Role of Quantiferon-TB Gold Assays in Detecting Latent Tuberculous Infection among Contacts of Active Tuberculous Patients *

Transcription

1 Med. J. Cairo Univ., Vol. 79, No. 1, June , Role of Quantiferon-TB Gold Assays in Detecting Latent Tuberculous Infection among Contacts of Active Tuberculous Patients * NARIMAN A. HELMY, M.D.***; SOMIA A. ESSA, M.D.****; AYMAN E. SALEM, M.D., FCCP*** and HAMED A.H. TOAIMA, M.Sc.** The Departments of Chest Diseases*** and Microbiology****, Faculty of Medicine, Cairo University and Giza Chest Hospital** Abstract The detection and treatment of latent tuberculosis (LTB) infection is a key strategy in the control of tuberculosis. Conducting the present study aimed at evaluating the efficacy of Quantiferon TB Gold in detecting latent TB infection in close family contacts of active tuberculous patients in comparison to the tuberculin skin test. This study was conducted on 91 cases; 31 males and 60 females. Tuberculin skin test (TST) was carried on 91 cases but 24 cases of those tested did not undergo a follow-up visit to read the diameter of the TST, so were considered lost subjects, while Quantiferon test (QFT) was carried on the same 91 cases, out of whom only 10 subjects had "indeterminate" results. So the available results for both tests refer only to 67 subjects out of the 91 who were included in the study. In 6 cases of these 67 subjects showed indeterminate results for Quantiferon. So actually we have comparable results for 61 cases from the 91 subjects. In the present study there was a statistically significant association between Quantiferon and Tuberculin tests. Twenty seven subjects of individuals who have positive Tuberculin showed positive Quantiferon, and this number represents (44.2%) of all cases. Thirteen cases of individuals that have negative tuberculin showed negative Quantiferon, and this number represents (21.3%) of all cases. So concordant results were obtained in (65.5%) of the studied subjects and were not affected by gender or age. Discordant positive QFT reactions and negative TST reactions occurred in 7 cases of individuals that have negative tuberculin, and this number represents (11.5%) of all persons. Discordant negative QFT reactions and positive tuberculin skin test reactions occurred in 14 cases of cases that have positive tuberculin, and this number represents (23%) of all persons. So discordant results were obtained in (34.4%) of the studied subjects and were not affected by gender or age. The percentage of subjects with quantiferon positivity increased with accordance to the increase in the TST induration diameters. Key Words: Latent Tuberculosis Infection (LTB) Tuberculin Skin Test (TST) Quantiferon. Correspondence to: Dr. Ayman E. Salem, The Department of Chest Diseases, Faculty of Medicine, Cairo University. Introduction THE detection and treatment of latent tuberculosis (LTB) infection is a key strategy in the control of TB [1]. Until recently, the only tool available to detect latent tuberculosis infection (LTBI) was the tuberculin skin test (TST). Although the TST is useful in clinical practice, it has several known limitations, including variable specificity, cross-reactivity with BCG vaccine and non-tuberculous mycobacterial (NTM) infection, and hence problems with reliability exist [2]. Because of advances in molecular biology and genomics, for the first time, an alternative has emerged in the form of a new class of T cell-based, on vitro assays that measure interferon gamma (IFN-y) released by sensitized T cells after stimulation by Mycobacterium tuberculosis antigens [3]. The Quantiferon-TB Gold test (QFT-G), as one of these assays called interferon gamma assay release (IGRA), is a whole-blood test for use as an aid in diagnosing Mycobacterium tuberculosis infection, including latent tuberculosis infection (LTBI) and tuberculosis disease. This test was approved by the U.S. Food and Drug administration (FDA) in 2005, CDC [4]. The Quantiferon-TB Gold test (QFT-G), (produced by Cellestis Limited Australia), is an invitro diagnosis test which measures the IFN-gamma released by T lymphocytes sensitized after incubation with two M. tuberculosis-specific antigens ESAT-6 and CEP-10 [5]. Both these antigens are absent from all BCG strains. The QFT-Gold offers a number of advantages compared with the TST, including increased spec- * Based on a Master thesis in Chest Diseases Conducted by the first author and supervised by the three other authors & presented to Faculty of Medicine, Cairo University. 169

