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Role of QuantiFERON-TB Gold antigen-specific IL-1β in diagnosis of active tuberculosis

Abstract

The main objective of the study was to evaluate whether in vitro QuantiFERON-TB Gold In-Tube (QFT-GIT) assay antigen-specific IL-1β, TNF-α, IL-2, IL-6, IL-8 and IL-12 (p40) production is associated with active TB. In a cohort of 77 pulmonary TB patients (PTB), 67 healthy household contacts (HHC) and 83 healthy control subjects (HCS), the antigen-specific cytokines levels were determined in supernatants generated from QFT-GIT tubes. Antigen-specific IL-1β levels were significantly higher in PTB than HHC and HCS. At a fixed cutoff point (1,108 pg/ml), IL-1β showed positivity of 62.33 % in PTB, 22.38 % in HHC and 22.89 % in HCS. Moreover, antigen-specific IL-1β assay can differentiate PTB and HHC (believed to be latently infected) (p < 0.0001). Like IL-1β, significantly higher levels of antigen-specific TNF-α were associated with PTB and displayed 43.63 % positivity in PTB. The antigen-specific IL-2 levels were associated both with PTB (54.54 %) and HHC (48.14 %). Other cytokines levels did not differ among the groups. Our results suggest that antigen-specific IL-1β can be used as a biomarker for active TB diagnosis as well as for differential diagnosis of PTB and LTBI.

Introduction

Human tuberculosis (TB) is an infectious disease caused by intracellular pathogen Mycobacterium tuberculosis. Despite the availability of highly efficacious treatment for decades, TB still remains the second leading cause of death from an infectious agent worldwide, accounting for an estimated 8.6 million new TB cases with 1.3 million deaths annually [1]. India accounted for an estimated one quarter (26 %) of all TB cases worldwide with the largest number of incident cases in 2012 (2.0–2.5 million) [1]. This situation is further aggravated by human immunodeficiency virus (HIV) co-infection and emergence of drug-resistant bacteria. Thus, early and accurate diagnosis and treatment of TB are the key strategies to control the global burden of TB.

The widely used bacteriological diagnostic tests for active TB, which are considered as the gold standard, have limitations—smear microscopy test is less sensitive and culture test is time-consuming. Though the nucleic acid amplification test is a rapid confirmatory test for active TB diagnosis, it has reduced sensitivity [2]. The recent exploration in diagnosis of TB infection is the development of in vitro interferon gamma (IFN-γ) release assays (IGRAs). IGRAs rely on the principle that sensitized T cells upon re-stimulation with M. tuberculosis-specific antigens produce IFN-γ. The antigens used in IGRAs such as early-secreted antigen target-6 (ESAT-6), culture filtrate protein-10 (CFP-10) and TB 7.7 (Rv2654) are present only in M. tuberculosis and are not present in vaccine strains of Bacille Calmette–Guerin (BCG) and most of the environmental mycobacteria.

Recent studies from TB non-endemic countries revealed that IGRAs are better correlated with risk factors for infection with M. tuberculosis [3, 4] than compared with tuberculin skin test (TST) and are more specific (90–98 %) [5]; they do not give false-positive results in BCG-vaccinated individuals [68]. Although IGRAs are mounted for LTBI diagnosis, numerous studies were conducted in both TB-endemic and non-endemic countries to evaluate IGRAs role in active TB diagnosis. In TB non-endemic countries, sensitivity of IGRAs is estimated to be high in diagnosis of active TB [9, 10].

Studies from high TB burden countries such as India revealed that the diagnostic potential of IGRAs is suboptimal in the diagnosis of active TB. Due to high prevalence of LTBI, specificity of IGRAs is found to be low [11]. The sensitivity of IGRAs is also low because of more indeterminate results that lead to lower detection rate, particularly in patients with severe TB and HIV co-infection [12, 13].

There is a hope to increase the diagnostic performance of IGRAs by measuring TB antigen-specific alternative or additional biomarkers, either alone or in combination with IFN-γ [11, 14, 15]. Moreover, identification of such biomarkers may also help in understanding the pathogenesis of the disease.

