Lab diag. tb
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The document discusses various laboratory methods for the diagnosis of Mycobacterium tuberculosis infection and tuberculosis, including: 1) Microscopic examination of sputum or other samples to look for acid-fast bacilli via staining techniques. 2) Culture-based techniques to isolate M. tuberculosis from samples on solid or liquid media over several weeks. 3) Biochemical and molecular tests to identify M. tuberculosis and determine drug resistance from cultures. 4) Immunological tests like the Mantoux test, interferon-gamma release assays, and ELISPOT to detect immune responses to M. tuberculosis antigens.
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Lab diag. tb
- 1. Hi ! Good Morning ! I am Mycobacterium tuberculosis.
- 2. LABORATORY DIAGNOSIS OF MYCOBACTERIUM TUBERCULOSIS
- 9. ZN Smear evaluation & AFB Report (Grading of smear) 4+ 01 10 or more 3+ 01 01 – 09 2+ 10 01 – 09 1+ 100 01 – 09 Doubtful; Repeat smear 300 01 – 02 AFB Not seen 300 0 Report No. of OIF No. of AFB
- 15. 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
- 16. 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
- 19. Slow growth 3-4 wk
- 20. 24-hr generation time AFB AFB AFB 24 hr
- 21. MYCOBACTERIAL COLONIES ON SOLID MEDIA
- 22. Colonies growing on media
- 26. BACTEC CONTINUOUS MONITORING SYSTEM
- 27. BLOOD CULTURE BOTTLES FOR BACTEC 9240,9120,9050.
- 28. SEPTI - CHECK
- 29. Radiolabeled palmitic acid AFB *C___ *C___ *C___ *C___
- 30. Detect growth with CO 2 AFB AFB AFB AFB AFB *CO 2 *CO 2 *C___ *C___
- 31. BIOCHEMICAL REACTIONS: + - +/- - - - - - - - M Africanum + - - +/- - - - - - - M bovis + + + +/- - + - + - + M tuberculosis UREASE TEST PYRAZI-NAMIDASE TSET GROWTH ON TCH TELLURI--TE REDUCTION TEST TWEEN 80 HYDRO--LYSIS TSET PEROX---IDASE TEST HOT CATAL---ASE TEST NITRATE REDUC---TION TEST ARYL-SULPH---ATASE TEST NIACIN TEST SPECIES
- 43. Whole Blood IFN- Assay QuantiFERON-TB Test Cellestis ESAT-6 CFP 10 Mitogen Control TMB COLOR Stage 1 Whole Blood Culture Stage 2 IFN-gamma ELISA Nil Control Incubate -> INF- from sensitized T-cells Draw blood + heparin Aliquot blood & add antigen Harvest plasma from above settled cells Measure [ IFN- ] in ‘Sandwich’ ELISA Computerized interpretation
- 44. Species Specificity of ESAT-6 and CFP-10
- 45. QFT Assay
- 46. 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-
- 47. 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
- 49. T-Spot. TB : “Six easy Steps” Oxford Immunotec Nil Control Positive Control Infection Infection
- 60. Nucleic acid amplification for mycobact. diagnosis Genus specific protocols Targeting genes code for 16S rRNA 65KDa hsp M.TB Complex specific is 6110 Other targets: Genes encoding 38 KDa MPB 64 mtp 40 PMT 64 Methods: Target amplification - PCR (TMA, LCR, SDA or signal amplification EG: QB amplification) PFYFFER G.E. J.INF. 1999, 39 , 21-26. TC/ICM 30
- 62. Mycobacteria (AFB) sample U AFB U
- 63. Extract 16S RNA U U 16S RNA
- 64. Probe for specific 16S RNA U U 16S RNA Probe Probe
- 65. Probe remains in sample U U 16S RNA Probe
- 66. Detector binds to probe ******* U ******* U 16S RNA Probe
- 67. Detector remains in sample ******* U U 16S RNA Probe
- 68. Detector identifies 16S RNA ******* VVVVV U U 16S RNA Probe
- 78. THANK YOU
Editor's Notes
- NEJM 7/19/01
- 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.