2 170 Role of Quantiferon-TB Gold Assays ificity in persons who have had a BCG vaccination, and elimination of the need for a second visit to read the TST [4]. The aim of the present study is to compare TST and QFT, in detection of LTBI in asymptomatic contacts of open tuberculous patients. Subjects and Methods This study was conducted in Giza Chest Hospital, where active tuberculous patients were admitted. The study was carried out between June (2009) and August (2009). Ninety one healthy close family contacts to newly diagnosed active tuberculous patients admitted in this hospital were enrolled randomly in the study. The participants consisted of 31 males and 60 females. The age of the participants ranged between 4 to 64 years. These persons were subjected to history taking. History of BCG vaccination was carefully inquired about. All of them definitely gave such history. Thorough clinical examination: General and local was done and proved clinically free. Plain chest X-ray was also done to assure that there was no detected abnormality. TST was performed according to the Mantoux technique. The Tuberculin test was performed by injecting intradermally mL of PPD on the forearm. In the absence of a papule; confirming the intradermal injection, the test was repeated immediately on the opposite forearm. The test is then read 72 hours later. A skin test results meant a raised (induration) at the point of administration. It was read by measuring the diameter of induration. Induration of 1 0mm or more in diameter was considered a positive result. Quantiferon TB Gold test (QFT), was simultaneously done by estimation of interferon gamma in whole blood in these subjects Quantiferon TB Gold tests were produced by (Cellestis Ltd, Australia) [5,6]. These tests were described by Brock, et al. [6]. The system uses two specialized blood collection tubes, one of them contains antigens representing certain M. tuberculosis proteins (ESAT)- 6, CFP-10, and TB-7.7 (p4) as well as negative (Nil) controls. Tubes were labeled properly. One ml of blood by venipuncture was introduced directly into each of the collection tubes. The tubes were vigorously shaked for 5 seconds (or 10 times) till frothing to ensure that the entire inner surface of the tube has been coated with blood. The tubes were incubated at 37 º C incubator as soon as pos- sible, and within 16 hours of collection. Incubation of the blood occurs in the tubes for 16 to 24 hours, after which plasma is harvested by centrifugation and tested for the presence of IFN- y by enzymelinked immunosorbent assay (ELISA) [5,6] : The Optical Density (OD) of each well was measured within 5 minutes of stopping the reaction using a microplate reader fitted with a 450nm filter and with a 620nm filter. OD values are used to calculate results. Quantiferon-TB Gold IT Analysis Software, used to analyse raw data and calculate results, is available from Cellestis [5,6]. The software performs a Quality Control assessment of the assay, generates a standard curve and provides a test result for each subject. Interpretation of QFT-G results is based on interferon-gamma (IFN- y) concentrations in test samples. A test is considered positive if IFN- y response T.B antigen tubes is significantly above the Nil IFN-y tube as negative control. If it is like the Nil value it is negative. An indeterminate result is considered when the result is neither positive nor negative. This was documented by a print-out report delivered from the system supplied by Cellestis Ltd, Australia) [5,6]. All the obtained data were collected and statistically analyzed. Statistical analysis: [SPSS, Inc, Chicago, IL USA] [7]. Qualitative data were presented as frequencies and percentages. Chi-square (X 2 ) test was used for studying the association between Quantiferon and Tubeculin. Quantitative data were presented as minimum, maximum, means and standard deviation (SD) values. Sensitivity, specificity and diagnostic accuracy of Quantiferon were calculated as follows: True positive Sensitivity (%) X 100 True positive + False negative True negative Specificity (%) X 100 False positive + True negative True positive + True negative Diagnostic accuracy (%) X 100 Total number An increase in sensitivity means a decrease in false negative cases, while an increase in specificity means a decrease in false positive cases.

