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
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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
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
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49. T-Spot. TB : “Six easy Steps” Oxford Immunotec Nil Control Positive Control Infection Infection
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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
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.
