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Diagnosing Tuberculosis Infection in the 21st Century^*

New Tools To Tackle an Old Enemy

^* From the Tuberculosis Immunology Group, Department of Respiratory Medicine, National Heart and Lung Institute, Wright-Fleming Institute of Infection and Immunity, Imperial College London, London, UK.

Correspondence to: Ajit Lalvani, DM, FRCP, Tuberculosis Immunology Group, Department of Respiratory Medicine, National Heart and Lung Institute, Wright-Fleming Institute of Infection and Immunity, Imperial College London, Norfolk Place, London W2, UK; e-mail: a.lalvani{at}imperial.ac.uk

Abstract

The century-old tuberculin skin test (TST) was until recently the only means of diagnosing latent tuberculosis infection (LTBI). Recent advances in mycobacterial genomics and human cellular immunology have resulted in two new blood tests that detect tuberculosis infection by measuring in vitro T-cell interferon (IFN)- release in response to two unique antigens that are highly specific for Mycobacterium tuberculosis but absent from bacille Calmette-Guérin (BCG) vaccine and most nontuberculous mycobacteria. One assay, the enzyme-linked immunospot (ELISpot) [T-SPOT.TB; Oxford Immunotec; Oxford, UK] enumerates IFN-–secreting T cells, while the other assay measures IFN- concentration in supernatant by enzyme-linked immunosorbent assay (ELISA) [QuantiFERON-TB Gold; Cellestis; Carnegie, Australia]. A large and growing clinical evidence base indicates that both tests are more specific than the skin test because they are not confounded by prior BCG vaccination. In active tuberculosis, ELISA has similar sensitivity to the skin test, while ELISpot is significantly more sensitive. Current cross-sectional evidence suggests that for diagnosis of LTBI, sensitivity of ELISA is similar to TST, while ELISpot appears more sensitive. High specificity will enable clinicians to avoid unnecessary preventive treatment in BCG-vaccinated persons without infection who commonly have false-positive TST results. High sensitivity could enable accurate targeting of preventive treatment to patients with infection at the highest risk of progression to active tuberculosis who frequently have false-negative TST results due to impaired cellular immunity. However, longitudinal studies are needed to define the predictive value of positive blood test results for progression to tuberculosis.

Key Words: assay • diagnosis • interferon- • latent tuberculosis infection • tuberculin skin test • tuberculosis

Although tuberculosis is a long-standing and expanding threat to public health with 8 to 10 million cases a year, our tools for diagnosis and prevention, now > 80 years old, are inadequate to contain the epidemic. Fortunately, in the last 5 years we have witnessed major advances in the diagnosis of tuberculosis infection, which represent the first tangible output of the last few decades of basic science tuberculosis research.¹²

Diagnosing Latent Tuberculosis Infection in the 20th Century

The biology of latent tuberculosis infection (LTBI) is poorly understood, and the condition has hitherto been defined by a positive tuberculin skin test (TST) result in an asymptomatic person exposed to tuberculosis with no clinical or radiographic signs of active tuberculosis. The clinical relevance of this definition is that it carries a small but significant forward risk of progression to active tuberculosis, which is significantly increased in persons with suppressed or immature (ie, young children) cellular immune systems. Unfortunately, the TST has poor specificity in bacille Calmette-Guérin (BCG)-vaccinated persons, low sensitivity in people with weakened cellular immunity, and several logistic drawbacks. So why has there been no better test for LTBI in the 110 years since Koch first developed the TST? The very low bacterial burden in LTBI makes it impossible directly to detect Mycobacterium tuberculosis and induces only a very weak humoral response, making serologic testing unreliable. The TST exploits the fact that LTBI induces a strong cell-mediated immune response by measuring the delayed-type hypersensitivity (DTH) response to intradermal inoculation of tuberculin purified protein derivative (PPD), a crude mixture of > 200 M tuberculosis proteins (Fig 1 , left). Given that DTH-induced cutaneous induration is not a sensitive marker of immune sensitization in immunosuppressed individuals with LTBI, and given that the DTH response to PPD is not specific for LTBI because of the antigenic cross-reactivity of PPD with BCG vaccination and environmental mycobacteria, how can we improve on the TST?

