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Tuberculosis (2005) 85, 137 145

Tuberculosis
http:/ /intl.elsevierhealth.com/journals/tube

Sensitivity of human gamma interferon assay and tuberculin skin testing for detecting infection with Mycobacterium tuberculosis in patients with culture positive tuberculosis
W.J. Brittona,b,, G.L. Gilbertc, J. Wheatleyd, D. Lesliee, J.S. Rothelf,1, S.L. Jonesf, P. Bradleyf
Centenary Institute of Cancer, Medicine and Cell Biology, Locked Bag No. 6, Newtown NSW 2042, Australia Department of Medicine (DO6), Central Clinical School, University of Sydney, Sydney 2006, Australia c Centre for Infectious Diseases and Microbiology, Institute of Clinical Pathology and Medical Research, Westmead Hospital, Westmead 2145, Australia d Department of Respiratory Medicine, Westmead Hospital, Westmead 2145, Australia e Faireld Hospital, Yarra Bend Rd, Faireld 3078, Australia f CSL Limited, Parkville 3052, Australia
b a

Accepted 16 June 2004

KEYWORDS
Tuberculosis; Interferon-gamma; Tuberculin skin test; Diagnostic; Sensitivity

Summary Setting: Nine university-afliated chest clinics in Australia. Objective: To evaluate the sensitivity of a whole blood human gamma-interferon assay (HGIA, QuantiFERONs-TB) for specic T lymphocyte responses and Tuberculin skin testing (TST) for the detection of Mycobacterium tuberculosis infection in subjects with culture-proven M. tuberculosis disease (TBCP). Design: Prospective testing of 129 patients with recent TBCP and 100 patients with non-tuberculosis lung disease (NTBLD). Results: Using a dened level of specic IFN-g production and TST X10 mm as positive cut-offs, the sensitivity of HGIA was 81% compared to 89% for TST (p 0:06). When positive responses in both TST and HGIA were combined, 96% of TB patients were detected. For the NTBLD group, 43% of whom were born overseas, 73% were negative for both the HGIA and TST. Prior immunization with M. bovis (bacille CalmetteGuerin) (BCG) or the type of TB had no effect on the sensitivities of the assays. For those treated for o2 months, the sensitivities for both assays were 84%, but for those treated for 42 months the sensitivity of TST (90%) tended to be higher

Corresponding author. Centenary Institute of Cancer, Medicine and Cell Biology, Locked Bag No. 6, Newtown NSW 2042, Australia.

Tel.: +61-2-9515-5210; fax: +61-2-9351-3968. E-mail address: wbritton@medicine.usyd.edu.au (W.J. Britton). 1 Current address: Cellestis Limited, Carnegie, Australia. 1472-9792/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.tube.2004.06.003

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138 W.J. Britton et al.
than for HGIA (81%) (p 0:07). The distribution of TST results in TB patients showed a broad peak between 10 and 25 mm, while the results in the HGIA were bimodal in both TB and NTBLD patients. Conclusion: HGIA may prove an alternative to skin testing for detecting M. tuberculosis infection in certain settings. r 2004 Elsevier Ltd. All rights reserved.

Introduction
The resurgence of tuberculosis (TB) in developed countries has refocused attention on Mycobacterium tuberculosis as one of the major bacterial pathogens worldwide. Currently nine million TB cases occur annually throughout the world, resulting in two million deaths, 20% of which are in children.1 Detecting infection with M. tuberculosis is important, not only in clinical situations where appropriate cultures cannot be obtained, but for detecting latent tuberculosis infection (LTBI) during the monitoring of healthcare workers, contact tracing and epidemiological studies. The measurement of cell-mediated immunity to mycobacterial products by skin testing has a long pedigree as a surrogate marker for infection with M. tuberculosis.2 Tuberculin skin testing (TST) with an intradermal injection of Puried Protein Derivative (PPD) from M. tuberculosis culture ltrate has been widely used in clinical epidemiological studies, and, as observed by Snider3 in 1982, no better method for detecting LTBI has yet been devised. Problems remain, however, with the practical utilization of TST, including the reliability of administration and reading of the TST.4 TST has a variable sensitivity in detecting active cases of TB with 1030% of such patients with being tuberculin negative.5 Moreover, cross reactivity with nontuberculous mycobacteria reduces the specicity of TST, especially at lower levels of reactivity. In addition there are logistic problems in the application of TST in contact tracing as it requires two visits, one to administer and one to read the response. Serological assays, although capable of detecting M. tuberculosis-specic antibodies, have insufcient sensitivity for detecting all cases of pulmonary TB and, in particular, subjects with subclinical M. tuberculosis infection.6 Historically, bovine TB has been diagnosed using the same principles as the TST in humans, utilizing skin testing with PPD prepared from M. bovis (PPDB). Although this has a satisfactory positive predictive value in herds with a high proportion of infected animals, in situations of low endemicity the interpretation is confounded by reactivity to non-tuberculous mycobacteria. To obviate these

