Evaluation of cytokine levels using QuantiFERON-TB Gold Plus in patients with active tuberculosis

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Evaluation of cytokine levels using QuantiFERON-TB Gold Plus in patients with active tuberculosis Maho Suzukawa , Keita Takeda , Shunsuke Akashi , Isao Asari , Masahiro Kawashima , Nobuharu Ohshima , Eri Inoue , Ryota Sato , Masahiro Shimada , Junko Suzuki , Akira Yamane , Atsuhisa Tamura , Ken Ohta , Shigeto Toma , Katsuji Teruya , Hideaki Nagai PII: DOI: Reference:

S0163-4453(20)30092-X https://doi.org/10.1016/j.jinf.2020.02.007 YJINF 4450

To appear in:

Journal of Infection

Accepted date:

10 February 2020

Please cite this article as: Maho Suzukawa , Keita Takeda , Shunsuke Akashi , Isao Asari , Masahiro Kawashima , Nobuharu Ohshima , Eri Inoue , Ryota Sato , Masahiro Shimada , Junko Suzuki , Akira Yamane , Atsuhisa Tamura , Ken Ohta , Shigeto Toma , Katsuji Teruya , Hideaki Nagai , Evaluation of cytokine levels using QuantiFERON-TB Gold Plus in patients with active tuberculosis, Journal of Infection (2020), doi: https://doi.org/10.1016/j.jinf.2020.02.007

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd on behalf of The British Infection Association.

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Highlights 

QFT-plus did not enhance cytokine production vs. QFT-3G.

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PDGF-BB was higher in the TB2 tube than the TB1 tube in QFT-plus.

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IL-6 and TNF-α were higher in the TB1 tube than the TB2 tube in QFT-plus.

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IP-10 in QFT-3G and QFT-plus were useful for diagnosing active tuberculosis.

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IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β were higher in tuberculosis patients than in the healthy controls.

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Title Page Title: Evaluation of cytokine levels using QuantiFERON-TB Gold Plus in patients with active tuberculosis Authors: Maho Suzukawaa, Keita Takedaa,b, Shunsuke Akashia, Isao Asaria, Masahiro Kawashimaa, Nobuharu Ohshimaa, Eri Inouea, Ryota Satoa, Masahiro Shimadaa, Junko Suzukia, Akira Yamanea, Atsuhisa Tamuraa, Ken Ohtaa,c, Shigeto Tomaa, Katsuji Teruyad and Hideaki Nagaia Affiliations: aNational Hospital Organization Tokyo National Hospital, Tokyo, Japan; b

Department of Basic Mycobacteriology, Graduate School of Biomedical Science,

Nagasaki University; cJapan Anti-Tuberculosis Association, Fukujuji Hospital, Tokyo, Japan; dNational Center for Global Health and Medicine, Tokyo, Japan. Corresponding author: Maho Suzukawa, Clinical Research Center, National Hospital Organization Tokyo National Hospital, 3-1-1 Takeoka, Kiyose-City, Tokyo 204-8585, Japan. E-mail: [email protected] Tel +81-42-491-2111; FAX: +81-42-491-2168

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Abstract Objectives A recently released new QuantiFERON (QFT) product, QFT TB Gold plus (QFT-plus), is optimized for both CD4 and CD8 responses and reported to have higher sensitivity compared to the former QFT-3G. Previously, using supernatants of QFT-3G, we and others have demonstrated that cytokines other than IFN-γ may be useful in diagnosing tuberculosis. The present study aimed to identify cytokines that are useful for accurately diagnosing active tuberculosis by using QFT-plus and compared the data to those with QFT-3G. Methods Eighty-three active tuberculosis patients and 70 healthy control subjects who were examined by QFT at Tokyo National Hospital from June 2017 to July 2018 were enrolled. QFT-3G and QFT-plus were performed according to the manufacturer’s instructions. At the same time, blood cell culture supernatants were collected and assayed for their cytokine levels using R&D Systems Luminex Assay and MAGPIX System. The levels of cytokines were compared between different antigen-containing

