Mol Diag Ther 2006; 10 (1): 57-63 1177-1062/06/0001-0057/$39.95/0

ADIS DIAGNOSTIC PROFILE

© 2006 Adis Data Information BV. All rights reserved.

T-SPOT™.TB An In Vitro Diagnostic Assay Measuring T-Cell Reaction to Mycobacterium tuberculosis-Specific Antigens Antona J. Wagstaff1 and Jean-Pierre Zellweger2 1 2

Adis International Inc., Yardley, Pennsylvania, USA University Medical Policlinic, Lausanne, Switzerland

Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 1. Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 2. Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59 3. Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 4. Clinical Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60 5. T-SPOT™.TB: Current Status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

Abstract ▲ The overall sensitivity of the RD1 ELISPOT assay (T-SPOT™.TB), which counts T cells sensitized to specific Mycobacterium tuberculosis peptide sequences, was 81–97% in populations with confirmed tuberculosis, in whom 39–78% had immunosuppressive conditions. ▲ In patients with confirmed tuberculosis, the respective RD1 ELISPOT versus tuberculin skin test (TST) sensitivities were 100% versus 89% in adults and 77% versus 35% in children (of whom 39% were HIV-positive). ▲ In contrast to that of the TST, the sensitivity of the RD1 ELISPOT assay was not significantly affected by age <36 months, or the immunological or nutritional status of the subjects. ▲ Specificity was 100% in two UK-based studies. Isolated false positives have been recorded in patients infected with non-tuberculous M. kansasii. ▲ A study investigating latent tuberculosis infection found no significant difference in results between HIV-positive and -negative participants for the RD1 ELISPOT assay, while the TST varied significantly with HIV status. ▲ Contact-tracing studies have demonstrated concordance between the RD1 ELISPOT assay and the TST of 65–89%. ▲ There is a significant correlation between a positive result and the degree of exposure to the index case for the RD1 ELISPOT assay, but not for the TST, in contact-tracing studies. ▲ Unlike the TST, the RD1 ELISPOT assay is not confounded by bacille Calmette-Gu´erin vaccination.

Features and properties of the RD1 ELISPOT (T-SPOT™.TB) assay

Diagnostic indication

Diagnosis of Mycobacterium tuberculosis infection

Assay characteristics

The RD1 ELISPOT assay is an in vitro enzyme-linked immunosorbent spot assay that detects activated T cells by capturing and visualizing interferon-γ after T-cell contact with specific M. tuberculosis antigenic peptides

The assay identifies individual activated T cells, resulting in high sensitivity for the identification of latent tuberculosis infection

Selected peptides from the RD1 proteins ESAT-6 and CFP 10 are used

ESAT-6 and CFP 10 occur in all tuberculous mycobacteria and in only three of the common non-tuberculous mycobacteria; they do not occur in the BCG vaccine

Assay results are available within 1 day

´ BCG = bacille Calmette-Guerin; CFP = culture filtrate protein; ELISPOT = enzyme-linked immunosorbent spot; ESAT = early secretory antigenic target; RD1 = M. tuberculosis genomic region of difference 1.

58

The tuberculosis species complex, comprising Mycobacterium tuberculosis, M. bovis, M. africanum, M. microti, and M. canettii, all cause tuberculosis, although M. tuberculosis is the primary pathogen in humans.[1] M. tuberculosis infection may manifest in various ways, depending on the host immune response, which in turn depends on factors such as age (children <4 years old are at high risk), underlying immune status, coexisting diseases and/or malnutrition, intake of corticosteroids and other immunosuppressive drugs, immunisation with the M. bovis bacille Calmette-Gu´erin (BCG) vaccine, the virulence of the organism, and the site of infection.[2] In immunocompetent individuals, tuberculosis mainly occurs in the lungs; however, worsening immune status is associated with an increased tendency for extrapulmonary involvement.[2] In most infected individuals, the bacillus is kept under immunological control as asymptomatic latent tuberculosis infection, but severe symptoms can occur in patients with heavy infections.[2] Systemic effects include fever, loss of appetite, weight loss, weakness, night sweats, malaise, increased peripheral blood leukocyte count, and anemia. Symptoms specific to the site of infection also occur (for example, cough, purulent sputum production, and chest pain in pulmonary tuberculosis). It has been estimated that active disease will develop in about 10% of those with latent tuberculosis infection over a lifetime (with the risk greatest in the first 2 years); treatment of latent disease is considered a necessary step in the elimination of tuberculosis.[2,3] Five years ago, it was estimated that up to 43% of the world’s population was infected with M. tuberculosis[2] and it is now known that the global burden of tuberculosis is increasing.[4] The WHO has suggested that this increase is linked with the increased global incidence of HIV infection.[2,4] It has been estimated that HIV-infected individuals are 60 times more likely to develop tuberculosis than immunocompetent individuals in industrialized countries, and six times more likely in other countries.[4] Latent tuberculosis infection progresses to active tuberculosis in most patients with concomitant HIV infection, and the presence of HIV infection also speeds the progression of active disease.[4,5]

