- Email: [email protected]
Evaluation of the performance of two tuberculosis interferon gamma release assays (IGRA-ELISA and T-SPOT.TB) for diagnosing Mycobacterium tuberculosis infection
Clinica Chimica Acta 479 (2018) 74–78
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Evaluation of the performance of two tuberculosis interferon gamma release assays (IGRA-ELISA and T-SPOT.TB) for diagnosing Mycobacterium tuberculosis infection ⁎
Linchuan Wanga, Xu-dong Tiana,1, Yan Yub, , Wei Chena, a b
T
⁎
The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, China Honghui Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Mycobacterium tuberculosis IGRA-ELISA T-SPOT.TB
Objectives: The IGRA-ELISA and T-SPOT.TB are widely used in China. The aim of the study was to evaluate the performance of the two assays in diagnosis Mycobacterium tuberculosis infection. Methods: Of the 3727 patients in the study, 204 underwent testing using both the T-SPOT.TB and IGRA-ELISA, 1794 were tested using the T-SPOT.TB only, and 1729 were tested using the IGRA-ELISA only. The positive rate and consistency of the two assays were analyzed, and their sensitivity and specificity for diagnosing active tuberculosis were compared. Results: There were no significant differences in the positive rate between the T-SPOT.TB test (25.8%) and IGRAELISA (28.6%), p = .065. The two assays were highly consistent, with a kappa value of 0.852 (p < .0001) and a total coincidence rate of 92.7%. For the diagnosis of active tuberculosis, the sensitivity and specificity values of the T-SPOT.TB test were 82.9% (107/129) and 78.6% (1309/1665), respectively, and those of IGRA-ELISA were 81.7% (94/115) and 75.2% (1214/1614), respectively. There were no significant differences in sensitivity (p > .05), but the specificity of the T-SPOT.TB test was slightly higher than that of IGRA-ELISA (p = .023). Conclusion: Both in terms of diagnosing M. tuberculosis infection and ruling out active tuberculosis, the performance of the IGRA-ELISA—a simple, almost labor-free assay that allows simultaneous processing of a very large number of samples—was well-matched with that of T-SPOT.TB test. However, IGRAs cannot be used as the only test to diagnose active tuberculosis.
1. Introduction China has the second highest burden of tuberculosis (TB) in the world; approximately 44.5% people in China are infected with M. tuberculosis [1]. The Fifth National TB Epidemiological Survey conducted in China in 2010 reported that the prevalence of active pulmonary TB (PTB) was 459, of smear positive PTB was 66, and of culture positive PTB was 119 per 100,000 people aged > 15 years [2]. As the National TB Control Programme (2001–2010) in China was performed effectively, the annual decline in the rate of PTB, smear positive PTB, and culture positive PTB was 0.2%, 9.0%, and 5.8%, respectively, from 2000 to 2010 [2]. However, the proportion of individuals with clinically asymptomatic PTB was significantly higher in 2010 than in 2000 (43.1% vs 12.1%, respectively). Moreover, and the cumulative number of PTB infections in China up to 2010 is approximately 4.99 million cases [2].
⁎
1
Treatment of autoimmune diseases such as rheumatoid arthritis with tumor necrosis factor (TNF)-α-inhibitors may result in progression of latent tuberculosis infection (LTBI) to active tuberculosis (ATB) [3–6]. Hence, it is necessary to screen for LTBI prior to initiating biologic therapy [3–8]. M. tuberculosis infection is difficult to diagnose as there is often a lack of explicit evidence. Tuberculin skin testing (TST) was commonly used for LTBI screening in the past. However, falsepositive or false-negative results are often observed, limiting its clinical application [9–11]. Interferon (IFN)-γ release assays (IGRAs), i.e., the Quantiferon-Gold® TB In-Tube (QFT-GIT) or T-SPOT.TB test, [8, 12, 13] are based on the finding that effector T-cells secrete IFN-γ when stimulated by Mycobacterium-specific RD1 antigens, such as early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) [14, 15]. Currently in China, the T-SPOT.TB and IGRA-enzyme linked immunosorbent assay (IGRA-ELISA)—based on the QFT-GIT—are the two
Corresponding authors. E-mail addresses: [email protected] (Y. Yu), [email protected] (W. Chen). These authors contributed equally to this work.
https://doi.org/10.1016/j.cca.2018.01.014 Received 22 June 2017; Received in revised form 12 November 2017; Accepted 8 January 2018 Available online 09 January 2018 0009-8981/ © 2018 Elsevier B.V. All rights reserved.
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
power function with six standards (12.5, 25, 50, 100, 200, and 400 pg/ mL). Thereafter, the IFN-γ values of the T- and N-tubes were calculated. If the optical density of the standard (400 pg/mL) was < 1.0 or if the correlation coefficient (R2) of the power function fitted curve is < 0.99, the test is invalid. If the value in the P-tube minus that in the N-tube was < 20, or if the N-tube value is > 400, the test is indeterminate. The test is considered positive if the value in the T-tube minus that in the N-tube is ≥14 and is ≥25% of the N-tube value. The test is considered negative if the value in the T-tube minus that in the Ntube is < 14, or if the value in the T-tube minus that in the N-tube is ≥14 but is < 25% of the N-tube value.
most important screening and diagnostic tests for M. tuberculosis infection. However, the reference intervals and test units of these two assays differ. In China, there is a concern about whether there are differences in performance of the two assays for the diagnosis of M. tuberculosis infection and active TB disease. As the T-SPOT.TB test and IGRA-ELISA are both used in our hospital, in this study, the performance of these two assays were analyzed and compared. 2. Materials and methods 2.1. Study population From November 2016 through February 2017, a total of 3727 patients with suspected M. tuberculosis infection at the First Affiliated Hospital of Xi'an Jiaotong University, were enrolled in the study. The median age was 55 years, the majority of participants (n = 2100 [56.3%]) were men. Peripheral blood samples from the participants were collected in lithium-heparin-coated tubes. Of these samples, 204 were tested using both the T-SPOT.TB and IGRA-ELISA, 1794 were tested using the T-SPOT.TB only, and 1729 were tested using the IGRAELISA only. The data used in the study were obtained from the LIS and HIS systems of the First Affiliated Hospital of Xi'an Jiaotong University. 244 patients were diagnosed with active TB if at least one of the following conditions were fulfilled: (i) smear-positive for acid-fast bacilli, n = 19 (3 cases were also TB PCR-positive), (ii) chest x-ray findings consistent with a radiological diagnosis of PTB, n = 53, (iii) granulomata or caseous necrosis observed on histopathological examination of biopsy specimens, n = 45, or (iv) despite the absence of explicit evidence of active TB on microscopy, chest x-ray, or histopathology, the clinical symptoms were consistent with active TB and responded to anti-TB treatment (n = 127). 129 and 115 of the patients underwent the T-SPOT.TB and IGRA-ELISA testing, respectively.
