Use of an Interferon- Release Assay To Diagnose Latent Tuberculosis Infection in Foreign-Born Patients*

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1 Original Research MYCOBACTERIAL DISEASE Use of an Interferon- Release Assay To Diagnose Latent Tuberculosis Infection in Foreign-Born Patients* Daniel Brodie, MD; David J. Lederer, MD, MS; Jade S. Gallardo, MD; Saumil H. Trivedi, MD; Joseph N. Burzynski, MD; and Neil W. Schluger, MD Background: The tuberculin skin test (TST) has a low specificity in the setting of bacille Calmette-Guérin (BCG) vaccination. Interferon- release assays (IGRAs) appear to be more specific but have not been validated in this population under routine clinical conditions. We sought to validate the routine clinical use of the T-SPOT.TB test (Oxford Immunotec; Oxford, UK), an IGRA, in a predominantly foreign-born population with a high rate of BCG vaccination. Methods: We compared the TST and the T-SPOT.TB test in 96 subjects at a New York City Department of Health tuberculosis clinic. We aimed to determine which test better predicted being a close contact of a case of active tuberculosis, a surrogate for latent tuberculosis infection. Results: A positive T-SPOT.TB test result was strongly associated with being a close contact of a case of active tuberculosis after adjustment for potential confounders (adjusted odds ratio, 2.9; 95% confidence interval, 1.1 to 7.3; p 0.03). A positive TST result was associated with being a contact only in subjects without BCG vaccination (p 0.02). The T-SPOT.TB test was more specific for being a close contact than the TST (p < 0.001). Specificity in BCG-vaccinated subjects was 3% for the TST compared with 70% for the T-SPOT.TB test (p < 0.001). Conclusions: The T-SPOT.TB test is superior in routine clinical use to the TST for identifying high-risk individuals among foreign-born populations with high rates of BCG vaccination. (CHEST 2008; 133: ) Key words: infection; interferon; tuberculosis; tuberculosis testing Abbreviations: BCG bacille Calmette-Guérin; CDC Centers for Disease Control and Prevention; CI confidence interval; DOHMH Department of Health and Mental Hygiene; IGRA interferon- release assay; LTBI latent tuberculosis infection; QFT QuantiFERON-TB Gold; TST tuberculin skin test Screening and treatment for latent tuberculosis infection (LTBI) are essential components of the strategy for reducing and eliminating tuberculosis in the United States. Current guidelines stress the targeting of screening and treatment to at-risk populations, which include both close contacts of cases of active tuberculosis and recent immigrants from high-prevalence countries. 1 These immigrants are of particular importance because more than one half of tuberculosis cases in the United States occur in this population, and strong evidence exists that most cases result from reactivation of latent infection. 2 LTBI is usually diagnosed by the tuberculin skin test (TST). Confounding by bacille Calmette-Guérin (BCG) vaccination (especially when administered after 1 year of age 3 ) is a significant limitation of skin testing. 4 Because the majority of individuals at high risk for LTBI, and by extension active tuberculosis, in the developed world have come from countries where they are vaccinated with BCG, the TST is least reliable and most problematic in those most in need of screening. Studies of LTBI in foreign-born patients, as diagnosed using the TST, indicate a very high frequency of positive results, yet active tuberculosis will develop in very few of those with a diagnosis made using the TST. It is likely that many of these test results are falsely positive due to BCG vaccination. Treatment of all such individuals is costly, carries a risk of significant side effects, and is obviously futile in those who do not have LTBI to begin with. Tests more specific for LTBI would improve the cost- CHEST / 133 / 4/ APRIL,

