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Publication Open Access Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting.(2015-01) Tong, Steven Y.C.; Holden, Matthew T.G.; Nickerson, Emma K.; Cooper, Ben S.; Köser, Claudio U.; Cori, Anne; Jombart, Thibaut; Cauchemez, Simon; Fraser, Christophe; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Janjira Thaipadungpanit; จันทร์จิรา ไทยผดุงพานิช; Maliwan Hongsuwan; มะลิวัลย์ หงษ์สุวรรณ; Day, Nicholas P.; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Parkhill, Julian; Peacock, Sharon J.; Peacock, Sharon J.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.colonized by more than one clade. One patient on each unit was the source of numerous transmission events, and deep sampling of one of these cases demonstrated colonization with a "cloud" of related MRSA variants. The application of whole-genome... of nosocomial infection occurs in resource-restricted settings where barriers to transmission are lower. Here, we study the flux and genetic diversity of MRSA on ward and individual patient levels in a hospital where transmission was common. We repeatedlyPublication Open Access Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei(2013-03) Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Dance, David A. B.; Vanaporn Wuthiekanun; Kaestli, Mirjam; Mayo, Mark; Warner, Jeffrey; Wagner, David M.; Apichai Tuanyok; Wertheim, Heiman; Cheng, Tan Yoke; Mukhopadhyay, Chiranjay; Puthucheary, Savithiri; Day,Nicholas P. J.; Steinmetz, Ivo; Currie, Bart J.; Peacock, Sharon J.; Direk Limmathurotsakul; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Hygiene; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunologyand categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected... of affordable and 'low-tech' methodology that is applicable in both developed and developing countries. CONCLUSIONS/SIGNIFICANCE: The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global mapPublication Open Access Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis.(2014-03-20) Houghton, Raymond L.; Reed, Dana E.; Hubbard, Mark A.; Dillon, Michael J.; Chen, Hongjing; Currie, Bart J.; Mayo, Mark; Sarovich, Derek S.; Theobald, Vanessa; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Gumphol Wongsuvan; Narisara Chantratita; นริศรา จันทราทิตย์; Peacock, Sharon J.; Hoffmaster, Alex R; Duval, Brea; Brett, Paul J.; Burtnick, Mary N.; AuCoin, David P.; AuCoin, David P.; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Hygiene, Mahidol-Oxford Tropical Medicine Research Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology, and Mahidol-Oxford Tropical Medicine Research Unit.Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.Publication Open Access Genetic diversity and microevolution of Burkholderia pseudomallei in the environment.(2008-02-20) Narisara Chantratita; นริศรา จันทราทิตย์; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Mongkol Vesaratchavest; มงคล เวสารัชเวศย์; Aunchalee Thanwisai; อัญชลี ฐานวิสัย; Premjit Amornchai; เปรมจิตร อมรชัย; Sarinna Tumapa; สรินนา ทุมาภา; Feil, Edward J.; Day, Nicholas P.; Peacock, Sharon J.; Peacock, Sharon J.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.BACKGROUND: The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined. METHODS AND FINDINGS: We undertook a study of BPublication Open Access NLRC4 and TLR5 each contribute to host defense in respiratory melioidosis.(2014-09-18) West, T. Eoin; Myers, Nicolle D.; Narisara Chantratita; นริศรา จันทราทิตย์; Wirongrong Chierakul; วิรงค์รอง เจียรกุล; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Miao, Edward A.; Hajjar, Adeline M.; Peacock, Sharon J.; Liggitt, H. Denny; Skerrett, Shawn J.; West, T. Eoin; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.; Mahidol University. Faculty of Tropical Medicine. Department of Clinical Tropical Medicine.; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology.; Mahidol University. Faculty of Tropical Medicine. Department of Tropical Hygiene.Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4(-/-) mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.