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1 Aals of Iteral Medicie Article Meta-aalysis: New Tests for the Diagosis of Latet Tuberculosis Ifectio: Areas of Ucertaity ad Recommedatios for Research Dick Mezies, MD, MSc; Madhukar Pai, MD, PhD; ad George Comstock, MD, DrPH Backgroud: Util recetly, the tuberculi ski test was the oly test for detectig latet tuberculosis (TB) ifectio, but 2 ex vivo iterfero- release assays (IGRAs) are ow commercially licesed. Purpose: To estimate sesitivity, specificity, ad reproducibility of IGRAs (commercial or research versios of QuatiFERON [QFT] ad Elispot) for diagosig latet TB ifectio i healthy ad immue-suppressed persos. Data Sources: The authors searched MEDLINE ad reviewed citatios of all origial articles ad reviews for studies published i Eglish. Study Selectio: Studies had evaluated IGRAs usig Mycobacterium tuberculosis specific atiges (RD1 atiges) ad overight (16- to 24-h) icubatio times. Referece stadards had to be clearly defied without kowledge of test results. Data Extractio ad Quality Assessmet: Specific criteria for quality assessmet were developed for sesitivity, specificity, ad reproducibility. Data Sythesis: Whe ewly diagosed active TB was used as a surrogate for latet TB ifectio, sesitivity of all tests was suboptimal, although it was higher with Elispot. No test distiguishes active TB from latet TB. Sesitivity of the tuberculi ski test ad IGRAs was similar i persos who were categorized ito cliical gradiets of exposure. Pooled specificity was 97.7 (95 CI, 96 to 99) ad 92.5 (CI, 86 to 99) for QFT ad for Elispot, respectively. Both assays were more specific tha the tuberculi ski test i samples vacciated with bacille Calmette Guéri. Elispot was more sesitive tha the tuberculi ski test i 3 studies of immuecompromised samples. Discordat tuberculi ski test ad IGRA reactios were frequet ad largely uexplaied, although some may be related to varied defiitios of positive test results. Reversio of IGRA results from positive to egative was commo i 2 studies i which it was assessed. Limitatios: Most studies used cross-sectioal desigs with the iheret limitatio of o gold stadard for latet TB ifectio, ad most ivolved small samples with a widely varyig likelihood of true-positive ad false-positive test results. There is isufficiet evidece o IGRA performace i childre, immue-compromised persos, ad the elderly. Coclusios: New IGRAs show cosiderable promise ad have excellet specificity. Additioal studies are eeded to better defie their performace i high-risk populatios ad i serial testig. Logitudial studies are eeded to defie the predictive value of IGRAs. A Iter Med. 2007;146: For author affiliatios, see ed of text. The tuberculi ski test is 1 of the few tests that has bee i use for early 100 years i cliical medicie (1). Therefore, it is ot surprisig that the test has importat limitatios. The tuberculi ski test uses a relatively crude mix of atiges from Mycobacterium tuberculosis. As a result, false-positive reactios ca occur because of previous bacille Calmette Guéri (BCG) vacciatio or sesitizatio to otuberculous mycobacteria. False-egative results o tuberculi ski tests ca occur because of severe illess, icludig active tuberculosis (TB), or immue suppressio, ofte due to HIV ifectio. Iitial testig ca affect results of subsequet tests because of aamestic recall of immuity (the booster effect). Errors i admiistratio or readig ca lead to icorrect results. The variability of the tuberculi ski test ca be miimal with welltraied persoel usig meticulous techiques (2 8), although such persoel are ot always available. Iterpretatio of test results requires a thorough uderstadig of the test. I the past decade, 2 ew T-cell based tests for diagosig latet TB ifectio have bee developed ad licesed for commercial distributio i may coutries (Appedix Table 1, available at Oe test, QuatiFERON (QFT)-TB Gold (Cellestis, Victoria, Australia), uses ezyme-liked immuosorbet assay to measure atige-specific productio of iterfero- (IFN- ) by circulatig T cells i whole blood. The other test, the T-SPOT.TB (Oxford Immuotec, Oxford, Uited Kigdom), uses the Elispot techique to measure peripheral blood moouclear cells that produce IFN-. Curret versios of both IFN- release assays (IGRAs) use more specific M. tuberculosis atiges ESAT-6, CFP-10, ad TB7.7. The gees ecodig these atiges are foud i the regio of differece 1 (RD1) of the M. tuberculosis geome, which is deleted from the geome of M. bovis BCG, ad certai otuberculous mycobacteria, such as M. avium. The U.S. Ceters for Disease Cotrol ad Prevetio (9) has recommeded that QFT replace the tuberculi ski See also: Prit Editors Notes Web-Oly Appedix Tables Appedix Figure CME quiz Coversio of figures ad tables ito slides America College of Physicias

