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Diagnostic Methods For Mycobacterium
 

Diagnostic Methods For Mycobacterium

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    Diagnostic Methods For Mycobacterium Diagnostic Methods For Mycobacterium Presentation Transcript

    • Diagnostic Methods By: Ravi Dhiman, M.Sc. (Agro-Biotechnology) Justus Liebig Universitat, Giessen Germany
    • Structure  Microscopic Examination (ZN, Flurochrome Staining)  Culture (Traditional, Rapid methods)  Interferon-Gamma Release Assays (IGRA)  ELISA  Nucleic acid amplification assays  LAMP
    • Microscopic Examination 1.Ziehl-Neelson Staining Ziehl-Neelsen staining is used to demonstrate the presence of the acid fast bacilli in a smear Appear as straight/curved rods (1-4μ x 0.20.8μ) singly, in pairs or in clumps The technique is simple, inexpensive Limited sensitivity (46-78%) but specificity is virtually 100%. M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain
    • 2.Fluorochrome Staining The smear may be stained by Auramine-O with or without rhodamine  Fluorochrome stained bacteria appear bright yellow against dark background when visualized using fluorescent microscope FS is not as specific for acid fast bacteria as ZN Staining Good for labs with high workload
    • Traditional Culture  More sensitive & can be positive even when bacterial load is low (10-100 bacilli/ml)  Three types of media are used: Egg based: Lowenstein Jensen & Petragnani Agar based: Middlebrook 7H10 or 7H11 Liquid based: Kirschner’s, Middlebrook 7H9  Growth is slow and takes 6-12 weeks  There after the same length of time is required for complete identification & sensitivity testing
    • Runyon Group Runyon Group Name Growth speed , Colony pigmentation Chromogen Chromogen production in production the dark during light I Photochromogens Slow Cream/buff, Orange/yellow in 6-12 weeks - + II Scotochromogens Slow Orange/ Yellow in 2-4 weeks + + III Non-photochromogens Slow Cream/ buff in 2-4 weeks - - IV Rapid growers - - Fast < 7days
    • Typical small, buff coloured colonies of M. tuberculosis on Lowenstein Jensen medium
    • Broth Based Rapid Culture Methods 1.BACTEC 460 ( Rapid Radiometric Culture System)  Specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C14 – labelled palmitic acid & PANTA antibiotic mixture  Growing mycobacteria utilize the acid, releasing radioactive CO2 which is measured as growth index (GI) in the BACTEC instrument  The daily increase in GI output is directly proportional to the rate & amount of growth in the medium *PANTA (Polymyxin B , Amphotericin B , Nalidixic acid , Trimethoprim ,Azlocillin )
    • BACTEC 460
    • 2.Mycobacterium Growth Indicator Tube (MGIT 960)  Tubes contains modified Middlebrook 7H9 broth base with OADC enrichment & PANTA antibiotic mixture  A fluorescent compound (which is sensitive to O2) is embedded in silicone on the bottom of the tubes containing broth  When mycobacterium grows, they deplete the dissolved oxygen in the broth & allow the indicator to fluoresce brightly in a 365nm UV light
    • The MGIT 960 System
    • Interferon-Gamma Release Assays (IGRA)  Two in-vitro interferon gamma assays used for diagnosis of latent TB infection :  T-Spot Test  Quanti-Feron TB gold Test  In-vitro interferon gamma assays are based on the principle that T cells of individuals sensitised with tuberculosis antigens produce interferon when they re-encounter mycobacterial antigens ESAT6 and CFP-10
    • ELISA ELISA is used as a diagnostic tool ELISA is based on a solid phase immunoassay that detects the presence of antigens or antibodies Liu & colleagues have developed a double antibody sandwich ELISA for the detection of Mpt64 It can detect as little as 0.01 mg/L of the target protein
    • Urinary LAM Antigen Test (Chemogen®)  Detects LAM antigen by ELISA in the urine  Lipoarabinomannan(LAM) is a complex glycolipid associated with cell wall of mycobacteria & is produced in substantial quantities by growing mycobacterium  Rapid (2.5 hrs)  Sensitivity: 80%  Strip test under development
    • Nucleic Acid Amplification Assays  NAA assays amplify MTBC-specific nucleic acid sequences using a nucleic acid probe  The sensitivity of the NAA assays currently in commercial use is at least 80% in most studies  Require as few as 10 bacilli from a given sample  Specificity NAA assays in the range of 98% to 99%
    • Loop-Mediated Isothermal Amplification  It is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity  LAMP is used for detection of M.tb complex, M.avium, and M.intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT) or on a solid medium
    • LAMP Advantages:  Due to its easy operation without sophisticated equipment, it will be simple enough to use in:  Small-scale hospitals  Primary care facilities  Clinical laboratories in developing countries
    • Summary Staining Culture ELISA NAA LAMP Sensitivity 46-78% 80-85% 80% 95% 97% Specificity 100% 98% 95% 98 -99% 99%
    • References  Tomotada Iwamoto, Toshiaki Sonobe and KozaburoHayashi.Loop-Mediated Isothermal Amplification for Direct Detection of Mycobacterium tuberculosis Complex, M. avium, and M. intracellulare in Sputum Samples. J. Clin. Microbiol. 2003, 41(6):2616  New Tools & Emerging Technologies for the Diagnosis of Active Tuberculosis and Drug Resistance. Dr. Madhukar Pai, MD, PhD McGill University, Montreal  Aliya Bekmurzayeva, Marzhan Sypabekova, Damira Kanayeva. Tuberculosis diagnosis using immunodominant, secreted antigens of Mycobacterium tuberculosis. Tuberculosis 93 (2013) 381e388  Madhukar Pai, Lee W Riley, and John M Colford Jr. Interferon- assays in the immunodiagnosis of tuberculosis: a systematic review