Tuberculosis Research and Treatment

Tuberculosis Research and Treatment / 2012 / Article
Special Issue

Tuberculosis Diagnostics in the New Millennium: Role in TB Identification and Control

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Clinical Study | Open Access

Volume 2012 |Article ID 768723 | 4 pages |

The Use of Interferon Gamma Release Assays in the Diagnosis of Active Tuberculosis

Academic Editor: Soumitesh Chakravorty
Received25 Nov 2011
Revised05 Jan 2012
Accepted06 Jan 2012
Published22 Jan 2012


Interferon gamma release assays (IGRAs) are in vitro immunologic diagnostic tests used to identify Mycobacterium tuberculosis infection. They cannot differentiate between latent and active infections. The cutoff suggested by the manufacturer is 0.35 IU/mL for latent tuberculosis. As IGRA tests were recently approved for the differential diagnosis of active tuberculosis, we assessed the diagnostic accuracy of the latest generation IGRA for detection of active tuberculosis in a low-incidence area in Germany. Our consecutive case series includes 61 HIV negative, Mycobacterium tuberculosis culture positive patients, as well as 234 control patients. The retrospective analysis was performed over a period of two years. In 11/61 patients with active tuberculosis (18.0%) the test result was <0.35 IU/mL, resulting in a sensitivity of 0.82. We recommend establishing a new cut-off value for the differential diagnosis of active tuberculosis assessed by prospective clinical studies and in various regions with high and low prevalence of tuberculosis.

1. Introduction

Tuberculosis (TB) remains a major public health problem affecting one-third of the world’s population [1, 2]. Diagnosis of TB is usually based on a combination of anamnestic symptoms, clinical presentation, radiological and pathological changes, bacteriological findings of acid/alcohol-fast bacilli, and molecular tests [3]. Definitive TB diagnosis is based on the detection of Mycobacterium tuberculosis (MTB) in the culture, which usually takes four to six weeks. For decades, tuberculin skin test (TST) has been used as diagnostic tool to support the physician’s decision process. With the introduction of interferon gamma release assays (IGRAs), a more specific method became available. Although primarily developed for the diagnosis of latent TB, clinicians have also been searching for improved diagnostic tools and explored IGRAs for the immunodiagnosis of active TB. In 2010, the Centers for Disease Control and Prevention (CDC) updated their guidelines for testing for TB infection, concluding that IGRAs “may be used instead of a tuberculin skin test in all situations in which the CDC recommends the tuberculin skin test as an aid in diagnosing M. tuberculosis infection” [4, 5].

Nevertheless, with the cutoff for the diagnosis of latent TB as given by the producers, pooled sensitivity for the diagnosis of culture positive TB did not exceed 80% in the most recent meta-analyses [6, 7]. The present case series constitutes one of the largest reports of latest generation IGRA used in culturally proven HIV-negative TB cases in a low-prevalence country. Our study is meant to help evaluate the cutoff for the IGRA in the differential diagnosis of TB.

2. Study Population and Methods

This is a retrospective study performed on inpatients of a regional hospital specialized in lung diseases (Lungenklinik Heckeshorn, Berlin). At least one IGRA is routinely performed on a blood sample of each TB-suspect patient, and every suspicious sample (smear, lymph node biopsy, pleural effusion, or biopsy) is routinely cultured for TB. All samples of patients included in this study were taken prior to initiation of antibiotic therapy. Over a two-year period (1/2008–1/2010), IGRA results of all MTB culture positive cases of active TB were analyzed. Patients with other lung diseases than TB, including negative history for MTB infection and without radiological findings suggestive of MTB infection in the past and with no signs of active disease, were chosen as control group. All HIV-positive patients were excluded from the database.

2.1. IGRA

We used the latest generation IGRA (QuantiFERON-TB Gold in-Tube, Cellestis, Carnegie, Australia), later referred to as QFT-GIT, on all samples. Peripheral blood samples were obtained by trained personnel in specific blood collection tubes following the manufacturer’s instructions. All blood samples were processed within 4 h of phlebotomy. Otherwise, the test was performed as previously described [8].

2.2. TB Culture

Specimens were stained, processed, and cultured by standard procedures in mycobacteriology [9]. The isolates were cultured for 4 weeks on Löwenstein-Jensen (L-J) medium at 37°C and tested for growth rate, pigment production, and by biochemical testing using standard methods [10, 11].