difculties, Wood and colleagues developed7 a simple in vitro assay in which whole blood from cattle was stimulated with PPD-B and the activation of antigen-specic T-cells was detected by the release of gamma-interferon (IFN-g). When compared with skin testing in a large cross-sectional study of cattle, the IFN-g assay was more sensitive than TST (94% versus 66%) in detecting M. bovis infection conrmed by post mortem dissection of the animal and microbiological analysis.7 Further, the IFN-g assay was more sensitive than serology for detecting M. bovis-infected cattle.8 More recently whole blood assays have been used to detect mycobacteria-specic responses in human TB and leprosy patients.9,10 When a human gamma-interferon assay (HGIA) employing for M. tuberculosis PPD was tested in TST-positive contacts of TB patients without clinical disease,11 the majority of infected individuals were detected with a sensitivity of 90% and specicity of 98% in TST-negative control subjects. Culture of M. tuberculosis provides the denitive proof of TB infection. Therefore, we have examined the sensitivities of the HGIA assay and TST in patients with mycobacterial culture-proven active TB and compared these in patients with non-tuberculous lung disease in a multi-center study in eastern Australia.

Materials and methods

Subjects
Patients admitted to or attending nine different teaching hospitals in Sydney and Melbourne were enrolled in the study between October 1994 and April 1996. Group A consisted of 129 patients in whom pulmonary or non-pulmonary TB had been conrmed by culture of M. tuberculosis from sputum or tissue samples and who had received anti-tuberculosis treatment for less than twelve months. HIV infection was excluded by serological testing. Group B consisted of 100 patients with nontuberculous lung disease (NTBLD) who had no past history of TB. These included patients with a wide range of lung diseases, including acute pneumonia

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Sensitivity of human gamma interferon assay and tuberculin skin testing (43), lung carcinoma (12) and other chronic lung diseases. Sputum from control subjects was obtained for microscopy and culture for mycobacteria whenever possible. Chest radiology was performed unless a recent chest X-ray was available. Demographic details, clinical history, current medications and radiological and pathological ndings were recorded. Patients were excluded if there was a past history of infection with non-tuberculous mycobacteria, if they had primary or secondary immunodeciency including infection with human immunodeciency virus (HIV) or if they were taking immunosuppressive medications or oral corticosteroids. The use of inhaled corticosteroids for asthma was permitted. Those who had a past history of a severe reaction to TST or had received TST within the last 8 weeks were also excluded. The study was approved by the Human Ethics Committee at each participating hospital. 139

Study design
Venous blood was collected from each subject into lithium heparin containers for the HGIA and for a full blood examination. The sample for HGIA was kept at room temperature and the assay performed within 12 h of collection. After the blood collection, TST was performed by intradermal injection of 10 international units of PPD from M. tuberculosis in 0.1 ml (CSL Limited, Melbourne, Australia) into the ventral surface of the forearm using a 26 or 27 gauge needle. The reaction was read between 48 and 72 h later and the diameter of induration recorded in millimetres. Patients with known TB were given 1 IU PPD in 0.1 ml initially to avoid serious reaction. If this was negative a repeat dose of 10 IU in 0.1 ml was given within seven days. Adverse reactions to the tuberculin skin tests were documented.

natants which were stored at 41 until tested. The levels of IFN-g in the four samples were determined by enzyme immuno-assay (EIA) employing two antiIFN-g monoclonal antibodies and using recombinant IFN-g as standard. The detection limit of the assay was 20 pg/ml. The HGIA was interpreted according to the manufacturers instructions. The level of IFN-g in each plasma sample was calculated in IUs from the standard curve and the % human response and % avian difference were calculated using the following formulae: % Human Responses HuPPD Negative=Mitogen Negative 100%; and % Avian Difference AvPPD Negative HuPPD Negative = HuPPD Negative 100%: Previous studies have determined the optimum cutoff for the assay to be % Human Response415% and % Avian Differenceo10% for a positive result for TB infection.11 The samples for HGIA were consecutively numbered with identication numbers and the patient results interpreted without knowledge of the clinical diagnosis.

