Home About us Editorial board Search Ahead of print Current issue Archives Submit article Instructions Subscribe Contacts Login 

 Table of Contents  
Year : 2014  |  Volume : 27  |  Issue : 2  |  Page : 269-273

Diagnostic value of the QuantiFERON-TB gold assay (T-SPOT.TB test) in patients with pulmonary tuberculosis after a full course of antituberculous therapy

Department of Chest Medicine, Menofiya University, Menofiya, Egypt

Date of Submission06-Apr-2013
Date of Acceptance28-Feb-2014
Date of Web Publication26-Sep-2014

Correspondence Address:
Shireen Mahmoud El-Sayed
MD, Mogama Elmahakemst, 51, Mansoura, Daqahliya
Login to access the Email id

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1110-2098.141674

Rights and Permissions

The study aims to clarify the effectiveness of the T-SPOT.TB test as a method of evaluation of antituberculous (anti-TB) treatment.
Among the various diagnostic tools of TB disease, the T-SPOT.TB test is the most advanced. It depends on the detection of interferon-g released from sensitized T cells when exposed to Mycobacterium tuberculosis antigens in vitro.
Materials and methods
The T-SPOT.TB test was carried out for two groups of individuals; the first group included 25 patients diagnosed previously by a positive culture of M. tuberculosis after a full course of anti-TB treatment. The second group included 10 healthy individuals.
Twenty-one patients (84%) of the first group had a positive T-SPOT.TB test, whereas only four patients (16%) had a negative T-SPOT.TB test. Of the 23 patients with a negative result by culture and microscopy, 19 (82.6%) had a positive T-SPOT.TB test.
The T-SPOT.TB test is a weak test for use during treatment monitoring. As conversion of IGRA results might be later than smear conversion results of clinical specimens, we should not consider the IGRA test as a surrogate marker of a cure or a predictor of disease relapse.

Keywords: IGRA, QuantiFERON, TB, T-SPOT, tuberculosis

How to cite this article:
Mansour OF, Azab NY, El-Helbawy RH, Eed EM, El-Sayed SM. Diagnostic value of the QuantiFERON-TB gold assay (T-SPOT.TB test) in patients with pulmonary tuberculosis after a full course of antituberculous therapy. Menoufia Med J 2014;27:269-73

How to cite this URL:
Mansour OF, Azab NY, El-Helbawy RH, Eed EM, El-Sayed SM. Diagnostic value of the QuantiFERON-TB gold assay (T-SPOT.TB test) in patients with pulmonary tuberculosis after a full course of antituberculous therapy. Menoufia Med J [serial online] 2014 [cited 2020 Apr 8];27:269-73. Available from: http://www.mmj.eg.net/text.asp?2014/27/2/269/141674

  Introduction Top

The WHO estimates that more than one-third of the world's population is infected with Mycobacterium tuberculosis [1]. Clinical and radiological examinations, combined with direct microscopic examination of sputum samples and culture of mycobacteria, are still the traditional tools for the diagnosis of tuberculosis (TB) infection [2].

Since 1910, the tuberculin skin test (TST) has been the only immunodiagnostic tool available for TB infection. It is based on in-vivo detection of delayed-type hypersensitivity to purified protein derivative, a mixture of antigens shared by several mycobacteria that gives rise to a skin reaction [3]. The main drawback of the TST is its poor specificity as previous BCG vaccination and non-TB mycobacterial exposure can lead to false-positive results, in addition to the need for a return visit and the possibility of operator-dependent variability in the placement and reading of the test [4].

QuantiFERON testing (T-SPOT.TB) is a type of enzyme-linked immunospot assay (ELISPOT) used for the diagnosis of TB. It was developed by researchers at the University of Oxford in the UK. The release of interferon-g (IFN-g), in response to stimulation with antigens and overall assessment of the antigen load on the immune system, can indicate the presence of subclinical disease [5].

The technique can be used to detect conditions of active and latent TB infection. This technique is especially useful because it is much faster (results within 24 h) than TST and more accurate [6].