2.2.3. Statistical analysis Statistical analyses were performed using SPSS, version 13.0 (serial number 5026743; SPSS Inc., Chicago, Illinois, USA). The sensitivity and specificity values of the two assays were compared using the chi-square test and the consistency of the two assays was evaluated using the kappa statistic. A p-value < .05 was considered statistically significant. 2.2.4. Ethics statement The study was deemed exempt from review by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University because of its retrospective nature and because the data used in the study were processed anonymously. 3. Results 3.1. The performance of the two tests In the T-SPOT.TB test group, 82.9% (107 of 129) of the patients with active TB and 21.4% (356 of 1665) of the patients without active TB had positive test results. In the IGRA-ELISA test group, 81.7% (94 of 115) of the patients with active TB and 24.8% (400 of 1614) of the patients without active TB had positive results (Table 1). In terms of diagnosing active TB, the T-SPOT.TB test had a sensitivity of 82.9% and a specificity of 78.6%, while the IGRA-ELISA had a sensitivity of 81.7% and a specificity of 75.2%. There was no significant difference in the sensitivity (χ2 = 0.061, p = .867) of these tests, but the specificity of the T-SPOT.TB test was slightly higher than that of the IGRA-ELISA (χ2 = 5.346, p = .023). The median values of the TSPOT.TB test and the IGRA-ELISA were 373.3 pg/mL and 88 SFCs, respectively, in the group with active TB; these values were significantly higher than those observed in the group without ATB (110.3 pg/mL and 20 SFCs, respectively; p < .0001) (Fig. 1).
2.2. Laboratory tests 2.2.1. Performing and interpreting the T-SPOT.TB test The T-SPOT.TB test (Oxford Immunotec Ltd., Abingdon, UK) should be performed within 6 h of obtaining the blood sample. The separation and incubation of peripheral blood mononuclear cells were conducted as previously reported [16]. If < 20 spot-forming cells (SFCs) are detected in the positive control well, the test is invalid. If > 10 SFCs are detected in the negative control well, the test is indeterminate. If either or both panel A (ESAT-6 antigen) and/or panel B (CFP-10 antigen) have 6 or more SFCs detected than are detected in the negative control, the test is positive. If both panel A and panel B have 5 or fewer SFCs detected than are detected in the negative control, the test is negative.
3.2. Analysis of positive test results
2.2.2. Performing and interpreting the IGRA-ELISA The IGRA-ELISA (WanTai Biological Pharmacy Enterprise Co, Ltd., Beijing, China) should be performed within 2 h of obtaining the blood sample. Blood samples were mixed by inverting the vacutainer 5 times, then 1 mL of blood was added to each of 3 heparinized tubes (T, N, and P tube): The T-tube contained ESAT-6 and CFP-10 antigens, the N-tube was the negative control, and the P-tube (positive control) contained phytohemagglutinin. The tubes were incubated at 37 °C for 20–24 h and were then centrifuged at 3000–5000 rpm for 10 min. The supernatant was used for the ELISA assay, performed using an automated instrument. The whole process was performed as follows: 50 μL supernatant and standards were added to the wells that contained anti-IFN-γ and the plate was incubated at 37 °C for 60 min. Then, 50 μL of horseradish peroxidase-labeled anti-IFN-γ was added to each well and the plate was again incubated at 37 °C for 60 min. After washing five times, the plate was incubated for 15 min at 37 °C with 100 μL of substrate; the reaction was stopped by the addition 50 μL of stop solution to each well. The optical densities were read at a wavelength of 450 nm. The relationship between the IFN-γ values and the optical densities was fitted using a
There were no significant differences in the positive rates between the T-SPOT.TB test (25.8%) and the IGRA-ELISA test (28.6%) group (χ2 = 3.398, p = .065) (Table 1). The positive results were divided into four groups. For the T-SPOT.TB test, these four groups were: 6–20 SFCs, group 1; 20–40 SFCs, group 2; 40–60 SFCs, group 3; and > 60 SFCs, group 4. For the IGRA-ELISA, the four groups were divided as follows: 14–100 pg/mL, group 1; 100–200 pg/mL, group 2; 200–300 pg/mL, group 3; and > 300 pg/mL, group 4. For the T-SPOT.TB test, the proportions of positive results in each group were 39.1% (group 1), 22.5% Table 1 The results of T-SPOT.TB and IGRA-ELISA for diagnosis ATB. Result
Positive Negative Total (n)
75
T-SPOT.TB (n = 1794)
IGRA-ELISA (n = 1729)
ATB
NATB
ATB
NATB
107 22 129
356 1309 1665
94 21 115
400 1214 1614
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
Fig. 1. The comparison of values between ATB group and NATB group in positive results for (A) T-SPOT.TB test and (B) IGRA-ELISA test. ATB was active tuberculosis; and NATB was non active tuberculosis.
Table 2 The analysis for positive results of T-SPOT.TB and IGRA-ELISA tests. Groups
Group Group Group Group
1 2 3 4
IGRA-ELISA positive(n = 494)
T-SPOT.TB positive(n = 463)
Range: pg/mL
Median (25%,75%)
n (%)
Range: SFCs
Median (25%,75%)
n (%)
14–100 100–200 200–300 > 300
43.85 (29.93, 69.98) 146.3 (119.38, 172.23) 259.05 (226.4, 279.48) 378.2 (327.85, 427.65)
188 (38.06) 92 (18.62) 120 (24.29) 94 (19.03)
6–20 20–40 40–60 > 60
11 (7, 15) 28.5 (24, 33) 47.5 (44, 53) 95 (77,134.75)
181 (39.1) 104 (22.46) 92 (19.87) 86 (18.57)
SPOT.TB test and the IGRA-ELISA. The number of positive and negative results were 95 and 109, respectively, using the T-SPOT.TB test, and were 88 and 116, respectively, using the IGRA-ELISA (Table 4). The positive, negative, and total coincidence rates between the two tests were 95.5% (84 of 88), 90.5% (105 of 116), and 92.7% (189 of 204), respectively. There was excellent consistency between the two tests with a kappa value of 0.852 (χ2 = 148.64, p < .0001). Of the 15 inconsistent results, 12 gave either a value of ≤15 SFCs on the T-SPOT.TB test or ≤80 pg/mL on the IGRA-ELISA; only three (with values of 31 SFCs, 38 SFCs, and 61 SFCs using T-SPOT.TB test) were negative on the IGRA-ELISA. When the values of the 84 samples that tested positive using both tests were analyzed, we found that the value change trend of the IGRA-ELISA was consistent with that of T-SPOT.TB test, and that the two curves intersected at approximately 50 pg/mL (IGRA-ELISA) and 30 SFCs (T-SPOT.TB). Below and above the intersection, the degree of IGRA-ELISA value increase was lower and higher, respectively than that of the T-SPOT.TB test (Fig. 3).