Wagstaff & Zellweger

benefit from preventive treatment. The first eliminates a source of transmission to the population; the second reduces the pool of potential future cases. This is particularly important in children, in whom the risks of progression or dissemination of the disease are increased,[8] possibly as a result of undeveloped immune systems.[9] Until recently, the only method of identifying subjects infected with latent M. tuberculosis was the tuberculin (Mantoux) skin test (TST) which uses purified protein derivative (PPD), a precipitant of M. tuberculosis culture supernatant containing >200 antigens, to produce a delayed (cellular) hypersensitivity response.[2] Many of the antigens in PPD occur in the widely used BCG vaccine and in common environmental mycobacteria. In the TST, PPD is injected as an antigen and the reaction of the immune system is assessed around 3 days later by the extent of the subsequent skin induration.[10] The TST is associated with several disadvantages, including the necessity for a second visit to the health clinic to assess the result (many subjects fail to return), interoperator variation as a result of subjective assessment of the test result, a booster phenomenon in which repeated testing can result in a false-positive result, low specificity as a result of the presence of PPD antigens in the BCG vaccine and non-tuberculous mycobacteria, and reduced sensitivity in immunocompromized patients (e.g. those with HIV infection or advanced tuberculosis, transplant recipients, those taking biological cytokine-inhibiting agents such as tumor necrosis factor inhibitors for rheumatoid arthritis or other autoimmune diseases, or malnourished individuals).[10-15] M. tuberculosis evokes a strong cell-mediated immune response in humans.[10] One of the main components of the response involves the formation of antigen-specific cytokine-secreting CD4 and CD8 T cells. Early secretory antigenic target-6 (ESAT-6) and culture filtrate protein (CFP) 10, secreted proteins encoded by the M. tuberculosis genomic segment region of difference 1 (RD1), are immunogenic in patients in whom M. tuberculosis infection has developed.[16,17]

Tuberculosis is most prevalent in developing countries, but dissemination of infection from these areas by immigration to countries with a previously low incidence is increasing.[2,4,6] Early detection and treatment of latent tuberculosis infection would help to stem the increase of the disease in developing countries and could also result in significant health and economic benefits in industrialized countries.[7]

Recently developed alternatives to the TST include assays measuring the interferon (IFN)-γ produced by these antigen-specific T cells in response to ESAT-6/CFP 10 antigenic stimulation, using in vitro enzyme-linked immunosorbent spot (ELISPOT) technology measuring the number of antigen-specific T cells, or ELISA technology measuring overall IFNγ production in whole blood.[11,18,19]

The first steps in resolving this major global problem are to accurately identify patients with active tuberculosis, so that treatment can begin, and individuals with latent infection who could

The focus of this profile is an ELISPOT assay that counts T cells sensitized to specific peptide sequences from ESAT-6 and CFP 10 in the RD1 M. tuberculosis genomic segment, herein

© 2006 Adis Data Information BV. All rights reserved.