View larger version (31K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Diagrammatic representation of TST, ELISpot, and ELISA for diagnosing M tuberculosis infection. TST (left): PPD is intradermally injected into the volar surface of the forearm, and induration of any delayed-type hypersensitivity response is measured 72 h later. ELISpot (center): Peripheral blood mononuclear cells (PMBCs), which includes T cells, are separated from the blood sample by density centrifugation, washed, counted, and then incubated with ESAT-6 and CFP-10 in a 96-well microtitre ELISpot plate for 16 to 20 h. If the patient has M tuberculosis infection, T cells will recognize the antigens and secrete IFN-. This cytokine is captured in the immediate vicinity of the cytokine-secreting T cell by antibodies specific for IFN- coated on the bottom of each well. The cytokine-bound antibodies are subsequently detected with another antibody conjugated to an enzyme that catalyzes a colorimetric reaction resulting in visible spots, where each spot represents the footprint of one T cell that responded to the antigens. These spot-forming cells are counted giving the frequency of M tuberculosis-specific T cells. ELISA (right): Whole blood from the patient is incubated with ESAT-6 and CFP-10 in a 24-well plate for 24 h. If the patient has M tuberculosis infection, T cells will recognize the antigens and secrete IFN-. The plate is centrifuged and the plasma transferred to a 96-well microtitre plate. IFN- in the plasma is captured by antibodies specific for IFN- coated on the bottom of each well. The cytokine-bound antibodies are subsequently detected with another antibody conjugated to an enzyme that catalyzes a colorimetric reaction. The optical density (OD) of each well is measured, and the concentration of IFN- is determined using a standard curve. Although a laboratory is needed to process the blood samples from patients, which optimally need to be processed within 8 to 16 h (within 8 h for ELISpot, 12 h for ELISA), multiple patient samples can be analyzed at the same time, interpretation of the tests is standardized, and the internal positive control allows assessment of the performance of each assay.

Advances in mycobacterial genomics in the late 1990s identified an M tuberculosis genomic segment that is deleted from all strains of BCG vaccine and most environmental mycobacteria, denoted region of difference-1.³ Two proteins encoded by this stretch of DNA, early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), are strong targets of T-helper type 1 T cells in patients with M tuberculosis infection.⁴⁵ Therefore, a T-cell response to these antigens could, in theory, serve as a specific marker of M tuberculosis infection. However, measuring antigen-specific T-cell responses has traditionally been confined to the research laboratory because it required specialized sterile tissue culture facilities, technical expertise, and radioisotopes. However, two methods now exist for rapid, convenient measurement of antigen-specific T-cell responses. The rapid ex vivo enzyme-linked immunospot (ELISpot) assay, developed in the late 1990s by Lalvani et al,⁶ counts individual antigen-specific T cells. T cells from individuals with M tuberculosis infection become sensitized to ESAT-6 or CFP10 in vivo; when the T cells re-encounter these antigens ex vivo in the overnight ELISpot assay, they release a cytokine, interferon (IFN)- (Fig 1, center). By the next morning, each such T cell gives rise to a dark spot, which is the "footprint" of an individual M tuberculosis-specific T cell⁴⁷ (Fig 1, center). The readout is thus the number of spots that are counted using a magnifying lens or automated reader. The principle that underpins ELISpot is that a highly sensitive T-cell assay using highly specific M tuberculosis antigens should result in a test with high diagnostic sensitivity and specificity (Fig 1, right).⁸ The alternate method is a whole-blood enzyme-linked immunosorbent assay (ELISA), which measures the IFN- concentration in the supernatant of a sample of diluted whole blood after 24 h of incubation with ESAT-6 and CFP-10.⁹ Originally developed in the 1980s for detecting tuberculosis in cattle, the assay was adapted for human use in the 1990s. Both assay platforms are sometimes collectively referred to as T cell-based IFN-–release assays and are now commercially available as quality-controlled, regulatory-approved diagnostic test kits.² The tests have some similarities (eg, both need to be processed within 12 h of obtaining a blood sample, and both provide results by the next day) and several differences (eg, ELISpot requires WBC separation, which makes it technically more complex to process but ensures a fixed number of WBCs in the assay, which is important for immunosuppressed populations), summarized in Table 1 .

A large and rapidly growing number of clinical studies with these assays have been published since 2001. The ELISpot evidence base is comprised predominantly of studies using the Lalvani ELISpot test, which has recently been developed into the T-SPOT.TB test (Oxford Immunotec; Oxford, UK) [Table 1]. Therefore, all published studies using either of these assay formats are reviewed here, and the assays are denoted as ELISpot. For QuantiFERON-TB Gold (Cellestis; Carnegie, Australia), studies using this ELISA platform with peptides or recombinant antigen have been included, and the assays are denoted as ELISA. A simpler, more convenient version of the whole-blood ELISA, known as QuantiFERON-TB In-tube (Cellestis), has recently been developed. However, at the time of writing, the size of the published evidence base with this method was very small, and it has not been substantially discussed here.