2.3. Statistical Analysis

Data were analyzed using STATA 12.0 (StataCorp, College Station, Texas, USA). The t-test for independent samples (two-tailed) was carried out to assess significance level of detected differences in both groups (IGRA test results, age).

3. Results

A total of 61 patients with active TB as ascertained through positive MTB culture were examined using QFT-GIT between 1/2008 and 1/2010 (see Table 1(a)). 53 patients presented with pulmonary TB, three patients with lymph node TB, and five patients with tuberculous pleurisy. The median QFT-GIT value for all TB patients was 3.46 IU/mL, ranging from 0.00 to 300 IU/mL. In 11 patients, the test result was <0.35 IU/mL (see Figure 1). Assuming the cutoff for latent TB suggested by the producer, these are false-negative (FN) test results (11/61 = 0.18; 18% FN). We calculated the sensitivity (the proportion of patients with active TB who are correctly identified as such) as 1-FN (=82%). Only in one patient four months after therapy with adalimumab, the result was 0.00 IU/mL, and in one other patient with lung cancer under concomitant chemotherapy, it was 0.02 IU/mL. All other patients showed values of 0.07 IU/mL or above. The median age of our patients was 46 years (range: 5–84 years), in the group with a test result ≥0.07 and <0.35 IU/mL, the median age was 80 years (range: 44–84 years), and, in the group testing ≥0.35 IU/mL, it was 36 years (range: 5–84 years).


MTB infectionMaleFemaleTotalMedian age, years (range)Median QFT, IU/mL (range)

Lung28255345 (4–83)2.40 (0,00–103,4)
Pleura23526 (17–76)2,45 (0,96–300)
Lymph node21365 (25–69)21,32 (3,78–86,8)

Total32296146 (5–84)3.46 (0,00–300)


DiagnosisMaleFemaleTotalMedian age, years (range)Median QFT, IU/mL (range)

Malignancy22234569 (39–90)0,01 (0,00–23,9)
Bronchitis27134064 (1–90)0,00 (0,00–1,84)
Pneumonia30174767 (1–99)0,00 (0,00–75)
Others534910262,5 (5–99)0,02 (0,00–103,4)

Total13210223466 (1–99)0,00 (0,00–103,4)


Mean95% CI Mean95% CI


Within the control group (see Table 1(b)), QFT-GIT values ranged from 0.00 to 103.4 IU/mL in one patient with pneumonia. The median was 0.00 IU/mL. In 50% of all cases (116/234), no Interferon gamma (IFN-γ) release was measured (0.00 IU/mL). In 51/234 patients (21%), test results were above 0.35 IU/mL. The median age in the control group was 66 years with a range from 1 to 99 years.

Patients in the study group were significantly younger than in the control group and had significantly higher QFT-GIT test results ( for both, see Table 1(c)).

4. Discussion

IGRAs are in vitro immunologic diagnostic tests to identify MTB infection. Latent and active infections are not differentiated. Several test systems are commercially available: QuantiFERON-TB Gold (QFT), QuantiFERON-TB Gold in-Tube (QFT-GIT), both Cellestis Limited, Chadstone, Australia, and T-SPOT.TB (Oxford Immunotec, Abingdon, UK). The QFT-GIT measures IFN-γ responses to the MTB specific antigens early secretory antigenic target 6 (ESAT-6), culture filtrate protein 10 (CFP-10), and Rv2654 (TB 7.7). Their use in clinical practice is more and more widespread. Whereas the test was initially conceived to support diagnosis of latent infection, an increasing body of evidence is published on its use in detection of active TB infection [1214]. More and more guidelines now include recommendations for or against the use of IGRAs in the differential diagnosis of active TB [15].

Several systematic reviews and meta-analyses have recently been published specifically on the diagnostic accuracy of IGRAs in active TB [6, 16, 17]. Strong heterogeneity between study populations is a major limitation of these meta-analyses, and most studies had small sample sizes. Overall, few studies were done with the latest generation QFT-GIT in areas of low endemicity such as Germany (2 studies in [17], 13 studies in [16]). Among those, even fewer restricted sensitivity analysis to patients with culturally proven MTB infection and confirmed HIV-negative status [12, 18].

