxoplasma lysate antigens from tachyzoites which are costly and hazardous. An alternative method for better antigen production involving the prokaryotic expression system was therefore used in this study. Recombinant dense granular protein, GRA2, was successfully cloned, expressed, and purified in Escherichia coli, BL21 (DE3) pLysS. The potential of this purified antigen for diagnosis of human infections was evaluated through western blot analysis against 100 human serum samples. Results showed that the rGRA2 protein has 100 and 61.5 % sensitivity towards acute and chronic infection, respectively, in T. gondii-infected humans, indicating that this protein is useful in differentiating present and past infections. Therefore, it is suitable to be used as a sensitive and specific molecular marker for the serodiagnosis of Toxoplasma infection in both humans and animals.
  17. Recombinant expression of the larval excretory-secretory antigen TES-120 of Toxocara canis in the methylotrophic yeast Pichia pastoris
    Fong MY, Lau YL
    Parasitol Res, 2004 Jan;92(2):173-6.
    PMID: 14655048
    A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris. Specificity of the recombinant TES-120 antigen produced by the yeast was investigated. Forty-five human serum samples from patients infected with different()parasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays. Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.
  18. Plasmodium knowlesi Duffy binding protein alpha region II (PkDBPαII) in clinical isolates from Peninsular Malaysia and Malaysian Borneo exhibit different immune responses in animal models
    Azlan UK, Cheong FW, Lau YL, Fong MY
    Parasitol Res, 2022 Dec;121(12):3443-3454.
    PMID: 36152079 DOI: 10.1007/s00436-022-07665-7
    Plasmodium knowlesi utilizes the Duffy binding protein alpha (PkDBPα) to facilitate its invasion into human erythrocytes. PkDBPα region II (PkDBPαII) from Peninsular Malaysia and Malaysian Borneo has been shown to occur as distinct haplotypes, and the predominant haplotypes from these geographical areas demonstrated differences in binding activity to human erythrocytes in erythrocyte binding assays. This study aimed to determine the effects of genetic polymorphisms in PkDBPαII to immune responses in animal models. The recombinant PkDBPαII (~ 45 kDa) of Peninsular Malaysia (PkDBPαII-H) and Malaysian Borneo (PkDBPαII-S) were expressed in a bacterial expression system, purified, and used in mice and rabbit immunization. The profile of cytokines IL-1ra, IL-2, IL-6, IL-10, TNF-α, and IFN-γ in immunized mice spleen was determined via ELISA. The titer and IgG subtype distribution of raised antibodies was characterized. Immunized rabbit sera were purified and used to perform an in vitro merozoite invasion inhibition assay. The PkDBPαII-immunized mice sera of both groups showed high antibody titer and a similar IgG subtype distribution pattern: IgG2b > IgG1 > IgG2a > IgG3. The PkDBPαII-H group was shown to have higher IL-1ra (P = 0.141) and IL-6 (P = 0.049) concentrations, with IL-6 levels significantly higher than that of the PkDBPαII-S group (P ≤ 0.05). Merozoite invasion inhibition assay using purified anti-PkDBPαII antibodies showed a significantly higher inhibition rate in the PkDBPαII-H group than the PkDBPαII-S group (P ≤ 0.05). Besides, anti-PkDBPαII-H antibodies were able to exhibit inhibition activity at a lower concentration than anti-PkDBPαII-S antibodies. PkDBPαII was shown to be immunogenic, and the PkDBPαII haplotype from Peninsular Malaysia exhibited higher responses in cytokines IL-1ra and IL-6, antibody IgM level, and merozoite invasion inhibition assay than the Malaysian Borneo haplotype. This suggests that polymorphisms in the PkDBPαII affect the level of immune responses in the host.
  19. The return of chloroquine-sensitive Plasmodium falciparum parasites in Jazan region, southwestern Saudi Arabia over a decade after the adoption of artemisinin-based combination therapy: analysis of genetic mutations in the pfcrt gene
    Madkhali AM, Abdulhaq AA, Atroosh WM, Ghzwani AH, Zain KA, Ghailan KY, et al.
    Parasitol Res, 2021 Nov;120(11):3771-3781.
    PMID: 34561749 DOI: 10.1007/s00436-021-07323-4
    This study investigated the polymorphism in the P. falciparum chloroquine resistance transporter (pfcrt) gene 11 years after chloroquine (CQ) cessation in Jazan region, southwestern Saudi Arabia. Two hundred and thirty-five P. falciparum isolates were amplified to detect mutations in the pfcrt gene. The pfcrt 76 T molecular marker for CQ resistance was detected in 66.4% (156/235) of the isolates, while the K76 CQ-sensitive wild type was detected in 33.6%. The pfcrt 74I and pfcrt 75E point mutations were each found to be present in 56.2% of isolates, while only four isolates (1.7%) were found to carry the pfcrt 72S mutation. Moreover, four pfcrt haplotypes were identified as follows: the CVIET triple-allele (56.2%), SVMET double-allele (1.7%) and CVMNT single-allele (8.5%) mutant haplotypes and the CVMNK wild haplotype (33.6%). The analysis also revealed significant associations between the prevalence of mutant pfcrt alleles and haplotypes and the age group, governorate and nationality of the patients as well as the parasitaemia level (p 
  20. Fulltext Genetic polymorphism of the thrombospondin-related apical merozoite protein (TRAMP) of Plasmodium knowlesi in Malaysia
    Ng YL, Lau YL, Hamid MHA, Jelip J, Ooi CH, Mudin RN, et al.
    Parasitol Res, 2023 Jan;122(1):195-200.
    PMID: 36378331 DOI: 10.1007/s00436-022-07716-z
    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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