There are two commercial assays available in the world. The first of these, called the QuantiFERON TB test Gold is available in the United States. The test works like this. First blood is drawn into an heparinized tube. The cells are separated and stimulated with various antigens including positive and negative controls. After incubation, sensitized T cells produce interferon that can be harvested and measured using an ELISA assay. The results are interpreted by a computer program.
When we measure a person’s TST result, we are measuring and indirect result of inflammation caused by injection of the PPD into the skin. In this figure, you can see that when an antigen presenting cell such as a macrophage encounters a T-cell, the lymphocyte produces a number of cytokines such as TNF-alpha, interferon-gamma, and various interleukins. These cytokines cause swelling and induration that is measured with a ruler. It is now possible to measure these cytokines directly from the blood. Interferon-gamm is the best cytokine to measure because it is produced in large amounts, is stable and thus easy to measure. Importantly it is also a critical cytokine in the immune response to mycobacterial infections.
Hi ! Good Morning ! I am Mycobacterium tuberculosis.
LABORATORY DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS
NEED FOR ACCURATE & EARLY DIAGNOSIS OF TUBERCULOSIS.
Tuberculosis mainly caused by M.tuberculosis.
Is a major health problem world wide.
Found in Neolithic remains.
Largest cause of “DEATHS” from a single infectious disease.
HENCE “ACCURATE” & “EARLY” diagnosis is required for its effective “MANAGEMENT”.
Simple, inexpensive & control the growth of contaminants
Twenty samples can be processed in 2 Hrs, with centrifuge capacity being the limiting factor
Sterilized NaOH can be kept for several weeks
The specimen exposure times must be strictly followed to prevent over kill of tubercle bacilli. The initial kill is independent of additional contributory factors such as heat build-up in the centrifuge and centrifugal efficiency
Specimen Sterile Non - Sterile Centrifuge & use sediment Liquefaction (N-acetyl-L- cystein) Decontamination NaOH Neutralization Buffer or H2O Centrifugation > 3000 X g Screen by AFB smear & inoculate media (one liquid & one solid) FLOW CHART OF SPECIMEN PROCESSING FOR ISOLATION OF MYCOBACTERIA
FLOW CHART CONTD. Screen by AFB smear & inoculate media (one liquid & one solid) Liquid Medium Solid Media MGIT BACTEC SEPTI-CHEK CMS Incubate At 37 ºC For 6 wks Incubate At 37ºC For 6 wks Incubate inverting At 37ºC For 8 wks Incubate At 37ºC For 6 wks Fluoresc- -ence detected Growth Index >10 Colonies or turbidity Growth detected Confirm by AFB smear Reinoculate on Solid media L J L J with RNA LJ with Pyruvic acid Incubate At 37ºC For 8 wks If growth Confirm on AFB smear
Flow Chart Identification of Tubercle bacilli & related Growth on LJ Medium Rapid growth within 7 days Growth on MacConkey Agar Aryl-sulphataes test Slow Growth Niacin test
+ ve M. smegmatis
- ve Type of growth + ve M. tuberculosis + ve M. Fortuitum complex Pigment Scanty Smooth Flat Colonies In Light Group I Photochromogens In Dark Group II Scotochromogens No Pigment Group III Non - Chromogens - ve BCG + ve M. bovis M.Kansasi M.intermedium M.Szulgai M.scrofulaceum M.Avium complex M.gastri
In Vivo and In Vitro Diagnostic Tests Antigen presenting cell Memory T-cell Presentation of mycobacterial antigens IFN- IFN- IL-8, etc. IL-8, etc. TNF- TNF-
Results and Interpretation MTB infection status cannot be determined as a result of impaired immunity and/or incorrect performance of the test INDETERMINATE No ESAT-6 or CFP-10 responsiveness detected M. tuberculosis unlikely NEGATIVE ESAT-6 and/or CFP-10 responsiveness detected M. tuberculosis infection likely POSITIVE INTERPRETATION RESULT