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tubes (3G Ag, TB1 and TB2 tubes), as well as between the patients and the control subjects. ROC curves were drawn, and the AUCs were calculated. Results Five cytokines, i.e., IL-2, IL-6, IL-8, IP-10 and MIP-1β, produced by human blood cells in three independent tubes containing different tuberculosis antigens were higher in the 3G Ag tube compared to both the TB1 and TB2 tubes. Further, when the TB1 and TB2 tubes were compared, TB2 showed greater production of only PDGF-BB, and less production of IL-6 and TNF-α. For diagnosing active tuberculosis, the levels of IP-10 were superior to the level of IFN-γ based on showing a larger AUC for ROC curves in our present study setting. Finally, the levels of IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β were distinctly different between the active tuberculosis patients and healthy controls. Conclusions In summary, there was no cytokine that was higher in the tubes of QFT-plus compared to the tube of QFT-3G, suggesting inferiority of QFT-plus antigens to 3G Ag in terms of elicitation of cytokine production. Our results also suggest the usefulness of cytokines

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that showed a significant difference between the active tuberculosis patients and the healthy controls—namely, IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β—for diagnosing tuberculosis, but the roles of these cytokines in the pathogenesis of tuberculosis need to be elucidated (UMIN000035253).

Key words: tuberculosis, QFT, QFT-3G, QFT-plus, IGRA, IP-10

Introduction Today, interferon-gamma release assays (IGRAs) are essential tools for detecting infection with Mycobacterium tuberculosis (M. tuberculosis), including for diagnosing latent tuberculosis infection (LTBI). IGRAs, including both QuantiFERON (QFT) and T-SPOT.TB, enable direct observation of the response of a patient’s blood cells to specific antigens derived from M. tuberculosis (1) (2). On the other hand, IGRAs were not useful for accurately discriminating between active tuberculosis (active TB) and LTBI (3). In previous studies, including ours, many cytokines in the supernatants of QFT have been proposed to be useful for differentiating between active TB and LTBI

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(4-7). We showed a combination of various cytokines, i.e., IL-2, IL-5, IL-10, IL-1RA and MCP-1, to be a good candidate for accurately identifying active disease and, importantly, those cytokines may indicate a difference in the pathogenesis of active TB and LTBI (4). Recently, a new QFT product, QuantiFERON TB Gold plus (QFT-plus), has become commercially available in Japan. QFT-plus uses two independent antigen tubes, TB1 and TB2. TB1 tube no longer contains the former antigen TB7.7, which has been deleted, and it now contains only ESAT-6 and CFP-10. According to recent reports, QFT-plus is superior to the previous QuantiFERON TB Gold (QFT-3G) for diagnosing aged patients and immunocompromised patients with active TB (8, 9). Needless to say, since tuberculosis has still not been eradicated, in spite of man’s long fight against M. tuberculosis, it would be very helpful if QFT-plus were able to achieve clearer, more accurate diagnoses of active TB. The present study aimed to elucidate the possible advantage of QFT-plus compared to QFT-3G in achieving accurate diagnosis of active TB by using cytokines produced by

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patients’ blood cells in response to specific antigens. The different responses to the original antigens in QFT-3G, TB1 and TB2 in QFT-plus were of special interest. Materials and Methods Subjects The study population comprised 83 patients diagnosed as active TB and 70 healthy control subjects. Subjects who were examined by QFT at Tokyo National Hospital from June 2017 through July 2018 were enrolled in this study. The control patients were examined by QFT as part of routine annual examinations of healthcare workers at Tokyo National Hospital. All the active TB patients underwent QFT at the time of diagnosis, before initiation of therapy. Active TB patients were defined as patients with abnormal radiologic findings suggestive of active pulmonary tuberculosis, with microbiologic confirmation of infection with M. tuberculosis by mycobacterial culture, acid-fast smear examination and/or transcription–reverse transcription concerted amplification (TRC) of sputum. All the active TB patients were treatment-naïve. No sputum specimens were examined for the healthy subjects, because they had almost no sputum.