2 effectiveness, safety, and overall effectiveness of screening and treatment programs. Interferon- release assays (IGRAs) detect T-cell responses to antigens, including ESAT-6 and CFP10, specific for Mycobacterium tuberculosis and notably absent from Mycobacterium bovis BCG. Consequently, IGRAs should offer greater specificity for LTBI than the TST, 5 and these tests should be of particular benefit in those who have received BCG. Current guidelines from the Royal College of Physicians of London recommend using the commercially available IGRAs, either the QuantiFERON-TB Gold (QFT) [Cellestis, Ltd; Victoria, Australia] or the T-SPOT.TB test (Oxford Immunotec; Oxford, UK), only in those patients with positive TST results. 6 Recommendations from the Centers for Disease Control and Prevention (CDC) written for the QFT allow for it to be used more broadly, in lieu of the TST in all circumstances in which the TST is currently used. 1 In the present study, we sought to validate the use of the T-SPOT.TB test as compared with the TST under routine clinical conditions in the United States. In an urban public health clinic setting, we studied a cohort that included both close contacts of cases of active tuberculosis and noncontacts, of whom most were recent immigrants with a high rate of BCG vaccination. Contact status was used as a surrogate for LTBI. We aimed to compare the use of the T-SPOT.TB test with the TST in detecting LTBI in high-risk individuals as well as discriminating LTBI from BCG vaccination. Specifically, we aimed to answer the following questions: first, what is the sensitivity of the T-SPOT.TB test for contact status as compared with the TST? Second, what is the specificity of the T- SPOT.TB test in this population, and how does it *From the Division of Pulmonary, Allergy, and Critical Care Medicine (Drs. Brodie, Lederer, Gallardo, Trivedi, and Schluger), Columbia University Medical Center; and New York City Department of Health and Mental Hygiene (Dr. Burzynski), Bureau of Tuberculosis Control, New York, NY. Equipment for this study was provided by Oxford Immunotec, Ltd, Oxford, UK. Dr. Brodie is supported in part by a grant from the Stony Wold- Herbert Fund, Inc. Dr. Schluger is supported in part by the National Institutes of Health/National Heart, Lung, and Blood Institute (K24 HL004074). Dr. Lederer is supported in part by grant RR from the National Institutes of Health. The authors have no financial or other potential conflicts of interests to disclose. Manuscript received July 23, 2007; revision accepted December 5, Reproduction of this article is prohibited without written permission from the American College of Chest Physicians ( org/misc/reprints.shtml). Correspondence to: Neil Schluger, MD, Columbia University Medical Center, 622 West 168th St, PH 8 East, Room 101, New York, NY 10032; ns311@columbia.edu DOI: /chest compare with the TST? And third, what are the odds of having a positive test result (either T-SPOT.TB test or TST) in contacts as compared with noncontacts? Study Subjects Methods and Materials This study was approved by the institutional review boards of the New York City Department of Health and Mental Hygiene (DOHMH) and the Columbia University Medical Center. All subjects provided written, informed consent. We enrolled 123 subjects from a New York City DOHMH tuberculosis clinic from September 8, 2005, through February 21, Those enrolled included contacts of cases of active tuberculosis ( contacts ) and subjects who were not contacts of patients with active tuberculosis ( noncontacts ) but who were administered a TST at the DOHMH for reasons other than contact status (primarily as a result of outside referral regardless of risk status). All cases of active tuberculosis were sputum culture positive for M tuberculosis. Contacts were identified by the DOHMH and classified according to their standardized protocol into close contacts (defined as 8 h of contact with a patient with active tuberculosis per week), and other than close contacts (defined as those with contact for 8 h/wk.) A TST was administered as part of the routine workup. Subjects with active tuberculosis were excluded. All TSTs were performed by the Mantoux technique using 5 tuberculin units. TSTs were administered and read by trained, experienced DOHMH personnel according to DOHMH protocol. A positive reaction to the TST was defined according to published CDC criteria that stratify results by risk groups. 