Supported by a grant from the Korea Healthcare Technology R&D Project (A080588), Ministry for Health, Welfare & Family Affairs, Republic of Korea.
Prospective evaluation of the clinical utility of interferon-γ assay in the differential diagnosis of intestinal tuberculosis and Crohn's disease†
Article first published online: 25 OCT 2010
Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.
Inflammatory Bowel Diseases
Volume 17, Issue 6, pages 1308–1313, June 2011
How to Cite
Kim, B. J., Choi, Y. S., Jang, B. I., Park, Y. S., Kim, W. H., Kim, Y. S., Jung, S.-A., Han, D. S., Kim, J. S., Choi, J. H., Choi, C. H., Jeen, Y. T., Cheon, J. H., Ye, B. D., Yang, S.-K. and Kim, Y.-H. (2011), Prospective evaluation of the clinical utility of interferon-γ assay in the differential diagnosis of intestinal tuberculosis and Crohn's disease. Inflamm Bowel Dis, 17: 1308–1313. doi: 10.1002/ibd.21490
- Issue published online: 10 MAY 2011
- Article first published online: 25 OCT 2010
- Manuscript Received: 18 AUG 2010
- Manuscript Accepted: 18 AUG 2010
- intestinal tuberculosis;
- Crohn's disease;
- interferon-γ assay;
- tuberculin skin test
Distinguishing intestinal tuberculosis (ITB) from Crohn's disease (CD) is challenging. This study prospectively evaluated the clinical utility of the QuantiFERON-TB gold test (QFT) in the differential diagnosis of ITB and CD, and compared it with the clinical utility of the tuberculin skin test (TST).
Patients with suspected ITB or CD on colonoscopic findings were enrolled from 13 hospitals in Korea between June 2007 and November 2008. A QFT and TST were performed. When the initial diagnosis was not confirmed, 2–3 months of empiric antituberculous therapy was administered.
In all, 128 patients were analyzed; 64 patients had ITB and 64 patients had CD. The median age of patients with ITB was greater than the patients with CD (47 years versus 31 years, P < 0.001). The positive rate for the QFT and TST (≥10 mm) in patients with ITB was significantly higher than patients with CD (67% versus 9% and 69% versus 16%, respectively; P < 0.001). The QFT and TST had good agreement (κ = 0.724, P < 0.001). The diagnostic validity of QFT in ITB had a 67% sensitivity, 90% specificity, 87% positive predictive value, and 73% negative predictive value. There was no difference in these parameters between the QFT and TST. The likelihood ratio for a positive QFT was higher than a positive TST in the diagnosis of ITB (7.1 and 4.4, respectively).
The QFT is a limited but useful diagnostic aid in combination with the TST in the diagnosis of ITB. (Inflamm Bowel Dis 2011;)
The global prevalence of Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), is ≈30%. TB is one of the major worldwide causes of morbidity and mortality.1, 2 Along with the increased incidence of TB, intestinal tuberculosis (ITB) has also increased in incidence. With the reemergence of TB in Western countries as a result of the acquired immunodeficiency syndrome (AIDS) epidemic3 and the emergence of Crohn's disease (CD) in Asian countries,4 possibly a result of Westernization, differentiating between ITB and CD is more important than ever. Unfortunately, it is difficult to differentiate ITB from CD because of similar clinical, pathologic, radiologic, and endoscopic findings.5, 6 Although the diagnosis of ITB can be confirmed when caseating granulomas or acid-fast bacilli are identified during the histologic examination of colonoscopic biopsy specimens or when MTB is isolated from the culture of biopsy specimens, these findings are positive in <50% of patients with ITB.6 Moreover, CD has no pathognomic findings. While various clinical, radiologic, endoscopic, and histologic parameters may aid in differentiating CD from ITB, these parameters are imperfect, and as treatment options differ, misdiagnosis has grave consequences.6, 7
The QuantiFERON-TB gold test (QFT; Cellestis, Victoria, Australia), an interferon (IFN)-γ release assay, is a blood test that measures the release of IFN-γ after stimulation in vitro by MTB antigens.8 The main advantage of this assay with respect to the tuberculin skin test (TST) is the lack of crossreaction with bacillus Calmette-Guérin (BCG) and most nontuberculous mycobacteria. QFT also eliminates the need for the patient to return for test reading in 48–72 hours. It has also been used to identify false-positive TSTs given its high specificity. In previous studies, this assay showed high sensitivity and specificity in patients with latent pulmonary TB and correlated better with exposure to MTB compared to TST.9 Recently, QFT has been used for the diagnosis of active TB, as well as latent TB infection (LTBI) in some clinical settings.10, 11 Most studies on this test have been performed on pulmonary TB.