(group 2), 19.9% (group 3), and 18.5% (group 4). The median TSPOT.TB test values were 11.0 (group 1), 28.5 (group 2), 47.5 (group 3), and 95 (group 4) SFCs. For the IGRA-ELISA, the proportions of positive results in each group were 38.1% (group 1), 18.6% (group 1), 24.3% (group 1), and 19.0% (group 1). The corresponding median values were 43.9, 146.3, 259.1, and 378.2 pg/mL, respectively (Table 2). Except for group 3, the proportion of positive results was similarly distributed between the two tests, p > .05 (Fig. 2A). To rule in patients with active TB, at cutoffs of 14, 100, 200, and 300 pg/mL, the positive predictive values of the IGRA-ELISA were 19.0%, 30.4%, 43.0%, and 96.8%, respectively. At cutoffs of 6, 20, 40, and 60 SFCs, the positive predictive values of the T-SPOT.TB test were 23.1%, 35.6%, 56.1%, and 98.9%, respectively (Table 3) (Fig. 2B). 3.3. The consistency of the two tests The blood samples of 204 patients were tested using both the T-
Fig. 2. The analysis of IGRAs positive results, (A) The proportion of group 1 to group 4 in T-SPOT.TB and IGRA-ELISA test positive results; (B) The positive predictive value of T-SPOT.TB and IGRA-ELISA test at cutoff 1, cutoff 2, cutoff 3 and cutoff 4. Group 1, 2, 3 and 4 for T-SPOT.TB were 6–20, 20–40, 40–60 and > 60SFCs; those for IGRA-ELISA were 14–100, 100–200, 200–300 and > 300 pg/mL; Cutoff 1, 2, 3, 4 for T-SPOT.TB were 6, 20, 40 and 60SFCs; those for IGRA-ELISA were 14, 100, 200 and 300 pg/mL.
76
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
Table 3 Diagnosis of active tuberculosis at different cutoffs. Diagnosis ATB
At different cutoffs for T-SPOT.TB
True positive: n False positive: n PPV: %
At different cutoffs for IGRA-ELISA
6
20
40
60
14
100
200
300
107 356 23.11
104 188 35.62
101 79 56.11
86 1 98.85
94 400 19.03
93 213 30.39
92 122 42.99
91 3 96.81
lymphocyte isolation, washing, and counting is not needed. The assay is simple and allows a very large number of samples to be processed simultaneously, thereby markedly reducing the labor required. In our hospital, 50–70 samples are processed for IGRA testing every workday; thus, in December 2016, the IGRA-ELISA started to be used as an alternative to the T-SPOT.TB test. In our study, both of the IGRA tests evaluated had a high sensitivity for diagnosing active TB; there were no significant differences in sensitivity between the two assays, consistent with previous reports [10–12, 14–17]. The specificity of T-SPOT.TB was slightly higher than that of IGRA-ELISA (78.6% vs 75.2%, p = .023). Data from previous reports [16, 18, 19] had shown an association between mycobacterial activity and the level of IFN-γ secretion. This was also confirmed by the analysis of the positive IGRA results in our study: The median values of the T-SPOT.TB and IGRA-ELISA test results were significantly higher in patients with active TB than in those without active TB (T-SPOT.TB: 373 vs 110.3 pg/mL, respectively, and IGRA-ELISA: 88 vs 20 SFCs, respectively, p < .0001). In our study, the positive rate of T-SPOT.TB was consistent with that of IGRA-ELISA (25.8% vs 28.6%, p = .065). The positive test results were divided into four groups. The proportions in each group, except group 3, were well-matched between the two assays (p > .05). The two assays were highly consistent, with a kappa value of 0.852 (p < .0001) and a total coincidence rate of 92.7%. Analysis of the 86 patients who
Table 4 The consistency evaluation for T-SPOT.TB and IGRA-ELISA. T-SPOT.TB
Positive (n) Negative (n) Total (n)
IGRA-ELISA
Total (n)
Positive (n)
Negative (n)
84 4 88
11 105 116
95 109 204
4. Discussion The clinical value of T-SPOT.TB test to diagnose M. tuberculosis infection has been demonstrated in other studies [5, 10, 12, 13, 16]. However, the process of the test is tedious. First, the sample should be centrifuged for 36 min, three times and washed twice to separate peripheral blood mononuclear cells, then, the peripheral blood mononuclear cells should be adjusted to volumes containing 2.5 × 106 cells per mL. The next process (ELISA and SFC counting) is also labor intensive. The IGRA-ELISA kit (WanTai Biological Pharmacy Enterprise Co, Ltd., Beijing, China) was approved for use as a diagnostic test for M. tuberculosis infection by the China Food and Drug Administration in 2012. In contrast to the T-SPOT.TB test, this assay makes Tcell incubation simple and quick to perform. The tedious process of
Fig. 3. The comparison of the value change trend between IGRA-ELISA and T-SPOT.TB test, the two curves crossed at approximately 50 pg/ml and 30 SFCs. Under the crossed point, the increase degree of IGRAELISA was slightly lower than that of TSPOT.TB, but above the crossed point, the increase degree of IGRA-ELISA was sharply higher than that of T-SPOT.TB.
77
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
tested positive using both the T-SPOT.TB test and the IGRA-ELISA showed that the value change trends of the two assays were consistent, and the curves intersected at approximately 50 pg/mL and 30 SFCs. Above the intersection, the degree of increase of the IGRA-ELISA value was markedly higher than that of T-SPOT.TB test. The T-SPOT.TB and QFT-IT tests are recommended to diagnose M. tuberculosis infection or to rule out active TB [7, 10, 12, 15, 20, 21], but whether they can be applied to diagnose active TB remains controversial as the assays cannot differentiate active TB from LTBI. In the study, both the T-SPOT.TB test and the IGRA-ELISA had a high sensitivity for diagnosing active TB, but a high level of IFN-γ was also detected in 199 patients with no evidence of active TB. Among these 199 cases, in 122 the IGRA-ELISA value was > 200 pg/mL and in 77 the TSPOT.TB test value was > 40 SFCs. In summary, we demonstrated that the IGRA-ELISA—a simple, almost labor-free assay that allows simultaneous processing of a very large number of samples—was well-matched with the T-SPOT.TB test for diagnosing M. tuberculosis infection. A cutoff value > 300 pg/mL for the IGRA-ELISA or > 60 SFCs for T-SPOT.TB test may be reliable to diagnose active TB. However, IGRA tests certainly cannot be used in isolation to diagnose active TB; other clinical and radiological information is required to establish the diagnosis of active TB.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Competing interests There are no conflicts of interest to declare.
