ª 2005 Lippincott Williams & Wilkins Curr Opin Pulm Med 11: ª 2005 Lippincott Williams & Wilkins.

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1 Utility of the antigen-specific interferon-g assay for the management of tuberculosis Keertan Dheda a,b, Zarir F. Udwadia c, Jim F. Huggett a, Margaret A. Johnson b and Graham A.W. Rook a Purpose of review The tuberculin skin test, now over a century old, is prone to reader variability, and outcomes are influenced by cross-reactivity with environmental mycobacteria, previous bacillus Calmette-Guerin (BCG) vaccination, and anergy in immunosuppressed individuals. More recently, T-cell-based interferon-g responses to Mycobacterium tuberculosis specific antigens have been investigated for their role in diagnosing latent tuberculosis infection. Recent findings We review the evidence supporting the utility of the interferon-g assay for the diagnosis of latent tuberculosis infection (LTBI) in low-prevalence countries. We discuss the principle of the test, technical factors related to performance, and its utility in active tuberculosis, in specialised subgroups such as immunocompromised patients, and its applicability in developing countries. Summary Compared with the tuberculin skin test, the antigen-specific interferon-g assay, when used in a standardised protocol (overnight incubation assay using a combination of two antigens) for the diagnosis of LTBI, has greater specificity in BCG-vaccinated individuals, displays a stronger association with exposure, and is less biased by environmental mycobacteria such as Mycobacterium avium. Prospective studies are required, however, to confirm that treating LTBI, as defined by the interferon-g assay, will reduce the tuberculosis burden in low-prevalence countries and whether interferon-g responses are predictive of those who have a high risk of progression to active tuberculosis. Further studies are required to address the utility of the interferon-g assay in specialised subgroups of patients, in developing countries, and as a marker of disease activity. Keywords early secreted antigenic target 6 kda, enzyme-linked immunosorbent assay, enzyme-linked immunosorbent spot-forming cell, immunodiagnosis, interferon-g, tuberculosis Curr Opin Pulm Med 11: ª 2005 Lippincott Williams & Wilkins. a Centre for Infectious Diseases and International Health, Royal Free & UCL Medical School, London, UK, b Department of Thoracic and HIV Medicine, Royal Free Hospital, London, UK, and c Hinduja Hospital and Research Centre, Bombay, India Correspondence to Keertan Dheda, Centre for Infectious Diseases and International Health, UCL and Royal Free Medical School, 46 Cleveland Street, London, W1T 4JF, UK Tel: ; fax: ; k.dheda@ucl.ac.uk This study was supported by a grant from the British Lung Foundation. Current Opinion in Pulmonary Medicine 2005, 11: Abbreviations BCG bacillus Calmette-Guerin CFP-10 culture filtrate protein 10 ELISA enzyme-linked immunosorbent assay ESAT-6 early secreted antigenic target 6 kda HBHA heparin-binding haemagglutinin LTBI latent tuberculosis infection LRTI lower respiratory tract infection TST tuberculin skin test ª 2005 Lippincott Williams & Wilkins Introduction Tuberculosis is a public health catastrophe resulting in one death every 15 seconds [1]; in high-burden countries almost two-thirds of new cases are coinfected with HIV [2]. In approximately 85% of cases the disease affects the lungs. It is estimated that a third of the world s population is infected with Mycobacterium tuberculosis. These individuals are asymptomatic and usually have normal chest radiographs, and until now, the only way to identify underlying M. tuberculosis infection was to use the tuberculin skin test (TST). The TST has a number of drawbacks, however, including reader variability, and these are further discussed below. Recently, peripheral blood derived T-cell interferon-g responses to M. tuberculosis specific antigens early secreted antigenic target 6 kda (ESAT-6) and culture filtrate protein 10 (CFP-10) have been investigated for the management of tuberculosis. This review outlines the principle of the test, related technical factors that modulate results, the available commercial assays, and performance outcomes of the test. We also discuss its utility for the diagnosis of latent tuberculosis infection (LTBI), as a tool to facilitate diagnosis in active tuberculosis, and applicability to those with underlying immunodeficiency. Finally we discuss applicability to highburden and resource-poor settings. Peer-reviewed data for this paper were identified by searches of the Medline and PubMed databases, up to and including October 2004, in all languages, using the search terms tuberculosis and ESAT-6, CFP-10, RD1, IFN-g, T-cell epitopes, and immunodiagnosis. Other sources were experts in the field, manufacturers, the references of retrieved articles, textbooks, and the files of the authors. We have noted a marked heterogeneity in the type of assay used enzyme-linked (ELISA) vs immunospot (ELISPOT) the type of antigen used (peptide vs protein), the number of antigens used (cocktails vs ESAT-6 and CFP-10 and purified protein derivative alone), and the type of test (in-house 195