To date, there has been a lack of studies that evaluate the IFN-γ release assay in patients with extrapulmonary TB, especially ITB in the differentiation from CD. Although there is a report describing the use of the QFT in the diagnosis of two cases of ITB,12 the value has not been formally studied in ITB. Therefore, the present study evaluated the clinical utility of the QFT in the differential diagnosis of ITB and CD, as compared with the clinical utility of the TST.
MATERIALS AND METHODS
Between June 2007 and November 2008 we prospectively enrolled patients who were suspected to have ITB or CD on initial work-up, as based on the results of colonoscopy from 13 general hospitals under the auspices of the Korean Association for the Study of Intestinal Disease in Korea. All the patients were >18 years of age and had subjective symptoms, including abdominal pain and/or diarrhea. Exclusion criteria included subjects with active pulmonary TB or a history of using anti-TB medications, pregnancy, use of immunosuppressant medications, subjects with neutropenia (<1000/mL) after chemotherapy or transplantation, and subjects with an alanine aminotransferase (ALT) >200 U/L.
A polyethylene glycol electrolyte lavage solution was used in all patients for bowel preparation before colonoscopy. The colonoscopic parameters in this study included endoscopic characteristics of ulcers, such as aphthous ulcers, longitudinal ulcers, or a cobblestone appearance. Biopsy specimens were obtained for hematoxylin and eosin staining, Ziehl-Neelsen and fluorescence smear for acid-fast bacilli (AFB), and Bactec culture for AFB. In some cases with uncertain diagnoses, additional radiologic studies, such as an abdominopelvic computed tomography (CT) or barium study were undertaken for differential diagnosis. When the initial diagnosis was not confirmed, 2–3 months of empiric antituberculous therapy with isoniazid, rifampicin, ethambutol, and pyrazinamide was administered. The final diagnosis was analyzed after follow-up because a confirmed diagnosis was not made at the initial work-up in many cases. The study was approved by the ethics committees of each institution and informed consent was obtained from all patients.
Twelve milliliters of peripheral blood were drawn from each patient for the QFT. The QFT test was performed in two stages according to the manufacturer's instructions. First, 1 mL of heparinized whole blood was incubated with aliquots of antigen-free control and antigen ESAT-6, CFP-10, or phytohemagglutinin for 16–24 hours at 37°C in a carbon dioxide incubator. Then, after overnight incubation, 200 μL of plasma was removed from each well and the concentration of IFN-γ was determined using the assay kit according to the manufacturer's instructions (Cellestis, Victoria, Australia). A QFT test value of 0.35 IU/mL was used as the cutoff for a positive response.
After collection of blood samples for the QFT, a TST was performed on the volar side of the forearm according to the Mantoux method using a 2-TU dose of purified protein derivative RT23 (Statens Serum Institut, Copenhagen, Denmark), and any induration was measured in millimeters after 48–72 hours using the ballpoint method. We used a 10-mm induration as a positive cutoff value for the TST.
Diagnostic Criteria for ITB and CD
The diagnosis of ITB was established when at least one of the following criteria was met: 1) histologic evidence of a caseating granuloma; 2) histologic demonstration of AFB; or 3) growth of MTB on tissue culture. For the diagnosis of CD, the Japanese diagnostic criteria were used.12
In patients in whom the differentiation between ITB and CD was uncertain, antituberculous therapy was tried for 2–3 months and the final diagnosis was made based on the clinical and endoscopic response to antituberculous therapy.6 Clinical response was determined by loss of subjective symptoms. Endoscopic response was determined by disappearance of ulcerations.
Data from the two groups were compared using Student's t-test, Pearson's chi-squared test, or Fisher's exact test. A P-value < 0.05 was considered statistically significant. Ninety-five percent confidence intervals (CIs) were estimated according to a binomial distribution. Concordance between test results from the TST and the QFT was assessed using κ coefficients (κ > 0.75, excellent agreement; κ < 0.4, poor agreement; and κ between 0.4 and 0.75, good agreement). The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), likelihood ratio of a positive test result (LR+), and likelihood ratio of a negative test result (LR−) for the diagnosis of ITB were calculated for each diagnostic test. Statistical analysis was conducted using PASW Statistics 17.0 (SPSS, Chicago, IL).
