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WELCOME TO THE 2011 - Harvard Initiative for Global Health ...

WELCOME TO THE 2011 - Harvard Initiative for Global Health ...

7) Construction and

7) Construction and evaluation of an M(X)DR-TB multiplex assay using LATE-PCR & thermalight probes L. Rice, J. Rice, S. Fishbein, L. Wangh, (Dept. of Biology Brandeis Univ., Waltham, MA); N. Kurpina, B. Kreiswirth (PHRI, Newark, NJ); N. Casali, F. Drobniewski (Blizard Institute, Queen Mary College, Univ. of London, UK); EM. Streicher, R. Warren (DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Stellenbosch Univ. Tygerberg, South Africa); Rohit Mistry (Smiths Detection Diagnostics, Watford, U.K.) Abstract: Background: We have constructed and are evaluating a highly informative, rapid, single-tube assay for M(X)DR tuberculosis using technologies invented at Brandeis University. Methods: LATE-PCR generates single-stranded amplicons; PrimeSafe enhances polymerase specificity and multiplexing; Thermalight probes make it possible to scan long DNA targets for mutations. Amplification occurs in standard fluorescent thermocyclers. All amplicons are analyzed simultaneously at end-point at temperatures below the annealing temperature. Quasar: Rifampin resistance (rpoB gene); Cal Red: Isoniazid and Ethambutol resistance (inhA promotor, embB gene); Cal Orange: Isoniazid and Fluoroquinolone resistance (katG gene, gyrA gene); Fam: Aminoglycoside and Streptomycin resistance (rrs1401, rrs904-908, rrs504-516). If a 5 th color channel is available in the PCR machine it can be used to detect geo-specific targets of interest. Current Results: Unique “fluorescent signatures” were obtained for all 75 different strains of rifampin resistant Mycobacterium tuberculosis tested, plus 7 drug-sensitive strains containing different neutral of mutations. Each of the predominant mutations for each of the other drug-resistant targets also has its own fluorescent signature in its own color and temperature space. In addition, the assay distinguishes M.tuberculosis, M.bovis, and M.africanum from each other and does not detect any NTM species. Finally, these assays can detect as little as ten bacterial genomes in a background of 10,000 human genomes. No false positives or negatives have thus far been observed. Conclusions: The single-tube assay we are constructing is robust, sensitive, and highly informative for M(X)DR-TB, MTBCs and NTMs. Supported in part by the TSB (UK) and Smiths Detection Diagnostics, Inc. Biography: Lisa M. Rice obtained her degree in Biology from Brandeis University in 2010, and has since worked for three years with Lawrence Wangh at Brandeis University developing various infectious disease diagnostic assays. She will attend Boston University Medical School for a Ph.D. in Molecular Medicine this fall. 11

WINNING POSTER ABSTRACTS AND PRESENTER BIOGRAPHIES 1) Using comparative lipidomics to identify new virulence factors of Mycobacterium tuberculosis E Layre 1 , R Edifor 2 , MY Hahn 3 , S Raman 4 , RN Husson 2 and DB Moody 1 1 Division of Rheumatology, Immunology and Allergy, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA. 2 Division of Infectious disease, Children’s Hospital, Harvard Medical School, Boston, USA. 3 Laboratory of Molecular Microbiology, School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, Korea. 4 NEIDL, Boston University, Boston, USA. Mycobacterium tuberculosis adapts to stress conditions encountered in host cell to establish a persistent infection, which involves remodeling metabolic pathways and upregulation of stress response pathways. A significant portion of the M. tuberculosis genome, including a large family of polyketide synthases genes (pks), is devoted to the production of a highly diverse repertoire of immunoregulatory lipids. We and others have shown that a member of RNA polymerase subunit, Sigma factor �L, is a virulence factor and induces the transcription of a group of 6 functionally uncharacterized polyketide synthases: the pks10 locus. Under unstressed laboratory growth conditions this locus is only weakly expressed, suggesting that it may be involved in the biosynthesis of infection-specific lipid virulence factors. We recently developed a broad lipidomic survey consisting in HPLC-MS analysis of M. tuberculosis total lipid extracts and bioinformatic tools for comparative and statistical analysis of multiple lipidomes, which allows rapid detection of all significantly changed ions and thereby links alterations in lipid composition to genetic modifications (Layre et al., Chem. Biol., 2011). Comparative lipidomic analysis of pks10 over-expressing M. tuberculosis strain, and wild type or pks10 deleted strains highlighted cell wall lipids only produced by the pks10 over-expressing strain currently under structural characterization. These results point to a role of �L in regulating the lipid profile of M. tuberculosis. The unbiased nature of the lipidomic screen offers the opportunity to identify new molecules that participate in cell surface remodeling and pathogenesis (NIH NIAID R01 AI 049313 and R01 AI 037901). Biography: Dr. Layre obtained her PhD in Biochemistry and Molecular Biology in 2008 from the Université Paul Sabatier de Toulouse (France). In 2009, she joined the laboratory of Branch Moody at Brigham and Women's Hospital-Harvard Medical School as a research fellow. 2) Oxidative escape of Mycobacterium tuberculosis from neutrophil killing Björn Corleis 1 , Daniel Korbel 1 Robert Wilson 3 , Johan Bylund 4 , Ronnie Chee 5 and Ulrich E. 1, 2 Schaible 1 Department of Immunology and Infection, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, WC1E 7HT, London, UK; 2 Research Centre Borstel, Molecular Infection Research, D-23845 Borstel, Germany; 3 University College London, Centre for Respiratory Research, Rayne Institute, WC1E 6JF London, UK, 4 Department of Rheumatology and Inflammation Research, The Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden; 5 Immunology, Royal Free Hospital London NW3 2QG, UK. Neutrophils are at the host defence front line against invading pathogens and among the first cells entering sites of infection, where they can kill pathogenic bacteria in an oxidative manner. The importance of NADPH-Oxidase activity in anti-bacterial host defence becomes evident in CGD (chronic granulomatous disease) patients, which suffer from massive bacterial infections due to non-functional oxygen-dependent bacterial killing. In pulmonary tuberculosis, neutrophils are predominant in lesions during the acute phase and represent the main cell population infected with M. tuberculosis. To reveal the function of neutrophils in host defence against 12

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