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Dilektaşlı Aslı, Erdem Elif, Budakoğlu İrem, Eyüboğlu Ö Füsun

Comparision of a T- cell - based assay (T-Spot. TB ) with tuberculin skin test in patients with latent and active tuberculosis. Dilektaşlı Aslı, Erdem Elif, Budakoğlu İrem, Eyüboğlu Ö Füsun Baskent University Faculty of Medicine Department of Pulmonary Diseases,

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Dilektaşlı Aslı, Erdem Elif, Budakoğlu İrem, Eyüboğlu Ö Füsun

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  1. Comparision of a T-cell-basedassay (T-Spot.TB)withtuberculin skin test in patientswithlatentandactive tuberculosis Dilektaşlı Aslı, Erdem Elif, Budakoğlu İrem, Eyüboğlu Ö Füsun Baskent University Faculty of Medicine Department of Pulmonary Diseases, Ankara, Turkey

  2. Latent Tuberculosis Infection • LTBI is defined as the presence of dormant Mycobacterium tuberculosis in an individual, and the infection is not clinically apparent • For LTBI diagnosis • Higher sensitivity and specifity than TST • Less cross-reactivity due to BCG and nontuberculous mycobacterial infection • Rapid and easier tests are needed Cristopher C.W. Diagnosis of Latent Tuberculosis Infection. JAMA; Jun 8, 2005; 293, 22; 2785–7.

  3. Serum Interferon-ReleaseAssays enduration IL-8 TCT/ in vivo TNF- IFN- skin Effector CD4T lymphocytes Antigen Memory T lymphocytes CD4 T lymphocytes IL-2 IFN-  release assays/in vitro ELISA QUANTIFERON ELISPOT T-SPOT.TB M.phages/DH M.phages/DH M.phages/DH IL-8 TNF- IFN- Memory CD4 T lymphocytes Memory T lymphocytes

  4. Which Antigens? • Region of Difference-1, a genomic segment of M. Tuberculosis, which is deleted from all strains of BCG vaccine and most environmental mycobacteria • Early secreted antigenic target 6 (ESAT–6) and culture filtrate protein 10 (CFP–10) antigens encoded by RD-1, are strong targets of T-helper type 1 cells • Therefore, a T-cell response to these specific antigens serve as specific markers of M.tuberculosis infection Lalvani A. Diagnosing Tuberculosis Infection in the 21st century. Chest 2007;131:1898-1906

  5. Localimmunodiagnosis of pulmonary TB by ELISPOT • BAL/Periferic Blood ESAT-6 spesific T-lymphocyte: 9.9 • BAL/Periferic Blood CFP-10 spesific T-lymphocyte : 8.9 Jafari C, Lange C. e al. Eur Respir J 2008:31:261-265

  6. AIM • To compare TST and ELISPOT (T-Spot. TB) in TB patients, contacts and healthy control subjects • To investigate whether a rapid diagnosis of active pulmonary TB can be established by enumeration of M. tuberculosis-specific T lymphocytes from induced sputum in routine clinical practice

  7. MATERIAL-METHOD • Group 1: Health-careworkerswithexposuretoM. Tuberculosis (n=30) • Group 2: Culture (+) TB patients (n=31) • Healthyvolunteerswith no TB contactand TB disease (n=30) TST T-SPOT.TB InducedsputumT-SPOT.TB in active TB patients

  8. T-SPOT.TB Methodology Plasma PKMNH Seperation jel Ficoll-Paque TM plus Red blood cells

  9. T-SPOT.TB Methodology

  10. T-SPOT.TB Methodology

  11. Induced Sputum Analysis • Well-standardizednon-invasivediagnosticproceduretoobtainlowerrespiratorytractsecretions in patientswithoutspontaneoussputumexpectoration • Expectorationafterinhalation of sterile hypertonicNaClbyultrasonicnebulisorfollowingsalbutamol • Sputum + sputalysin (1,4- dithiothreitol) • Evaluation of viabilityand TCC • Santrifugation (790xg, 10’, 4C) • Cytospinpreperationbycytosantrifuge (22xg- 6’) • Evaluation of DCC • MNC isolationbyFicoll • Incubationwith ESAT-6 ve CFP-10 (T-SPOT.TB) Filtration Paggiaro P, Spanevello A. et al. Sputum induction: methods and safety. An Atlas of Induced Sputum. Parthenon Publishing, UK, 2004. p11-21.