Mol Diag Ther 2006; 10 (1)

T-SPOT™.TB: Adis Diagnostic Profile

referred to as the RD1 ELISPOT assay (T-SPOT™.TB).1 The term RD1 ELISPOT thus encompasses both the T-SPOT™.TB assay and premarketing versions using identical technology. 1. Technology In the RD1 ELISPOT assay, peripheral blood mononuclear cells comprising antigen-presenting cells and CD4+ and CD8+ T cells from the subject to be tested are placed in an ELISPOT plate. ● Amino acid sequence length and overlap of M. tuberculosisspecific peptides ESAT-6 and CFP 10 have been chosen to maximize the antigenic response.[20] T cells that recognize the antigens respond by producing cytokines, including IFNγ.[10] ● Anti-IFNγ antibodies subsequently capture and fix the released IFNγ to the bottom of the ELISPOT plate well.[10] The results are available on the following day; each resultant spot, revealed by the addition of a dye, corresponds to the specific reaction of an individual T cell to the antigen. ● Infection with M. tuberculosis is indicated if there are >6 spots relative to the negative control.[21] ● The highly specific antigen sets used in the RD1 ELISPOT assay are absent from most environmental mycobacteria and from the BCG vaccine.[10] The RD1 ELISPOT assay is capable of identifying infections with the tuberculous mycobacteria M. tuberculosis, M. bovis, and M. africanum.[21] Only three of the nontuberculous mycobacteria (M. marinum, M. szulgai, and M. kansasii) carry the antigens used in the RD1 ELISPOT, with subsequent potential for a false-positive test. ●

2. Sensitivity Overall Sensitivity

The overall sensitivity of the RD1 ELISPOT assay ranged from 81% to 97% in patients with confirmed active tuberculosis and mixed HIV status in three studies based in Germany, the UK, and Zambia.[20,22,23] ● A sensitivity rate of 97% was seen for the RD1 ELISPOT assay in a German study of 72 adults with confirmed active tuberculosis; 48% had concomitant immunosuppressive conditions including 2% with HIV infection.[22] Sixty-three patients had positive cultures for M. tuberculosis (n = 60), M. bovis (n = 2), or M. africanum (n = 1); the rest were assessed using clinical symptoms, radiography, histology, and/or a positive TST. Duration of current antituberculous chemotherapy ranged from 2 days to 4 months (median 2 weeks), plus one outlier of >4 years. ●

1

59

Two patients with culture-confirmed M. tuberculosis pulmonary disease in this study had spot counts below the cutoff level for the RD1 ELISPOT assay. These patients had been receiving chemotherapy for tuberculosis for >4 years and 30 days, respectively, which may account for the negative results, since positive ELISPOT results are known to decline with therapy as the numbers of M. tuberculosis bacilli present in the body declines. Neither had signs of immunosuppression.[22] ● An overall sensitivity of 92% was seen in 50 adult Zambian patients (78% HIV-positive) with active tuberculosis diagnosed by clinical symptoms, radiography, or presumptive sputum-smear acid-alcohol-fast bacilli microscopy, all of whom were untreated or had received <1 month of treatment.[20] ● The sensitivity of the RD1 ELISPOT assay was 81% in a single-blind study of 57 South African children (aged 22 months to 8 years, either untreated or treated for <1 month) with active tuberculosis confirmed by culture (68%) or by the less specific (non-M. tuberculosis acid-fast bacilli may be wrongly identified) sputum-smear acid-fast microscopy (32%).[23] Of these children, 42 had been tested for HIV status, and 52% of these were HIVpositive. ●

Comparative Sensitivity ● In two studies making the comparison, the RD1 ELISPOT assay was more sensitive than the TST in patients with confirmed tuberculosis.[22,23] ● In a subgroup of 45 adults with confirmed tuberculosis who received both tests in the German study, all were positive for the RD1 ELISPOT assay (100% sensitivity) but five (all had pulmonary tuberculosis) were negative according to the TST (89% sensitivity).[22] ● Fifteen of 43 tested children with confirmed tuberculosis had a positive reaction to the TST (sensitivity 35%) in the South African study.[23] The sensitivity of the RD1 ELISPOT assay in these 43 children was 77%; 53% had positive RD1 ELISPOT and negative TST results. ● Among children with confirmed or highly probable tuberculosis who received both tests in this study (n = 116), the sensitivity of the RD1 ELISPOT assay was 82%, while that of the TST was 63% (p = 0.001).[23] ● A positive RD1 test (using ESAT-6) was significantly more predictive of future reactivation of tuberculosis than a positive TST in a study of healthy household contacts of tuberculosis patients in Ethiopia.[24]

The use of trade names is for product identification only and does not imply endorsement.