7,8 Inclusion and Exclusion Criteria Subjects were 5 years old. Subjects with active tuberculosis or nontuberculous mycobacteria infection or who had previously been treated for LTBI, active tuberculosis, or nontuberculous mycobacteria were excluded. Subjects with known HIV infection were excluded. TST testing must have taken place within 6 months of enrollment and T-SPOT.TB testing. A history of a positive TST result 6 months prior to enrollment also led to the exclusion of the subject because they were not retested if previously positive. Study Procedure After consent was obtained, demographic information and medical history were recorded on a standard questionnaire. Race and ethnicity were self-identified. Subjects were considered to have a history of BCG vaccination if they came from a country where BCG is routinely administered and either reported a history of BCG or had a characteristic vaccination scar. All patient information remained anonymous, identified only by a unique patient code, and was stored in locked files. All blood samples were marked with the patient code and were not identifiable by any other means. Samples were kept at room temperature and processed in our laboratory within8hbyoneofthree technicians all of whom were trained to run the T-SPOT.TB test by the manufacturer. The T-SPOT.TB test was performed according to manufacturer specifications. Spots were counted manually with a magnifying glass, but final results were based on counts checked by an automated plate reader after shipment to the manufacturer, Oxford Immunotec, Ltd. Additional detail on the method is provided in an online data supplement. Assay results and ques- 870 Original Research

3 tionnaire data were entered into a clinical database. The study sponsor played no role in data collection, analysis, or interpretation and was not involved in the writing or submitting of this article for publication. Statistical Analysis Continuous variables were summarized by mean and SD. Categorical variables were summarized by frequency and percentage and compared using 2 tests. We used logistic regression to estimate odds ratios for the associations between contact status and TST or T-SPOT.TB test results. Statistically significant interaction terms (p 0.05) were retained in the model. Multivariate models included terms for BCG vaccination status, age, HIV status (negative or unknown), and race/ethnicity. In lieu of a true gold standard for the diagnosis of LTBI, we used tuberculosis exposure (ie, contact status) as the standard for determining the operating characteristics of the TST and the T-SPOT.TB test. The overall agreement,, sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of each test as predictors of close contact status were estimated from contingency tables; p values 0.05 were considered statistically significant. Statistical software (SAS 9.1; SAS Institute; Cary, NC) was used for all analyses. Role of the Sponsor Oxford Immunotec Ltd. provided the required consumables including cell preparation tubes (Becton Dickinson; Franklin Lakes, NJ) and venipuncture systems, training in the use of the assay, the T-SPOT.TB assay kits, and automated plate reading. In addition, they paid a portion of the salary of one technician for 2 years. Excluded Subjects Results We enrolled 123 subjects, of whom 27 were excluded. Four subjects never returned to have their TST results read. Three subjects were identified after enrollment who had positive TST results 6 months prior, and a repeat TST was not done as per protocol. Two subjects were determined to be HIV positive. One subject had been previously treated for LTBI but did not recall this information until after enrollment. Disseminated M bovis infection was diagnosed in one patient. Phlebotomy was unsuccessful in two subjects. Eight subjects had indeterminate tests. Technical difficulties with the T-SPOT.TB test occurred in six subjects. Study Subjects The characteristics of the 96 subjects included in the study are presented in Table 1. Fifty-six subjects were close contacts. Among the close contacts, 52 subjects were exposed to patients with active tuberculosis who were themselves acid-fast bacilli smear positive. Three additional subjects were other than close contacts. Each had minimal contact with index active tuberculosis cases. Their TST results