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The protocol for this study was reviewed and approved by the National Hospital Organization Tokyo National Hospital’s Institutional Ethical Review Board (IRB) (approval number 170043). Written informed consent was obtained from all the study participants. QFT-3G and QFT-plus QFT-3G and QFT-plus were performed according to the manufacturer’s instructions. Briefly, blood was drawn by venipuncture and then incubated at 37˚C for 16-24 hours with either tuberculosis-specific antigens, i.e., QFT-3G antigen, TB1 antigen in QFT-plus and TB2 antigen in QFT-plus, or a mitogen as a positive control, or without stimulation as a negative control (Nil). The culture supernatants were collected and used to quantitate IFN- by an enzyme-linked immunosorbent assay using the QFT system. The QFT results were judged according to the manufacturer’s instructions.

Multiple Cytokine Assay

Supernatants remaining from QFT were frozen at -20°C for as long as one year at Tokyo National Hospital and subsequently used for this study. The levels of cytokines

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in the QFT supernatants were analyzed using an R&D Systems Luminex Assay (R&D Systems, Minneapolis, MN, USA) and MAGPIX System (Luminex, Austin, TX, USA) according to the manufacturers’ instructions. The analyzed cytokines were IFN-, IL-1RA, IL-2, IL-5, IL-6, CXCL8/IL-8, IL-10, IL-12p70, CXCL10/IP-10, CCL2/MCP-1, CCL4/MIP-1, PDGF-BB, CCL5/RANTES and TNF-. The supernatants were diluted 30x prior to assaying for IL-1RA, CXCL8/IL-8, CXCL10/IP-10, PDGF-BB, CCL2/MCP-1 and RANTES. Standard curves were generated using serial dilutions of the assay standards for quantification. The xPONENT 4.2 Software for MAGPIX was used for bead acquisition and analysis of median fluorescence intensity. The concentrations of the released cytokines in each tube were calculated by subtracting the value of the Nil (includes no antigen) tube. If a negative value was obtained, the value was recorded as zero.

Statistical Analyses Continuous variables were expressed as medians with interquartile ranges. Overall comparisons between the groups were performed with 1-way ANOVA followed by post

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hoc Bonferroni comparisons, and P values were determined. A two-tailed P-value of less than 5% was considered statistically significant. Correlations between variables were analyzed using Spearman’s test. Linear regression analyses were also performed. We constructed receiver operating characteristic (ROC) curves, and the area under each ROC curve (AUC) was calculated. All statistical analyses were performed using GraphPad Prism version 5.0 for MAC OS X (GraphPad Software, San Diego, USA). Results Baseline characteristics of the participants Table 1 shows the baseline characteristics of the participants. The active TB patients had a mean age of 58.1 ± 20.8 years and consisted of 55 males and 28 females, whereas the healthy control subjects were 32.1 ± 7.9 years old and consisted of 14 males and 56 females. These different demographics seemed to have been due to the way we recruited the healthy control subjects: they were all hospital workers at our facility with no history of tuberculosis who had annual checkups to exclude infection with M. tuberculosis. They were significantly younger and included significantly more females compared with the active TB patient group.

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Cytokine levels in different antigen tubes for active TB patients We first compared the levels of IFN-γ measured by the MAGPIX system and the enzyme-linked immunosorbent assay used by the QFT system (Figure 1). Spearman’s correlation analysis showed significant correlations for the levels of IFN-γ in TB1 tube (r = 0.87, p < 0.0001), TB2 tube (r = 0.83, p < 0.0001) and 3G Ag tube (r = 0.82, p < 0.0001). Figure 2 shows the cytokine levels in the three different QFT tubes. The differences in IFN-γ levels among the tubes were not statistically significant (TB1: 314.1, 95% CI 222.7–405.6; TB2: 446.4, 95% CI 250.6-642.2; 3G Ag: 413.4, 95% CI 267.8-559), which was in line with previous reports. Among the cytokines measured, the levels of the following were significantly higher in the QFT-3G antigen tube: IL-2 (TB1: 65.31, 95% CI 44.22–86.41; TB2: 69.29, 95% CI 43.8–94.78; 3G Ag: 92.87, 95% CI 63.21–122.5), IL-6 (TB1: 204.8, 95% CI 139.9–269.7; TB2: 109, 95% CI 54.41– 163.6; 3G Ag: 547.5, 95% CI 287.7–807.2), IL-8 (TB1: 7621, 95% CI 4506-10736; TB2: 6826, 95% CI 3925-9726; 3G Ag: 12425, 95% CI 7142-17708), IP-10 (TB1: 7354, 95% CI 5731-8976; TB2: 7885, 95% CI 6190-9851; 3G Ag: 8591, 95% CI 6679-10503)