Apart from IFN-γ, the pro-inflammatory cytokines such as interleukin (IL)-1beta (β), tumor necrosis factor-alpha (TNF-α), IL-2, IL-6, IL-8 and IL-12 are mainly associated with mycobacterial infections. All these chosen pro-inflammatory cytokines are probably involved in leukocyte activation, formation and maintenance of granulomas during M. tuberculosis infection [1620]. Many studies had demonstrated that the levels of these cytokines are elevated in active TB patients compared with the healthy controls [15, 2129].

Hence, in the present study, we evaluated the diagnostic performance of these pro-inflammatory cytokines to diagnose active TB in India, an endemic country for TB. We also compared the results of these cytokines with IGRA to determine whether the diagnostic performance of IGRA could be improved by addition of these new biomarkers.

Materials and methods

Study subjects

The study was approved by Institutional Ethical Committee of National Institute for Research in Tuberculosis (NIRT), Chennai. All the study subjects were informed about the study procedure and written consent was obtained from the volunteers before recruitment into the study. Individuals with previous history of TB; those who underwent TST in the past 16 months; those with silicosis, end-stage renal disease and leukemia/lymphoma; those under immunosuppressive therapy; or those with HIV infection were excluded from the study.

The study subjects were categorized into three groups: (1) Healthy control subjects (HCS) were apparently free from TB symptoms and did not have close family contact of active TB. Since our setting is endemic for TB, radiological examination and sputum smear microscopy were done to rule out the suspicion of active TB. (2) Healthy household contacts (HHC) were recruited from the families where there was at least one sputum-positive pulmonary tuberculosis patient (PTB) (index case) living in the same household, for at least 3 months immediately preceding the start of treatment of index case, who had high probability to be infected. (3) Active pulmonary TB patients (PTB) were recruited from Revised National Tuberculosis Control Program (RNTCP) centers. The presence of active TB was defined as positive for sputum smear microscopy and/or M. tuberculosis in culture and/or abnormality suggestive of TB in chest X-ray. The patients with smear- and culture-negative results but who had abnormalities on chest X-ray, which was suggestive of active TB, were given broad-spectrum antibiotics for 3 weeks. The non-responders were then treated with anti-tuberculous therapy (ATT) and followed up for at least 2 months. If the subject responded to ATT, then he/she was considered to have PTB as per RNTCP guidelines [30].

Tuberculin skin test (TST)

The 2 TU (tuberculin unit) of purified protein derivative RT23 (Statens Serum Institute, Copenhagen, Denmark) was injected intradermally by Mantoux method, and the induration was measured between 48 and 72 h after TST injection by trained professionals. The cutoff point for TST positivity was considered as >10 mm for this study [31].

Interferon gamma release assay (IGRA)

As described previously [11], IGRA was performed using QuantiFERON-TB Gold In-Tube (QFT-GIT) (Cellestis, a company of Qiagen GmBH) kit as per the manufacturer’s instructions. Briefly, one ml of blood was taken in each of the three tubes and precoated with TB antigen, phytohemaglutinin for the positive control or nil antigen for the negative control. The tubes were incubated for 16–24 h at 37 °C, 5 % CO2 atmosphere, and supernatant, i.e., plasma, was collected after centrifugation and stored at −30 °C until the assay was done. QFT-GIT-ELISA was carried out using the supernatants as per the manufacturer’s instructions. The test results were interpreted as per kit guidelines, using the software provided by the manufacturer.

Determination of IL-1β, TNF-α, IL-2, IL-6, IL-8 and IL-12 (p40)

The levels of these cytokines were estimated in the plasma acquired from the QFT-GIT assay tubes. As per the manufacturer’s instructions, cytokine levels were measured by using BD opt-EIA Kits (BD Biosciences, Franklin Lakes, NJ, USA).

Statistical analysis

Statistical analysis was performed by using GraphPad Prism software version 5.0 (GraphPad software, CA, USA). The median value for each group was determined and compared among the groups using Kruskal–Wallis statistical test. The proportion of positivity between the tests was compared using Fisher’s exact test. In all instances, a p < 0.05 was considered as statistically significant. Receiver operating characteristic (ROC) curves were used to determine the cutoff points yielding the highest combined specificity and sensitivity, and discriminative ability was evaluated by the area under the ROC curve (AUC).

