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Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus
J Infect Chemother xxx (xxxx) xxx
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original Article
Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus Mizue Tsuyuzaki a, Hidetoshi Igari b, *, Nao Okada a, Kiminori Suzuki a a b
Chiba Foundation for Health Promotion and Disease Prevention, Chiba, Japan Division of Infection Control, Chiba University Hospital, Chiba, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 September 2019 Received in revised form 18 December 2019 Accepted 26 January 2020 Available online xxx
Recent contacts with active TB (tuberculosis) patients were screened for latent tuberculosis infection (LTBI) because of their greater relative risk for developing active TB. QuantiFERON®-TB Gold Plus (QFTPlus) offers two TB-specific antigen tubes (TB1 and TB2). TB1 elicits CD4 T-cell responses, and TB2 is designed to elicit both CD4 and CD8 T-cell responses. These mechanisms could be useful for estimating the role of CD8 T-cell immune responses to TB-specific antigens. To estimate the QFT-Plus capability to diagnose LTBI, a prospective cross-sectional study was conducted. A total of 412 TB contacts (median age 44 years) were enrolled. The positivity rates of QFT-Plus, TB1 and TB2 were 7.5%, 6.3% and 7.2%, respectively. TB2 showed a higher positivity rate compared to TB1, but without significant difference. The interferon (IFN)-g productions of TB1 and TB2 were well correlated (r ¼ 0.934, P < 0.001). The ratio of IFN-g production between TB1 and TB2 showed a median (interquartile range) of IFN-g[QFT-Plus TB2]/IFNg[QFT-Plus TB1] of 1.09 (0.91e1.36). CD8 T-cell immune response to TB-specific antigens varied among subjects. CD8 T-cell potentially boosts IFN-g productions in QFT-Plus and results in the detection of more persons with LTBI. However, there was no significant difference in the positivity rates of QFT-Plus TB1 and TB2 in our TB contact investigation. The contribution of CD8 T-cells might be small for the diagnosis of LTBI. The analysis of IFN-g production in both TB1 and TB2 would lead to further analysis of the TB immune response, and especially that caused by CD8 T-cells. © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: QuantiFERON TB-Plus CD4 T-cell CD8 T-cell Interferon-g Tuberculosis specific antigen
1. Introduction Persons coming in contact with active pulmonary tuberculosis (TB) patients are at increased risk of acquiring TB infection. The relative risk (RR) of developing active TB from latent TB infection (LTBI) was analyzed [1]. RR of LTBI persons in contact with active TB patients within two years was estimated to be 15-fold. Usually, they are recommended for screening for LTBI. If they are diagnosed with LTBI, they are recommended to receive treatment to prevent the development of active TB. LTBI is defined as a state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens without
* Corresponding author. Division of Infection Control, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba, 260-8677, Japan. E-mail addresses: [email protected] (M. Tsuyuzaki), igari_h@ chiba-u.jp (H. Igari), [email protected] (N. Okada), kimi.suzuki@ nifty.com (K. Suzuki).
evidence of clinically manifested active TB, according to World Health Organization Guidelines [2]. Interferon-g (IFN-g) release assays (IGRAs), which estimate cell-mediated immune responses to TB-specific antigens, are a diagnostic tool for LTBI. Actually, QuantiFERON®-TB Gold in-tube (QFT-GIT) (Cellestis/Qiagen, Carnegie, Australia) is one of the IGRAs that have been approved for LTBI diagnosis. QuantiFERON®-TB Gold Plus (QFT-Plus) is a newly introduced QFT, which uses two TB-specific antigen tubes, TB1 and TB2. TB1 is designed to elicit a CD4 T-cell response, like QFT-GIT. TB2 was newly designed to elicit immune responses driven by both CD4 and CD8 T-cells. This mechanism would be expected to increase the sensitivity for LTBI diagnosis compared with QFT-GIT. We also revealed a significantly increased positivity rate with QFT-Plus compared with QFT-GIT in the TB contact investigation [3]. We had a concern regarding the function of CD8 T-cells associated with immune responses to TB-specific antigens. We therefore conducted a secondary analysis of a former study [3]. We hypothesized that a comparison of the IFN-g productions with TB1 and
https://doi.org/10.1016/j.jiac.2020.01.011 1341-321X/© 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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M. Tsuyuzaki et al. / J Infect Chemother xxx (xxxx) xxx
TB2 in QFT-Plus would explain the differences in immune responses elicited by CD4 and CD8 T-cells. Especially the role of CD8 T-cells could be estimated in the immune response of TB-contact persons.
responding to TB-specific antigens were logarithmically transformed. The correlations of IFN-gproduction were analyzed by linear regressions. A P-value <0.05 was considered statistically significant.
