For reprint orders, please contact Diagnosis of latent. Expert Rev. Anti Infect. Ther. 3(6), (2005)

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1 Review For reprint orders, please contact Diagnosis of latent Mycobacterium tuberculosis infection: is the demise of the Mantoux test imminent? James S Rothel and Peter Andersen CONTENTS Latent tuberculosis: a précis of the global perspective Why do we need an improved diagnostic test for latent tuberculosis infection? Immune response to tuberculosis infection: impact on diagnostic tests Mycobacterium tuberculosis-specific antigens Interferon-γ-based tests: how are they performed & what are their technical pros & cons? Clinical performance Expert commentary Five-year view Key issues References Affiliations Author for correspondence Cellestis Limited, 1046A Dandenong Road, Carnegie, Melbourne, Victoria, Australia Tel.: Fax: jim_rothel@cellestis.com KEYWORDS: CFP-10, diagnosis, ESAT-6, interferon-γ, Mantoux, Mycobacterium tuberculosis, TST, tuberculosis Tuberculosis is responsible for more then 2 million deaths worldwide each year and vies with HIV as the world s most fatal infectious disease. In many developing countries, attempts to control the spread of infection rely solely on identification and treatment of those with active disease, ignoring subclinical infection. However, in developed countries, large efforts are also expended to identify and give prophylactic drugs to people with latent tuberculosis infection. Until recently, the 100-year-old tuberculin skin test (Mantoux) has been the only available diagnostic test for latent tuberculosis infection, despite its many well-known limitations. Advances in scientific knowledge have led to the development of tests for tuberculosis that measure the production of interferon-γ by T-cells stimulated in vitro with Mycobacterium tuberculosis-specific antigens. These interferon-γ tests are highly specific and unaffected by prior Bacille Calmette Guérin vaccination or immune reactivity to most atypical mycobacteria. They are more sensitive than the tuberculin skin test in detecting people with active tuberculosis, and their results correlate more closely with M. tuberculosis exposure risk factors than the tuberculin skin test in people likely to have latent tuberculosis infection. Science has caught up with one of the oldest diagnostic tests still in use worldwide, and the adoption of new, tuberculosisspecific interferon-γ-based tests should move us one step closer to better control of this insidious pathogen. Expert Rev. Anti Infect. Ther. 3(6), (2005) Latent tuberculosis: a précis of the global perspective The World Health Organization (WHO) estimates that approximately a third of the world s population is infected with Mycobacterium tuberculosis, the vast majority with latent tuberculosis infection (LTBI) [1], albeit an estimate made with imperfect tools and data. Unlike active, symptomatic tuberculosis (TB), people with LTBI are not infectious to others and are asymptomatic. It is a widely quoted dogma that approximately 5% of immunocompetent people with LTBI progress to smearpositive active TB disease within 2 years of infection, and another 5% over their lifetime. However, this estimate uses the tuberculin skin test (TST; Mantoux) as the standard for LTBI, which is prone to false-positive reactions, and the rate of progression for those with true LTBI may be much higher. Whatever the true rate of progression may be, substantial evidence demonstrates that identification and treatment of LTBI with anti-m. tuberculosis chemoprophylaxis reduces the probability of progression to active TB by up to 80% [2]. Control efforts based on LTBI treatment, however, are largely limited to developed countries. The majority of people with LTBI, and also those found with active TB, are in the developing countries of Africa, Asia, the Pacific and the Americas [1]. In these TB-endemic areas, current TB control activities focus on the detection and treatment of people with active TB disease. Identification and treatment of / Future Drugs Ltd ISSN

2 Rothel & Andersen people with LTBI is largely seen as beyond the capabilities of the limited resources available in these countries. However, with significant funding and cheaper supplies of antiretroviral drugs recently made available for the treatment of HIV infection in developing countries, there is a cogent argument to be made for the testing and treatment of LTBI, especially as recent studies have demonstrated significant health benefit outcomes when HIV and LTBI are both treated [3,4]. TB is the most common cause of death in HIV-positive people in these environments, and the risk of progression from LTBI to active TB is estimated at 10% per year for HIV-positive people [5]. In many developed nations, extensive TB control efforts have led to significant reductions in the rate of active TB. In the USA, the rate of active TB has decreased from ten out of 100,000 in the early 1990s, when TB control efforts were stepped up, to five out of 100,000 patients in 2003 [6]. Unsurprisingly, the proportion of people identified with active TB who were born outside of the USA dramatically increased, with more than 50% of TB cases in 2004 being foreign born [6]. Despite this substantial decline in TB rates, it is estimated by the US Centers for Disease Control and Prevention (CDC) albeit based on the TST that million people in the USA have LTBI [7]. This large pool of infected people, along with those immigrating to the USA from TB-endemic regions, are at risk of developing active TB and spreading the infection to others. For this reason, the USA and many other industrialized countries routinely screen people at risk of being exposed to M. tuberculosis and offer treatment to them if they test positive for LTBI. However, although the epidemiologic reasoning of treating LTBI is clear, the success of programs to test and treat populations at TB risk in developed countries has been limited by the lack of a definitive diagnostic test for LTBI. The US Institute of Medicine in its 2000 statement on TB control reported that the greatest needs in the USA are new diagnostic tools for the more accurate identification of individuals who are truly infected and who are also at risk of developing tuberculosis [8]. Availability of a more accurate test than the TST (or Mantoux) would clearly enhance TB control efforts in developed nations and perhaps, with time, also in developing countries. Why do we need an improved diagnostic test for latent tuberculosis infection? Robert Koch s discovery of the tubercle bacillus in 1890 led to the preparation of a concentrated filtrate from heat-killed bacilli, which he referred to as tuberculin, and was later adapted