2 Diagosis of Latet Tuberculosis Ifectio Article test, whereas the U.K. Natioal Istitute for Cliical Excellece has suggested that IGRAs are useful adjucts to the tuberculi ski test (10). Iterfero release assays have several importat advatages over the tuberculi ski test: Testig requires oly 1 patiet visit ad these assays are ex vivo tests, which reduce the risk for adverse effects ad elimiate potetial boostig whe testig is repeated. However, IGRAs have importat disadvatages, icludig higher material cost, eed for a equipped laboratory, ad a requiremet to draw blood with subsequet careful hadlig to maitai viability of lymphocytes. Although boostig will ot occur, the variability of these tests whe repeated after moths or years, such as i serial testig of exposed populatios, has ot bee well studied. The greatest disadvatage is the lack of prospective data regardig the future risk for active TB i persos with positive results o IGRAs. This has bee established for differet-sized tuberculi ski test reactios i may large-scale cohort ad experimetal studies (11 20), which permits the estimatio of risk for disease ad beefit of therapy. I this review, we compared sesitivity, specificity, ad reproducibility of commercially available IGRAs (or ihouse equivalet tests) with the tuberculi ski test for diagosig latet TB ifectio i healthy ad immuesuppressed adults ad childre. Because there is o gold stadard for testig latet TB ifectio, we estimated sesitivity from studies of 1) patiets with active TB (who must have ifectio), 2) persos i cotact with patiets with active TB who were categorized ito gradiets of exposure, ad 3) cocordace of IGRA ad the tuberculi ski test. We estimated specificity from studies of healthy persos with a very low likelihood of exposure. Reproducibility was estimated from studies of repeated IGRAs, with or without treatmet for active or latet TB. METHODS Search Strategy We coducted a MEDLINE search for articles published betwee 1966 ad October 2006 o 4 December 2005 ad updated the search o 2 July 2006 ad 31 October Search terms icluded TB ifectio, or TB disease, AND Quatifero, or Elispot, or iterfero-gamma assays, or iterfero-gamma release assays, or T cell assays, AND ESAT-6, or CFP10, or RD1 atiges. The searches were limited to huma studies published i Eglish. We idetified additioal studies from the referece lists of articles idetified i this maer ad performed a had search of all issues of the Iteratioal Joural of Tuberculosis ad Lug Disease that were published over the past 5 years. We cotacted the authors of some studies for clarificatio. Study Selectio Studies were icluded if they used QFT or Elispot tests with overight (16 to 24 h) i vitro icubatio of Cotext Do ew ex vivo iterfero- release assays (IGRAs) detect latet tuberculosis (TB) more accurately tha tuberculi ski tests (TSTs)? Cotributio This meta-aalysis of 59 studies foud that o test distiguished active TB from latet TB, o test had high sesitivity, IGRAs were more specific tha the TST i populatios vacciated with bacille Calmette Guéri (BCG), ad the results of IGRAs ad the TST were frequetly discordat. Cautios Studies had may limitatios, icludig o gold stadard for diagosig latet TB. Implicatios New IGRAs have good specificity ad show promise for detectig latet TB, particularly i BCG-vacciated patiets. The Editors peripheral blood lymphocytes stimulated with RD1 atiges. This meat research, i-house, or commercial versios of QFT or Elispot tests that used ESAT-6 with or without CFP-10 ad with or without TB7.7. Commercial versios that were icluded were the QuatiFERON-TB Gold (QFT-G), QuatiFERON-TB Gold I-Tube (QFT- IT), ad the