Usefulness of interferon-gamma release assays for diagnosing TB infection and problems with these assays

J Infect Chemother (2009) 15:143–155 DOI 10.1007/s10156-009-0686-8

© Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2009

REVIEW ARTICLE

Toru Mori

Usefulness of interferon-gamma release assays for diagnosing TB infection and problems with these assays

Received: February 16, 2009

Abstract The specificity of the tuberculin skin test (TST) in the diagnosis of tuberculosis infection is seriously compromised because of extensive use of the bacille CalmetteGuérin (BCG) vaccination. The interferon-gamma release assay (IGRA), a new diagnostic using Mycobacterium tuberculosis-specific antigens has been introduced in response to these needs. In this review, published findings on the performance of the QuantiFERON-TB (QFT), one of the IGRA formats, are summarized and discussed. In addition to its high specificity, the QFT has considerably high sensitivity, comparable with or superior to that of the TST, if applied to patients with active tuberculosis as a surrogate of latent tuberculosis infection. When applied to patients with immunosuppression, such as aging patients, or those with HIV infection, those with immunosuppressive drug therapies, or those with renal hemodialysis, QFT is shown to be more robust than the TST. As regards the dynamics of QFT responses to chemotherapy, there are many reports showing a decrease in responses during the treatment, which indicates the possibility that QFT could be used as a tool for monitoring the progress of treatment. However, there are discordant reports that warrant further study.

specificity of the TST is seriously compromised, and its usefulness is limited. The importance of contact investigation and treatment of latent TB infection (LTBI) has been emerging; moreover, the need for accurate diagnosis of TB infection is growing, especially for monitoring and infection control in TB prevention among healthcare workers. A new diagnostic tool based on the quantification of interferongamma, the interferon-gamma release assay (IGRA), has been introduced in response to these needs. Currently, two IGRA formats have been officially approved in many countries. These are the QuantiFERON-TB Gold (QFT-G; Cellestis, Carnegie, Australia) and the T-Spot-TB (Oxford Immunotec, Oxford, UK) diagnostics. This review summarizes the findings regarding some important aspects of the diagnostics and discusses how successfully these methods are responding to expectations, and which questions must still be addressed. Because the experience of the author is limited to the use of the QFT-G (i.e., a whole blood interferon gamma enzyme-linked immunosorbent assay [ELISA]), findings from studies using mainly this format are included for review.

Diagnostic performance Key words Tuberculosis · Interferon-gamma · Diagnostics · QuantiFERON

Introduction Since its discovery by Robert Koch in the late nineteenth century, the tuberculin skin test (TST) has been exclusively employed as a tool for the diagnosis of tuberculosis (TB) infection. However, in Japan, where the bacille CalmetteGuérin (BCG) vaccination has been used extensively, the T. Mori (*) Research Institute of Tuberculosis, Japan Anti-Tuberculosis Association, 3-1-24 Matsuyama, Kiyose, Tokyo 204-8533, Japan Tel. +81-42-493-5711; Fax +81-42-492-4600 e-mail: [email protected]

Sensitivity While the IGRA aims to diagnose LTBI, no gold standard of LTBI has been established. Active TB, typically confirmed primarily by bacteriology, is widely employed as a surrogate of LTBI. Out of a collection of numerous reports, Pai and O’Brien1 recently selected reports of observation of patients with considerably homogeneous characteristics for a meta-analysis. With slight modification and the addition of new reports to this analysis, this review summarizes observations of the performance of the QFT-G and the QFT-G in Tube (QFT-GIT; Cellestis. The synthesis analysis was completed with the use of Metadisc software.2 A total of 27 reports are included, involving 503 TB patients, most of whom were adults with bacteriological confirmation. Most reports are from non-TB-endemic

144 Sensitivity (95% CI)

Fig 1. Sensitivity of QuantiFERON-TB Gold (Cellestis, Carnegie, Australia) (QFT-G) in adult tuberculosis (TB) patients. CI, Confidence interval

Katiyar et al. 2008 (3) Mori et al. 2004 (4) Kang et al. 2007 (5) Kobashi et al. 2006 (6) Ravn et al. 2005 (7) Kobashi et al. 2008(8) Goletti et al. 2006 (9) Harada et al. 2008 (10) Bartu et al. 2008 (11) Kobashi et al. 2008 (12) Kobashi et al. 2008 (13) Soysal et al. 2008 (14) Nishimura et al. 2008 (15) Bua et al. 2007 (16) Kang et al. 2005 (17) Aoki et al. 2006 (18) Pai et al. 2007 (19) Ferrara et al. 2006 (20) Lee et al. 2006 (21) Mazurek et al. 2007 (22) Dewan et al. 2007 (23)

0

0.2

0.4 0.6 Sensitivity

0.8

0.95 0.88 0.87 0.86 0.85 0.85 0.83 0.81 0.81 0.81 0.79 0.77 0.77 0.77 0.76 0.76 0.73 0.71 0.70 0.67 0.62

(0.87 - 0.99) (0.81 - 0.93) (0.76 - 0.94) (0.73 - 0.94) (0.72 - 0.94) (0.77 - 0.90) (0.61 - 0.95) (0.72 - 0.89) (0.68 - 0.91) (0.67 - 0.91) (0.59 - 0.92) (0.68 - 0.85) (0.66 - 0.86) (0.58 - 0.90) (0.63 - 0.86) (0.62 - 0.87) (0.60 - 0.84) (0.49 - 0.87) (0.59 - 0.79) (0.54 - 0.78) (0.45 - 0.78)

Pooled Sensitivity = 0.80 (0.78 to 0.82) Chi-square = 48.53; df = 20 (p = 0.0004) 1 Inconsistency (I-square) = 58.8 %

Sensitivity (95% CI)

Fig 2. Sensitivity of QuantiFERON-TB Gold inTube (Cellestis) (QFT-GI-T) in adult TB patients

Harad et al.. 2008 (10) Palazzo et al.. 2008 (24) Dominguez et al.. 2008 (25) Pai et al.. 2007 (26) Tsiouris et al.. 2006 (27) Adetifa et al.. 2007 (28)

0

0.2

0.4 0.6 Sensitivity

countries, and the proportion of HIV-infected patients is not high. A forest plot of the QFT-G tests 21 reports3–23 is presented in Fig. 1. The range of observed sensitivity was 62% to 95%, the pooled value was 80%, and the 95% confidence interval (CI) was 78% to 82%. The lowest sensitivity was reported by Dewan et al.23 from the United States. They observed bacteriologically confirmed TB patients, 8% of whom were HIV-positive. No other particular factor to lower sensitivity was identified. The same is true for the report by Mazurek et al.,22 who reported the next lowest sensitivity. In their report, the only significant factor associated with lower test positivity was being Hispanic, and the related meaning of that factor is obscure. Figure 2 summarizes six reports10,24–28 of the QFT-GIT, a new system of the QFT-G using a third antigen, TB7.7, in addition to ESAT-6 and CFP-10. The range of sensitivity was 64% to 93%; the pooled sensitivity was 74%, with a 95% CI of 69% to 78%. It appears that the QFT-G is more sensitive than QFT-GIT, possibly due to the difference in patient characteristics observed for each format of the test.

0.8

0.93 0.82 0.79 0.73 0.65 0.64

(0.85 - 0.97) (0.57 - 0.96) (0.63 - 0.90) (0.60 - 0.84) (0.57 - 0.72) (0.52 - 0.75)

Pooled Sensitivity = 0.74 (0.69 to 0.78) Chi-square = 32.37; df = 5 (p = 0.0000) 1 Inconsistency (I-square) = 84.6 %

According to Harada et al.,10 who made a head-to-head comparison of the two tests, the sensitivity of QFT-GIT was 93%, while that of QFT-G was 83%, the latter being significantly lower at P = 0 0.006. This result is plausible, given that the additional Mycobacterium TB-specific antigen TB7.7 is included in the QFT-GIT system. From the standpoint of performance in LTBI diagnosis, a comparison of the TST with the purified protein derivative (PPD) may be very relevant, because the TST has an almost established status for that purpose. A head-to-head comparison between QFT-G and TST by Mori et al.4 revealed QFT-G to be more sensitive than TST (91% vs 63%; P = 0.00). Many other studies report similar results. As a whole, Pai and O’Brien1 reported a pooled sensitivity of 77%, with a 95% CI of 71% to 82% for TST. However, this comparison should be interpreted with care, and it is difficult to expand it to LTBI, because IGRA seems to be more robust to immunosuppression than the TST, which is likely to have been used to varying extents in patients with active TB but is less likely to have been used in LTBI subjects.

