No influence of haemodialysis on interferon production in the QuantiFERON-TB Gold-In-Tube test

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1 ORIGINAL ARTICLE JN EPHROL 24( 2011; 05) : DOI: /JN No influence of haemodialysis on interferon production in the QuantiFERON-TB Gold-In-Tube test Martine Hoogewerf 1, Greet J. Boland 2, Andy I.M. Hoepelman 1, Walther H. Boer 3, Tania Mudrikova 1 1 Department of Internal Medicine & Infectious Diseases, University Medical Center Utrecht, Utrecht - The Netherlands 2 Department of Medical Microbiology and Virology, University Medical Center Utrecht, Utrecht - The Netherlands 3 Department of Nephrology & Hypertension, University Medical Center Utrecht, Utrecht - The Netherlands Abstract Background: Immunodeficiency in end-stage renal disease (ESRD) can be aggravated by haemodialysis (). This results in an increased incidence of reactivation of tuberculosis (TB) in patients. The tuberculin skin test to detect a latent TB infection (LTBI) has its limitations in these patients because of a high rate of false negative results due to anergy of T cells. Data on the influence of on the performance of interferon-gamma release assays are limited. The aim of this study was to determine the effect of on the performance of the QuantiFERON-TB Gold (QFT-G) assay in ESRD patients before, during and after the session. Methods: In patients older than 18 years without immunosuppressive medication or other immunocompromising conditions, the QFT-G assay was performed just before starting, 30 minutes after start and immediately after the finish of the session. Results: Twenty patients were included. No statistically significant differences were found in interferongamma production in the nil- and antigen tubes between pre-, during and after. In 1 patient the predialysis result was indeterminate (one of 60 samples, 1.67%). In all 3 patients with a history of LTBI, the QFT-G test tube results were positive at all time points. In the other 16 patients, all test tubes showed negative results. Conclusions: The QFT-G assay could be a useful test for the evaluation of the immunological response against Mycobacterium tuberculosis in patients. The time point of blood sampling does not seem to affect the interpretation of test results. Key words: Haemodialysis, Interferon-gamma release assay, QuantiFERON-TB Gold test Introduction Immunodeficiency is a well-known consequence of endstage renal disease (ESRD) (1), which further can be aggravated by haemodialysis (). Both the cellular and humoral defense are affected, resulting in higher morbidity and mortality from infectious causes in these patients. One of the consequences of immunodeficiency is the increased incidence of reactivation of tuberculosis (TBC). The incidence of active TBC in patients compared with the general population ranges in different studies from 8- to 25-fold (2-4). For this reason it is important to have reliable tools to detect latent tuberculosis infection (LTBI) in this group. The widely used tuberculin skin test (TST) has its limitations in these patients because of a high rate of false negative results due to anergy of T cells, reaching up to 44% (5, 6). The more recently introduced interferon-γ release assays (IGRAs) have shown improved diagnostic accuracy for LTBI also in high-risk patients (7, 8). The US Food and Drug Administration has approved the QuantiFERON- TB Gold (QFT-G) assay, as well as the T-SPOT.TB assay, for determination whether a patient has been exposed to or is infected with Mycobacterium tuberculosis. However, information about the interpretation of the results of these tests in immunocompromised patients such as those requiring, is still limited (3, 4, 7, 9-14). IGRAs are based on detection of interferon-γ (IFN-γ) release by T cells in response to restimulation of previously in vivo sensitized T lymphocytes with M. tuberculosis specific antigens. The intact lymphocyte function is crucial for reliable results with IGRAs. The cellular immunodeficiency in patients can lead to the increased incidence of so-called indeterminate (i.e. not interpretable) results, which were reported to occur frequently in immunocompromised populations 2011 Società Italiana di Nefrologia - ISSN

