Comparative Analysis of Mycobacterium Tuberculosis-Specic Antigens and T-Cell Mitogen-Stimulated Interferon Gamma Production in Hemodialysis Patients and Healthy Individuals

Background: The incidence of Tuberculosis (TB) is higher in patients undergoing chronic hemodialysis than in the general population; further, both the incidence and development of active TB from latent TB infection (LTBI) are considerably higher in patients undergoing hemodialysis than in any other group. The QuantiFERON-TB Gold In-Tube (QFT-GIT) assay is one of the most commonly used interferon gamma (IFN-γ) release assays (IGRAs) currently. We aim to know T-cell function of hemodialysis patients and the possible QFT false negatives. Methods: In order to analyze the MTB-specic antigen-stimulated IFN-γ release in patients on hemodialysis, the QFT-GIT test was used in this study. A total of 84 hemodialysis patients and 52 healthy subjects were enrolled from Kosin University Gospel Hospital and Catholic University of Pusan, Busan, Republic of Korea; their whole blood samples were collected and used for the present study. Results: The positivity results of the IGRAs in hemodialysis patients and normal subjects were 34/84 (40.4%) and 4/52 (7.6%), respectively. The median value of MTB-specic antigen-stimulated IFN-γ in hemodialysis patients with LTBI and non-LTBI status in hemodialysis patients, healthy individuals were 1.85 IU/mL (4.44ng/mL) and 0.028 IU/mL (0.067 ng/mL) and 0.255 IU/mL (0.612 ng/mL) respectively. In addition, Of the 34 LTBI status in hemodialysis patients, 14 (41.2%) had T-cell mitogen-stimulated IFN-γ levels of 10 or less, and 20 (58.8%) had 10 or more T-cell mitogen-stimulated IFN-γ. Moreover, Of the 49 non-LTBI status in hemodialysis patients, 19 (38.8%) and 30 (61.2%) respectively. On the other hand, there was no low level of T-cell mitogen-stimulated IFN-γ in the healthy individuals. Conclusion: This

challenge of how to optimize the evaluation of diagnostics for TB infection, for which there is currently no accurate diagnostic assessment, needs to be addressed [5].
The two main immunodiagnostic methods currently used for the diagnosis of LTBI are the tuberculin skin test (TST) [6] and the interferon gamma (IFN-γ) release assays (IGRAs) [7]. These tests work on the principle of in vivo and ex vivo cell-mediated immunity, respectively [8]. The TST is an intradermal injection of tuberculin puri ed protein derivatives (PPD) to identify the whole MTB Ag-speci c immune response, delayed-type hypersensitivity. Nevertheless, it has been reported that the accuracy measures of TST are often confounded by the Bacillus Calmette Guérin (BCG) vaccination and nontuberculous mycobacteria (NTM) infections [9]. IGRA is an ex vivo whole blood test of the T-cell immune response that measures the T-cell release of IFN-γ, an in ammatory cytokine, following stimulation by Ags speci c to  [10]. MTB-speci c Ags used in IGRAs, such as early secretory antigen target 6 (ESAT-6) and culture ltrate protein 10 (CFP-10), and TB 7.7 are not present in all M. bovis BCG strains or major environmental mycobacteria, NTMs, and are therefore speci c for MTB and respond to T-cells that have had MTB bacteria in their blood [11][12]. The IGRAs have shown signi cant potential to reduce false positive results, particularly among BCG-vaccinated lowrisk populations [10]. However, IGRAs have also been known to have limited sensitivity for immunocompromised individuals and young children. Furthermore, IGRAs cannot differentiate between active TB disease and LTBI [13].
End stage renal disease (ESRD) is known to compromise cellular immune function, and patients on hemodialysis have been shown to have T-cell dysfunction, including abnormal IFN-γ levels, considered to have a pivotal role in controlling LTBI. In fact, among ESRD patients, those on hemodialysis had a higher prevalence of TB, compared to other patient groups. According to Min et al., The probability of being infected with active TB during hemodialysis or peritoneal dialysis among ESRD patients was increased 4.44 ratio for hemodialysis patients and 5.17 ratio for peritoneal dialysis patients respectively. [14]. In ESRD patients and hemodialysis patients, there is a possibility that T cell dysfunction related to adaptive immunity may occur unlike normal healthy persons. It is therefore likely that the results of the IGRA test for diagnosing LTBI and the interpretation of the results are likely to be affected.
The aim of the present study was to evaluate the effectiveness of the diagnosis of LTBI using IGRA in a group at high risk for contracting active TB, whole blood samples from a total of 84 hemodialysis patients and 52 healthy non-TB subjects were collected and analyzed by currently available IGRA testing, QuantiFERON TB Gold In-Tube (QFT-GIT) assay (Qiagen, Hilden, Germany). Characteristics of MTBspeci c Ag and T-cell mitogen (PHA) stimulated IFN-γ release were compared between hemodialysis patients and healthy non-TB subjects.