2 196 Infectious diseases vs standardized protocol and reagents). These factors, particularly the latter, may account for interlaboratory variability and are important for valid comparison between investigators. Consequently, for practical and meaningful interpretation of tests that are available to the clinician or other interested readers, we have focused on assays that use standardized protocols incorporating both ESAT-6 and CFP-10 antigens and that use incubation times of 24 hours or less, unless otherwise stated. Antigen-specific interferon-g assay and the tuberculin skin test Latent tuberculosis infection is classically diagnosed by performing a TST, which measures cell-mediated immunity as a delayed-type hypersensitivity reaction to purified protein derivative (a mixture of M. tuberculosis specific and nonspecific antigens). The TSTsuffers from a number of problems that interferon-g assays are designed to solve (Table 1). Furthermore, due to anergy, the TST is often unhelpful in individuals with chronic organ dysfunction, diabetes, malnourished or debilitated patients, and those taking immunosuppressive treatments. Diagnosis of LTBI by the new test is based upon a vigorous T-cell interferon-y response, measured by ELISPOT assay or ELISA, to relatively M. tuberculosis specific antigens, ESAT-6 and CFP- 10 (Fig. 1). Both the proteins are encoded on the region of difference (RD1) domain, a region associated with virulence and attenuation of bacillus Calmette-Guerin (BCG) [3 ]. An ESAT-6 receptor has not been described and the precise function of these secreted proteins remains unclear (reviewed by Brodin et al. [3 ]). Their expression is absent in BCG and most environmental mycobacteria except Mycobacterium marinum, Mycobacterium szulgai,andmycobacterium kansasii; homologues of these proteins are also found in the genome of Mycobacterium leprae [4]. There are two commercial assays available, which both measure overnight interferon-g responses (<24 hours) to overlapping ESAT-6 and CFP-10 peptides. The T SPOT TB assay (Oxford Immunotec, Oxford, UK, see oxfordimmunotec.com) is an ELISPOT assay that uses peripheral blood mononuclear cells and has European CE Mark approval. QuantiFERON-TB Gold (Cellestis, Victoria, Australia) is an ELISA using whole blood and has both European CE Mark and American Food and Drug Administration approval. The older QuantiFERON- TB, which measured interferon-g responses to purified protein derivative, is now superseded by Quanti- FERON-TB Gold, which is also available in a fieldfriendly version, QuantiFERON-TB Gold In-Tube, although there are no published data using the latter version (see Latent tuberculosis infection The lack of a gold standard for diagnosing LTBI was a problem that investigators faced when trying to determine performance outcomes of the interferon-g assay. It is assumed that sensitivity should be at least as good as, or better than, in active tuberculosis, which tends to have lower interferon-g levels due to the immunosuppressive effect of disease itself and trafficking of cells out of the peripheral blood compartment [5]. Based on this arguable assumption, the sensitivity was determined to be between 78 and 100% when both ESAT-6 and CFP-10 antigens were used in either an ELISPOT or ELISA overnight incubation assay [6 9,10 ]. The number of subjects per study varied between eight [7] and 118 [10 ]. Specificity was determined by studying T-cell interferon-g responses in developed-world study subjects who were asymptomatic and at low risk for LTBI. Specificity in these populations when both antigens were used, either in overnight ELISPOTor ELISA format, was % [6 8,10 ]. Study subjects varied from 22 [7] to 216 [10 ]. Compared with the TST, the specificity of interferon-g assay was considerably higher in BCG-vaccinated individuals [7,11]; 89 vs 53% and 94 vs 55% in two studies, respectively. Mori et al. [10 ] studied 216 BCG-vaccinated nurses who had no risk factors for LTBI; specificity of the TST was considerably lower than the interferon-g assay (35 vs 98%); in Table 1. Comparison of factors impacting upon utility of the tuberculin skin test and interferon-g assay Parameter Tuberculin skin test Interferon-g assay Specificity Cross-reactivity with BCG and environmental bacteria Relatively Mycobacterium tuberculosis specific Workload Requires return visit for which attendance is poor Single visit Chemoprophylaxis Casts a wide net; may result in overtreatment May avoid unnecessary treatment and toxicity Subjectivity Results dependent on observer and technique and cross-reactivity with BCG or other bacteria Provides yes / no answer Booster phenomenon Yes No Cost High in developed countries; low in developing countries Affordable in developed countries Longitudinal efficacy data Plentiful Limited (1 study) Other factors Prone to breakage of cold chain and syringe reuse in resource-poor setting Requires basic laboratory expertise BCG, bacillus Calmette-Guerin.