Studies of test performance in active tuberculosis have used culture-confirmed tuberculosis or clinically highly probable tuberculosis as a "gold standard." Diagnostic specificity of both blood tests is higher than TST because they are not confounded by prior BCG vaccination. Both tests have higher diagnostic sensitivity than the TST in active tuberculosis; and the sensitivity of ELISpot, which ranges from 83 to 97%,^410111213 is significantly higher than the 70 to 89% sensitivity^912131415 of ELISA (Table 2 ).

The strategy for diagnosis and treatment of LTBI is based on targeted tuberculin testing, which targets persons at highest risk of progression from LTBI to active tuberculosis.²⁸ This includes all recently infected people (ie, tuberculosis contacts) and people with suppressed or immature (ie, young children) cellular immune systems. Tables 2345 summarize the performance of the tests in immunocompetent contacts, but what of the high-risk groups in whom the TST has poor sensitivity associated with impaired or immature cellular immunity?

There is only one study¹⁷ of LTBI comparing a blood test with the TST in infants that found that ELISpot results correlated better with tuberculosis exposure than TST results. The clinical relevance of positive ELISpot results in infancy is highlighted by the example of a ELISpot-positive, TST-negative infant who progressed to active disease 2 years after intense neonatal exposure to multidrug-resistant tuberculosis.²⁴ In older children, ELISpot results correlated better with tuberculosis exposure than the TST¹⁶; in older teenagers without BCG vaccination, ELISA performed similarly to the TST.²⁰ However, high levels of discordance between ELISA and the TST in a study²⁵ of younger children at high risk for LTBI suggest that the sensitivity of ELISA for LTBI is probably lower than the TST. For active tuberculosis, a prospective study²⁶ of 293 children with suspected tuberculosis in South Africa found that the diagnostic sensitivity of the ELISpot was much higher than the TST and was independent of HIV coinfection, malnutrition, and age < 3 years, all factors that adversely affect sensitivity of TST. Overall sensitivity of ELISpot in these children with a high prevalence of HIV coinfection and malnutrition was 83%, and rose to 91% when combined with TST results.²⁶

In HIV-coinfected adult tuberculosis patients in Zambia, ELISpot maintained a high diagnostic sensitivity of 92%⁵; in children with suspected tuberculosis, ELISpot results were independent of HIV coinfection status.²⁶ A study²⁷ of adults with HIV infection using QuantiFERON-TB Gold In-Tube (Cellestis), a simpler more convenient version of QuantiFERON-TB Gold, found that test results were associated with known risk factors for LTBI or history of tuberculosis, but no comparison with TST was made. A study²⁸ of asymptomatic South African adults from a region of high prevalence for tuberculosis and HIV found that although the rates of positive TST results were much reduced in HIV-positive vs HIV-negative subjects, rates of positive ELISpot and ELISA results did not vary significantly by HIV infection status, although there was a trend toward higher rates of positive ELISpot results. Most subjects had relatively high CD4 counts (median, 392). Studies in the setting of other types of immunosuppression are scarce, but emerging evidence indicates that ELISpot is robust to hematologic malignancy-associated immunosuppression and some types of iatrogenic immunosuppression, including corticosteroids and azathioprine.²

Reliability of the Blood Tests in Routine Use

Indeterminate results can arise for a variety of reasons, but the most significant cause is a failed positive control, which usually reflects underlying cellular immune suppression. Indeterminate results are quite common with ELISA in routine practice, ranging from 12 to 21%,^12152529 and are associated with young age (< 5 years), old age (> 80 years),²⁵ and immunosuppression due to a variety of causes, including HIV infection, in which indeterminate results become more common with lower CD4 counts.²⁷ In contrast, failed positive controls are rare with ELISpot, including infants (0 of 41 indeterminate results),¹⁷ children, and HIV-coinfected persons (0 of 60 adults with active tuberculosis or LTBI),⁵ for which rates of indeterminate results are unaffected by low CD4 counts.³⁰

Direct Comparison of the Blood Tests

Studies directly comparing the two blood tests in the same study populations have recently been published. In patients with active tuberculosis, three reports¹²¹³³¹ compared blood tests in a total of 134 patients; the sensitivity of ELISpot was 83 to 95% compared to 74 to 83% for ELISA, and this difference was statistically significant in the largest study.¹³ Two studies compared the tests head-to-head in persons at risk of LTBI; in a low-prevalence country, close contacts of pulmonary tuberculosis were significantly more likely to have positive ELISpot results than ELISA results,¹² and a similar, but nonsignificant trend was observed in adults at risk of LTBI by virtue of residence in a high prevalence area.²⁸ This finding is consistent with the studies presented in Table 2 suggesting that ELISpot correlates better with tuberculosis exposure than TST and ELISA.