[14]
Acknowledgments None
[15]
Ethical approval [16]
Not required References
[17]
[1] H. Duanmu, Report on fourth national epidemiological sampling survey of tuberculosis, Chin. J. Tuberc. Respir. Dis. 25 (1) (2002) 3–7. [2] H. Duanmu, The prevalence of pulmonary tuberculosis in a national survey across China in 2010, Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chin. J. Tuberc. Respir. Dis. 35 (9) (2012) 665–668. [3] R. Diel, B. Hauer, R. Loddenkemper, B. Manger, K. Krüger, Recommendations for tuberculosis screening before initiation of TNF-alpha-inhibitor treatment in rheumatic diseases, Pneumologie 68 (5) (2009) 411–416. [4] A. Balbir-Gurman, M. Lidgi, O. Elkayam, Update of the guidelines of the Israeli
[18]
[19]
[20]
78
Association of Rheumatology for the prevention of tuberculosis in patients treated with TNF-alpha blockers, Harefuah 153 (6) (2014) 359–361. S. Girlanda, P. Mantegani, E. Baldissera, P. Aiello, M. Ratti, M.G. Sabbadini, C. Fortis, ELISPOT-IFN-γ assay instead of tuberculin skin test for detecting latent Mycobacterium tuberculosis infection in rheumatic patients candidate to anti-TNF-α treatment, Clin. Rheumatol. 29 (10) (2010) 1135–1141. D. Vassilopoulos, N. Stamoulis, E. Hadziyannis, A.J. Archimandritis, Usefulness of enzyme-linked immunospot assay (Elispot) compared to tuberculin skin testing for latent tuberculosis screening in rheumatic patients scheduled for anti-tumor necrosis factor treatment, J. Rheumatol. 35 (7) (2008) 1271–1276. C.M. Denkinger, K. Dheda, M. Pai, Guidelines on interferon-γ release assays for tuberculosis infection: concordance, discordance or confusion? Clin. Microbiol. Infect. 17 (6) (2011) 806–814. G.H. Mazurek, K. Castro, Updated Guidelines for Using Interferon Gamma Release Assays to Detect Mycobacterium tuberculosis Infection-United States, 2010, MMWR, Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports, 59(RR-5) (2010), pp. 1–25. T. Ozekinci, E. Ozbek, Y. Celik, Comparison of tuberculin skin test and a specific Tcell-based test, T-Spot.TB, for the diagnosis of latent tuberculosis infection, J. Int. Med. Res. 35 (5) (2007) 696–703. H. SiMsek, S. Alpar, N. Ucar, F. Aksu, I. Ceyhan, A. Gozalan, S. Cesur, M. Ertek, Comparison of tuberculin skin testing and T-SPOT.TB for diagnosis of latent and active tuberculosis, Jpn. J. Infect. Dis. 63 (2) (2010) 99–102. A. Soysal, D. Toprak, M. Koc, H. Arikan, E. Akoglu, M. Bakir, Diagnosing latent tuberculosis infection in haemodialysis patients: T-cell based assay (T-SPOT. TB) or tuberculin skin test? Nephrol. Dial. Transplant. 27 (4) (2012) 1645–1650. Y.A. Kang, H.W. Lee, S.S. Hwang, S.W. Um, S.K. Han, Y.S. Shim, J.J. Yim, Usefulness of whole-blood interferon-γ assay and interferon-γ enzyme-linked immunospot assay in the diagnosis of active pulmonary tuberculosis, Chest 132 (3) (2007) 959–965. C.C. Lai, C.K. Tan, S.H. Lin, C.H. Liao, Y.T. Huang, P.R. Hsueh, Diagnostic performance of whole-blood interferon-γ assay and enzyme-linked immunospot assay for active tuberculosis, Diagn. Microbiol. Infect. Dis. 71 (2) (2011) 139–143. D. Goletti, D. Vincenti, S. Carrara, O. Butera, F. Bizzoni, G. Bernardini, M. Amicosante, E. Girardi, Selected RD1 peptides for active tuberculosis diagnosis: comparison of a gamma interferon whole-blood enzyme-linked immunosorbent assay and an enzyme-linked immunospot assay, Clin. Diagn. Lab. Immunol. 12 (11) (2005) 1311–1316. P. Ravn, M.E. Munk, Å.B. Andersen, B. Lundgren, J.D. Lundgren, L.N. Nielsen, A. Kokjensen, P. Andersen, K. Weldingh, Prospective evaluation of a whole-blood test using Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis, Clin. Diagn. Lab. Immunol. 12 (4) (2005) 491–496. L. Wang, Y. Yu, W. Chen, J. Feng, J. Wang, H. Zhao, L. Ma, B. Yang, Y. Ma, P. Dang, Evaluation of the characteristics of the enzyme-linked immunospot assay for diagnosis of active tuberculosis in China, Clin. Vaccine Immunol. 22 (5) (2015) 510–515. S.Y. Park, K. Jeon, S.W. Um, O.J. Kwon, E.S. Kang, W.J. Koh, Clinical utility of the QuantiFERON-TB gold in-tube test for the diagnosis of active pulmonary tuberculosis, Scand. J. Infect. Dis. 41 (11–12) (2009) 818–822. P.C. Chang, P.H. Wang, K.T. Chen, Use of the QuantiFERON-TB gold in-tube test in the diagnosis and monitoring of treatment efficacy in active pulmonary tuberculosis, Int. J. Environ. Res. Public Health 14 (3) (2017) 236. J.P. Janssens, P. Roux-Lombard, T. Perneger, M. Metzger, R. Vivien, T. Rochat, Quantitative scoring of an interferon-gamma assay for differentiating active from latent tuberculosis, Eur. Respir. J. 30 (4) (2007) 722–728. V. Bartu, M. Havelkova, E. Kopecka, QuantiFERON-TB gold in the diagnosis of active tuberculosis, J. Int. Med. Res. 36 (3) (2008) 434–437.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/cca
Evaluation of the performance of two tuberculosis interferon gamma release assays (IGRA-ELISA and T-SPOT.TB) for diagnosing Mycobacterium tuberculosis infection ⁎
Linchuan Wanga, Xu-dong Tiana,1, Yan Yub, , Wei Chena, a b
T
⁎
The First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi Province, China Honghui Hospital, Xi'an Jiaotong University, Xi'an, Shaanxi Province, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Mycobacterium tuberculosis IGRA-ELISA T-SPOT.TB
