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

Baskent University Faculty of MedicineDepartment of Pulmonary Diseases,

Ankara, Turkey

Cristopher C.W. Diagnosis of Latent Tuberculosis Infection. JAMA; Jun 8, 2005; 293, 22; 2785–7.

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

ELISPOTT-SPOT.TB

ELISAQUANTIFERON

Serum Interferon- Release Assays

Effector CD4T lymphocytes

IL-2

CD4 T lymphocytes

Memory CD4 T lymphocytes

M.phages/DH

Antigen

M.phages/DH

TNF- IFN-IL-8

Memory T lymphocytes

IFN- release assays/in vitro

TCT/ in vivoTNF- IFN-

IL-8

M.phages/DH Memory T lymphocytes

enduration

skin

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

Which Antigens?

Lalvani A. Diagnosing Tuberculosis Infection in the 21st century. Chest 2007;131:1898-1906

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

Local immunodiagnosis of pulmonary TB by ELISPOT

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

1. Group 1: Health-care workers with exposure to M. Tuberculosis (n=30)

2. Group 2: Culture (+) TB patients (n=31)

3. Healthy volunteers with no TB contact and TB disease (n=30)

TST

T-SPOT.TB

Induced sputumT-SPOT.TB in active TB patients

Plasma

PKMNH

Red blood cells

Seperation jel

Ficoll-Paque TM plus

T-SPOT.TB Methodology

T-SPOT.TB Methodology

T-SPOT.TB Methodology

Induced Sputum Analysis

Evaluation of viability and TCC

Santrifugation (790xg, 10’, 4C)

Cytospin preperation by cytosantrifuge (22xg- 6’)

Evaluation of DCC

Expectoration after inhalation of sterile hypertonic NaCl by ultrasonic nebulisor following salbutamol

Sputum + sputalysin (1,4- dithiothreitol)

MNC isolation by Ficoll

Incubation with ESAT-6 ve CFP-10 (T-SPOT.TB)

Paggiaro P, Spanevello A. et al. Sputum induction: methods and safety. An Atlas of Induced Sputum. Parthenon Publishing, UK, 2004. p11-21.

Filtration

Well-standardized non-invasive diagnostic procedure to obtain lower respiratory tract secretions in patients without spontaneous sputum expectoration

Solid organ and bone marrow transplants

15 mg/day prednisone /equavelent therapy for at least 1 month

Chronic renal/liver failure

Malignancy

DM

MATERIAL-METHODExclusion Criteria

RESULTS

VariablesPulmonary

TB

Health-care workers with MTB

exposure

Healthy volunteers

Total

Age 41.8 ± 17.5 28.5 ± 3.8 37.2 ± 12.8 35.9 ± 13.8

Sex (F/M) 16:15 17:13 20:10 53:38

BCG vaccination (%)

83.9 96.7 100 93.4

TST (mm) 11.8 ± 5.7 17.1 ± 4.8 14.2 ± 5.3 14.3 ± 5.7

Number (n) 31 30 30 91

Demographic Features

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-TB Sensitivity and Specifity

Sensitivity(% )

Specifity(% )

PPV (% )

NPV(% )

TST (≥ 15 mm)

38.1 42.5 25.8 56.7

T-SPOT.TB 93.1 76.7 79.4 92

TST:tuberculin skin test, PPV: positive predictive value, NPV:negative predictive value

Group 1: Health-care workers with exposure to MTB

TSTT.SPOT-TB

positive (n)

T.SPOT-TB Negative

(n)

T.SPOT-TB undefined

(n)

Totaln (%)

≤5 mm 6-14 mm≥ 15mm

--

12

-5

11

--2

-5 (16.7%)

25 (83.3%)

Toplam 12 (40%) 16 (53.3%) 2 (6.7%) 30 (100%)83.3% LTBI

40%LTBI

Agreement between two tests was low (=0,28, p=0.33)

ELISPOT TST15mm

OR (%95 CI) p OR (%95 CI) p

Sex Female Male

111.0 (1.02-126.44) 0.05

11.0 (0.13-8.21)