were 20 mm, 10 mm, and Table 1 Characteristics of Close Contacts and Control Subjects* Variables Close Contacts 0 mm, respectively. We conservatively chose to group these three subjects with our noncontact control subjects (control group). Age, gender, foreign birth, race/ ethnicity, and HIV status were similar between close contacts and control subjects. Among close contacts, 63% were TST positive, 45% were T-SPOT.TB test positive, and 68% were BCG vaccinated. Among control subjects, 78% were TST positive, 25% were T-SPOT.TB test positive, and 75% were BCG vaccinated. Agreement Between the T-SPOT.TB and TSTs Overall agreement between the T-SPOT.TB and the TST was 64% (95% confidence interval [CI], 54 to 74%), and was 0.33 (95% CI, 0.19 to 0.48). Among BCG-vaccinated subjects, overall agreement and were 56% (95% CI, 43 to 68%) and 0.22 (95% CI, 0.06 to 0.37), respectively. Among those not BCG vaccinated, overall agreement and were 82% (95% CI, 68 to 96%) and 0.64 (95% CI, 0.38 to 0.91), respectively. Both overall agreement and were significantly better among subjects without BCG vaccination compared to subjects with BCG vaccination (p 0.02 for each measure). Associations Between Contact Status and the Results of the T-SPOT.TB and TSTs Control Subjects Subjects 56 (58) 40 (42) Age, yr Gender Male 39 (70) 20 (50) Female 17 (30) 20 (50) Foreign birth 41 (73) 32 (80) Race/ethnicity Hispanic (all races) 34 (61) 23 (58) Non-Hispanic black 13 (23) 9 (23) Non-Hispanic white 8 (14) 5 (13) Non-Hispanic Asian 1 (2) 3 (8) T-SPOT.TB test result Positive 25 (45) 10 (25) Negative 31 (55) 30 (75) TST result Positive 35 (63) 31 (78) Negative 21 (38) 9 (23) TST size 13.5 (0 18.5) 14 (10 20) BCG Vaccinated 38 (68) 30 (75) Unvaccinated 18 (32) 10 (25) HIV Negative 29 (52) 26 (65) Unknown 27 (48) 14 (35) *Data are presented as No. (%), mean SD, and median (interquartile range). In univariate analyses, neither the T-SPOT.TB test nor the TST were significantly associated with contact CHEST / 133 / 4/ APRIL,

4 Table 2 Odds Ratios for a Positive Test Result Among Close Contacts vs Control Subjects Variables Odds Ratio 95% CI status (Table 2). However, after adjustment for age, HIV status (negative or unknown), BCG vaccination status, and race/ethnicity, being a close contact was strongly associated with a positive T-SPOT.TB test (adjusted odds ratio, 2.9; 95% CI, 1.1 to 7.4, p 0.03). In this model, BCG vaccination was not significantly associated with a positive T-SPOT.TB test (adjusted odds ratio, 1.5; 95% CI, 0.5 to 4.2; p 0.47). The association between TST result and contact status varied by BCG vaccination status (p for interaction). Among BCG-vaccinated subjects, close contacts were less likely than control subjects to have a positive TST (adjusted odds ratio, 0.1; 95% CI, 0.01 to 0.5; p 0.01). However, among subjects without BCG vaccination, close contacts were more likely than control to have a positive TST result (adjusted odds ratio, 9.1; 95% CI, 1.2 to 67; p 0.03). Diagnostic Characteristics of the T-SPOT.TB and TSTs p Value T-SPOT.TB test Unadjusted Adjusted* TST Unadjusted Adjusted BCG vaccinated only BCG unvaccinated only *Adjusted for BCG vaccination status, age, HIV status, and race/ ethnicity; p value for interaction between contact status and BCG vaccination p 0.001, contact status vs BCG vaccination; therefore, the model adjusted for age, HIV status, and race/ethnicity is presented stratified by BCG vaccination status. The diagnostic characteristics of the T-SPOT.TB and TST are shown in Table 3. The T-SPOT.TB test was more specific for contact status than the TST (75% vs 23%, p 0.001). Importantly, this difference was even more pronounced among BCGvaccinated subjects (70% vs 3%, p 0.001). In contrast, the tests had similar specificities among subjects without BCG vaccination. The T-SPOT.TB test and the TST had similar sensitivities for contact status regardless of BCG vaccination status. Among BCG-vaccinated subjects, the T-SPOT.TB test had a higher negative predictive value (50% vs 6%, p 0.002) and a higher accuracy (56% vs 35%, p 0.02). Test characteristics were similar for both tests among BCG-unvaccinated subjects. Indeterminate Results and Technical Difficulties Eight subjects