Objectives: The IGRA-ELISA and T-SPOT.TB are widely used in China. The aim of the study was to evaluate the performance of the two assays in diagnosis Mycobacterium tuberculosis infection. Methods: Of the 3727 patients in the study, 204 underwent testing using both the T-SPOT.TB and IGRA-ELISA, 1794 were tested using the T-SPOT.TB only, and 1729 were tested using the IGRA-ELISA only. The positive rate and consistency of the two assays were analyzed, and their sensitivity and specificity for diagnosing active tuberculosis were compared. Results: There were no significant differences in the positive rate between the T-SPOT.TB test (25.8%) and IGRAELISA (28.6%), p = .065. The two assays were highly consistent, with a kappa value of 0.852 (p < .0001) and a total coincidence rate of 92.7%. For the diagnosis of active tuberculosis, the sensitivity and specificity values of the T-SPOT.TB test were 82.9% (107/129) and 78.6% (1309/1665), respectively, and those of IGRA-ELISA were 81.7% (94/115) and 75.2% (1214/1614), respectively. There were no significant differences in sensitivity (p > .05), but the specificity of the T-SPOT.TB test was slightly higher than that of IGRA-ELISA (p = .023). Conclusion: Both in terms of diagnosing M. tuberculosis infection and ruling out active tuberculosis, the performance of the IGRA-ELISA—a simple, almost labor-free assay that allows simultaneous processing of a very large number of samples—was well-matched with that of T-SPOT.TB test. However, IGRAs cannot be used as the only test to diagnose active tuberculosis.
1. Introduction China has the second highest burden of tuberculosis (TB) in the world; approximately 44.5% people in China are infected with M. tuberculosis [1]. The Fifth National TB Epidemiological Survey conducted in China in 2010 reported that the prevalence of active pulmonary TB (PTB) was 459, of smear positive PTB was 66, and of culture positive PTB was 119 per 100,000 people aged > 15 years [2]. As the National TB Control Programme (2001–2010) in China was performed effectively, the annual decline in the rate of PTB, smear positive PTB, and culture positive PTB was 0.2%, 9.0%, and 5.8%, respectively, from 2000 to 2010 [2]. However, the proportion of individuals with clinically asymptomatic PTB was significantly higher in 2010 than in 2000 (43.1% vs 12.1%, respectively). Moreover, and the cumulative number of PTB infections in China up to 2010 is approximately 4.99 million cases [2].
⁎
1
Treatment of autoimmune diseases such as rheumatoid arthritis with tumor necrosis factor (TNF)-α-inhibitors may result in progression of latent tuberculosis infection (LTBI) to active tuberculosis (ATB) [3–6]. Hence, it is necessary to screen for LTBI prior to initiating biologic therapy [3–8]. M. tuberculosis infection is difficult to diagnose as there is often a lack of explicit evidence. Tuberculin skin testing (TST) was commonly used for LTBI screening in the past. However, falsepositive or false-negative results are often observed, limiting its clinical application [9–11]. Interferon (IFN)-γ release assays (IGRAs), i.e., the Quantiferon-Gold® TB In-Tube (QFT-GIT) or T-SPOT.TB test, [8, 12, 13] are based on the finding that effector T-cells secrete IFN-γ when stimulated by Mycobacterium-specific RD1 antigens, such as early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) [14, 15]. Currently in China, the T-SPOT.TB and IGRA-enzyme linked immunosorbent assay (IGRA-ELISA)—based on the QFT-GIT—are the two
Corresponding authors. E-mail addresses: [email protected] (Y. Yu), [email protected] (W. Chen). These authors contributed equally to this work.
https://doi.org/10.1016/j.cca.2018.01.014 Received 22 June 2017; Received in revised form 12 November 2017; Accepted 8 January 2018 Available online 09 January 2018 0009-8981/ © 2018 Elsevier B.V. All rights reserved.
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
power function with six standards (12.5, 25, 50, 100, 200, and 400 pg/ mL). Thereafter, the IFN-γ values of the T- and N-tubes were calculated. If the optical density of the standard (400 pg/mL) was < 1.0 or if the correlation coefficient (R2) of the power function fitted curve is < 0.99, the test is invalid. If the value in the P-tube minus that in the N-tube was < 20, or if the N-tube value is > 400, the test is indeterminate. The test is considered positive if the value in the T-tube minus that in the N-tube is ≥14 and is ≥25% of the N-tube value. The test is considered negative if the value in the T-tube minus that in the Ntube is < 14, or if the value in the T-tube minus that in the N-tube is ≥14 but is < 25% of the N-tube value.
most important screening and diagnostic tests for M. tuberculosis infection. However, the reference intervals and test units of these two assays differ. In China, there is a concern about whether there are differences in performance of the two assays for the diagnosis of M. tuberculosis infection and active TB disease. As the T-SPOT.TB test and IGRA-ELISA are both used in our hospital, in this study, the performance of these two assays were analyzed and compared. 2. Materials and methods 2.1. Study population From November 2016 through February 2017, a total of 3727 patients with suspected M. tuberculosis infection at the First Affiliated Hospital of Xi'an Jiaotong University, were enrolled in the study. The median age was 55 years, the majority of participants (n = 2100 [56.3%]) were men. Peripheral blood samples from the participants were collected in lithium-heparin-coated tubes. Of these samples, 204 were tested using both the T-SPOT.TB and IGRA-ELISA, 1794 were tested using the T-SPOT.TB only, and 1729 were tested using the IGRAELISA only. The data used in the study were obtained from the LIS and HIS systems of the First Affiliated Hospital of Xi'an Jiaotong University. 244 patients were diagnosed with active TB if at least one of the following conditions were fulfilled: (i) smear-positive for acid-fast bacilli, n = 19 (3 cases were also TB PCR-positive), (ii) chest x-ray findings consistent with a radiological diagnosis of PTB, n = 53, (iii) granulomata or caseous necrosis observed on histopathological examination of biopsy specimens, n = 45, or (iv) despite the absence of explicit evidence of active TB on microscopy, chest x-ray, or histopathology, the clinical symptoms were consistent with active TB and responded to anti-TB treatment (n = 127). 129 and 115 of the patients underwent the T-SPOT.TB and IGRA-ELISA testing, respectively.
