Acid-fast bacilli (AFB) staining of sputum has a sensitivity of only 50~60%, and cultures for tuberculosis (TB) diagnosis usually require several weeks for results. Moreover, diagnosis and treatment decisions may be difficult in case with clinical suspicion of TB and negative AFB sputum smears. In this case, the rapid diagnosis of TB classically relies on the results of different methods, including the tuberculin skin test (TST), chest radiography, nucleic acid amplification (NAA) test of Mycobacterium tuberculosis (M.TB) and/or pathological examinations, although TST has a disadvantage of poor specificity in bacille Calmette-Guerin (BCG) vaccinated individuals and in those living in high TB prevalence countries1. Meanwhile, interferon-gamma release assays (IGRAs), what measure and interpret interferon-gamma (IFN-γ) that is produced by immune responses to early secreted antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10), has advantages of higher specificity than TST, no need to revisit the hospital after the test, and no boosted effect in a repeated conduct2. Since the Food and Drug Administration (FDA) announced that QuantiFERON-TB GOLD test (QFT-G; Cellestis Ltd., Carnegie, Victoria, Australia) can be alternatively used for TB diagnosis instead of TST, QuantiFERON-TB Gold In-Tube test (QFT-GIT; Cellestis Ltd.) and T-SPOT. TB test (Oxford Immunotec, Abingdon, UK) have been proven to be used as supportive tests along with other tests for the diagnosis of active pulmonary TB as well as latent TB infection (LTBI)3,4.

QFT-GIT, which has been developed to improve the limitation of QFT-G that should be performed within several hours after blood collection, enables direct test using a special tubes containing antigens without pipetting or handling, and its sensitivity has been improved due to the addition of TB7.7 to TB antigens4. The rate of indeterminate results, significantly associated with elderly and immunocompromised patients, has been reported to be 5~40% in QFT-G, thereby causing clinical difficulty in the interpretation of test results. Whereas, such high rate of indeterminate results in QFT-G is anticipated to decrease in QFT-GIT. However, there have been few studies performing a detailed investigation of indeterminate results with clinical effectiveness in QFT-GIT with active TB patients5. In addition, in the case of elderly patients with TB, the positive rate of AFB smears is even lower due to difficulty in appropriate expectoration of sputum. Thus, a supportive method is required to improve diagnostic yield in elderly patients6, but the results of IGRAs should be carefully interpreted due to decrease in the immunologic status associated with aging7. In Korea, studies on the interpretation of the results of QFT performed on elderly TB patients have been insufficiently conducted so far. Therefore, we evaluated the risk factors including age distribution that can influence indeterminate and false negative results when using the QFT-GIT in routine clinical practice in a single institution.

This study was performed on 77 patients with clinically suspected active TB, of whom 44 patients were confirmed to have active TB (34 patients with culture-proven TB, 24 patients with positive NAA test of M.TB). The confirmed TB patients consisted of 39 patients with pulmonary TB, 1 patient with miliary TB, 3 patients with TB pleurisy, and 1 patient with TB lymphadenitis. The 33 control group consisted of 20 patients with bacterial pneumonia, 3 patients with NTM lung disease, 1 patient with lung cancer, and 9 patients with fibrotic TB sequelae. The median age of the TB group was shown to be 48.5 (IQR, 41.0~66.0), and their male to female ratio was shown to be 31 (70.5%):13 (29.5%). Meanwhile, the median age of the control group was shown to be 62.0 (IQR, 50.5~70.5), and their male to female ratio was shown to be 22 (66.7%):11 (33.3%). No difference in the age and gender was found between the TB and control groups (Table 2).

The result of QFT-GIT was shown to be positive in 23 cases (52.3%), negative in 16 cases (36.4%), and indeterminate in 5 cases (11.4%) in the TB group, whereas the result of TST was shown to be positive (>10 mm) in 26 cases (59.1%), and negative in 14 cases (31.8%). Meanwhile, the result of QFT-GIT was shown to be positive in 7 cases (21.2%), negative in 19 cases (57.6%), and indeterminate in 7 cases (21.2%) in the control group, whereas the result of TST was shown to be positive in 8 cases (24.2%), and negative in 22 cases (66.7%) (Table 3).

