Comparison of Quantiferon-TB Gold With Tuberculin Skin Test for Detecting Latent Tuberculosis Infection Prior to Liver Transplantation
The study was presented in part at the American Transplant Congress 2007, May 5–9, San Francisco, California, USA.
* Corresponding author: Oriol Manuel, firstname.lastname@example.org
Screening for latent tuberculosis infection (LTBI) is recommended prior to organ transplantation. The Quantiferon-TB Gold assay (QFT-G) may be more accurate than the tuberculin skin test (TST) in the detection of LTBI. We prospectively compared the results of QFT-G to TST in patients with chronic liver disease awaiting transplantation. Patients were screened for LTBI with both the QFT-G test and a TST. Concordance between test results and predictors of a discordant result were determined. Of the 153 evaluable patients, 37 (24.2%) had a positive TST and 34 (22.2%) had a positive QFT-G. Overall agreement between tests was 85.1% (κ= 0.60, p < 0.0001). Discordant test results were seen in 12 TST positive/QFT-G negative patients and in 9 TST negative/QFT-G positive patients. Prior BCG vaccination was not associated with discordant test results. Twelve patients (7.8%), all with a negative TST, had an indeterminate result of the QFT-G and this was more likely in patients with a low lymphocyte count (p = 0.01) and a high MELD score (p = 0.001). In patients awaiting liver transplantation, both the TST and QFT-G were comparable for the diagnosis of LTBI with reasonable concordance between tests. Indeterminate QFT-G result was more likely in those with more advanced liver disease.
The rate of tuberculosis (TB) in solid organ transplant recipients has been estimated to be 50-fold higher than in the general population (1). Management of TB is challenging in solid organ transplant recipients. First, clinical manifestations can be atypical in immunocompromised patients and, therefore, delays in diagnosis are common (2). Second, therapy against Mycobacterium tuberculosis infection may be associated with significant toxicity and potential drug interactions with immunosuppressive agents (1,3).
The guidelines of the American Society of Transplantation (AST) recommend that all candidates for organ transplantation should undergo tuberculin skin testing (TST) regardless of the Bacillus Calmette-Guérin (BCG) vaccination status (4). However, the performance of TST for the diagnosis of latent tuberculosis infection (LTBI) in transplant candidates has been estimated to be suboptimal due to false-negative results due to anergy (3). In a retrospective study of 547 liver transplant recipients, all six patients who developed TB disease posttransplant had a previous negative TST (3). Moreover, none of the patients with a positive TST developed TB disease, regardless of whether they had received isoniazid prophylaxis or not.
Quantiferon-TB Gold (QFT-G) is an interferon-γ (IFN- γ) release assay approved by Health Canada and the US Food and Drug Administration for the diagnosis of LTBI (5). The test is based on the measurement of a cell-mediated immune response against a mixture of synthetic peptides simulating two proteins present in M. tuberculosis: early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10). After incubation of whole blood with the mixture of synthetic peptides, an ELISA detects the release of IFN-γ from lymphocytes. Because the target peptides are secreted by M. tuberculosis and M. bovis, but not by strains present in the BCG vaccine, nor by most nontuberculous mycobacteria, QFT-G may correlate better with exposure to M. tuberculosis than the TST. A third generation QFT-G test, known as the QFT-G In-Tube assay, includes a third TB-specific antigen known as TB7.7. This third generation assay is approved in Canada for the diagnosis of LTBI although it is currently not available in the United States. The comparative sensitivity and specificity of the newer generation assay with the previous version in not known.
There is limited published literature on the performance of the QFT-G in immunocompromised persons. A recent study compared the QFT-G and TST in patients with a variety of immunodeficiencies that were suspected of having TB (6). In this setting, the QFT-G appeared to perform better than the TST. To our knowledge, there is no published data on the performance of QFT-G in patients with end-stage liver disease. Improvements in diagnosis of LTBI in patients awaiting transplantation would lead to more targeted prevention strategies. Therefore, we performed a prospective study to assess the performance of the QFT-G In-Tube assay compared with the standard TST in patients with end-stage liver disease awaiting liver transplantation.