2.2.3. Statistical analysis Statistical analyses were performed using SPSS, version 13.0 (serial number 5026743; SPSS Inc., Chicago, Illinois, USA). The sensitivity and specificity values of the two assays were compared using the chi-square test and the consistency of the two assays was evaluated using the kappa statistic. A p-value < .05 was considered statistically significant. 2.2.4. Ethics statement The study was deemed exempt from review by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University because of its retrospective nature and because the data used in the study were processed anonymously. 3. Results 3.1. The performance of the two tests In the T-SPOT.TB test group, 82.9% (107 of 129) of the patients with active TB and 21.4% (356 of 1665) of the patients without active TB had positive test results. In the IGRA-ELISA test group, 81.7% (94 of 115) of the patients with active TB and 24.8% (400 of 1614) of the patients without active TB had positive results (Table 1). In terms of diagnosing active TB, the T-SPOT.TB test had a sensitivity of 82.9% and a specificity of 78.6%, while the IGRA-ELISA had a sensitivity of 81.7% and a specificity of 75.2%. There was no significant difference in the sensitivity (χ2 = 0.061, p = .867) of these tests, but the specificity of the T-SPOT.TB test was slightly higher than that of the IGRA-ELISA (χ2 = 5.346, p = .023). The median values of the TSPOT.TB test and the IGRA-ELISA were 373.3 pg/mL and 88 SFCs, respectively, in the group with active TB; these values were significantly higher than those observed in the group without ATB (110.3 pg/mL and 20 SFCs, respectively; p < .0001) (Fig. 1).
2.2. Laboratory tests 2.2.1. Performing and interpreting the T-SPOT.TB test The T-SPOT.TB test (Oxford Immunotec Ltd., Abingdon, UK) should be performed within 6 h of obtaining the blood sample. The separation and incubation of peripheral blood mononuclear cells were conducted as previously reported [16]. If < 20 spot-forming cells (SFCs) are detected in the positive control well, the test is invalid. If > 10 SFCs are detected in the negative control well, the test is indeterminate. If either or both panel A (ESAT-6 antigen) and/or panel B (CFP-10 antigen) have 6 or more SFCs detected than are detected in the negative control, the test is positive. If both panel A and panel B have 5 or fewer SFCs detected than are detected in the negative control, the test is negative.
3.2. Analysis of positive test results
2.2.2. Performing and interpreting the IGRA-ELISA The IGRA-ELISA (WanTai Biological Pharmacy Enterprise Co, Ltd., Beijing, China) should be performed within 2 h of obtaining the blood sample. Blood samples were mixed by inverting the vacutainer 5 times, then 1 mL of blood was added to each of 3 heparinized tubes (T, N, and P tube): The T-tube contained ESAT-6 and CFP-10 antigens, the N-tube was the negative control, and the P-tube (positive control) contained phytohemagglutinin. The tubes were incubated at 37 °C for 20–24 h and were then centrifuged at 3000–5000 rpm for 10 min. The supernatant was used for the ELISA assay, performed using an automated instrument. The whole process was performed as follows: 50 μL supernatant and standards were added to the wells that contained anti-IFN-γ and the plate was incubated at 37 °C for 60 min. Then, 50 μL of horseradish peroxidase-labeled anti-IFN-γ was added to each well and the plate was again incubated at 37 °C for 60 min. After washing five times, the plate was incubated for 15 min at 37 °C with 100 μL of substrate; the reaction was stopped by the addition 50 μL of stop solution to each well. The optical densities were read at a wavelength of 450 nm. The relationship between the IFN-γ values and the optical densities was fitted using a
There were no significant differences in the positive rates between the T-SPOT.TB test (25.8%) and the IGRA-ELISA test (28.6%) group (χ2 = 3.398, p = .065) (Table 1). The positive results were divided into four groups. For the T-SPOT.TB test, these four groups were: 6–20 SFCs, group 1; 20–40 SFCs, group 2; 40–60 SFCs, group 3; and > 60 SFCs, group 4. For the IGRA-ELISA, the four groups were divided as follows: 14–100 pg/mL, group 1; 100–200 pg/mL, group 2; 200–300 pg/mL, group 3; and > 300 pg/mL, group 4. For the T-SPOT.TB test, the proportions of positive results in each group were 39.1% (group 1), 22.5% Table 1 The results of T-SPOT.TB and IGRA-ELISA for diagnosis ATB. Result
Positive Negative Total (n)
75
T-SPOT.TB (n = 1794)
IGRA-ELISA (n = 1729)
ATB
NATB
ATB
NATB
107 22 129
356 1309 1665
94 21 115
400 1214 1614
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
Fig. 1. The comparison of values between ATB group and NATB group in positive results for (A) T-SPOT.TB test and (B) IGRA-ELISA test. ATB was active tuberculosis; and NATB was non active tuberculosis.
Table 2 The analysis for positive results of T-SPOT.TB and IGRA-ELISA tests. Groups
Group Group Group Group
1 2 3 4
IGRA-ELISA positive(n = 494)
T-SPOT.TB positive(n = 463)
Range: pg/mL
Median (25%,75%)
n (%)
Range: SFCs
Median (25%,75%)
n (%)
14–100 100–200 200–300 > 300
43.85 (29.93, 69.98) 146.3 (119.38, 172.23) 259.05 (226.4, 279.48) 378.2 (327.85, 427.65)
188 (38.06) 92 (18.62) 120 (24.29) 94 (19.03)
6–20 20–40 40–60 > 60
11 (7, 15) 28.5 (24, 33) 47.5 (44, 53) 95 (77,134.75)
181 (39.1) 104 (22.46) 92 (19.87) 86 (18.57)
SPOT.TB test and the IGRA-ELISA. The number of positive and negative results were 95 and 109, respectively, using the T-SPOT.TB test, and were 88 and 116, respectively, using the IGRA-ELISA (Table 4). The positive, negative, and total coincidence rates between the two tests were 95.5% (84 of 88), 90.5% (105 of 116), and 92.7% (189 of 204), respectively. There was excellent consistency between the two tests with a kappa value of 0.852 (χ2 = 148.64, p < .0001). Of the 15 inconsistent results, 12 gave either a value of ≤15 SFCs on the T-SPOT.TB test or ≤80 pg/mL on the IGRA-ELISA; only three (with values of 31 SFCs, 38 SFCs, and 61 SFCs using T-SPOT.TB test) were negative on the IGRA-ELISA. When the values of the 84 samples that tested positive using both tests were analyzed, we found that the value change trend of the IGRA-ELISA was consistent with that of T-SPOT.TB test, and that the two curves intersected at approximately 50 pg/mL (IGRA-ELISA) and 30 SFCs (T-SPOT.TB). Below and above the intersection, the degree of IGRA-ELISA value increase was lower and higher, respectively than that of the T-SPOT.TB test (Fig. 3).