Specificity depends highly on the definition of the control group. Pai et al. reported a pooled specificity of 99% among non-BCG vaccinated and 96% among BCG-vaccinated low-risk groups [17]. According to a recent meta-analysis that did not restrict studies on specificity to low-risk groups [6], a situation more compatible to our clinical setting, the specificity of QFT-GIT was only 0.79 (95% CI 0.75–0.82). In our study, 51 out of 234 control patients (21%) showed test results above 0.35 IU/mL, indicative of latent TB according to the producer. However, this finding is also consistent with the expected number of false positives assuming a specificity of around 0.8 according to Sester et al. [6]. The retrospective design of our study does not allow any conclusion about the test specificity in our study population.

Sensitivity in these studies was also found to be highly dependent on the study population, notably local TB prevalence, and ranged from 0.58 in a high-prevalence country [19] to 1.00 in a low-prevalence country [20], when QFT-GIT was assessed. Diel et al. found a pooled sensitivity of 0.84 (95% CI 0.81–0.87) when including only developed countries [16], consistent with the results published by Sester et al. (0.77, 95% CI 0.75–0.80) [6].

In our consecutive case series of 61 TB culture positive, HIV-negative patients in a low-prevalence setting in Germany, we found a sensitivity of 82.0% (95% CI: 0.696, 0.902) for the QFT-GIT, when the cutoff recommended for the diagnosis of latent TB was used (<0.35 IU/mL). In a low-prevalence country such as Germany with a TB prevalence of 5.4/100,000 inhabitants [21], case finding is an outstanding priority in the management of this disease. Therefore, highly sensitive test systems are needed for screening purposes. If the use of IGRA in the differential diagnosis of active TB is recommended, at least under certain conditions, then the cut-off point for active TB should be lower compared to latent TB, as previously discussed by Davidow [22]. Prospective clinical studies in different defined regions, with high- and low-prevalence of active TB, should be performed to evaluate the use of IGRAs in the diagnostic workup of TB patients. The cutoff for the detection of latent TB does not seem applicable for that purpose.

5. Conclusions

We suggest that the cutoff for the use of IGRA in the differential diagnosis of active TB in low incidence settings be reevaluated. Further prospective studies including clinical criteria for TB are needed to determine a new cutoff for active TB.

Authors’ Contribution

H. Mauch, N. Schönfeld, and S. M. Vesenbeckh designed the study, T. Bergmann and S. Wagner managed data acquisition and compilation, N. Schönfeld and S. M. Vesenbeckh analysed the data, T. T. Bauer and S. M. Vesenbeckh did statistical analysis, S. M. Vesenbeckh wrote the paper, and T. T. Bauer, H. Mauch, H. Rüssmann, and N. Schönfeld reviewed and edited the manuscript.


This work was funded by HELIOS Klinikum Emil von Behring and received support by DZK (Deutsches Zentralkomitee zur Bekämpfung der Tuberkulose) and OHH (Stiftung Oskar-Helene-Heim).

Conflict of Interests

The authors declare that there is no conflict of interests.


