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.
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-Guerin (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?
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Translation From Bench to Bedside
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.’5 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 immu-nospot (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)-7 (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 cell”7 (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). The alternate method is a whole-blood enzyme-linked immunosorbent assay (ELISA), which measures the IFN-7 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-y-release assays and are now commercially available as quality-controlled, regulatory-approved diagnostic test kits. 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.
Rapidly Expanding Clinical Evidence Base in the Last 5 Years
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 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.
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.
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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%, is significantly higher than the 70 to 89% sensitivity”of ELISA (Table 2).
Demonstrating superiority of a new test for LTBI is more difficult than for active tuberculosis because there is no “gold standard” reference test. Thus, it is not possible to measure directly the sensitivity and specificity of a new test for LTBI. However, since airborne transmission of M tuberculosis is promoted by increasing duration and proximity of contact with an infectious case, a key determinant of infection is the amount of time spent sharing room air with the source case. Hence, if a new test is indeed a more sensitive and specific test, it should correlate more closely with level of exposure to M tuberculosis than the TST and should be independent of BCG vaccination status. Five studies with ELISpot and two studies with ELISA have exploited this principle to compare the diagnostic accuracy of the TST in contact investigations (Table 3). The ELIS-pot studies were prospective and comprised two community-based contact investigations and three point-source institutional outbreaks, involving a total of 1,136 contacts. ELISpot results, but not TST results, were independent of BCG vaccination status, indicating higher specificity (Table 4). Overall, ELISpot correlated better with tuberculosis exposure than TST, suggesting higher sensitivity for diagnosis of LTBI (Table 5). Another study of 369 contacts in an institutional outbreak, using a related but different ELISpot format, gave similar results. In the smaller number of contact investigations using ELISA, this assay was also independent of BCG vaccination status but correlation of ELISA results with tuberculosis exposure was generally similar to the TST. Quantitative estimates of diagnostic specificity can be generated by studying BCG-vaccinated individuals at an ultralow risk of LTBI due to absence of epidemiologic risk factors tuberculosis exposure. Four studies using ELISpot in 127 participants, and two studies’14 using ELISA in 315 participants show that both assays have very high specificity, approaching 100%.
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-7. This cytokine is captured in the immediate vicinity of the cytokine-secreting T cell by antibodies specific for IFN-7 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-7. The plate is centrifuged and the plasma transferred to a 96-well microtitre plate. IFN-7 in the plasma is captured by antibodies specific for IFN-7 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-7 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.
Table 1—Key Features of the New Blood Tests for Tuberculosis Infection
|Variables||ELISpot (T-SPOT.TB)||ELISA (QuantiFERON-Gold)|
|Antigens||ESAT-6 and CFP10||ESAT-6 and CFP10|
|Positive internal control||Yes||Yes|
|Potential for boosting effect in repeated tests||No||No|
|Need for return visit||No||No|
|Time required for results, h||16-20||16-24|
|Readout units||IFN-7 spot-forming cells||International units of IFN-7|
|Test substrate||Peripheral blood mononuclear cells||Whole blood|
|Outcome measure||Number of IFN-7-producing T cells||Serum concentration of IFN-7 produced by T cells|
|Readout system||Enumeration of spots by naked eye, magnifying lens, or automated reader||Measurement of optical density values using an automated reader|
|Regulatory approvals||Europe, Canada, US Food and Drug Administration pending||Europe, Canada, US Food and Drug Administration pending|
Table 2—Published Studies on Diagnostic Performance of the New Blood Tests for Sensitivity in Active Tuberculosis in Immunocompetent Adults
|ELISpot (T-SPOTTB)*||ELISA (QuantiFERON-Gold)i|
|Study Design||Subjects’ No.||Sensitivity’ %||Study Design||Subjects’ No.||Sensitivity’ %|
|Case control||47||96||Case control||54||81|
Table 3—Published Studies on Diagnostic Performance of the New Blood Tests for Sensitivity in LTBI
|ELISpot (T-SPOT.TB)t||ELISA (QuantiFERON-Gold)j|
|Correlation With Tuberculosis Exposure||Study Design||
|Correlation With Tuberculosis Exposure|
|Contact tracing||535||Contact tracing||125||Same as TST|
|Contact tracing||413||Case control||48||Higher than TST|
|Contact tracing||88||Higher than TST§|
Table 4—Published Studies on Diagnostic Performance of the New Blood Tests for Specificity in BCG-Vaccinated Exposed Contacts
|ELISpot (T-SPOT.TB)*||ELISA (QuantiFERON-Gold)f|
|Study Design||Subjects, No.||Effect of BCG||Study Design||Subjects, No.||Effect of BCG|
|Contact tracing||467||Independent of BCG status!||Contact tracing||309||Independent of BCG status|
Table 5—Published Studies on Diagnostic Performance of the New Blood Tests for Specificity in BCG-Vaccinated Unexposed Control Subjects
|ELISpot (T-SPOT.TB)*||ELISA (QuantiFERON-Gold)i|
|Study Design||Subjects, No.||Specificity, %||Study Design||Subjects, No.||Specificity, %|
|Case control||28||100||Case control||216||98|
|Case control||40||100||Case control||99||96|