(group 2), 19.9% (group 3), and 18.5% (group 4). The median TSPOT.TB test values were 11.0 (group 1), 28.5 (group 2), 47.5 (group 3), and 95 (group 4) SFCs. For the IGRA-ELISA, the proportions of positive results in each group were 38.1% (group 1), 18.6% (group 1), 24.3% (group 1), and 19.0% (group 1). The corresponding median values were 43.9, 146.3, 259.1, and 378.2 pg/mL, respectively (Table 2). Except for group 3, the proportion of positive results was similarly distributed between the two tests, p > .05 (Fig. 2A). To rule in patients with active TB, at cutoffs of 14, 100, 200, and 300 pg/mL, the positive predictive values of the IGRA-ELISA were 19.0%, 30.4%, 43.0%, and 96.8%, respectively. At cutoffs of 6, 20, 40, and 60 SFCs, the positive predictive values of the T-SPOT.TB test were 23.1%, 35.6%, 56.1%, and 98.9%, respectively (Table 3) (Fig. 2B). 3.3. The consistency of the two tests The blood samples of 204 patients were tested using both the T-
Fig. 2. The analysis of IGRAs positive results, (A) The proportion of group 1 to group 4 in T-SPOT.TB and IGRA-ELISA test positive results; (B) The positive predictive value of T-SPOT.TB and IGRA-ELISA test at cutoff 1, cutoff 2, cutoff 3 and cutoff 4. Group 1, 2, 3 and 4 for T-SPOT.TB were 6–20, 20–40, 40–60 and > 60SFCs; those for IGRA-ELISA were 14–100, 100–200, 200–300 and > 300 pg/mL; Cutoff 1, 2, 3, 4 for T-SPOT.TB were 6, 20, 40 and 60SFCs; those for IGRA-ELISA were 14, 100, 200 and 300 pg/mL.
76
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
Table 3 Diagnosis of active tuberculosis at different cutoffs. Diagnosis ATB
At different cutoffs for T-SPOT.TB
True positive: n False positive: n PPV: %
At different cutoffs for IGRA-ELISA
6
20
40
60
14
100
200
300
107 356 23.11
104 188 35.62
101 79 56.11
86 1 98.85
94 400 19.03
93 213 30.39
92 122 42.99
91 3 96.81
lymphocyte isolation, washing, and counting is not needed. The assay is simple and allows a very large number of samples to be processed simultaneously, thereby markedly reducing the labor required. In our hospital, 50–70 samples are processed for IGRA testing every workday; thus, in December 2016, the IGRA-ELISA started to be used as an alternative to the T-SPOT.TB test. In our study, both of the IGRA tests evaluated had a high sensitivity for diagnosing active TB; there were no significant differences in sensitivity between the two assays, consistent with previous reports [10–12, 14–17]. The specificity of T-SPOT.TB was slightly higher than that of IGRA-ELISA (78.6% vs 75.2%, p = .023). Data from previous reports [16, 18, 19] had shown an association between mycobacterial activity and the level of IFN-γ secretion. This was also confirmed by the analysis of the positive IGRA results in our study: The median values of the T-SPOT.TB and IGRA-ELISA test results were significantly higher in patients with active TB than in those without active TB (T-SPOT.TB: 373 vs 110.3 pg/mL, respectively, and IGRA-ELISA: 88 vs 20 SFCs, respectively, p < .0001). In our study, the positive rate of T-SPOT.TB was consistent with that of IGRA-ELISA (25.8% vs 28.6%, p = .065). The positive test results were divided into four groups. The proportions in each group, except group 3, were well-matched between the two assays (p > .05). The two assays were highly consistent, with a kappa value of 0.852 (p < .0001) and a total coincidence rate of 92.7%. Analysis of the 86 patients who
Table 4 The consistency evaluation for T-SPOT.TB and IGRA-ELISA. T-SPOT.TB
Positive (n) Negative (n) Total (n)
IGRA-ELISA
Total (n)
Positive (n)
Negative (n)
84 4 88
11 105 116
95 109 204
4. Discussion The clinical value of T-SPOT.TB test to diagnose M. tuberculosis infection has been demonstrated in other studies [5, 10, 12, 13, 16]. However, the process of the test is tedious. First, the sample should be centrifuged for 36 min, three times and washed twice to separate peripheral blood mononuclear cells, then, the peripheral blood mononuclear cells should be adjusted to volumes containing 2.5 × 106 cells per mL. The next process (ELISA and SFC counting) is also labor intensive. The IGRA-ELISA kit (WanTai Biological Pharmacy Enterprise Co, Ltd., Beijing, China) was approved for use as a diagnostic test for M. tuberculosis infection by the China Food and Drug Administration in 2012. In contrast to the T-SPOT.TB test, this assay makes Tcell incubation simple and quick to perform. The tedious process of
Fig. 3. The comparison of the value change trend between IGRA-ELISA and T-SPOT.TB test, the two curves crossed at approximately 50 pg/ml and 30 SFCs. Under the crossed point, the increase degree of IGRAELISA was slightly lower than that of TSPOT.TB, but above the crossed point, the increase degree of IGRA-ELISA was sharply higher than that of T-SPOT.TB.
77
Clinica Chimica Acta 479 (2018) 74–78
L. Wang et al.
tested positive using both the T-SPOT.TB test and the IGRA-ELISA showed that the value change trends of the two assays were consistent, and the curves intersected at approximately 50 pg/mL and 30 SFCs. Above the intersection, the degree of increase of the IGRA-ELISA value was markedly higher than that of T-SPOT.TB test. The T-SPOT.TB and QFT-IT tests are recommended to diagnose M. tuberculosis infection or to rule out active TB [7, 10, 12, 15, 20, 21], but whether they can be applied to diagnose active TB remains controversial as the assays cannot differentiate active TB from LTBI. In the study, both the T-SPOT.TB test and the IGRA-ELISA had a high sensitivity for diagnosing active TB, but a high level of IFN-γ was also detected in 199 patients with no evidence of active TB. Among these 199 cases, in 122 the IGRA-ELISA value was > 200 pg/mL and in 77 the TSPOT.TB test value was > 40 SFCs. In summary, we demonstrated that the IGRA-ELISA—a simple, almost labor-free assay that allows simultaneous processing of a very large number of samples—was well-matched with the T-SPOT.TB test for diagnosing M. tuberculosis infection. A cutoff value > 300 pg/mL for the IGRA-ELISA or > 60 SFCs for T-SPOT.TB test may be reliable to diagnose active TB. However, IGRA tests certainly cannot be used in isolation to diagnose active TB; other clinical and radiological information is required to establish the diagnosis of active TB.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Competing interests There are no conflicts of interest to declare.
