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 ...
Microscopic Examination
1.Ziehl-Neelson Staining
Ziehl-Neelsen staining is used to
demonstrate the presence of the acid f...
2.Fluorochrome Staining
The smear may be stained by
Auramine-O with or without rhodamine

 Fluorochrome
stained
bacteria...
Traditional Culture
 More sensitive & can be positive even when bacterial load is
low (10-100 bacilli/ml)
 Three types o...
Runyon
Group

Runyon Group Name

Growth speed , Colony pigmentation Chromogen Chromogen
production in production
the dark
...
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 me...
BACTEC 460
2.Mycobacterium Growth Indicator Tube
(MGIT 960)
 Tubes contains modified Middlebrook 7H9 broth base with

OADC enrichmen...
The MGIT 960 System
Interferon-Gamma Release Assays (IGRA)
 Two in-vitro interferon gamma assays used for diagnosis of
latent TB infection :
...
ELISA
ELISA is used as a diagnostic
tool
ELISA is based on a solid phase

immunoassay

that

detects

the

presence of a...
Urinary LAM Antigen Test (Chemogen®)
 Detects LAM antigen by ELISA in
the urine
 Lipoarabinomannan(LAM) is a
complex gly...
Nucleic Acid Amplification Assays
 NAA assays amplify MTBC-specific nucleic acid sequences

using a nucleic acid probe
 ...
Loop-Mediated Isothermal Amplification
 It is a novel nucleic acid
amplification method in which
reagents react under iso...
LAMP
Advantages:

 Due to its easy operation without sophisticated equipment, it will
be simple enough to use in:
 Small...
Summary
Staining

Culture

ELISA

NAA

LAMP

Sensitivity

46-78%

80-85%

80%

95%

97%

Specificity

100%

98%

95%

98 -...
References
 Tomotada Iwamoto, Toshiaki Sonobe and KozaburoHayashi.Loop-Mediated Isothermal
Amplification for Direct Detec...
Diagnostic Methods For Mycobacterium
Diagnostic Methods For Mycobacterium
Diagnostic Methods For Mycobacterium
Diagnostic Methods For Mycobacterium
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Diagnostic Methods For Mycobacterium

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Diagnostic Methods For Mycobacterium

  1. 1. Diagnostic Methods By: Ravi Dhiman, M.Sc. (Agro-Biotechnology) Justus Liebig Universitat, Giessen Germany
  2. 2. Structure  Microscopic Examination (ZN, Flurochrome Staining)  Culture (Traditional, Rapid methods)  Interferon-Gamma Release Assays (IGRA)  ELISA  Nucleic acid amplification assays  LAMP
  3. 3. 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
  4. 4. 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
  5. 5. 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
  6. 6. 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
  7. 7. Typical small, buff coloured colonies of M. tuberculosis on Lowenstein Jensen medium
  8. 8. 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 )
  9. 9. BACTEC 460
  10. 10. 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
  11. 11. The MGIT 960 System
  12. 12. 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
  13. 13. 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
  14. 14. 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
  15. 15. 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%
  16. 16. 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
  17. 17. 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
  18. 18. Summary Staining Culture ELISA NAA LAMP Sensitivity 46-78% 80-85% 80% 95% 97% Specificity 100% 98% 95% 98 -99% 99%
  19. 19. 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

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