Patients and Methods
Patients with chronic liver disease followed at the preliver transplant outpatient clinic at the Toronto General Hospital, Toronto, Canada, and the University of Alberta Hospital, Edmonton, Canada, were eligible for inclusion in the study if they were older than 18 years and if they were on the waiting list for liver transplantation. Exclusion criteria included a previous history of immediate hypersensitivity to the TST, a previous severe local ulceration with TST, suspected active TB and inability to provide informed consent. Patients were included between October 2006 and June 2007. The study was approved by the local Institutional Review Boards and all study participants provided informed consent. The trial was registered at http://www.clinicaltrials.gov registration number NCT00402402.
Tuberculin skin test
New liver transplant candidates underwent a TST as routinely performed at each institution. If a patient was already on the transplant waiting list and had not previously had their routine TST, this was performed. If patients on the waiting list had a TST in the previous month, a repeat TST was not required as long as the result was recorded in the medical record. The same procedure for performing the TST was followed at both institutions. In detail, the TST was performed on the volar side of the forearm according to the Mantoux method using 5 IU of PPD RT-23 (7). Using a 27 gauge, one-quarter inch needle, 0.1 mL volume was injected intradermally, thereby raising a pale wheal 6–10 mm. The investigator read the result of the TST 48–72 h later. Any induration was measured using the ballpoint method (8). According to the AST guidelines, a test was considered positive if there was ≥5 mm of induration at 48–72 h. A secondary analysis using a cut-off of 10 mm was done to test the concordance of TST with the QFT-G assay. No anergy testing was performed with the TST.
Interferon-γ release assay
The QFT-G In-Tube assay was performed according to the manufacturer's instructions (Cellestis Ltd., Carnegie, Victoria, Australia). One milliliter of whole blood was drawn in each of three separate test tubes: one containing no antigen (nil control), one with TB antigens (ESAT-6, CFP-10 and TB7.7) and one with phytohemagglutinin (mitogen or positive control). The three tubes were incubated for 16–24 h at 37°C. Following incubation, the tubes were centrifuged and plasma removed from each tube and placed in a plasma storage container. These containers were then frozen at –70°C and IFN-γ measurement by ELISA according to the manufacturer's instructions was subsequently performed in batch testing.
A result of ≥0.35 IU/mL of IFN-γ in the TB antigen tube minus the negative control tube was considered a positive result. If the level was less than this and the mitogen control was positive (≥0.5 IU/mL), a negative result was recorded. If the level in both the TB antigen and mitogen tube was less than the threshold for positive, or the level in the nil tube was >8.0 IU/mL, then an indeterminate result was recorded (according to the manufacturer's instructions).
Assessment of the risk of latent tuberculosis infection
As no gold standard exists for the diagnosis of LTBI, the clinical risk of LTBI was assessed at the time of recruitment. High risk was defined as those patients with any one of the following defined risk factors for LTBI: (a) previous contact with a patient with TB; (b) prolonged stay or born in a country where TB is prevalent (>10 cases/100 000 inhabitants) (9); (c) occupational exposure; (d) prior history of LTBI or active TB; and (e) chest radiograph changes consistent with old TB. All other patients were defined as low risk for LTBI. Subjects were questioned about prior BCG vaccination, but were not screened for the presence of a BCG vaccination scar due to the potential for confusion with smallpox vaccination scar and because BCG vaccination is known not to elicit a scar in up to 25% of people (10).
Concordance between TST and QFT-G test results was assessed using κ coefficients, where κ values >0.75 represented excellent agreement, 0.4–0.75 represented fair to good agreement, and <0.4 represented poor agreement. A logistic regression model was used to describe the independent variables associated with test discordances. We also assessed the variables associated with an indeterminate result of the QFT-G assay. All analyses were performed using SPSS software (SPSS version 14.0, Chicago, IL) and a p-value <0.05 was considered to be statistically significant.