by von Pirquet as the intradermal TST in 1907 [9,10]. Since then, and until quite recently, the TST has been the sole tool for detecting infection with M. tuberculosis. Despite its limited specificity and subjective nature, the TST does have value in detecting people with both past and present infection, and allows an epidemiologic approach to TB control; thus, the TST has persisted as a diagnostic aid for the control of TB. However, many factors, other than prior infection, may adversely influence the outcome of the test. The major factors that influence the specificity of the TST are Bacille Calmette Guérin (BCG) vaccination, prior exposure of the subject to nontuberculous mycobacteria (NTM) [11 13], and the inability to distinguish current infection from previous, resolved infection. In countries where BCG is widely used, as many as 60 90% of people with low risk of true M. tuberculosis infection can be tested falsely positive by the Mantoux test [14,15]. The subjective nature of both placing and reading the TST has a large effect on specificity and sensitivity [16 18]. Other variables include the source of tuberculin used [19,20] and the various interpretation methods and induration/erythema size diagnostic cutoffs used worldwide. Measurement of induration is open to reader variability, especially for the untrained operator [21]. The logistics of performing the TST, especially the need for people to return 2 3 days after their tuberculin injection to have their reaction measured, creates additional problems, and return rates are often poor. The TST is also falsely negative in a significant proportion of people with M. tuberculosis infection, especially in infants, the elderly, immunosuppressed, HIV-positive and malnourished people, and so on. The lack of sensitivity in such groups further limits the utility of the test. Clearly, there is a pressing need for a more definitive test for LTBI than the TST: one that is objective, unaffected by BCG vaccination and reactivity to NTM, has good sensitivity in the immunosuppressed and is logistically friendly, as well as cost effective. Targeted testing for latent tuberculosis infection, with intention to treat The USA probably has the most extensive LTBI screening program of any nation. The policy promoted by the CDC and other relevant US groups, termed targeted testing, is to screen those individuals at increased risk of being infected, and to discourage testing in low-risk individuals [2]. Examples of groups that are routinely screened for TB infection are healthcare workers, the incarcerated, homeless, HIV-positive individuals and, in particular, those in contact with active TB cases. Inherent in this policy is the intent to treat those that test positive. In the USA, people testing positive for LTBI are normally offered prophylactic chemotherapy, usually with 9 months of daily isoniazid. Outside of the USA, in practice, most other developed countries limit their LTBI screening to people at extremely high risk, notably recent contacts of active TB cases. Due to the use of BCG in many of these countries, and the resultant lack of confidence in the TST for routine diagnosis in vaccinated people, more extensive skin testing is considered a low-value activity. Additionally, many national policies do not advocate the use of isoniazid in people older than 40 years of age (varies widely between countries), predominantly due to the risk of serious side effects due to the hepatotoxicity of the drug that increases with age [22]. The advent of new diagnostic tests (see below) that are unaffected by BCG vaccination stimulate a need for the developed countries of the world to reappraise their TB control policies. By screening and treating infected people in high-tb risk groups, far more efficient TB control is possible. 982 Expert Rev. Anti Infect. Ther. 3(6), (2005)

3 Is the demise of the Mantoux test imminent? One obvious area where targeted testing with a highly specific test could be of great benefit is in screening immigrants and refugees prior to, or on entry into the country. In many industrialized nations, a significant and growing proportion of active TB cases occur in people recently arriving from high-tb burden countries. BCG vaccination is commonplace in such countries, and thus, screening for LTBI has been problematic when the TST was the only available method. Many nations therefore resort to chest x-ray for screening immigrants and refugees, an inexact method that will detect overt active pulmonary TB, but will not detect those with LTBI nor nonpulmonary disease. As more than 50% of people found with active TB in many developed countries (e.g., USA: 53.7% in 2004 [23]; UK: 67% in 2002 [24]; and Australia: 85.4% in 2003 [25]) were born outside those countries, it is apparent that identifying those with LTBI and treating them at entry would result in a substantial decline in TB disease rates. The development of new diagnostic tests for LTBI that are unaffected by BCG status makes this a possibility. Immune response to tuberculosis infection: impact on diagnostic tests It is not in the scope of this article to review the literature on immune response to TB infection, but it is important for us to understand what component(s) of the immune response are best suited to diagnose the different stages of TB infection and disease. M. tuberculosis is an intracellular pathogen primarily residing within the phagosome of macrophages and, as such, antigens are primarily presented in conjunction with major histocompatibility complex (MHC) class II to CD4 + T-cells, but also with MHC class I to CD8 + T-cells [26]. It has long been recognized that cell-mediated immune (CMI) responses predominate with TB infection; specifically a Type-1 T-cell response as characterized by production of interferon (IFN)-γ [27]. For those people that progress to active disease, there is a trend for the CMI response to move towards a Th-2-type response and the production of interleukin (IL)-4, -5 and -10 [28]. During the latent phase of infection, when mycobacteria are largely within macrophages, little, if any, free unprocessed antigen can be expected to leave the macrophage and be available to B-cells to stimulate a humoral immune response, although antigen is presented by infected macrophages to stimulate a CMI response. Thus, measurement of CMI response, rather than antibody responses, to M. tuberculosis antigens is likely to provide a sensitive means of detecting LTBI. However, as a patient develops active TB and the bacterial and antigenic load increases, antibody responses may become more robust and measurable. This leads to the possibility that a specific antibody-based test may be able to assist in diagnosis of active TB as distinct