Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigen-specific interferon γ assay

G Matulis; P Jüni; P M Villiger; S D Gadola

doi:10.1136/ard.2007.070789

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Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigen-specific interferon γ assay

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The theoretical advantages of the ELISPOT assay based T-SPOT.TB test
San San Aye
Published on: 8 January 2008
PHA concentration in IGRA assays
Christopher J Granger
Published on: 1 August 2007

Published on: 8 January 2008
The theoretical advantages of the ELISPOT assay based T-SPOT.TB test
- San San Aye, Research Co-ordinator
Dear Editor,
After carefully reading this article and other related references, weagree that detection of M tuberculosis using interferon gamma release assays (IGRA) is more sensitive and specific than the classic Tuberculin Skin Test (TST) for the detection of Latent TB Infection (LTBI) in patients receiving immunosuppressive therapy. As the authors indicate, there are two commercially available IGRAs, an Enzyme Link...
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Dear Editor,
After carefully reading this article and other related references, weagree that detection of M tuberculosis using interferon gamma release assays (IGRA) is more sensitive and specific than the classic Tuberculin Skin Test (TST) for the detection of Latent TB Infection (LTBI) in patients receiving immunosuppressive therapy. As the authors indicate, there are two commercially available IGRAs, an Enzyme Linked Immunosorbent Assay (ELISA), QuantiFeron TB-Gold, and an Enzyme Linked Immunospot (ELISPOT) assay, T-SPOT.TB.
It has been shown that the ELISPOT methodology can be up to 200 times more sensitive than ELISA. T-SPOT.TB requires just a few spots to give a positive result (one spot = one effector T cell), so just a few activated T cells in a sample can provide a positive result. Many more T cells are required to provide a measurable change in cytokine concentration by ELISA.
In addition to the methodological advantages of ELISPOT over ELISA, the T-SPOT.TB protocol has some procedural differences that produce a more reliable result. T-SPOT.TB has a washing step that removes the source of interfering background signals, which reduces sensitivity and produces indeterminate results. This washing step is absent in the ELISA based QuantiFeron TB-Gold.
In the T-SPOT.TB procedure, the Peripheral Blood Mononuclear Cells (PBMCs) are counted. This step ensures there are an adequate number of cells and that each time the assay is performed there is the same number of cells present, which standardizes the assay. This step maintains the high sensitivity in immunocompromised and immunosuppressed patients. This step is also absent in the ELISA based QuantiFeron TB-Gold.
In conclusion, we believe that T-SPOT.TB is a novel in vitro diagnostic test that uniquely identifies the number of T cells specific to Mycobacterium tuberculosis antigens and therefore is better equipped to identify LTBI, particularly in immunosuppressed populations.
References
1. Tanguay and Killion. Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine and Cytokine Research 1994;13:259–263.
2. G Matulis, P Jüni, P M Villiger, S D Gadola. Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigen-specific interferon assay. Ann Rheum Dis 2008;67:84-90.
3. www.oxfordimmunotec.com
4. www.cellestis.com
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Conflict of Interest:
None declared.
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Published on: 1 August 2007
PHA concentration in IGRA assays
- Christopher J Granger, Director, Professional Relations
Dear Editor, In the article by Matulis et al, published online on 20 July 2007 the authors state in their discussion that “Compared with the ELISA test used in our study the IFN-gamma ELISPOT test was previously reported to have lower rates of indeterminate results (1). These differences may, among other reasons, be explained by differences in the concentrations of the mitogen PHA between the two test systems”. Can the authors...
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Dear Editor, In the article by Matulis et al, published online on 20 July 2007 the authors state in their discussion that “Compared with the ELISA test used in our study the IFN-gamma ELISPOT test was previously reported to have lower rates of indeterminate results (1). These differences may, among other reasons, be explained by differences in the concentrations of the mitogen PHA between the two test systems”. Can the authors please provide the PHA concentrations used in the QuantiFERON-TB Gold and IFN-gamma ELISPOT (T-SPOT.TB) assays and explain if they are indeed different.
We agree with the authors statement “the elevation of PHA concentrations to lower the rates of indeterminate results will not per se lead to a more accurate test: T-lymphocytes may respond to the strong unspecific stimulus, but not to tuberculosis specific peptides”. The authors continue “the performance of IFN gamma assays using higher PHA concentrations in the positive control reaction of the test may therefore be overestimated.” The authors are suggesting that an assay system that elevates PHA concentrations will have lower indeterminate rates. However, this would also allow the assay to report results in individuals who may not respond to tuberculosis-specific peptides. In some cases these subjects will be falsely negative. Therefore, following the author’s logic, an assay system where the PHA concentration has been increased to lower the numbers of indeterminates would result in lower sensitivity.
The authors imply that the IFN-ELISPOT assay system (T-SPOT.TB) has been modified in this way, which would explain the previously reported lower rates of indeterminate results compared to the IFN-ELISA system (QuantiFERON TB Gold). However, if this were the case, the lower indeterminate rates associated with the T-SPOT.TB system would also result in a reduced ability to identify infected subjects (lower sensitivity). However, previous studies have shown that T-SPOT.TB has BOTH lower indeterminates AND higher sensitivity than QFN-Gold1 (1,2,3).
This is consistent with the fact that the ELISPOT assay methodology is orders of magnitude more sensitive than the ELISA methodology (4,5,6) in detecting antigen-specific T cell response.
In conclusion, indeterminate results should be reviewed along with the sensitivity of a particular assay system since increasing the PHA concentration in order to reduce the rate of indeterminate results will directly decrease the sensitivity of that assay.
References
1. Ferrara G, Losi M, D’Amico R, Roversi P, Piro, Meacci M, Meccugni B, Marchetti Dori I, Andreani A, Bergamini B, Mussini C, Rumpianesi F, Fabbri L, Richeldi L (2006) Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study. Lancet 2006;367:1328–34.
2. Lee JY, Choi HJ, Park I-N, Hong S-B, Oh Y-M, Lim C-M, Lee SD, Koh Y, Kim WS, Kim DS, Kim WD, and Shim TS. Comparison of two commercial interferon-ã assays for diagnosing Mycobacterium tuberculosis infection. Eur Respir J 2006;28:24–30.
3. Goletti D, Carrara D, Vincenti D, Saltini D, Busi Rizzi E, Schinina V, Ippolito G, Amicosante M and Girardi E. Accuracy of an immunediagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study. Clin Microbiol Infect 2006 10.1111/j.1469-0691.
4. Tanguay S, Killion JJ. Direct comparison of ELISPOT and ELISA-based assays for detection of individual cytokine-secreting cells. Lymphokine Cytokine Res 1994;13(4):259-63.
5. Tassignon J, Burny W, Dahmani S, Zhou L, Stordeur P, Byl B, De Groote D. Monitoring of cellular responses after vaccination against tetanus toxoid: comparison of the measurement of IFN-gamma production by ELISA, ELISPOT, flow cytometry and real-time PCR. 1. J Immunol Methods 2005;305(2):188-98. Epub 2005 Aug 31.
6. Ekerfelt C. Ernerudh J. Jenmalm MC. Detection of spontaneous and antigen-induced human interleukin-4 responses in vitro: comparison of ELISPOT, a novel ELISA and real-time RT-PCR. J Immunol Methods 2002;260(1-2):55-67.
Chris Granger is an employee of Oxford Immunotec, the manufacturer of T-SPOT.TB.
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Conflict of Interest:
None declared.
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