© 2006 Adis Data Information BV. All rights reserved.

Mol Diag Ther 2006; 10 (1)

60

Wagstaff & Zellweger

RD1 ELISPOT

* HIV-positive HIV-negative *

TST

Age <36 months Age >36 months Malnourished Not malnourished

**

0

10

20

30

40

50

60

70

80

90

100

Sensitivity (%)

Fig. 1. Comparative sensitivity of the RD1 ELISPOT assay and the tuberculin skin test (TST). In a cohort of South African children with confirmed or highly probable tuberculosis,[23] the sensitivity of the RD1 ELISPOT assay and the TST were compared according to HIV status (n = 30 HIV-positive, n = 103 HIVnegative), age (n = 54 <36 months, n = 79 >36 months), and nutritional status (n = 59 very malnourished, n = 71 not malnourished). ELISPOT = enzymelinked immunosorbent spot; RD1 = Mycobacterium tuberculosis genomic region of difference 1; * p ≤ 0.01, ** p < 0.001 vs corresponding TST group. ● A meta-analysis of studies investigating the RD1 ELISPOT assay, an RD1 ELISA assay (Quantiferon®-TB Gold), and the TST found 96%, 85%, and 70% sensitivity, respectively, in culture-confirmed, otherwise healthy patients.[25]

Effect of Concurrent Health Disorders ● In contrast to the TST, the sensitivity of the RD1 ELISPOT assay was not significantly affected by the immunological status, age, or nutritional status of the children in the South African study (figure 1).[23] ● All 11 (100%) HIV-negative and 35 of 39 (90%) HIV-positive patients in the Zambian trial had a positive response with the RD1 ELISPOT assay.[20] Since the diagnosis of active tuberculosis was uncertain in one of the four HIV-positive patients who did not respond to any of the peptides in the assay, the sensitivity may have actually been 92% in HIV-positive patients (94% overall). ● In the same study,[20] 11 (100%) and 28 (72%) HIV-negative and -positive patients, respectively, responded to PPD in an ex vivo ELISPOT assay (number BCG-vaccinated not reported).

3. Specificity One hundred percent specificity was demonstrated for the RD1 ELISPOT assay in 40 healthy, low-risk, adult subjects (83% BCGvaccinated) based in the UK.[20,26] While no ESAT-6- or CFP ●

© 2006 Adis Data Information BV. All rights reserved.

10-specific T cells were found in this cohort, 33 (83%) responded positively to PPD in an ELISPOT assay.[20,26] ● A specificity rate of 100% was also seen for the RD1 ELISPOT assay in another UK-based study of 18 healthy, low-risk, HIVnegative adult subjects.[27] ● Of 14 patients with non-tuberculous lung disease investigated in another trial,[22] three reacted positively to both the RD1 ELISPOT assay and the TST. Two were subsequently found to have non-tuberculous M. kansasii infection. Three patients who were negative for both RD1 ELISPOT and TST were subsequently found to have M. xenopii (n = 2) or M. avium (n = 1) infection, and two who were RD1 ELISPOT-negative and TST-positive had no signs of active tuberculosis. 4. Clinical Use Identification of Latent Tuberculosis Infection

The prevalence of latent tuberculosis infection has been investigated using the RD1 ELISPOT assay in adults in Zambia[20] and India,[26] and in patients with HIV infection based in the UK.[27] ● Among 75 healthy Zambian adults with normal chest radiography and no past history or symptoms suggestive of tuberculosis, 76% had evidence of BCG vaccination and 21 (28%) were HIVpositive.[20] A positive response to the RD1 ELISPOT assay was Mol Diag Ther 2006; 10 (1)