had indeterminate tests. None of the eight were known to have an immunosuppressive condition and none were receiving immunosuppressive medications. Four of the eight subjects were BCG vaccinated, and six subjects were TST positive. Technical difficulties with the T-SPOT.TB test occurred in six subjects. Five subjects had excessive spots in the nil control. Only one of the five subjects was available for repeat testing, and the repeat demonstrated excessive spots in the nil control once again. One additional subject had too low a cell count for interpretation. Discussion We have demonstrated that, compared with TST, the T-SPOT.TB test has improved specificity in identifying a group of subjects at high risk for progression to active tuberculosis, namely, close contacts of cases of active tuberculosis. Our study demonstrates the superiority of the T-SPOT.TB test over the TST under routine clinical conditions in a population critical to tuberculosis control: foreignborn persons from high-prevalence countries with high rates of BCG vaccination. These findings should Table 3 Diagnostic Characteristics of the T-SPOT.TB and TST as Predictors of Close Contact Status Variables Sensitivity (95% CI) Specificity (95% CI) Positive Predictive Value (95% CI) Negative Predictive Value (95% CI) Accuracy (95% CI) Entire cohort T-SPOT.TB 45 (31 59) 75 (59 87)* 71 (54 85) 49 (36 62) 57 (47 67) TST 62 (49 75) 23 (11 38) 53 (40 65) 30 (15 49) 46 (36 56) BCG vaccinated only T-SPOT.TB 45 (29 62) 70 (50 85)* 65 (44 83) 50 (34 66)* 56 (43 68)* TST 61 (44 76) 3 (0 17) 44 (30 59) 6 (0 30) 35 (24 47) BCG unvaccinated only T-SPOT.TB 44 (21 69) 90 (56 99) 89 (52 99) 47 (24 71) 61 (41 79) TST 67 (41 87) 80 (44 97) 86 (57 98) 57 (29 82) 71 (51 87) *p 0.05 for comparison with TST. 872 Original Research

5 be widely applicable to the many outpatient clinics serving similar populations. As a recent metaanalysis 5 points out, prior studies of the T-SPOT.TB test have suggested improved specificity as compared with the TST, especially in BCGvaccinated individuals. Our study adds strength to these earlier findings by demonstrating good performance characteristics of the test in a population that included both close contacts and noncontacts. Shams et al 9 reported similar findings in a large cohort of contacts using a scoring system which had not been previously validated for determining the likelihood of being a close contact. Interestingly, our study demonstrated similar performance characteristics for the T-SPOT.TB test as in the study by Shams et al, 9 with a much simpler scheme for judging closeness of contact. Another study 10 that examined both contacts and others was largely hospital based and does not reflect routine outpatient clinical practice. The improved specificity of the T-SPOT.TB test is especially important because it could decrease unnecessary treatment in those who are not actually infected. This would avoid costs to the health-care system (as well as the individual patient) for medication, follow-up, and management of complications. Given that there is no gold standard for the diagnosis of LTBI, determining test characteristics of any novel diagnostic, as compared with the TST, becomes problematic. Specificity is something of an exception. A test could be considered superior in specificity if its results appeared to be independent of BCG vaccination status. Since BCG vaccination status is easily ascertainable by the investigator (or the clinician), a reasonable estimate of specificity may be obtained. Sensitivity is more difficult to determine. Contacts are imperfect surrogates for the diagnosis of LTBI. Because not all contacts are actually infected with M tuberculosis, it is difficult to precisely estimate the sensitivity of any new test. Nevertheless, since it is well documented that the risk of LTBI among contacts increases with increasing exposure to a source case, 4,11,12 or increasing amounts of time spent in situations of known risk for acquiring LTBI (eg, incarceration), the odds of having LTBI correlate with degree of exposure. 