In total, 12 patients (15.6%) showed indeterminate result on the QFT-GIT and all of them were due to positive control failure. They included 5 patients with TB, 3 patients with bacterial pneumonia, and 4 patients with fibrotic TB sequelae. Four patients among the non-TB group had the presence of previous TB scar, which indicated the possibility of LTBI. The median TB Ag-Nil and the median Mitogen-Nil were shown to be 0.015 (IQR, -0.03~0.045) and 0.095 (IQR, 0.0825~0.275) in the indeterminate group, of whom 58.3% (7 cases) showed negative TST results, and 33.3% (3 patients with TB and 1 patient with non-TB group) showed positive TST results. Among the 4 patients with positive TST results in indeterminate group, 1 patient had a previous history of TB treatment, and the patient was diagnosed with reactivated pulmonary TB at the time of colon cancer surgery. The other patient had active pulmonary TB with a history of BCG vaccination. Another patient died of respiratory failure caused by multidrug-resistant TB 20 days after mechanical ventilator management at the intensive care unit (ICU). Another patient in non-TB group who had a previous history of TB treatment had bipolar disorder and was hospitalized in the ICU for 15 days with severe sepsis.

The median age of the indeterminate group was shown to be 70.5 years (IQR, 55.5~76.0), which was significantly higher than that of the determinate group (51.0, IQR 41~66.0) (p=0.011). The age distribution of patients showing indeterminate results on QFT-GIT test is shown in Figure 1. Indeterminate results were significantly more frequent among the age bracket of 65 years or higher (25.0% with indeterminate results vs. 18.4% with determinate results) and age bracket of 75 years or higher (33.3% vs. 7.6%). In the comparison of previous disease history, bronchial asthma (25.0% vs. 3.1%, p=0.004), hypertension (58.3% vs. 21.5%, p=0.008), malignant disease (25.0% vs. 4.6%, p=0.007), history of organ transplantation and steroid administration (8.3% vs. 0%, p=0.019) were significantly higher in the indeterminate group than in the determinate group. The history of ICU stay was shown in 7 patients (58.3%) within the indeterminate group, which was significantly higher than that of the determinate group (18.5%, 12/65) (p=0.003). However, the median hospital stay of the indeterminate group was shown to be 15.0 (IQR, 5.0~40.0), which showed no difference with that of the determinate group (16.5; IQR, 7.0~38.0). The death rate of the indeterminate group was shown to be 25.0% (3 patients), which was higher than that of the determinate group (7.7%, 5/65), but was not statistically significant (p=0.071). In multivariate analysis, in the cases of age of 65 years or higher (odds ratio [OR], 11.29; p=0.026), bronchial asthma (OR, 51.66; p=0.028), malignant diseases (OR, 5.90; p=0.078) and history of ICU stay (OR, 15.47; p=0.018), the frequency was shown to be higher in the indeterminate group than in the determinate group (Table 4).

In the blood tests, the lymphocytopenia (0.7; IQR, [0.4~1.1]×10³/mm³ vs. 1.3; IQR, [0.9~1.7]×10³/mm³; p=0.013), particularly CD4 lymphocytopenia (147.0; IQR, 84.9~390.5/mm³ vs. 583.5; IQR, 332.3~849.0/mm³; p=0.021), CRP elevation (2.7; IQR, 1.8~11.8 mg/dL vs. 1.2; IQR, 0.3~5.0 mg/dL; p=0.014), and hypoproteinemia (6.6; IQR, 5.9~6.8 g/dL vs. 7.2; IQR, 6.5~7.6 g/dL; p=0.026) were significantly associated with indeterminate laboratory findings. However, no difference in the radiologic finding and extension of lesion was found between the two groups.