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and MIP-1β (TB1: 2194, 95% CI 1407-2981; TB2: 2113, 95% CI 1197-3030; 3G Ag: 3568, 95% CI 2419-4717). Comparing TB1 and TB2, only the level of PDGF-BB was significantly higher in TB2 than in TB1 (TB1: 163.1, 95% CI 43.42-282.8; TB2: 299.7, 95% CI 137-462.5; 3G Ag: 349.9, 95% CI 100.9-598.8), and interestingly, IL-6 (TB1: 204.8, 95% CI 139.9-269.7; TB2: 109, 95% CI 54.41-163.6; 3G Ag: 547.5, 95% CI 287.7-807.2) and TNF- (TB1: 201.6, 95% CI 140.1-263; TB2: 115.3, 95% CI 68.8-161.8; 3G Ag: 223.4, 95% CI 60.79-386.1) were significantly higher in TB1 than in TB2.

The level of IP-10 in TB2 tube as well as 3G-Ag tube may be a useful marker for diagnosing active tuberculosis Next, ROC curves were drawn for the cytokine levels in the three independent antigen tubes in an attempt to distinguish active tuberculosis patients from uninfected individuals (Fig. 3 and Table 2). The highest AUC among all the cytokines in all antigen tubes was 0.955, for IP-10 in the TB2 tube (Fig. 3 and Table 2). Table 2 shows the AUCs of the ROCs drawn for IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β in

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which the AUCs were significantly high. All of these cytokines showed significant elevation from the baseline (Nil) upon stimulation with 3G Ag except for IL-2 (Supplemental Figure 1). The cut-off points were chosen as values having the highest likelihood ratio. Among all the cytokines and antigens, the level of IP-10 in the TB1 tube showed the highest sensitivity of 86.75%, and specificity of 98.57%, followed by the IP-10 level in the 3G-Ag tube, which showed a sensitivity of 84.34% and specificity of 98.57%. We also compared the cytokine levels in the three independent antigen tubes from patients with tuberculosis and the healthy control subjects (Fig. 5). As revealed by the ROCs, IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β showed a clear difference between the two groups. Discussion In the present study, we found that the production of IL-2, IL-6, IL-8, IP-10 and MIP-1β cytokines by human blood cells in three independent tubes containing different tuberculosis antigens was higher in the 3G Ag tube compared to both the TB1 and TB2 tubes. Furthermore, when the TB1 and TB2 tubes were compared, TB2 showed greater

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production of only PDGF-BB, and lesser production of IL-6 and TNF-α. For diagnosis of active tuberculosis, the level of IP-10 was superior to the level of IFN-γ based on showing a larger AUC for the ROCs in our present study setting. Finally, the levels of IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β were distinctly different between the active tuberculosis patients and healthy controls, strongly suggesting involvement of these cytokines in the pathogenesis of active tuberculosis. Evidence to date has shown that both CD4- and CD8-positive antigen-specific T cells secrete IFN-γ in response to tuberculosis antigens (10, 11). Now, with the addition of a CD8 cytotoxic T-cell stimulating peptide to QFT-plus, researchers and clinicians expect this new assay tool to be even more useful for diagnosis of active tuberculosis. CD8 T cells are important in controlling the bacterial load, and these cells increase when mycobacterium is replicating and decrease during treatment (12). A previous study revealed that when TB therapy is completed, the CD8 T-cell response decreases (16). However, other studies reported equivalent sensitivity with both QFT-3G and QFT-plus (13-15). Our present study adds support to those observations that QFT-plus shows no significant superiority in regard to any of the measured cytokines in the