T-SPOT.TB. For studies assessig sesitivity, the study sample had to have active TB or cotact with a perso with active TB ad a clearly defied gradiet of exposure of at least 2 categories (such as high or low exposure). For studies assessig specificity, the populatio had to be healthy, lifelog residets of low-icidece coutries with a average age youger tha 40 years without occupatioal, travel, or other exposure to TB. I studies assessig cocordace of 2 or more tests, all tests had to be doe simultaeously i all persos. We excluded studies that performed sequetial testig, i which the secod test was doe oly i persos selected o the basis of the results of the first test. Studies assessig reproducibility had to perform at least 2 tests i the same persos. Two idepedet reviewers assessed titles ad abstracts to select studies for the review. A list of excluded studies is available from the authors. Extractio of Data ad Assessmet of Study Quality Oe reviewer reviewed all studies ad abstracted data regardig test characteristics ad study quality (Appedix Tables 2 through 6, available at A secod reviewer assessed a radom sample of 20 of the full-text articles to determie cocordace i assessmet of data quality ad accuracy of abstracted data. We assessed 2 quality criteria i all studies: The techicias performig 6 March 2007 Aals of Iteral Medicie Volume 146 Number 5 341

3 Article Diagosis of Latet Tuberculosis Ifectio Figure 1. Forest plot of studies estimatig sesitivity of the 3 tests i patiets with active tuberculosis as a surrogate for latet tuberculosis ifectio. Poit estimates for sesitivity ad 95 CIs are show. QFT QuatiFERON; TST tuberculi ski test March 2007 Aals of Iteral Medicie Volume 146 Number 5

4 Diagosis of Latet Tuberculosis Ifectio Article Figure 2. Forest plot of studies estimatig specificity of the 3 tests i populatios at very low risk for latet tuberculosis ifectio. Poit estimates for specificity ad 95 CIs are show. BCG bacille Calmette Guéri; QFT QuatiFERON; TST tuberculi ski test. the tuberculi ski test ad IGRAs were blided to the patiets cliical status, other test results ad timig of IGRA relative to the tuberculi ski test, ad treatmet (if applicable). I all studies estimatig sesitivity, the cliical gold stadard had to be determied without kowig or usig results of the tests that were beig evaluated. I additio, high-quality studies of active TB (as a surrogate for latet TB ifectio) cofirmed the diagosis microbiologically or histologically. High-quality studies of reproducibility had high rates of participatio of eligible persos, ad high rates of completio of testig protocols ad assessed treatmet adherece or outcomes i studies of treated persos. Data Sythesis ad Aalysis Methods of studies estimatig sesitivity amog patiets with active TB as a surrogate for latet TB ifectio ad estimatig specificity i low-risk populatios were similar eough to allow pooled estimates. I particular, the same diagostic thresholds were used for i-house assays ad commercial assays. Differet cut-poits were used for the tuberculi ski test; these were aalyzed separately. For each study, we calculated sesitivity or specificity (ad 95 CIs) ad summarized the results i forest plots. A fixed-effects meta-aalysis was doe by usig Meta-DiSc, versio 1.2 (Ramo y Cajal Hospital, Madrid, Spai) (21): Studies were weighted by total sample size to pool (summarize) estimates of sesitivity ad specificity across the studies. Presece of statistically sigificat heterogeeity across studies was evaluated by usig the chi-square test for heterogeeity. To accout for heterogeeity due to betwee-study variability, we corrected for overdispersio. We did ot pool estimates of test sesitivity o the basis of gradiets of exposure, cocordace amog highrisk persos, ad effects of immue suppressio or treat- 6 March 2007 Aals of Iteral Medicie Volume 146 Number 5 343