Statistical analysis
Differences in the proportions of patients positive in the different assays were determined by McNemars test for paired proportions with binomial distribution. The level of correlation between the HGIA and TST results in individual patients was tested by Spearman rank correlation coefcient.

Results
Patient characteristics
Group A consisted of 129 patients in whom TB infection was proven by positive culture for M. tuberculosis. The most common form of TB was pulmonary (59%) followed by TB lymphadenitis in 25% of patients and the remainder had TB in other organs. The sensitivities of the HGIA and TST were calculated in these patients with culture-proven TB (TBCP). Group B consisted of 100 patients with nonTB lung disease diagnosed after careful clinical and radiological assessment. Sputum culture was available in 40% of these patients and was negative. Data from these NTBLD patients were used to determine the rate of positivity of HGIA and TST in this population. The mean age of TB patients was 14 years younger than that of NTBLD patients and the distribution of males and females was similar in both groups. TB patients were more likely to have been born outside Australia, particularly in countries with a high prevalence of TB (Table 1). There

Human gamma interferon assay

The HGIA (QuantiFERONs-TB, CSL Ltd., Parkville, Australia, now Cellestis Ltd, Carnegie, Australia) was performed according to the manufacturers instructions in three different laboratories in the participating centers.9 Briey 1 ml of heparinized blood was transferred into each of four separate wells of a 24 well tissue culture plate. Then three drops of PPD from human M. tuberculosis (HuPPD), PPD from M. avium (AvPPD), negative control or mitogen (phytohaemagglutinin) were added to the individual wells. The samples were cultured at 371 in a humidied atmosphere for 1624 h before collection of the plasma super-

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140 was a wide spectrum of lung diseases in the nontuberculosis group with about half being infective.
Table 1 Demographic characteristics of patients with culture-proven tuberculosis (TBCP) and nontuberculosis lung disease (NTBLD). Characteristics Sex Male Female Age p30 3060 460 Mean7SD Country of birth Australia Other TBCP n 129 65 (50%) 64 (50%) 38 (29%) 72 (56%) 19 (15%) 41.8716.7 5 (4%) 124 (96%) NTBLD n 100 56 (56%) 44 (44%) 10 (10%) 48 (48%) 42 (42%) 55.6717.8 57 (57%) 43 (43%) 78 (78%) 19 (19%) 3 (3%)

W.J. Britton et al.

HGIA and TST responses

The distribution of tuberculin reactions in TB patients showed a broad peak between 10 and 25 mm, while in the NTBLD patients the pattern differed with the majority of subjects being zero and a minority with reactions X10 mm (Fig. 1). By contrast, the IFN-g responses to human PPD were more bimodal in both groups (Fig. 1). There was a moderate correlation between TST (mm) and specic IFN-g released, expressed as % Human Responses (Spearman r s 0:59; po0:0001). The sensitivity of the IFN-g response to human PPD was compared at various levels of the % Human Response in the HGIA assay, maintaining the % Avium Difference at less than 10%. At the recommended ratio of HuPPD to mitogen-induced IFN-g responses of 415%, 81% of TB patients were positive in HGIA with 27% of the NTBLD subjects also positive (Table 2A). The sensitivity of TST was compared at three levels of positivity (Table 2B). TST responses X10 mm were associated with a sensitivity of 89% for diagnosis of proven TB disease and identied 27% of the NTBLD subjects as positive. When the subjects with positive responses in either assay were combined, 124/129 (96%) of the TB patients were detected. Using the same combination of positive TST and HGIA, 37% of the NTBLD subjects were positive to both or either of the tests.
100

TB risk in country of birth* Low 18 (14%) Medium 76 (59%) High 32 (27%)

*

The risk for tuberculosis was based on the prevalence rates of tuberculosis in various countries as published by the World Health Organization.34 Countries were categorized according to their TB prevalence rate as Low, Medium or High risk based on incidence rates of o25, 25100 or 4100 per 100,000, respectively.

