Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment

I-Nae Park; Tae Sun Shim

doi:10.3904/kjim.2016.199

Korean J Intern Med > Volume 32(2); 2017 > Article

Park and Shim: Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment

Original Article

Pulmonology

Korean J Intern Med 2017;32(2):302-308.

Published online: December 13, 2016

DOI: https://doi.org/10.3904/kjim.2016.199

Qualitative and quantitative results of interferon-γ release assays for monitoring the response to anti-tuberculosis treatment

I-Nae Park¹, Tae Sun Shim²

¹Department of Internal Medicine, Inje University Seoul Paik Hospital, Seoul, Korea

²Division of Pulmonology and Critical Care Medicine, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Korea

Correspondence to I-Nae Park, M.D. Department of Internal Medicine, Inje University Seoul Paik Hospital, 9 Mareunnae-ro, Jung-gu, Seoul 04551, Korea Tel: +82-2-2270-0004 Fax: +82-2-2285-2286 E-mail: eanee@hanmail.net

Received June 27, 2016 Revised July 29, 2016 Accepted October 20, 2016

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background/Aims

The usefulness of interferon-γ release assays (IGRAs) in monitoring to responses to anti-tuberculosis (TB) treatment is controversial. We compared the results of two IGRAs before and after anti-TB treatment in same patients with active TB.

Methods

From a retrospective review, we selected patients with active TB who underwent repeated QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) and T-SPOT.TB (Oxford Immunotec) assays before and after anti-TB treatment with first-line drugs. Both tests were performed prior to the start of anti-TB treatment or within 1 week after the start of anti-TB treatment and after completion of treatment.

Results

A total of 33 active TB patients were included in the study. On the QFN-Gold test, at baseline, 23 cases (70%) were early secreted antigenic target 6-kDa protein 6 (ESAT-6) or culture filtrate protein 10 (CFP-10) positive. On the T-SPOT. TB test, at baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Most of patients remained both test-positive after anti-TB treatment. Although changes in interferon-γ release responses over time were highly variable in both tests, there was a mean decline of 27 and 24 spot-forming counts for ESAT-6 and CFP-10, respectively on the T-SPOT.TB test (p < 0.05 for all).

Conclusions

Although limited by the small number of patients and a short-term follow-up, there was significant decline in the quantitative result of the T-SPOT. TB test with treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate range.

Keywords: Interferon-gamma release tests; Tuberculosis; Therapeutics

INTRODUCTION

One third of the world’s population is infected with Mycobacterium tuberculosis, and tuberculosis (TB) ranks a leading cause of death among infectious diseases worldwide. In 2014, 9.6 million people fell ill with TB, an estimated 480,000 people developed multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) has been reported by 105 countries by 2015 [1]. An estimated 9.7% of people with MDR-TB have XDR-TB. Response to anti-TB treatment in patients with bacteriologically confirmed TB is monitored principally by serial bacteriologic examinations [2], whereas responses in patients with clinically diagnosed TB, including cases diagnosed on the basis of X-ray abnormalities or suggestive histology and extrapulmonary cases without laboratory confirmation, are usually monitored clinically and/or radiographically. Therefore, new monitoring tools for TB treatment are needed to improve TB treatment strategies in some clinical or experimental settings.

The search for improved tools for detection of latent TB infection has led to the development of in vitro assays based on interferon-γ production. Commercial ex vivo interferon-γ release assays (IGRAs) have been approved as indirect tests for M. tuberculosis infection (including infection resulting in active disease) when used in conjunction with risk assessment, radiography, and other medical and diagnostic evaluations [3], QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited, Carnegie, Australia), QuantiFERON-TB Gold In-tube (QFT-GIT, Cellestis Limited, Carnegie, Australia), and T-SPOT. TB (Oxford Immunotec, Oxford, UK) [4-7]. These assays use the M. tuberculosis-specific antigenic peptides, early secreted antigenic target 6-kDa protein 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), which are not present in Bacille Calmette-Guérin (BCG) strains and most nontuberculous mycobacteria, making them highly specific for detecting TB infection.

