Tb sure ultra short july 2019

Diagnosis of tuberculosis by direct demonstration of the pathogen or by indirect demonstration of cell mediated immunity through activation of CD 4 and / or CD 8 T lymphocytes.

1. Recent Advances in Laboratory Diagnosis of Tuberculosis Prof. Ashok Rattan, MD, MAMS, Common Wealth Fellow, SEARO TA, INSA DFG, WHO Lab Director (CAREC/PAHO) Advisor : Pathkind Labs Knowledge Forum, R & D and Quality,

2. A disease of great antiquity Fossile bones 9000 BC show evidence Egyptian mummies: Spinal caries 2400 BC 1500 BC mention of consumption in India Hippocrates 460 – 370 BC recognized phthisis (felt it was heredity) Tuberculosis, Phthisis

3. Since its discovery 137 years ago TB has claimed more than 1 billion lives Which is more than those claimed by (all of the below) Malaria Small Pox HIV/AIDS Cholera P lague I nfluenza

4. Commonest method for diagnosis of TB 1885 2019 A change in mind set is required Mark Twain said, “ You cannot keep doing the same thing and expect different results”

5. Tuberculosis Clinically suspected ? Pulmonary or Extra Pulmonary ?

6. Improving detection CO 2 production O 2 consumption Radiometric Fluorescent Colourimetric Pressure sensor Decrease TAT by half 14 C Palmitate  14 CO 2

7. Speed Of Growth Of M.tuberculosis (MGIT 960 vs L J Media) 1 st wk 2 nd wk 3 rd wk 4 th wk 5 th wk 6 th wk 166 132 23 55 46 23 63 13 48 2 24 391 204 Data generated in 2007 – 08 by Dr Bansidhar Tarai under guidance of Dr Ashok Rattan during their tenure in Religare SRL Reference Lab, Gurgaon, unpublished Time-to-Detection in Culture Predicts Risk of Mycobacterium tuberculosis Transmission: Matthew K. O’Shea et al. [UK] Clinical Infectious Diseases 2014;59(2):177–85 TTD < 9 days identifies patients at high risk of transmitting tuberculosis and is superior to sputum smear .

8. Molecular Biology Detection on clinical samples In the past: Polymerase Chain Reaction (PCR) Lab made tests Variable performance High cross contamination Inhibitors in clinical samples Real Time PCR [Cepheid] Closed system Simple sample processing Bacteria are made inactivated, no chance of infection Rapid results [2 hours] Detection of M tb and Rif Resistance [surrogate marker for MDR] Extensively field tested in different parts of the world 30 million cartridges used Cost subsidized for developing countries by Bill Clinton Foundation

9. Gene Xpert MTB/RIF WHO endorsed Cartridge based, closed RT PCR for Detection of MTB & RIF Resistance (as surrogate for MDR TB) Contains Internal quality controls for Sample Processing (SPC) Probe Check (PCC) Amplifies a 192 bp region of MTB rpoB gene RIF resistance is detected by 5 overlapping molecular beacon probes that are complementary to entire 81 bp region of RRDR of rpo B gene

10. Gene Xpert Report Gene Xpert MTB/RIF is Positive / Negative Mycobacterium tuberculosis DNA Detected / Not Detected Quantitation High / Medium / Low / Very Low Rifampicin Resistance Present / Absent Result: MTB Quantitation Result Ct Range High < 16 Medium 16 – 22 Low 22 – 28 Very Low 28 Rifampicin result result types , when MTB is DETECTED Rifampicin resistance Detected : a mutation in the rpoB gene has been detected that falls within the valid delta Ct setting. Rifampicin resistance Not detected : no mutation in the rpoB has been detected Rifampicin resistance Indeterminate : The MTB concentration was very low and resistance couldnot be detected.