from LTBI. FIGURE 1 depicts the generalized immune response of individuals during the course of infection. As soon as 2 weeks after infection with M. tuberculosis infection, a CMI response can develop. This CMI response is associated with both delayed Response Mycobacterial load Infection Latent TB Disease Cell-mediated response Antibody Figure 1. Spectrum of the immune response during the course of infection with Mycobacterium tuberculosis. Time type hypersensitivity (DTH) responses as measured by the Mantoux test, and production of Type 1 T-cell cytokines (IFN-γ). This response is maintained throughout the course of the infection, although it may wane for people who progress to active TB [29 31]. Mycobacterial load remains low during the latent phase of infection, and for practical purposes is undetectable, and then steadily grows for those that progress to active disease. Similarly, M. tuberculosis-specific antibody responses are usually undetectable during LTBI, but are found in a significant number of people with active TB, likely due to an increased antigenic load [32]. Thus, it is clear that a rational path for an effective new diagnostic test for LTBI is the measurement of a M. tuberculosis-specific Type 1 T-cell response, of which IFN-γ is an appropriate marker. Mycobacterium tuberculosis-specific antigens The tuberculin purified protein derivative (PPD) used in the Mantoux test is an undefined cocktail of proteins and protein fragments, of which some are specific for M. tuberculosiscomplex organisms, but the vast majority have homologs or are shared with many environmental mycobacteria and BCG vaccine strains. It is largely for this reason that the Mantoux test has poor specificity, especially in BCG-vaccinated individuals. There is an obvious need for antigens more specific than PPD if TB diagnostics were to improve. Relatively recent advances in immunologic and molecular biology knowledge and techniques have led to the identification of proteins that are essentially specific for M. tuberculosis complex organisms and are absent from BCG strains. The identification and potential use of these antigens for diagnosis of TB infection has previously been reviewed [33]. Two proteins from M. tuberculosis, ESAT-6 and CFP-10, stand out as suitable antigens for test monitoring T-cell immune responses to infection, and these were identified by immunologic response to purified protein [33]. These two proteins are encoded by the RD1 section of the M. tuberculosis genome, which is absent in all BCG vaccine strains. The genes encoding ESAT-6 and CFP-10 are also absent from most 983

4 Rothel & Andersen environmental mycobacteria, with the exceptions of Mycobacterium szulgai, Mycobacterium marinum and Mycobacterium kansasii [33]. A homolog of ESAT-6 exists in Mycobacterium leprae, but identity is only 36% at the protein level, although the possibility remains that in some populations, M. leprae infection may generate immunologic cross-reactions [34,35]. The genes encoding ESAT-6 and CFP-10 are on the same operon, under control of the same promoter, and are actively secreted from M. tuberculosis, after which they form a dimer [36]. Thus, although ESAT-6 and CFP-10 are two separate poly, to the immune system they are likely to be presented at the same time, in the same molar amounts and are likely seen as one antigen. Genome sequencing provides other candidate antigens for a specific TB diagnostic assay. The M. tuberculosis, BCG and M. avium genomes have been sequenced, allowing identification of other genes of candidate antigens absent from BCG and most environmental mycobacteria [37,38]. A number of these antigens have been evaluated for their diagnostic potential and one, TB7.7 (Rv2654), has been shown to be highly specific for TB [38,39]. The gene for TB7.7 has been mapped to a region on the M. tuberculosis genome called RD11, which is a phage insertion unique to M. tuberculosis. Like ESAT-6 and CFP-10, RD11 is absent from all M. bovis BCG strains [37] and Basic Local Alignment Search Tool (BLAST) searches against available mycobacterial gene databases (M. tuberculosis, M. bovis, M. leprae, M. marinum, M. avium, Mycobacterium paratuberculosis, Mycobacterium smegmatis and Mycobacterium ulcerans) have shown that the region is also absent from all non-m. tuberculosis complex mycobacterial genomes searchable at this time. The new diagnostic tests for LTBI described below (Quanti- FERON -TBGold [QFT; Cellestis Ltd] and TSpot-TB [SPOT; Oxford Immunotec]) measure IFN-γ responses of T-cells when stimulated with ESAT-6 and CFP-10. The In Tube version of the QFT test also includes a peptide from the internal region of TB7.7, termed TB7.7 (p4) (AWRTAAVELARALVRAVA) for maximal sensitivity. Purification of ESAT-6, CFP-10 and other antigens from M. tuberculosis culture for use in commercial tests is impractical, and hence the question arises of what is the best source of these antigens. Both recombinant protein and overlapping peptide formulations of the ESAT-6 and CFP-10 antigens have been used in clinical studies of early versions of the QFT and SPOT tests, and in other systems measuring IFN-γ responses. A number of publications have demonstrated equivalence between recombinant and peptide antigen formulations [40 45], although in stark contrast, a study involving an early version of the SPOT test found considerable discord between the two forms of antigen [46]. In this regard, it is important to note that even low levels of host expression protein contaminants of recombinant antigen preparations can severely affect specificity, allied by the potential effects of Toll-like receptor stimulants, such as lipopolysaccharide, potentially present in research-quality recombinant protein preparations. However, if highly purified recombinant ESAT-6 and CFP-10 antigens are used, responses are commensurate with those elicited by overlapping. If overlapping peptide antigen formulations are employed in assays, their size is of importance. Larger greater than or equal to 15 mer of high purity are likely to stimulate only CD4 + T-cell responses in in vitro assays. If smaller are used (or with lower purity and thus, contaminants of smaller fragments of the parent peptide), CD8 + T-cell responses will also be stimulated due to the smaller binding directly to the MHC class I cleft. No study published to date has used ESAT-6/CFP-10 size-optimized for MHC class I loading in a diagnostic assay, although measuring CD8 + responses remains a possible adaptation. The QFT assay uses overlapping of mer in size and of greater than 90% purity by high-performance liquid chromatography [14]. For the commercial SPOT test, a recent publication states that two pools of overlapping, representing ESAT-6 and