Study design and locations
This study targeted patients receiving hemodialysis for approximately one year, from March 2017 to June 2018. A total of 84 hemodialysis patients and 52 healthy subjects were enrolled from Kosin University Gospel Hospital and Catholic University of Pusan, Busan, Republic of Korea, and their whole blood samples were collected and used for the present study. All participants provided written informed consent. The Institutional Ethics Committee of Catholic University of Pusan and Kosin University Gospel Hospital approved the study (approval numbers CUPIRB-2017-036 and KUGH 2017-11-042). Results of IGRA, MTB-speci c Ag stimulated IFN-γ release, and T-cell mitogen (PHA)-stimulated IFN-γ release in the collected whole blood samples were compared between the two groups, hemodialysis patients and healthy non-TB subjects. Plasma was isolated from whole blood after MTB-speci c antigen stimulation and stored at -70℃ until use.

Data interpretation ofinterferon gamma release assay
The test results were interpreted using GFT-GIT ELISA software (version no. 2.43; Cellestis LTD. Victoria, Australia) and the cut-offs for diagnosis in the manufacturer's instructions were used. The test result was considered positive if the IFN-γ level in the sample well, after stimulation with ESAT-6, CFP-10, and TB 7.7 was 0.35 IU/ml (0.42 ng/mL), irrespective of the result for the positive control well. The test was considered negative if the IFN-γ level was 0.35 IU/ml (0.42 ng/mL) and the IFN-γ level of the positive control well was 0.35 IU/ml (0.42 ng/mL). The test result was considered indeterminate if the IFN-γ level was 0.35 IU/ml (0.42 ng/mL) in both antigen wells and 0.5 IU/ml (0.6 ng/mL) in the mitogen control well.

Statistical analysis
Statistical analysis was performed using GraphPad prism, version 5.00 (GraphPad Software, San Diego, CA, USA). The signi cant difference in the results between the hemodialysis patients and the healthy individuals, which were predicted to be weaker, was calculated. In particular, the value of mitogen control, which is coated with a peptide called PHA that stimulates T-cells nonspeci cally to secrete IFN-γ, is calculated as the difference between the hemodialysis patients and the healthy individuals, with 95% con dence intervals (CIs), and the values of these groups were compared using the unpaired t-test. A P value less than 0.05 was considered statistically signi cant.

Characteristics of hemodialysis patients and healthy individuals
The characteristics of the patients involved in this study are shown in Table 1. The mean age of hemodialysis patients was 61.1 years (range 31-90) and the male-to-female ratio was 43:41 (51.2%:48.8%), while the mean age of the healthy individuals was 23.9 years (range 22-35) and the male-to-female ratio was 23:29 (44.2%:55.8%). The age of the hemodialysis patients was considerably higher than that of the healthy individuals. In addition, only 57 (67.9%) of the hemodialysis patients had received BCG vaccine, while all the healthy subjects had received the vaccine. Cases reporting having been in contact with tuberculosis patients were 13 (15.4%) of the hemodialysis patients, and there were only 6 (7.1%) patients with radiological lesions among the hemodialysis patients.

Comparison of MTB-speci c antigen-stimulated IFN-γ production between hemodialysis patients and healthy individuals
The median values of MTB-speci c Ag-stimulated IFN-γ in hemodialysis patients with LTBI and non-LTBI status in hemodialysis patients, healthy individuals were 1.85 IU/mL (4.44 ng/mL) and 0.028 IU/mL (0.067 ng/mL) and 0.255 IU/mL (0.612 ng/mL) respectively (Fig. 1). These results showed that the IFN-γ value was signi cantly higher in hemodialysis patients with LTBI; however, the T-cell mitogen-stimulated IFN-γ value was much higher in healthy individuals. Based on these results, we con rmed that T-cell function in hemodialysis patients was reduced as compared to healthy subjects (Fig. 2). Table 3 shows that in hemodialysis patients with LTBI, the rate of IFN-γ stimulated with TB antigen was higher than 0.5 IU/mL (1.2 ng/mL) in 30 cases (88.2%) and lower than 0.5 IU/mL (1.2 ng/mL) in 4 cases (11.8%). On the other hand, in healthy individuals there were 4 cases (7.6%) and 48 cases (92.3%) respectively.