11. The First Choice Diagnostic Laboratory

12. Antibodies tests From Agglutination to SAFA all techniques have been tried Cannot differentiate between infection and disease Positive even in healthy individuals WHO and GOI have banned the use of Antibody tests

13. In Vitro Diagnostic Tests : Cell Mediated Immunity Antigen presenting cell Memory T-cell Presentation of mycobacterial antigens IFN-  IFN-  IL-8, etc. IL-8, etc. TNF-  TNF-  Andersen P, et al. Lancet 2000;356:1099

14. E arly S ecreted A ntigenic T arget 6 & C ulture F ilterate P rotein 10

15. 3 rd Generation Quantiferon TB Gold IT (in Tube method) Grey cap tube (Nil control) Red cap tube (TB Ag) Purple cap tube (Mitogen control) Collect 1 ml blood into each tube and mix well Transfer to Lab ASAP F or detection of Latent TB, has negative predictive value

16. Attribute Tuberculin Skin Test (Mantoux Test) IGRA Type In vivo In vitro Sample Injected into skin (intradermal) 5 mL of Blood in Lithium Heparin tube Visits 2 1 Antigen used PPD ESAT 6 & CFT 10 from Mtb RD1 region Cross reaction with BCG Yes No Use For diagnosis of LTBI Latent TB & now recent exposure of contacts or active TB Results Read after 48 or 72 hours ELISA test after 24 hours of exposure Reading Subjective Objective Booster effect Yes No Cost Low High

17. CD 8 CD 8+ cells assessed in adults with LTBI or active TB disease by FACS CD 8+ cells found more frequently in PTB (67%), ETB (37%) than LTBI (15%) CD 8+ cells found in high frequency in Smear + than Smear - patients

18. Cellular Immunity plays an important role in containing Tuberculosis

19. CD8 T-cells Suppress Mycobacteria tuberculosis growth Kill infected cells Directly lyse intracellular Mycobacteria TB specific CD8 T cells that produce IFN- Ύ have been More frequently detected in those with active TB disease vs latent infection Associated with recent exposure Detectable in active TB subjects with HIV co-infection & young children

20. We are calling this test TB Sure to avoid any confusion TB SURE

21. © In conclusion , QFT-Plus and QFT-GIT assays showed a substantial agreement and similar accuracy for active TB detection . Interestingly, a higher proportion of the LTBI subjects responded concomitantly to TB1 and TB2 compared to those with active TB, whereas a selective TB2 response associated with active TB .

22. HIV + HIV --

23. 108 TB patients; 73% HIV + ve ; 90 were TB Sure + ve ; 11 Negative & 7 indeterminate at initiation Sensitivity 83%

24. TB SURE TB Sure®

25. Caution A NEGATIVE TB Sure® result doesnot preclude the possibility of Mycobacterium tuberculosis infection or disease 1. Sample may have been collected before development of CMI response 2. Hetrophil antibodies or non specific IFN- Ύ production from other inflammatory conditions may mask specific response to ESAT 6 or CFP 10. A POSITIVE TB Sure® shouldnot be the sole or definitive basis for determining infection or disease with M tuberculosis . It should be accompanied by addition clinical and laboratory investigations.

26. TB SURE TB Sure

27. Greater Specificity for M tuberculosis exposure with TB Sure®

28. Quantiferon TB Gold Plus [TB Sure®] Is a blood test, provides indirect evidence of infection Can detect LTBI as well as recent exposure & active infection Can be used for monitoring of response to treatment [0, 3 & 6 Month] Not effected by HIV status [till CD4 count is < 100 /mm 3 Doesnot detect Drug resistance Gene Xpert & MGIT culture [Gene xpert combo] is preferred if bacteria containing sample can be submitted e.g. sputum, pus, fluid, biopsy, csf

29. Tuberculosis Clinically suspected ? Pulmonary or Extra Pulmonary ?

30. TB Sure® Reports Possible Negative Mitogen TB 1- N TB 2- N TB 2 – TB 1 Report IU / mL < 8 > 0.5 < 0.35 < 0.35 < 0.5 Reference Range 0.05 4.08 0.10 0.12 0.02 Infection with M tuberculosis unlikely 0.05 7.61 4.14 3.16 -- 0.92 LTBI 0.20 1.10 4.26 9.34 5.09 Active disease / Recent Infection 0.2 0.2 0.2 0.2 Indeterminate 8.2 > 10 > 10 > 10 ? Indeterminate