CFP-10, are employed, although their size is not quoted [47]. Publications on early versions of the enzyme-linked immunospot test assay () refer to both recombinant antigens and 15-mer overlapping in various and numerous formulations [48 50]. Interferon-γ-based tests: how are they performed & what are their technical pros & cons? Wood and colleagues were the first to develop a diagnostic test for LTBI based on the detection of IFN-γ responses [51,52]. The test was developed for use in cattle, and involved overnight incubation of whole heparinized blood with a PPD from M. bovis and the subsequent detection of IFN-γ in the plasma samples. This bovine test was the forerunner to new diagnostic tests for TB in humans, and established that IFN-γ responses were measurable with less than 1 day of antigen stimulation. A number of testing formats measuring IFN-γ production have been developed in the research setting, and many of these have recently been reviewed [53,54]. However, the present review will be limited to the two tests that are currently commercially available, the QFT and SPOT tests, and will cover studies using two different versions of the commercial QFT test (described below) and studies evaluating earlier research versions of the SPOT test. Both versions of the QFT test are CE marked, and the 24-well culture plate form, as described by Mori and colleagues [14], is approved by the US Food and Drug Administration (FDA) and Japan s Ministry of Health, Labour and Welfare, thereby allowing it to be used worldwide. At the time of writing, the SPOT test is CE marked, allowing its use in Europe and in other countries where regulatory approval is not required. Both the QFT and SPOT tests rely on the detection of IFN-γ produced by T-cells when stimulated with ESAT-6 and CFP-10. Both tests also utilize a saline negative control and a mitogen (phytohemagglutinin) positive control, the latter serving as a measure of the immune status of the individual being tested, as well as a check that the test has been performed correctly. There may be some confusion in the literature as three commercial versions of the QuantiFERON-TB test exist. The first version, QuantiFERON-TB, measured by enzyme-linked immunosorbent assay (ELISA), IFN-γ responses in 1-ml 984 Expert Rev. Anti Infect. Ther. 3(6), (2005)

5 Is the demise of the Mantoux test imminent? aliquots of whole blood incubated overnight in 24-well culture plates with tuberculin PPD and M. avian PPD, as well as a negative control and a mitogen positive control. The QFT (i.e., QuantiFERON-TB Gold) test differs in that it measures responses to ESAT-6 and CFP-10 rather than the PPDs, and utilizes a more sensitive ELISA for the detection of IFN-γ [14]. The latest version of the test, QFT In Tube, involves incubation of blood and stimulation antigens directly in the blood collection tubes. ESAT-6 and CFP-10, along with a peptide from TB 7.7, are included in the one tube. The methodology for the two tests is shown in FIGURE 2, where the In Tube version of QFT is shown. As can be seen from FIGURE 2, the primary differences between QFT and the SPOT tests are the initial sample preparation (whole blood vs. purified lymphocytes) and the readout QuantiFERON-TB Gold Collect 1 ml of blood into each of three tubes: nil control, ESAT-6/CFP-10/TB7.7(P4) & mitogen Incubate for h at 37 C CO 2 not required Harvest plasma (centrifuge tubes 15 min optional). Can store samples for up to 8 weeks at 4 C Add plasma & conjugate to ELISA plate Incubate for 120 min at room temp Wash microtiter plate & add substrate for 30 min Add stop reagent & read optical densities Software calculates & prints results D A Y O N E D A Y T W O Figure 2. Comparison of testing methods for the QuantiFERON -TB Gold In Tube and T-SPOT-TB tests. ELISA: Enzyme-linked immunosorbent assay; PBMC: Peripheral blood mononuclear cells. method for IFN-γ production (amount of IFN-γ produced, as measured by ELISA vs. number of cells secreting IFN-γ, as measured by ). The QFT In Tube test uses whole blood, and the blood collection tubes also serve as the incubation vessels, as they are precoated with the control and TB-specific antigens. This results in a logistic and labor-saving advantage, and means that the test can be performed anywhere in which a 37 C incubator is available. Once the blood tubes have been incubated, they can be stored or shipped at between 2 and 27 C for up to 3 days without centrifugation, or can be stored for up to 8 weeks at 2 8 C if centrifuged. This allows for batching of samples, and shipping samples to a remote laboratory for the ELISA phase of the test. The SPOT test uses peripheral blood mononuclear cells (PBMC), preferentially purified using T Spot-TB Vacutainer CPT tubes that are best centrifuged within 2 h of collection [101], therefore requiring a suitably equipped laboratory with skilled technicians within relatively short access. A microtiter plate washer is required in the IFN-γ detection phase for both tests, a microtiter optical density plate reader for QFT, and an reader or microscope for the SPOT test. Every method has the virtues of its vices. The whole-blood method of QFT does not use counted lymphocytes, but measures responses from the cells contained in 1 ml of unfractionated blood, with incubation with antigens occurring in homogenous plasma. The PBMC purification and quantification steps involved with the SPOT test are a labor and logistic constraint, but theoretically allow standardization of methodology, even across the immunosuppressed, although this would require the PBMC purified from an immunosuppressed patient to have the same cell populations as those from an immunologically normal individual. The QFT test has the theoretical benefit that it measures responses from lymphocytes present in 1 ml of blood (1 3 million cells for an immunocompetent individual), rather than the 250,000 employed in the SPOT test. Additionally, the QFT test measures the IFN-γ response to ESAT-6, CFP-10 and TB7.7 in the one tube, allowing for at least additive responses, which can result in improved sensitivity [45]. In conclusion, the QFT and SPOT tests measure the same component of the Collect 4 8 ml of blood in lymphocyte separation tube Centrifuge for 30 min Collect PBMC layer & add to 10 ml sterile culture media Wash PBMC twice by resuspension & centrifugation (7 min) Stain & count viable cells. Adjust to cells/ml in culture media Add cells & antigens (ESAT-6, CFP-10, nil control & mitogen) to coated microtiter plate Incubate for h at 37 C. CO 2 incubator required Wash off cells & add conjugate to plate. Incubate for 60 min at room temp Wash microtiter plate & add precipitating substrate for 7 min Wash plate, dry for 3 h & count spots visually or using an plate reader Software with reader calculates results, or perform manually 985