Results

Table 1 shows the clinical characteristics of study groups. A total of 227 study subjects were prospectively recruited, including 77 PTB, 67 HHC and 83 HCS. Subjects, based on smear microscopy test and/or culture and/or chest X-ray results, were included in PTB. QFT-GIT test did not show any indeterminate results in HHC and HCS groups, but it showed three indeterminate results in PTB group. The recent studies recommend considering the indeterminate results as negatives for sensitivity calculation, since the indeterminate results do not have any clinical significance [32]. Thus, in the present study, we considered these three indeterminate results as negative and proceeded for further analysis.

Table 1 Clinical characteristics of study groups

Comparison of IL-1β level among the groups

The antigen-specific IL-1β levels were obtained by subtracting the levels of IL-1β of unstimulated from antigen-stimulated levels of IL-1β. The median values of TB antigen-specific IL-1β were 1,300 pg/ml (IQR 540.4–2,631) in PTB, 479.2 pg/ml (IQR 169.3–1,100) in HHC and 355.6 pg/ml (IQR 116.0–1,097) in HCS. There was a significant difference between PTB and other two groups (HHC and HCS), but no significant difference between HHC and HCS (Fig. 1).

Fig. 1
figure1

Comparison of antigen-specific IL-1β levels among the study groups. Each dot represents a study subject and horizontal line represents the median value of IL-1β levels. In all the cases, p < 0.05 was considered as significant. *** p < 0.0001; ns not significant (p > 0.05)

We further compared the TB antigen-specific IL-1β level in all the study groups stratified by QFT-GIT results. The QFT-GIT positives in the HHC and HCS were regrouped as “QFT +ve with no TB” group and QFT-GIT negatives were considered as “QFT-ve with no TB” group. We found higher levels of TB antigen-specific IL-1β in PTB group (median 1,300 pg/ml) followed by “QFT +ve with no TB” (median 479.2 pg/ml) group, and lower levels were observed in “QFT −ve with no TB group” (median 358.7 pg/ml). Based on Kruskal–Wallis test, there was a significant difference between PTB and other two groups (p < 0.0001). However, no significant difference was observed in antigen-specific levels of IL-1β between “QFT +ve with no TB” and “QFT −ve with no TB” groups (Fig. 2).

Fig. 2
figure2

Antigen-specific IL-1β levels among the study groups, stratified by QFT-GIT status. Elevated levels of IL-1β were mainly associated with PTB. *** p < 0.0001; ns not significant (p > 0.05)

Altogether, these data suggested that unlike QFT-GIT, antigen-specific IL-1β secretion was mainly associated with PTB and not with HHC and HCS.

Determination of cutoff point for antigen-specific IL-1β

Since IL-1β secretion was associated with PTB, the diagnostic performance of TB antigen-specific IL-1β was evaluated by ROC curve analysis. To find the optimum cutoff point for IL-1β, we considered all PTB patients as “diseased” group and QFT-GIT-negative and TST-negative HCS as “control” group (since our country is endemic to TB, we had taken both QFT-GIT-negative and TST-negative HCS alone as true controls to fix the cutoff value). The AUC for antigen-specific IL-1β was 0.7361 (95 % CI 0.6560–0.8163), and 1,108 pg/ml was chosen as the optimum cutoff point (based on Yuden’s Index method). Those above the cutoff value were considered as positives, and those below the cutoff value were considered as negatives.

Table 2 shows the percentage of positivity of both IL-1β and QFT-GIT tests in all the three groups. Out of 77 PTB, IL-1β test was positive in 48 (62.33 %; 95 % CI 51.25–73.40) and QFT-GIT was positive in 57 (74.02 %; 95 % CI 63.9–84) PTB, and this difference was not significant (p = 0.166). However, in HHC group out of 67, IL-1β test was positive only in 15 (22.38 %; 95 % CI 12.12–32.63), whereas QFT-GIT was positive in 43 (64.17 %; 95 % CI 52.37–75.96) HHC, and this difference was statistically significant (p = 0.0001). In case of HCS, out of 83 tested, 19 (22.89 %; 95 % CI 13.65–32.12) HCS were positive for IL-1β and 14 (16.86 %; 95 % CI 8.62–25) HCS were positive for QFT-GIT, and this difference was not significant (p = 0.43) (Fig. 3).