Whether the level of IFN-g release after the completion of a full anti-TB treatment can assess the response to treatment or not is being studied as this would prevent overdiagnosis of already-treated cases (when using TST) and would also aid follow-up of the response to treatment as it detects the antigenic load [7].

This study aimed to evaluate the T-SPOT.TB test as an indicator of the effectiveness of the anti-TB treatment and to monitor the response of active cases to the treatment.

  Materials and methods Top

This study was carried out in the Chest and Medical Microbiology and Immunology Departments, Faculty of Medicine, Menofiya University Hospitals, on two groups of patients. The first group included 25 patients (15 men and 10 women) diagnosed previously with active TB disease [on the basis of clinical and radiological examination, the TST, direct microscopic examination of sputum samples or other materials (bronchoalveolar lavage or pleural fluid if needed), and/or culture for mycobacteria] who received a full course of anti-TB therapy. The second group (control group) included 10 healthy individuals.

After an informed consent was provided, the following was performed for all patients: a full assessment of medical history, full clinical examination, a plain chest radiography, computed tomography of the chest, TST, full laboratory investigations, Ziehl-Neelsen staining, and culturing of sputum samples on Lowenstein-Jensen (L.J.) media.

The T-SPOT.TB test depends on in-vitro detection of IFN-g released by isolated previously sensitized T cells of the patient when they are exposed to specific TB antigens, ESAT-6 (early secreted antigen target-6) and CFP10 (culture filtrate protein-10).

The T-SPOT.TB test kit was supplied by Oxford Immuntec (Oxford, UK). The test was carried out and interpreted according to the supplier's protocol.

  1. Specimen collection and handling: Venous blood (5 ml) was obtained from each patient after a full 6-month course of anti-TB treatment and collected in cell preparation tubes.
  2. Cell isolation: The cell preparation tubes were centrifuged and then a pipette tip was inserted down through the plasma to aspirate the cloudy peripheral blood mononuclear cell layer.
  3. Cell washing: Cell washing with media was performed to remove any extracellular cytokine and therefore reduce background.
  4. Plate set up and incubation: The T-SPOT.TB assay requires four wells to be used for each patient sample. A, nil control well; B, panel A (ESAT-6) well; C, panel B (CFP10) well; and D, positive control well. The plate contains 96 wells that can process up to 24 patient samples. A volume of 100 ml of the patient's final cell suspension (containing 250 000 cells) was added to each of the four wells to be used as a patient sample.
  5. Spot development and counting: The number of distinct, dark blue spots on the membrane of each well was counted and recorded.
  6. Results interpretation:

    1. The result is 'positive' if (panel A-nil control) and/or (panel B-nil control) is at least six spots.
    2. The result is 'negative' if (panel A-nil control) and (panel B-nil control) is five or less spots.
    3. The result is 'borderline' if (panel A-nil control) and (panel B-nil control) are five or six spots.

  Results Top

Only four patients (16%) were T-SPOT.TB test negative, whereas 21 patients (84%) were T-SPOT.TB test positive after a full course of anti-TB treatment [Table 1]. In comparison with other post-treatment diagnostic tools, the TST was positive in 13 patients (52%), Ziehl-Neelsen sputum staining was positive in only two patients (8%), and sputum culture was positive in only two patients (8%) [Table 2]. [Table 3] shows a comparison between pretreatment and post-treatment detection of TB in terms of Ziehl-Neelsen staining and sputum culturing.
Table 1: Comparison of post-treatment cases and controls in the T-SPOT.TB test

Click here to view
Table 2: Comparison of the T-SPOT.TB test and other post-treatment diagnostic parameters

Click here to view
Table 3: Comparison of pretreatment and post-treatment cases in sputum culture and Ziehl-Neelsen results

Click here to view

Of 23 patients negative by culture and microscopy, 19 (82.6%) had a positive T-SPOT.TB test, indicating that this test may not be useful to monitor TB treatment.