100

(A)
80 60 40 Frequency of response 20 0 0 60 1-4 5-9 10-14 15-19 20-24 25-29 30-34 35+

(C)
80 60 40 20 0 0 60 1-4 5-9 10-14 15-19 20-24 25-29 30-34 35+

TB-CP TST

TB-CP HGIA

(B) NTB-LD TST

(D) NTB-LD HGIA

40

40

20

20

0 0 1-4 5-9 10-14 15-19 20-24 25-29 30-34 35+ Tuberculin Skin Test Reaction (mm)

0 0 1-4 5-9 10-14 15-19 20-24 25-29 30-34 35+ HGIA (%HuPPD Response)

Figure 1 The distribution of Tuberculin skin test (TST) responses (mm) in culture-proven TB patients (TBCP) (Panel A) and non-TB lung disease patients (NTBLD) (Panel B) and of human IFN-g responses to PPD (% HuPPD Response) in TBCP (Panel C) and NTBLD patients (Panel D).

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Sensitivity of human gamma interferon assay and tuberculin skin testing 141

Table 2 Proportions of patients with culture-proven TB positive in the human gamma interferon assay (A) and with tuberculin skin testing (B), and proportions of patients with non-TB lung disease negative to the two tests at the same levels of response. (A) Human gamma-interferon assay % HuPPD response TBCP patients test positive* (%) 40 45 410 415 420 425 430 (B) Tuberculin skin test TST response (mm) X5 X10 X15
* y

NTBLD patients test negativey (%) 54 63 70 73 76 76 77 (54%) (63%) (70%) (73%) (76%) (76%) (77%)

117 111 108 105 102 99 94

(91%) (86%) (84%) (81%) (79%) (77%) (73%)

TBCP patients test positive* (%) 123 (95%) 115 (89%) 90 (70%)

NTBLD patients test negativey (%) 66 (66%) 73 (73%) 83 (86%)

Number and proportion of TBCP patients (n 129) test positive. Number and proportion of NTBLD patients (n 100) test negative.

Table 3 The agreement between TST and HGIA results using HGIA response to human PPD 415% and various levels of the TST for patients with culture positive TB and non-TB lung disease. Group HGIA (X15%) Tuberculin skin test X5 mm + TBCP TBCP NTBLD NTBLD + + 103 20 19 15 2 4 8 58 X10 mm + 96 19 17 10 9 5 10 63 X15 mm + 76 14 12 2 29 10 15 71

The level of agreement between HGIA and TST was examined using the HGIA response 415% as positive and three levels of tuberculin reactions for both TB patients and non-TB subjects (Table 3). When the % human response in the HGIA at 415% and the TST at X10 mm were compared, the level of agreement of the assays was 78.3%. There was a trend for the sensitivity of TST (89%) to be higher than for HGIA (81%) (w2 3:57; p 0:06). There was no difference in the proportion of NTBLD subjects positive in either assay (w2 0:05; p 0:82). When comparing the HGIA at 415% with the TST at X15 mm, the sensitivity of the HGIA (81%) was signicantly higher than for the TST (70%) (w2 4:56; p 0:03), but more NTBLD subjects were HGIA positive (27%) than TST positive (14%) at this level (w2 8:47; p 0:004).

Effect of previous BCG immunization on HGIA and TST reactivity

BCG status was determined by the presence or absence of a BCG scar for 90 of the TBCP patients and 73 of NTBLD subjects. Of these TB patients 46/ 90 (51%) had a BCG scar compared to 33/73 (45%) of the NTBLD patients. The HGIA and TST responses were compared in BCG positive and negative subjects (Table 4). At a TST response X10 mm, the sensitivity of TST in BCG-positive patients was 87%, while the sensitivity for the HGIA was also 87% (w2 0; p 1:0). In BCG-negative TB patients, the sensitivity for TST X10 mm was 91% and for the HGIA was 77% (w2 3:0; p 0:08). When TBCP patients with either or both assays positive were combined, the proportion positive was 96% in those

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142 W.J. Britton et al.