Discussion
TB is one of the top 10 causes of death in the world, and the leading cause from a single infectious agent (ranking above HIV/AIDS) [7]. Millions of people continue to fall sick with TB each year [15]. Therefore, there is a need for a method to quickly diagnose those with a high risk of TB. Delayed diagnosis in this situation is an important issue contributing to the high burden and transmission of TB in developing countries [16].
In a previous study, the performance of QFT-GIT was not excellent in patients with impaired immune function, and the results were unclear [17]. Recently, the relationship between TB and the immune status of patients has been revealed, and directions for diagnosis based on the immune status have been suggested [18]. Another study has shown that IGRA testing is useful in identifying LTBI in patients with rheumatic disease, one of the groups at high risk of TB [19]. The population undergoing hemodialysis is expanding and is suggested as a high priority group for LTBI treatment, given their increased TB risk [20].
Immunologically, renal disease (RD) is associated with some disorders in both innate and adaptive immune systems in such a way that there is coexistence of both immune activation and immune suppression [21]. Immune response to MTB infection is primarily mediated by T-cells [22].
Recent studies have shown that hemodialysis patients have a higher infection rate because their immune system is weakened [23]. This study investigated the risk of TB in patients undergoing hemodialysis and in healthy individuals and compared them with LTBI status indicated by QFT-GIT. The LTBI positive rate was signi cantly higher in the hemodialysis patients than in the healthy individuals. Table 3 shows that more IFN-γ is secreted in T-cells that have been exposed to TB in hemodialysis patients than in the   (Fig. 3-A). On the other hand, none of the healthy individuals had T-cell mitogen(PHA)-stimulated IFN-γ levels below 10 ( Fig. 3-B). healthy individuals. In hemodialysis patients whose mitogen-nil values were less than 10 IU/mL (24 ng/mL), the immune responses of the T-cells were lower than those of the healthy individuals. As shown in Table 4, in the healthy individuals, no patients with a T-cell mitogen-stimulated IFN-γ level lower than 10 were observed. However, in hemodialysis patients with LTBI, 20 out of 34 (58.8%) showed lower than 10. In particular, non-LTBI status in hemodialysis patients, 30 out of 49 showed lower than 10. These results suggest that T-cell mitogen-stimulated IFN-γ levels are lower than 10 and that T-cell mediated immunity is lowered, resulting in a false negative [24]. Other studies have reported alteration of adaptive immune system for ESRD patients. For example, in the ESRD patient group, the vaccination response to TB or the tuberculin skin test to diagnose LTBI resulted in weak or no response [25]. Compared with the results of this study, the QFT-GIT assay also shows similar results for ESRD patients.
Impaired cellular immunity may explain anergy and high rates of false negative results of IGRA in hemodialysis patients [26]. In fact, many ESRD patients and hemodialysis patients are known to have symptoms of uremia-induced immune system and acquired immune system, resulting in various infections and complications [25]. In vitro studies have shown that uremic milieu caused by ESRD reduces T cell function and population. It also has been shown to in uence the formation of a variety of pro-in ammatory cytokines, TNF-α, IL-12 or INF-γ, which are secreted by T cells [27][28]. The results of this study also con rm that the IFN-γ concentration is decreased during stimulation by mitogen, possibly due to uremic milieu in ESRD patients. Therefore, further studies on the correlation between serum concentration of urea levels and QFT-GIT assay results are needed. For the ESRD patients, it may be possible to use the serum concentration of urea as an auxiliary index in the interpretation of the QFT-GIT assay results.
In this study, among the negative cases in hemodialysis patients there may be positives, that is, there are likely to be false negatives due to the immune response of T-cells being reduced. These results suggest the possibility of false negatives in the QFT-GIT assay using enzyme-linked immunosorbent assays to measure the antigen-speci c production of IFN-γ using T-cells in whole blood. Thus, interpretation of IGRA results among hemodialysis patients should be undertaken with caution before treatment for LTBI.

Conclusion
The reliability of QFT-GIT in patients with T-cell-mediated immune responsiveness is not high; additional analyses using alternative immune markers or in ammatory markers, and qRT-PCR that simultaneously detects the mRNA expressions of TB-related proin ammatory immune biomarkers may increase the reliability of the test and be more helpful for the rapid screening and detection of groups at high risk for TB, including hemodialysis patients. Furthermore, screening for LTBI using nonimmunologic methods may help combat TB, given that the immune system has already been compromised.

Declarations
Acknowledgement This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1C1B108888 and -2020R1C1007169) and the Brain Busan 21 Plus project.

Funding
This research did not receive any speci c grant from funding agencies in the public, commercial, or not for pro t sectors.

Availability of data and materials
The data analysed during this study are included in this paper. Some of the datasets are available from the corresponding author upon reasonable request Author`s contributions Heechul Park conceptualized, designed the paper, analyzed, wrote and drafted the paper. Heechul park, Ye Na Kim, Sung-Bae Park, Junseong Kim, Jaewon Lim and Jungho Kim collected the data and reviewed the paper critically. Sunghyun Kim and Ji Young Park also analyzed the data. All authors read and approved the nal paper.

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