Although to date there have been several reports about the clinical usefulness of IGRA in the diagnosis of active TB and monitoring the response to TB treatment [8-14], there is no report on a direct comparison of two IGRAs for monitoring the response to TB treatment in Korea. Therefore, we evaluated the qualitative and quantitative IGRA results measured by both QFN-Gold and T-SPOT. TB assays before and after anti-TB treatment in same patients with active TB.

METHODS

Subjects and study design

From a retrospective review, we selected patients with active TB who underwent repeated QFN-Gold and T-SPOT.TB assays before and after anti-TB treatment with first-line drugs, from June 2004 through December 2005 at the Asan Medical Center (Seoul, Korea). Active TB was defined as bacteriologically confirmed or clinically diagnosed cases of TB by 2013 WHO revised definition. Pulmonary tuberculosis (PTB) refers to any bacteriologically confirmed or clinically diagnosed case of TB involving the lung parenchyma or the tracheobronchial tree. A patient with both PTB and extrapulmonary tuberculosis (EPTB) classified as a case of PTB. EPTB refers to any bacteriologically confirmed or clinically diagnosed case of TB involving organs other than the lungs.

Finally, patients who completed the treatment or who were cured were enrolled in the study. In all cases, both tests were performed prior to the start of treatment or within 1 week after the start of treatment (T₀) and after completion of treatment (T₁), respectively. This study was part of a larger study comparing the abilities of tuberculin skin tests (TST) and ex vivo IGRAs to detect TB infection and to monitor the response to anti-TB treatment [15]. All patients provided written informed consent, and the study protocol was approved by the Institutional Review Board of Asan Medical Center.

QuantiFERON-TB Gold assay

The QFN-Gold assay was performed according to the manufacturer’s instructions [15]. Immediately before TST, 4 mL of peripheral venous blood was collected into a heparinized tube. Within 4 hours of collection, 1 mL aliquots of blood were mixed with 3 drops each of one of the following provided solutions: saline (negative control), phytohemagglutinin (mitogenic positive control), ESAT-6 peptides or CFP-10 peptides. The samples were incubated at 37°C for 20 hours and then frozen until analysis. T cell responses were determined by quantitative measurement of plasma interferon-γ levels using the enzyme-linked immunosorbent assay technique. For each subject, the negative control value was subtracted from the values obtained from the antigen-stimulated plasma samples. An interferon-γ concentration of 0.35 IU/mL was used as the cut-off for a positive response as recommended by a manufacturer, regardless of the result of the positive control (mitogen well). The result was considered “negative” if the response to the specific antigens was < 0.35 IU/mL and if the interferon-γ level of the positive control was ≥ 0.5 IU/mL. The result was considered “indeterminate” if both antigen-stimulated samples were negative and the value of the positive control was < 0.5 IU/mL.

T-SPOT.TB assay

T-SPOT.TB analysis was performed according to the manufacturer’s instructions [15-17]. Immediately before TST, peripheral venous blood samples were collected and processed within four hours. The samples were incubated for 20 hours, and the number of spots was counted using an automated AID ELISPOT Reader (AID GmbH, Strasbourg, Germany). ESAT-6- or CFP-10-stimulated wells with at least six spots more than the negative control well, which had ≤ 5 spots, were scored as positive. When the negative control well had 6 to 10 spots, the result was defined as positive when either the ESAT-6- or CFP-10-stimulated well contained at least twice as many spots. If the negative control well had more than 10 spots, the result was considered “indeterminate.”

Statistical methods

Statistical analyses were performed using SPSS version 10.0 (SPSS Inc., Chicago, IL, USA). Data are expressed as mean ± SD for continuous variables, and percentages for categorical variables. Paired numeric data were compared using Student t test. All tests of significance were two sided; p values < 0.05 were considered statistically significant.

