By: Ravi Dhiman, M.Sc. (Agro-Biotechnology)
Justus Liebig Universitat, GiessenGermany
Diagnostic Methods
Structure
Microscopic Examination (ZN, Flurochrome Staining)
Culture (Traditional, Rapid methods)
Interferon-Gamma Release Assays (IGRA)
ELISA
Nucleic acid amplification assays
LAMP
Microscopic Examination
M. tuberculosis appearing as bright red bacilli (rods) in a sputum smear stained with the Ziehl-Neelsen stain
1.Ziehl-Neelson StainingZiehl-Neelsen staining is used to demonstrate the presence of the acid fast bacilli in a smear Appear as straight/curved rods (1-4μ x 0.2-0.8μ) singly, in pairs or in clumps
The technique is simple, inexpensive
Limited sensitivity (46-78%) but specificity is virtually 100%.
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
RunyonGroup
Runyon Group Name Growth speed , Colony pigmentation Chromogen production in the dark
Chromogen production during light
I Photochromogens SlowCream/buff,Orange/yellow in 6-12 weeks
- +
II Scotochromogens SlowOrange/ Yellow in 2-4 weeks
+ +
III Non-photochromogens SlowCream/ 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
ELISAELISA 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