145 Specificity (95% CI)

Fig. 3. Specificity of QFT-G (either QFT-G or QFT-GIT, mostly in adult TB patients)

Taggart et al.. 2006 (30) Kobashi et al.. 2006 (6) Bua et al.. 2007 (16) Palazzo et al.. 2008 (24) Mazurek et al.. 2007 (31) Soborg et al.. 2007 (33) Mori et al.. 2004 (4) Ravn et al.. 2005 (7) Franken et al.. 2007 (32) Kang et al.. 2005 (17) Brock et al.. 2001 (29) Lee et al.. 2006 (21)

0

0.2

0.4 0.6 Specificity

Specificity Figure 3 presents the results of a synthesis of reports on QFT-G and QFT-GI-T.4,6,7,16,17,21,24,29,30–33 The range of observed specificity was 92% to 100% and the pooled specificity was 98% with a 95% CI of 97% to 99%. Needless to say, specificity must be based on the observation of infection-free subjects. Thus, the study subjects in these observations were healthy adults with no known exposure to infection, which, however, may have allowed contamination with infected subjects to some extent. This is especially true in Korea17,21 and Japan,4,6 which have an intermediate burden of TB. The report of Lee et al.,21 which had a relatively low specificity, involved medical students in Korea. A specificity figure based on TST-negative subjects was adopted, while the specificity for all subjects was still lower, at 89%. However, the report of Brock et al.29 included 19 BCG-vaccinated subjects, of whom 2 were QFT-G-positive reactors. If the BCG-vaccinated subjects were excluded, the specificity would be calculated as 100%. In their meta-analysis, Pai and O’Brien1 found that the specificity of QFT-G and QFT-GIT was slightly higher in subjects with a BCG vaccination history, so that the pooled specificity was 96%, compared with 99% in nonvaccinated subjects. However, it is possible that this is because a BCG vaccination history is more common in areas with higher TB prevalence; hence, there is a higher chance of infection. The pooled specificity of the TST was as high as 97% in populations not vaccinated with BCG, while it was very low (59%) in vaccinated populations (Pai and O’Brien).1 Thus, as applied to general populations with a mix of vaccinated and unvaccinated individuals, the specificity of the TST may be variable. For example, in Japan, where BCG vaccination has been extensively practiced (e.g., repeated revaccination accompanied by TST), the specificity of the TST is still lower than 14% (Mori et al.).4 Apart from the observation of patients with pulmonary TB, Song et al.34 applied QFT-G to extrapulmonary cases, where 43% were confirmed by bacteriology or histology. The QFT-positive rate was 72% (31/43), with a 95% CI of

0.8

1.00 1.00 1.00 1.00 1.00 0.99 0.98 0.97 0.97 0.96 0.94 0.92

(0.96 - 1.00) (0.89 - 1.00) (0.79 - 1.00) (0.75 - 1.00) (0.99 - 1.00) (0.95 - 1.00) (0.95 - 0.99) (0.87 - 1.00) (0.93 - 0.99) (0.90 - 0.99) (0.80 - 0.99) (0.85 - 0.97)

Pooled Specificity = 0.98 (0.97 to 0.99) Chi-square = 35.17; df = 11 (p = 0.0002) 1 Inconsistency (I-square) = 68.7 %

56% to 85%. The rate was 94% (17/18; 95% CI, 73% to 100%) for lymphadenitis, but it was as low as 45% (5/11;, 17% to 77%) for bone TB. From Japan, Ariga et al.35 reported the use of QFT-G applied to TB serosites, including pleurisy, peritonitis, and pericarditis. The QFT-positive rate of 28 patients whose diagnosis of TB origin was confirmed bacteriologically was 77.8%, with a 95% CI of 57.7% to 91.4%. Interestingly, they found that the QFT-positive rate for serositis cases of nontuberculous origin that were concurrently tested was 70.2% (95% CI, 55.1% to 82.7%), but these cases could be accurately differentiated from TB by determining the interferon-gamma response of lymphocytes in cavitary fluid to specific antigens. In order to confirm the validity of IGRAs as a diagnostic of LTBI, rather than active TB disease, no way other than the accumulation of findings exists to indicate the compatibility of the test results with situational evidence of infection, such as intensity of exposure. This type of study has already been reported by many authors with the scale of the studies varying. For example, in Germany36 a total of 1989 persons in contact with 123 index cases were tested with IGRAs (QFT-GI-T and T-Spot-TB). Of the 812 contacts with positive TST (5 mm or greater), the positive rate was 30.2% for QFT-GI-T and 28.7% for T-Spot-TB. The test results were strongly associated with exposure risk factors, including age, smear positivity of the index cases, presence of cough, cumulative exposure time, and country of birth. This study demonstrated that if subjects with positive results in both IGRAs were assumed to have true LTBI, the TSTdefined LTBI could be reduced by 74%. If a stricter cutoff of 10 mm were to be applied considering the history of BCG vaccination, the number of those diagnosed with LTBI would be reduced to 248 (31%), while 28% of true LTBI cases would be missed. An outbreak of TB in a military facility in Switzerland was reported.37 One of the soldiers in a troop was diagnosed with smear-positive TB after several months of coughing and after medical examinations. A total of 168 contacts were categorized in the order of closeness of exposure as: (a) those in the same troop sharing a dormitory

146

IGRA in children In TB in children bacteriology is not always useful for diagnosing active disease; therefore, the diagnosis of infection can play an important role. Thus, the TST has been a useful adjunct for the diagnosis of pediatric TB. IGRA use in children has been limited so far, and cellular immune responses are generally considered to be weak in children; therefore, many countries’ guidelines (e.g., those in the United States,43 the United Kingdom,44 the Netherlands,45 Switzerland,46 Canada,47 France,48 and Japan49) currently recommend that IGRAs be used very carefully in children.

Median

0.519

0.684

0

1

0.756

0.802

0.979

2

1

0

-1

-2

LOG(MN)

(sleeping facility), (b) those in the same troop not sharing a dormitory, (c) staff and patients of the hospital, and (d) soldiers in other troops. The QFT-GIT-positive rates were (a) 14/15 (93%), (b) 4/20 (20%), (c) 5/22 (22.7%), and (d) 11/111 (9.9%), correlating well with degree of exposure. Of note was the high rate of 93% in the most heavily exposed group, suggesting the possibility of high sensitivity of the test. In a Japanese university, one student developed TB, and then nine of his classmates developed the disease.38 TST and QFT-G were performed on 220 classmates of the index case, along with 240 students in other classes. Almost 100% of the tested students had been BCG-vaccinated in the past, and TST positivity was 93.2% for the contacts and 72.3% for the noncontacts. The QFT-G was positive in 33% of the contacts, and only 1% of the noncontacts, indicating a clear distinction between the two groups. The stronger the TST reaction, the higher the QFT-G positivity; however, QFTG-positives were detected also among those with weak TST reactions. In Japan, a teacher in a cram school, teaching students between the ages of 10 and 24 years, developed TB with coughing and was diagnosed after 3 months of being in contact with the children in a one-on-one teaching system.39 TB was diagnosed in 63 of the children and school staff members. The QFT-G-positive rate was 70/90 (78%) for the children in close contact, which was clearly higher than 16/32 (50%) for other contacts. The children were grouped according to frequency of contact with this teacher as: (a) more than four times a week, (b) one to three times a week, and (c) occasionally. A clear parallel was observed between this grouping and the QFT-G positivity: (a) 12/12 (100%), (b) 57/76 (75%), and (c) 9/21 (43%). All of these cases indirectly confirm that a positive QFT test reflects LTBI soon after infection, possibly with high sensitivity. However, the validity of the dichotomous theory of infection is questionable, considering the discrepancy with the TST (e.g., TST-positive and QFT-negative cases), the dynamic transition of the QFT response (see section below: “Dynamics of IGRA responses”) and the possible relationship between the intensity of the response and the risk of future clinical breakdown.40–42 This question is an important issue with IGRA that needs to be addressed in more detail in future.

-3

-4

2 Age (yo)

3

4

Fig. 4. Interferon-γ (IFN-γ) response to mitogen in children by age. Correlation r = 0.112; P = 0.10. yo, Years old

Figure 4 depicts the relationship between age and response to mitogen in QFT-G for 14 000 subjects (Harada et al., 2008 unpublished data;). The response was indeed lower in younger subjects, but not to a great extent. The frequency of those with a mitogen response lower than 5.0 IU/ml was 3.1% for children aged 0 to 1 year, 2.1% for those aged 2 to 9 years, 1.6% for those aged 10 to 14 years, and 0.8% for those aged 15 to 18 years. Connell et al.50 applied QFT-G to children younger than 18 years who had been exposed to TB infection and found that the number of children with indeterminate test results was larger among the younger subjects. Lighter et al.51 also reported that the response to mitogen depended on age, so that the interferon-gamma level for those younger than 24 months was half that of those 5 years old or older. They proposed an alternative cutoff for children younger than 2 years (e.g., 0.26 IU/ml instead of the usual 0.35 IU/ml); this modification elevated the positive rate by 17%. However, Lewinsohn et al.60 compared the responses to specific antigens between children younger than 2 years and older ones and found no difference. Several actual observational studies50,52–59 of QFT-G sensitivity in pediatric TB patients have been reported, although the scale of each study was small (Fig. 5). Unlike observational studies of adults, these studies included patients whose diagnoses were based not on bacteriology or histology, but on clinical findings; as a result, the observed sensitivity might have been lower than expected, as was reported by Okada et al.59 However, the pooled sensitivity of 82% was rather high and was comparable to that for adults. The study by Russo55 included children aged 0 to 14 years, but the sensitivity remained high enough, at 88%, even for those aged 0 to 5 years. This result is in agreement with that of a report61 of a newborn with congenital TB who was tested with QFT-G 18 days after birth and demonstrated a positive result.