2 Hoogewerf et al: Haemodialysis and QuantiFERON-TB Gold test (5%-40%) when compared with the immunocompetent individuals (0-5%) (11, 15-17). A few studies have been published that describe indeterminate results in ESRD patients varying from 2.5% to 40% (6, 10-14, 18, 19). The procedure itself could be one of the possible mechanisms in the impairment of the lymphocyte function, as membranes may affect mononuclear cell apoptosis (20). However, the data on the influence of on the performance of IGRAs are limited. One study has described reduced IFN-γ production in patients on low-flux, and as far as we know, only 1 other study has investigated the effects of itself on the QFT-G test results (19, 21). The aim of our study was to determine the effect of on the qualitative and quantitative performance of the QFT-G assay (test performance and amount of IFN-γ production) in ESRD patients before, during and after the session. Subjects and methods Study population This study was performed in an university hospital (University Medical Center Utrecht) in The Netherlands which is a lowendemic TBC country. Patients treated with for ESRD were eligible for this study. Patients younger than 18 years, those using immunosuppressive medication or having other immunocompromising conditions, signs of an acute infection or other intercurrent medical problems were excluded. All patients were treated with regular 3 times a week; the duration of each session was between 4 and 5 hours. The study was approved by the medical ethics committee of the University Medical Center of Utrecht. All patients signed an informed consent form before blood sampling. Sample collection and QuantiFERON-TB Gold-In- Tube test QuantiFERON-TB Gold-In-Tube test (QFT-G; Cellestis, Darmstadt, Germany) was used, which consists of 3 tubes: a mitogen tube coated with phytohaemagglutinin (a positive control), a sample tube coated with specific TB antigens (ESAT-6, CFP-10 and TB7.7; test tube) and a nil tube without any antigens or mitogens (a negative control). The QFT-G test was performed according to the instructions of the manufacturer. In each of the 3 tubes, 1 ml of whole blood was added, which was drawn at 3 different time points: just before starting the, 30 minutes after start and immediately after the finish of the session. The tubes were incubated for hours at 37 C and then centrifuged; the plasma was removed from each tube and frozen at -20 C. IFN-γ measurement was subsequently performed by ELISA technique batchwise. According to the manufacturer s instructions, the results were considered positive if the IFN-γ level in the TB antigen tube minus the nil tube was 0.35 IU/mL, and negative if this result was 0.35 IU/mL and if the level of the positive control minus the level of the nil tube was 0.5 IU/ ml. The test was considered indeterminate, if the IFN-γ level in the positive control tube minus the nil tube was 0.5 IU/mL and the level in the TB antigen tube minus the nil tube was 0.35 IU/mL at the same time. Statistical analysis SPSS, version 16 (SPSS Inc, Chicago, IL, USA) was used for the statistical analysis. The intraindividual and interindividual variations in test results from blood drawn before, during and after were evaluated in a quantitative manner (OD value minus background), and statistical analysis was performed using ANOVA, after checking for normal distribution. A paired t-test was used to evaluate the significance of the differences between the 3 time points. A p value less than 0.05 was considered statistically significant. Results Twenty patients (14 men and 6 women) were included in this study. Patient characteristics are summarised in Table I. Three patients had been diagnosed with latent TBC in the past: 2 of them had a positive TST result and both were treated prophylactically with isoniazid; another patient had a positive ELISpot-TB result but refused prophylactic treatment. No statistically significant differences were found between the pre-, during and after values when the IFN-γ production in the nil tubes and in the antigen tubes were evaluated. When comparing the results of the mitogen tubes, IFN-γ production was significantly higher after the session compared with predialysis or during the session, although mitogen production before and during dialysis was also sufficient for the interpretation of the results. Nineteen of the 20 patients showed interpretable results at all 3 tested time points; in 1 patient the predialysis result was indeterminate and thus not interpretable (1 of the 60 samples, 1.67%) (Fig. 1). In all 3 patients with a history of LTBI, the QFT-G test tube results were positive at all 3 time points (Fig. 2). In the other 16 patients, the test tube showed a negative result at all time Società Italiana di Nefrologia - ISSN

3 JN EPHROL 24( 2011; 05) : Fig. 1 - Mitogen-induced IFN-γ release before, during and after haemodialysis (). Fig. 2 - TB antigen induced IFN release before, during and after haemodialysis () (patients with a history of latent TB infection shown with squares and triangles). TABLE I PATIENT CHARACTERISTICS Mean age, years (range) 56.3 (25-87) Male/female 14/6 Diabetes mellitus, no. (%) 7 (35%) Meantime on, months (range) 19 (0-66)* History of positive TST, no. (%) 3 (15%) Cause of ESRD Diabetic nephropathy 6 Hypertensive nephropathy 3 Focal segmental glomerulosclerosis 2 Polycystic kidney disease 2 Ureteral stones 1 Membranous nephropathy 1 Reflux nephropathy 1 Unknown 4 ESRD = end-stage renal disease; = haemodialysis; TST = tuberculin skin test. *Four unknown. points. In the 1 patient with an indeterminate result before start of, the other 2 were negative. In Table II, the means, standard deviations, medians, minimum and maximum values of the IFN-γ production in the 3 different test tubes at 3 different time points are shown. Discussion In recent years, many studies have aimed to determine the place of IGRAs in the diagnosis of active and latent TBC, mostly in comparison with the TST. Evaluation of the performance of the new in vitro methods seems especially important in patients with immunodeficiency as these persons have worse prognosis after developing active TBC. The aim of the present study was to evaluate the influence of on the performance of the QFT-G test when sampling blood before, during and after a single session. We did not observe any difference in the interpretation of the QFT- G test results at the predefined time points in 19 patients (95%); in 1 patient, the predialysis test result was indeterminate and changed to determinate during and after dialysis. Moreover, IFN-γ production in the mitogen tube was highest after compared with the values before and during. From a practical point of view, this means that blood for this test can be drawn from the dialysis line after the start or just before finishing the dialysis session, and that patients do not have to undergo an additional venipuncture. The incidence of indeterminate results was very low in our group of patients (1 of 60 samples, 1.7%); this rate is com Società Italiana di Nefrologia - ISSN