Characteristics of the study population
A total of 170 patients were enrolled in the study between October 2006 and June 2007. No patient was excluded because of a prior severe reaction to the TST. In eleven patients the TST was either not performed (n = 1) or not read (n = 10). In three patients the QFT-G was not performed. In three additional patients both the TST and the QFT-G were not performed because the patients did not return for testing. Thus, complete results were available for 153 patients. None of the patients had a TST within the previous 12 months. The TST and the QFT-G was performed simultaneously in 146/153 patients. In seven patients, due to difficulties in obtaining blood, QFT-G was performed a median of 2 days after the TST (range 1–7 days). Patients' clinical characteristics are shown in Table 1. The majority of patients (82%) had been previously vaccinated with the BCG vaccine. All participants had a negative test result for human immunodeficiency virus (HIV). One patient had been previously treated for active pulmonary TB, and three patients had been treated for LTBI. Based on clinical and radiologic assessment, 69/153 patients (45.1%) were considered high-risk for LTBI.
Table 1. Patients' clinical characteristics
|Age (years; mean ± SD)||54.6 ± 8.2|
| Caucasian||124 (81%) |
| Afro–American||5 (3%)|
| Asian||20 (13%)|
| Other||4 (3%)|
| Chronic hepatitis C||73 (48%)|
| Alcoholic liver disease||32 (21%)|
| Chronic hepatitis B||15 (10%)|
| Primary sclerosing cholangitis||6 (4%)|
| Primary biliary cirrhosis||1 (1%)|
| Other||26 (17%)|
|MELD score (mean ± SD.)||13.6 ± 4.4|
|Current immunosuppresion||5 (3%)|
|BCG vaccination1||116/142 (82%)|
|High risk for TB2||69 (45%)|
| Contact with patient with TB||12 (8%) |
| Born or stay in a country with high TB prevalence||50 (33%)|
| Work or stay in prison, hospital or homeless shelter||14 (9%) |
| Chest X-ray with changes consistent with old TB||2 (1%)|
| History of latent tuberculosis infection||3 (2%)|
| Previous active tuberculosis||1 (1%)|
Agreement between the tuberculin skin test and the quantiferon-TB gold assay
Overall, the TST was positive (≥5mm) in 37/153 patients (24.2%) and the QFT-G was positive in 34/153 (22.2%). A total of 12/153 (7.8%) patients had an indeterminate result for the QFT-G (see details below). When a cut-off of 10 mm was used, the TST was positive in 27/153 patients (17.6%). As shown in Table 2, the concordance between the QFT-G and the TST was 85.1%, κ= 0.60 (p < 0.001) (cut-off ≥5 mm; excluding 12 patients with an indeterminate result for QFT-G). When a cut-off of 10 mm was used to define the positivity of the TST, concordance was 82.2%, κ= 0.48 (p < 0.001) (Table 3).
Table 2. Agreement between tuberculin skin test (TST), cut-off ≥5 mm and Quantiferon-TB Gold in tube assay (QTF-G), excluding patients with a QFT-G indeterminate result
|QFT-G positive|| 25 (17.7%×3)|| 9 (6.4%×3)|| 34 (24.1%×3)|
|QFT-G negative||12 (8.5%×3)|| 95 (67.4%×3)||107 (75.9%×3)|
|Total|| 37 (26.2%×3)||104 (73.8%×3)||141 (100%×3)|
| %||85.1% || || |
| κ Coefficients||0.60 || || |
| p-value||<0.001 || || |
Table 3. Agreement between tuberculin skin test (TST), cut-off ≥10 mm and quantiferon-TB Gold in tube assay (QTF-G), excluding patients with a QFT-G indeterminate result
|QFT-G positive||18 (12.8%×3)|| 16 (11.3%×3)|| 34 (24.1%×3)|
|QFT-G negative||9 (6.4%×3)|| 98 (69.6%×3)||107 (75.9%×3)|
|Total||27 (19.1%×3)||114 (80.9%×3)||141 (100%×3)|
| %||82.2% || || |
| κ Coefficients||0.48 || || |
| p-value||<0.001 || || |
Twenty-one of 153 patients (13.7%) had a discordant result between the TST and the QFT-G. Of these, 12 patients had a positive TST but negative QFT-G. Analysis of clinical and radiological criteria in these patients suggested that 9/12 were high risk and 3/12 were low risk for LTBI. Of the 21 patients with a discordant result, 9 had a positive QFT-G but a negative TST. Analysis of clinical and radiological criteria suggested that 4/9 were high risk and 5/9 were low risk for LTBI. No specific factors were predictive of a discordant result including prior BCG vaccination (p = 0.82).