2. Material and methods
3. Results
2.1. Study design and settings
412 subjects (male 220, female 192) were enrolled. Median age was 44 years (range 9e76). None was diagnosed with active pulmonary TB by chest X-ray (Table 1). The number of index cases was 140, and their details are shown in Table 1. Sputum smear-positive cases were 77.1%, and chest X-ray cavity formations were 47.9%. The positivity rates of QFT-Plus, QFT-Plus TB1, and QFT-Plus TB2 were 7.5% (95% confidence interval (CI), 5.4e10.5), 6.3% (95% CI, 4.3e9.1), and 7.2% (95% CI, 5.1e10.2), respectively (Table 2). There were no indeterminate cases. There was no significant difference in positivity rate between TB1 and TB2 (McNemar's test). Comparing TB1 and TB2 results, 406 (98.5%) were concordant and the k coefficient (kappa coefficient) was 0.885 (Table 3). The distributions of IFN-g productions responding to TB-specific antigens were shown in Fig. 1. The histograms of TB1 and TB2 in QFT-Plus were quite similar, showing only minor differences (Fig. 1). After analyzing the differences between IFN-g[QFT-Plus TB1] and IFN-g[QFT-Plus TB2] (log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])), we drew the histogram (Fig. 2). Normal distribution was ruled out by Shapiro-Wilk test, and we calculated the median (interquartile range: IQR) of 0.038 (0.041e0.133) (Table 4). Then we reversely converted the value of (log10(IFN-g[QFT-Plus TB2]) log10(IFN-g[QFT-Plus TB1])). The median (IQR) of IFN-g[QFT-Plus TB2]/IFNg[QFT-Plus TB1] was equivalent to 1.09 (0.91e1.36) (Table 4). Although the IFN-g production varied among subjects, TB2 produced 1.09 times IFN-g in comparison with TB1. The relation of IFN-g productions in both TB1 and TB2 was analyzed by scatter plot (Fig. 3). The IFN-g productions in TB1 and TB2 were well correlated. (r ¼ 0.934, P < 0.001) However, variations of IFN-g productions were mainly observed in a range lower than 0.46, which was equivalent to 0.35IU/mL after reverse transformation from the logarithmic value. Actually, IFN-g production is judged after subtracting the negative control value. Then, this dashed line in the figure represents the approximate guideline for QFT-Plus diagnosis.
We have already published an analysis of the difference between QFT-GIT and QFT-Plus. The current work was an additional study for analyzing differences between TB1 and TB2 in QFT-Plus [3]. Thus, this study is a secondary analysis of our earlier study. The subjects were persons listed for TB contact investigations. We examined the positivity rates by QFT-GIT and QFT-Plus. We also analyzed IFN-g production in response to TB-specific antigens, and its effects on the QFT diagnosis. A cross-sectional study was conducted at the Chiba Foundation of Health Promotion and Disease Prevention (CFHPDP) in Japan. CFHPDP is a non-political organization founded to support public health programs. TB contact investigation is one of these programs, and it is commissioned by the Chiba-city local government. A total of 412 subjects were prospectively enrolled between July 2015 and March 2018. The Japanese Infection Law regulates that physicians who diagnose TB patients are bound to immediately report to regional health centers, public health offices operated by local governments. Our subjects had experienced close contacts with active TB patients and were judged to be at higher risk of acquiring TB infection, and therefore were recommended for contact investigations. The usual contact investigation consists of chest X-ray and IGRAs. We conducted QFT-Plus, in addition to QFT-GIT. QFT-Plus had not been approved at the time of the study. We purchased QFTPlus from QIAGEN (Hilden, Germany) by legal import procedure and ethical approval. Peripheral venous blood was collected for QFT-Plus, in addition to QFT-GIT, at the clinic of CFHPDP. Blood samples for QFT-Plus and QFT-GIT were immediately processed at the CFHPDP laboratory, and assessed according to the manufacturers’ instructions. Sampling tubes for QFT-Plus consisted of four tubes (TB1, TB2, negative control, positive control). The TB1 tube contains TB-specific antigen eliciting CD4 T-cells, and the TB2 tube contains TB-specific antigens eliciting both CD4 T-cells and CD8 T-cells. QFT-Plus was interpreted according to the supplier's instructions. The IFN-g values for TB1 and TB2 were calculated by subtracting IFN-g in negative control from IFN-g in the TB antigens tubes (TB1 and TB2), with a value of 0.35 IU/ml being considered a positive result in both TB1 and TB2. If the IFN-g value was <0.35 IU/ ml and mitogen control was 0.5 IU/ml, the test was interpreted as being negative. If the IFN-g value was <0.35 IU/ml and mitogen control was <0.5 IU/ml, the result was considered as being indeterminate. If the negative control was >8.0 IU/ml, the result was considered as being indeterminate. TB contacts volunteered to participate in the study and provided written consent following written and oral descriptions of the study. This study was approved by the Institutional Review Board of CFHPDP, and was conducted in accordance with the principles of the Declaration of Helsinki. The study was funded by CFHPDP. 2.2. Statistical analysis JMP® Pro 13.0.0 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses. McNemar's test was used to compare paired proportions in IGRAs. Agreement between TB1 and TB2 was quantified using k coefficients. The values of IFN-g productions
4. Discussion QFT-Plus revealed a higher positivity rate in TB2 than in TB1, although not significant in the present study. TB1 and TB2 in QFTPlus revealed excellent concordancy in this study. Our main concern was regarding the role of CD8 T-cells in immune responses
Table 1 Characteristics of subjects and index case information.
Information associated with contact persons Total number Age (yr); median (range) Male Previous TB treatment history Days after final contact; median (IQR) Information associated with index cases Number of index cases Chest X-ray cavity Sputum smear examination: acid-fast bacilli Negative 1þ 2þ 3þ
n
(%)
412 44 220 4 99
(976) (53.4) (1.0) (84e138)
140 67
(47.9)
32 43 39 26
(22.9) (30.6) (27.9) (18.6)
TB: tuberculosis, IQR: interquartile range.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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Table 2 Results of QFT-Plus.
QFT-Plus TB1 TB2
Positive
(%) [95%CI]
Negative
(%)
31 26 30
(7.5) [5.4e10.5] (6.3) [4.3e9.1] (7.2) [5.1e10.2]
381 386 382
(92.5) (93.7) (92.8)
No subject was judged as indeterminate. There was no significant difference in the percentage of positive between TB1 and TB2. QFT-Plus: QuantiFERON-TB Gold Plus, CI: confidence interval, ns: not significant.