  Discussion Top

In a study carried out by Domínguez et al. [8], there was a significant decrease in the proportion of positive responders to both IFN-g tests during the treatment compared with that at the beginning of the therapy. The decrease in IFN-g released and in the count of specific responder T cells was significant. The effect of anti-TB treatment was observed in both pulmonary and extrapulmonary TB cases. These results strengthen the idea of the use of these tests for monitoring the response to the treatment.

However, in the current study, 19 out of 23 (82.6%) patients who were negative by culture and microscopy were T-SPOT.TB test positive, indicating that the test is a weak test for use during treatment monitoring.

In agreement with our findings, Millington et al. had reported that the sole utilization of IFN-g secretion by T cells is an insufficient biomarker of antigen load and clinical disease status [9]. In Domínguez's study, the results obtained individually for the patients monitored at the beginning of and during the treatment confirmed the variation in responses between individuals. Furthermore, it has also been observed that the response increases after the completion of treatment than during treatment [8].

In agreement with our results, the persistence of high levels of CFP10-specific T cells in patients after a curative treatment has recently been described by Fox and colleagues. In fact, they observed that CFP10 responder T cells were higher in active TB cases than in cases with a latent TB infection. In addition, 6 months after completing anti-TB treatment, the rates of these T cells were similar to those in the healthy contacts. This differing immunogenicity of CFP10 could reflect the nature of T-cell epitopes, and indicates that CFP10 responses could be more reliant on CD8-T cells in active TB than in contacts [10].

Previous studies on the influence of active TB disease or latent TB infection treatment on the T-cell response to MTB-specific antigens, using the ELISPOT method, showed decreases in T-cell response to ESAT-6 with the treatment of active TB disease in a small number of patients. Nicol et al. [11] reported an early increase in ELISPOT responses to ESAT-6 and CFP10, followed by a decrease after 3 and 6 months of active TB disease treatment in ten children. Aiken et al. [9] showed decreases in ESAT-6 and CFP10 ELISPOT counts after successful TB treatment 12 months after diagnosis in 89 patients with TB disease in Gambia.

Some hypotheses have been proposed to explain the negative results of the IFN-g assays in immunocompetent patients with active TB at the beginning of the treatment reported by some studies. This could be because of the release of anti-inflammatory cytokines by peripheral blood mononuclear cells and the temporary depression of T-cell responsiveness or it could be attributed to the recruitment of specific T cells during active TB. Antigen-specific cells clonally expand and migrate to the site of infection [12],[13].

The increased IFN-g production after treatment with these assays likely reflects the fact that antigen-specific lymphoproliferation is inhibited by the nonspecific immunosuppression associated with active untreated TB. Therefore, other measures of T-cell function will be required in addition to the IFN-g. In this sense, promising results have been obtained on studying the dynamic relationship between IFN-g and interleukin-2 profiles [14].

Using a QFT-2G assay in which whole blood was incubated with ESAT-6 and CFP10 peptide, Kobashi et al. [15] showed a transitional decrease in the IFN-g-positive response rate for ESAT-6 and/or CFP10 peptide after 6 months (48%) and 12 months (33%). The positive response on the QFT-2G assay was persistent in the remaining patients despite treatment.

They concluded that anti-TB treatment induced a significant decrease in T-cell responses to ESAT-6 and CFP10 antigen as measured by both IFN-g assays for patients with active TB disease until the treatment was completed. The quantitative responses to CFP10 antigen on both IFN-g assays indicate that it may be a useful monitoring marker of clinical efficacy for active TB disease treatment [15].

Also, they found a differential effect of active TB disease treatment on separate T-cell responses to ESAT-6 and CFP10. The significant decrease in response to CFP10 following TB disease treatment is consistent with the hypothesis that there is clearing of this antigen as the mycobacterial load decreases with treatment, although a poor corresponding decrease in the response to ESAT-6 could reflect true persistence of the antigen or host immune response to the antigen despite treatment because of memory T cells [15].

Similar findings have been reported by other investigators, and it has been postulated that the ESAT-6 antigen may play a role in protective immunity. Evidence for the protective role of ESAT-6 against TB infection has also been found in the mouse TB model [16]. Chee et al. [17] postulated that T-cell response to ESAT-6 may persist as a 'scar' of previously treated or 'quiescent' infection, whereas that to CFP10 may be more indicative of 'active' infection because this response appears to be influenced by TB treatment.