5 Article Diagosis of Latet Tuberculosis Ifectio Table 1. Summary of Sesitivity from Pooled Estimates from All Studies* Variable Studies, Sesitivity (95 CI) Chi-Square Test for Heterogeeity Tuberculi ski testig All studies ( ) 61.4 (0.001) Size of reactio, mm ( ) 23.5 (0.001) ( ) 18.0 (0.01) ( ) Sample Pediatric ( ) 17.4 (0.01) Adult ( ) 35.7 (0.001) QuatiFERON All studies ( ) 38 (0.001) Atiges ESAT-6 oly ( ) ESAT-6/CFP ( ) 20.9 (0.001) ESAT-6/CFP ( ) 6.8 (0.05) ad TB7.7 Sample Pediatric ( ) 11.0 (0.01) Adult ( ) 32.5 (0.001) Elispot or T-SPOT.TB All studies ( ) 57.3 (0.001) Atiges ESAT ( ) 0.8 (NS) ESAT-6/CFP ( ) 51.7 (0.001) Sample Pediatric ( ) 3.0 (0.08) Adult ( ) 17.1 (0.001) * Patiets with active tuberculosis were used as surrogates for latet tuberculosis. NS ot sigificat. All 95 CIs are corrected for overdispersio. met, because of the heterogeeity of ascertaimet ad classificatio of exposure ad study samples. Role of the Fudig Source No fudig sources directly supported the study. Dr. Mezies receives salary support from the Fods de la Recherche e Saté duquébec, which had o role i the coduct of or i the decisio to publish the study. RESULTS Studies Idetified A total of 275 studies were idetified from PubMed, of which 83 were selected for full-text review (Appedix Figure, available at From these origial articles ad reviews, 21 additioal articles were idetified ad 6 more were idetified at the time of the last update, for a total of 110 studies for full review. Of these studies, 52 were excluded because they did ot meet the review criteria, leavig 58 studies icluded i this review. All studies used cross-sectioal desigs, except for 8 that performed serial testig i cohorts. Studies raged from fewer tha 10 participats to more tha 500 participats. The expected prevalece of latet TB ifectio ad the prevalece ad policy of BCG vacciatio also varied widely. Twety-two studies evaluated sesitivity of IGRA amog ewly diagosed patiets with active TB as a surrogate for latet TB ifectio. Of these studies, 3 tested both IGRAs ad the tuberculi ski test, 12 tested 1 IGRA ad the tuberculi ski test, ad 7 tested 1 IGRA oly. Specificity of IGRA i populatios at very low risk for latet TB ifectio was assessed i 11 studies, of which 6 performed the tuberculi ski test. Te studies compared a sigle IGRA with the tuberculi ski test i cotacts with a gradiet of exposure, ad 14 studies examied cocordace of IGRA with the tuberculi ski test. Serial testig was performed i 7 treated cohorts ad i 1 additioal utreated cohort. Five studies examied the impact of immue suppressio o IGRA performace; 3 of these compared results with those of the tuberculi ski test. Sesitivity Usig Tuberculosis as a Surrogate for Latet Ifectio Figure 1 shows that all 3 tests, particularly the tuberculi ski test, have suboptimal sesitivity amog patiets with ewly diagosed, active TB. Pooled estimates of sesitivity were lowest for the tuberculi ski test, higher for QFT, ad highest for Elispot. However, oly 3 studies, with a total of 143 participats, coducted direct head-tohead comparisos (22 24). There were some differeces i sesitivity betwee studies usig differet versios of QFT. QuatiFERON with ESAT-6 aloe was used oly oce i a older study with earlier assay ad atiges. This study reported substatially lower sesitivity but higher specificity (Figure 2). Sesitivity of QFT-G was somewhat higher tha with QFT-IT (Tables 1 ad 2). Sesitivities of Elispot assays that icorporated ESAT-6 aloe were similar to those of assays with ESAT-6 plus CFP-10. The IGRA test procedures were well stadardized, but tuberculi ski test procedures were ot. As show i Table 1, differet cutpoits were used, ad this had some effect o sesitivity. I 4 studies, which used a dose of tuberculi that was 50 or less tha the recommeded amout (26 28, 85), sesitivity of the tuberculi ski test averaged 0.63 (rage, 0.46 to 0.80) compared with a sesitivity of 0.73 (rage, 0.62 to 0.84) i studies that used a tuberculi dose recommeded by the World Health Orgaizatio (2). Sesitivity Usig a Gradiet of Exposure as a Idicator of the Likelihood of Latet Tuberculosis Ifectio Table 3 summarizes studies that compared IGRA ad the tuberculi ski test with a cliically defied gradiet of exposure. It is difficult to esure equivalece of the exposure categories because each study characterized exposure differetly. Nevertheless, overall fidigs were similar. The prevalece of positive results o IGRA ad the tuberculi ski test was highest i the most-exposed groups. I the less-exposed groups, the prevalece of positive results o the tuberculi ski test was higher tha that of IGRA i studies that ivolved populatios that had received BCG vacciatio at a older age (30, 31). I 1 study, the expected prevalece of latet TB ifectio was 30 i the March 2007 Aals of Iteral Medicie Volume 146 Number 5