T-SPOT™.TB: Adis Diagnostic Profile

61

seen in 37 (69%) of the HIV-negative and nine (43%) of the HIVpositive subjects (figure 2).[20] TST results were available for 49 subjects in this study.[20] A positive TST result occurred in 28 of 35 (80%) HIV-negative subjects and 5 of 14 (36%) HIV-positive subjects (p < 0.01) [figure 2]. Twenty-four (69%) and six (43%), respectively, in this group had positive RD1 ELISPOT results. ●

The response to TST in this study was greater in the 19 HIVnegative subjects with a positive response to both RD1 ELISPOT and TST than in the nine with a negative response to RD1 ELISPOT; eight of these nine had a BCG vaccination scar.[20] Three HIV-positive subjects had positive RD1 ELISPOT results and negative TST results, indicating that the RD1 ELISPOT assay can pick up latent infection in patients with HIV-associated cutaneous anergy (failure of the positive control). ●

A positive response to the RD1 ELISPOT assay was seen in 80% of 100 healthy, ethnically diverse adults from urban India with normal chest radiography, no history of tuberculosis, and no features of HIV infection.[26] Although 98% responded to PPD in an ELISPOT test, the response was less potent than that to ESAT-6/CFP 10.[26] ●

Latent tuberculosis infection was diagnosed in two of 29 asymptomatic HIV-positive individuals using the RD1 ELISPOT assay (both were immigrants with previous exposure) and in one of 19 healthy, low-risk, HIV-negative controls in a study in the UK.[27] ●

HIV-negative HIV-positive RD1 ELISPOT (n = 75)

TST (n = 49)

*

PPD ELISPOT (n = 75)

0

10

20

30 40 50 60 70 Positive response (%)

80

90

100

Fig. 2. Comparative results for diagnosis of latent tuberculosis in healthy Zambian adults.[20] Diagnostic tests included the RD1 ELISPOT assay using ESAT-6 and CFP 10 peptides, a PPD ELISPOT test and the TST. CFP = culture filtrate protein; ELISPOT = enzyme-linked immunosorbent spot; ESAT = early secretory antigenic target; PPD = mycobacterial purified protein derivative; RD1 = Mycobacterium tuberculosis genomic region of difference 1; TST = tuberculin skin test; * p < 0.01 vs HIV-negative patients. © 2006 Adis Data Information BV. All rights reserved.

In this study, the performance of the RD1 ELISPOT assay was not correlated with CD4+ counts, CD8+ counts, the CD4+ : CD8+ ratio, or the plasma viral load.[27] A positive correlation was, however, noted between response and total peripheral CD3+ counts. Although the response was slightly lower in HIV-positive patients with CD4+ counts <200 cells/μL than in those with higher counts, in vitro anergy occurred in only one HIV-positive patient (who had a CD4+ count >200 cells/μL) and in none of the HIVnegative controls. ●

Contact Tracing

Contact-tracing studies using the RD1 ELISPOT assay have investigated contacts of index cases in a UK school,[28] a maternity unit in Italy,[29] a residential institution for alcoholics in Switzerland,[30] and in community-based studies in adults in the US[31] and children in Turkey.[32] ● The RD1 ELISPOT assay and the TST were used to screen 535 school contacts of a UK student diagnosed with pulmonary tuberculosis after 9 months of a chronic cough.[28] Concordance between the tests was 89%; 121 students were positive to both tests, 26 were positive only to RD1 ELISPOT, and 34 were positive only to TST. ● After spending 4 days in a maternity unit in Italy, a woman was diagnosed with sputum smear-positive cavitary multidrug-resistant tuberculosis, resulting in possible nosocomial exposure to 41 infants and 47 adults, and nosocomial and household exposure to four additional adults.[29] These contacts were screened using RD1 ELISPOT, PPD ELISPOT, and TST tests 11 weeks after exposure. ● Of the 92 participants in this study, 17 responded positively to RD1 ELISPOT, 34 to PPD ELISPOT, and four to TST; concordance between RD1 ELISPOT and PPD ELISPOT was 68%, while that between RD1 ELISPOT and TST was 82%.[29] Only two subjects were positive to all three tests. All neonates had evaluable results. ● The RD1 ELISPOT assay and the TST were compared in 50 residents and 41 staff a mean of 12 weeks after about a month’s exposure to an index case with sputum smear-positive pulmonary tuberculosis in a residential institution for alcoholics in Switzerland.[30] Positive RD1 ELISPOT or TST results were seen in 15% and 44% of participants, respectively. Concordance was 65%; 11 subjects were positive to both tests, three were positive only to RD1 ELISPOT, and 29 were positive only to TST. ● In all three studies, there was a strong correlation between a positive result and the degree of exposure to the index case for the RD1 ELISPOT assay (figure 3).[28-30] ● The correlation between a positive result and degree of exposure to the index case was significantly greater for the RD1 Mol Diag Ther 2006; 10 (1)