In terms of unresolved problems in the interpretation of IGRAs on serial testing, one is the definition of IFN-g assay test conversion or reversion. The optimal threshold to distinguish these events from nonspecific variations is not known for either IGRA test. The manufacturer has not made any recommendation on the use of the tests to monitor TB treatment [18].

Veerapathran et al. [19] recommended a 30% increase in the cutoff level in the response as a true conversion on examining active TB patients. On the basis of these reports, we may have to reconsider the cutoff levels of IFN-g assays with respect to the positive response, especially in patients with active TB disease during anti-TB treatment.

Some authors have speculated that reversion may be not only be because of a reduction in IFN-g-producing antigen-specific effector T cells during effective treatment, but that it may also reflect biological fluctuations in IFN-g production or variability in laboratory procedures. In addition, they may be influenced by the life cycle of M. tuberculosis, with phases of active replication and the dormant state, associated with variations in the amount of secreted antigens such as ESAT-6 and CFP10. Anti-TB therapy may restore active T-cell response, as noted in vivo in the TB paradoxic reaction, leading to sustained IFN-g production even in the presence of a lower bacterial burden [20].

Reversion of IGRA results has been shown in two studies: among 82 HIV-negative cases of TB treated successfully in Gambia, 44 (55%) were ELISPOT negative 12 months after diagnosis [9]. In Cape Town, 17 of 21 (81%) HIV-negative patients who had completed TB treatment successfully were IGRA negative [21]. Our data do not confirm these high reversion rates: 5.5% of patients showed reversion in their T-SPOT.TB during the course of treatment and none during the following 6 months. Furthermore, patients sampled at the end of treatment and 6 months later had, respectively, 93 and 98% of positive T-SPOT.TB results.

  Conclusion Top

Post-treatment follow-up of patients with a history of active TB disease by IGRA testing is a weak test for use during treatment monitoring. As conversion of IGRA results might be later than smear conversion results of clinical specimens, we should not consider the IGRA test as a surrogate marker of a cure or a predictor of disease relapse.

  Acknowledgements Top

I would like to thank my parents and family for encouraging me to complete this study. Also, I would like to thank my professors for guiding me and supporting my work.

Conflicts of interest

None declared.