In the seven patients where the QFT-G was performed shortly after the TST (and therefore whose QFT-G could potentially be positive due to ‘boosting’), only a single patient had a positive QFT-G (also positive by TST). The remaining six were all negative for the TST and either negative (n = 5) or indeterminate (n = 1) by QFT-G. In the four patients treated for LTBI or active TB, all had a positive TST and three patients had a positive QFT-G. The patient with discordant result was a 48-year-old female who was treated for LTBI with isoniazid for 6 months in 1986. Her TST was positive with a 14-mm induration and her QFT-G was negative.
High-risk versus low-risk patients
As no gold standard exists for the diagnosis of LTBI, patients were classified based on clinical and radiological criteria into high-risk (69/153; 45.1%) and low-risk (84/153; 54.9%) categories. Performance characteristics of both tests were analyzed in each category of patients. In high-risk patients, the TST was positive in 29/69 (42.0%) and the QFT-G was positive in 24/69 (34.8%). In low-risk patients, the TST was positive in 8/84 patients (9.5%) and the QFT-G was positive in 10/84 patients (11.9%). Both tests were significantly more likely to be positive in the high-risk group compared to the low-risk group (p < 0.01). Overall concordance between tests appeared to be similar in both categories: for the low-risk group concordance was 90.0%, κ= 0.50; p < 0.001 and for the high-risk group concordance was 78.7%, κ= 0.57; p < 0.001.
Quantiferon-TB Gold assay indeterminate results
As noted previously, 12/153 patients (7.8%) had an indeterminate result of the QFT-G test. In all cases, both results of TB antigen tube and mitogen tube (i.e. the positive control tube) were lower than the threshold for positivity. All 12 of these patients also had a negative TST. Four patients were at low risk for TB and eight patients were at high risk for TB. Univariate analysis of factors associated with an indeterminate result for the QFT-G (Table 4) showed that a low lymphocyte count (p = 0.002), a high model for end-stage liver disease (MELD) score (p = 0.006) and a negative TST (p = 0.04) were significantly associated. Multivariate analysis showed that a low lymphocyte count (OR: 0.06, 95% CI: 0.01–0.52, p = 0.01) and a high MELD score (OR: 1.28, 95% CI: 1.10–1.48, p = 0.001) remained significant factors for an indeterminate result of the QFT-G assay.
Table 4. Univariate analysis of risk factors for indeterminate result of the Quantiferon-TB Gold in tube assay (QTF-G)
|Chronic C hepatitis (%)||4 (33.3)||68 (49.3)||0.30|
|MELD score||17.8||13.3|| 0.006|
|Lymphocyte count (cell/mL)||0.66||1.16|| 0.002|
|High-risk for TB (%)|| 8 (66.7)|| 61 (44.2)||0.13|
|TST negative (%)||12 (100)||104 (77.8)||0.04|
|BCG vaccination (%)1||10/11 (91.0)||106/131 (80.9)||0.70|
Several studies have compared IFN-γ release assays with the TST in the general population, children, health-care workers and in countries with a high incidence of TB (11–17). However, few studies have compared both tests in immunocompromised patients (6,18–20). To our knowledge, this is the first study using an IFN-γ release assay in the diagnosis of LTBI in patients with end-stage liver disease. Our study shows that in patients awaiting liver transplantation, the QFT-G in-tube assay was comparable to the TST in the diagnosis of LTBI. Moderate correlation was observed between the two tests. A possible confounder in studies comparing IFN-γ release assays with the TST is the potential boosting effect if the TST is administered shortly before the QFT-G assay is performed. We do not feel this was a significant consideration in our study, because the majority of patients had both tests simultaneously and only a single positive QFT-G result was observed in the seven patients where the QFT-G was performed after the TST.