Summary of Clinical Data

The conclusions from the published studies to date are that neither blood test is confounded by BCG vaccination and both are therefore more specific than the TST. Both tests are more sensitive than the TST in active tuberculosis, with ELISpot having higher sensitivity than ELISA. ELISpot also performs well in HIV-coinfected tuberculosis patients and young children with tuberculosis, but published data with ELISA in these patient populations are lacking. In LTBI, ELISpot probably has higher sensitivity than the TST, while ELISA seems to have similar sensitivity to the TST. In young children, ELISpot appears to have higher sensitivity than the TST, while ELISA may have lower sensitivity than the TST. Unlike the TST, both tests appear relatively robust to HIV coinfection in people with relatively high CD4 counts, with a trend toward more positive results by ELISpot. Indeterminate results are common with ELISA and are strongly associated with immunosuppression, young age, and old age, while indeterminate results with ELISpot are rare in all risk groups studied to date.

From Clinical Trials to Clinical Practice

What should clinicians do with blood test results in practice? Current national guidelines³²³³ recommend the use of blood tests for diagnosis of LTBI and as an adjunct for the evaluation of patients with suspected tuberculosis. But national guidelines vary, reflecting residual uncertainty about the clinical status of persons with discordant TST and blood test results. Current European guidelines, of which those published by the UK National Institute of Health and Clinical Excellence³² (NICE) are the first example, recommend the use of the new blood tests in the following groups: (1) individuals at risk of LTBI who have tested positive by TST, and (2) individuals for whom the TST is unreliable (ie, those with cellular immune suppression who commonly produce false-negative TST results). In contrast, current US guidelines³³ suggest up-front use of a blood test in all groups, as a direct replacement for the TST. Thus, in Europe, if guidelines are followed, TST-negative, blood test-positive individuals should only be identified among immunosuppressed populations. In the absence of a "gold standard" of asymptomatic M tuberculosis infection, it is difficult to ascertain whether a TST-negative, blood test-positive result represents M tuberculosis infection. However, the high sensitivity of ELISpot and its robustness to cellular immune suppression mean that people with recent tuberculosis contact who have suppressed or immature immune systems and test TST negative but ELISpot positive are likely to have false-negative TST results and should be considered to be infected. The high risk of progression to tuberculosis disease in such individuals in any case requires clinicians to have a low threshold for diagnosis and treatment of LTBI. However, an unknown number of immunocompetent individuals with negative TST results who might have tested positive by blood test will not receive treatment under current European guidelines; these individuals would be treated in the United States because they would only have the blood test performed. This raises the question as to whether such persons are truly infected, whether they have a significant risk of progression to active tuberculosis, and whether they stand to benefit from treatment. The key determinant of net treatment benefit will be the overall population risk of progression to tuberculosis disease in blood test-positive contacts. This is why longitudinal natural history studies with hard clinical outcomes (ie, development of tuberculosis) are urgently required and have been called for by the World Health Organization, NICE, and the Centers for Disease Control and Prevention. In the meantime, two contrasting sets of guidelines on either side of the Atlantic seem likely to remain in place until the prognostic power of these tests in recent tuberculosis contacts is elucidated. Another reason for the differing recommendations between the United States and the United Kingdom lies in health economic considerations. Several studies,³⁴³⁵ including that of NICE,³² show that the blood tests are overall more cost-effective than skin tests, but the NICE analysis found that the most cost-effective model in the United Kingdom is to reserve the blood tests for confirmation of LTBI in people who have tested positive by the TST, except in people for whom the TST is unreliable.

As immune-based assays, the new blood tests cannot distinguish between latent and active infection; and rapid, sensitive tests for active tuberculosis that improve on sputum smear microscopy are urgently needed. However, since M tuberculosis infection is a prerequisite for tuberculosis disease, the T-cell–based tests can play a useful role in evaluation of patients with suspected tuberculosis because a negative result in a test of high diagnostic sensitivity could help rapidly to rule out tuberculosis.²⁶ A further increase in the sensitivity of T-cell IFN- release assay (TIGRA), for example, by incorporation of novel M tuberculosis-specific antigens, could potentially enable reliable and rapid exclusion of tuberculosis.³⁶ Positive blood test results can also guide clinical decision making in low-prevalence countries if judiciously interpreted.³⁷ Application of the Lalvani ELISpot with clinical specimens from disease sites in patients with tuberculous pleuritis³⁸ and sputum smear-negative pulmonary tuberculosis³⁹ has shown high sensitivity and high specificity for active tuberculosis, and larger studies are now required to confirm these results in routine practice.