147 Sensitivity (95% CI)

Fig. 5. Sensitivity of QFT (either QFT-G or QFT-G-IT) in child TB patients

Connel et al. 2006 (51) Molicotti et al. 2008 (52) Detjen et al.. 2007 (53) Connel et al. 2008 (54) Russo et al. 2007 (55) Herrmann et al. 2009 (56) Takamatsu et al. 2008 (57) Dogra et al. 2006 (58) Okada et al. 2008 (59)

0

0.2

0.4 0.6 Sensitivity

In young children, a diagnostic of infection with high sensitivity is very seriously needed to allow the early introduction of preventive therapy and careful follow up. However, the absence of a gold standard for LTBI hampers direct proof. Okada et al.59 reported that in an observational study of children (younger than 5 years) who were household contacts in Cambodia, the QFT-G correlated well with the TST (kappa = 0.674), and the QFT-G-positive rate was higher in the contacts of subjects with a higher smear positivity score. This result is similar to that in a report by Dogra et al.58 from India. Using the TST and QFT-G in TB suspects aged 1 to 12 years, they found high agreement between the two tests, and the agreement became higher when the cutoff of the TST was changed from 5 mm to 10 mm. They also found a higher positive rate for those exposed in the household than for those without exposure. Similar surveys have been conducted by Lewinsohn et al.60 (household contacts aged 5 to 14 years, Uganda), and by Tsiouris et al.63 (children aged 5 to 15 years, South Africa). In Nigeria, Nakaoka et al.63 compared TST and QFT-G findings between child (aged 14 years or younger) contacts of smear-positive subjects and contacts of smear-negative subjects, together with noncontact controls. Both the TST and QFT-G exhibited higher positivity in the contacts of smear-positive subjects than in the contacts of smearnegative subjects. Interestingly, the positive rates were higher for QFT-G than for the TST in all groups, which is a unique finding of their study, while many other studies report a higher TST-positive rate that could be ascribed to the influence of BCG vaccination history or environmental mycobacteria. Connell et al.54 tested child patients having different risks of TB infection, using the TST and QFT-G in the United States. Results indicated that QFT-G positivity correlated positively with the level of infection risk. At the same time, they noticed that there were several subjects with positive TST and negative QFT-G results, which may indicate the infection was not detected by QFT-G, and they proposed that it would be relevant to study the long-term prognosis of these subjects with discrepant results in terms of future clinical development. Lighter et al.51 also found that the QFT-GIT-positive rate in children at a hospital in New

0.8

1.00 1.00 0.93 0.89 0.89 0.78 0.77 0.63 0.53

(0.66 - 1.00) (0.72 - 1.00) (0.76 - 0.99) (0.52 - 1.00) (0.73 - 0.97) (0.60 - 0.91) (0.58 - 0.90) (0.24 - 0.91) (0.29 - 0.76)

Pooled Sensitivity = 0.82 (0.75 to 0.87) Chi-square = 23.26; df = 8 (p = 0.0030) 1 Inconsistency (I-square) = 65.6 %

York correlated well with infection-related risk factors, although some TST-positive reactors had negative QFT tests. In South Korea, where the BCG vaccination is widely used, 136 TB patient contacts aged 0 to 14 years were subdivided into three groups, according to intensity of contact: (a) close, (b) not close, and (c) no contact with subjects with positive TST, and they were administered QFT-GI-T.64 The positive rates were (a) 19%, (b) 7%, and (c) 2%, indicating a correlation with the epidemiological situation. In this study, another 91 children who were under workup with the suspicion of TB were also tested; their positive rate was 5%, and the patients who were diagnosed with TB had a positivity of 80% (4/5). Takamatsu et al.57 conducted a multicenter study in Japan on the diagnostic performance of IGRA in children. The child contacts who were judged by experienced pediatricians as very likely to have been infected with TB, based on history of exposure and TST, tested GFT-G-positive at a very low rate. Therefore, Takamatsu et al. concluded that in diagnosing TB infection in children, the TST should be prioritized. The TST is emphasized as a top priority for diagnosing TB infection, especially for children younger than 5 years, who run a higher risk of clinical development of the disease. The recommendations in many Western countries are largely in agreement with this conclusion. Regarding comparison of the performance of the TST and the QFT-G, recently the difference in qualitative aspects has been the focus, apart from the difference in sensitivity. For example, one argument contends that while QFT acts only on the effector cells in the peripheral blood, the longer incubation time for the TST would make it possible to mobilize the central memory cells, resulting in a stronger response.65 Also, the QFT involves only the Th1 system producing interferon-gamma, but in children this Th1 system is immature, while, alternatively, the Th2 system is active and could be more likely to be detected by the TST.66 Moreover, as indicated at the end of the section “Diagnostic performance”, we are left with the important question as to the difference these qualitative aspects of the infected host may make in the risk of clinical breakdown.

148

Diagnostic performance of IGRA in immunosuppressed hosts For an individual with immunosuppression and LTBI, preventive chemotherapy is recommended as treatment in many countries, for such a person runs a higher risk of clinical TB development in the future.67,68 In order to decide whether or not to treat LTBI, it is necessary to confirm the existence of infection, which has been done exclusively with the TST so far. However, existing immunosuppression interferes with the TST.69 Thus, it is important to determine the test performance of the QFT-G in subjects with such a problem. Ferrara et al.70 used the QFT-G for 318 patients with different diagnoses in a general hospital in Italy. Of these patients, 21.4% were judged as “indeterminate,” due to a low response to mitogen. The possible causes of this indeterminate result included immunosuppressive therapy (26/65), malignancy (8/41), very young (<3 years) or old (<80 years) age (11/37), HIV infection (2/7), and chronic renal failure (4/10). Kobashi et al.71 also used the TST and the QFT-G to test a total of 252 patients with immunosuppression (74 with malignancy, 72 undergoing immunosuppressive therapy, 52 with diabetes, 50 with renal failure, and 4 with HIV). The overall positive rate was significantly higher for the QFT-G (78.1%) than for the TST (50.0%). Indeterminate cases were seen in 13% (32/252), more commonly in those patients receiving immunosuppressive therapy, especially those with lymphocytopenia. Many TST-negative and QFT-positive cases were found among those with a past history of TB chemotherapy, and it was assumed that the immunocompromised subjects’ responses to QFT could be better maintained than their responses to TST. Thus, the problem of infection diagnosis is that it is disturbed by the anergy of the host. For TST, the anergy inflates the number of negative reactors; however, QFT-G can report these cases as low responders to mitogen (i.e., “indeterminate”), which is a unique strength of this test. Discussion will focus mainly on the sensitivity of the QFT-G, in comparison with the TST, in aged subjects, those with HIV infection, hosts who are immunocompromised due to other causes, and subjects undergoing hemodialysis.

Old age Mori et al.4 reported a significant decrease in the QFT-Gpositive rate in older TB patients. The positive rate of those aged 60 to 69 years was 96%; that for those aged 70 to 79 years was 91%; and that for those aged 80 years or older was 80%. However, the TST-positive rates in these groups were 72%, 67%, and 17%, respectively, and the extent of the decrease with age was more marked than that in the QFT-G. According to Harada et al. (2008, unpublished) “indeterminate” cases due to low mitogen response were seen in 1.30% of tested subjects aged 20 to 59 years, in

4.04% of those aged 60 to 79 years, and in 7.47% of those aged 80 years or older. These results suggest a nonspecific weakening of the immune capacity due to aging. Kobashi et al.8 found a QFT-G-positive rate of 77% for older TB patients (>80 years of age) and a rate of 87% for younger ones (nonsignificant difference). The TST-positive rate was 70% for older TB patients and 27% for younger ones. This significant difference indicates that the QFT-G response is less affected by aging than the TST response. The frequency of the “indeterminate” category was 17% for older patients and 9% for younger patients, but this difference was not significant. These observations assume aging to be a factor in the decrease in immune response; but LTBI involves other factors, such as a waning of response as a consequence of the time lapse after infection. Mori et al.72 observed that, in Japan, the QFT-G-positive rate was 9.8% in healthy subjects aged 60 to 69 years, while the prevalence of TB infection was estimated at 53%. This wide discrepancy could be ascribed to the waning of the QFT-G response, along with the time lapse after the establishment of infection. This result is also related to the dynamics of IGRA responses (see section: “Dynamics of IGRA responses” below); however, it is important to realize that an older subject’s QFT result is complicated by various factors.