6 Rothel & Andersen immune response, and probably with similar sensitivity for detecting IFN-γ responses, although this question remains unresolved, especially in the immunosuppressed. Clinical performance There is no definitive method of determining if a person has LTBI, and thus, there is no gold standard with which to compare new diagnostic assays. Comparison of a new test s results with those of the TST is an inappropriate method for determining performance, as highlighted by the many deficiencies of the TST discussed above. Indeed, for a new test to be an improvement over current practices, results should be discordant with those of the TST for a significant number of individuals. With no gold standard for LTBI, investigators have generally estimated diagnostic test performance parameters by: Specificity in people with no identified risk factors of M. tuberculosis exposure Sensitivity in patients with confirmed active TB and also as surrogates for those with LTBI Correlation of test results with markers of M. tuberculosis exposure in people at risk of LTBI (e.g., contacts and healthcare workers) A large number of clinical studies of the QFT test and research versions of the SPOT test have been conducted, and those studies published are shown in TABLE 1, and discussed below. Specificity Specificity of both the QFT and SPOT test for M. tuberculosis infection is very high and approaches 100%. As would be expected from the specificity of the antigens used in both tests, BCG vaccination is not a confounding factor. To investigate the specificity of QFT, Mori and colleagues tested 216 Japanese students at low risk of M. tuberculosis infection, all of whom had received BCG vaccination, with the 24-well culture plate version of QFT [14]. Specificity was found to be 98.1%, with only four of the students being QFT positive, assuming all of the subjects were indeed uninfected. A large study in over 500 low-risk US Navy recruits has been reported in the FDA-approved product insert of the QFT test and showed a specificity of 99.8%, assuming the one patient found positive was not infected [102]. There are no published studies directly evaluating the specificity of the commercially available SPOT test. However, a number of studies have investigated specificity of different assays, forerunners to the SPOT test, which measured responses to individually, rather than in pools, and also to recombinant antigens. Lalvani and colleagues tested 40 low-risk UK residents with an test and found that all (100%) tested negative [48]. This study measured IFN-γ responses to mer representing ESAT-6 and CFP-10, as well as to recombinant ESAT-6. A later paper from the same group reported these same results [55]. Two earlier papers looked at the specificity of an test measuring responses to ESAT-6 alone (and using a different cut-off to the SPOT test) and found that none of the 58 people tested in the two studies were positive [49,56]. Sensitivity for active disease A number of studies have been conducted in patients with culture-confirmed M. tuberculosis infection to both directly estimate the test sensitivities for active TB, and also as a surrogate for sensitivity for LTBI. It should be noted that neither the QFT nor SPOT tests can differentiate between active TB disease and LTBI. In examining sensitivity, some important variables have to be considered. Ongoing treatment for TB can alter IFN-γ responses [56,57]. For this reason, TB suspects (subsequently confirmed) should be tested prior to initiation of treatment. Previous tuberculin skin testing boosts existing T-cell reactions to both tuberculin and, in animal models, ESAT-6. Whilst placing a TST on an uninfected person does not induce an IFN-γ response to ESAT-6 or CFP-10 [58], injection of tuberculin, which contains small amounts of ESAT-6 and CFP-10, is known to boost an existing IFN-γ response in cattle with TB, and is likely to do the same in humans [59]. A final point to consider is the time taken from collection of blood to initiation with the antigens for IFN-γ production. Viability of PBMC, in regard to IFN-γ production, decreases with time in vitro before encountering antigen [47]. However, despite this phenomenon, a realistic timeframe should be used in clinical studies if a real world estimate of sensitivity is to be obtained. Mori and colleagues found the sensitivity of the 24-well culture plate version of the QFT test to be 89%, with 105 out of 118 patients testing positive [14]. Only 50 out of the 76 (65.8%) patients who had also received a TST were positive at 5 mm of induration. A study in Denmark found 41 out of 48 (85.4%) TB patients were positive in a test that differed from the QFT test in that recombinant ESAT-6 and CFP-10 were used rather than overlapping 25-mer [60]. A recent study by Kang and colleagues found 44 out of 54 (81.5%) patients with active TB (positive culture or clinical diagnosis) were QFT positive, whereas the TST detected 42 (77.8%) at 5 mm of induration [61]. Taken together, these studies estimate the sensitivity of the QFT 24-well culture plate test at 86.4% (190 out of 220). Although no sensitivity data are yet published for the In Tube test, two independent studies indicate an increased sensitivity over the 24-well culture plate version of QFT-Gold (PERS. COMM. G JENKIN & N HARADA). One recent publication investigated the sensitivity of the commercial version of the SPOT test for active TB [47], and a number of studies have been conducted with the earlier versions of the test, using different antigen formats and different test cutoffs. Lalvani and colleagues investigated 47 TB patients with an assay utilizing eight 15-mer from ESAT-6, and found that 45 (95.7%) were positive under the criteria used at the time [49]. Using a similar assay, Pathan and colleagues demonstrated that 23 out of 25 (92%) cultureconfirmed patients tested positive, as did ten out of 11 (91%) 986 Expert Rev. Anti Infect. Ther. 3(6), (2005)