Table 3 Results of TB1 and TB2 in QFT-Plus. TB2
TB1 Total
Positive Negative
Total
Positive
Negative
25 5 30
1 381 382
26 386 412
The k coefficient was 0.885, and the concordant rate of TB1 and TB2 was high (n ¼ 406, 98.5%). QFT-Plus: QuantiFERON-TB Gold Plus.
to TB-specific antigens. Both TB1 and TB2 induce a CD4 T-cell response, although the CD8 T-cell response is mainly due to TB2 stimulation, which is largely associated with active TB [4]. The present study was not able to demonstrate the distinct CD8 T-cell response, as would be estimated in TB2. Then, we further analyzed the IFN-g productions in TB1 and TB2. The difference of IFN-g productions in TB1 and TB2 would be a marker for estimating both CD4 T-cell and CD8 T-cell immune responses. The median (IQR) of IFN-g[QFT-Plus TB2]/IFN-g[QFT-PlusTB1] was equivalent to 1.09 (0.91e1.36) (Table 4). The histogram in Fig. 2 shows that more than half of the subjects produced equal or more IFN-g in TB2. However, some subjects revealed an opposite result, producing lower amounts of IFN-g compared with TB1. We also analyzed IFN-g productions as showed in the scatter graph of Fig. 3. The IFN-g productions in TB1 and TB2 were closely correlated. We speculated that there would be personal variations in the CD8 T-cell function in response to TB-specific antigens. A study has reported that CD8 Tcell response was observed in LTBI as well as in active TB [5]. Even in consideration of such evidence, the role of CD8 T-cells is restricted in contact investigations without active TB cases, such as in the present study. In general, our subjects demonstrated immune response mainly caused by CD4 T-cells.
Fig. 2. Differences of interferon-g productions responding to TB-specific antigens, Histograms were drawn after calculating the differences between IFN-g[QFT-Plus TB1] and IFN-g[QFT-Plus TB2] (log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])). Then we reversely converted the value of (log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])). The median (IQR) of IFN-g[QFT-Plus TB2]/IFN-g[QFT-Plus TB1] was equivalent to 1.09 (0.91e1.36). Although IFN-g production varied among subjects, TB2 produced 1.09 times IFN-g in comparison to TB1. QFT-Plus: QuantiFERON-TB Gold Plus, QFT-GIT: QuantiFERON-TB Gold In-Tube. IFN: interferon, IQR: inter quartile range.
Table 4 Ratio of interferon-g production between TB-specific antigen tubes.
log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus IFN-g[QFT-Plus TB2]/IFN-g[QFT-Plus TB1]
TB1])
median
(IQR)
0.038 1.09a
(0.041e0.133) (0.91e1.36)a
TB: tuberculosis, IQR: interquartile range, IFN: interferon, QFT-Plus: QuantiFERONTB Gold Plus. a These values were transformed reversely from the values of log10 (IFN-g).
QFT-Plus is a LTBI diagnostic tool with more sensitivity in comparison with QFT-GIT [6e11]. Our previous study also partially supported these results [3]. Horne et at conducted a study comparing QFT-Plus with QFT-GIT in active TB patients [12]. Higher IFN-g production in TB2 compared to TB1 was the major reason. This effect might increase the QFT-Plus positivity rate and boost the QFT-Plus sensitivity. In spite of this, they concluded that QFT-Plus had similar sensitivity to QFT-GIT in adult patients with active TB. This variation of IFN-g productions would boost the positivity rate in TB2. Some subjects revealed conversion to positive even when
Fig. 1. Distribution of interferon-g production responding to TB-specific antigen tubes of TB1 and TB2 in QFT-Plus, The distributions of IFN-g productions are shown. IFN-g productions responding to TB-specific antigens were logarithmically transformed. The histograms of TB1 and TB2 in QFT-Plus were quite similar, showing only minor differences. QFTPlus: QuantiFERON-TB Gold Plus, IFN: interferon, TB: tuberculosis.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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M. Tsuyuzaki et al. / J Infect Chemother xxx (xxxx) xxx
Fig. 3. Relation of interferon-g production between TB1 and TB2 in QFT-Plus. The IFNg productions of TB1 and TB2 were also strongly correlated (P < 0.01). Variation of IFNg productions was mainly observed at a range lower than 0.46, which was equivalent to 0.35IU/mL after reverse transformation from the logarithmic value. Black circles and white circles indicate subjects finally diagnosed as QFT-Plus positive and negative, respectively. Longitudinal line and horizontal line are 0.46. The value of 0.46 is equivalent to 0.35IU/mL after reverse transformation from the logarithmic value. QFTPlus: QuantiFERON-TB Gold Plus, IFN: interferon.