Characteristics of Patients
In all, 165 patients with suspected ITB or CD on colonoscopy were prospectively enrolled in this study. Of the 165 patients included in this study, five patients were lost to follow-up before the diagnosis was confirmed and 25 patients in whom the diagnosis was changed from ITB or CD to other diseases during follow-up were excluded: 19 of them were finally diagnosed as nonspecific enterocolitis, three were Behçet's colitis, and three were infectious colitis. Seven patients with indeterminate QFT results were also excluded. Therefore, 37 patients were excluded from the analysis (Fig. 1). The data analyses were performed on 128 patients (72 men and 56 women); ITB was diagnosed in 14 patients and CD was diagnosed in 50 patients based on the initial work-up, but 64 patients who had suspected ITB or CD based on endoscopic findings, but had not met the diagnostic criteria, were administered empirical antituberculous therapy for 2–3 months. Among the 64 patients, 50 and 14 patients were confirmed to have ITB and CD, respectively, based on the clinical and endoscopic response to antituberculous therapy.
As a consequence, there were 64 patients with a final diagnosis of ITB and 64 patients with a final diagnosis of CD. The male-to-female ratio was 30:34 in patients with ITB and 42:22 in patients with CD (P = 0.003). The median age at the time of diagnosis was 47 years (range, 21–79 years) in patients with ITB and 31 years (range, 19–68 years) in patients with CD (P < 0.001).
Diagnosis of ITB
In 14 of the 64 patients with ITB (21.9%), the confirmed diagnosis was made at the initial work-up by one or more of the following methods: histologic evidence of caseating granulomas in eight patients, histologic demonstration of AFB in three patients, growth of MTB on tissue culture in four patients. In the remaining 50 patients (78.1%), the diagnosis of ITB was confirmed after a period of follow-up based on the patient's response to antituberculous therapy.
The patients with ITB were separated into two groups: microbiologically diagnosed group versus clinically diagnosed group with assumption of ITB based on treatment response. In comparison between the two groups, there was no significant difference in age, sex, endoscopic finding, TST result, and QFT result (P > 0.05).
Diagnosis of CD
At the time of the initial work-up, 50 patients fulfilled the Japanese diagnostic criteria for CD. A total of 14 patients (21.9%) with assumed CD underwent anti-TB treatment (10 men, 4 women; median age, 27 years; range 18–68 years). Ten patients had negative TST, whereas 13 patients had negative QFT. There was no documented response to this therapy.
Results of the QFT and TST
A comparison of the results for the TST and QFT test based on the final diagnosis in patients with ITB and CD is shown in Table 1. As shown in Table 1, both positive QFT and TST results were more prevalent in patients with ITB than patients with CD.
|No. of subjects||64||64|
|TST ≥ 10 mm (%)||44 (68.8%)||10 (15.6%)||<0.001|
|QFT (+) (%)||43 (67.2%)||6 (9.4%)||<0.001|
The results of the QFT were positive in 49 (38.3%) of 128 patients, 43 of whom (87.8%) were diagnosed with ITB; the results of the QFT were negative in 79 patients (61.7%), 21 of whom (26.6%) were diagnosed with ITB. Eleven of 14 patients (78.6%) with histologic evidence of caseating granulomas, histologic demonstration of AFB, and growth of MTB on tissue culture had positive QFT results. Interestingly, among 25 patients excluded without an eventual diagnosis of ITB or CD, negative QFT results were represented in 20 patients (80.0%).
When using 10 mm of induration as the cutoff for a positive TST, results were positive in 54 patients (42.2%), 44 of whom (81.5%) were diagnosed with ITB. Negative TST results were recorded in 74 patients (57.8%), 20 of whom (27.0%) were diagnosed with ITB.
Table 2 shows the concordance between the QFT and TST to be in good agreement (κ = 0.724, P < 0.001).
As for the QFT result in patients with ITB, 43 of 64 patients (67.2%) showed positive QFT whereas 21 (32.8%) showed negative QFT. When the patients with ITB were classified into two groups (positive QFT group versus negative QFT group), endoscopic findings favoring ITB and positive TST in the positive QFT group was more than those in the negative QFT group (P = 0.03 and P < 0.01, respectively). Otherwise, there was no statistical difference between the two groups in age, sex, and improvement in response to the empirical therapy (P > 0.05, respectively).