Table 2 Diagnostic performance of IL-1β and QFT-GIT assays
Fig. 3
figure3

Comparison of IL-1β assay performance with QFT-GIT in different study groups. The performance of IL-1β assay was similar to QFT-GIT in both PTB and HCS. However, the positivity of IL-1β assay was less compared to QFT-GIT, indicating IL-1β assay was not influenced by LTBI unlike QFT-GIT. ns not significant

Altogether, these results emphasizing that as a single biomarker IL-1β showed similar positivity like QFT-GIT to diagnose PTB as diseased group and HCS as disease-free control group. Moreover, IL-1β showed significantly lower positivity than QFT-GIT in HHC (p = 0.0001), indicating unlike IFN-γ measured in QFT-GIT, antigen-specific IL-1β secretion was not influenced by latent TB infection.

Comparison of TNF-α levels among the groups

Apart from IL-1β, we also measured TNF-α levels in TB antigen-stimulated plasma samples. TB antigen-specific TNF-α levels were significantly higher in PTB (ranged 0.1–1,329 pg/ml) than HHC (ranged 0.1–154.2 pg/ml) and HCS (ranged 0.1–119.8 pg/ml) (Fig. 4), and no differences between the other study groups by pairwise comparisons. Analysis of ROC curve was done as described previously for IL-1β. At a fixed cutoff point 10.62 pg/ml, TNF-α showed a positivity of 43.63 % (24 out of 55) in PTB, 7.4 % (4 out of 54) in HHC and 1.81 % in HCS (1 out of 55) (AUC = 0.7924; 95 % CI 0.7044–0.8804). As compared to IL-1β or QFT-GIT or smear microscopy test, TNF-α showed lesser positivity to diagnose PTB as diseased.

Fig. 4
figure4

Antigen-specific TNF-α levels among the study groups. The levels of TNF-α were significantly higher in PTB than HHC and HCS. Kruskal–Wallis test was used to find out the differences among the study groups. ***p < 0.0001; ns not significant (p > 0.05)

Comparison of IL-2, IL-6, IL-8 and IL-12 (p40) levels among the groups

The secretion of TB antigen-specific IL-2 level was significantly higher in PTB (ranged 0.1–3,367 pg/ml) and HHC (ranged 0.1–2,360 pg/ml) than HCS (ranged 0.1–576.2 pg/ml) (p < 0.0001), whereas no significant difference was observed between PTB and HHC (Fig. 5). Using ROC curve analysis, 30.14 pg/ml was chosen as the optimum cutoff point for IL-2 (AUC = 0.7428; 95 % CI 0.6482–0.8374). At this cutoff value, IL-2 showed a positivity of 54.54 % in PTB (30 out of 55), 48.14 in HHC (26 out of 54) and 9.09 % in HCS (5 out of 55). The median values of TB antigen-specific levels of IL-6 were 433.4 pg/ml (IQR 132.2–952.9) in PTB, 361.1 pg/ml (IQR 120.2–1,098) in HHC and 416.1 pg/ml (103.0–922.6) in HCS. We did not find any significant difference in TB antigen-specific levels of IL-6 among the groups (Fig. 6).

Fig. 5
figure5

Comparison of antigen-specific IL-2 levels among the study groups. Significantly elevated levels of antigen-specific IL-2 were observed both in PTB and HHC compared to HCS. ***p < 0.0001; ns not significant (p > 0.05)

Fig. 6
figure6

Comparison of antigen-specific IL-6 levels among the study groups. Significant differences were not observed in antigen-specific IL-6 levels among the study groups

In case of IL-8, TB antigen-specific IL-8 level was observed to be high in HHC (median 474.8 pg/ml, IQR 244.9–978.8) when compared to PTB (394.7 pg/ml, IQR 157.5–817.2) and HCS (median 285.8 pg/ml, IQR 113.8–621.2). But the difference was also not significant between the groups (Fig. 7). After stratified by QFT-GIT results also, antigen-specific IL-6 and IL-8 were not significantly differentiating the study groups.

