Accurately identifying those with tuberculosis infection and disease is crucial to initiative to reduce related deaths.
Recent mathematical models have estimated that, in 2014, the global burden for individuals demonstrating tuberculosis (TB) infection was 1.7 billion people, and that 9.6 million people developed active TB disease, resulting in 1.5 million deaths.1,2 Of the 1.7 billion people infected with TB, 5-10%, who are HIV negative, will progress to active disease during their lifetime.3 A number of risk factors for progression have been identified and include HIV, type 2 diabetes, young age, alcohol use, smoking and the use of immunosuppressants for autoimmune diseases.4-9
Despite a low rate of transmission, the disease remains highly lethal.10 TB has emerged as the leading cause of death for any single pathogen.11 The WHO has issued an End TB Strategy initiative with the goals of a 95% reduction in the number of TB deaths and a 90% reduction in TB incidence rate compared with 2015 by the year 2035.12 To achieve this goal, there must be the capacity to accurately identify those who have been infected with TB (TB infection, TBI) and those who have active TB disease.
Detecting TB Infection
The ability to identify individuals infected with TB originated with the Mantoux test—tuberculin skin test (TST)—approximately 100 years ago. The test is performed by intradermal placement of TB antigens that will initiate a delayed type hypersensitivity reaction within 48-72 hours in TB infected individuals. Accurate interpretation of the test requires precise measurement of the induration, knowledge of the individual’s pre-test probability of infection and health status.13 The TST requires few reagents, no sample manipulation and has the perception that it is cost-effective. Therefore, it is frequently used in resource-poor settings. Limitations of the test include cross-reactivity with Bacillus Calmette-Guérin (BCG), used for vaccines, as well as previous exposure to non-tuberculous mycobacteria (NTM).14,15 A reading of the test is also dependent on the individual’s compliance, as they need to return 48-72 hours following TST placement.
The need for a test with less cross-reactivity gave rise in the early 2000s to two tests that use in vitro assessment of an immune response to selected TB antigens. The two tests have a similar concept, which is to quantify T lymphocyte generation of interferon gamma (IFNγ) following stimulation with two TB antigens that are not present in BCG or in most NTM. The tests differ in the approach as one, T-SPOT.TB, uses an ELISPOT platform, and one, QuantiFERON Gold-In-Tube, uses an ELISA platform. The two interferon-gamma release assays (IGRAs) differ from the TST in that they are assessing selective responsiveness by T effector cells to TB antigens in contrast to the TST which demonstrates responsiveness based on activation of both T effector and T memory cells stimulated by a large number of TB antigens.16 The selective nature of the IGRAs in most reports have resulted in methodologies which have increased sensitivity and specificity compared with the TST, in addition to a reported increased compliance.17
The IGRA approaches are quite different. The ELISA test utilizes whole blood collected in three tubes that contain either TB antigens (ESAT-6, CFP10 and TB 7.7), PHA as a positive control, or no antigen as a negative control. The tubes are incubated overnight and then plasma isolated and assessed for IFNγ production by a standard ELISA technique.18 This process results in stimulation of TB-sensitized cells contained in the 1cc whole blood used for each tube.
The ELISPOT assay uses a different approach whereby peripheral blood mononuclear cells (PBMCs) are isolated from whole blood using density centrifugation, washed and then resuspended to effect a concentration of 250,000 cells/well. This results in a normalized number of cells in each well, independent of the number of cells contained in each 1cc of whole blood prior to isolation.