Table 4 The inuence of prior BCG immunization, as determined by the presence or absence of a BCG scar, on the sensitivity of the HGIA (415% to HuPPD) and TST (X10 mm) tests in culture-positive TB patients and on the proportions of non-tuberculosis lung disease patients test negative. Group TBCP TBCP NTBLD NTBLD
* y

BCG scar (n) Yes (46) No (44) Yes (33) No (40)

HGIA positive 40 35 9 7

% TBCP positive* 87 77

% TBCP negativey

TST positive 40 40 10 7

% TBCP positive* 87 91

% NTBLD negativey

73 83

70 83

Proportion of TBCP patients with and without BCG scar with a positive result in HGIA or TST. Proportion of NTBLD patients with and without BCG scar with a negative result in HGIA or TST.

with a BCG scar and 98% in those without a scar. Therefore, prior BCG immunization did not appear to affect the sensitivity of TST or HGIA for detecting M. tuberculosis infection in these TB patients. The effect of BCG immunization on the responses in the two assays was also examined in NTBLD patients (Table 4). In BCG recipients, 70% and 73% of this group were TST or HGIA negative respectively (w2 0:14; p 0:71). Of the NTBLD patients without a BCG scar, 83% were negative for both assays. The proportion of the NTBLD group negative to both tests was 61% in BCG recipients and 75% in those without a BCG scar.

Pulmonary and non-pulmonary tuberculosis

There were no signicant differences in the sensitivity of the HGIA in pulmonary (80%, n 76) and non-pulmonary (83%, n 53) TB or of the sensitivity of TST (X10 mm) in pulmonary (93%) and non-pulmonary (83%) TB.

Length of treatment and reactivity

The length of treatment appeared to affect the sensitivity of the assays in the TB patients. For those treated for less than 2 months, the sensitivities of the HGIA (84%) and TST (84%) were the same; but for those treated more than 2 months, the sensitivity of HGIA was 81% and TST 90% (p 0:06).

Discussion
Both the HGIA and TST recognize T cell responses to mycobacterial antigens and, as such, are indirect measures of infection with M. tuberculosis. One difculty in comparing the sensitivities of the two assays for detecting this is determining the gold

standard for M. tuberculosis infection. In this study, we utilized patients with culture proven active TB as a population to compare the sensitivities of these assays. The in vitro IFN-g response to PPD, corrected for response to mitogen, had a sensitivity of 81% for detecting culture-proven infection with M. tuberculosis, as compared to sensitivity of 89% for a TST result X10 mm. This appeared inuenced by therapy as the sensitivities for HGIA and TST were similar (84%) in recently diagnosed patients, but tended to be higher for TST (90%) than HGIA (81%) in those treated for more than 2 months. This is consistent with the previously observed sensitivity of 90% for the HGIA to detect TST positivity in healthy subjects with past exposure to M. tuberculosis.11 The rate of positivity for the HGIA and TST were the same in these particular control subjects, who had lung disease from causes other than TB. The overlap between the TST- and HGIA-positive TB patients was not complete, so that if positive responses in either assay were combined 96% of TB patients were detected (Table 3). There were no signicant differences in the sensitivity of either the TST or HGIA between patients with pulmonary and non-pulmonary TB. Older studies prior to widespread HIV infection estimated the sensitivity of TST in patients with active TB to range from 70% to 91%.1214 There are difculties in comparing the various studies because of differences in patient selection and the doses and types of Tuberculin used,3 however clearly a subset of patients with untreated TB are TST negative. For example, 25% of 220 patients with pulmonary TB were negative (o10 mm) when tested with 5 U of Tuberculin, and 39% of the negative responders remained negative when tested with a higher dose of 250 U.14 Three factors may contribute to reduced TST or IFN-g responses in active TB. First, medical factors, such as protein malnutrition, other infections and corticosteroid or immuno-suppressive therapy, may cause transient or permanent anergy.2 Patients