In the TB group, comparisons among the group aged 65 years or higher (elderly patients, n=12) and the group aged less than 65 years (younger patients, n=32), no significant difference in the test results and demographic characteristics was found between the two groups. However, hypertension among underlying diseases was significantly higher in the elderly patients than in the younger patients (p=0.013). Thirty-six point four percent (n=4) of the elderly patients were shown to be positive response in TST, whereas 78.5% (n=22) of the younger patients were shown to be positive response in TST (p=0.019). In addition, the QFT-GIT positive rate was shown to be 54.5% (n=24) in the younger patients, but only 8.3% (n=1) in the elderly patients, which was significantly lower in the elderly patients than in the younger patients (p=0.000). The rate of the indeterminate (33.3%, n=4) and the false negative results (58.3%, n=7) of QFT-GIT were higher in the elderly patients than in the younger patients (Table 5).

No lymphocytopenia (<500/mm³) was shown in 16 patients (36.4%) who were false negative in QFT-GIT within the TB group. The 16 patients had 2 cases (12.5%) of radiologically extensive lesion, 9 cases (56.3%) of intermediate severity, and 5 cases (31.3%) of mild severity. In addition, in the 21 patients (47.7%) who were false negative or indeterminate in QFT-GIT within the TB group, the result of multivariate analysis showed that age of 65 years or higher (OR, 65.87; 95% confidence interval [CI], 3.812~114.0; p=0.004) and history of ICU stay (OR, 41.90; 95% CI, 2.33~752.4; p=0.011) were risk factors.

In this study, the result of QFT-GIT showed that 15.6% of the subjects were adjudged indeterminate due to positive control failure. The median age of the indeterminate group was 70.5 years, which was significantly higher than that of the determinate group (51.0 years). Indeterminate result was significantly higher in the patients with bronchial asthma and a history of ICU stay. In addition, the results of blood tests showed that the lymphocytopenia, including CD4 lymphocytopenia, CRP elevation, and hypoproteinemia were higher in the indeterminate group. The rate of indeterminate results of QFT-GIT was 11.4% in the TB group, and that of false negative results was 36.4%, which was higher than that of TST (31.8%). In the TB group aged 65 years or higher, the indeterminate results (33.3%) and false negative results (58.3%) accounted for 91.7%, which showed that QFT-GIT alone had a limitation of being used for diagnosis of TB. According to the national statistics reported by the KNTA9, the rate of new TB cases for the past 10 years has been increasing. The rate of new TB cases of the elderly group aged 65 years of higher has been rapidly increasing from 11.5% in 2001 to 22.9% in 2004, and 27.2% in 2008. The possibility of the occurrence of TB is anticipated to increase in the elderly who have aging, various concurrent diseases, and resultant decrease in the immunologic status. In previous study in Taiwan, elderly patients with pulmonary TB were more likely to present with negative sputum smears and had more pleural effusion but fewer cavity findings on chest radiography. The prognosis is poor for the elderly TB patients with lower body weight, coexisting medical diseases, and extensive radiographic disease6. In other study in Korea, difference in AFB smear or culture positive rate between the elderly and the younger has not been observed9. However, it was suggested that the diagnostic yield of the elderly TB patients could be improved by applying methods such as the induced sputum using a saline solution nebulizer or bronchial washing fluid using bronchoscopy10. Potentially infectious or fatal patients should undergo diagnosis and treatment via clinical findings and radiologic examination, although they have negative results in sputum AFB smears or cultures. However, as it is not easy to diagnose and treat active TB in this case, NAA test of M.TB, pathologic examination, radiologic examination, TST and IGRAs are required. However, as the function of the T lymphocytes and amplification reaction decrease in the elderly patients6, the results of not only TST but also IGRAs should be carefully interpreted in elderly patients with TB.