7 Is the demise of the Mantoux test imminent? Table 1. Published studies on versions of the QFT-Gold and SPOT tests. Test used Antigen(s) n Outcomes Ref. ESAT-6 REC Sensitivity 95% (n = 47) Specificity 100% (n = 26) in low risk and 91% (n = 47) in patients with non-tb infections 85% of subjects at high risk for LTBI were positive (n = 26) ESAT-6 REC Sensitivity 92% (n = 36) Specificity 100% (n = 32) in low risk Number of reactive T-cells dropped with treatment in active TB cases (n = 12) ESAT-6 REC Specificity 100% in low risk (n = 40) High prevalence of positive responses in healthy adults in India (80 out of 100) Number of reactive T-cells dropped with treatment in active TB cases (n = 5) QuantiFERON- TB Gold ESAT-6 REC + CFP-10 ESAT-6 REC + CFP-10 ESAT-6 REC + CFP-10 ESAT-6/CFP Responses more closely correlated than the TST (Heaf test) with degree of exposure in TB contacts Not affected by BCG vaccination 124* Sensitivity for HIV coinfected, 92% (n = 38) Similar rates of test positivity in healthy Zambian subjects with and without HIV (n = 75) 535 Responses more closely correlated than the TST (Heaf test) with degree of exposure in TB contacts TST results associated with BCG vaccination 334 Sensitivity 89% (n = 118) Specificity in BCG vaccinated low-risk subjects 98% (n = 216) QuantiFERON ESAT-6/CFP-10 REC 125 Responses associated with exposure in TB contacts Responses closely correlated with TST results in non-bcg vaccinated QuantiFERON ESAT-6/CFP-10 REC 1 Detected TB missed by TST, chest x-ray, smear and culture [68] QuantiFERON- TB Gold T SPOT-TB ESAT-6/CFP-10 ESAT-6 REC + CFP-10 REC + ESAT-6 REC + CFP-10 REC + ESAT-6 REC + CFP-10 ESAT-6 REC + CFP-10 REC + ESAT-6/CFP Responses associated with level of exposure in TB contacts Specificity of TST poor in BCG vaccinated low risk 88 Responses more closely correlated than the TST (Mantoux) with degree of exposure in TB contacts 1 Detected subclinical TB missed by the TST [73] 293 More sensitive than the TST in diagnosing active TB in children Less affected than the TST by HIV coinfection, malnutrition or age <3 years 413 Appeared at least as sensitive as the TST for detecting LTBI in contacts TST was associated with BCG status 72 Sensitivity 97.2%, (following prior TST) [47] QuantiFERON ESAT-6/CFP-10 REC 121 Sensitivity 85% (n = 48) Sensitivity for extrapulmonary TB 92% (n = 13) useful tool for assisting with active TB diagnosis *Also included what appears to be previously reported data from 40 low-risk subjects. Recombinant ESAT-6/CFP-10 rather than the used in QFT-Gold. Not all were tested with QFT-Gold. BCG: Bacille Calmette-Guérin; : Enzyme-linked immunospot; LTBI: Latent tuberculosis infection; REC: Recombinant; TST: Tuberculin skin test (Mantoux) [49] [56] [48] [70] [54] [50] [14] [63] [65] [46] [62] [58] [60] 987

8 Rothel & Andersen Table 1. Published studies on versions of the QFT-Gold and SPOT tests. Test used Antigen(s) n Outcomes Ref. QuantiFERON- TB Gold QuantiFERON- TB Gold QuantiFERON- TB Gold QuantiFERON- TB Gold ESAT-6/CFP-10 ESAT-6/CFP- 10/TB7.7(p4) Peptides. In Tube format ESAT-6/CFP-10 ESAT-6/CFP Better indicator of LTBI and active TB infection than the TST Specificity of TST was poor % TST positive and 40% QFT-Gold positive in high-risk healthcare workers in India Agreement with TST was high (81.4%) 318 Feasible for routine hospital use Patients receiving immunosuppressive drugs often nonreactive positive control and also TST negative Suggests positive control is a marker for immune status 462 Responses associated with level of exposure in TB contacts Specificity of TST poor in BCG vaccinated low risk *Also included what appears to be previously reported data from 40 low-risk subjects. Recombinant ESAT-6/CFP-10 rather than the used in QFT-Gold. Not all were tested with QFT-Gold. BCG: Bacille Calmette-Guérin; : Enzyme-linked immunospot; LTBI: Latent tuberculosis infection; REC: Recombinant; TST: Tuberculin skin test (Mantoux) [61] [67] [64] [69] with lymph node TB (many clinically diagnosed) [56]. A study in Zambia found all 11 (100%) HIV-negative TB patients were positive to an test measuring responses to both ESAT-6 and CFP-10 [55]. In the same study, 23 out of 25 (92%) HIV-positive TB patients tested positive. Liebeschuetz and colleagues investigated an test employing pools of ESAT-6 and CFP-10, along with recombinant ESAT-6 in 57 children in South Africa with active TB [62]. They found an overall sensitivity for of 80.7% (46 out of 57) in culture-confirmed TB cases, compared with that for the TST of 39.5%. However, this sensitivity estimate must be interpreted with the knowledge that a number of children were less than 3 years of age, and a number were HIV positive. The published study on the SPOT test found a sensitivity of 97% in 72 patients with active TB [47]. However, this excellent sensitivity estimate may in part be related to the prior use of the TST in these subjects, who received a TST, on average, 10 days prior to collection of blood for the SPOT test. Although only limited published information exists for the most recent versions of the two tests, the available data supports the sensitivity of the QFT and SPOT tests being significantly higher than that for the TST (even when using a 5-mm cutoff) and suggests both tests have an achievable sensitivity of approximately 90 95% for untreated active TB in immunocompetent patients. There is a possibility that sensitivities of both tests may be lower in young children and those severely immunocompromised, but early results are promising. Further studies are required to investigate these groups. It is an important aspect of most of the above studies that the TB patients investigated have received little or no chemotherapy at the time of testing. There is a growing body of evidence demonstrating that IFN-γ responses to ESAT-6 and CFP-10 in assays with short incubation periods (<24 h) decline, and in many cases disappear, with successful treatment of patients [56,57]. Decrease in IFN-γ responses to the TB-specific antigens in the QFT and SPOT assays may be due to measurement of responses from effector T-cells, which require the presence of antigen to maintain activation, rather than memory T-cells, which can persist in the absence of antigen [54]. Both assay systems utilize incubation periods of a maximum of 24 h; generally insufficient time for central memory T-cells to produce significant levels of IFN-γ. This opens up an exciting possibility that the QFT and SPOT tests may be useful tools for monitoring success of treatment in the future, however, more data from long-term studies are required before this use can be verified. Diagnosis of latent tuberculosis infection A large number of studies have been conducted using the QFT or tests in people at risk of having LTBI, and a number of these have been published. These studies have generally involved screening contacts of active TB cases, where the association between test results and the level of exposure of contacts to the index case(s) is assessed, or healthcare workers, where results are compared with measurable risk factors for nosocomial transmission of M. tuberculosis. Numerous studies using QFT have been completed and recently published, and most of these have investigated the test performance for detecting LTBI [14,60,61,63 69]. Brock and colleagues compared QFT (using recombinant antigens rather than ) and TST in a high school contact investigation in Denmark [63]. In total, 85 of the contacts had not received BCG vaccination, and this enabled a direct comparison of QFT and TST results without the TST being corrupted by BCG responses. In these contacts, there was excellent agreement between the two tests (94%), demonstrating that QFT detected LTBI with at least similar efficacy to the TST. A case study from Denmark reported that in a blinded study, QFT detected LTBI in a patient who was TST negative and developed active TB after immunosuppressive therapy [68]. 988 Expert Rev. Anti Infect. Ther. 3(6), (2005)