0.96

Age 1.15 (0.87-1.53) 0.33 1.13 (0.86-1.49) 0.39

BCG positive negative

10 (0-1.32) 0.90

10 (0.0-4.59) 0.91

Time of exposure 1-199 hr 200-399 hr ≥ 400 hr

17.690.01

(0.41-145.63)(0-1.2)

0.170.90

17.690.01

(0.0-1.47)(0.0-4.59)

0.900.91

TSTT.SPOT-TB

positive (n)

T.SPOT-TB negative

(n)

T.SPOT-TB undefined

(n)

Totaln (%)

≤ 5 mm

6-14 mm

≥15 mm

3

16

8

-

2

-

2

-

-

5 (16.1%)

18(58.1%)

8(25.8%)

Total 27 (87%) 2 (6.5%) 2 (6.5%) 31 (100%)

Group 2: TST and T-SPOT.TB in Active TB Patients

Moderate agreemnet between two tests was defined (=0,55, p=0.36)

TCC x 106

Viability(%)

Macrophages (%)

PMNL (%)

Lymphocytes(%)

Eosinophils (%)

Epithelial cells(%)

Median

(min-max)

3.96

(1-24)

80

(50-90)

22

(11-95)

62

(2-82)

8

(1-41)

2

(1-4)

8

(1-22)

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 T.SPOT-TB

SerumT.SPOT-TB

+(n)

Serum T.SPOT-TB

-(n)

Serum T.SPOT-TB undefined

(n)

Totaln (%)

IS T-SPOT.TB +

IS T-SPOT.TB –

IS T-SPOT.TB undefined

5

2

19

1

-

1

1

-

-

7 (24.1%)

2 (6.9%)

20 (69%)

Total 26 2 1 29

(100%)

Induced Sputum and Serum T.SPOT-TB Results

IS: Induced sputum

TSTT.SPOT-TB

positive(n )

T.SPOT-TB negative

(n )

Total n (%)

≤ 5 mm6-14 mm≥ 15 mm

124

-149

1 (3.4%)16 (53.3%)13 (43.3%)

Total 7 (23.3%) 23 (76.7%) 30

Group 3: TST and T-SPOT.TB in Healthy Volunteers

43.3%LTBI

23.3% 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

ELISPOT (T-SPOT.TB)

n Sensitivity, %

Meier T. et. al. (Eur J Clin Microbiol Infect Dis 2005)

72 97

Lalvani A. et. al. (Am J Respir Crit Care Med 2001)

47 96

Pathan AA. et. al. (J Immunol 2001) 36 92

Ferrara G. et.al. (Lancet 2006) 24 83

Lee JY. et. al. (Eur Respir J 2006) 87 96.6

Eyüboğlu FÖ. et al. 61 93

ELISPOT (T-SPOT.TB)

n Specifity%

Pathan AA. et. al. (J Immunol 2001)

28 100

Lalvani A.et al. (J Infect Dis 2001) 40 100

Lalvani A. et. al. (Am J Respir Crit Care Med 2001)

26 100

Lee JY. et al. (Eur Respir J 2006) 218 84.7

Eyüboğlu FÖ. et al. 61 77

The lower specifity of T-SPOT.TB results in this study could be explained by high exposure to nontuberculous mycobacterial species in our country

High negative predictive value of the T-Spot.TB test suggests that this test could be reliable in exclusion of TB in active TB suspects

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.TB results 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

Induced Sputum and T.SPOT-TB

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

Laboratory infrastructure and this reserach was founded by

The Scientific and Technological Research Council of Turkey

ACKNOWLEDGEMENTS

Elif Erdem, our laboratory technician

Dr. Şeref Özkara, Dr. Nilgün Kalaç

Dr. Filiz Duyar Ağca, Dr. Onur Aksu Ceyhan and Dr. Keriman

Altunay

WHO Global tuberculosis control report, 2008

TST IFN- Release Assays

Sensitivity 75-90% 75-95%

Specifity 70-95% 90-100 %

Antigens PPD ESAT-6, CFP-10 (RD-1)

Cross-reactivity with BCG Yes Less likely

Cross-reactivity with NTM Yes Less likely, limited evidence

Boosting phenomenon yes no

Patient visits to complete testing

two one

Material costs low Moderate-high

Laboratory infrastructure needed

no yes

Trained personnel required yes yes

Time to obtain a result 2-3 days 1-2 days

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.