3 Nariman A. Helmy, et al. 171 PV+ is the probability that a person has the disease given that his test is positive, while PV is the probably that a person is disease-free given that his test negative. The significance level was set at p<0.05. Statistical analysis was performed with SPSS 16.0 (Statistical Package for Scientific Studies) for Windows. Student's t-test was used to compare between ages of subjects with positive and negative Quantiferon. Kappa statistic was used to measure the agreement (Concordance) between Quantiferon and Tuberculin. Kappa values can be interpreted as follows: Poor (0.2), fair ( ), moderate (0.41). Results This study was conducted on 91 cases; 31 males and 60 females (Table 1). Their ages ranged between 4 and 64 years (mean age of 34.9 ± 16.9 years) (Table 2). History taking revealed that all subjects admitted being BCG vaccinated, as it is compulsory by law in Egypt. All of them had no significant past medical illness or concomitant immunosuppressive diseases. General and local clinical examination of the subjects revealed no significant abnormality. All individuals had normal physical examination and their chest X-ray showed no abnormality. The Tuberculin test was carried on 91 cases but 24 cases of them did not present for a followup visit to read the diameter of the test, so were considered lost subjects. Quantiferon test on the other hand, was carried on 91 cases but it was "indeterminate" in 10 subjects. The sample showed a mean age of 34.9 ± 16.9 years with a minimum of 4 years and a maximum of 64 years. Tuberculin Skin Test (TST): A positive TST result was defined as induration diameter of 10mm or more. Forty-five subjects (49.5%) showed positive tuberculin skin test, 22 subjects (24.1 %) showed negative test while in 24 subjects (26.4%) the test was performed but they did not come back to read the test. So were considered lost subjects (Table 3). Tuberculin test values showed a mean of ± 5.13mm induration with a minimum of 3mm and a maximum of 35mm (Table 5). Quantiferon (QFT): Forty-five subjects (49.5%) showed positive Quantiferon test, 36 subjects (39.6%) showed negative test while the test was indeterminate in 10 subjects (11%) (Table 6). QFT +ve cases showed statistically significantly higher TST diameter than QFT ve cases (Table 7). Association between Quantiferon positivity and induration diameter of TST: Quantiferon positivity increases with increased induration diameter of TST but this difference was not statistically significant (Table 8). 4 cases were found to have Tuberculin 5<10 induration of mm from them one subject only was positive to Quantiferon. 8 cases were found to have tuberculin 10<15 induration and out of them 5 subjects only were positive to Quantiferon. 15 cases were found to have tuberculin 15<20 induration of mm from them 9 subjects only were positive to Quantiferon, and 2 subjects had indeterminate results for Quantiferon. 14 cases were found to have tuberculin 20<25 induration and out of them 9 subjects only were positive to Quantiferon and 1 subject had indeterminate result for Quantiferon. 4 cases were found to have tuberculin 25<30 induration and out of them 3 subjects only were positive to Quantiferon. 1 case was found to have tuberculin 30<35 induration of mm and this case had indeterminate result for Quantiferon. 1 case only was found to have tuberculin and this case was positive to Quantiferon. Kappa statistic was used to measure such agreement (Concordance) between Quantiferon and Tuberculin. Kappa values were interpreted as follows: Poor (0.2), fair ( ), moderate ( ), good ( ) and very good ( ). A value of 1 indicates perfect agreement. Table (9) shows that in the whole sample, there was a statistically significant fair agreement (Kappa=0.283) between the two tests. In males, there was non-statistically significant fair agreement (Kappa=0.342) between the two tests.