3 Interferon-g assays and tuberculosis Dheda et al. 197 TB exposure than did TST, suggesting increased sensitivity with ELISPOT [13 ]. Furthermore, ELISPOT, but not TST, was independent of BCG vaccination status indicating higher specificity than TST. The authors suggest that the higher specificity of the interferon-g assay allows for more precise targeting of antituberculosis treatment in low-prevalence countries. Concordance between the TST and interferon-g assay was 89% [13 ] and was similar in other studies using overnight incubation with both antigens (74 94%) [7,15 ]. There is a paucity of data on assay reproducibility and on changes in responses over time and with treatment of LTBI. Figure 1. The methods used for the two commercially available interferon-g assays. In the T SPOT TB ELISPOT assay (a), peripheral blood mononuclear cells (PBMCs) are separated from blood and incubated with antigen in plates of which the wells are precoated with an antibody that captures the interferon-g. For the QuantiFERON-TB Gold ELISA assay (b), the antigens are added to whole blood, and the supernatant is subsequently assayed for interferon-g using a sensitive enzyme-linked immunosorbent assay. Thus the former yields the total number of cells secreting interferon-g, while the latter yields the total quantity of interferon-g released. Numerous variants of these protocols are possible. two-thirds of evaluable study subjects the TST measured more than 10 mm and in a third more than 15 mm. Although there is no gold standard for LTBI, we know that the risk of LTBI is closely related to proximity and duration of exposure to infectious pulmonary tuberculosis cases. Investigators have therefore used precisely quantified TB exposure as a surrogate gold standard for LTBI [12,13,14]. Ewer et al. [13 ], in the largest such study, using an overnight ELISPOT assay incorporating both antigens and performed 8 weeks following exposure in a school tuberculosis outbreak, found that ELISPOT correlated significantly better with duration and proximity of Interferon-g assays have also been investigated in other mycobacterial infections. Most patients with clinically active M. marinum and M. kansasii disease had positive interferon-g responses to either ESAT-6 or CFP-10 antigens [10,16]. Positive responses were also found in individuals with high environmental mycobacterial exposure, such as flower sellers and tropical fish tank owners [16]. It has been shown in low-burden countries that a significant proportion of positive TSTs are due to M. avium infection [17,18]. Interferon-g assays remain negative, however, in the face of proven M. avium disease [19]. M. leprae homologues of M. tuberculosis CFP-10 [20] and ESAT-6 [21], designated L-ESAT-6 and L-CFP-10, induce interferon-g from T cells of patients with leprosy, active tuberculosis [20 22], and also in healthy volunteers from Brazil where leprosy is endemic [21]. Consequently, the standardised interferon-g assays will need validation in populations who come from or reside in countries where both tuberculosis and leprosy are endemic and where there is a high environmental mycobacterial load [23]. Available data indicate the interferon-g-based prevalence of LTBI to be 80% in Mumbai, India [9] and 30% in the Gambia [15 ]. In the only published study that prospectively evaluated outcomes, an interferon-g response to ESAT-6 was strongly predictive for the subsequent development of active disease in 24 Ethiopian subjects with LTBI over a 2-year follow-up period, whereas an interferon-g response to purified protein derivative was not [24]. Collectively these data indicate that, when a combination of antigens are used (ESAT-6 and CFP-10) in either overnight ELISA or ELISPOT format, interferon-g assays have a high sensitivity for diagnosing presumed LTBI, and agreement between the TST and interferon-g assays is satisfactory. Nevertheless, in the absence of a gold standard, the absolute sensitivity of the interferon-g assay, and the TST for that matter, for diagnosing LTBI remains unclear. When compared with the TST, however, interferon-g assays have greater specificity in BCG-vaccinated subjects and in the case of T-SPOT TB display a stronger association with TB exposure. Interferon-g assays are less biased by environmental mycobacteria like M. avium, which is responsible for significant number of false-positive TST

4 198 Infectious diseases reactions [18]. This specificity will facilitate more accurate targeting of treatment for LTBI. To what extent a heavy burden of environmental mycobacteria and M. leprae influence the performance of interferon-g assays requires further study [4,23]. Despite the shortcomings of the TST, large studies have shown that treatment of LTBI, as defined by the TST, substantially reduces the risk of developing active disease [25 27]. Consequently, the important clinical question is whether treatment of those identified as having LTBI by the interferon-g assay will reduce the incidence of subsequent clinical tuberculosis. Secondly, relevant to both industrialized and developing nations, it remains to be determined whether interferon-g responses are predictive of those who have a risk of progression to active tuberculosis. Long-term and appropriately powered prospective studies are required and currently underway to answer these