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