With regard to the QFT result in patients with CD, 6 of 64 patients (9.4%) showed positive QFT whereas 58 (90.6%) showed negative QFT. When the patients with CD were classified into two groups (positive QFT group versus negative QFT group), positive TST in the positive QFT group was more than that in the negative QFT group (P < 0.01). Otherwise, there was no statistical difference between the two groups in age, sex, endoscopic findings favoring CD, and improvement in response to the empirical therapy (P > 0.05).
Diagnostic Validity of QFT and TST in ITB
The sensitivities of the QFT and TST in the diagnosis of ITB were 67% (95% CI, 54%–78%) and 68% (95% CI, 55%–79%), respectively, and the specificities were 90% (95% CI, 80%–96%) and 84% (95% CI, 72%–91%). There were no differences in sensitivity and specificity between the QFT and TST. The PPV and NPV of the QFT in the diagnosis of ITB were statistically similar to the TST. When positive QFT and TST results were combined, the results were statistically similar to a single test (Table 3).
|Sensitivity (95% CI)||Specificity (95% CI)||Positive Predictive Value (95% CI)||Negative Predictive Value (95% CI)|
When patients who had been treated empirically were excluded from the analysis, the sensitivity, specificity, PPV, and NPV of the QFT test in the diagnosis of ITB were 78% (95% CI, 48%–94%), 90% (95% CI, 77%–96%), 68% (95% CI, 41%–87%), and 93% (95% CI, 81%–98%), respectively. By comparison, the sensitivity, specificity, PPV, and NPV of the TST in the diagnosis of ITB were 85% (95% CI, 56%–97%), 88% (95% CI, 74%–95%), 66% (95% CI, 41%–85%), and 95% (95% CI, 83%–99%), respectively.
The LR+ of the QFT was greater than the TST in the diagnosis of ITB (7.1 and 4.4, respectively); when the QFT and TST were combined, however, the LR+ was 7.6. The LR− of the QFT was 0.3, which was similar to the TST in the diagnosis of ITB (Table 4).
Finally, we analyzed the diagnostic validity of QFT and TST in patients whose diagnoses were unclear at initial work-up. The 68 patients were diagnosed as ITB or CD, based on the culture of MTB (n = 4) or the response to empirical treatment (n = 64). In this group, the sensitivity, specificity, PPV, NPV, LR+, and LR− of the QFT test in the diagnosis of ITB were 64% (95% CI, 50%–76%), 92% (95% CI, 64%–99%), 97% (95% CI, 83%–99%), 40% (95% CI, 24%–59%), 9.0 (1.4–60.6), and 0.3 (0.3–0.6), respectively. By comparison, the sensitivity, specificity, PPV, NPV, LR+, and LR− of the TST in the diagnosis of ITB were 66% (95% CI, 52%–78%), 71% (95% CI, 42%–90%), 90% (95% CI, 75%–96%), 35% (95% CI, 19%–55%), 2.3 (0.1–6.0), and 0.4 (0.3–0.7), respectively. NPV of TST and QFT in this subgroup was lower than in total patients. On the other hand, PPV and LR+ in this subgroup were higher than in total patients.
This study is the largest trial evaluating the clinical usefulness of the QFT in differentiating ITB from CD among an immunocompetent population. For better comprehension, we compared the results of the QFT with the TST in patients with ITB or CD. As a result, the QFT was demonstrated to have 67% sensitivity and 90% specificity for diagnosing ITB. However, there was no significant difference in the clinical utility of the QFT and TST in the diagnosis of ITB, although the LR+ of the QFT was greater than the TST. These results suggest that the TST and QFT are useful in the differential diagnosis of ITB and CD, but these tests have some limitations.
ITB and CD are chronic granulomatous diseases of the intestine.13, 14 Although these diseases have similar clinical manifestations, the courses of the diseases are quite different. ITB can be completely cured if diagnosed early and treated appropriately. In contrast, CD is not curable and recurrence is common. In addition, steroids and other immunosuppressive agents or biologic drugs, which are very useful in the treatment of patients with CD, can be hazardous if used to treat patients with ITB.15, 16
Although TB remains a common problem in underprivileged areas of the world and less common in the developed world, the geography of the disease is changing, primarily because of immigration, human immunodeficiency virus infections, and the development of multidrug-resistant strains of TB.1–3, 6, 7 At the same time, the incidence of CD in areas that are endemic for TB has increased.4 In Korea, where TB is relatively prevalent, it has been reported that 40%–45% of patients with CD received a therapeutic trial of antituberculous medication before they were finally diagnosed with CD because the differentiation from ITB was difficult.17 Hence, in many cases a trial of antituberculosis therapy may often be prescribed before the definite diagnosis of CD is made. We also tried empirical antituberculous therapy and diagnosed ITB based on the response to the therapy in the patients in whom the differentiation between ITB and CD was uncertain. Of course, the potential alteration of the disease course in CD due to antituberculous treatment exists. Furthermore, potential spontaneous remission in patients with CD regardless of antituberculous treatment should be considered. However, we thought the possibility of misdiagnosis was very low. Likewise, 65% of patients with CD were misdiagnosed with ITB in one study conducted in China.18
To find a solution for establishing the differential diagnosis, clinical, radiologic, colonoscopic, and histopathologic methods or parameters have been studied, but no satisfactory method has been proposed.