RESULTS

Baseline characteristics

A total of 33 active TB patients were included in the analysis; their baseline demographic and clinical data are summarized in Table 1. Active TB was diagnosed bacteriologically in 25 patients (76%) and clinically in eight patients (24%). Of bacteriologically confirmed TB, 21 had M. tuberculosis cultured from their specimens. In 20 of these 21 patients, the isolates were sensitive to all anti-TB medications; the isolate from one patient showed isoniazid (INH) monoresistance. Clinical TB patients had high adenosine deaminase level in pleural effusion or typical CT findings, including tree-in-bud appearance accompanying lung nodules, suggesting active TB; these lesions improved after anti-TB treatment. All patients completed anti-TB treatment successfully with first-line anti-TB medication; 32 patients completed treatment in 6 months and one patient with INH monoresistance in 9 months. Both interferon-γ tests were performed at baseline (T₀) and treatment completion (T₁), with a mean interval of 6.9 ± 0.9 months between T₀ and T₁.

Interferon-γ release assay

Fig. 1 shows qualitative change in QFN-Gold test results for patients with active TB before and after anti-TB treatment. At baseline, 23 cases (70%) were ESAT-6 or CFP-10 positive. Of these, three cases (13%) were ESAT-6 and CFP-10 negative after completion of anti-TB treatment. Fig. 2 shows qualitative change in T-SPOT. TB results for patients with active TB before and after anti-TB treatment. At baseline, 31 cases (94%) were ESAT-6 or CFP-10 positive. Of these, three cases (10%) were ESAT-6 and CFP-10 negative after completion of anti-TB treatment. Fig. 3 shows the quantitative changes in the interferon-γ level in QFN-Gold test between before treatment and after treatment. Eighteen cases (55%) had decreased in their interferon-γ level using ESAT-6. Also, 18 cases (55%) had decreased in their interferon-γ level using CFP-10. Fig. 4 shows the quantitative changes in the number of spot-forming cells (SFCs) in T-SPOT. TB test between before treatment and after treatment. Twenty-three cases (70%) had decreased in their number of SFCs using ESAT-6. Twenty-two cases (67%) had decreased in their number of SFCs using CFP-10. The mean value of interferon-γ level (QFN-Gold test) including all cases remained unchanged on a comparison test results between before and after treatment. However, the mean value of number of SFCs (T-SPOT.TB test) significantly decreased on a comparison test results between before and after treatment (Table 2).

DISCUSSION

Although this report presents short-term follow-up results of two commercial blood tests based on small number of patients, this study is the first report on a comparison of two IGRAs for monitoring the response to TB treatment in Korea, where the incidence of TB is within the intermediate range (90 per 100,000) and BCG vaccination is mandatory [18].

The aim of the present study was to evaluate the results of two IGRAs before and after anti-TB treatment in same patients with active TB. We hypothesized that interferon-γ release would decrease as a response to anti-TB treatment in active TB patients. We have shown here that positive rates for QFN-Gold test and T-SPOT. TB test result remain unchanged after anti-TB treatment, although there was a significant reduction in mean ESAT-6 and CFP-10 T-SPOT.TB counts.