A recent meta-analysis of studies comparing TST with IFN-γ release assays showed that the agreement between tests was generally good, with κ values between 0.57 and 0.70; this is similar to the results we observed in our pretransplant population (21). A true determination of test performance is hampered by the lack of a gold standard for the diagnosis of LTBI.
Although these assays have not been evaluated in transplant candidates, they have been assessed, to a limited degree, in other groups of immunocompromised patients. In HIV positive individuals, QFT-G and TST were comparable in a study by Luetkemeyer et al., but the number of positive results was low (18). A low CD4+ cell count was associated with an indeterminate result of the QFT-G assay, a finding that was also seen in another study with HIV positive persons in Denmark (19).
The IFN-γ release assay has also been assessed in patients with rheumatoid arthritis before starting an anti-TNF-α treatment. In this population, commonly receiving concurrent immunosuppressive therapy, the incidence of an indeterminate result with such assays is approximately 10% (20). Similarly, Kobashi et al. have evaluated the QFT-G in immunocompromised persons and noted 13% indeterminate results (6). These are similar to the 7.8% incidence of indeterminate results observed with our study. Our data suggest that a higher MELD score and a lower lymphocyte count are risk factors for an indeterminate result. We were unable to compare this to the rate of anergy with the TST, because no anergy testing was done based on current guidelines and the limitations of anergy testing (7,22). However, all patients with an indeterminate QFT-G also had a negative TST. As many of these patients were clinically at high-risk of LTBI, it is likely the TST was falsely negative in at least a proportion of those with an indeterminate QFT-G.
BCG vaccination status has been shown to be a risk factor for having a discordant result between the TST and the QFT-G (21). In our study, BCG vaccination was not associated with a discordant result between tests. In current clinical practice, BCG vaccination status in generally is not taken into account when interpreting TST results in adults and this approach is supported by our data. It has been shown that the TST response wanes rapidly when BCG vaccine is given at birth. In a study evaluating the impact of the BCG vaccine on the TST in workers at a university hospital in Switzerland, the BCG status was clearly relevant in persons younger than 40 years old, but was not significant in older persons (23). Almost half of the population of our study came from countries where BCG was given at birth, and a majority of patients of Canadian origin were vaccinated 40–50 years before the TST was performed. Although we cannot exclude a recall bias in some patients when asked about BCG vaccination status, it is likely that BCG vaccine had little impact on the results of the TST in our population.
In conclusion, our study demonstrates that the QFT-G and TST performed similarly for the diagnosis of LTBI in a population with end-stage liver disease awaiting transplantation. This QFT-G has not been evaluated for the detection of active TB in this population. However, for detection of LTBI, we suggest it would be reasonable to use either test as a pretransplant screening tool for these patients. Occasional discordant results may occur and optimal sensitivity may involve the development of better assays. False negative results occur with both tests and we show that the advanced liver disease is associated with indeterminate test results. Similar to general population, the main limitation of studies comparing the IFN γ-release assay and the TST is the lack of a gold standard for the diagnosis of latent TB infection.
We thank Dr. Stephen Jones (Cellestis Ltd., Carnegie, Victoria, Australia) for critical review of the manuscript and helpful discussions. Dr. Manuel is the recipient of a transplant infectious diseases fellowship from The Transplantation Society/Hoffman-LaRoche. Quantiferon-TB Gold In-Tube kits were provided by Cellestis Ltd., Carnegie, Victoria, Australia.