9,12,13 A test that is superior to the TST in sensitivity would therefore be more likely to be positive in a contact than a noncontact. Sensitivity in the context of our study therefore refers to sensitivity for contact status, and there was no statistically significant difference between the tests in their sensitivity for contact status. We found that there were fewer positive T-SPOT.TB test results than positive TST results in those deemed close contacts. However, the clinical significance of this finding is unclear and may reflect the fact that some of those who were deemed close contacts were not truly infected but had false-positive TST results due to prior BCG vaccination. To confirm that suspicion, a study similar to ours but using a largely BCG-unvaccinated population would need to be undertaken. Although no single, large study of this kind has been done, a comprehensive metaanalysis that includes smaller studies of this type concluded that the sensitivity of the IGRAs and the TST were in fact similar. 5 Another factor that may have contributed to the discrepancy is the possibility that the TST detects both new infection and remote infection in which the IGRAs may only detect the latter. The finding of a negative association between the TST and close contact status in our analysis of BCG-vaccinated subjects almost certainly reflects abundant false-positive results with skin testing in these subjects and reinforces the impression of confounding of the TST by BCG. Clearly, the strong association between the TST and contact status in subjects without BCG vaccination shows its utility in this setting. However, in BCG-vaccinated individuals, the TST appears to be highly inaccurate as a predictor of contact status. In actuality, identifying every person with LTBI is not important clinically because only a small percentage of such persons will ever go one to have active tuberculosis, and most will not benefit (and may be harmed) by treatment. A strategy aimed at identifying those with LTBI who are most at risk of progression to active TB is a useful one in evaluating a new diagnostic test. In that regard, we have shown that in a population such as ours, the T-SPOT.TB test is superior to the TST. One potential limitation of our study was the collection of blood samples for T-SPOT.TB testing regardless of the timing of TST. There is a theoretical concern that the IGRAs may be boosted by TST placement. A recent report 14 suggests that this may be an issue with the QFT but not with the T-SPOT.TB test. Other investigators 9,15 have similarly found no boosting effect with the ELISPOT test. Nonetheless, it is an issue that requires further study. Another issue that this raises is that of conversions and reversions of the TSTs during the period of time between the TST and the T-SPOT.TB test. While this window of time is rather small with regard to this phenomenon, an effect cannot be ruled out. Both the TST and the T-SPOT.TB test rely to a degree on an intact immune response. It is therefore imperative that they be tested in populations with impaired immune function, particularly in patients with HIV given the disease burden of TB in this population. In our study, we took care to avoid mixing these populations because this might have clouded the interpretation of our results. We CHEST / 133 / 4/ APRIL,

6 therefore excluded patients with known HIV. Despite this, eight subjects had indeterminate test results, defined as an inadequate response to the mitogen-positive control. Interestingly, six of the eight subjects had positive TST results (two subjects with unknown HIV status, and four subjects who were HIV negative by report), which argues against the presence of complete anergy in these subjects. However, anergy is a highly variable phenomenon, and it is possible that antigens present in the Mantoux reagent other than ESAT6 and CFP10 elicited a skin test response not provoked by the T-SPOT.TB assay. The cutoff values for the TST were interpreted according to the current guidelines as indicated in the Materials and Methods section. The results would not have been affected by stratifying the analysis using different TST size cutoffs because only two contacts were TST positive with 10 mm of induration (5 mm and 6 mm, respectively), and no noncontacts were deemed negative in the 10- to 14-mm range (low risk). Currently, the QFT is recommended