9 Is the demise of the Mantoux test imminent? A study in college students in Japan exposed to a teacher with pulmonary TB found that there was little difference between TST positivity rates in close contacts versus other students, most likely attributable to BCG vaccination and revaccination. In contrast, 46% of the close contacts tested and only 7% of the other students were QFT positive [65]. This study demonstrates the association of QFT results with level of exposure, and again highlights that BCG does not confound the test. A recent publication by Kang and colleagues reported on BCGvaccinated subjects with no exposure, some exposure and close contact with active TB cases [61]. They found TST positivity in the three groups of 51, 60 and 71%, compared with 4, 10 and 44% for QFT. The TST was clearly corrupted by BCG, and QFT results were more closely associated with M. tuberculosis exposure risk. A study of over 700 healthcare workers at a rural hospital in India found strong agreement (81.4%) between QFT (In Tube version) and TST, and both tests estimating a prevalence of LTBI of approximately 40%. Results for both tests were significantly associated with age and years in the healthcare worker industry, surrogate markers of increased risk of LTBI [67]. Ferrara and colleagues reported on the use of QFT in a hospital setting in Italy [64]. They found that QFT was feasible for routine use, and detected more patients with confirmed TB than the TST. The patients tested were largely from respiratory, hematologic, oncology, infectious diseases, pediatric and internal medicine wards. There were a significant number of patients (68 out of 318 [21%]) who failed to respond to the test s mitogen positive control, and were therefore deemed test indeterminate. These indeterminate results were significantly associated with negative TST responses and immunosuppression due to medical conditions and treatments. This study highlights the benefit of the mitogen positive control when testing patients with possible immunosuppression. In a person unable to respond in an immunologic test, the TST would, by definition, be negative. This is a potentially dangerous outcome because an immunosuppressed but infected patient is more likely to have or develop TB disease. The mitogen positive control therefore provides an indication of the immune status of the individual being tested, and will yield an indeterminate result in a person who is significantly immunosuppressed. This should be helpful information for the clinician. As mentioned above, there are no publications directly assessing the SPOT test s performance for detecting LTBI. However, a number of publications have directly investigated this when using earlier versions of the test with different antigen formats and cut offs [46,48 50,54,55,58,66]. Two studies investigated the use of the test (incorporating ESAT-6 and recombinant antigen only) in household contacts of active TB patients. Pathan and colleagues reported that 23 out of 27 contacts were positive [56], and the same group reported in a separate publication that, for what appear to be 26 of the same people with the same data, 22 were positive [49]. Another study from the same group found that 80% of healthy Indian executives were ELI- SPOT positive (ESAT-6 and CFP-10 were used) [48]. This is an extremely high percentage of positive results from what would be considered a lower risk group in India, and raises possible concerns about the test s specificity in this study, but does suggest that the test is likely identifying LTBI. Collectively, the data from these studies indicates that the test detects people with LTBI, with likely high sensitivity. Definitive studies have been reported with the use of ELI- SPOT variations in contact investigations [50,70]. Lalvani and colleagues, using an measuring responses to recombinant ESAT-6 only, tested 50 contacts of confirmed pulmonary TB patients graded into four groups based on their level of exposure [70]. There was a highly significant association between positive responses and exposure (odds ratio [OR] 9.0) and also a significant association with TST responses (OR 1.9). was not associated with BCG status, whereas TST was highly associated (OR 12.1), clearly demonstrating the specificity of ESAT-6 IFN-γ responses. A second study investigated an test (employing recombinant ESAT-6 and 12 different peptide pools of ESAT-6 and CFP-10 ) in over 500 secondary school children with possible long-term exposure to a fellow student with pulmonary TB [50]. Contacts were again assigned to four groups based on their level of exposure to the index case. results were significantly more closely associated with proximity and length of exposure than with the TST, and unlike the TST, were not associated with BCG vaccination status. These two studies demonstrate that IFN-γ responses to ESAT-6 and CFP-10 are more likely to detect those people with LTBI than the TST. The consensus from published studies of QFT and early versions of the SPOT test is that both tests detect people with LTBI with a sensitivity at least commensurate to that of the TST, and with a high level of specificity, unaffected by BCG vaccination status. Expert commentary Detection and treatment of LTBI is crucial if we are to move towards better control of TB in developed nations. Current TB control efforts have been hampered by imperfect diagnostic tools for LTBI. As a result of this diagnostic limitation, many countries have struggled to provide effective screening programs when the cost versus benefit of identification and treatment of suspected TB infection may be unclear. New diagnostic tests measuring IFN-γ responses to M. tuberculosis-specific antigens are considerably more accurate and objective than the 100-yearold skin test, and can improve control efforts and individual clinical outcomes by targeting treatment to those truly carrying a current infection. The new tests carry a number of advantages, of which the greatest is probably the elimination of the distorting effects of BCG vaccination on testing regimens; important as this vaccination is common, particularly in those most at risk of TB infection. Science has caught up with one of the oldest diagnostic tests still in use around the world, and the adoption of new, TB-specific, IFN-γ-based tests will move us into an era where control of TB through targeting LTBI is both cost effective and commonplace