Several authors have made a direct comparison between the T-SPOT.TB and the QFN-Gold in the diagnosis of active TB in the same study population [15,19-21]. Although there are few differences between the sensitivities of the tests in these studies, the sensitivities are in the same range, with the T-SPOT.TB being more sensitive than QFN-Gold. The impact of the anti-TB treatment on the interferon-γ tests in reducing the response to the specific antigens has been described elsewhere. During TB treatment, the number of specific T cells decreases progressively, suggesting that the frequency of ESAT-6-specific T cells is related to antigen load [22,23]. It has been proposed that the interferon-γ could be used to monitor the patients’ response to the treatment [8]. Although there have been several reports regarding the clinical usefulness of IGRA in the monitoring the response to TB treatment, there has not been any report on a direct comparison of two IGRAs for monitoring the response to TB treatment in Korea. In one Japan study [24], they compared of QFN-Gold test and T-SPOT.TB test in the same patients with active TB disease: positive results (for ESAT-6 and/or CFP-10) of both QFN-Gold test results and T-SPOT.TB test results decreased from 81% and 88% (before treatment), respectively, to 41% and 49% (12 months later). However, in our study, we showed that positive responses on both IGRAs were persistent after treatment. The reason might be long duration of exposure in TB endemic country and shorter follow-up period. However, in Norway study from a TB low-endemic country, most patients with latent TB infection were still QFT-TB positive after preventive therapy [25]. Therefore, further research is needed to better understand why T cell responses stay persistently elevated after treatment.

Although there was failure of reversion on the T-SPOT. TB test in our study, 23 cases (70%) had decreased number of SFCs using ESAT-6 and 22 cases (67%) had decreased number of SFCs using CFP-10. Also there was a mean decline of 27 and 24 SFCs for ESAT-6 and CFP-10, respectively (p < 0.05 for all). In a previous report on the T-SPOT.TB assay in Gambia [9], 82 cases (96%) successfully completed treatment: 44 (55%) were ESAT-6 and/or CFP-10 negative at 12 months, 17 (21%) were TST negative. Also, 64 (78%) had an ESAT-6 count decrease, 60 (73%) cured cases had a CFP-10 count decrease. There was a mean decline of 44 and 25 SFCs for ESAT-6 and CFP-10, respectively (p < 0.001 for all). They insist that a repeat T-SPOT.TB test at 12 months reflects the efficacy of a course of anti-TB treatment even in a TB endemic tropical setting. These findings were consistent with those of our study except for reversion rate.

In our study, changes in interferon-γ over time were highly inconsistent (Fig. 3) and median and the mean value of interferon-γ level (QFN-Gold test) remained unchanged on comparing the test results between before and after treatment. Pai et al. [10] reported that at baseline, 44 of 60 patients (73%) were positive by the QFN-Gold test and at the treatment completion, 31 of 39 patients (79%) were positive. Also, the average of interferon-γ level declined slightly during treatment, but it was not significant as in our results.

In conclusion, although limited by the small number of patients and a short-term follow-up, there was significant decline in the quantitative result of the T-SPOT. TB test with treatment. However, both commercial IGRAs may not provide evidence regarding the cure of disease, in Korea, a country where the prevalence of TB is within the intermediate range.

KEY MESSAGE

1. Although there was significant decline in the quantitative result of the T-SPOT.TB test with treatment, most of patients remained both test-positive after anti-tuberculosis (TB) treatment.

2. Interferon-γ release assays may not provide evidence regarding the cure of disease in Korea, a country where the prevalence of TB is within the intermediate-range.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Figure 1.

Change in the QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) test results for patients with active tuberculosis (TB) before and after anti-TB treatment. TST, tuberculin skin test; PTB, pulmonary tuberculosis; EPTB, extrapulmonary tuberculosis.

Figure 2.

Change in the T-SPOT.TB (Oxford Immunotec) test results for patients with active tuberculosis (TB) before and after anti-TB treatment. TST, tuberculin skin test; PTB, pulmonary tuberculosis; EPTB, extrapulmonary tuberculosis.

Figure 3.

Paired before and after treatment quantitative interferon-γ (IFN-γ) level on the QuantiFERON-TB Gold (QFN-Gold, Cellestis Limited) test using (A) early secreted antigenic target 6-kDa protein 6 and (B) culture filtrate protein 10.

Figure 4.

Paired before and after treatment quantitative interferon-γ level in on the T-SPOT.TB (Oxford Immunotec) test using (A) early secreted antigenic target 6-kDa protein 6 and (B) culture filtrate protein 10.

Table 1.