[14]
Acknowledgments None
[15]
Ethical approval [16]
Not required References
[17]
[1] H. Duanmu, Report on fourth national epidemiological sampling survey of tuberculosis, Chin. J. Tuberc. Respir. Dis. 25 (1) (2002) 3–7. [2] H. Duanmu, The prevalence of pulmonary tuberculosis in a national survey across China in 2010, Zhonghua jie he he hu xi za zhi = Zhonghua jiehe he huxi zazhi = Chin. J. Tuberc. Respir. Dis. 35 (9) (2012) 665–668. [3] R. Diel, B. Hauer, R. Loddenkemper, B. Manger, K. Krüger, Recommendations for tuberculosis screening before initiation of TNF-alpha-inhibitor treatment in rheumatic diseases, Pneumologie 68 (5) (2009) 411–416. [4] A. Balbir-Gurman, M. Lidgi, O. Elkayam, Update of the guidelines of the Israeli
[18]
[19]
[20]
78
Association of Rheumatology for the prevention of tuberculosis in patients treated with TNF-alpha blockers, Harefuah 153 (6) (2014) 359–361. S. Girlanda, P. Mantegani, E. Baldissera, P. Aiello, M. Ratti, M.G. Sabbadini, C. Fortis, ELISPOT-IFN-γ assay instead of tuberculin skin test for detecting latent Mycobacterium tuberculosis infection in rheumatic patients candidate to anti-TNF-α treatment, Clin. Rheumatol. 29 (10) (2010) 1135–1141. D. Vassilopoulos, N. Stamoulis, E. Hadziyannis, A.J. Archimandritis, Usefulness of enzyme-linked immunospot assay (Elispot) compared to tuberculin skin testing for latent tuberculosis screening in rheumatic patients scheduled for anti-tumor necrosis factor treatment, J. Rheumatol. 35 (7) (2008) 1271–1276. C.M. Denkinger, K. Dheda, M. Pai, Guidelines on interferon-γ release assays for tuberculosis infection: concordance, discordance or confusion? Clin. Microbiol. Infect. 17 (6) (2011) 806–814. G.H. Mazurek, K. Castro, Updated Guidelines for Using Interferon Gamma Release Assays to Detect Mycobacterium tuberculosis Infection-United States, 2010, MMWR, Recommendations and reports: Morbidity and mortality weekly report. Recommendations and reports, 59(RR-5) (2010), pp. 1–25. T. Ozekinci, E. Ozbek, Y. Celik, Comparison of tuberculin skin test and a specific Tcell-based test, T-Spot.TB, for the diagnosis of latent tuberculosis infection, J. Int. Med. Res. 35 (5) (2007) 696–703. H. SiMsek, S. Alpar, N. Ucar, F. Aksu, I. Ceyhan, A. Gozalan, S. Cesur, M. Ertek, Comparison of tuberculin skin testing and T-SPOT.TB for diagnosis of latent and active tuberculosis, Jpn. J. Infect. Dis. 63 (2) (2010) 99–102. A. Soysal, D. Toprak, M. Koc, H. Arikan, E. Akoglu, M. Bakir, Diagnosing latent tuberculosis infection in haemodialysis patients: T-cell based assay (T-SPOT. TB) or tuberculin skin test? Nephrol. Dial. Transplant. 27 (4) (2012) 1645–1650. Y.A. Kang, H.W. Lee, S.S. Hwang, S.W. Um, S.K. Han, Y.S. Shim, J.J. Yim, Usefulness of whole-blood interferon-γ assay and interferon-γ enzyme-linked immunospot assay in the diagnosis of active pulmonary tuberculosis, Chest 132 (3) (2007) 959–965. C.C. Lai, C.K. Tan, S.H. Lin, C.H. Liao, Y.T. Huang, P.R. Hsueh, Diagnostic performance of whole-blood interferon-γ assay and enzyme-linked immunospot assay for active tuberculosis, Diagn. Microbiol. Infect. Dis. 71 (2) (2011) 139–143. D. Goletti, D. Vincenti, S. Carrara, O. Butera, F. Bizzoni, G. Bernardini, M. Amicosante, E. Girardi, Selected RD1 peptides for active tuberculosis diagnosis: comparison of a gamma interferon whole-blood enzyme-linked immunosorbent assay and an enzyme-linked immunospot assay, Clin. Diagn. Lab. Immunol. 12 (11) (2005) 1311–1316. P. Ravn, M.E. Munk, Å.B. Andersen, B. Lundgren, J.D. Lundgren, L.N. Nielsen, A. Kokjensen, P. Andersen, K. Weldingh, Prospective evaluation of a whole-blood test using Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis, Clin. Diagn. Lab. Immunol. 12 (4) (2005) 491–496. L. Wang, Y. Yu, W. Chen, J. Feng, J. Wang, H. Zhao, L. Ma, B. Yang, Y. Ma, P. Dang, Evaluation of the characteristics of the enzyme-linked immunospot assay for diagnosis of active tuberculosis in China, Clin. Vaccine Immunol. 22 (5) (2015) 510–515. S.Y. Park, K. Jeon, S.W. Um, O.J. Kwon, E.S. Kang, W.J. Koh, Clinical utility of the QuantiFERON-TB gold in-tube test for the diagnosis of active pulmonary tuberculosis, Scand. J. Infect. Dis. 41 (11–12) (2009) 818–822. P.C. Chang, P.H. Wang, K.T. Chen, Use of the QuantiFERON-TB gold in-tube test in the diagnosis and monitoring of treatment efficacy in active pulmonary tuberculosis, Int. J. Environ. Res. Public Health 14 (3) (2017) 236. J.P. Janssens, P. Roux-Lombard, T. Perneger, M. Metzger, R. Vivien, T. Rochat, Quantitative scoring of an interferon-gamma assay for differentiating active from latent tuberculosis, Eur. Respir. J. 30 (4) (2007) 722–728. V. Bartu, M. Havelkova, E. Kopecka, QuantiFERON-TB gold in the diagnosis of active tuberculosis, J. Int. Med. Res. 36 (3) (2008) 434–437.