Wagstaff & Zellweger

Residential alcoholic institute Maternity unit in in Switzerland Italy (n = 92) (n = 91)

School in UK (n = 535)

62

RD1 ELISPOT TST

RD1 ELISPOT TST

Most exposure Least exposure

RD1 ELISPOT TST 0

10

20

30

40

50

60

70

Positive response (%)

Fig. 3. Rate of positive response by intensity of tuberculosis exposure among contacts assessed by the RD1 ELISPOT assay and the tuberculin skin test (TST). The figure outlines the correlation between a positive result and the degree of exposure to the index case in three studies (in a school,[28] a maternity unit,[29] and a residential institution for alcoholics[30]). For the school and maternity unit studies, results for the two groups with closest contact and the two with least contact have been combined (to form most and least exposure groups) for comparative purposes. ELISPOT = enzyme-linked immunosorbent spot assay; RD1 = Mycobacterium tuberculosis genomic region of difference 1.

ELISPOT assay than for the TST across the four stratified groups in the school study (p < 0.05).[28] In the maternity unit study, the odds of a positive result for each increase across the four stratified groups increased significantly for the RD1 ELISPOT and PPD ELISPOT tests (both p < 0.05) but there was no significant correlation for the TST.[29] When the contacts were stratified by low or high exposure to the index case in the study conducted in the residential institution for alcoholics, the results of the RD1 ELISPOT assay were significantly correlated with exposure (p < 0.05) but there was no such correlation for the TST.[30] ●

The response to the TST, but not to the RD1 ELISPOT assay, was strongly linked with the prevalence of BCG vaccination in all three studies.[28-30] ●

● The likelihood of having latent tuberculosis was estimated via a contact score quantifying exposure to and infectiousness of the index case in 413 contacts (49% BCG-vaccinated) of 72 US patients with culture-proven pulmonary tuberculosis in a community-based study.[31] The relationship between these scores and results of the RD1 ELISPOT assay and the TST was analysed. A positive assay result was obtained in 39% with the RD1 ELISPOT and 50% with the TST.

For both the RD1 ELISPOT assay and the TST in this study, the correlation increased significantly with rising contact score (p < 0.001 and p = 0.01, respectively).[31] A positive TST result was significantly more likely than a positive RD1 ELISPOT result among foreign-born, BCG-vaccinated contacts. ●

© 2006 Adis Data Information BV. All rights reserved.

In another community-based study, the prevalence of tuberculosis infection was assessed in 979 Turkish children aged ≤16 years (median 7 years; 79% BCG-vaccinated) who were household contacts of 414 adults with sputum smear-positive pulmonary tuberculosis.[32] The RD1 ELISPOT assay and the TST revealed positive results in 43% and 51%, respectively. Of the 13 children with active tuberculosis at enrolment, 12 were positive by RD1 ELISPOT, and 11 were positive by TST. ● Risk factors for M. tuberculosis infection in this study that were significantly associated with positive results for both RD1 ELISPOT and TST after multivariate analysis included increasing number of index patients in the household, being the child of an index patient versus a more distant relationship, and increasing age.[32] ● The only risk factor differentiating the tests was the absence of a BCG scar, which was significantly associated with a positive RD1 ELISPOT result (p < 0.0001 vs TST).[32] Concordance between the RD1 ELISPOT and TST assays was higher in children without a BCG scar (κ = 0.76) versus those with a scar (κ = 0.50). ●