they were negative in TB1. However, IFN-g productions in TB1 and TB2 were closely correlated, thus these conversions were likely observed around cut-off. Then, the effect on the increase of sensitivity was small even in the introduction of TB2 for the estimation of CD8 T-cell response. Variability of the IFN-g value in QFT-GIT has been observed in a range between 0.25 and 0.59 IU/mL [13]. Conversion and reversion were frequently observed in serial tests in QFT-GIT. Others have claimed a wider variability in a range between 0.2 and 0.7IU/mL [14,15]. Variability has already been observed in QFT-Plus [15]. In our study, the number of subjects with reliable positive diagnosis with an IFN-g value of 0.6 IU/mL was 25. Six other subjects were diagnosed as positive with an IFN-g value of <0.6 IU/mL, and their positive might convert to negative in repeated tests. We need further discussion regarding the judgement of the variability zone even if a higher positivity rate in QFT-Plus would be driven by TB2. The LTBI diagnosis procedure is proposed to be based on not only interpretation of the cut-off value but also on epidemiological and clinical assessment [16]. Therefore, we need to be cautious when interpreting QFT-Plus results with an IFN-g value of <0.6 IU/mL. The difference between TB1 and TB2 might be positively correlated with the antigenic load in active TB patients [17]. Barcellini et al. speculated that a TB2TB1 difference of >0.6 IU/mL meant a higher antigenic burden in LTBI, leading to the prediction of developing active TB [18]. Ten subjects met this criterion in the present study, but 6 of the 10 were located in the IQR of (log10(IFNg[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])). TB2TB1 difference of >0.6IU/mL might be a possible variation of IFN-g production responding to TB-specific antigens after logarithmic transformation. Thus, we are skeptical about the meaning of a TB2TB1 difference of >0.6 IU/mL as a predictive marker for developing active TB from LTBI. QFT-GIT, the former generation QFT, is an excellent diagnostic tool with higher sensitivity, and a further
increase of sensitivity in QFT-Plus would be limited, especially in Japan [9]. The immune responses to TB-specific antigens would change in the phases of TB infection, active TB infection, and LTBI [4,17]. IFN-g production has been mainly used for the judgement of QFT both in QFT-GIT and QFT-Plus. QFT-Plus provides additional opportunity for analyzing the immune response caused by CD8 Tcells. The analysis of IFN-g production in both TB1 and TB2 would lead to further analysis of the TB immune response, and especially that caused by CD8 T-cells. We estimated the IFN-g production after logarithmic transformation, as was adopted in our previous study [3]. This method has not yet been in general usage. We can take note of the distribution of IFN-g production at a glance in Fig. 1. The difference of logarithmically transformed IFN-g production between TB2 and TB1 was also estimated as the ratio of IFN-g production between TB2 and TB1. In this process, we deduced the function of CD8 T-cells in response to TB-specific antigens in the present study. The introduction of logarithmic transformation of IFN-g production is our trial, and we expect it to be re-estimated or criticized by others. There are some limitations to this study. It was conducted in a single facility. The number of subjects in QFT-Plus positive was also limited. The factors associated with QFT-Plus results, including TB1 and TB2, were not analyzed sufficiently owing to the limited information provided by regional health centers operated by the local government. CD8 T-cell immune responses were analyzed based on the condition of the QFT-plus kit. The modification of dose and combination of TB specific antigens would elicit other immune responses in TB contact screening. 5. Conclusions IFN-g production in TB1 and TB2 were correlated well, and IFN-g production in TB2 was 1.09 times that in TB1 and varied among subjects. Then, CD8 T-cells potentially boost IFN-g production in QFT-Plus. However, there was no significant difference in the positivity rate in QFT-Plus TB1 and TB2 according to the TB contact investigation. The contribution of CD8 T-cells might be small for the diagnosis of LTBI in the present contact investigation. The analysis of IFN-g production in both TB1 and TB2 would lead to further analysis of the TB immune response, and especially that caused by CD8 T-cells. Financial support This work was supported by the Chiba Foundation of Health Promotion and Disease Prevention. Authorship statement All authors meet the ICMJE authorship criteria. All authors have seen and approved the manuscript, and contributed significantly to the work. Authors’ contributions Mizue Tsuyuzaki: Conducted examinations, data analysis. Hidetoshi Igari: Study planning, data analysis. Nao Okada: Conducted examinations, data analysis Kiminori Suzuki Study planning, data analysis. Declaration of Competing Interest All authors: No potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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Acknowledgement I would like to express my gratitude to J. Yamaguchi of the regional health center operated by the Chiba-city government for his support. References [1] Landry J, Menzies D. Preventive chemotherapy. Where has it got us? Where to go next? Int J Tubercul Lung Dis 2008;12:1352e64. [2] World Health Organization. Latent TB Infection: Updated and consolidated guidelines for programmatic management. http://www.who.int/tb/ publications/2018/latent-tuberculosis-infection/en/. [Accessed 3 July 2018]. [3] Tsuyuzaki M, Igari H, Okada N, Suzuki K. Variation of interferon-g production between QFT-Plus and QFT-GIT in contact investigation. Respir Investig 2019;57:561e5. [4] Petruccioli E, Chiacchio T, Pepponi I, Vanini V, Urso R, Cuzzi G, et al. First characterization of the CD4 and CD8 T-cell responses to QuantiFERON-TB plus. J Infect 2016;73:588e97. [5] Lee MR, Chang CH, Chang LY, Chuang YC, Sun HY, Wang JT, et al. CD8 response measured by QuantiFERON-TB Gold Plus and tuberculosis disease status. J Infect 2019;78:299e304. [6] Sotgiu G, Saderi L, Petruccioli E, Aliberti S, Piana A, Petrone L, et al. QuantiFERON TB Gold Plus for the diagnosis of tuberculosis: a systematic review and meta-analysis. Infect 2019;79:444e53. [7] Pourakbari B, Mamishi S, Benvari S, Mahmoudi S. Comparison of the QuantiFERON-TB Gold Plus and QuantiFERON-TB Gold In-Tube interferon-g release assays: a systematic review and meta-analysis. Adv Med Sci 2019;64: 437e43. [8] Ryu MR, Park MS, Cho EH, Jung CW, Kim K, Kim SJ, et al. Comparative evaluation of QuantiFERON-TB gold in-tube and QuantiFERON-TB gold plus in diagnosis of latent tuberculosis infection in immunocompromised patients. J Clin Microbiol 2018;56. e00438-18.