  12. MATERIAL-METHOD ExclusionCriteria • Solid organ and bone marrow transplants • 15 mg/day prednisone /equavelent therapy for at least 1 month • Chronic renal/liver failure • Malignancy • DM

  13. RESULTS

  14. DemographicFeatures TST results were interpretated according to Turkish National Tuberculosis Dispensary , Ministry of Health of Turkey Values are expressed as median±SD or n (%) . F:female, M:male, BCG: Bacille Calmette-Guérin, TS: tuberculin skin test

  15. TST ve T.SPOT-TBSensitivityandSpecifity TST:tuberculin skin test, PPV: positive predictive value, NPV:negative predictive value

  16. Group 1: Health-care workers with exposure to MTB 40% LTBI 83.3% LTBI Agreement between two tests was low (=0,28, p=0.33)

  17. Group 2: TST and T-SPOT.TB in Active TB Patients Moderate agreemnet between two tests was defined (=0,55, p=0.36)

  18. Differential Cell Counts in Induced Sputum Samples (n=29) *TCC: total hücre sayısı, min: minumum değer, max: maksimum değer **Induced sputum differential cell counts in healthy adults : TCC 4.13X106/g, Macrophages: 60.8%, Neutrophils36.7%, Eosinohils: 0.00% , Lymphocytes 0.50%, Epithelial cells: 0.30%

  19. Induced Sputum and Serum T.SPOT-TB Results IS: Induced sputum

  20. Group 3: TST and T-SPOT.TB in Healthy Volunteers 23.3% LTBI 43.3% LTBI Agreement between two tests was poor (=0,14, p=0.4)

  21. DISCUSSION • Health-care workers and healthy volunteers were more likely to be diagnosed as LTBI on the basis of TST than T-Spot.TB TST:83.3%,T-Spot.TB:40%; TST:43.3%,T-Spot.TB:23.3% • Diagnosing LTBI on the basis of T-Spot.TB rather than TST results in a decrease of costs and side effects due to unnecesseray LTBI treatment

  22. Thelowerspecifity of T-SPOT.TB results in thisstudycould be explainedbyhighexposuretonontuberculousmycobacterialspecies in ourcountry • Highnegativepredictivevalue of the T-Spot.TB test suggeststhatthis test could be reliable in exclusion of TB in active TB suspects

  23. Induced Sputum and T.SPOT-TB • Induced sputum T-Spot.TB results were undefined in 69% of active TB patients. This can be explained by lower lymhocyte count in induced sputum compared to PBMC (1/10) • Positive induced sputum T-SPOT.TBresults support the diagnosis of active pulmonary TB • Improving T-SPOT.TB technique in induced sputum samples may lead to a decrease in undetermined results of this method

  24. CONCLUSION • The sensitivity and specifity of the T-Spot.TB is greater than TST in diagnosis of active TB • T-Spot.TB test allows a more rapid exclusion of TB in suspected cases than TST • T-Spot.TB offers a more accurate approach than TST in identification of individuals who have LTBI

  25. LaboratoryinfrastructureandthisreserachwasfoundedbyTheScientificandTechnologicalResearchCouncil of Turkey ACKNOWLEDGEMENTS • Elif Erdem, ourlaboratorytechnician • Dr. Şeref Özkara, Dr. Nilgün Kalaç • Dr. Filiz Duyar Ağca, Dr. Onur Aksu Ceyhan and Dr. KerimanAltunay

  26. WHO Global tuberculosis control report, 2008

  27. There are three potential outcomes of infection of the human host in Mycobacterium tuberculosis. a | The frequency of abortive infection resulting in spontaneous healing is unknown, but is assumed to be minute. b | In the immunocompromised host, disease can develop directly after infection. c | In most cases, mycobacteria are initially contained and disease develops later as a result of reactivation. The granuloma is the site of infection, persistence, pathology and protection. Effector T cells (including conventional CD4+ and CD8+ T cells, and unconventional T cells, such as T cells, and double-negative or CD4/CD8 single-positive T cells that recognize antigen in the context of CD1) and macrophages participate in the control of tuberculosis. Interferon- (IFN- ) and tumour-necrosis factor- (TNF- ), produced by T cells, are important macrophage activators. Macrophage activation permits phagosomal maturation and the production of antimicrobial molecules such as reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI). LT- 3, lymphotoxin- 3.

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