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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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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
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.
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
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
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
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
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
T-SPOT.TB Methodology Plasma PKMNH Seperation jel Ficoll-Paque TM plus Red blood cells
Induced Sputum Analysis • Well-standardizednon-invasivediagnosticproceduretoobtainlowerrespiratorytractsecretions in patientswithoutspontaneoussputumexpectoration • Expectorationafterinhalation of sterile hypertonicNaClbyultrasonicnebulisorfollowingsalbutamol • Sputum + sputalysin (1,4- dithiothreitol) • Evaluation of viabilityand TCC • Santrifugation (790xg, 10’, 4C) • 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.
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
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
TST ve T.SPOT-TBSensitivityandSpecifity TST:tuberculin skin test, PPV: positive predictive value, NPV:negative predictive value
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)
Group 2: TST and T-SPOT.TB in Active TB Patients Moderate agreemnet between two tests was defined (=0,55, p=0.36)
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%
Induced Sputum and Serum T.SPOT-TB Results IS: Induced sputum
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)
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
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
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
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
LaboratoryinfrastructureandthisreserachwasfoundedbyTheScientificandTechnologicalResearchCouncil of Turkey ACKNOWLEDGEMENTS • Elif Erdem, ourlaboratorytechnician • Dr. Şeref Özkara, Dr. Nilgün Kalaç • Dr. Filiz Duyar Ağca, Dr. Onur Aksu Ceyhan and Dr. KerimanAltunay
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.