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[9] Takasaki J, Manabe T, Morino E, Muto Y, Hashimoto M, Iikura M, et al. Sensitivity and specificity of QuantiFERON-TB gold plus compared with QuantiFERON-TB gold in-tube and T-SPOT.TB on active tuberculosis in Japan. J Infect Chemother 2018;24:188e92. [10] Chien JY, Chiang HT, Lu MC, Ko WC, Yu CJ, Chen YH, et al. QuantiFERON-TB gold plus is a more sensitive screening tool than QuantiFERON-TB gold intube for latent tuberculosis infection among older adults in long-term care facilities. J Clin Microbiol 2018;56. e00427-18. [11] Theel ES, Hilgart H, Breen-Lyles M, McCoy K, Flury R, Breeher LE, et al. Comparison of the QuantiFERON-TB gold plus and QuantiFERON-TB gold intube interferon gamma release assays in patients at risk for tuberculosis and in health care workers. J Clin Microbiol 2018;56:e00614e8. [12] Horne DJ, Jones BE, Kamada A, Fukushima K, Winthrop KL, Siegel SAR, et al. Multicenter study of QuantiFERON®-TB Gold Plus in patients with active tuberculosis. Int J Tubercul Lung Dis 2018;22:617e21. [13] Metcalfe JZ, Cattamanchi A, McCulloch CE, Lew JD, Ha NP, Graviss EA. Test variability of the QuantiFERON-TB gold in-tube assay in clinical practice. Am J Respir Crit Care Med 2013;187:206e11. [14] Nienhaus A, Ringshausen FC, Costa JT, Schablon A, Tripodi D. IFN-g release assay versus tuberculin skin test for monitoring TB infection in healthcare workers. Expert Rev Anti Infect Ther 2013;11:37e48. [15] Knierer J, Gallegos Morales EN, Schablon A, Nienhaus A, Kersten JF. QFT-Plus: a plus in variability? - evaluation of new generation IGRA in serial testing of students with a migration background in Germany. J Occup Med Toxicol 2017;12:1. [16] Fong KS, Tomford JW, Teixeira L, Fraser TG, van Duin D, Yen-Lieberman, et al. Challenges of interferon-g release assay conversions in serial testing of health-care workers in a TB control program. Chest 2012;142:55e62. [17] Barcellini L, Borroni E, Brown J, Brunetti E, Codecasa L, Cugnata F, et al. First independent evaluation of QuantiFERON-TB Plus performance. Eur Respir J 2016;47:1587e90. [18] Barcellini L, Borroni E, Brown J, Brunetti E, Codecasa L, Cugnata F, et al. First evaluation of QuantiFERON-TB Gold Plus performance in contact screening. Eur Respir J 2016;48:1411e9.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
Contents lists available at ScienceDirect
Journal of Infection and Chemotherapy journal homepage: http://www.elsevier.com/locate/jic
Original Article
Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus Mizue Tsuyuzaki a, Hidetoshi Igari b, *, Nao Okada a, Kiminori Suzuki a a b
Chiba Foundation for Health Promotion and Disease Prevention, Chiba, Japan Division of Infection Control, Chiba University Hospital, Chiba, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 September 2019 Received in revised form 18 December 2019 Accepted 26 January 2020 Available online xxx
Recent contacts with active TB (tuberculosis) patients were screened for latent tuberculosis infection (LTBI) because of their greater relative risk for developing active TB. QuantiFERON®-TB Gold Plus (QFTPlus) offers two TB-specific antigen tubes (TB1 and TB2). TB1 elicits CD4 T-cell responses, and TB2 is designed to elicit both CD4 and CD8 T-cell responses. These mechanisms could be useful for estimating the role of CD8 T-cell immune responses to TB-specific antigens. To estimate the QFT-Plus capability to diagnose LTBI, a prospective cross-sectional study was conducted. A total of 412 TB contacts (median age 44 years) were enrolled. The positivity rates of QFT-Plus, TB1 and TB2 were 7.5%, 6.3% and 7.2%, respectively. TB2 showed a higher positivity rate compared to TB1, but without significant difference. The interferon (IFN)-g productions of TB1 and TB2 were well correlated (r ¼ 0.934, P < 0.001). The ratio of IFN-g production between TB1 and TB2 showed a median (interquartile range) of IFN-g[QFT-Plus TB2]/IFNg[QFT-Plus TB1] of 1.09 (0.91e1.36). CD8 T-cell immune response to TB-specific antigens varied among subjects. CD8 T-cell potentially boosts IFN-g productions in QFT-Plus and results in the detection of more persons with LTBI. However, there was no significant difference in the positivity rates of QFT-Plus TB1 and TB2 in our TB contact investigation. The contribution of CD8 T-cells might be small for the diagnosis of LTBI. The analysis of IFN-g production in both TB1 and TB2 would lead to further analysis of the TB immune response, and especially that caused by CD8 T-cells. © 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Keywords: QuantiFERON TB-Plus CD4 T-cell CD8 T-cell Interferon-g Tuberculosis specific antigen
1. Introduction Persons coming in contact with active pulmonary tuberculosis (TB) patients are at increased risk of acquiring TB infection. The relative risk (RR) of developing active TB from latent TB infection (LTBI) was analyzed [1]. RR of LTBI persons in contact with active TB patients within two years was estimated to be 15-fold. Usually, they are recommended for screening for LTBI. If they are diagnosed with LTBI, they are recommended to receive treatment to prevent the development of active TB. LTBI is defined as a state of persistent immune response to stimulation by Mycobacterium tuberculosis antigens without
* Corresponding author. Division of Infection Control, Chiba University Hospital, 1-8-1 Inohana, Chuo-Ku, Chiba, 260-8677, Japan. E-mail addresses: [email protected] (M. Tsuyuzaki), igari_h@ chiba-u.jp (H. Igari), [email protected] (N. Okada), kimi.suzuki@ nifty.com (K. Suzuki).