HIV infection Raby et al.73 studied QFT-G-IT performance in TB patients in Zambia. The QFT-positive rate was 84% in HIV-negative patients, while that in HIV-positive patients was 63%. At the same time, the frequencies of indeterminate cases were 3% and 17%. When the number of CD4 cells was below 100/ mm3, the QFT-positive rate decreased to 23%, and the indeterminate cases increased to 46%. In contrast, the TST-positive rate in HIV patients was 55%, which was slightly lower than the QFT rate (P = 0.06). In their study of the QFT-GIT in bacteriologically confirmed TB cases, Vincenti et al.74 reported a positive rate of 85% (11/13), excluding anergy cases, compared to 46% for the TST. According to Nagai et al.,75 the QFT-positive rate in Japanese HIV/TB patients was 77% (10/13), and the TST-positive rate was 15% (2/13). A synthesis of the results of these studies, with the addition of the study by Ferrara et al.,70 is presented in Fig. 6. The range of reported sensitivity was 63% to 85%; the pooled sensitivity was 70% (95% CI, 60% to 79%). For TST, the pooled sensitivity was 45% (95% CI, 35% to 56%), results which also confirmed that QFT has higher sensitivity and is less affected by HIV infection than the TST. As for QFT performance on LTBI in HIV infection, Talati76 tested 706 HIV-infected subjects with the QFT-G and TST in the United States. The positive rate was 4.5% for QFT-G and 2.8% for the TST, and QFT-G detected more LTBI cases. QFT-G detected 3.9 times more indeterminate cases, with the number of CD4 cells below 200/ mm3, than cases with 200/ mm3 or more CD4 cells. Luetkemeyer et al.77 tested subjects in the HIV registry of San Francisco

149 Fig. 6. Sensitivity of interferongamma release assays (IGRAs) in HIV-infected TB patients. Tspot TB (Oxford Immunotec, Oxford, UK) was used in study 1, QFT-GI-T in study 2, and QFT-G in all the other studies

with the QFT-G and TST. The agreement of the two tests was as low as kappa = 0.37, and among those testing positive with either test, only 8/29 (28%) were positive with both tests. In New York, Jones et al.78 found that 10 (5%) of 200 HIV-infected subjects were QFT-G indeterminate, and all of them had a CD4 cell count of less than 200/mm3. The QFT-G positivity was 5.5%, and the TST positivity was 6.5%; the kappa value was low, at 0.38. The risk factors of LTBI were found to be more closely correlated with the QFT-G results than with the TST results. Having applied the QFT-G-IT to 590 HIV-infected subjects (average age of 43 years) in Denmark, Brock et al.79 found that 27 (4.6%) were positive and 78% had a risk factor (e.g., history of residing in highly TB-endemic area, history of exposure to a TB patient, or his/her own TB treatment history), and these factors correlated well with QFT positivity. They concluded that even in HIV-infected subjects, QFT could effectively indicate LTBI. In Germany, Stephan et al.80 reported a QFT-G-positive rate of 20.0%, compared with a TST-positive rate of 12.8%, with a kappa value of 0.335, among 286 HIV-infected outpatients in a university hospital. Similarly, in Chile, Balcells et al.81 found that the QFT-GIT-positive rate was 14.8% and the TST positivity rate was 10.9% in 116 HIV-infected persons; the agreement between the two tests was relatively good at kappa 0.59. They also observed that the potential LTBI risk factors (e.g., contact history and history of TB) correlated more strongly with the QFT than with the TST. Among the TST-negative reactors, 8.2% were QFT-positive, with a CD4 cell count lower than the count in those who were negative for both the TST and QFT, suggesting that the QFT was less likely than the TST to be affected by immunosuppression. Many studies have indicated that the QFT seems to be less influenced by immunosuppression due to HIV infection than the TST, but not enough information is available to explain this finding. Hammond et al.82 applied a direct ex vivo enzyme-linked immunoassay with PPD, ESAT-6, and CFP-10 to HIV-infected subjects at different stages of immunosuppression. The result was that when the CD4 cell count in the peripheral blood decreased, the interferongamma response to PPD was lowered, while no change was observed in the responses to ESAT-6 or to CFP-10. This finding warrants further studies to clarify the mechanism that causes this difference between the two tests.

Immunosuppressive therapy Some diseases require long-term administration of medications that inhibit cellular immunity. These diseases include malignancies, collagen diseases, autoimmune diseases, and nephrotic syndrome. Corticosteroids elevate TB risk.83 Recently, tumor necrosis factor (TNF)-alpha blockers have been used for rheumatic diseases, and the resulting development of TB has attracted attention.84,85 In patients treated with corticosteroids and those receiving TNF-alpha blockers, the use of preventive therapy is indicated for those with LTBI.86 Matulis et al.87 compared the QFT-GIT and TST in 142 patients being treated for chronic inflammatory diseases with immunosuppressive therapies, in terms of TB infection diagnosis. These patients had taken corticosteroids and other disease-modifying antirheumatic drugs (DMARDs) in addition to TNF-alpha blockers. Matulis et al. found that with the use of these agents, the QFT better reflected TB infection exposure than the TST, and that agreement between the QFT and TST was poor. They also found that steroids and ordinary DMARDs did not affect the QFT response, but QFT positivity did decrease with TNF-alpha blocker use. Chen et al.88 conducted a prospective study on 43 patients with rheumatic diseases who had been treated with adalimumab, and administered the TST and QFT-G. At the start of the treatment, 8 (18.6%) were TST-positive, and they were given isoniazid (INH) treatment. Of the remaining 35 negative TST reactors, 2 developed active TB soon after the start of treatment. Another 6 stopped adalimumab treatment for other reasons. Thus, 27 initially TST-negative patients completed the 12-month course of therapy. Of these subjects, 10 (37.0%) exhibited TST conversion; 2 were QFT-positive; and 1 developed TB. Of the 17 patients who remained TST-negative, 1 turned QFT-positive and later developed TB. Overall, of the initial 43 patients, 4 (9.3%) developed TB after the start of the therapy. Chen et al. concluded that the use of the TST alone for monitoring TB risk was not reliable due to anergy, and combining the TST with QFT was useful. Inflammatory bowel disease is another condition for which TNF-alpha blockers are indicated. Schoepfer et al.89 compared the QFT-GIT and TST in the monitoring of LTBI. They observed 212 subjects: 44 healthy subjects; and

150

114 with Crohn’s disease, 44 with colitis ulcerosa, and 10 with unclassified colitis. The TST-positive rate was 18% in the 168 patients and 43% in the 44 healthy controls, clearly demonstrating the inhibition of the reaction. When the study was limited to BCG-vaccinated subjects, the positive rates were 23% and 52%, respectively In contrast, the QFT positivity rates were 8% (14/168), and 9% (4/44), indicating the negligible influence of the therapy on the test. Chronic renal failure Patients undergoing hemodialysis due to chronic renal failure run a high risk of TB,90 which again requires preventive therapy for LTBI. Winthrop et al.91 tested 100 patients with end-stage renal failure who received hemodialysis in a facility in the United States. They conducted TST, QFT, and enzyme-linked immunosorbent spot (ELISPOT) assays after 1 patient developed smear-positive TB. The tested patients included those with and without contact with the index case, and many of them originated from high-TBprevalence countries. The positive rates of QFT and ELISPOT correlated well with the presence of contact with the index case, while that of the TST did not. This result suggested that the IGRAs reflected TB infections in patients with hemodialysis; therefore, they were useful for TB infection diagnosis in such patients. Because IGRA-positive and TST-negative cases were more common in contacts, Winthrop et al. assumed that immunosuppression due to renal failure influenced IGRAs less markedly than it influenced the TST.

Lee et al.92 compared the QFT-G, T-Spot-TB, and TST in 32 patients with end-stage renal failure and the same number of healthy controls, matched by age. The QFT-Gpositive rate was significantly different between patients (12.5%) and controls (40.0%). Results indicated that 41% of the patients and 16% of the controls exhibited a TST reaction with no induration; of these patients, 2 (6.3%) were indeterminate on QFT. The controls included no indeterminate cases. Again, QFT appeared more robust to immunosuppression than the TST. In this study, both the patients and controls were followed up for the subsequent 2 years, and 2 patients developed active TB.

Dynamics of IGRA responses Temporal changes in IGRA responses are important in terms of clarifying the dynamics of the cellular immunity mechanism, and for using IGRA response as a parameter of treatment progress or for evaluating the risk of the future development of active TB. Here we discuss studies of the potential value of IGRA response as a parameter of treatment progress. If the IGRA response level is assumed to reflect the current antigen load or the proliferation of bacilli in a host, then treatment with medication, or a successful immune process, can lower the IGRA response. In contrast, if the treatment fails or immunity is disrupted, the response can be restored or elevated. Some observations correspond to these hypotheses, as seen in Table 1. Ulrichs et al.,94 Al-

Table 1. Dynamics of IGRA responses Author, year

Ref. no.

Method

Treatment of active tuberculosis (TB) Ulrichs et 93 ESAT-6, lysed al., 2000 MTB, ELISPOT Lalvani et al., 2001 Pathan et al., 2001

94

ESAT-6, ELISPOT

95

ESAT-6, ELISPOT, 14-h culture

Al-Attiyah et al., 2003 Carrara et al., 2004

96

97

Various antigens, cell proliferation, IFN-gamma ESAT-6, ELISPOT

Ferrand et al., 2005

98

ESAT-6, ELISPOT

Kobashi et al., 2006

99

QFT-G

Subjects

Findings

Germany: TB patients

Comparison was done at 0–5 days and 60 days of treatment. T-cell response to ESAT-6 was elevated during treatment, suggesting improved immune capability. The response continuously decreased during 4 to 20 weeks of treatment in 5 culture-positive patients. Number of responding cells decreased in every group., down to 38% of the initial level by 34 weeks of treatment, i.e. 5.4% per week.