4 172 Role of Quantiferon-TB Gold Assays In subjects less than 40 years of age, there was non-statistically significant fair agreement (Kappa= 0.223) between the two tests. In subjects 40 years of age and above there was non-statistically significant fair agreement (Kappa= 0.263) between the two tests. Association between Quantiferon and Tuberculin: In 24 of these 91 subjects where the TST was performed, the subjects did not undergo a followup visit or they came back after more than 72 hours when the test was scheduled to be read. For these patients the result of the TST was not taken into consideration. So only 67 subjects underwent both tests and had available results for both tests. 6 cases of these 67 comparable subjects showed indeterminate results for Quantiferon and were thus excluded. So actually we have comparable results for 61 cases from the 91 subjects (as shown in Table 10). After exclusion of indeterminate cases there were 41 cases with positive Tuberculin and 20 cases with negative Tuberculin. As regards Quantiferon, there were 34 cases with positive Quantiferon and 27 cases with negative Quantiferon. There was a statistically significant association between Quantiferon and tuberculin test. Twenty seven cases with positive Tuberculin showed positive Quantiferon. Thirteen cases with negative Tuberculin showed negative Quantiferon (Table 10). Calculating the sensitivity and specificity according to statistical rolls as mentioned in methodology, it was found that sensitivity, (%) was 65.9%; specificity (%) was 65%; and diagnostic accuracy (%) would be 65.6%. Table (1): Sex distribution (n=91). Gender Frequency Percentage Male Female 60 } 67 } Total Table (2): Descriptive statistics of age (n=91). Age Tuberculin Frequency Percentage Positive 45 } Negative 22 } Lost (missed) Total Table (4): Descriptive data of tuberculin skin test as regards the diameter of induration (n=67). Induration diameter of TST (mm) Less than 5mm 5 to <10 10 to <15 15 to 20 >20 Mean ± 50 (years) 34.9± 16.9 Minimum Number of cases Percentage Total Maximum Table (3): Frequency and percentages of tuberculin test results in the studied group (n=91). Table (5): Descriptive statistics of tuberculin test (n=67). Tuberculin test Mean ± SD Minimum Maximum mm induration 18.86± Table (6): Frequency and percentages of Quantiferon test (QFT) results in the studied group (n=91). Quantiferon Frequency Percentage Positive 45 } Negative 36 } Indeterminate Total Table (7): Statistical comparison between Quantiferon (QFT) positive and Quantiferon negative as regards TST diameter (Student t-test) (n=81). TST QFT +ve QFT (n=45) ve QFT (n=36) * statistically significant. Range TST positivity Mean ± SD 16.5± ± Student t -test (p) 0.010* Table (8): The relation between the induration diameter of TST (n=46) and frequency of Quantiferon positivity (n=28) (Chi-square test). Induration diameter of TST Frequency of QFN positivity Percentage p-value 5 to <10 (n=4) 10 to <15 (n=8) 15 to <20 (n=15) 20 & more (n=19) } Total QFN +ve

5 Nariman A. Helmy, et al. 173 Table (9): Concordance (agreement) between Quantiferon and Tuberculin (Kappa test). Item Kappa value p-value In the whole sample * Males Female Age <40 years Age 40 years and more * Significant at p<0.05. Table (10): Frequency, percentages and results of Chi-square test for the association between Quantiferon and Tuberculin (n=61). Quantiferon Tuberculin Positive QFT (n=34) Negative QFT (n=27) Positive TST (n=41) Negative TST (n=20) Frequency % Frequency % p-value 0.023* Total (n=61) * Significant at p<0.05. Discussion Because infection is a necessary prerequisite for active disease, a sensitive test for Mycobacterium tuberculosis (MTB) infection would help to rule out a diagnosis of active TB, particularly in immunosuppressed patients. However, due to its poor sensitivity, a negative tuberculin skin test (TST) in these patients is almost invariably clinically unhelpful and therefore not recommend by current guidelines [8]. The second major drawback of TST is its low specificity. Because protein-purified derivative (PPD) is a culture filtrate of tubercle bacilli containing over 200 antigens shared with the Bacilli Calmette-Guerin (BCG) vaccine and most nontuberculous mycobacteria [9] individuals vaccinated with BCG but not infected with MTB can give falsely positive using the tuberculin test. BCG is vaccination is compulsory by law in Egypt in early childhood. On the other hand, the Quantiferon-TB Gold test (QFT-G) is a whole-blood test for use as an aid in diagnosing Mycobacterium tuberculosis infection, including latent tuberculosis infection (LTBI) and tuberculosis (TB) disease. This test was approved by the U.S. Food and Drug Administration (FDA) in 2005 as stated by CDC, [4]. It represents interferon gamma release assay (IGRA), by sensitized lymphocytes to special mycobacterium tuberculosis not shared by BCG strain and hence not affected by previous BCG vaccination as is compulsory by law in Egypt. In the US, the center for disease control and prevention (CDC) has recommended that QFT-G can be used in place of the TST for all indications, including screening of contacts, immigrants, and health care workers [5]. Conducting the present study aimed at evaluating the efficacy of Quantiferon TB Gold in detecting latent TB infection in close family contacts of active open tuberculous patients in