evidence of clinically manifested active TB, according to World Health Organization Guidelines [2]. Interferon-g (IFN-g) release assays (IGRAs), which estimate cell-mediated immune responses to TB-specific antigens, are a diagnostic tool for LTBI. Actually, QuantiFERON®-TB Gold in-tube (QFT-GIT) (Cellestis/Qiagen, Carnegie, Australia) is one of the IGRAs that have been approved for LTBI diagnosis. QuantiFERON®-TB Gold Plus (QFT-Plus) is a newly introduced QFT, which uses two TB-specific antigen tubes, TB1 and TB2. TB1 is designed to elicit a CD4 T-cell response, like QFT-GIT. TB2 was newly designed to elicit immune responses driven by both CD4 and CD8 T-cells. This mechanism would be expected to increase the sensitivity for LTBI diagnosis compared with QFT-GIT. We also revealed a significantly increased positivity rate with QFT-Plus compared with QFT-GIT in the TB contact investigation [3]. We had a concern regarding the function of CD8 T-cells associated with immune responses to TB-specific antigens. We therefore conducted a secondary analysis of a former study [3]. We hypothesized that a comparison of the IFN-g productions with TB1 and
https://doi.org/10.1016/j.jiac.2020.01.011 1341-321X/© 2020 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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TB2 in QFT-Plus would explain the differences in immune responses elicited by CD4 and CD8 T-cells. Especially the role of CD8 T-cells could be estimated in the immune response of TB-contact persons.
responding to TB-specific antigens were logarithmically transformed. The correlations of IFN-gproduction were analyzed by linear regressions. A P-value <0.05 was considered statistically significant.
2. Material and methods
3. Results
2.1. Study design and settings
412 subjects (male 220, female 192) were enrolled. Median age was 44 years (range 9e76). None was diagnosed with active pulmonary TB by chest X-ray (Table 1). The number of index cases was 140, and their details are shown in Table 1. Sputum smear-positive cases were 77.1%, and chest X-ray cavity formations were 47.9%. The positivity rates of QFT-Plus, QFT-Plus TB1, and QFT-Plus TB2 were 7.5% (95% confidence interval (CI), 5.4e10.5), 6.3% (95% CI, 4.3e9.1), and 7.2% (95% CI, 5.1e10.2), respectively (Table 2). There were no indeterminate cases. There was no significant difference in positivity rate between TB1 and TB2 (McNemar's test). Comparing TB1 and TB2 results, 406 (98.5%) were concordant and the k coefficient (kappa coefficient) was 0.885 (Table 3). The distributions of IFN-g productions responding to TB-specific antigens were shown in Fig. 1. The histograms of TB1 and TB2 in QFT-Plus were quite similar, showing only minor differences (Fig. 1). After analyzing the differences between IFN-g[QFT-Plus TB1] and IFN-g[QFT-Plus TB2] (log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])), we drew the histogram (Fig. 2). Normal distribution was ruled out by Shapiro-Wilk test, and we calculated the median (interquartile range: IQR) of 0.038 (0.041e0.133) (Table 4). Then we reversely converted the value of (log10(IFN-g[QFT-Plus TB2]) log10(IFN-g[QFT-Plus TB1])). The median (IQR) of IFN-g[QFT-Plus TB2]/IFNg[QFT-Plus TB1] was equivalent to 1.09 (0.91e1.36) (Table 4). Although the IFN-g production varied among subjects, TB2 produced 1.09 times IFN-g in comparison with TB1. The relation of IFN-g productions in both TB1 and TB2 was analyzed by scatter plot (Fig. 3). The IFN-g productions in TB1 and TB2 were well correlated. (r ¼ 0.934, P < 0.001) However, variations of IFN-g productions were mainly observed in a range lower than 0.46, which was equivalent to 0.35IU/mL after reverse transformation from the logarithmic value. Actually, IFN-g production is judged after subtracting the negative control value. Then, this dashed line in the figure represents the approximate guideline for QFT-Plus diagnosis.
We have already published an analysis of the difference between QFT-GIT and QFT-Plus. The current work was an additional study for analyzing differences between TB1 and TB2 in QFT-Plus [3]. Thus, this study is a secondary analysis of our earlier study. The subjects were persons listed for TB contact investigations. We examined the positivity rates by QFT-GIT and QFT-Plus. We also analyzed IFN-g production in response to TB-specific antigens, and its effects on the QFT diagnosis. A cross-sectional study was conducted at the Chiba Foundation of Health Promotion and Disease Prevention (CFHPDP) in Japan. CFHPDP is a non-political organization founded to support public health programs. TB contact investigation is one of these programs, and it is commissioned by the Chiba-city local government. A total of 412 subjects were prospectively enrolled between July 2015 and March 2018. The Japanese Infection Law regulates that physicians who diagnose TB patients are bound to immediately report to regional health centers, public health offices operated by local governments. Our subjects had experienced close contacts with active TB patients and were judged to be at higher risk of acquiring TB infection, and therefore were recommended for contact investigations. The usual contact investigation consists of chest X-ray and IGRAs. We conducted QFT-Plus, in addition to QFT-GIT. QFT-Plus had not been approved at the time of the study. We purchased QFTPlus from QIAGEN (Hilden, Germany) by legal import procedure and ethical approval. Peripheral venous blood was collected for QFT-Plus, in addition to QFT-GIT, at the clinic of CFHPDP. Blood samples for QFT-Plus and QFT-GIT were immediately processed at the CFHPDP laboratory, and assessed according to the manufacturers’ instructions. Sampling tubes for QFT-Plus consisted of four tubes (TB1, TB2, negative control, positive control). The TB1 tube contains TB-specific antigen eliciting CD4 T-cells, and the TB2 tube contains TB-specific antigens eliciting both CD4 T-cells and CD8 T-cells. QFT-Plus was interpreted according to the supplier's instructions. The IFN-g values for TB1 and TB2 were calculated by subtracting IFN-g in negative control from IFN-g in the TB antigens tubes (TB1 and TB2), with a value of 0.35 IU/ml being considered a positive result in both TB1 and TB2. If the IFN-g value was <0.35 IU/ ml and mitogen control was 0.5 IU/ml, the test was interpreted as being negative. If the IFN-g value was <0.35 IU/ml and mitogen control was <0.5 IU/ml, the result was considered as being indeterminate. If the negative control was >8.0 IU/ml, the result was considered as being indeterminate. TB contacts volunteered to participate in the study and provided written consent following written and oral descriptions of the study. This study was approved by the Institutional Review Board of CFHPDP, and was conducted in accordance with the principles of the Declaration of Helsinki. The study was funded by CFHPDP. 2.2. Statistical analysis JMP® Pro 13.0.0 (SAS Institute Inc., Cary, NC, USA) was used for statistical analyses. McNemar's test was used to compare paired proportions in IGRAs. Agreement between TB1 and TB2 was quantified using k coefficients. The values of IFN-g productions
4. Discussion QFT-Plus revealed a higher positivity rate in TB2 than in TB1, although not significant in the present study. TB1 and TB2 in QFTPlus revealed excellent concordancy in this study. Our main concern was regarding the role of CD8 T-cells in immune responses
Table 1 Characteristics of subjects and index case information.