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Sensitivity of human gamma interferon assay and tuberculin skin testing receiving corticosteroids or immuno-suppressive drugs were not included in this study. The most important confounder is co-infection with HIV.15 In the current study HIV was excluded and did not impact on the sensitivity of the HGIA or TST. In a separate study of intravenous drug users, about half of whom were HIV seropositive, the HGIA was more sensitive than TST for detecting reactivity to PPD.16 The second factor inhibiting T cell responses is active TB itself, which is associated with a temporary immuno-suppressive effect.17 CD4+ T cell numbers in the peripheral blood are reduced in patients with active TB18 and in vitro proliferative responses of T cells to mitogens or PPD are less than in matched TST positive contacts.17 This effect is probably mediated by cytokines, such as transforming growth factor-b19 and may be induced by mycobacterial cell wall components including lipo-arabinomannan.20 Reduction in bacterial load with therapy may lead to the re-emergence of skin test responses.5 In this study there was a trend for TST responses to improve more than HGIA responses with treatment, as was also reported by Mazurek and colleagues.21 This is consistent with the observation that M. tuberculosis specic IFN-g responses may remain low or absent for a year in some TB patients following successful completion of therapy,22 while TST responses are restored more rapidly.23 The third factor is error in the intradermal administration of the Tuberculin and reading of the skin test.4 This is unlikely to be a signicant problem in the current study as the skin testing was conducted by experienced specialist nurses within the community TB control program, however it is an important issue in the general use of TST. The sensitivity of the HGIA was examined at various cut-off levels (Table 2). Analysis of receiver operator curves for the sensitivity and specicity of the HGIA in over 900 TST positive subjects without active TB and in TST negative subjects without exposure to TB demonstrated that the IFN-g response to PPD at the 15% level provided optimal discrimination.11 Although the majority of patients were positive in both assays, there were small proportions reactive in only one assay (Table 3). Therefore, when results from both assays were combined, the sensitivity for detecting M. tuberculosis infection was increased to 96%. In bovine TB the sensitivity for detecting microbiologically proven TB was also increased by combining both assays.8 The rate of positivity of the HGIA and TST in the population of non-TB pulmonary patients was relatively high, presumably because of their previous exposure to M. tuberculosis. Nearly half of the NTBLD group were born outside Australia, and 50% of these in countries of high to medium TB prevalence 143

rates (Table 1). This rate of positivity compares with the specicity of the HGIA in individuals with no known exposure to TB of 97.6%.11 In a recent multi-center study of subjects at risk of LTBI and active TB, sponsored by the Centers for Disease Control and Prevention (CDC),21 the level of agreement between the HGIA and TST was 83.5% as compared to the 78.3% in this study. This degree of concordance may vary depending on the population studied. In a separate evaluation of patients with possible active TB,24 the sensitivity of the HGIA for conrmed TB was 77%, and the agreement with TST was 86% for TST positive patients and 81% for TST negative patients. There was a moderate correlation between the extent of induration of the TST and the IFN-g level in the current (r s 0:59) and other studies.24 The frequency distribution for the individual HGIA and TST responses differed. The pattern of TST responses in TB patients resembled a normal distribution around a mean of 1519 mm, similar to that observed for Mantoux testing in other studies. By contrast, the distribution of HGIA responses in TB patients was less uniform in distribution with a strong discrimination between positive and negative responses (Fig. 1). The biological determinants of both assays, although similar are not equivalent, as illustrated by the retention of delayed type hypersensitivity (DTH) to PPD in IFN-g gene knock out mice.25 The source of IFN-g is largely antigenspecic activated/memory CD4+ T cells, although CD8+ T cells may also respond with rapid IFN-g responses.26 The major application of both the TST and the HGIA is not in the diagnosis of active TB, but in the recognition of LTBI during contact tracing or the surveillance of people deemed at high risk for TB exposure, such as healthcare workers. In this study HGIA had a slightly reduced sensitivity compared to TST in patients with active TB, but this difference may be attributable to effects of anti-TB chemotherapy as there was no difference in sensitivity for patients who had received less than 2 months of treatment. In a study of intravenous drug users HGIA was found to be more sensitive than TST.18 Advantages of HGIA over TST are that it can be conducted at a single visit and the results are rapidly available within 24 h if necessary. The problem of skin tested subjects returning for reading is highlighted by one community study in which 60% of children screened for TB infection failed to return for reading.27 The reliability of the HGIA can be ensured by standardization of the IFN-g ELISA and incorporation of a mitogen as a positive control to detect subjects with cellular unresponsiveness. By contrast, the measurement of skin test