questions. Utility in HIV: tuberculosis coinfection The risk of active tuberculosis doubles in the first year of HIV coinfection [28], and the risk of developing active disease in those who have LTBI is approximately 10% per year [29]. HIV tuberculosis coinfected individuals have reduced survival [30] and are at higher risk for subsequent opportunistic infections [31,32]. Treating LTBI in the HIV-positive patient substantially reduces the subsequent risk of tuberculosis [33]. Consequently treatment of LTBI is recommended and is suggested by a TST exceeding 5 mm [34,35]. Meaningful utility of the TST, however, is hampered by anergy in HIV-positive and other immunocompromised individuals [36] and this worsens with increasing immunosuppression [37]. The interferon-g assay may circumvent this problem; however, published studies that have enrolled HIV-positive subjects are limited [8,9,38,39,40]. Chapman and coworkers [8] studied 39 Zambian HIV TB coinfected patients with advanced immunosuppression (mean lymphocyte count ; /L blood); sensitivity was 90% in patients with active tuberculosis, and three of 14 Zambian adults with LTBI had a positive interferon-g assay but negative TST findings [8]. These preliminary data suggest that the interferon-g assay may be more sensitive than the TST in HIV-positive individuals with LTBI. Whether this will confer benefit in HIV-positive individuals, however, requires confirmation in well-powered prospective studies as those anergic by the TSTappear not to be at high risk for developing active tuberculosis [41,42]. It remains unclear to what extent anergy will influence the interpretation of the interferon-g assay. Is this test useful for evaluation of patients with active tuberculosis? In patients with active disease, even when HIV seroprevalence rates are low and there is good laboratory infrastructure, only ;60% of treated patients have microbiologically confirmed disease [43]. Therefore in day-to-day clinical practice the TST is frequently used as additional evidence of presumptive disease. The TST is unhelpful, however, in at least half the cases; if negative it may be due to anergy (10 25% of cases [44]) and if positive may represent a false positive in up to a third of cases [45]. Moreover, in children, immunocompromised patients, and those with extrapulmonary tuberculosis, bacterial counts may be low and sample acquisition difficult; consequently diagnosis is problematic for the clinician and health care costs increase. A negative interferon-g assay may be a convenient rule out test for tuberculosis if the diagnostic sensitivity of the assay is sufficiently high, e.g. ;95%. This would be especially useful when investigating pulmonary infiltrates or an extrapulmonary clinical presentation where tuberculosis is part of the differential diagnosis (fungal infections, nocardia, immunocompromised individuals, or foreign-born persons living in Western countries). Significantly, the sensitivity of the IFN-g ELISPOT was 96% in active TB but results were negative in patients with non-tuberculous pulmonary diseases including sarcoidosis [46,47]. A negative test, furthermore, may prompt a more vigorous search for alternative diagnoses. Prospective studies are required to evaluate interferon-g assays for their utility as rule out tests in patients in whom tuberculosis is part of the differential diagnosis. As interferon-g assays do not distinguish between LTBI and active disease, their use as a rule in test becomes tenuous. A positive interferong assay result can help to support a presumptive diagnosis of active TB where the clinical index of suspicion is high but other diagnostic tests are negative. Here a positive result can be of value in initiating empiric treatment [46,47]. Richeldi et al. [48] described a case of multidrug-resistant tuberculosis in an asymptomatic man with a cavitating lesion on CT scan who had a negative TST and positive ELISPOT interferon-g assay. Monitoring of disease activity and efficacy of antituberculosis treatment The test relies on the principle that effector lymphocytes, which have very recently encountered antigen in vivo, will produce interferon-g within hours when they encounter M. tuberculosis specific antigens. By contrast, memory T cells are more likely to require several days in the presence of antigen before interferon-g is produced [49 51]. Therefore incubation of whole blood or peripheral blood mononuclear cells is for 24 hours or less (16 20 hours for T-SPOT TB, hours for QuantiFERON-TB Gold). This is to ensure that effector rather than memory T-cell responses are being measured. If longer incubation times are used, it is possible for infection that has been treated or resolved to yield positive tests. This may explain the discordant results of studies that evaluated the change in interferon-g responses with antituberculosis treatment. Studies that show substantially decreased interferon-g responses with treatment used short incubation times (ELISPOT with <24 hours in two studies and #40 hours