  1. C. Dye et al., “Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project,” the Journal of the American Medical Association, vol. 282, no. 7, pp. 677–686, 1999. View at: Google Scholar
  2. World Health Organization, “Tuberculosis,” 2011, View at: Google Scholar
  3. World Health Organization, “Treatment of tuberculosis: guidelines for national programmes,” WHO/CDS/TB/2003.313, Geneva, Switzerland, 2003. View at: Google Scholar
  4. G. H. Mazurek, J. Jereb, A. Vernon, P. LoBue, S. Goldberg, and K. Castros, “Updated guidelines for using interferon gamma release assays to detect Mycobacterium tuberculosis infection—United States, 2010,” Morbidity and Mortality Weekly Report, vol. 59, no. RR-5, pp. 1–25, 2010. View at: Google Scholar
  5. L. Barclay, “CDC issues updated guidelines for testing for tuberculosis infection,” View at: Google Scholar
  6. M. Sester, G. Sotgiu, C. Lange et al., “Interferon-γ release assays for the diagnosis of active tuberculosis: a systematic review and meta-analysis,” European Respiratory Journal, vol. 37, no. 1, pp. 100–111, 2011. View at: Publisher Site | Google Scholar
  7. J. Z. Metcalfe, C. K. Everett, K. R. Steingart et al., “Interferon-γ release assays for active pulmonary tuberculosis diagnosis in adults in low-and middle-income countries: systematic review and meta-analysis,” Journal of Infectious Diseases, vol. 204, supplement 4, pp. S1120–S1129, 2011. View at: Publisher Site | Google Scholar
  8. B. Kampmann, E. Whittaker, A. Williams et al., “Interferon-γ release assays do not identify more children with active tuberculosis than the tuberculin skin test,” European Respiratory Journal, vol. 33, no. 6, pp. 1374–1382, 2009. View at: Publisher Site | Google Scholar
  9. E. D. Roberts, E. W. Koneman, and Y. K. Kim, “Mycobacterium,” in Manual of Clinical Microbiology, H. W. J. J. Balows, K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy, Eds., pp. 304–339, Washington, DC, USA, 1991. View at: Google Scholar
  10. P. T. Kent and G. P. Kubica, Public Health Mycobacteriology—A Guide for the Level III Laboratory, vol. 86, U. S. Department of Health and Human Services Publication (CDC), 1985.
  11. V. V. Levy-Frebault and F. Portaels, “Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species,” International Journal of Systematic Bacteriology, vol. 42, no. 2, pp. 315–323, 1992. View at: Google Scholar
  12. A. K. Detjen, T. Keil, S. Roll et al., “Interferon-γ release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis,” Clinical Infectious Diseases, vol. 45, no. 3, pp. 322–328, 2007. View at: Publisher Site | Google Scholar
  13. L. Bianchi, L. Galli, M. Moriondo et al., “Interferon-gamma release assay improves the diagnosis of tuberculosis in children,” Pediatric Infectious Disease Journal, vol. 28, no. 6, pp. 510–514, 2009. View at: Publisher Site | Google Scholar
  14. V. Bartu, M. Havelkova, and E. Kopecka, “QuantiFERON-TB gold in the diagnosis of active tuberculosis,” Journal of International Medical Research, vol. 36, no. 3, pp. 434–437, 2008. View at: Google Scholar
  15. C. M. Denkinger, K. Dheda, and M. Pai, “Guidelines on interferon-γ release assays for tuberculosis infection: concordance, discordance or confusion?” Clinical Microbiology and Infection, vol. 17, no. 6, pp. 806–814, 2011. View at: Publisher Site | Google Scholar
  16. R. Diel, R. Loaddenkemper, and A. Nienhaus, “Evidence-based comparison of commercial Interferon-γ Release assays for detecting active TB a metaanalysis,” Chest, vol. 137, no. 4, pp. 952–968, 2010. View at: Publisher Site | Google Scholar
  17. M. Pai, A. Zwerling, and D. Menzies, “Systematic review: T-cell-based assays for the diagnosis of latent tuberculosis infection: an update,” Annals of Internal Medicine, vol. 149, no. 3, pp. 177–184, 2008. View at: Google Scholar
  18. R. Palazzo, F. Spensieri, M. Massari et al., “Use of whole-blood samples in in-house bulk and single-cell antigen-specific gamma interferon assays for surveillance of Mycobacterium tuberculosis infections,” Clinical and Vaccine Immunology, vol. 15, no. 2, pp. 327–337, 2008. View at: Publisher Site | Google Scholar
  19. K. Baba, S. Sørnes, A. A. Hoosen et al., “Evaluation of immune responses in HIV infected patients with pleural tuberculosis by the QuantiFERON TB-Gold interferon-gamma assay,” BMC Infectious Diseases, vol. 8, article no. 35, 2008. View at: Publisher Site | Google Scholar
  20. I. Sauzullo, F. Mengoni, M. Lichtner et al., “In vivo and in vitro effects of antituberculosis treatment on mycobacterial interferon-γ T cell response,” PLoS One, vol. 4, no. 4, Article ID e5187, 2009. View at: Publisher Site | Google Scholar
  21. Robert Koch Institut, “RKI-Bericht zur epidemiologie der tuberkulose in deutschland für 2009,” 2011; View at: Google Scholar
  22. A. L. Davidow, “Interferon-gamma release assay test characteristics depend upon the prevalence of active tuberculosis,” International Journal of Tuberculosis and Lung Disease, vol. 13, no. 11, pp. 1411–1415, 2009. View at: Google Scholar

Copyright © 2012 Silvan M. Vesenbeckh et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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