5. T-SPOT™.TB: Current Status The T-SPOT™.TB assay is currently available in the EU and is awaiting approval by the US FDA. In contrast to the TST, clinical studies have demonstrated high sensitivity and specificity for this assay, with little dependence on the immunological status of the subject and no confounding of the result by previous BCG vaccination. Mol Diag Ther 2006; 10 (1)

T-SPOT™.TB: Adis Diagnostic Profile

Disclosure During the peer-review process, the manufacturer of the diagnostic assay under review was also offered an opportunity to comment on this article; changes based on any comments received were made on the basis of scientific and editorial merit.

References 1. Zink AR, Sola C, Reischl U, et al. Characterization of Mycobacterium tuberculosis complex DNAs from Egyptian mummies by spoligotyping. J Clin Microbiol 2003 Jan; 41 (1): 359-67 2. American Thoracic Society. Diagnostic standards and classification of tuberculosis in adults and children. Am J Respir Crit Care Med 2000; 161: 1376-95 3. Dooley KE, Sterling TR. Treatment of latent tuberculosis infection: challenges and prospects. Clin Chest Med 2005 Jun; 26 (2): 313-26 4. Corbett EL, Watt CJ, Walker N, et al. The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 2003 May 12; 163 (9): 1009-21 5. Burman WJ. Issues in the management of HIV-related tuberculosis. Clin Chest Med 2005 Jun; 26 (2): 283-94 6. Broekmans JF, Migliori GB, Rieder HL, et al. European framework for tuberculosis control and elimination in countries with a low incidence: recommendations of the World Health Organization (WHO), International Union Against Tuberculosis and Lung Disease (IUATLD) and Royal Netherlands Tuberculosis Association (KNCV) Working Group. Eur Respir J 2002 Apr; 19 (4): 765-75 7. Khan K, Muennig P, Behta M, et al. Global drug-resistance patterns and the management of latent tuberculosis infection in immigrants to the United States. N Engl J Med 2002 Dec 5; 347 (23): 1850-9 8. Feja K, Saiman L. Tuberculosis in children. Clin Chest Med 2005 Jun; 26 (2): 295-312 9. Lewinsohn DA, Gennaro ML, Scholvinck L, et al. Tuberculosis immunology in children: diagnostic and therapeutic challenges and opportunities. Int J Tuberc Lung Dis 2004 May; 8 (5): 658-74 10. Hoffmann M, Vernazza P, Fierz W. Diagnostik der latenten Tuberkulose im neuen Millenium. Schweiz Med Forum 2003 Dec; (50): 1244-7 11. Pai M, Riley LW, Colford JM, et al. Interferon-γ assays in the immunodiagnosis of tuberculosis: a systematic review. Lancet Infect Dis 2004 Dec; 4: 761-76 12. Lalvani A. Spotting latent infection: the path to better tuberculosis control. Thorax 2003 Nov; 58 (11): 916-8 13. Benito N, Sued O, Moreno A, et al. Diagnosis and treatment of latent tuberculosis infection in liver transplant recipients in an endemic area. Transplantation 2002 Nov 27; 74 (10): 1381-6 14. Bieber J, Kavanaugh A. Consideration of the risk and treatment of tuberculosis in patients who have rheumatoid arthritis and receive biologic treatments. Rheum Dis Clin North Am 2004 May; 30 (2): 257-70