Fig. 7
figure7

Comparison of antigen-specific IL-8 levels among the study groups. Significant differences were not observed in antigen-specific IL-8 levels among the study groups

The baselines as well as TB antigen-specific IL-12 (p40) levels did not differ significantly among the all three groups. Perhaps, in most of the study subjects, the levels of this cytokine were not in detectable range (data not shown).

Discussion

Early and accurate diagnosis of TB is the major challenge in TB control programs. Since M. tuberculosis is an intracellular pathogen, various in vitro T cell-based assays are emerging for TB diagnosis. Upon infection with M. tuberculosis, host immune cells initiate secretion of numerous cytokines against various antigens of bacteria, which further proceed by various pathways to offer protection to the host. However, the pattern of cytokine secretion may be different among active TB, latently infected and HCS. Finding such differentially expressed cytokines will be a useful approach in the field of TB diagnosis. One such cytokine is IFN-γ (differential expression observed between M. tuberculosis infected and uninfected individuals), and based on this cytokine, IGRAs are newly emerged diagnostic tests which have better correlation to infection with M. tuberculosis. But IGRAs cannot distinguish active TB from latent infection; thus, its utility for active TB diagnosis is low in TB-endemic countries, where most of the people are believed to be latently infected with M. tuberculosis [3335]. Thereby, as a part of search for improved diagnostic measures, various studies have focused on screening large array of antigen-specific biomarkers other than IFN-γ. In the present study, we have screened six potential biomarkers, such as TB antigen-specific IL-1β, IL-6 IL-8, TNF-α, IL-2 and IL-12(p40).

IL-1β is a pro-inflammatory cytokine, secreted mainly by monocytes, macrophages and dendritic cells [36, 37]. The synthesis and secretion of IL-1β is a highly regulated multi-step process; live M. tuberculosis actively modulates signaling pathways by up-regulating the transcription of the IL-1β mRNA and pro-IL-1β formation and enhances IL-1β secretion into the external environment [38]. Thus, in TB patients, IL-1β expression is observed to be elevated [39] and as described by Law et al. [21], there is a 5- to 20-fold increased release of IL-1β by broncho-alveolar cells lavaged from the involved sites in PTB patients compared with normal individuals. The production of IL-1β in peripheral blood monocytes is high in PTB than in control subjects [40], and reduced IL-1β expression was observed with improved chest radiography in patients with pulmonary TB [41]. So far, very few studies exist to examine the role of antigen-specific IL-1β in TB diagnosis [42, 43]. In the present study, we observed that the levels of IL-1β in TB antigen-stimulated plasma are high in PTB than HHC (who are more prone to develop LTBI) and HCS.

Compared to IFN-γ (measured in QFT-GIT assays), IL-1β might be a more suitable biomarker for active TB diagnosis because of the following reasons: (1) QFT-GIT is able to diagnose both active TB disease and LTBI, which leads to its reduced utility for active TB diagnosis. In case of IL-1β, elevated levels of TB antigen-specific IL-1β are mainly associated with PTB and not with HHC; thus, IL-1β can identify active TB disease and not LTBI. Of 67 HHC, IL-1β assay was positive only in 15 (22.38 %), whereas QFT-GIT showed 43 (64.17 %) positives, and this difference between two assays in percentage of positivity is highly significant (p = 0.0001). Thus, when compared to IFN-γ, as a single biomarker, IL-1β can effectively identify PTB alone. (2) In our earlier studies, we have observed that the positivity of QFT-GIT is higher among elder subjects (age >45 years) than the younger, and detection rate is lower in children (age <4 years) [44]. (3) Moreover, the diagnostic performance of QFT-GIT is estimated to be low in patients with advanced radiological extent of pulmonary TB [45]. However, IL-1β secretion is not influenced by risk factors such as age, sex, severity and pulmonary involvement of disease. In TB-endemic countries such as India, it is very important to identify a biomarker which will be useful to predict the breakdown of LTBI to active TB disease. IL-1β may be one of the suitable surrogate biomarkers to fulfill this strategy, since elevated levels of IL-1β are mainly associated with PTB. Further studies are required in this direction to evaluate role of antigen-specific IL-1β in prognosis of LTBI to active TB.