4 Hoogewerf et al: Haemodialysis and QuantiFERON-TB Gold test TABLE II IFN-γ PRODUCTION IN 3 QFT-G TUBES AT 3 DIFFERENT COLLECTION TIME POINTS Nil before Nil (IU/ ml) during Nil (IU/ ml) after TB AG before TB AG during TB AG after Mitogen before Mitogen during Mitogen after Number Mean SD Median Min Max F and p value, ANOVA p Value paired t-test F=3.76, p=0.15 F=2.00, p=0.38 F=17.3, p= Comparison B-D D-A B-A B-D D-A B-A B-D D-A B-A A = after haemodialysis; B = before haemodialysis; D = during haemodialysis; mitogen = phytohaemagglutinin tube; Nil = no antigen present; SD = standard deviation; TB AG = tuberculosis antigen tube. parable with that in the immunocompetent population (15-17). In immunocompromised persons, higher frequencies of indeterminate test results were reported (11). Indeterminate test results in the study population of Kobashi et al (11) were significantly associated with higher age, presence of an immunocompromising condition or immunosuppressive treatment, lymphocytopaenia and hypoalbuminaemia. The exclusion of patients with immunosuppressive treatment and immunocompromising condition other than ESRD in our study could possibly be an explanation for the low percentage of indeterminate results. Another reason could be the lower mean age of the patients in our group compared with that of Kobashi et al (56.3 vs years). Hursitoglu et al in their population excluded patients requiring immunosuppressive therapy as well, and they also found a low percentage of indeterminate results (3.6%) (19). Similar to our study design, the design of the study by Hursitoglu et al compared in a high endemic setting the predialysis and postdialysis IFN-γ production levels in 56 patients requiring. They found an obvious reduction in IFN-γ production levels in the TB antigen tube after the dialysis session. In contrast to our study, 59% of patients had a positive reaction in the test tube before dialysis. They suggested to perform the blood collection for QFT-G before the start of session (19). Our results do not support this conclusion. In our study population, all 3 patients with a history of LTBI showed a positive QFT-G test result. Several studies have compared the QFT-G test, the T-SPOT-TB and the TST in the detection of latent TBC in patients with ESRD (3, 4, 11, 13). Although the absence of a gold standard to diagnose LTBI precludes any definite conclusion, in these studies the QFT-G test seemed to be the most accurate way to identify patients truly infected with M. tuberculosis. Using this test in patients with ESRD can bypass the problems associated with anergy in response to TST or cross-reactivity in patients with a bacille Calmette-Guérin (BCG) vaccination in the past. The most important limitation of our study is the small number of patients included because of the quite strict inclusion criteria (no immunosuppressive medication or intercurrent medical conditions which could influence lymphocyte functions) Società Italiana di Nefrologia - ISSN

5 JN EPHROL 24( 2011; 05) : In conclusion, we did not find any evidence of impaired performance of the QFT-G test when blood was collected during or after, compared with the results before. In addition, the frequency of indeterminate results was low in this population with no other immunocompromising conditions besides the ESRD and. Taken together, these findings suggest that the QFT-G test could be a useful test for the evaluation of the immunological response against M. tuberculosis in patients without intercurrent problems. The time point of blood sampling does not seem to affect the interpretation of test results. Financial support: No financial support. Conflict of interest statement: None declared. Address for correspondence: Tania Mudrikova Department of Internal Medicine & Infectious Diseases University Medical Center Utrecht Heidelberglaan 100 PO Box NL-3508 GA Utrecht, The Netherlands T.Mudrikova@umcutrecht.nl References 1. Descamps-Latscha B, Jungers P, Witko-Sarsat V. Immune system dysregulation in uremia: role of oxidative stress. Blood Purif. 2002;20: Simon TA, Paul S, Wartenberg D, Tokars JI. Tuberculosis in hemodialysis patients in New Jersey: a statewide study. Infect Control Hosp Epidemiol. 1999;20: Triverio PA, Bridevaux PO, Roux-Lombard P, Niksic L. Interferon-gamma release assays versus tuberculin skin testing for detection of latent tuberculosis in chronic haemodialysis patients. Nephrol Dial Transplant. 2009;24: Lee SS, Chou KJ, Su IJ, Chen YS. High prevalence of latent tuberculosis infection in patients in end-stage renal disease on hemodialysis: comparison of QuantiFERON- TB GOLD, ELISPOT, and tuberculin skin test. Infection. 2009;37: Shankar MS, Aravindan AN, Sohal PM, Kohli HS. The prevalence of tuberculin sensitivity and anergy in chronic renal failure in an endemic area: tuberculin test and the risk of post-transplant tuberculosis. Nephrol Dial Transplant. 2005;20: Smirnoff M, Patt C, Seckler B, Adler JJ. Tuberculin and anergy skin testing of patients receiving long-term hemodialysis. Chest. 1998;113: Richeldi L. An update on the diagnosis of tuberculosis infection. Am J Respir Crit Care Med. 2006;174: Menzies D, Pai M, Comstock G. Meta-analysis: new tests for the diagnosis of latent tuberculosis infection: areas of uncertainty and recommendations for research. Ann Intern Med. 2007;146: Richeldi L, Losi M, D Amico R. Performance of tests for latent tuberculosis in different groups of immunocompromised patients. Chest. 2009;136(1): Società Italiana di Nefrologia - ISSN

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