Clinical characteristics of 33 patients with active tuberculosis

Characteristic	Total (n = 33)	Positive at T₀ on QFN-Gold (n = 23)	Positive at T₀ on T-SPOT. TB (n = 31)
Age, yr	46.1 ± 15.0	46.1 ± 16.4	46.3 ± 15.0
Sex, male:female	19:14	13:10	18:13
History of TB treatment	3/29 (10)	2/20 (10)	3/27 (11)
Presence of BCG scar	17/29 (59)	10/20 (50)	16/27 (59)
TST, mm	11.6 ± 8.9 (0–37)	14.0 ± 8.9 (0–37)	12.1± 8.9 (0–37)
TST ≥ 10 mm	22 (67)	18 (78)	21 (68)
No. of patients with risk factor for immunosuppression	11 (33)	6 (26)	10 (32)
Uncontrolled DM	5 (15)	3 (13)	5 (16)
Cancer on chemotherapy	2 (6)	2 (9)	2 (6)
Kidney transplantation	1 (3)	0	1 (3)
Liver transplantation	1 (3)	1 (3)	1 (3)
On prednisolone > 1 mon	2 (6)	0	1 (3)
PTB:EPTB	31:2	22:1	29:2
Bacteriological TB^a:clinical TB^b	25:8	17:6	23:8

Values are presented as mean ± SD, number (%), or mean ± SD (range).

TB, tuberculosis; BCG, Bacille Calmette-Guérin; TST, tuberculin skin test; DM, diabetes mellitus; PTB, pulmonary tuberculosis; EPTB, extrapulmonary tuberculosis.

^a A bacteriologically confirmed TB case is one from whom a biological specimen is positive by smear microscopy, culture or TB polymerase chain reaction.

^b A clinically diagnosed TB case is one who does not fulfil the criteria for bacteriological confirmation but has been diagnosed with active TB by a clinician who has decided to give the patient a full course of TB treatment. This definition includes cases diagnosed on the basis of X-ray abnormalities or suggestive histology and extrapulmonary cases without laboratory confirmation.

Table 2.

Change in the IFN-γ level and the number of SFCs of patients with active TB during anti-TB treatment

	IFN-γ level, IU/mL			No. of SFCs
	T₀^a	T₁^b	p value	T₀^a	T₁^b	p value
ESAT-6	2.7 ± 6.3	2.9 ± 7.8	NS	58.9 ± 53.6	31.9 ± 7.43	0.011
CFP-10	1.0 ± 2.7	1.5± 4.8	NS	48.7± 59.8	25.0 ± 39.6	0.029

Values are presented as mean ± SD.

IFN-γ, interferon-γ SFC, spot-forming cell; TB, tuberculosis; ESAT-6, early secreted antigenic target 6-kDa protein 6; CFP-10, culture filtrate protein 10.

^a T₀: before or within 1 week after the start of anti-TB treatment.

^b T₁: after completion of treatment.

REFERENCES

1. World Health Organization. Global Tuberculosis Report 2015. Geneva: World Health Organization, 2015.

2. World Health Organization. Treatment of Tuberculosis: Guidelines for National Programmes. 3rd ed. Geneva: World Health Organization, 2003.

3. Mazurek GH, Jereb J, Vernon A, et al. Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosis infection: United States, 2010. MMWR Recomm Rep 2010;59:1–25.

4. Liebeschuetz S, Bamber S, Ewer K, Deeks J, Pathan AA, Lalvani A. Diagnosis of tuberculosis in South African children with a T-cell-based assay: a prospective cohort study. Lancet 2004;364:2196–2203.

5. Mazurek GH, Jereb J, Lobue P, Iademarco MF, Metchock B, Vernon A, et al. Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR Recomm Rep 2005;54:49–55.

6. Meier T, Eulenbruch HP, Wrighton-Smith P, Enders G, Regnath T. Sensitivity of a new commercial enzyme-linked immunospot assay (T SPOT-TB) for diagnosis of tuberculosis in clinical practice. Eur J Clin Microbiol Infect Dis 2005;24:529–536.