The cells are divided into four wells and incubated overnight with TB antigens ESAT-6 and CFP-10 in separate wells, PHA as a positive control and no antigen as a negative control. Interferon gamma produced by antigen-sensitized cells is captured by plate-bound anti-IFNγ antibody and the number of IFNγ-producing cells are quantified by elucidation of IFNγ-mediated spots for each well.19
The different methodological approaches result in different sensitivities. In the recent U.S. Preventive Services Task Force Recommendation Guidelines, they state that pooled analyses indicate a sensitivity of 90% (16 studies/984 subjects), specificity of 95% (5 studies/1810 subjects) for T-SPOT.TB, and a sensitivity of 80% (24 studies/2321 subjects), specificity of 97% (4 studies/2053 subjects) for QuantiFERON Gold-In-Tube.20
Inherent in conducting an in vitro assay using whole blood is the importance of blood handling prior to performing the test. This is particularly important when the blood is collected several hours prior to setting up the assay. Studies have indicated that unless isolated from granulocytes (neutrophils), T lymphocytes begin to lose responsiveness to antigen stimulation approximately 8 hours following venipuncture.21
Loss of responsiveness can be attributed, at least in part, to neutrophil-generated hydrogen peroxide, which alters the ability of the T lymphocyte to produce cytokines such as IFNγ.22 Both IGRA tests have incorporated steps that prolong the responsiveness of the T lymphocytes. The QuantiFERON test prolongs T lymphocyte responsiveness by drying the antigens onto the sides of the tubes rather than suspending them in a liquid. This change allows for 16 hours between venipuncture and initiation of incubation for 16-24 hours.18
Extending Blood Storage
While the Oxford Immunotec T-SPOT.TB test is available as a kit, similar to QuantiFERON Gold-In-Tube, most tests run in the U.S. utilize a centralized laboratory to process the cells and conduct the ELISPOT, which for most draw sites, requires an overnight shipment of the blood sample.19,23
To address the issue of granulocyte contamination, Oxford Immunotec has incorporated an FDA-approved step in enriching PBMC designed to reduce granulocytes, even those with reduced density due to degranulation.19 The step includes the addition of a reagent, T-Cell Xtend, an antibody complex that binds to both red blood cells and CD66b, a protein expressed on activated granulocytes.26 By virtue of binding to both cell types, the antibody acts to bridge the cells, creating a complex that now has sufficient density during gradient centrifugation. The result is a highly enriched PBMC preparation with typically less than 2% contaminating granulocytes.27
This level of granulocyte contamination does not appear to affect cell responsiveness to TB antigen stimulation as a number of clinical studies have identified very high congruency when comparing T-SPOT.TB test results using T lymphocytes from freshly isolated blood or from blood stored up to 32 hours prior to isolation of T lymphocytes.28,29,30
One study included numerous subjects with increased risk factors for TB reported overall agreement for the paired tests was 95.4% (288/302; range 92.3-97.4%), indicating that, even in immunocompromised individuals, the addition of T-Cell Xtend can prolong T cell responsiveness without negatively affecting test results.30
Maintaining Test Sensitivity
While congruency tests have indicated comparable data for fresh versus stored blood, the better assessment of preserving T lymphocyte responsiveness is to determine whether this process affects overall test sensitivity. A number of studies have demonstrated test sensitivity greater than 93%, with two including immunocompromised subjects—HIV+ and aged populations.31,32 To accomplish the “End TB Initiative,” a screening strategy must exist to help identify the 1.7 billion individuals who constitute the world’s reservoir of TB. Blood tests have proven to be more sensitive and reliable than the TST, but require time to allow for blood transportation and processing prior to loss of T lymphocyte responsiveness. Prolonging T lymphocyte responsiveness provides the needed flexibility for when blood samples cannot be processed within 8 hours of venipuncture.
- Houben RMGJ, Dodd PJ. The Global Burden of Latent Tuberculosis Infection: A Re-estimation Using Mathematical Modelling. PLoS Med. 2016;13(10).
- WHO. Global Tuberculosis Report 2015. 20th ed. Geneva; 2015.
- Vynnycky, E. and Fine, PE. The natural history of tuberculosis: the implications of age-dependent risks of disease and the role of reinfection. Epidemiol. Infect. 119:183-201, 1997.
- Havlir, DV. Getahun, H. Sanne, I. and Nunn P. Opportunities and challenges for HIV care in overlapping HIV and TB epidemics. JAMA 300:423-430, 2008.
- Jeon, CY, and Murray, MB. Diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies. PLoS Med. 5:e152, 2008.
- Dodd, PJ, Sismanidis C, and Seddon JA. Global burden of drug-resistant tuberculosis in children: a mathematical modelling study. Lancet Infect. Dis. 16:1193-1201, 2016
- Rehm, J, et al. The association between alcohol use, alcohol use disorders and tuberculosis (TB). A systematic review. BMC Public Health 9:450, 2009.
- Bates, MN, et al. Risk of tuberculosis from exposure to tobacco smoke: a systematic review and meta-analysis. Arch. Intern. Med. 167:335-342, 2007.
- Singanayagam A, Manalan K, Sridhar S, et al. Evaluation of screening methods for identification of patients with chronic rheumatological disease requiring tuberculosis chemoprophylaxis prior to commencement of TNF-alpha antagonist therapy. Thorax. 2013;68(10):955-961.