  References Top

1.Dye C, Maher D, Weil D, et al. Targets for global tuberculosis control. Int J Tuberc Lung Dis 2006; 10 :460-462.  Back to cited text no. 1
2. Dinnes J, Deeks J, Kunst H, et al. A systematic review of rapid diagnostic tests for the detection of tuberculosis infection. Health Technol Assess 2007; 11 :190-196.  Back to cited text no. 2
3. Jasmer RM, Nahid P, Hopewell PC. Clinical practice: latent tuberculosis infection. N Engl J Med 2002; 347 :1860-1866.  Back to cited text no. 3
4. Chen J,Su X, Zhang Y, et al. Novel recombinant RD2- and RD11 encoded Mycobacterium tuberculosis antigens are potential candidates for diagnosis of tuberculosis infections in BCG-vaccinated individuals. Microb Infect 2009; 11 :876-885.  Back to cited text no. 4
5. Liao CH, Chou CH, Lai CC, et al. Diagnostic performance of an enzyme-linked immunospot assay for interferon-G in extrapulmonary tuberculosis varies between different sites of disease. J Infect 2009; 59 :402-408.  Back to cited text no. 5
6. Kunst H. Diagnosis of latent tuberculosis infection: the potential role of new technologies. Respir Med 2006; 100 :2098-2106.  Back to cited text no. 6
7. Kabeer B. IP-10 response to RD1 antigens might be a useful biomarker for monitoring tuberculous therapy. Infect Dis 2011; 11 :135-139.  Back to cited text no. 7
8. Domínguez J, Ruiz-Manzano J, De Souza-Galvao M, et al. Comparison of two commercially available gamma interferon blood tests for immunodiagnosis of tuberculosis. Clin Vaccine Immunol 2008; 15 :168-171.  Back to cited text no. 8
9. Aiken AM, Hill PC, Fox A, et al. Reversion of the ELISPOT test after treatment in Gambian tuberculosis cases. Infect Dis 2006; 6 :66.  Back to cited text no. 9
10.Fox A, Jeffries DJ, Hill PC, et al. ESAT-6 and CFP-10 can be combined to reduce the cost of testing for Mycobacterium tuberculosis infection, but CFP-10 responses associate with active disease. Trans R Soc Trop Med Hyg 2007; 101 :691-698.  Back to cited text no. 10
11.Nicol MP, Pienaar D, Wood K, et al. Enzyme-linked immunospot assay responses to early secretory antigenic target 6, culture filtrate protein 10, and purified protein derivative among children with tuberculosis: implication for diagnosis and monitoring of therapy. Clin Infect Dis 2005; 40 :1301-1308.  Back to cited text no. 11
12.Chen X, Zhou B, Li M, et al. CD4 (+) CD25 (+) FoxP3 (+) regulatory T cells suppress Mycobacterium tuberculosis immunity in patients with active disease. Clin Immunol 2007; 123 :50-59.  Back to cited text no. 12
13.Barnes PF, Lu S, Abrams JS, et al. Cytokine production at the site of disease in human tuberculosis. Infect Immun 1993; 61 :3482-3489.  Back to cited text no. 13
14.Millington KA, Innes JA, Hackforth S, et al. Dynamic relationship between IFN-gamma and IL-2 profile of Mycobacterium tuberculosis-specific T cells and antigen load. J Immunol 2007; 178 :5217-5226.  Back to cited text no. 14
15.Kobashi Y, Mouri K, Obase Y, et al. Clinical evaluation of QuantiFERON TB-2G test for immunocompromised patients. Eur Respir J 2005; 30 :945-950.  Back to cited text no. 15
16.Ulrichs T, Anding P, Kaufmann SHE, et al. Numbers of IFN-g producing cells against ESAT-6 increase in tuberculosis patients during chemotherapy. Int J Tuberc Lung Dis 2000; 4 :1181e3.  Back to cited text no. 16
17.Chee CBE, KhinMar KW, Gan SH, et al. Latent tuberculosis infection treatment and T-cell responses to Mycobacterium tuberculosis-specific antigens. Am J Respir Crit Care Med 2007; 175 :282-287.  Back to cited text no. 17
18.Kobashi Y,Mouri K, Yagi S, et al. Transitional changes in T-cell responses to Mycobacterium tuberculosis-specific antigens during treatment. J Infect 2009; 58 :197-204.  Back to cited text no. 18
19.Veerapathran A, Joshi R, Goswami K, et al. T-cell assays for tuberculosis infection: deriving cut-offs for conversions using reproducibility data. J Infect 2008; 26 :1850.  Back to cited text no. 19
20.Herrmann JL, Belloy M, Porcher R, et al. Temporal dynamics of interferon-gamma responses in children evaluated for tuberculosis. PLoS One 2009; 4 :130.  Back to cited text no. 20
21.Dheda K, Pooran A, Pai M, et al. Interpretation of Mycobacterium tuberculosis antigen-specific IFN-gamma release assays (T-SPOT.TB) and factors that may modulate test results. J Infect 2007; 5:169-173.  Back to cited text no. 21


  [Table 1], [Table 2], [Table 3]

This article has been cited by
1 The pros and cons of the QuantiFERON test for the diagnosis of tuberculosis, prediction of disease progression, and treatment monitoring
Elvis Ndukong Ndzi,Celine Nguefeu Nkenfou,Luc Christian Gwom,Nadine Fainguem,Joseph Fokam,Yone Pefura
International Journal of Mycobacteriology. 2016;
[Pubmed] | [DOI]


Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
Access Statistics
Email Alert *
Add to My List *
* Registration required (free)

  In this article
Materials and me...
Article Tables

 Article Access Statistics
    PDF Downloaded128    
    Comments [Add]    
    Cited by others 1    

Recommend this journal