The TST has been extensively studied in cases of pulmonary TB. The value of this test is unknown for ITB, and the diagnostic value of this test varies according to the population that is being tested.
Recently, a whole-blood IFN-γ assay based on the MTB-specific antigens, early secreted antigenic target 6 (ESAT-6), and culture filtrate protein 10 (CFP-10) has been introduced for the diagnosis of latent TB infection. The whole-blood IFN-γ assay, the QFT, is based on the production of inflammatory cytokines by T cells previously sensitized to mycobacterial antigens when they encounter ESAT-6 and CFP-10. In Korea, the positive rates of QFT and TST were reported to be 8.4% and 21.4%, respectively, in subjects with low risk for TB, which were similar to those in our CD patients.10
In the current study, the LR+ of the QFT was much higher in ITB than pulmonary TB (7.1 versus 1.76).11 However, the sensitivity of the QFT as well as the TST in the diagnosis of ITB was lower and the specificity was higher compared with previously reported data on active pulmonary TB. We are unable to explain the difference in results. As it is difficult to confirm a diagnosis of extrapulmonary TB, the diagnostic performance for the detection of extrapulmonary TB has not been well defined19; the data are limited, involve a small number of patients by subgroup analyses for extrapulmonary TB,20–23 and the results have shown sensitivities for the diagnosis of extrapulmonary TB ranging from 14%–80%. The overall sensitivity and specificity of the assay for detection of extrapulmonary TB, except ITB in Koreans, were 69% and 82%, respectively.23 Considering the poor sensitivity of the QFT in our study, we suggest that the QFT result has a lower sensitivity in extrapulmonary TB than in pulmonary TB. To resolve this problem, it is necessary to use other IFN-γ assays, such as the T-SPOT.TB test (Oxford Immunotec, Oxford, UK) and the QuantiFERON-TB Gold in tube (Cellestis), which have a higher sensitivity for the diagnosis of TB.22, 24 The T-SPOT.TB test in particular has a high sensitivity (94%) for extrapulmonary TB, although 50% of the enrolled patients were immunocompromised.25
Although previous studies have reported a higher sensitivity for the QFT relative to that of the TST in pulmonary TB, the sensitivity and specificity of the QFT and TST were not significantly different in our study in patients with ITB. However, this does not mean that the usefulness of QFT is not superior to that of TST. The LR+ of QFT was greater than the TST. When it comes to the patients whose initial diagnosis was unclear, the difference in LR+ between QFT and TST became greater. This fact suggests that QFT has higher probability of ITB than TST, especially in patients whose diagnosis was not clear initially. It is well known that the sensitivity and specificity of a test has limited clinical usefulness, as the sensitivity and specificity cannot be used to estimate the probability of disease in an individual patient.26, 27 According to a recent Italian study,29 the combined use of the IFN-γ assay and the TST may allow exclusion of tuberculosis. However, the combination of QFT and TST results did not increase the diagnostic utility in the current study.
The advent of QFT provides an important new indirect tool for the clinician to assist in the diagnosis of ITB. However, a positive QFT result is not a diagnostic standard for ITB; the QFT has a supplementary role in the differential diagnosis between ITB and CD.
In conclusion, this study confirmed that the QFT is a limited but useful diagnostic aid for the diagnosis of ITB.
- 4An update on the epidemiology of inflammatory bowel disease in Asia. Am J Gastroenterol. 2008; 103: 3167–3182., , , et al.Direct Link:
- 8Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection, United States. MMWR Recomm Rep. 2005; 54: 49–55., , , et al.
- 16Risk of tuberculosis is higher with anti-tumor necrosis factor monoclonal antibody therapy than with soluble tumor necrosis factor receptor therapy: the three-year prospective French research axed on tolerance of biotherapies registry. Arthritis Rheum. 2009; 60: 1884–1894., , , et al.