Effect of Treatment on Blood Test Results

The impact of treatment on blood test results has been studied for ELISpot in tuberculosis patients and, more recently, in persons with LTBI. In general, the magnitude of ELISpot responses declines significantly with treatment in both active tuberculosis¹⁰²³ and LTBI,⁴⁰⁴¹ raising the interesting possibility that the quantitative, dynamic readout of ELISpot might be used to monitor treatment response. This would be particularly useful in LTBI, for which there are no clinical or laboratory parameters for monitoring effect of treatment. However, there is considerable interindividual variation in the rate of decline of the response, and only a minority of individuals become negative after completing treatment. Therefore, the assay cannot be used for treatment monitoring or as a "test of cure." However, simultaneous measurement of interleukin-2 and IFN- secretion by M tuberculosis-specific T cells correlates well with treatment and opens a new paridigm for monitoring.⁴³ Next-generation T-cell–based tests measuring dual cytokines promise to provide more clinically useful information about in vivo bacterial burden and clinical status than existing IFN-–only based assays.⁴³

The fact that treatment of LTBI significantly impacts on the IFN- ELISpot response indicates that there is a dynamic equilibrium between the frequencies of M tuberculosis-specific T cells measured by the assay and the status of dormant tubercle bacilli in vivo. Thus, positive ELISpot results very probably reflect the presence of latent but still-viable bacilli in vivo, and this suggests that positive results must be associated with a forward risk of progression to tuberculosis. However, confirmation of this postulate will have to await the results of longitudinal natural history studies with clinical outcomes, and such studies will have to address several ethical and logistical challenges. Such studies might reveal that T-cell–based blood tests have better predictive value for subsequent development of tuberculosis than TST, thereby unraveling the clinical significance of discordant test results (eg, blood test-positive, TST-negative individuals) as a marker of latent infection with significant risk of progression to active disease.²⁴

Longitudinal studies⁴⁰ of contacts with precisely defined exposure in point-source outbreaks have identified a group of TST-negative subjects who have transiently positive ELISpot results. This raises the intriguing possibility that some TST-negative contacts may acquire, and spontaneously clear, a transient M tuberculosis infection.⁴²

Impact on Clinical Practice Today

The high specificity of both blood tests will lessen the number of persons inappropriately treated on the basis of false-positive TST results caused by prior BCG vaccination. This represents an enabling step for tuberculosis elimination programs in low prevalence countries where, as the prevalence of LTBI declines, an increasing proportion of positive TST results are falsely positive. High diagnostic sensitivity in young children (as evidenced for ELISpot) and immunosuppressed populations (as evidenced for ELISpot and to a lesser extent ELISA) will allow more accurate targeting of persons with latent infection at highest risk of progression to tuberculosis. However, longitudinal studies with clinical outcomes are urgently needed to define the predictive value of positive TIGRA results for subsequent development of active tuberculosis; such data would provide a clear mandate for basing treatment decisions on TIGRA results.

Footnotes

Abbreviations: BCG = bacille Calmette-Guérin; CFP-10 = culture filtrate protein-10; DTH = delayed-type hypersensitivity; ELISA = enzyme-linked immunosorbent assay; ELISpot = enzyme-linked immunospot; ESAT-6 = early secretory antigenic target-6; IFN = interferon; LTBI = latent tuberculosis infection; NICE = National Institute of Health and Clinical Excellence; PPD = purified protein derivative; TIGRA = T-cell interferon- release assay; TST = tuberculin skin test

Professor Lalvani is a named inventor for several patents underpinning T-cell–based diagnosis filed by the University of Oxford since 1996. Regulatory approval and commercialization of the Lalvani ELISpot (T-SPOT.TB) has been undertaken by a spin-out company of the University of Oxford (Oxford Immunotec Ltd.; Oxford, UK), in which Professor Lalvani has a share of equity and to which he acts as scientific advisor in a nonexecutive capacity. The University of Oxford has a share of equity in Oxford Immunotec Ltd.

Professor Lalvani is supported by a Wellcome Senior Research Fellowship in Clinical Science.

Received for publication October 9, 2006. Accepted for publication February 2, 2007.

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