7. Arend SM, Thijsen SF, Leyten EM, et al. Comparison of two interferon-gamma assays and tuberculin skin test for tracing tuberculosis contacts. Am J Respir Crit Care Med 2007;175:618–627.

8. Lalvani A. Counting antigen-specific T cells: a new approach for monitoring response to tuberculosis treatment? Clin Infect Dis 2004;38:757–759.

9. Aiken AM, Hill PC, Fox A, et al. Reversion of the ELISPOT test after treatment in Gambian tuberculosis cases. BMC Infect Dis 2006;6:66.

10. Pai M, Joshi R, Bandyopadhyay M, et al. Sensitivity of a whole-blood interferon-gamma assay among patients with pulmonary tuberculosis and variations in T-cell responses during anti-tuberculosis treatment. Infection 2007;35:98–103.

11. Ribeiro S, Dooley K, Hackman J, et al. T-SPOT.TB responses during treatment of pulmonary tuberculosis. BMC Infect Dis 2009;9:23.

12. Lee SW, Lee CT, Yim JJ. Serial interferon-gamma release assays during treatment of active tuberculosis in young adults. BMC Infect Dis 2010;10:300.

13. Chee CB, KhinMar KW, Gan SH, et al. Tuberculosis treatment effect on T-cell interferon-gamma responses to Mycobacterium tuberculosis-specific antigens. Eur Respir J 2010;36:355–361.

14. Chiappini E, Fossi F, Bonsignori F, Sollai S, Galli L, de Martino M. Utility of interferon-γ release assay results to monitor anti-tubercular treatment in adults and children. Clin Ther 2012;34:1041–1048.

15. Lee JY, Choi HJ, Park IN, et al. Comparison of two commercial interferon-gamma assays for diagnosing Mycobacterium tuberculosis infection. Eur Respir J 2006;28:24–30.

16. Lalvani A, Pathan AA, McShane H, et al. Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells. Am J Respir Crit Care Med 2001;163:824–828.

17. Mori T, Sakatani M, Yamagishi F, et al. Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens. Am J Respir Crit Care Med 2004;170:59–64.

18. World Health Organization. Tuberculosis [Internet]. Geneva: World Health Organization, c2016. [cited 2016 Nov 14]. Available from: http://www.who.int/tb/en/.

19. Ferrara G, Losi M, D’Amico R, et al. Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study. Lancet 2006;367:1328–1334.

20. Kang YA, Lee HW, Hwang SS, et al. Usefulness of wholeblood interferon-gamma assay and interferon-gamma enzyme-linked immunospot assay in the diagnosis of active pulmonary tuberculosis. Chest 2007;132:959–965.

21. Chee CB, Gan SH, Khinmar KW, et al. Comparison of sensitivities of two commercial gamma interferon release assays for pulmonary tuberculosis. J Clin Microbiol 2008;46:1935–1940.

22. Pathan AA, Wilkinson KA, Klenerman P, et al. Direct ex vivo analysis of antigen-specific IFN-gamma-secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J Immunol 2001;167:5217–5225.

23. Ferrand RA, Bothamley GH, Whelan A, Dockrell HM. Interferon-gamma responses to ESAT-6 in tuberculosis patients early into and after anti-tuberculosis treatment. Int J Tuberc Lung Dis 2005;9:1034–1039.

24. Kobashi Y, Mouri K, Yagi S, et al. Clinical evaluation for diagnosing active TB disease and transitional change of two commercial blood tests. Scand J Infect Dis 2008;40:629–634.

25. Dyrhol-Riise AM, Gran G, Wentzel-Larsen T, Blomberg B, Haanshuus CG, Morkve O. Diagnosis and follow-up of treatment of latent tuberculosis; the utility of the QuantiFERON-TB Gold In-tube assay in outpatients from a tuberculosis low-endemic country. BMC Infect Dis 2010;10:57.