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144 induration in humans is subject to considerable variability and requires experienced personnel for the delivery and reading of the test. Intra-observer and inter-observer variation in reading induration diameter results in 5% of readings being 2.73.7 mm greater or less than the rst reading, sufcient to affect the clinical interpretation of the result.4 Disadvantages of the HGIA may be the higher costs of the assay and the laboratory requirements, however the total cost of TST screening itself may be underestimated.28 Prior exposure to non-tuberculous mycobacteria may reduce the specicity of the TST for detecting LTBI, particularly for reactions from 5 to 14 mm.29 An advantage of the HGIA is that it allows the testing of specic responses to different mycobacteria. For example, children with M. avium infection have stronger IFN-g responses to M. avium soluble antigens rather than M. tuberculosis, consistent with M. avium being the causative organism.30 In the CDC study, 21% of subjects with a positive TST to PPD and negative HGIA responded to M. avium antigens, suggesting the skin test reactivity was detecting infection with non-tuberculous mycobacteria in these subjects.12 Some major protein antigens are restricted to virulent M. tuberculosis and M. bovis and absent from the vaccine strain BCG,31 and cellular immune responses to these develop during infection with virulent strains. Inclusion of M. tuberculosis-specic antigens, such as ESAT-6 and CFP-10 in the HGIA improved the discrimination between M. tuberculosis-infected subjects and recipients of BCG vaccination,3233 although responses to these antigens were also detected in some non-tuberculous mycobacterial infections. The clinical utility of the HGIA with ESAT-6 and CFP-10 is being assessed. Recently published guidelines for the utilization of HGIA33 recommend its use for screening those at risk for LTBI, but not as yet in the evaluation of subjects with suspected active TB or contacts of infectious TB patients, because of the lack of data comparing the HGIA and TST in subjects with active TB. This study demonstrates that there is no signicant difference in the sensitivity of the HGIA and TST for detecting active TB, especially in patients with limited or no treatment, and suggests that the HGIA may be a suitable alternative to the TST in these situations. W.J. Britton et al. Hoy, Faireld; Associate Professor J. Turnbridge, Monash Medical Centre; Dr. H. Tiechtahl, Western; Dr. D. Hart, St Vincents; Dr. D. Spelman, Alfred; and Dr. A. Street, Royal Melbourne Hospital, Melbourne, Victoria; and Dr. C. Mukerjee, Dr. G. Marks, Sr. S. Simpson, Liverpool Hospital; Dr. P. Corte, Dr. H. Goldberg, Royal Prince Alfred; and staff at Westmead Hospital, Sydney, NSW. We are grateful to D. Dickeson, C. Feng and K. Dickson for technical assistance. The study was supported nancially by CSL Limited. Conict of Interest Statement: J.S.R, S.L.J. and P.B. were employees of CSL Limited when this study was conducted. J.S.R. is now an employee of Cellestis Ltd. who produce the QuantiFerons assay.

References
1. Dye C, Sheele S, Dolin P, Pathania V, Raviglione MC. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring project. J Am Med Assoc 1999;282:67786. 2. Britton WJ, Garsia RJ. Mycobacterial infections. In: Bradley J, McCluskey J editors. Clinical immunology. Oxford: Oxford University Press; 1997. p. 48398. 3. Snider DEJ. The tuberculin skin test. Am Rev Respir Dis 1982;125(3, Pt 2):10818. 4. Pouchot J, Grasland A, Collet C, Coste J, Esdaile JM, Vinceneux P. Reliability of tuberculin skin test measurement. Ann Intern Med 1997;1126:2104. 5. Dunlap NE, Briles DE. Immunology of tuberculosis. Med Clin North Am 1993;77:123551. 6. Bothamley GH. Serological diagnosis of tuberculosis. Eur Respir J 1995;20:676s88s. 7. Wood PR, Corner LA, Rothel JS, et al. Field comparison of the interferon-gamma assay and the intradermal tuberculin test for the diagnosis of bovine tuberculosis. Aust Vet J 1991;68:28690. 8. Wood PR, Corner LA, Rothel JS, et al. A eld evaluation of serological and diagnostic test for bovine tuberculosis. Vet Microbiol 1992;31:719. 9. Desem N, Jones S. Development of a human interferon-g enzyme immunoassay and its comparison with tuberculin skin testing for the detection of Mycobacterium tuberculosis infection. Clin Diagn Lab Immunol 1998;5:5315. 10. Weir RE, Morgan AR, Britton WJ, Butlin CR, Dockrell HM. Development of a whole blood assay to measure T cell responses to leprosy: a new tool for immuno-epidemiological eld studies of leprosy immunity. J Immunol Methods 1994;176:93101. 11. Streeton J, Desem N, Jones S. Sensitivity and specicity of a gamma interferon blood test for tuberculosis infection. Int J Tuberc Lung Dis 1998;2:44350. 12. Holden M, Dubin MR, Diamond PH. Frequency of negative intermediate strength tuberculin sensitivity in patients with active tuberculosis. N Engl J Med 1971;285:15069. 13. Marmorstein BL, Scheinhorn DJ. The role of nontuberculous mycobacterial skin test antigens in the diagnosis of mycobacterial infections. Chest 1975;67:3204. 14. Nash DR, Douglass JE. Anergy in active pulmonary tuberculosis. A comparison between positive and negative reactors

Acknowledgement
We thank the following physicians and staff at these hospitals for recruiting patients for the study: Dr. J.

