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Publication Open Access Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum(2007-09-15) Prapaporn Boonma; Peter R Christensen; Rossarin Suwanarusk; Ric N Price; Bruce Russell; Usa Lek-Uthai; อุษา เล็กอุทัย; Prapaporn Boonma; Usa Lek-Uthai; Mahidol University. Faculty of Public Health. Department of Parasitology.standard may result in apparent false positive results by superior techniques. Future studies should consider using more than one established molecular methods as a new gold standard to assess novel malaria diagnostic kits and PCR assays.Publication Open Access Suppression of Plasmodium falciparum by serum collected from a case of Plasmodium vivax infection(2008-06-26) Nagao, Yoshiro; Kimura-Sato, Masako; Porntip Chavalitshewinkoon-Petmitr; พรทิพย์ เพ็ชรมิตร; Supatra Thongrungkiat; สุพัตรา ทองรุ่งเกียรติ; Polrat Wilairatana; พลรัตน์ วิไลรัตน์; Ishida, Takafumi; Peerapan Tan-ariya; Souza, J. Brian de; Srivicha Krudsood; ศรีวิชา ครุฑสูตร; Sornchai Looareesuwan; ศรชัย หลูอารีย์สุวรรณ; Nagao, Yoshiro; Mahidol University. Faculty of Tropical Medicine.. falciparum. Cross-reactive antibodies against P. falciparum antigens, and cytokines were measured in the sera. RESULTS: Significant growth inhibitory effects upon P. falciparum cultures (maximally 68% inhibition as compared to pre-illness average) werePublication Open Access Biochemical and functional characterization of Plasmodium falciparum DNA polymerase δ(2016) Jitlada Vasuvat; Atcha Montree; Sangduen Moonsom; Ubolsree Leartsakulpanich; Songsak Petmitr; Federico Focher; Wright, George E.; Porntip Chavalitshewinkoon‑Petmitr; Mahidol University. Faculty of Tropical Medicine. Department of ProtozoologyS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1 were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE... and identified by LC–MS/MS. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 11 compounds were tested on PfPolδ-cat activity and on in vitro parasite growthPublication Open Access Sequence variation of PfEMP1-DBLalpha in association with rosette formation in Plasmodium falciparum isolates causing severe and uncomplicated malaria(2009-08-04) Natharinee Horata; ณัฐริณี หอระตะ; Thareerat Kalambaheti; ธารีรัตน์ กะลัมพะเหติ; Craig, Alister; Srisin Khusmith; ศรีสิน คูสมิทธิ์; Srisin Khusmith; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology.BACKGROUND: Rosetting and cytoadherence of Plasmodium falciparum-infected red blood cells have been associated with severity of malaria. ICAM-1 and CD36 are the main host cell receptors, while PfEMP1-DBLalpha is a major parasite ligand, which can contribute to rosette formation. This study is aimed at demonstrating whether the highly polymorphic PfEMP1-DBLalpha sequences occurring among Thai isolates causing severe and uncomplicated malaria are associated with their ability to form rosettes and reflected the clinical outcome of the patients. METHODS: Two hundred and ninety five PfEMP1-DBLalpha sequences from Thai clinical isolates causing severe and uncomplicated malaria were evaluated by sequencing and direct comparison using the specific text string analysis functions in Microsoft Excel and Perl. The relationships between the PfEMP1-DBLalpha sequences were also analysed by network analysis. The binding abilities of parasitized red blood cells (PRBCs) to CD36, wild type ICAM-1, ICAM-1Kilifi and ICAM-1S22/A under static condition were included. RESULTS: Two hundred and eighty one non-identical amino acid sequences were identified (< 95% sequence identity). When the distributions of semi-conserved features (PoLV1-4 and sequence group) within the rosetting domain PfEMP1-DBLalpha were observed, close similarity was found between isolates from the two disease groups. The sequence group 1 representing uncomplicated malaria was significantly different from the sequence group 3 representing the majority of severe malaria (p = 0.027). By using a simple non-phylogenetic approach to visualize the sharing of polymorphic blocks (position specific polymorphic block, PSPB) and cys/PoLV among DBLalpha sequences, the sequence group 1 was split from the other five sequence groups. The isolates belonging to sequence group 5 gave the highest mean rosetting rate (21.31%). However, within sequence group 2 and group 6, the isolates causing severe malaria had significantly higher rosetting rate than those causing uncomplicated malaria (p = 0.014, p = 0.007, respectively). CONCLUSION: This is the first report of PfEMP1-DBLalpha analysis in clinical Thai isolates using semi-conserved features (cys/PoLV and PSPBs). The cys/PoLV group 5 gave the highest rosetting rate. PfEMP1-DBLalpha domains in Thai isolates are highly diverse, however, clinical isolates from severe and uncomplicated malaria shared common sequences.Publication Open Access A potential role for interleukin‑33 and γ‑epithelium sodium channel in the pathogenesis of human malaria associated lung injury(2015) Sumate Ampawong; Urai Chaisri; Parnpen Viriyavejakul; Panote Prapansilp; Grau, Georges E; Turner, Gareth D. H.; Emsri Pongponratn; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Pathologywith or without PE. Results: The expression of leukocyte sub-set antigens, bronchial interleukin (IL)-33, γ-epithelium sodium channel (ENaC), aquaporin (AQP)-1 and -5, and control cytokeratin staining was quantified in the lung tissue of severe malaria patientsPublication Open Access An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.