For countries with high BCG vaccination rate and high TB morbidity, IGRAs rather than TST is particularly anticipated to be more helpful for diagnosis of TB infection. However, despite the usefulness of IGRAs in the diagnosis of LTBI, its usefulness in the diagnosis of active TB is still controversial11,12. A recently published meta analysis reported that the sensitivity of blood IGRAs was higher than that of TST for active TB, but its specificity was lower than that of TST, which showed a limitation in the discrimination between LTBI and active TB. The sensitivity and specificity of blood QFT-GIT was reported to be 80% and 79%, respectively. However, the indeterminate rate was also reported to reach 14.9% in adults1. QFT is considered limited if the result is indeterminate due to a difficulty in interpretation. As its frequency is higher than expected, the indeterminate result was reported to be 11% in a study conducted on healthy medical professionals13, most of whom were indeterminate due to positive control failure. Indeterminate result with positive control failure was associated with the decreased number and activity of T lymphocytes or the failure of IFN-γ synthesis in T lymphocytes. In addition, the indeterminate result mainly occurred in patients with human immunodeficiency virus infection, malignant disease and renal dysfunction, which indicated the possibility of abnormal immune system13. Ferrara et al.14 and Kobashi et al.5 reported that 21% and 10.8% of patients who underwent QFT-G were indeterminate, respectively, and that in addition to receive the immunosuppressive drugs, age of 80 years or higher, lymphocytopenia, and malnutrition such as hypoproteinemia were the risk factors of the indeterminate results.

Because the QFT-GIT contains additional TB-specific antigens (TB7.7), it is speculated that the sensitivity is higher and indeterminate result is lower. However, consistent with the results of previous studies using the QFT-G test, the rate of indeterminate results was 15.6% (12 cases) in the total subjects, and 11.4% (5 cases) in the TB group, which was lower than that of the non-TB group in our study. Indeterminate results were due to positive control failure in all 12 patients, and their median age was 70.5 years, which was significantly higher than that of the determinate group (51.0 years). In particular, no patient was adjudged indeterminate in the patients in their 40s or lower, and 67% of the indeterminate group were patients in their 70s or higher, showing a clear difference in the age bracket. The result of a Japanese study showed that no significant difference was found between TST (70%) and QFT-G (87%) in active TB patients aged less than 80 years, but that QFT positive rate (76%) was significantly higher than TST positive rate (27%) in active TB patients aged 80 years or higher. Thus, it was suggested that QFT could be more useful than TST if the number of lymphocytes is normal in the elderly patients15. In our study, however, both TST positive rate (36.4% vs. 78.5%) and QFT-GIT positive rate (8.3% vs. 54.5%) were significantly lower in the elderly patients aged 65 years or higher than in the patients aged less than 65 years within the TB group. In addition, the results of indeterminate and false negative rate in the elderly patients aged 65 years or higher were 33.3% and 58.3%, respectively, which accounted that 91.7% in elderly patient was not diagnostic with QFT-GIT. Unlike indeterminate results due to positive control failure, QFT-GIT negative result refers to the cases of insufficient or no secretion of IFN from T cells that are stimulated by TB antigens despite its proper response to control antigens. It mainly occurs by the non-responsive state of cellular immune system, but may occur in the normal immune system. In addition, false negative result in QFT was reported to more occur in the cases of small amount of TB pathogen in the cultured sputum, small range of the lesion, and extrapulmonary TB such as TB pleurisy than in the case of lymphocytopenia15. But in our study, the patients with false negative QFT-GIT had more advanced disease (radiologically extensive to intermediate severity in 68.7%) and most of whom were pulmonary TB except 2 miliary TB (13.3%) and 1 TB pleurisy (6.7%).