comparison to tuberculin skin test, in an Egyptian population. This study involved 91 subjects, all of them were close family contacts of patients having active pulmonary tuberculosis. All individuals had normal clinical parameters and chest X-rays. In the present study there was a statistically significant association between Quantiferon and Tuberculin tests. Table (8) shows that twenty seven cases out of subjects that have positive Tuberculin showed positive Quantiferon, and this number represents (44.2%) of all cases. Table (10) shows that thirteen cases out of subjects that have negative tuberculin showed negative Quantiferon, and this number represents (21.3%) of all cases. So concordant results were obtained in (65.5%) of the studied subjects and were not affected by gender or age. Discordant positive Quantiferon as a mean for of Interferon gamma release assay reactions and negative tuberculin skin test reactions occurred in 7 cases out of cases that have negative tuberculin, and this number represents (11.5%) of all persons. Discordant negative Quantiferon reactions and positive tuberculin

6 174 Role of Quantiferon-TB Gold Assays skin test reactions occurred in 14 cases out of cases that have positive tuberculin, and this number represents (23%) of all persons. So discordant results were obtained in (34.4%) of the studied subjects and were not affected by gender or age. The percentage of patients with Quantiferon positivity increased with accordance to the increase in the TST induration diameters (Table 7). The results of this study are in agreement with Connell, et al. [10] compared the performance of TST and Quantiferon in a total of 75 subjects who were contacts of active tuberculous patients, 49% of them were BCG vaccinated. They found that concordant results were obtained in 64% of subjects and discordant results were obtained in 36% of the studied group. The results of this study also are in agreement with Kang, et al., [11] who compared the performance of TST and Quantiferon in a total of 120 subjects who were close and casual contacts of persons with tuberculosis, 73% of them were BCG vaccinated at an age older than infancy. They found that concordant results were obtained in 53% of subjects and discordant results were obtained in 47% of the studied group. The results of this study were matching with the results of Lee, et al. [12] who studied the concordance of tuberculin and Quantiferon in a total of 131 healthy students. Discordant positive IGRA reactions and negative tuberculin skin test reactions occurred in 6% of the studied individuals. Discordant negative IGRA reactions and positive tuberculin and occurred in 19% of the studied individuals. The results of the present study did not agree with Harada, et al. [13] who compared the performance of TST and Quantiferon in a total of 304 health care workers; 91 % of them were BCG vaccinated. They found that concordant results were obtained in 14% of subjects and discordant results were obtained in 86% of the studied group! The present results were also not matching with Brock, et al. [6] who compared the performance of TST and Quantiferon in a total of 45 contacts of persons having active pulmonary tuberculosis, none of them was BCG vaccinated. Concordant results were obtained in 93% of cases while in only 7% discordant results were obtained. The higher concordance here may be attributed to the absence of BCG vaccination and this confirms the concept that BCG vaccination affects greatly performance of TST. In 3 studies Kang, et al. [11] ; Herada, et al. [13] and Diel, et al. [14] discordance was greater in persons with BCG vaccination than in those who were not vaccinated. Further studies are needed to compare the performance of Quantiferon in BCG and non-bcg vaccinated people as elucidated by scar presence and not by mere history taking. Conclusions and Recommendations: Conclusions: Quantiferon-TB Gold can be used for the diagnosis of latent tuberculous infections particularly in high risk groups and BCG vaccinated subjects where the results of Tuberculin skin test are inconclusive. The Quantiferon-TB Gold assays (QFT) have operational advantages over the tuberculin skin test because no return visit is required; QFT results are available by the next day, and repeated testing does not cause boosting. Quantiferon-TB Gold test, like the TST, is not meant to adequately differentiate infection associated with TB disease form LTBI. A positive result diagnosing LTBI should be followed by further medical and diagnostic evaluation to exclude TB disease. Recommendations: Extending the same work on larger number of cases. More studies are needed that compare the sensitivity of the tuberculin skin test with QFT in HIVinfected and other immune-compromised groups, intravenous drug users, diabetics, and pediatric and elderly populations. More data are needed to understand discordant tuberculin skin test and QFT reactions, including the effect of changes in cutpoints, the role of non-tuberculous mycobacteria, and time to conversion after exposure and infection. Further studies to investigate the possibility of occurrence of active pulmonary tuberculosis in subjects who proved to be positive for Quantiferon TB Gold. Further studies to compare the result of Quantiferon in BCG and non-bcg vaccinated people as evidenced by scar presence and not mere history taking.