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supernatants, including the cytokine levels in the TB2 tube. TB7.7, the only antigen retained from QFT-3G for the development of QFT-plus, was first identified as a tuberculosis antigen specifically recognized by T cells (16). TB7.7 induced production of IFN-γ by subjects infected with tuberculosis, but not by those vaccinated with bacilli Calmette-Guerin (16). Since the response of blood cells in producing all the cytokines measured in the present study was equivalent or greater with QFT-3G antigen, TB7.7 may have been an important antigen for reactivity in QFT by subjects with active tuberculosis. Still, since the TB1 tube contains long peptides derived from ESAT-6 and CFP-10 that induce a specific CD4 T-cell response, while the TB2 tube contains shorter peptides that stimulate CD8 T cells in addition to the same long peptides as in TB1, QFT-plus may be superior for patients with certain conditions, such as immune suppression resulting from CD4 T-cell impairment due to HIV-infection. As previously reported, with ROC analyses using the values for IFN-γ, the AUC values of TB1 and TB2 did not show any significant difference from that of the conventional QFT-3G, so it can be said that the diagnostic performance of QFT-plus

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was as accurate as that of QFT-3G (14). The cytokines determined in the present study to be significantly useful for diagnosis of active tuberculosis were mostly similar to the cytokines we identified in our previous study (4), except for IL-5 and IL-10, which were very low in the present study, probably due to the different assays used in the two studies. We previously showed that a combination of IL-2, IL-5, IL-10, IL-1RA and MCP-1 may be useful for distinguishing active tuberculosis from latent infection (4). In both of our studies, IL-2, IL-1RA and MCP-1 seemed to be useful for diagnosing active tuberculosis and discriminating between active tuberculosis and LTBI. Indeed, IL-2 released from mycobacterial antigen-stimulated blood cells is reported to be significantly higher in active tuberculosis (7, 17), and IL-1RA has been proposed as a plasma biomarker for tuberculosis (18-20). Similarly, the plasma levels of MCP-1 were reported to reflect the stages of tuberculosis infection (21), and genetic variants of MCP-1 have long been reported to be associated with susceptibility to tuberculosis (22). There are also studies from other groups—although the data are only for QFT-3G supernatants—showing the usefulness of similar cytokines, i.e., IL-2, IL-1RA, IP-10 and MIP-1β, for diagnosing infection with Mycobacterium tuberculosis (23, 24).

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Collectively, these inflammatory cytokines may play important roles in the pathogenesis of tuberculosis. Among the cytokines tested in the present study, IP-10 had been considered to be useful for diagnosing active tuberculosis and had also received close attention as a qualitative cytokine in the pathogenesis of tuberculosis (25-28). IP-10 is a chemokine produced by antigen-presenting cells in response to IFN-γ, and it induces migration of T cells to inflammatory sites. Interestingly, type I/II interferon signaling was elevated 18 months before tuberculosis was diagnosed in M. tuberculosis-infected individuals (29), suggesting the importance of IP-10 in the pathogenesis of tuberculosis. Recent reports further explained the role of IP-10 in inhibiting the growth of mycobacteria (30) and the important interplay between IP-10 and its natural inhibitor, dipeptidyl dipeptidase 4 enzyme (DPP4), at sites of tuberculosis (31). In addition, similar clinical results were reported by another group, showing IP-10 cut-offs of 1174 pg/ml for TB1 and 928.8 pg/ml for TB2 identified active TB with 86% sensitivity and 94% specificity, and they suggested IP-10 as an alternative biomarker to IFN-γ in QFT-plus (32). Our present findings support their hypothesis in an even larger population.