63

antigens ESAT-6 and culture filtrate protein 10 and to mixtures of synthetic peptides. Infect Immun 2000 Jun; 68 (6): 3314-21 18. Lazarevic V, Pawar S, Flynn J. Measuring T-cell function in animal models of tuberculosis by ELISPOT. Methods Mol Biol 2005; 302: 179-90 19. Doherty TM, Demissie A, Menzies D, et al. Effect of sample handling on analysis of cytokine responses to Mycobacterium tuberculosis in clinical samples using ELISA, ELISPOT and quantitative PCR. J Immunol Methods 2005 Mar; 298 (1-2): 129-41 20. Chapman ALN, Munkanta M, Wilkinson KA, et al. Rapid detection of active and latent tuberculosis infection in HIV-positive individuals by enumeration of Mycobacterium tuberculosis-specific T cells. AIDS 2002 Nov 22; 16 (17): 2285-93 21. Oxford Immunotec. T-SPOT™.TB [online]. Available from URL: http://www. oxfordimmunotec.com [Accessed 2005 Nov 21] 22. Meier T, Eulenbruch H-P, Wrighton-Smith P, et al. Sensitivity of a new commercial enzyme-linked immunospot assay (T SPOT-TB) for diagnosis of tuberculosis in clinical practice. Eur J Clin Microbiol Infect Dis 2005 Aug; 24 (8): 529-36 23. Liebeschuetz S, Bamber S, Ewer K, et al. Diagnosis of tuberculosis in South African children with a T-cell-based assay: a prospective cohort study. Lancet 2004 Dec 18; 364 (9452): 2196-203 24. Doherty T, Demissie A, Olobo J, et al. Immune responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 signal subclinical infection among contacts of tuberculosis patients. J Clin Microbiol 2002 Feb; 40 (2): 704-6 25. Kelly DW. Interferon-γ assays for the immunodiagnosis of tuberculosis: a quantitative meta-analysis [data on file]. Abingdon, UK: Oxford Immunotec Ltd, 2005 26. Lalvani A, Nagvenkar P, Udwadia Z, et al. Enumeration of T cells specific for RD1-encoded antigens suggests a high prevalence of latent Mycobacterium tuberculosis infection in healthy urban Indians. J Infect Dis 2001 Feb 1; 183 (3): 469-77 27. Dheda K, Lalvani A, Miller RF, et al. Performance of a T-cell-based diagnostic test for tuberculosis infection in HIV-infected individuals is independent of CD4 cell count. AIDS 2005; 19 (17): 2038-41 28. Ewer K, Deeks J, Alvarez L, et al. Comparison of T-cell-based assay with tuberculin skin test for diagnosis of Mycobacterium tuberculosis infection in a school tuberculosis outbreak. Lancet 2003 Apr 5; 361 (9364): 1168-73 29. Richeldi L, Ewer K, Losi M, et al. T cell-based tracking of multidrug resistant tuberculosis infection after brief exposure. Am J Respir Crit Care Med 2004 Aug 1; 170 (3): 288-95 30. Zellweger J-P, Zellweger A, Ansermet S, et al. Contact tracing using a new T-cellbased test: better correlation with tuberculosis exposure than the tuberculin skin test. Int J Tuberc Lung Dis 2005 Nov; 9 (11): 1242-7 31. Shams H, Weis SE, Klucar P, et al. Enzyme-linked immunospot and tuberculin skin testing to detect latent tuberculosis infection. Am J Respir Crit Care Med 2005 Nov 1; 172 (9): 1161-8

15. Gardam M, Iverson K. Rheumatoid arthritis and tuberculosis: time to take notice. J Rheumatol 2003 Jul; 30 (7): 1397-9

32. Soysal A, Millington KA, Bakir M, et al. Effect of BCG vaccination risk of Mycobacterium tuberculosis infection in children with household tuberculosis contact: a prospective community-based study. Lancet 2005 Oct 22; 366 (9495): 1443-51

16. Wilkinson KA, Wilkinson RJ, Pathan A, et al. Ex vivo characterization of early secretory antigenic target 6-specific T cells at sites of active disease in pleural tuberculosis. Clin Infect Dis 2005 Jan 1; 40 (1): 184-7

Correspondence: Antona J. Wagstaff, Adis International Inc., 770 Township

17. Arend SM, Geluk A, van Meijgaarden KE, et al. Antigenic equivalence of human T-cell responses to Mycobacterium tuberculosis-specific RD1-encoded protein

© 2006 Adis Data Information BV. All rights reserved.

Line Road, Yardley, PA 19067, USA. E-mail: [email protected]

Mol Diag Ther 2006; 10 (1)