5 Interferon-g assays and tuberculosis Dheda et al. 199 in the other) [9,46,52 ], whereas those that showed equivalent or increasing responses all had considerably longer incubation times (ELISA with ;3 days in one study and 5 or 6 days in the other four studies) [5,53 56]. Carrara et al. [50] showed that previously present interferon-g ELI- SPOT responses were absent in most patients after 3 months of therapy; significantly, the patients who retained responses were those who still had microbiologic evidence of active disease [52 ]. The interferon-g assay therefore may become a useful adjunct to assess relapse in the symptomatic patient [50]. The potential advantages for managing multidrug-resistant tuberculosis are obvious. The current benchmark for assessing the efficacy of new immunotherapeutic agents is clinical cure and failure to relapse at 2 years. If the preliminary data [52 ] are borne out by larger studies, then the interferon-g assay may serve as a useful marker of disease activity that will expedite the selection and evaluation of new immunotherapeutic agents. It may also facilitate the search for correlates of protective immunity as the previous TST-based distinction between infected and uninfected individuals was hazy. There are currently no studies directly comparing the effect of incubation times on interferon-g responses after antituberculosis treatment, and prospective studies evaluating how responses change in relation to antituberculosis treatment are urgently required. Will this test be useful in developing countries? Whilst diagnosing and treating LTBI is an essential component of tuberculosis control in the developed world, it is clearly a less important strategy in the developing world. There are several reasons for this. Here, the high annual risk of infection and large pediatric burden imply substantial ongoing transmission; the priority of health care systems is the treatment of large numbers of active cases that promote ongoing spread. A substantial proportion of the population has LTBI and the lifetime risk of these patients developing active disease may be as low as 5% [57]. Thus, until a tuberculosis program can provide short-course chemotherapy with 90% completion rates, it would be fruitless to consider the added burden of LTBI diagnosis and treatment [58]. Having made these important provisos, the newer interferon-g-based assays probably do have applications in the developing world, especially because the TST is even less reliable here. BCG at birth, a universal practice in most developing countries, more than doubles the relative risk of a false-positive TST [59]. Furthermore there is a high environmental mycobacterial burden, and technical problems like the requirement for a cold chain, improper storage, and shortages of syringes and needles are all more acutely felt. Finally, patients often fail to show up for the repeat visit needed to read the TST; of the 1028 TSTs done over 1 year at a large private hospital in Bombay, 42% of patients failed to show up for their reading despite paying for the test themselves. Consequently, the utility of the newer tests would be restricted to the following specific situations in the developing world: epidemiologic surveillance; HIV tuberculosis coinfection; children with tuberculosis; and malnourished tuberculosis patients. Epidemiologic surveillance Accurate determination of the prevalence of latent infection in a community is essential for an improved understanding of the epidemiology of tuberculosis and to guide tuberculosis control strategies. Diagnosing latent tuberculosis infection and tuberculosis in HIV-positive patients Highly active antiretroviral therapy is inaccessible to much of the developing world; however, treatment of LTBI is one of the few interventions proven to reduce morbidity in the absence of antiretroviral therapy [60]. An interferon-g ELISPOT assay utilising both ESAT-6 and CFP-10 antigens was more sensitive than the TST in a cohort of Zambian patients [8]. Whether the interferong assay can accurately target preventive therapy to this group requires further study. Children with tuberculosis In the developing world, children carry a large proportion of the tuberculosis burden and rates of tuberculosis HIV coinfection are increasing. Acquisition of sputum or other biologic samples is challenging and treatment is often empiric. When exposed to an index case, rates of infection with M. tuberculosis are high [61]. LTBI has a high risk of progression to active disease (40% in infants under 2 years old and 24% in children under 5 years old), usually within 12 months of infection [62]. This often takes the form of miliary tuberculosis or meningitis and therefore carries a high mortality. The interferon-g assays may therefore be useful in this group, in whom the TST has poor sensitivity and specificity. In KwaZulu Natal, where tuberculosis is endemic and there is a high prevalence of malnutrition (28%) and HIV (30%), Liebeschuetz and coworkers [38 ] found the interferon-g ELISPOT assay (overnight assay using both antigens) more sensitive than the TST in 293 children with suspected tuberculosis. In children with cultureconfirmed or highly probable tuberculosis, the interferon-g assay was more sensitive than TST (83 vs 62%, n = 133 in both groups and 81 vs 35% if only culture-proven cases were considered, n = 57). Significantly, the ELISPOT would have allowed an earlier diagnosis and commencement of treatment in the 52% of children whose findings were smear negative but culture positive. Thus the assay proved to be a sensitive, relevant, and robust new diagnostic test in the real world setting.

