Browsing by Author "Mirjam Kaestli"

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    Development and validation of burkholderia pseudomallei-specific real-time pcr assays for clinical, environmental or forensic detection applications
    (2012-05-18) Erin P. Price; Julia L. Dale; James M. Cook; Derek S. Sarovich; Meagan L. Seymour; Jennifer L. Ginther; Emily L. Kaufman; Stephen M. Beckstrom-Sternberg; Mark Mayo; Mirjam Kaestli; Mindy B. Glass; Jay E. Gee; Vanaporn Wuthiekanun; Jeffrey M. Warner; Anthony Baker; Jeffrey T. Foster; Patrick Tan; Apichai Tuanyok; Direk Limmathurotsakul; Sharon J. Peacock; Bart J. Currie; David M. Wagner; Paul Keim; Talima Pearson; Northern Arizona University; Menzies School of Health Research; Translational Genomics Research Institute; Centers for Disease Control and Prevention; Mahidol University; James Cook University, Australia; Genome Institute of Singapore; University of Cambridge
    The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel ( & 2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community. © 2012 Price et al.
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    The genetic and molecular basis of O-antigenic diversity in Burkholderia pseudomallei lipopolysaccharide
    (2012-01-01) Apichai Tuanyok; Joshua K. Stone; Mark Mayo; Mirjam Kaestli; Jeffrey Gruendike; Shalamar Georgia; Stephanie Warrington; Travis Mullins; Christopher J. Allender; David M. Wagner; Narisara Chantratita; Sharon J. Peacock; Bart J. Currie; Paul Keim; Northern Arizona University; Menzies School of Health Research; Mahidol University; Translational Genomics Research Institute
    Lipopolysaccharide (LPS) is one of the most important virulence and antigenic components of Burkholderia pseudomallei, the causative agent of melioidosis. LPS diversity in B. pseudomallei has been described as typical, atypical or rough, based upon banding patterns on SDS-PAGE. Here, we studied the genetic and molecular basis of these phenotypic differences. Bioinformatics was used to determine the diversity of genes known or predicted to be involved in biosynthesis of the O-antigenic moiety of LPS in B. pseudomallei and its near-relative species. Multiplex-PCR assays were developed to target diversity of the O-antigen biosynthesis gene patterns or LPS genotypes in B. pseudomallei populations. We found that the typical LPS genotype (LPS genotype A) was highly prevalent in strains from Thailand and other countries in Southeast Asia, whereas the atypical LPS genotype (LPS genotype B) was most often detected in Australian strains (~13.8%). In addition, we report a novel LPS ladder pattern, a derivative of the atypical LPS phenotype, associated with an uncommon O-antigen biosynthesis gene cluster that is found in only a small B. pseudomallei sub-population. This new LPS group was designated as genotype B2. We also report natural mutations in the O-antigen biosynthesis genes that potentially cause the rough LPS phenotype. We postulate that the diversity of LPS may correlate with differential immunopathogenicity and virulence among B. pseudomallei strains. © 2012 Tuanyok et al.
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    Systematic Review and Consensus Guidelines for Environmental Sampling of Burkholderia pseudomallei
    (2013-01-01) Direk Limmathurotsakul; David A.B. Dance; Vanaporn Wuthiekanun; Mirjam Kaestli; Mark Mayo; Jeffrey Warner; David M. Wagner; Apichai Tuanyok; Heiman Wertheim; Tan Yoke Cheng; Chiranjay Mukhopadhyay; Savithiri Puthucheary; Nicholas P.J. Day; Ivo Steinmetz; Bart J. Currie; Sharon J. Peacock; Mahidol University; Mahosot Hospital; Nuffield Department of Clinical Medicine; Royal Darwin Hospital; James Cook University, Australia; Northern Arizona University; Oxford University Clinical Research Unit; DSO National Laboratories; Kasturba Medical College, Manipal; Duke-NUS Graduate Medical School Singapore; Ernst-Moritz-Arndt-Universitat Greifswald; University of Cambridge
    Background: Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling. Methods/Principal Findings: An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and 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 to 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 provision 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 map of environmental B. pseudomallei. © 2013 Limmathurotsakul et al.