Information associated with contact persons Total number Age (yr); median (range) Male Previous TB treatment history Days after final contact; median (IQR) Information associated with index cases Number of index cases Chest X-ray cavity Sputum smear examination: acid-fast bacilli Negative 1þ 2þ 3þ
n
(%)
412 44 220 4 99
(976) (53.4) (1.0) (84e138)
140 67
(47.9)
32 43 39 26
(22.9) (30.6) (27.9) (18.6)
TB: tuberculosis, IQR: interquartile range.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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Table 2 Results of QFT-Plus.
QFT-Plus TB1 TB2
Positive
(%) [95%CI]
Negative
(%)
31 26 30
(7.5) [5.4e10.5] (6.3) [4.3e9.1] (7.2) [5.1e10.2]
381 386 382
(92.5) (93.7) (92.8)
No subject was judged as indeterminate. There was no significant difference in the percentage of positive between TB1 and TB2. QFT-Plus: QuantiFERON-TB Gold Plus, CI: confidence interval, ns: not significant.
Table 3 Results of TB1 and TB2 in QFT-Plus. TB2
TB1 Total
Positive Negative
Total
Positive
Negative
25 5 30
1 381 382
26 386 412
The k coefficient was 0.885, and the concordant rate of TB1 and TB2 was high (n ¼ 406, 98.5%). QFT-Plus: QuantiFERON-TB Gold Plus.
to TB-specific antigens. Both TB1 and TB2 induce a CD4 T-cell response, although the CD8 T-cell response is mainly due to TB2 stimulation, which is largely associated with active TB [4]. The present study was not able to demonstrate the distinct CD8 T-cell response, as would be estimated in TB2. Then, we further analyzed the IFN-g productions in TB1 and TB2. The difference of IFN-g productions in TB1 and TB2 would be a marker for estimating both CD4 T-cell and CD8 T-cell immune responses. The median (IQR) of IFN-g[QFT-Plus TB2]/IFN-g[QFT-PlusTB1] was equivalent to 1.09 (0.91e1.36) (Table 4). The histogram in Fig. 2 shows that more than half of the subjects produced equal or more IFN-g in TB2. However, some subjects revealed an opposite result, producing lower amounts of IFN-g compared with TB1. We also analyzed IFN-g productions as showed in the scatter graph of Fig. 3. The IFN-g productions in TB1 and TB2 were closely correlated. We speculated that there would be personal variations in the CD8 T-cell function in response to TB-specific antigens. A study has reported that CD8 Tcell response was observed in LTBI as well as in active TB [5]. Even in consideration of such evidence, the role of CD8 T-cells is restricted in contact investigations without active TB cases, such as in the present study. In general, our subjects demonstrated immune response mainly caused by CD4 T-cells.
Fig. 2. Differences of interferon-g productions responding to TB-specific antigens, Histograms were drawn after calculating the differences between IFN-g[QFT-Plus TB1] and IFN-g[QFT-Plus TB2] (log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])). Then we reversely converted the value of (log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])). The median (IQR) of IFN-g[QFT-Plus TB2]/IFN-g[QFT-Plus TB1] was equivalent to 1.09 (0.91e1.36). Although IFN-g production varied among subjects, TB2 produced 1.09 times IFN-g in comparison to TB1. QFT-Plus: QuantiFERON-TB Gold Plus, QFT-GIT: QuantiFERON-TB Gold In-Tube. IFN: interferon, IQR: inter quartile range.
Table 4 Ratio of interferon-g production between TB-specific antigen tubes.
log10(IFN-g[QFT-Plus TB2])log10(IFN-g[QFT-Plus IFN-g[QFT-Plus TB2]/IFN-g[QFT-Plus TB1]
TB1])
median
(IQR)
0.038 1.09a
(0.041e0.133) (0.91e1.36)a
TB: tuberculosis, IQR: interquartile range, IFN: interferon, QFT-Plus: QuantiFERONTB Gold Plus. a These values were transformed reversely from the values of log10 (IFN-g).