- Andrews, JR. et al. Risk of progression to active tuberculosis following reinfection with Mycobacterium tuberculosis. Clin. Infect. Dis. 54:784-791, 2012.
- Giorgia Sulis, Rosella Centis, Giovanni Sotgiu, Lia D’Ambrosio, et al. Recent developments in the diagnosis and management of tuberculosis. NPJ Prim Care Respir Med. 26: 16078, 2016.
- WHO. The END TB Strategy. Global strategy and targets for tuberculosis prevention, care and control after 2015. Published 2013.
- Farhat M, Greenaway C, Pai M, Menzies D. False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria? The International Journal of Tuberculosis and Lung Disease.10(11):1192–1204, 2006.
- Menzies D, Gardiner G, Farhat M, Greenaway C, Pai M. Thinking in three dimensions: a web-based algorithm to aid the interpretation of tuberculin skin test results. The International Journal of Tuberculosis and Lung Disease.12(5):498–505, 2008.
- Pai M, Riley LW, Colford JM Jr. Interferon-gamma assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis.4(12):761-776, 2004.
- Mack U, Migliori GB, Sester M, et al. LTBI: latent tuberculosis infection or lasting immune responses to M. tuberculosis? A TBNET consensus statement. Eur Respir J.33(5):956-973, 2009.
- Wrighton-Smith P, Sneed L, Humphrey F, Tao X, Bernacki E. Screening health care workers with interferon-gamma release assay versus tuberculin skin test: impact on costs and adherence to testing (the SWITCH study). J Occup Environ Med.54(7):806-815, 2012.
- Qiagen. QuantiFERON®-TB Gold (QFT®) ELISA Package Insert 2013.
- Oxford Immunotec. Oxford Immunotec Ltd. T-Cell Xtend Package Insert PI-TT.610-US-V5 Abingdon, UK. 2014.
- Bibbins-Domingo K, Grossman DC, Curry SJ, et al. Screening for Latent Tuberculosis Infection in Adults: US Preventive Services Task Force Recommendation Statement. JAMA.316(9):962-969, 2016.
- Bull M, Lee D, Stucky J, et al. Defining Blood Processing Parameters for Optimal Detection of Cryopreserved Antigen-Specific Responses for HIV Vaccine Trials. J Immunol Methods. 322(1-2):57-69, 2007.
- Schmielau J, Finn OJ. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients. Cancer Res. 61(12):4756-4760, 2001.
- McKenna KC, Beatty KM, Vicetti MR, Bilonick RA. Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function. J Immunol Methods.341(1-2):68-75, 2009.
- Lewis SL, Van Epps DE, Chenoweth DE. Analysis of density changes and chemotactic receptors of leukocytes from chronic hemodialysis and peritoneal dialysis patients. Blood Purif. 5(2-3):138-54,1987.
- Jan Schmielau J. and Finn O. Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of T-cell function in advanced
- cancer patients. Cancer Research 61:4756–4760, 2001.
- Futosi K, Fodor S, Mócsai A. Neutrophil cell surface receptors and their intracellular signal transduction pathways. Int Immunopharmacol. Nov;17(3):638-50, 2013.
- Ivan J. Fuss, Marjorie E. Kanof, Phillip D. Smith, Heddy Zola. Isolation of Whole Mononuclear Cells from Peripheral Blood and Cord Blood. Current Protocols in Immunology, Unit 7.1, 2009.
- Talbot EA, Maro I, Ferguson K, et al. Maintenance of Sensitivity of the T-SPOT.TB Assay after Overnight Storage of Blood Samples, Dar es Salaam, Tanzania. Tuberc Res Treat.;2012:345290, 2012.
- Higuchi K, Sekiya Y, Igari H, Watanabe A, Harada N. Comparison of specificities between two interferon-gamma release assays in Japan. Int J Tuberc Lung Dis.16(9):1190-1192, 2012.
- Wang SH, Stew SS, Cyktor J, Carruthers B, Turner J, Restrepo BI. Validation of increased blood storage times with the T-SPOT.TB assay with T-Cell Xtend reagent in individuals with different tuberculosis risk factors. J Clin Microbiol.50(7):2469-2471, 2012.
- Rajagopalan S. Tuberculosis in Older Adults. Clin Geriatr Med. 32(3):479-91, 2016.
- Bae W, Park KU, Song EY, et al. Comparison of the Sensitivity of QuantiFERON-TB Gold In-Tube and T-SPOT.TB According to Patient Age. Shams H, ed. PLOS ONE. 11(6):e0156917, 2016.