(2014-07-31) Tan, Jeslin J. L.; Capozzoli, Monica; Sato, Mitsuharu; Wanitda Watthanaworawit; วนิตดา วัฒนวรวิทย์; Ling, Clare L.; Mauduit, Marjorie; Malleret, Benoît; Grüner, Anne-Charlotte; Tan, Rosemary; Nosten, François H.; Snounou, Georges; Rénia, Laurent; Ng, Lisa F. P.; Rénia, Laurent; Ng, Lisa F. P.; Mahidol University. Faculty of TropicalMedicine. Mahidol-Oxford Tropical Medicine Research Unit. Shoklo Malaria Research UnitTropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.Publication Open Access Characterization of within-host Plasmodium falciparum diversity using next-generation sequence data.(2012) Auburn, Sarah; Campino, Susana; Miotto, Olivo; Djimde, Abdoulaye A.; Zongo, Issaka; Manske, Magnus; Maslen, Gareth; Mangano, Valentina; Alcock, Daniel; MacInnis, Bronwyn; Rockett, Kirk A.; Clark, Taane G.; Doumbo, Ogobara K.; Ouédraogo, Jean Bosco; Kwiatkowski, Dominic P.; Auburn, Sarah; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Research Unit.Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the F(WS) metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and F(WS) approaches. F(WS) statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10(-5)). The F(WS) metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the F(WS) metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity.Publication Open Access Liver changes in severe Plasmodium falciparum malaria: histopathology, apoptosis and nuclear factor kappa B expression.(2014-03-17) Parnpen Viriyavejakul; พรรณเพ็ญ วิริยเวชกุล; Vasant Khachonsaksumet; วสันต์ ขจรศักดิ์สุเมธ; Chuchard Punsawad; ชูชาติ พันธ์สวัสดิ์; Parnpen Viriyavejakul; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Pathology.; Mahidol University. Center for Emerging and Neglected Infectious Diseases.BACKGROUND: Liver involvement in severe Plasmodium falciparum infection is commonly a significant cause of morbidity and mortality among humans. The clinical presentation of jaundice often reflects a certain degree of liver damage. This study investigated the liver pathology of severe P. falciparum malaria as well as the regulation and occurrence of apoptosis in cellular components of formalin-fixed, paraffin-embedded liver tissues. METHODS: The liver tissues used in the study came from patients who died from P. falciparum malaria with hyperbilirubinaemia (total bilirubin (TB)≥ 51.3 μmol/L or 3 mg/dl) (12 cases), P. falciparum malaria without hyperbilirubinaemia (TB<51.3 μmol/L) (10 cases); and patients who died due to accidents, whose liver histology was normal (the control group) (10 cases). The histopathology of the liver tissue was studied by routine histology method. Caspase-3 and nuclear factor kappa B (NF-κB) p65 expressions were determined using immunohistochemistry. RESULTS: The severity of liver histopathology, occurrence of apoptosis and NF-κB p65 activation in P. falciparum malaria were associated with higher TB level. Significant correlations were found between NF-κB p65 expression and apoptosis in Kupffer cells and lymphocytes in the portal tracts. CONCLUSIONS: Hyperplastic Kupffer cells and portal tract inflammation are two main features found in the liver tissues of severe P. falciparum malaria cases. In addition, NF-κB is associated with Kupffer cells and lymphocyte apoptosis in severe P. falciparum malaria.Publication Open Access Activation of nuclear factor kappa B in peripheral blood mononuclear cells from malaria patients(2012-06-10) Chuchard Punsawad; ชูชาติ พันธ์สวัสดิ์; Srivicha Krudsood; ศรีวิชา ครุฑสูตร; Yaowapa Maneerat; เยาวพา มณีรัตน์; Urai Chaisri; อุไร ไชยศรี; Noppadon Tangpukdee; นพดล ตั้งภักดี; Emsri Pongponratn; เอี่ยมศรี พงศ์พนรัตน์; Kwannan Nantavisai; Rachanee Udomsangpetch; รัชนีย์ อุดมแสงเพ็ชร; Parnpen Viriyavejakul; พรรณเพ็ญ วิริยเวชกุล; Parnpen Viriyavejakul; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Pathology; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Hygiene; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical Medicine; Mahidol University. Faculty of Science. Department of Pathobiology.BACKGROUND: Malaria parasites and their products can activate a