10 Rothel & Andersen Five-year view The TST in its various guises (Mantoux, Heaf and Tine) is, to our knowledge, the oldest diagnostic test still in routine use. It has been a useful test over the decades of its life, and has undoubtedly assisted with TB control worldwide. However, science has overtaken the TST, and highly specific and sensitive tests are now available as an alternative. Despite the large and rapidly growing body of evidence that these IFN-γ tests are superior to the TST, there are still hurdles to be overcome before the medical community widely accepts them as the standard. A concern for some is that long-term prospective studies, following large populations of IFN-γ test-negative and -positive patients exposed to infection, are a requirement for widespread use. Such studies are ethically difficult as they presume withholding preventive therapy from people who have definitive evidence of infection, and moreover, it is known that people with IFN-γ responses to ESAT-6 and CFP-10 can progress to TB disease [68,69,71]. This includes people who are TST negative. Given this knowledge and the proven superior performance over the TST, it is inappropriate to wait to use the IFN-γ-based tests until this information is available. Prospective information to fill in the final picture will come from studies already underway and with experience in routine practice. What is sure is that the QFT and SPOT tests detect people with current M. tuberculosis infection, an obvious prerequisite for progression to active disease, but not in itself a predictor of progression to active disease. Progression is an outcome dependent on various clinical, environmental and even iatrogenic factors that affect immune status, many of which may not even be present at the time of testing. There is currently only limited data available in the literature on the use of QFT and SPOT assays in some select groups, such as children, HIV-positive and other immunosuppressed individuals. More clinical data will become available in the near future, as many studies with the QFT and SPOT tests in these population groups are underway. However, all available data show that the new tests more accurately identify true TB infection and/or disease than the TST in immunosuppressed and young children. It thus seems inappropriate not to use QFT and SPOT tests in these groups, where we know the TST is of limited clinical value. Another of the hurdles is related to the fact that the widespread use of the TST as the sole test for LTBI has created an acceptance by many that it must be correct, thereby ignoring the effects of tuberculin boosting, as well as interference with non-tb mycobacteria and BCG, and the other shortcomings of the test. Experience of the TST is extensive, and this allows the illusion that such experience can somehow compensate and adjust for its failings. Cost is another significant issue, especially with TB, where people incorrectly view the TST as an inexpensive procedure, as tuberculin PPD and a syringe may only cost a few dollars. However, the true costs include the labor component of the test, the need for two visits to a healthcare worker and the repeat testing and lack of results for people who don t have their test read. Add to this the costs associated with screening those falsely TST positive for active TB (chest x-ray or clinical visit), costs associated with unneeded chemoprophylaxis, liver function tests, and so on, and the TST is suddenly an expensive screening test. This was well illustrated by Lambert and colleagues, who estimated that the cost of TST in a healthcare worker ranged from US$ for hospitals and US$ for health departments in screening programs in four hospitals and two public health departments in the USA [72]. Many of these cost factors are deleted by a single visit and accurate blood test. Finally, regrettably, mere resistance to change is another major hurdle, as is reluctance to introduce a new test to the laboratory with the associated logistic problems. However, with all the recent progress described in the present review, the necessary questions for healthcare providers are, how important is getting the correct answer from a diagnostic test for LTBI? How can we prescribe chemoprophylaxis to LTBI based on a TST result we know is often wrong, and knowing the significant compliance burden to the individual and the common side effects of isoniazid? Key issues Identification and treatment of people with latent tuberculosis infection (LTBI) is important for enhanced control of the disease. The tuberculin skin test (TST) has poor specificity, especially in Bacille Calmette Guérin (BCG)-vaccinated people, making identification of those truly infected problematic, and poor sensitivity in immunocompromised individuals. A number of antigens highly specific for Mycobacterium tuberculosis complex organisms, absent from BCG and most non-tb mycobacteria, have been identified. Cell-mediated immune responses are the most relevant for detecting LTBI. Highly specific in vitro diagnostic tests measuring interferon-γ responses to these antigens have been developed and have achieved regulatory approval for use. The IFN-γ tests are more specific, and are better indicators of true M. tuberculosis infection than the TST. Availability of the IFN-γ tests will likely result in improved control of TB in the developed world and eventually in developing countries. Development of new drugs and regimens for treating LTBI will enhance the overall utility of these new diagnostic tests. 990 Expert Rev. Anti Infect. Ther. 3(6), (2005)

11 Is the demise of the Mantoux test imminent? The answer to these questions will pave the road for the demise of the TST and the widespread use of the novel specific IFN-γ tests. In addition to being widely adopted in the developed world, these tests will probably also be increasingly used in developing countries in several situations (i.e., HIV-positive patients and children). Combined with new and better drugs and regimens for LTBI, making prophylaxis easier and more acceptable to the subject, significant inroads will be made into improving TB control. References Papers of special note have been highlighted as: of interest of considerable interest 1 World Health Organization. Global Tuberculosis Control: Surveillance, Planning, Financing. WHO report 2005, Geneva, Switzerland (2005). 2 Targeted tuberculin testing and treatment of latent tuberculosis infection. 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