UK and India: TB patients UK: Adenopathy and pulmonary TB (culture-negative or -positive) Kuwait: Culturepositive patients Italy: Eighteen patients with active TB

Italy: Healthy subjects and patients with pulmonary TB (soon after start of treatment and after completion) Japan: TB patients

The pretreatment response to all antigens was low. At 2 months of treatment a significant improvement in response was seen. The response had increased further by 6 months. All patients were positive at the beginning. After 3 months 13/18 converted to negative. The conversion was seen in 13/13 of good bacteriological responders, and in 0/5 of nonresponders. The nonresponders had clinically serious disease and were malnourished from the beginning. No consistent change was seen for PPD. In 18-h culture the difference in IFN-gamma-spot-forming cell was significant between healthy and patients, and not significant between pre- and post-treatment. In 6-day culture the difference was higher in the order of post-treatment > pretreatment > healthy. QFT was positive in 86% in the beginning, with 10% indeterminate and 4% negative. The positivity decreased after the start of treatment: 69% at 4 months, 50% at 6 months, 43% at 9 months, and 33% at 12 months.

151 Table 1. Continued Author, year

Ref. no.

Method

Subjects

Findings

Aiken et al., 2006

100

ESAT-6,CFP-10 ELISPOT

Gambia: TB patients

Pai et al., 2006

101

QFT-G

India: TB patients (intermediate or severe disease)

3

QFT-G

India: TB patients

ELISPOT was positive in 82% pretreatment and 55% had converted to negative at 12 months of treatment. In cured patients 73% showed conversion, and response to CFP-10 and ESAT-6 decreased in 73% and 78%, respectively. In all of 4 noncured patients the test remained positive. The positive rate was 44/60 (73%) on diagnosis,. 38/47 (81%) after 2 months, and 31/39 (79%) at the completion of treatment. The change was variable among individuals; the trend was generally downward, but not significant. The test was done at the start, and repeated at 2 months and 6 months of treatment. The positive rate changed from 94.7% to 54.2% to 47.9%. The response declined in 77.6% of patients during the first 2 months of treatment. QFT at 2 months of treatment was a significant and independent predictor of bacteriological conversion. QFT-G positivity was 81% pretreatment, 80% at 3 months, 57% at 6 months, 47% at 9 months, and 41% at 12 months of treatment. T-Spot positivity was 88%, 85%, 62%, 55%, and 49%, respectively. Mean QFT-G response level and mean TSpot SFC count also showed similar declining trends. The QFT positive rate and mean response level continued to decline until 48 weeks of treatment. The response at 48 weeks was lower in those who showed bacteriological conversion by 55 days than in those who failed to do so. After 48 weeks the response remained constant or tended to increase slightly by 120 weeks.

Katiyar et al. 2008

Kobashi et al., 2008

12

QFT-G, T-Spot

Japan: TB patients

Takayanagi et al., 2009

18

QFT-G

Japan: TB patients

Treatment of latent TB infection Pai et al., 101 QFT–GIT 2006

India: Positive health care workers

Wilkinson et al., 2006

102

Immigrants to UK indicated for chemoprophylaxis

Ewer et al., 2006

103

ELISPOT, ESAT6, CFP-10, 38kDa antigen, alpha-crystallin, PPD ESAT-6, CFP-10, ELISPOT

Higuchi et al., 2008

104

QFT-G

Japan: Contacts positive on QFT

Franken et al., 2008

105

QFT–GIT

Holland: Contacts with positive TST

UK: Contacts

Attiyah et al.,96 and Ferrand et al.98 observed an increase in IGRA response during treatment of active TB; however, Pai et al.19 observed no significant change. Many other studies3,12,18,94,97–100 reported a general downward trend during chemotherapy. Most studies on preventive therapy or treatment of LTBI101–105 reported a decrease in the IGRA response; however, the extent of this decrease was variable. Wilkinson et al.102 reported that the IGRA response rose soon after the introduction of treatment, and then it decreased with time. The discrepancy among findings may be due to differences in incubation times of blood cells after stimulation.

After 1 year positivity was seen in 9/10, but the level was reduced. After 2 years the positivity remained at 9/10, but the response was further reduced. One patient who had a relatively low level converted to negative. All subjects had positive TST pretreatment, and positive ELISPOT to at least one RD1 antigen. The response increased transiently at 2 to 6 weeks of treatment (average, 26 ± 4 days), and declined subsequently by 82 ± 6 days. In TST-reactors the response declined continuously during and after treatment, by 18 months, at a rate of 68% per year on average. In positive reactors who were not treated, no change was seen. Of the initially positive subjects, 25% converted to negative after treatment. Other subjects also showed declining trends. The geometric mean of IFN-gamma also declined: ESAT-6, 1.398→ 0.362; CFP-10, 0.312→0.120 (unit: IU/mL). By 18 months after treatment there was no more change. There was no change in the level in 4 subjects who did not take medications. The positive rate was 50% pretreatment, 38% at 6 months, 44% at 12 months, and 41% at 18 months. Those with a higher value stayed at a higher level, those with a lower value stayed at a lower level, and those who had an intermediate value showed wide variation.

For example, Ferrand et al.98 observed the ELISA response to ESAT-6 in BCG-vaccinated healthy individuals, in patients with pulmonary TB in the early stage of treatment, and in those having completed treatment. Cell culture was performed for 18 h and for 6 days. The extent of the interferon-gamma response was in the order of healthy < early treatment < treatment completion, for both the 18-h and the 6-day cultures. However, the difference was greater (statistically significant) for the 6-day culture than for the 18-h culture. Pai et al.19 observed that, at the time of diagnosis of active TB, the QFT-G-positive rate was 44/60 (73%) and

152

then it became 38/47 (81%); at the time of treatment completion, it was 31/39 (79%). No significant change was observed in the positive rate overall, but various changes of conversion and reversion were observed individually. The average level of interferon-gamma showed a downward but insignificant trend. Aoki et al.18 closely followed the QFT-G responses of 50 bacteriologically confirmed TB patients during and after treatment. The response in terms of positive rate and average level of interferon-gamma clearly decreased until the 48th week of treatment; the positivity changed from 76% to 31%. Patients who exhibited bacteriological conversion by the end of the second month demonstrated a lower level of interferon-gamma response after 48 weeks than those who exhibited no bacteriological conversion. Also, the trend of the decrease in the interferongamma response was steeper in those who had rapid bacteriological conversion. From week 48 through week 120 of treatment, the response remained at an almost constant level or increased slightly. In this study, 2 patients experienced relapse of the disease, and their QFT-G was negative at the time of completion of first-time therapy. Katiyar et al.3 observed QFT-G responses in 76 patients with active pulmonary TB. At the start of chemotherapy, the QFT-positive rate was 94.7%; 2 months later, it was 54.2%; and 6 months after the start of chemotherapy, it was 47.9%. The response level decreased from the onset through the end of the treatment in 78% of the patients, and in the remaining 22% it remained constant. At the end of the treatment, 8 patients remained bacteriologically positive; they were all initially QFT-positive, and their response level had either increased, or decreased only slightly and remained at a considerably high level. A multivariate analysis revealed that the QFT-G result at 2 months correlated significantly with the sputum culture results. Of interest, Kobashi et al.12 observed the longitudinal course of QFT-G responses in 50 patients with bacteriologically confirmed TB. At the beginning, QFT-G was positive in 86% of the subjects (indeterminate in 10% and negative in 4%). Two months after the start of chemotherapy, the positive rate was up to 90% (similar to that reported by Wilkinson et al.102 in a study of chemoprophylaxis), and then decreased to 69% at 4 months, 50% at 5 months, 43% at 9 months, and 33% at 12 months. As for the treatment of LTBI, Higuchi et al.104 observed the contacts of a patient with nosocomial infection. The 27 patients were diagnosed with LTBI with QFB-G and completed 6-month treatment. Of these patients, 25% exhibited negative conversion, and their response level for interferongamma also decreased (mean value of ESAT-6 decreased from 1.398 to 0.362 IU/ml; mean value of CFP-10 decreased from 0.312 to 0.120 IU/ml). The follow up was extended until 18 months after the completion of treatment, and revealed that there was no longer a change in positivity. In addition, in 4 patients who did not take medication because of early side effects or refusal on other grounds, no significant change was observed in the response level after 4 months, compared with the initial QFT-G test. In contrast with these findings, Pai et al.101 from India reported the robustness of QFT-GI-T responses, after fol-

