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Publication Open Access Comparison of two multilocus sequence based genotyping schemes for Leptospira species.(2011-11) Ahmed, Ahmed; Janjira Thaipadungpanit; จันทร์จิรา ไทยผดุงพานิช; Siriphan Boonsilp; ศิริพรรณ บุญศิลป์; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Nalam, Kishore; Spratt, Brian G.; Aanensen, David M.; Smythe, Lee D.; Ahmed, Niyaz; Feil, Edward J.; Hartskeer, Rudy A.; Peacock, Sharon J.; Ahmed, Ahmed; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology.; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit.; Mahidol Univeristy. Faculty of Medicine Siriraj Hospital. Medical Proteomics Unit, Office for Research and Development.BACKGROUND: Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. METHODS AND FINDINGS: A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5-96.5) vs. 93.5 (95% CI 88.6-98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. CONCLUSIONS: This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.Publication 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 Immunologyto occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provisionPublication Open Access Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis(2011-12-13) Siriphan Boonsilp; Janjira Thaipadungpanit; จันทร์จิรา ไทยผดุงพานิช; Premjit Amornchai; เปรมจิตต์ อมรชัย; Vanaporn Wuthiekanun; Wirongrong Chierakul; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Day, Nicholas P.; Peacock, Sharon J.; Janjira Thaipadungpanit; Mahidol University. Faculty of Tropical Medicine. Mahidol-Oxford Tropical Medicine Research Unit. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium. METHODS: We evaluated our hypothesis during aPublication 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 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.Publication Open Access Burkholderia pseudomallei is spatially distributed in soil in northeast Thailand(2010-06-01) Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Narisara Chantratita; นริศรา จันทราทิตย์; Gumphol Wongsuvan; กำพล วงษ์สุวรรณ; Premjit Amornchai; เปรมจิตร อมรชัย; Nicholas P.J. Day; Peacock, Sharon J.; 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 Immunology.BACKGROUND: Melioidosis is a frequently fatal infectious disease caused by the soil dwelling Gram-negative bacterium Burkholderia pseudomallei. Environmental sampling is important to identify geographical distribution of the organism and related risk of infection to humans and livestock. The aim of this study was to evaluate spatial distribution of B. pseudomallei in soil and consider the implications of this for soil sampling strategies. METHODS AND FINDINGS: A fixed-interval sampling strategy was used as the basis for detection and quantitation by culture of B. pseudomallei in soil in two environmental sites (disused land covered with low-lying scrub and rice field) in northeast Thailand. Semivariogram and indicator semivariogram were used to evaluate the distribution of B. pseudomallei and its relationship with range between sampling points. B. pseudomallei was present on culture of 80/100 sampling points taken from the disused land and 28/100 sampling points from the rice field. The median B. pseudomallei cfu/gram from positive sampling points was 378 and 700 for the disused land and the rice field, respectively (p = 0.17). Spatial autocorrelation of B. pseudomallei was present, in that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count. Ranges of spatial autocorrelation in quantitative B. pseudomallei count were 11.4 meters in the disused land and 7.6 meters in the rice field. CONCLUSIONS: We discuss the implications of the uneven distribution of B. pseudomallei in soil for future environmental studies, and describe a range of established geostatistical sampling approaches that would be suitable for the study of B. pseudomallei that take account of our findings.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.Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infection. Whole-genome sequencing of MRSA has been used to define phylogeny and transmission in well-resourced healthcare settings, yet the greatest burden 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 repeatedly screened all patients on two intensive care units for MRSA carriage over a 3-mo period. All MRSA belonged to multilocus sequence type 239 (ST 239). We defined the population structure and charted the spread of MRSA by sequencing 79 isolates from 46 patients and five members of staff, including the first MRSA-positive screen isolates and up to two repeat isolates where available. Phylogenetic analysis identified a flux of distinct ST 239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids encoding antiseptic and antimicrobial resistance determinants. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were 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 sequencing and analysis provides novel insights into the transmission of MRSA in under-resourced healthcare settings and has relevance to wider global health.Publication Open Access Common TLR1 genetic variation is not associated with death from melioidosis, a common cause of sepsis in rural Thailand(2014-01-02) Narisara Chantratita; นริศรา จันทราทิตย์; Sarunporn Tandhavanant; ศรัณย์พร ตัณฑวนันท์; Myers, Nicolle D.; Wirongrong Chierakul; วิรงค์รอง เจียรกุล; Vanaporn Wuthiekanun; วรรณพร วุฒิเอกอนันต์; Weera Mahavanakul; Direk Limmathurotsakul; ดิเรก ลิ้มมธุรสกุล; Peacock, Sharon J.; West, T. Eoin; Narisara Chantratita; Mahidol University. Faculty of Tropical Medicine. Department of Microbiology and Immunology; 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 Tropical HygieneMelioidosis, infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is a common cause of sepsis in northeast Thailand. In white North Americans, common functional genetic variation in TLR1 is associated with organ failure and death from sepsis. We hypothesized that TLR1 variants would be associated with outcomes in Thais with melioidosis. We collated the global frequencies of three TLR1 variants that are common in white North American populations: rs5743551 (-7202A/G), rs4833095 (742A/G), and rs5743618 (1804G/T). We noted a reversal of the minor allele from white North American subjects to Asian populations that was particularly pronounced for rs5743618. In the Utah residents of European ancestry, the frequency of the rs5743618 T allele was 17% whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic association study in 427 patients with melioidosis to determine the association of TLR1 variation with organ failure or death. We genotyped rs5743551 and rs4833095. The variants were in high linkage disequilibrium but neither variant was associated with organ failure or in-hospital death. In 300 healthy Thai individuals we further tested the association of TLR1 variation with ex vivo blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly associated with blood cytokine responses induced by Pam3CSK4. We identified additional common variation in TLR1 by searching public databases and the published literature and screened three additional TLR1 variants for associations with Pam3CSK4-induced responses but found none. We conclude that the genetic architecture of TLR1 variation differs substantially in southeast Asians compared to other populations and common variation in TLR1 in Thais is not associated with outcome from melioidosis or with altered blood responses to Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory host response and determinants of sepsis in southeast Asian populations.