by the CDC. 1 CDC recommendations do not make mention of the T-SPOT.TB test since it is not yet approved by the US Food and Drug Administration. At this time, we cannot say whether the results of this study can be extrapolated to the QFT. Further comparisons of the two tests with the TST and with each other are needed. Also, our study did not address the cost-effectiveness of the T-SPOT.TB test in this setting. However, several studies have concluded that the IGRAs are indeed likely to be cost-effective. In conclusion, we have demonstrated that compared with skin testing, the T-SPOT.TB test has greatly improved specificity in identifying a group of subjects at high risk for progression to active tuberculosis. The T-SPOT.TB test proved robust in the setting of routine outpatient clinic conditions, suggesting wide applicability. Further testing is needed in immunocompromised subjects and subjects at the extremes of age. Longitudinal studies are needed to verify the significance of a positive T-SPOT.TB test with regard to risk for active tuberculosis. ACKNOWLEDGMENT: We gratefully acknowledge the support of the Stony Wold-Herbert Fund, Inc. References 1 Taylor Z, Nolan CM, Blumberg HM. Controlling tuberculosis in the United States: recommendations from the American Thoracic Society CDC, and the Infectious Diseases Society of America. MMWR Recomm Rep 2005; 54: Geng E, Kreiswirth B, Driver C, et al. Changes in the transmission of tuberculosis in New York City from 1990 to N Engl J Med 2002; 346: Farhat M, Greenaway C, Pai M, et al. False-positive tuberculin skin tests: what is the absolute effect of BCG and non-tuberculous mycobacteria? Int J Tuberc Lung Dis 2006; 10: Brodie D, Schluger NW. The diagnosis of tuberculosis. Clin Chest Med 2005; 26: , vi 5 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: National Collaborating Centre for Chronic Conditions. Tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control. London, UK: Royal College of Physicians, Targeted tuberculin testing and treatment of latent tuberculosis infection. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, July This is a Joint Statement of the American Thoracic Society (ATS) and the Centers for Disease Control and Prevention (CDC). This statement was endorsed by the Council of the Infectious Diseases Society of America (IDSA), September Am J Respir Crit Care Med 2000; 161:S221 S247 8 Screening for tuberculosis and tuberculosis infection in highrisk populations: recommendations of the Advisory Council for the Elimination of Tuberculosis. MMWR Recomm Rep 1995; 44: Shams H, Weis SE, Klucar P, et al. Enzyme-linked Immunospot and tuberculin skin testing to detect latent tuberculosis infection. Am J Respir Crit Care Med 2005; 172: Ferrara G, Losi M, D Amico R, et al. Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study. Lancet 2006; 367: Lutong L, Bei Z. Association of prevalence of tuberculin reactions with closeness of contact among household contacts of new smear-positive pulmonary tuberculosis patients. Int J Tuberc Lung Dis 2000; 4: Ewer K, Deeks J, Alvarez L, et al. Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet 2003; 361: Arend SM, Thijsen SF, Leyten EM, et al. Comparison of two interferon- assays and tuberculin skin test for tracing tuberculosis contacts. Am J Respir Crit Care Med 2007; 175: Naseer A, Naqvi S, Kampmann B. Evidence for boosting Mycobacterium tuberculosis-specific IFN- responses at 6 weeks following tuberculin skin testing. Eur Respir J 2007; 29: Richeldi L, Ewer K, Losi M, et al. Repeated tuberculin testing does not induce false positive ELISPOT results. Thorax 2006; 61: Wrighton-Smith P, Zellweger JP. Direct costs of three models for the screening of latent tuberculosis infection. Eur Respir J 2006; 28: Dewan PK, Grinsdale J, Liska S, et al. Feasibility, acceptability, and cost of tuberculosis testing by whole-blood interferon- assay. BMC Infect Dis 2006; 6:47 18 Diel R, Nienhaus A, Lange C, et al. Cost-optimisation of screening for latent tuberculosis in close contacts. Eur Respir J 2006; 28: Diel R, Wrighton-Smith P, Zellweger JP. Cost-effectiveness of interferon- release assay testing for the treatment of latent tuberculosis. Eur Respir J 2007; 30: Original Research