Numerous studies are conducted to appraise the diagnostic utility of TB antigen-specific TNF-α, since it plays a central role in the control and protection against M. tuberculosis like IFN-γ. Single-positive TNF-α M. tuberculosis-specific CD4 + T cells is a new tool for the rapid diagnosis of PTB [23]. In consistence with this report and some other studies, we also manifested that TB antigen-specific TNF-α levels are associated with PTB and not with HHC or HCS; however, TNF-α has depicted less positivity in PTB (43.63 %) compared with QFT-GIT assay. Moreover, the levels of TNF-α were found to be very low compared with other cytokines and; in most of the study subjects, the secretion of TNF-α is not in measurable quantity. Wang et al., also observed low level of TB antigen-specific TNF-α secretion during short-term culture conditions [25]. A study from West African cohort reported that production of TB antigen-specific TNF-α can discriminate between active TB disease and latent infection, where they have used long-term culture conditions [24]. Thus, further studies are required to investigate the usefulness of TNF-α in PTB diagnosis by using long-term culture conditions.

IL-2 has a central role in activation and expansion of T cells. IL-2 has been shown to limit mycobacterial replication, possibly by macrophage activation via interferon-mediated pathways or directly by the development of cytotoxic T lymphocytes recognizing mycobacterial antigens [46]. In our study, we have evaluated the role of IL-2 in TB diagnosis. In agreement with other studies, our results also demonstrate that antigen-specific IL-2 levels are high in PTB and HHC and low in HCS. However, the positivity of TB antigen-specific IL-2 assay is low in PTB (54.54 %) as well as in HHC (48.14 %) compared with QFT-GIT. The other antigen-specific cytokine levels include IL-6, IL-8 and IL-12 (p40) did not show any significant difference among the study groups.

This study concludes that there is a need for reliable biomarkers to diagnose TB, and measuring multiple cytokines could enhance the diagnostic potential of QFT-GIT test to diagnose PTB. To our knowledge, this is the first study from a TB-endemic country, showing that TB antigen-specific IL-1β levels were associated only with PTB, but not with HHC or HCS. However, IL-1β may not be a stand-alone diagnostic test for active TB. IL-β test in conjunction with the existing tests may enhance active TB diagnosis. As a single biomarker, IL-1β can differentiate LTBI and active disease. Further studies are required to evaluate the diagnostic performance of IL-1β alone or in combination with QFT-GIT in smear-negative TB as well as HIV–TB patients. The role of IL-1β in monitoring TB treatment as well as breakdown of LTBI to active disease has to be investigated.

The present study also displayed that antigen-specific TNF-α levels were associated with PTB and IL-2 levels were associated with PTB and HHC. Thus, further studies are required to investigate the usefulness of TNF-α and IL-2 in TB diagnosis. TB antigen-specific IL-6, IL-8 and IL-12(p40) may not be good biomarkers for TB diagnosis.

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Acknowledgments

The authors wish to thank all the patients participated in this study. Authors also thank the clinicians, social workers and health visitors who helped to collect samples from study patients. Ms. Maddineni Prabhavathi expresses her gratitude to Council of Scientific and Industrial Research (CSIR), New Delhi, India, for providing senior research fellowship.

Conflict of interest

The authors declare that they have no competing interests.

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Correspondence to Alamelu Raja.

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Prabhavathi, M., Kabeer, B.S.A., Deenadayalan, A. et al. Role of QuantiFERON-TB Gold antigen-specific IL-1β in diagnosis of active tuberculosis. Med Microbiol Immunol 204, 567–574 (2015). https://doi.org/10.1007/s00430-014-0382-x

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Keywords

  • Tuberculosis
  • QuantiFERON-TB Gold assay
  • Diagnosis
  • Interleukin-1beta
  • Tumor
  • Necrosis factor