lowing up healthcare workers’ tests with the TST and QFTGI-T in chemoprophylaxis for subjects positive to either test. This observation revealed ten QFT-G-positives, and all but one had a high level of response. The positive rate remained at 9/10 from the start to the end of treatment, although the response level generally decreased. Pai et al. concluded that preventive treatment with INH may have reduced the response, but the extent of reduction was slight, indicating the robustness of the QFT response. Pai et al.101 assumed that this result was due to four reasons. (a) In such a high prevalence area as India, the QFT-response is so high that a decrease may not be noticeable and may not be sufficient to reverse the positive test. (b) Nursing students are more likely to be exposed repeatedly, and 6-month treatment is not adequate to clear the bacilli. (c) Resistance to INH is common (10%) in India. (d) As a result of reinfection and ongoing infection, some effector T cells are continuously stimulated, and the T-cell response may persist robustly, in spite of the decrease in antigen burden brought about by chemotherapy. At the same time, such confounding factors as malnutrition, infection with parasites or nontuberculous mycobacteria, and BCG vaccination must be taken into consideration. Dheda et al.106 conducted systematic discussions and experiments concerning the factors that may be related to these variable results. They assumed that disagreement among observations could be due to differences in assay methods, lack of standardization of the assays, differences in incubation times (1 day vs several days), differences in antigens (synthetic peptides vs recombinant proteins), or possible contamination with endotoxins; and they tested these possibilities with experiments. An extended (5-day) ELISPOT cell culture for 17 patients during the later period of chemotherapy indicated that IGRA did not reverse the test to negative. Assuming that the lytic activity of the ESAT-6 protein on the cell membrane may activate the receptor or the path to recognize pathogens, ESAT-6 protein was applied to IGRA-negative patients; however, it failed to elicit a positive result. Moreover, negative reactors were challenged with lipopolysaccharide-added ESAT-6/ CFP-10 peptides in ELISPOT, but even in the 5-day culture no effect of the addition was observed. However, during and after treatment, all of the IGRA-negative patients exhibited a robust antigen-specific recall response in the tritium-thymidine incorporation assay, suggesting that in this phase of the treatment non-interferon-gammaproducing memory T cells may become predominant. However, the persistence of IGRA positivity after treatment should be investigated to address the possibility of effect of an existing exogenous reinfection of TB and environmental mycobacterial infections. It is expected that such studies will provide some insight into the IGRA response in old infections or the negative response in the early stage of infection. Also, such studies may, in turn, provide an important clue to another major challenge of IGRA research, the relationship between the level of the IGRA response and the risk of future clinical development of TB, an aspect that has not been covered in this review.

153

References 20. 1. Pai M, O’Brien R. Review of T-cell–based assays for the diagnosis of latent tuberculosis infection. Ann Intern Med 2008;149:1–12. 2. MetaDiSc software, version 1.4: Hospital Ramon y Cajal, Madrid Spain 3. Katiyar SK, Sampath A, Bihari S, Mamtani M, et al. Use of the QuantiFERON-TB Gold In-Tube test to monitor treatment efficacy in active pulmonary tuberculosis. Int J Tuber Lung Dis 2008;2:1146–52. 4. Mori T, Sakatani M, Yamagishi F, Takashima T, Kawabe Y, Nagao K, et al. Specific detection of tuberculosis infection: an interferon-gamma-based assay using new antigens. Am J Respir Crit Care Med 2004;170:59–64. 5. Kang YA, Lee HW, Hwang SS, Um SW, Han SK, Shim YS, et al. Usefulness of whole-blood interferon-gamma assay and interferon-gamma enzyme linked immunospot assay in the diagnosis of active pulmonary tuberculosis. Chest 2007;132:959–65. 6. Kobashi Y, Obase Y, Fukuda M, Yoshida K, Miyashita N, Oka M. Clinical reevaluation of the QuantiFERON TB-2G test as a diagnostic method for differentiating active tuberculosis from nontuberculous mycobacteriosis. Clin Infect Dis 2006;43:1540– 6. 7. Ravn P, Munk ME, Andersen AB, Lundgren B, Lundgren JD, Nielsen LN, et al. Prospective evaluation of a whole-blood test using Mycobacterium tuberculosis- specific antigens ESAT-6 and CFP-10 for diagnosis of active tuberculosis. Clin Diagn Lab Immunol 2005;12:491–6. 8. Kobashi Y, Mouri K, Yagi S, Obase Y, Miyashita N, Okimoto N, et al. Clinical utility of the QuantiFERON TB-2G test for elderly patients with active tuberculosis. Chest. 2008. DOI 10.1378/chest.07-1995. 9. Goletti D, Carrara S, Vincenti D, Saltini C, Rizzi EB, Schinina V, et al. Accuracy of an immune diagnostic assay based on RD1 selected epitopes for active tuberculosis in a clinical setting: a pilot study. Clin Microbiol Infect 2006;12:544–50. 10. Harada N, Higuchi K, Yoshiyama T, Kawabe Y, Fujita A, Sasaki Y, et al. Comparison of the sensitivity and specificity of two whole blood interferon-gamma assays for M. tuberculosis infection. J Infect 2008;56:348–53. 11. Bartu V, Havelkova M, Kopecka E. QuantiFERON®-TB Gold in the diagnosis of active tuberculosis. J Int Med Res 2008;36: 434–7. 12. Kobashi Y, Mouri K, Yagi S, Obase Y, et al. Clinical evaluation for diagnosing active TB disease and transitional change of two commercial blood tests. Scand J InfectDis 2008;40:629–34. 13. Kobashi Y, Mouri K, Yagi S, Obase Y, Fukuda M, Miyashita N, et al. Usefulness of the QuantiFERON TB-2G test for the differential diagnosis of pulmonary tuberculosis. Intern Med 2008; 47:237–43. 14. Soysal A, Torun T, Efe S, Gencer H, Tahaoglu K, Bakir M. Evaluation of cut-off values of interferon-gamma-based assays in the diagnosis of M. tuberculosis infection. Int J Tuberc Lung Dis 2008;12:50–6. 15. Nishimura T, Hasegawa N, Mori M, Takebayashi T, Harada N, Higuchi K, et al. Accuracy of an interferon-gamma release assay to detect active pulmonary and extra-pulmonary tuberculosis. Int J Tuberc Lung Dis 2008;12:269–74. 16. Bua A, Molicotti P, Delogu G, Pirina P, Mura MS, Madeddu G, et al. QuantiFERON TB Gold: a new method for latent tuberculosis infection. New Microbiol 2007;30:477–80. 17. Kang YA, Lee HW, Yoon HI, Cho B, Han SK, Shim YS, et al. Discrepancy between the tuberculin skin test and the wholeblood interferon gamma assay for the diagnosis of latent tuberculosis infection in an intermediate tuberculosis-burden country. JAMA 2005;293:2756–61. 18. Aoki M, Aman K, Mitarai S, et al. Sequential analysis of QuantiFERON-TB Gold test results in active tuberculosis patients: changes in values over time and timing of negative conversion Kekkaku 2006;81:277 (in Japanese). 19. Pai M, Joshi R, Bandyopadhyay M, Narang P, et al. Sensitivity of a whole-blood interferon-gamma assay among patients with pulmonary tuberculosis and variations in T-cell responses during

21.

22.

23. 24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35. 36.

anti-tuberculosis treatment. Infection 2007;35:98–103, DOI 10.1007/s15010-007-6114-z Ferrara G, Losi M, D’Amico R, Roversi P, Piro R, Meacci M, et al. Use in routine clinical practice of two commercial blood tests for diagnosis of infection with Mycobacterium tuberculosis: a prospective study. Lancet 2006;367:1328–34. Lee JY, Choi HJ, Park IN, Hong SB, Oh YM, Lim CM, et al. Comparison of two commercial interferon-gamma assays for diagnosing Mycobacterium tuberculosis infection. Eur Respir J 2006;28:24–30. Mazurek GH, Weis SE, Moonan PK, Daley CL, Bernardo J, Lardizabal AA, et al. Prospective comparison of the tuberculin skin test and two whole-blood interferon-gamma release assays in persons with suspected tuberculosis. Clin Infect Dis 2007; 45:837–45. Dewan PK, Grinsdale J, Kawamura LM. Low sensitivity of a whole-blood interferon-gamma release assay for detection of active tuberculosis. Clin Infect Dis 2007;44:69–73. Palazzo R, Spensieri F, Massari M, Fedele G, Frasca L, Carrara S, et al. Use of whole-blood samples in in-house bulk and singlecell antigen-specific gamma interferon assays for surveillance of Mycobacterium tuberculosis infections. Clin Vaccine Immunol 2008;15:327–37. Dominguez J, Ruiz-Manzano J, De Souza-Galvao M, Latorre I, Mila C, Blanco S, et al. Comparison of two commercially available gamma interferon blood tests for immunodiagnosis of tuberculosis. Clin Vaccine Immunol 2008;15:168–71. Pai M, Joshi R, Bandyopadhyay M, Narang P, Dogra S, Taksande B, et al. Sensitivity of a whole-blood interferon-gamma assay among patients with pulmonary tuberculosis and variations in T-cell responses during anti-tuberculosis treatment. Infection 2007;35:98–103. Tsiouris SJ, Coetzee D, Toro PL, Austin J, Stein Z, El-Sadr W. Sensitivity analysis and potential uses of a novel gamma interferon release assay for diagnosis of tuberculosis. J Clin Microbiol 2006;44:2844–50. Adetifa IM, Lugos MD, Hammond A, Jeffries D, Donkor S, Adegbola RA, et al. Comparison of two interferon gamma release assays in the diagnosis of Mycobacterium tuberculosis infection and disease in The Gambia. BMC Infect Dis 2007;7:122. Brock I, Munk ME, Kok-Jensen A, Andersen P. Performance of whole blood IFN-gamma test for tuberculosis diagnosis based on PPD or the specific antigens ESAT-6 and CFP-10. Int J Tuberc Lung Dis 2001;5:462–7. Taggart EW, Hill HR, Ruegner RG, Litwin CM. Evaluation of an in vitro assay for interferon gamma production in response to the Mycobacterium tuberculosis- synthesized peptide antigens ESAT-6 and CFP-10 and the PPD skin test. Am J Clin Pathol 2006;125:467–73. Mazurek GH, Zajdowicz MJ, Hankinson AL, Costigan DJ, Toney SR, Rothel JS, et al. Detection of Mycobacterium tuberculosis infection in United States Navy recruits using the tuberculin skin test or whole-blood interferon gamma release assays. Clin Infect Dis 2007;45:826–36. Franken WP, Timmermans JF, Prins C, Slootman EJ, Dreverman J, Bruins H, et al. Comparison of Mantoux and QuantiFERON TB Gold tests for diagnosis of latent tuberculosis infection in Army personnel. Clin Vaccine Immunol 2007;14:477–80. Soborg B, Andersen AB, Larsen HK, Weldingh K, Andersen P, Kofoed K, et al. Detecting a low prevalence of latent tuberculosis among health care workers in Denmark detected by M. tuberculosis specific IFN-gamma whole-blood test. Scand J Infect Dis 2007;39:554–9. Song KH, Jeon JH, Park WB, Kim SH, et al. Usefulness of the whole-blood interferon-gamma release assay for diagnosis of extrapulmonary tuberculosis. Diagn Microbiol Infect Dis 2009; 63:182–7. Ariga H, Kawabe Y, Nagai H, Kurashima A, et al. Diagnosis of active tuberculous serositis by antigen-specific interferon-γ response of cavity fluid cells. Clin Infect Dis 2007:45:1559–67. Diel R, Loddenkemper R, Meywald-Walter K, Gottschalk R, Nienhaus A. Comparative performance Tuberculin Skin Test, Quantiferon-Gold-in Tube Assay, T-Spot.TB in contact investigations for tuberculosis. Chest. 2008 DOI 10.1378/chest.082048.