QFT-Plus is a LTBI diagnostic tool with more sensitivity in comparison with QFT-GIT [6e11]. Our previous study also partially supported these results [3]. Horne et at conducted a study comparing QFT-Plus with QFT-GIT in active TB patients [12]. Higher IFN-g production in TB2 compared to TB1 was the major reason. This effect might increase the QFT-Plus positivity rate and boost the QFT-Plus sensitivity. In spite of this, they concluded that QFT-Plus had similar sensitivity to QFT-GIT in adult patients with active TB. This variation of IFN-g productions would boost the positivity rate in TB2. Some subjects revealed conversion to positive even when
Fig. 1. Distribution of interferon-g production responding to TB-specific antigen tubes of TB1 and TB2 in QFT-Plus, The distributions of IFN-g productions are shown. IFN-g productions responding to TB-specific antigens were logarithmically transformed. The histograms of TB1 and TB2 in QFT-Plus were quite similar, showing only minor differences. QFTPlus: QuantiFERON-TB Gold Plus, IFN: interferon, TB: tuberculosis.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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Fig. 3. Relation of interferon-g production between TB1 and TB2 in QFT-Plus. The IFNg productions of TB1 and TB2 were also strongly correlated (P < 0.01). Variation of IFNg productions was mainly observed at a range lower than 0.46, which was equivalent to 0.35IU/mL after reverse transformation from the logarithmic value. Black circles and white circles indicate subjects finally diagnosed as QFT-Plus positive and negative, respectively. Longitudinal line and horizontal line are 0.46. The value of 0.46 is equivalent to 0.35IU/mL after reverse transformation from the logarithmic value. QFTPlus: QuantiFERON-TB Gold Plus, IFN: interferon.
they were negative in TB1. However, IFN-g productions in TB1 and TB2 were closely correlated, thus these conversions were likely observed around cut-off. Then, the effect on the increase of sensitivity was small even in the introduction of TB2 for the estimation of CD8 T-cell response. Variability of the IFN-g value in QFT-GIT has been observed in a range between 0.25 and 0.59 IU/mL [13]. Conversion and reversion were frequently observed in serial tests in QFT-GIT. Others have claimed a wider variability in a range between 0.2 and 0.7IU/mL [14,15]. Variability has already been observed in QFT-Plus [15]. In our study, the number of subjects with reliable positive diagnosis with an IFN-g value of 0.6 IU/mL was 25. Six other subjects were diagnosed as positive with an IFN-g value of <0.6 IU/mL, and their positive might convert to negative in repeated tests. We need further discussion regarding the judgement of the variability zone even if a higher positivity rate in QFT-Plus would be driven by TB2. The LTBI diagnosis procedure is proposed to be based on not only interpretation of the cut-off value but also on epidemiological and clinical assessment [16]. Therefore, we need to be cautious when interpreting QFT-Plus results with an IFN-g value of <0.6 IU/mL. The difference between TB1 and TB2 might be positively correlated with the antigenic load in active TB patients [17]. Barcellini et al. speculated that a TB2TB1 difference of >0.6 IU/mL meant a higher antigenic burden in LTBI, leading to the prediction of developing active TB [18]. Ten subjects met this criterion in the present study, but 6 of the 10 were located in the IQR of (log10(IFNg[QFT-Plus TB2])log10(IFN-g[QFT-Plus TB1])). TB2TB1 difference of >0.6IU/mL might be a possible variation of IFN-g production responding to TB-specific antigens after logarithmic transformation. Thus, we are skeptical about the meaning of a TB2TB1 difference of >0.6 IU/mL as a predictive marker for developing active TB from LTBI. QFT-GIT, the former generation QFT, is an excellent diagnostic tool with higher sensitivity, and a further
increase of sensitivity in QFT-Plus would be limited, especially in Japan [9]. The immune responses to TB-specific antigens would change in the phases of TB infection, active TB infection, and LTBI [4,17]. IFN-g production has been mainly used for the judgement of QFT both in QFT-GIT and QFT-Plus. QFT-Plus provides additional opportunity for analyzing the immune response caused by CD8 Tcells. The analysis of IFN-g production in both TB1 and TB2 would lead to further analysis of the TB immune response, and especially that caused by CD8 T-cells. We estimated the IFN-g production after logarithmic transformation, as was adopted in our previous study [3]. This method has not yet been in general usage. We can take note of the distribution of IFN-g production at a glance in Fig. 1. The difference of logarithmically transformed IFN-g production between TB2 and TB1 was also estimated as the ratio of IFN-g production between TB2 and TB1. In this process, we deduced the function of CD8 T-cells in response to TB-specific antigens in the present study. The introduction of logarithmic transformation of IFN-g production is our trial, and we expect it to be re-estimated or criticized by others. There are some limitations to this study. It was conducted in a single facility. The number of subjects in QFT-Plus positive was also limited. The factors associated with QFT-Plus results, including TB1 and TB2, were not analyzed sufficiently owing to the limited information provided by regional health centers operated by the local government. CD8 T-cell immune responses were analyzed based on the condition of the QFT-plus kit. The modification of dose and combination of TB specific antigens would elicit other immune responses in TB contact screening. 5. Conclusions IFN-g production in TB1 and TB2 were correlated well, and IFN-g production in TB2 was 1.09 times that in TB1 and varied among subjects. Then, CD8 T-cells potentially boost IFN-g production in QFT-Plus. However, there was no significant difference in the positivity rate in QFT-Plus TB1 and TB2 according to the TB contact investigation. The contribution of CD8 T-cells might be small for the diagnosis of LTBI in the present contact investigation. The analysis of IFN-g production in both TB1 and TB2 would lead to further analysis of the TB immune response, and especially that caused by CD8 T-cells. Financial support This work was supported by the Chiba Foundation of Health Promotion and Disease Prevention. Authorship statement All authors meet the ICMJE authorship criteria. All authors have seen and approved the manuscript, and contributed significantly to the work. Authors’ contributions Mizue Tsuyuzaki: Conducted examinations, data analysis. Hidetoshi Igari: Study planning, data analysis. Nao Okada: Conducted examinations, data analysis Kiminori Suzuki Study planning, data analysis. Declaration of Competing Interest All authors: No potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.
Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011
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Please cite this article as: Tsuyuzaki M et al., Role of CD8 T-cell in immune response to tuberculosis-specific antigen in QuantiFERON-TB Gold Plus, J Infect Chemother, https://doi.org/10.1016/j.jiac.2020.01.011