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On the other hand, the level of IP-10 is also useful as a biomarker for other diseases, especially viral infections (33-37). Thus, an antigen-specific response is indispensable for specific diagnosis of tuberculosis, and QFT makes that possible. Also, since the blood level of IP-10 was reported to be elevated in patients with tuberculosis and to decrease during anti-tuberculosis therapy (25, 27), it may be useful for evaluating the infectious status of tuberculosis. Moreover, IP-10 in the QFT-supernatant has been proposed as a useful marker for tuberculosis, especially in immunocompromised individuals, including HIV-infected patients (26, 28). Since IP-10 can be easily assayed even in dried plasma spots on filter paper (38), it may become an inexpensive and handy clinical examination target that would be useful for diagnosing tuberculosis. Our present study has several limitations. First of all, the backgrounds of the patients, including age and gender, were significantly different between the study groups due to the way the healthy subjects were recruited. So, in order to check the effect of age and gender differences on cytokine production in QFTs, we analyzed the relationships between age/gender and the levels of cytokines in both groups. We found no significant relationship between the age and cytokine levels (Supplemental Figure 2)

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as well as no difference between males’ and females’ cytokine levels except for the levels of IL-6 (Supplemental Figure 3). Thus, at least in our present study settings, we believe the differences in age and sex between the two groups had minimal effect on the results. Secondly, we did not fully determine the background status of immunosuppression and comorbidities among the patients. However, since it was reported that HIV infection did not reduce the sensitivity of QFT-plus for active TB detection (39), any inclusion of patients with immunosuppression may not have greatly influenced our findings. There is a need for more precise studies that evaluate the usefulness of cytokines in the supernatant of QFT-plus by comparison of active TB patients with relevant control subjects by stratification for the background factors. In summary, we found that production of each of IL-2, IL-6, IL-8, IP-10 and MIP-1β was higher in the 3G Ag tube compared to both the TB1 and TB2 tubes, while IL-6 and TNF-α were higher in TB1 compared to TB2, and PDGF-BB was higher in TB2 compared to TB1. For diagnosis of active tuberculosis, IP-10 showed larger AUCs than IFN-γ did. Finally, the levels of IFN-γ, IL-1RA, IL-2, IP-10, MCP-1 and MIP-1β in the QFT supernatant were higher for the active tuberculosis patients compared to the

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healthy controls. Our findings suggest the usefulness of assaying cytokines in the QFT supernatant for diagnosis of tuberculosis, but the roles of these cytokines in the pathogenesis of tuberculosis still need to be elucidated. Acknowledgments The authors thank Mses. Sayaka Igarashi and Miyako Seto for their skilled technical assistance. The authors also greatly appreciate Mses. Mariko Yoshizawa and Taeko Kawabe for their excellent secretarial work. This project was supported by AMED under Grant Number JP18fk0410016 h0001 to KT and JSPS KAKENHI Grant Number 18K08461 to HN. Conflicts of interest The authors have no conflicts to disclose.

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26

Tables Table 1. Clinical and demographic characteristics of the participants. Group

All

Active TB

Healthy

p-value

Control N

153

83

70

Male, N (%)

69 (45.1)

55 (66.3)

14 (20)

< 0.0001

Age (y) (IQR)

46.2 (29

58.1 (43 – 77)

32.1 (26

< 0.0001

– 62) Origin (%)

Japan

149

– 37) 79 (95.2)

70 (100)

(97.4)

Disease type (%)

Philippines

2 (1.3)

2 (2.5)

0 (0)

Nepal

1 (0.7)

1 (1.2)

0 (0)

Bhutan

1 (0.7)

1 (1.2)

0 (0)

Pulmonary TB

78 (94.0)

including pleuritis Pulmonary TB +

3 (3.6)

military TB Pulmonary TB +

1 (1.2)

urinary tract tuberculosis Pulmonary TB +

1 (1.2)

tuberculous meningitis Clinical

Diabetes mellitus

18 (21.7)

complications

HIV infection

1 (1.2)

related to

Corticosteroid use

1 (1.2, facial

immunity (%)

nerve paralysis)

QFT-3G positive, N (%)

70 (45.8)

70 (84.3)

0 (0)

T-SPOT positive, N (%)

76 (49.7)

76 (91.6)

0 (0)

27

N.A., not applicable; IQR, interquartile range.