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Sensitivity of human gamma interferon assay and tuberculin skin testing
and an evaluation of 5 and 250 TU skin test doses. Chest 1980;77:327. Huebner RE, Villarno ME, Snider DE. Tuberculin skin testing and the HIV epidemic. J Am Med Assoc 1992;267:40910. Converse P, Jones SL, Astemborski J, Vlahov D, Graham N. Comparison of tuberculin interferon-g assay with tuberculin skin test in high risk adults: effect of human immunodeciency virus infection. J Infect Dis 1997;176:14450. Toossi Z, Ellner JJ. Mechanisms of anergy in tuberculosis. Curr Top Microbiol Immunol 1996;215:22138. Saenghirunvattana S. CD4+ T counts with a course of antituberculous therapy in healthy and HIV-infected patients. J Med Assoc Thailand 1996;79:2468. Hirsch CS, Hussain R, Toossi Z, Dawood G, Shahid F, Ellner JJ. Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production. Proc Natl Acad Sci USA 1996;93:31938. Dahl KE, Shiratsuchi H, Hamilton BD, Ellner JJ, Toossi Z. Selective induction of transforming growth factor beta in human monocytes by lipoarabinomannan of Mycobacterium tuberculosis. Infect Immun 1996;64:399405. Mazurek GH, LoBue PA, Daley CL, Bernardo J, Lardizabal AA, Bishai WR, Iademarco MF, Rothel JS. Comparison of a wholeblood interferon gamma assay with tuberculin skin testing for detecting latent Mycobacterium tuberculosis infection. J Am Med Assoc 2001;286:17407. Ellner JE. The immune response in human tuberculosis: implications for tuberculosis control. J Infect Dis 1997;176:13519. Rooney JJ, Crocco JA, Kramos, Lyons H. Further observations on tuberculin reaction in active tuberculosis. Am J Med 1976;60:51722. Pottumarthy S, Morris AJ, Harrison AC, Wells VC. Evaluation of the tuberculin gamma interferon assay: potential to replace the Mantoux skin test. J Clin Microbiol 1999;37:322932. Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon-g in resistance to

145

15. 16.

26.

27.

17. 18.

28.

19.

29.

20.

30.

21.

31.

22.

32.

23.

33.

24.

34.

25.

Mycobacterial tuberculosis infection. J Exp Med 1993;178:224954. Lalvani A, Brookes RSH, Britton WJ, Hill AVS, McMichael AJ. Rapid effector function in CD8+ memory T-cells. J Exp Med 1997;186:85965. Serwint JR, Hall BS, Baldwin RM, Virden JM. Outcomes of annual tuberculosis screening by Mantoux test in children considered to be at high risk: results from one urban clinic. Pediatrics 1997;99:52933. Lambert L, Rajbhandary S, Qualls N, Budnick L, Catanzaro A, Cook S, Daniel-Cuevas L, Garber E, Reves R. Costs of implementing and maintaining a tuberculin skin test program in hospitals and health departments. Infect Control Hosp Epidemiol 2003;24:814. von Reyn CF, Horsburgh CR, Olivier KN, et al. Skin test reactions to Mycobacterium tuberculosis puried protein derivative and Mycobacterium avium sensitin among health care workers and medical students in the United States. Int J Tuberc Lung Dis 2001;5:11228. Davidson PM, Creati L, Wood PR, Roberton DM, Hosking CS. Lymphocyte production of gamma-interferon as a test for non-tuberculous mycobacterial lymphadenitis in childhood. Eur J Ped 1993;152:315. Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 1996;178:127482. Johnson PDR, Stuart R, Grayson ML, Olden D, Clancy A, Ravn P, Andersen P, Britton WJ. Tuberculin-PPD, MPT-64 and ESAT6 stimulated gamma interferon responses for the diagnosis of tuberculosis infection in humans. Clin Diagn Lab Immunol 1999;6:9347. Brock I, Munk ME, Kok-Jensen A, Andersen P. Performance of whole blood IFN-gamma test for tuberculosis diagnosis based on PPD or the specic antigens ESAT-6 and CFP-10. Int J Tuberc Lung Dis 2001;5:4627. Mazurek GH, Villarino ME. Guidelines for using the QuantiFERON-TB test for diagnosing latent Mycobacterium tuberculosis infection. MMWR Recomm Rep 2003;52(RR-2):158.