154 37. Kipfer B, Reichmuth M, Buechler M, Meisels C, Bodmer T. Tuberculosis in a Swiss army training camp: contact investigation using an interferon gamma release assay. Swiss Med Wkly 2008; 138:267–72. 38. Funayama K, Tsujimoto A, Mori M, Yamamoto H, et al. Usefulness of QuantiFERON-TB-2G in contact investigation of a tuberculosis outbreak in a university. Kekkaku 2005;80:527–34. 39. Fukazawa K. Application and problems of QuantiFERON®TB2G for tuberculosis control programs. 1. Tuberculosis outbreak in a cram school. Kekkaku 2007;82:53–9. 40. Diel R, Loddenkemper R, Meywald-Walter K, Niemann S, Nienhaus A. Predictive value of a whole blood IFN-gamma assay for the development of active tuberculosis disease after recent infection with Mycobacterium tuberculosis. Am J Respir Crit Care Med 2008;177:1164–70. 41. Higuchi K, Harada N, Fukazawa K, Mori T. Relationship between whole-blood interferon-gamma responses and the risk of active tuberculosis. Tuberculosis (Edinb) 2008;88:244–8. 42. Andersen P, Doherty TM, Pai M, Weldingh K. The prognosis of latent tuberculosis: can disease be predicted? Trends Mol Med 2007;13:175–82. 43. Mazurek G, Jereb J, LoBue P, et al. Guidelines for using the QuantiFERON-TB Gold test for detecting Mycobacterium tuberculosis infection: United States. MMWR Recomm Rep 2005; 54:49–55. 44. National Institute for Health and Clinical Excellence. Tuberculosis: clinical diagnosis and management of tuberculosis, and measures for its prevention and control. Clinical Guideline 33, National Collaborating Centre for Chronic Conditions, London March 2006. 45. Canadian Tuberculosis Committee (CTC): Updated recommendations on interferon gamma release assays for latent tuberculosis infection. An Advisory Committee Statement (ACS). Can Commun Dis Rep 2008;34:1–13. 46. Drobniewski F, Cobelens F, Zellweger JP. Use of gammainterferon assays in low- and medium-prevalence countries in Europe: a consensus statement of a Wolfheze Workshop organised by KNCV/EuroTB, Vilnius Sept 2006. Euro Surveill 2007;12: E070726.2. 47. Haute authorite de Sante. Test de detection de la production d’interferon γ pour de diagnostic de infections tuberculeuses. 2006;1–50, Paris. 48. Lungenliga Schweiz: Handbuch Tuberkulose. 2007. Bern, pp 19–26. 49. Committee of TB Prevention. Japanese Society of Tuberculosis. Recommendations for use of QuantiFERON Gold in the diagnosis of tuberculosis infection. Kekkaku 2006;81:393–7. 50. Connell TG, Curtis N, Ranganathan SC, et al. Performance of a whole blood interferon gamma assay for detecting latent infection with Mycobacterium tuberculosis in children. Thorax 2006;61: 616–20. 51. Lighter J, Rigaud M, Eduardo M, Peng CH, Pollack H. Latent tuberculosis diagnosis in children by using the QuantiFERONTB Gold In-Tube Test. Pediatrics 2009;123:30–7. DOI: 10.1542/peds.2007-3618. 52. Molicotti P, Bua A, Mela G, Olmeo P, et al. Performance of QuantiFERON-TB Testing in a tuberculosis outbreak at a primary school. J Pediatr 2008;152:585–6. 53. Detjen AK, Keil T, Roll S, Hauer B, Mauch H, Wahn U, et al. Interferon gamma release assays improve the diagnosis of tuberculosis and nontuberculous mycobacterial disease in children in a country with a low incidence of tuberculosis. Clin Infect Dis 2007;45:322–8. 54. Connell TG, Ritz N, Paxton GA, Buttery JP, Curtis N, et al. A three-way comparison of tuberculin skin testing, QuantiFERONTB Gold and T-SPOT. TB in children. PLoS One 2008;3: e2624. 55. Russo C. Agreement between classic diagnostic procedures and the new interferon-gamma release assay for detection of TB disease in children. In: Rethinking the epidemiology of tuberculosis infection. Vancouver, February 21, 22, 2007. 56. Herrmann JL, Belloy M, Porcher R, Simonney N, et al. Temporal dynamics of interferon gamma responses in children evaluated for tuberculosis. PLoS One. 2009;4:e4130. Epub 2009 Jan 6.

57. Takamatsu I, Study Group of QFT in Pediatrics. Multicenter study of QuantiFERON in child tuberculosis. In: Kato PI, editor. A study of Mycobacterium tuberculosis: 2007 Report of Emerging and Re-emerging Infectious Disease Research Program. Tokyo: Ministry of Health, Labour and Welfare; 2008. 58. Dogra S, Narang P, Mendiratta DK, et al. Comparison of a whole blood interferon-γ assay with tuberculin skin testing for the detection of tuberculosis infection in hospitalized children in rural India. J Infect 2007;54:267–76. 59. Okada K, Mao TE, Mori T, Miura T, et al. Performance of an interferon-gamma release assay for diagnosing latent tuberculosis infection in children. Epidemiol Infect 2008;136:1179–87. 60. Lewinsohn DA, Zalwango S, Stein CM, Mayanja-Kizza H, Okwera A, et al. Whole blood interferon-gamma responses to Mycobacterium tuberculosis antigens in young household contacts of persons with tuberculosis in Uganda. PLoS One 2008;3:e3407. Epub 2008 Oct 15. 61. Connell T, Bar-Zeev N, Curtis N. Early detection of perinatal tuberculosis using a whole blood interferon-γ release assay. Clin Infect Dis 2006;42:482–485. 62 Tsiouris SJ, Austin J, Toro P, Coetzee D, Weyer K, Stein Z, ElSadr WM. Results of a tuberculosis-specific IFN-gamma assay in children at high risk of tuberculosis infection. Int J Tuberc Lung Dis 2006;10:939–41. 63. Nakaoka H, Lawson L, Squire SB, et al. Risk for tuberculosis among children. Emerg Infect Dis 2006;12:1383–8. 64. Chun JK, Kim CK, Kim HS, Jung GY, et al. The role of a whole blood interferon γ assay for the detection of latent tuberculosis infection in bacille Calmette–Guérin vaccinated children. Diagn Microbiol Infect Dis doi:10.1016/j.diagmicrobio.2008.08.022. 65. Leyten EMS, Arend SM, Prins C, Cobelens FGJ, et al. Discrepancy between Mycobacterium tuberculosis-specific gamma interferon release assays using short and prolonged in vitro incubation. Clin Vacc Immunol 2007;14:880–5, doi:10.1128/CVI.00132–07. 66. Lewinsohn D, Gennaro M, Scholvinck D. Tuberculosis immunology in children: diagnostic and therapeutic challenges and opportunities. Int J Tuberc Lung Dis 2004;8:658–74. 67. ATS/CDC Statement Committee on Latent Tuberculosis Infection: Targeted tuberculin testing and treatment of latent tuberculosis infection. MMWR 2000;49:1–54. 68. Prevention Committee of Japanese Society of Tuberculosis, Japanese College of Rheumatology. Recommendation for more active use of chemoprophylaxis of tuberculosis (in Japanese). Kekkaku 2004;79:747–8. 69. American Thoracic Society, CDC: The tuberculin skin test. Am Rev Respir Dis 1981;124:356–63. 70. Ferrara G, Losi M, Meacci M, Meccugni B, Piro R, Roversi P, et al. Routine hospital use of a new commercial whole blood interferon-γ assay for the diagnosis of tuberculosis infection. Am J Respir Crit Care Med 2005;172:631–5. 71. KobashiY, Mouri K, Obase Y, Fukuda M, Miyashita N, Oka M. Clinical evaluation of QuantiFERON TB-2G test for immunocompromised patients. Eur Respir J. 2007; doi: 10.1183/ 09031936.0004. 72. Mori T, Harada N, Higuchi K, Sekiya Y, Uchimura K, Shimao T. Waning of the specific interferon-gamma response after years of tuberculosis infection. Int J Tuberc Lung Dis 2007;11:1021–5. 73. Raby E, Moyo M, Devendra A, Banda J, De Haas P, Ayles H, Godfrey-Faussett P. The effects of HIV on the sensitivity of a whole blood IFN-c release assay in Zambian adults with active tuberculosis. PLoS One 2008;3:e2489. 74. Vincenti D, Cararra S, Butera FO, et al. Response to region of difference 1 (RD1) epitope in human immunodeficiency virus (HIV)-infected individuals enrolled with suspected active tuberculosis: a pilot study. Clin Exp Immunol 2007;150:91–8. 75. Nagai H, Kawabe Y, Ariga H, Shigeyama F, et al. Usefulness of a whole blood interferon-gamma assay (QuantiFERON-TB 2G) for detecting tuberculosis infection in HIV-infected persons (in Japanese). Kekkaku 2007;82:635–40. 76. Talati NJ. Comparison of tuberculin skin test and interferongamma release assays for diagnosis of latent TB infection among HIV positive persons. In: Rethinking the epidemiology of tuberculosis infection. Vancouver, February 21, 22, 2007. 77. Luetkemeyer AF, Charlebois ED, Flores LL, Bangsberg DR, Deeks SG, Martin JN, Havlir DV. Comparison of an interferon-