Table 2. AUCs of ROCs drawn for cytokine levels in each antigen tube. Cytokine Tube

AUC

Std. Error

Cut-off

Sensitivity

Specificity

point

(%)

(%)

(95% CI)

(95% CI)

75.9

98.57

(0.848 -

(65.27 -

(92.3 -

0.953)

84.62)

99.96)

75.9

98.57

(0.856 -

(65.27 -

(92.3 -

0.957)

84.62)

99.96)

75.9

98.57

(0.882 -

(65.27 -

(92.3 -

0.971)

84.62)

99.96)

61.45

98.57

(0.734 -

(50.12 -

(92.3 -

0.877)

71.93)

99.96)

56.63

98.57

(0.779 -

(45.29 -

(92.3 -

0.907)

67.47)

99.96)

5.01

98.57

(0.761 -

(41.74 -

(92.3 -

0.893)

64.07)

99.96)

36.14

98.57

(0.622 -

(25.88 -

(92.3 -

0.788)

47.43)

99.96)

36.14

98.57

(0.645 -

(25.88 -

(92.3 -

0.805)

47.43)

99.96)

(95% CI) IFN-γ

TB1

TB2

0.901

0.907

3G Ag 0.926

IL-1Ra

TB1

TB2

0.806

0.843

3G Ag 0.827

IL-2

TB1

TB2

0.705

0.725

0.027

0.026

0.023

0.036

0.032

0.033

0.042

0.041

21

27

49.5

7644

8200

7906

59.5

56

28

3G Ag 0.765

IP-10

TB1

TB2

38.55

98.57

(0.690 -

(28.07 -

(92.3 -

0.840)

49.88)

99.96)

86.75

98.57

(0.878 -

(77.52 -

(92.3 -

0.976)

93.19)

99.96)

83.13

98.57

(0.922 -

(73.32 -

(92.3 -

0.988)

90.46)

99.96)

84.34

98.57

(0.913 -

(74.71 -

(92.3 -

0.99)

91.39)

99.96)

34.94

98.57

(24.8 - 46.19)

(92.3 -

0.927

0.955

3G Ag 0.952

MCP-1

TB1

0.787

0.038

0.025

0.017

0.020

0.037

74

465.5

891

639.5

8670

(0.714 0.860) TB2

0.829

40.96

98.57

(0.764 -

(30.28 -

(92.3 -

0.894)

52.31)

99.96)

37.35

98.57

(0.689 -

(26.97 -

(92.3 -

0.841)

48.66)

99.96)

30.12

98.57

(0.660 -

(20.53 -

(92.3 -

0.816)

41.18)

99.96)

31.33

98.57

(0.620 -

(21.59 -

(92.3 -

0.784)

42.44)

99.96)

27.71

98.57

(0.6 -

(18.45 -

(92.3 -

0.769)

38.62)

99.96)

3G Ag 0.765

MIP-1β

TB1

TB2

99.9)

0.738

0.702

3G Ag 0.684

0.033

0.039

0.040

0.042

0.043

9263

10237

2513

1592

3735

29

Figure Legends

Figure 1. Correlation between the results from MAGPIX and QFT assay. Plots of the levels of IFN-γ in the supernatants from three antigen tubes, i.e., TB1, TB2 and 3G Ag measured by the MAGPIX system and QFT assay. Linear regression analysis was performed, and the results are shown in the figure (mean and 95% CI).

30

Figure 2. Cytokine levels in three different antigen tubes in QFT for active tuberculosis patients. The levels of 13 cytokines in TB1, TB2 and 3G Ag tubes for active tuberculosis patients (n = 83). * P < 0.05, ** P < 0.01 and *** P < 0.001. Bars represent means, and error bars represent the SD.

31

Figure 3. ROC curves of three antigen tubes for tuberculosis diagnosis. ROC curves comparing the diagnostic accuracy of QFT-3G and QFT-plus for 13 cytokines in the supernatants of three antigen tubes for diagnosing active TB vs. healthy controls.

Figure 4. Cytokine levels in three different antigen tubes in QFT for active

32

tuberculosis patients and healthy controls. The levels of 13 cytokines in TB1 and TB2 tubes in QFT-plus and QFT-3G (3G Ag) for active tuberculosis patients (TB, n = 83) and healthy control subjects (HC, n = 70). * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001 between the active tuberculosis and healthy control subjects. Bars represent means, and error bars represent the SD.