6 200 Infectious diseases Malnourished tuberculosis patients Malnutrition is a potent risk factor for reactivation of LTBI in diabetes and silicosis [63]. In India, 50% of children under the age of 4 years are estimated to be malnourished; the TST is often nonreactive in this group even in the face of active disease. Preliminary evidence suggests that the interferon-g assay may overcome this problem. Considerably more malnourished children had a positive interferon-g ELISPOT assay compared with the TST (78 vs 44%) [38 ]. Current priorities of tuberculosis programs, limited infrastructure, and consideration of resistance patterns [64] dictate that treatment of LTBI is not currently feasible in developing countries. Nevertheless in the special groups of patients referred to, this test would retain considerable value. Although concerns have been raised about laboratory expertise and infrastructure, the tests have been performed by local physicians with no laboratory experience and only a week s training using microscope, centrifuge, and incubator [8,9,38 ], and further studies are underway in India, Turkey, and Zimbabwe (Ajit Lalvani, personal communication). Nevertheless, compared with developed countries where costs of maintaining a TST program are not trivial and may exceed that of the interferon-g assay [65], limited financial resources remain a major obstacle to the widespread use of this test in developing nations. Further studies are needed to establish the sensitivity and specificity of these tests at multiple geographic locations among patients of different ethnicities. Other tuberculosis-specific antigens that appear promising for immunodiagnosis A further attraction of the interferon-g assays is that once the technology is set up, it can be improved as new antigens become available that allow the assay to ask different questions. For instance, when using ESAT-6 and CFP-10 the assay indicates the presence of live bacteria in the donor but does not distinguish between disease and latency. If the kit were supplemented with an antigen that evokes responses in one group but not the other, the test would become significantly more useful. The heparin-binding haemagglutinin (HBHA) of M. tuberculosis may be such an antigen. HBHA is involved in the binding of M. tuberculosis to pulmonary epithelial cells (but not to macrophages), and without it the bacteria do not disseminate [66]. It has emerged that cells from individuals with latent infection release interferon-g in response to HBHA, whereas cells from patients do not [67]. The patients response returns following treatment, the reverse of the trend seen using ESAT-6 and CFP-10. Thus the ratio of the response to HBHA divided by the response to ESAT-6 discriminates strongly between latency and progressive infection. The issue of the incubation time required to achieve this result has not been fully addressed, so it is not clear whether the HBHA-recognising cells are effector cells or memory cells, or both. It should be possible, however, to design a combined kit that measures the response to both antigens in adjacent wells. Such an assay would be remarkably useful. In addition, recently identified TB specific antigens encoded by RD1 may hold promise for further increasing the diagnostic sensitivity of IFN-g assays [68]. Conclusion The antigen-specific interferon-g assay has revolutionised the diagnosis of LTBI in low-prevalence countries. It retains specificity in those who are BCG vaccinated or have a false-positive TST due to environmental mycobacteria. Prospective studies will be needed, however, to confirm a reduction in active tuberculosis when interferon-gdefined LTBI is treated and whether this assay will identify those who have a high likelihood of progression to active disease. Two standardised commercially available tests will make future comparisons between studies considerably more meaningful. Future work will also have to address the utility of this test in HIV-positive subjects and other immunosuppressed individuals, as a rule out test for active tuberculosis, in developing countries, and as a marker of disease activity. Finally, exciting new antigens are likely to improve the utility of the current interferon-g-based assays. Acknowledgements The authors thank P. Wrighton-Smith, J. Rothel, and A. Lalvani for their input. 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