155

78.

79.

80.

81.

82. 83. 84. 85. 86.

87.

88.

89.

90. 91.

92.

{gamma} release assay to tuberculin skin testing in hiv-infected individuals. Am J Respir Crit Care Med 2007;75:737–42. Jones S, de Gijsel D, Wallach FR, Gurtman AC, Shi Q, Sacks H. Utility of QuantiFERON-TB Gold in-tube testing for latent TB infection in HIV-infected individuals. Int J Tuberc Lung Dis 2007;11:1190–5. Brock I, Ruhwald M, Lundgren B, Westh H, Mathiesen LR, Ravn P. Latent tuberculosis in HIV positive, diagnosed by the M. tuberculosis specific interferon-γ test. Respir Res 2006;7:56 doi:10.1186/1465-9921-7-56. Stephan C, Wolf T, Goetsch U, Bellinger O, Nisius G, et al. Comparing QuantiFERON- tuberculosis gold, T-SPOT tuberculosis and tuberculin skin test in HIV-infected individuals from a low prevalence tuberculosis country. AIDS 2008;22:2471–9. Balcells ME, Perez CM, Chanqueo L, Lasso M, et al. A comparative study of two different methods of the detection of latent tuberculosis in HIV-positive individuals in Chile. Int J Inf Dis 2008; doi: 10.1016/j.ijid.2008.03.005. Hammond AS, McConkey SJ, Hill P, Crozier S, et al. Mycobacterial T cell responses in HIV-infected patients with advanced immunosuppression. J Infect Dis 2008;197:295–9. Hans Rider HL, Cauthen GM, Comstock GW, Snider DE. Epidemiology of tuberculosis in the United States. Epidemiol Rev 1989;11:79–98. Keane J, Gershon S, Wise RP, et al. Tuberculosis associated with infliximab, a tumor necrosis factor-alpha neutralizing agent. N Engl J Med 2001;345:1098–104. Matsumoto T, Tanaka T, Kawase I. Infliximab for rheumatoid arthritis in a patient with tuberculosis. New Engl J Med 2006; 355:740–1. Centers for Disease Control and Prevention. Tuberculosis associated with blocking agents against tumor necrosis factor-alpha – California, 2002–2003. MMWR Morb Mortal Wkly Rep 2004; 53:683–6. Matulis G, Jüni P, Villiger PM, Gadola SD. Detection of latent tuberculosis in immunosuppressed patients with autoimmune diseases: performance of a Mycobacterium tuberculosis antigenspecific interferon gamma assay. Ann Rheum Dis 2008;67:84–90. Epub 2007. Chen DY, Shen GH, Hsieh TY, Hsieh CW, Lan JL. Effectiveness of the Combination of a whole-blood interferon-gamma assay and the tuberculin skin test in detecting latent tuberculosis infection in rheumatoid arthritis patients receiving adalimumab therapy. Arthritis Rheum 2008;59:800–6. Schoepfer AM, Flogerzi B, Fallegger S, Schaffer T, et al. Comparison of interferon-gamma release assay versus tuberculin skin test for tuberculosis screening in inflammatory bowel disease. Am J Gastroenterol 2008;103:1–8. Inamoto H. Tuberculosis in dialysis patients. 3. Epidemiology of pulmonary tuberculosis. Kekkaku 1987;57:477–81. Winthrop KL, Nyendak M, Calvet H, Oh P, et al. InterferonRelease assays for diagnosing Mycobacterium tuberculosis infection in renal dialysis patients. Clin J Am Soc Nephrol 2008;3:1357–63. Lee SSJ, Chou KJ, Su IJ, Chen YS, et al. High Prevalence of latent tuberculosis infection in patients in end-stage renal disease on hemodialysis: comparison of QuantiFERON-TB GOLD, ELISPOT, and tuberculin skin test. Infection 2008. DOI 10.1007/s15010-008-8082-3.

93. Ulrichs T, Anding R, Kaufmann SH, Munk ME. Numbers of IFN-gamma-producing cells against ESAT-6 increase in tuberculosis patients during chemotherapy. Int J Tuberc Lung Dis 2000;4:1181–3. 94. Lalvani A, Nagvenkar P, Udwadia Z, Pathan AA, Wilkinson KA, Shastri JS, 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;183:469–77. 95. Pathan AA, Wilkinson KA, Klenerman P, McShane H, Davidson RN, Pasvol G, et al. Direct ex vivo analysis of antigen-specific IFN gamma- secreting CD4 T cells in Mycobacterium tuberculosis-infected individuals: associations with clinical disease state and effect of treatment. J Immunol 2001;167:5217–25. 96. Al-Attiyah R, Mustafa AS, Abal AT, Madi NM, Andersen P. Restoration of mycobacterial antigen-induced proliferation and interferon-gamma responses in peripheral blood mononuclear cells of tuberculosis patients upon effective chemotherapy. FEMS Immunol Med Microbiol 2003;38:249–56. 97. Carrara S, Vincenti D, Petrosillo N, Amicosante M, Girardi E, Goletti D. Use of a T-cell-based assay for monitoring efficacy of antituberculosis therapy. Clin Infect Dis 2004;38:754–56. 98. Ferrand RA, Bothamley GH, Whelan A, Dockrell HM. Interferon-gamma responses to ESAT-6 in tuberculosis patients early into and after anti-tuberculosis treatment. Int J Tuberc Lung Dis 2005;9:1034–9. 99. Kobashi Y, Obase Y, Fukuda M, Yoshida K, et al. Clinical reevaluation of the QuantiFERON TB-2G test as a diagnostic method for differentiating active tuberculosis from nontuberculous mycobacteriosis. Clin Infect Dis 2006;43:1540–6. 100. Aiken AM, Hill PC, Fox A, McAdam KP, Jackson-Sillah DJ, Lugos MD, et al. Reversion of the ELISPOT test after treatment in Gambian tuberculosis cases. BMC Infect Dis 2006;6:66. 101. Pai M, Joshi R, Dogra S, Mendiratta DK, Narang P, Dheda K, Kalantri S. Persistently elevated T cell interferon-γ responses after treatment for latent tuberculosis infection among health care workers in India: a preliminary report. J Occup Med Toxicol. 2006;1:7 doi:10.1186/1745-6673-1-7. 102. Wilkinson KA, Kon OM, Newton SM, Meintjes G, Davidson RN, Pasvol G, Wilkinson RJ. Effect of treatment of latent tuberculosis infection on the T cell response to Mycobacterium tuberculosis antigens. J Infect Dis 2006;193:354–9. 103. Ewer K, Millington KA, Deeks JJ, Alvarez L, et al. Dynamic antigen-specific T-cell responses after point-source exposure to Mycobacterium tuberculosis. Am J Respir Crit Care Med 2006;174:831–9. 104. Higuchi K, Harada N, Mori T. Interferon-γ responses after isoniazid chemotherapy for latent tuberculosis. Respirology 2008; 13:468–72. 105. Franken WPJ, Arend SM, Thijsen SFT, Bouwman JJM, et al. Interferon-gamma release assays during follow-up of tuberculin skin test-positive contacts. Int J Tuberc Lung Dis 2008;12: 1286–94. 106. Dheda K, Pooran A, Pai M, Miller RF, Lesley K, Booth HL, et al. Interpretation of Mycobacterium tuberculosis antigen-specific IFN-γ release assays (T-SPOT-TB) and factors that may modulate test results. J Infect 2007;55:169–73.