specific immune response by stimulating cytokine production in the host's immune cells. Transcription nuclear factor kappa B (NF-κB) is an important regulator for the control of many pro-inflammatory genes, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF). The activation and expression of NF-κB p65 in peripheral blood mononuclear cells (PBMCs) of malaria patients were investigated and correlated with the levels of IL-10 and TNF to study the nature of NF-κB p65 and its linkage to inflammatory cytokines. METHODS: The sample group comprised 33 patients admitted with malaria caused by Plasmodium vivax (n = 11), uncomplicated Plasmodium falciparum (n = 11), and complicated Plasmodium falciparum (n = 11). Peripheral blood was collected at admission and on day 7 for PBMC isolation. Healthy subjects were used as a control group. The expressions of NF-κB p65 in the PBMCs from malaria patients and the plasma levels of IL-10 and TNF were measured by using enzyme-linked immunosorbent assay (ELISA). The immunofluorescence technique was used to determine NF-κB nuclear translocation. RESULTS: At admission, patients with P. vivax and uncomplicated P. falciparum had significantly elevated phospho-NF-κB p65 levels in the PBMCs compared with those of healthy controls. However, patients with complicated P. falciparum malaria had decreased levels of phospho-NF-κB p65. On day 7 post-treatment, significantly increased phospho-NF-κB p65 was found in the PBMCs of patients with complicated P. falciparum, compared with healthy controls. The plasma level of IL-10 was elevated in day 0 in patients with complicated P. falciparum malaria and was found to be negatively correlated with phospho-NF-κB p65 level (rs = -0.630, p = 0.038). However, there was no correlation between phospho-NF-κB p65 expression and TNF level in patients with complicated P. falciparum malaria. CONCLUSIONS: This is the first report demonstrating alterations in NF-κB p65 activity in the PBMCs of malaria patients. The altered lower features of NF-κB p65 in the PBMCs of patients with complicated P. falciparum at admission could be due to a suppressive effect of high IL-10 associated with complicated P. falciparum malaria.Publication Open Access Population genetic analysis of Plasmodium falciparum parasites using a customized Illumina GoldenGate genotyping assay.(2011) Campino, Susana; Auburn, Sarah; Kivinen, Katja; Zongo, Issaka; Ouedraogo, Jean-Bosco; Mangano, Valentina; Djimde, Abdoulaye; Doumbo, Ogobara K.; Kiara, Steven M.; Nzila, Alexis; Borrmann, Steffen; Marsh, Kevin; Michon, Pascal; Mueller, Ivo; Siba, Peter; Jiang, Hongying; Su, Xin-Zhuan; Chanaki Amaratunga; Socheat, Duong; Fairhurst, Rick M.; Mallika Imwong; มัลลิกา อิ่มวงศ์; Anderson, Timothy; Nosten, Francois; White, Nicholas J.; Gwilliam, Rhian; Deloukas, Panos; MacInnis, Bronwyn; Newbold, Christopher I.; Rockett, Kirk; Clark, Taane G.; Kwiatkowski, Dominic P.; Campino, Susana; Fitzgerald, J. Ross; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford University Research Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical Medicine.; Mahidol University. Faculty of Tropical Medicine. Department of Molecular Tropical Medicine and Genetics.The diversity in the Plasmodium falciparum genome can be used to explore parasite population dynamics, with practical applications to malaria control. The ability to identify the geographic origin and trace the migratory patterns of parasites with clinically important phenotypes such as drug resistance is particularly relevant. With increasing single-nucleotide polymorphism (SNP) discovery from ongoing Plasmodium genome sequencing projects, a demand for high SNP and sample throughput genotyping platforms for large-scale population genetic studies is required. Low parasitaemias and multiple clone infections present a number of challenges to genotyping P. falciparum. We addressed some of these issues using a custom 384-SNP Illumina GoldenGate assay on P. falciparum DNA from laboratory clones (long-term cultured adapted parasite clones), short-term cultured parasite isolates and clinical (non-cultured isolates) samples from East and West Africa, Southeast Asia and Oceania. Eighty percent of the SNPs (n = 306) produced reliable genotype calls on samples containing as little as 2 ng of total genomic DNA and on whole genome amplified DNA. Analysis of artificial mixtures of laboratory clones demonstrated high genotype calling specificity and moderate sensitivity to call minor frequency alleles. Clear resolution of geographically distinct populations was demonstrated using Principal Components Analysis (PCA), and global patterns of population genetic diversity were consistent with previous reports. These results validate the utility of the platform in performing population genetic studies of P. falciparum.