In previous studies reporting that the indeterminate rates of QFT-G and QFT-GIT varied from 0.1 to 40%, patients receiving immunosuppressive drugs and immunocompromised patients were mainly included in the subjects7,14-19. Whereas, in this study, immunocompromised patients, patients receiving immunosuppressive drugs, and patients with end-stage renal disease accounted for only 7.8% of the total subjects, but the rate of indeterminate result was 15.6%, which was not low. The main reason for this result is likely that our study population had many elderly and severely ill patients. The high indeterminate rate in the elderly patients is currently accepted to be associated with lymphocytopenia and malnutrition, as suggested by Kobashi et al.7. QFT test depends on inflammatory cytokines synthesized from T lymphocytes that are activated by TB-specific antigens. As the amount of IFN-γ produced from the sensitized T cells is measured via ELISA in QFT test, IFN-γ production is likely to decrease in the case of lymphocyte insufficiency18. In addition, Ferrara et al.14 reported that the rate of indeterminate result was higher in patients with the negative TST results, compared with the TST positive results (29% vs. 7%). In our study, the rate of indeterminate result was higher in the patients with negative TST results (19.4%) than positive TST patients (11.8%). In addition, 58.3% of the indeterminate group had a history of ICU stay. In the cases of lymphocytopenia, particularly CD4 lymphocytopenia, CRP elevation, and hypoproteinemia in the blood tests were significantly associated with indeterminate results with the positive control failure. This result may also occur in patients with chronic disease and patients requiring ICU stay as well as in patients receiving immunosuppressive drugs. Thus, it is likely that difference in the immune responses of the elderly, chronic disease, and severely-ill patients could have affected the result. In addition, Kim et al.20 reported that the quantitative amount of IFN-γ secretion and the number of CD4+T cells secreting IFN-γ were inversely proportional to the severity of pulmonary TB, which was consistent with the results of previous studies. They also reported that the limited sensitivity of QFT was shown in severe pulmonary TB due to the high indeterminate rate (19.0%) in QFT20. In this study, however, due to the small number of TB patients within the indeterminate group, no correlation of indeterminate results with the extent of TB lesion was found.

We should consider the limitations of this study. Although a number of publications have demonstrated that excessive administration of blood samples to the tube may dilute IFN-γ concentration, the survived T cells may be lost due to the delay after blood collection, and the rate of indeterminate result decreased by approximately 3% due to manual vortexing before cell culture13, our study includes no information with variables of QFT-GIT. So, the increased rate of indeterminate result due to the errors of the test itself cannot be excluded. In addition, the time from blood collection to culturing was not checked in this study, despite the principle that blood collection should be conducted within 2 hours after delivery to the examination organization. Furthermore, although re-sampling is recommended to exclude the above test errors in the case of the positive control failure14, the re-sampling was not performed in this study. Thus, it is not clear whether non-responsive IFN-γ was attributable to test errors, or it was a transient phenomenon, or non-responsive IFN-γ can be reversible with recovery from TB. Second, the numbers of the elderly patients with active TB and indeterminate group were insufficient to analyze within group. Third, BCG vaccination after birth is mandatory in Korea, and if the response to TST is negative at the time of senior in elementary school, BCG vaccination has been commonly administered again until 1997. However, it is uncertain to identify BCG vaccination status in a clinical practice, so we evaluated the status of vaccination with examination based on vaccination scar in some patients.

In this study, the sensitivity and specificity of QFTGIT were 52.3% and 57.6%, respectively, which were lower than those of previous studies. That is, compared to the previous studies, the rates of false negative and indeterminate result were higher in this study. This is likely to be attributable to the higher proportion of severely-ill patients such as patients with chronic diseases and elderly patients, and patients requiring ICU stay and the differences in TB morbidity. In Korea, IGRAs are recommended for screening patients before immunosuppressive therapy with anti-tumor necrosis factor-α and for confirming TB infection in anyone who is TST-positive after contact with a patient with active pulmonary TB. However, IGRAs have not been recommended for use in the diagnosis of active TB. Nevertheless, based on previous study results, frequent use IGRAs in routine clinical practice suggesting a negative test result could rapidly exclude TB from a differential diagnosis11. As it is, however, impossible to discriminate LTBI and active pulmonary TB via QFT in countries with a high burden of TB, the use of QFT test for the diagnosis of active pulmonary TB has a specific concern in Korea with a modest morbidity rate of TB. In addition, in the cases of elderly patients, severely-ill patients requiring ICU stay, lymphocytopenia, CRP elevation and hypoproteinemia, indeterminate results that is not useful for diagnosis, or false negative results may be shown. Thus, a precaution should be given in the interpretation of the results. A multicenter IGRAs study, which is conducted on elderly Korean patients with active TB, is required, and a further study is also required to investigate the mechanism of indeterminate or false negative results.