7 Nariman A. Helmy, et al. 175 References 1- SMIEJA M.J., MARCHETTI C.A., COOK D.J. and SM- AILL F.M.: Isoniazid for preventing tuberculosis in non- HIV infected persons. Cochrane Database Syst. Rev., MENZIES R.I.: Tuberculin skin testing. In Tuberculosis: A comprehensive international approach Edited by: Reichman LB, Hershfield ES, New York, Marcel Dekker; , PAI M., KALANTRI S. and DHEDA K.: New tools and emerging technologies for the diagnosis of tuberculosis: Part 1. Latent tuberculosis. Expert Rev. Mol. Diagn., 6 (3): , Center for Disease Control and Prevention (CDC): TB Elimination, Diagnosis of Tuberculosis Disease. WWW. cdc.go/tb, MAZUREK G.H., JEREB J., LOBUE P., IADEMARCO M.F., METCHOCK B. and VERNON A.: Guidelines for using the Quantiferon-TB Gold test for detecting Mycobacterium tuberculosis infection United States. MMWR Recomm. Rep., 54 (RR-15): 49-55, BROCK I., WELDINGH K., LILLEBAEK T., FOLLMAN F. and ANDERSEN P.: Comparison of a new specific blood test and the skin test in tuberculosis. Am. J. Respir. Crit. Care Med., 170: 65-9, Statistical Package for Scientific Studies (SPSS): Inc. Chicago, Illinois, USA, American Thoracic Society: Diagnosis standard and classification of tuberculosis in adults and children. Am. J. Respir. Dis. Crit. Care Med., 161: , HUBNER R.E., SCHEIN M.F. and BASS J.B. JR.: The tuberculin skin test. Clin. Infect. Dis., 17: , CONNELL T., et al.: Early detection of perinatal TB using a whole blood interferon-release assay. Clinical Infect. Dis., 42: e82-e5, KANG Y.A., LEE H.W., YOON H.I., CHO B., HAN S.K., SHIM Y.S. and YIM J.J.: Discrepancy between the tuberculin skin test and the whole-blood interferon gamma assay for the diagnosis of latent tuberculosis infection in an intermediate tuberculosis-burden country. JAMA, 293: , LEE J.Y., CHOI H.J., PARK I.N., HONG S.B., OH Y.M., LIM C.M., et al.: Comparison of two commercial interferon-gamma assays for diagnosing Mycobacterium tuberculosis infection. Eur. Respir. J., 28: (PMID: ), HARADA N., NAKAJIMA Y., HIGUCHI K., SEKIYA Y., ROTHEL J. and MORI T.: Screening for tuberculosis infection using whole-blood interferon-gamma and Mantoux testing among Japanese healthcare workers. Infect. Control Hosp. Epidemiol., 27: 442-8, (PMID: ), DIEL R., NIENHAUS A., LANG C., MEYWALD- WALTER K., FORSSBOHM M. and SCHABERG T.: Tuberculosis contact investigation with a new specific blood test in a low-incidence population containing a high proportion of BCG-vaccinated persons. Respir. Res., 7: 77, (PMID: ), 2006.