6 Diagosis of Latet Tuberculosis Ifectio Article least-exposed group, yet oly 4 had a positive result o IGRA (30). Specificity of Tests I all studies of healthy populatios cosidered at very low risk for latet TB ifectio, IGRA with RD1 atiges have had high specificity (Figure 2). Pooled average specificity was 97.7 ad 92.2 i QFT ad Elispot, respectively (Tables 1 ad 2), ad was uaffected by BCG vacciatio. A limitatio of these studies is that persos at low risk could still have had latet TB ifectio, but this could ot be verified because of the absece of a true gold stadard for testig for latet TB ifectio. Discordace betwee Iterfero- Release Assays ad Tuberculi Ski Test: Reflectio of Ucertaity o Sesitivity ad Specificity of These Tests Tables 4 ad 5 summarize studies of dual tuberculi ski testig ad IGRA testig of populatios cosidered at risk for TB ifectio. I 3 studies, discordace was greater i persos with BCG vacciatio tha i those who were ot vacciated (30, 32, 33). I 2 studies i which very few persos had bee BCG vacciated, almost half of all persos with positive results o tuberculi ski test had egative results o IGRA (34, 35). Discordat positive IGRA reactios ad egative tuberculi ski test reactios occurred i 6 to 7 of all persos ad accouted for 23 of all positive results o QFT ad 26 of all positive results o Elispot. I 3 studies summarized i Table 6, agreemet betwee QFT-G ad Elispot was good: values were 0.57 to Of ote, Elispot results were more ofte positive tha those of QFT, which is cosistet with higher sesitivity or lower specificity as oted earlier. I a fourth study (80), QFT-G was compared with QFT-IT. Cocordace was moderate ( 0.5): QFT-IT had a greater prevalece of positive reactios. The differeces i these 2 versios of the same test may reflect poor reproducibility uder field coditios, higher sesitivity of QFT-IT because of the additio of the third atige (TB7.7), or that QFT-IT was more sesitive because of its greater techical simplicity. Serial Testig Very few published studies have assessed reproducibility of IGRAs. I studies coducted by the maufacturer, the test-related coefficiet of variatio for the QFT was 8.7 by usig replicate serum samples from well-characterized patiets (37). Amog healthy voluteers from Gambia i whom Elispot was repeated 1 week apart, 12 iitially had egative results ad oe had coversio to positive results, but of the 11 who iitially had positive results, 1 (9) reverted to havig egative results (38). I a study of health care workers i Idia who were tested 18 moths apart, 11.6 had QFT coversio, but 24 of those with iitial positive results o QFT reverted to havig egative results (39). QuatiFERON reversio was associated with egative results o the iitial tuberculi ski test or a iitial QFT result close to the maufacturer s suggested cut-poit for a positive result. As see i Table 7, i 4 studies of patiets treated for active TB, serial Elispot assays decreased i 3 studies (38, 40, 41), icreased after 1 moth i a fourth study, the decreased with cotiued therapy (42). I patiets who were treated for latet TB ifectio, IGRA results did ot chage i 2 studies (26, 43) but reverted to egative results Table 2. Summary of Specificity from Pooled Estimates from Studies of Persos at Very Low Risk for Tuberculosis Ifectio* Groupig Studies, Specificity (95 CI) Chi-Square Test for Heterogeeity Tuberculi ski testig All studies ( ) BCG vacciatio Not vacciated ( ) 4.0 NS Vacciated ( ) Criteria Positive 10 mm ( ) Positive 15 mm ( ) QuatiFERON All studies ( ) ESAT ( ) 0 ESAT-6 ad CFP ( ) BCG vacciatio Not vacciated ( ) 0 Vacciated ( ) Elispot or T-SPOT.TB All studies ( ) * BCG bacille Calmette Guéri; NS ot sigificat. I 1 study (30), data for 2 tuberculi ski test cut-poits are give. I each of 2 studies (68, 69), 2 differet very-low-risk populatios were tested. These were couted as separate studies. 6 March 2007 Aals of Iteral Medicie Volume 146 Number P Value

7 Article Diagosis of Latet Tuberculosis Ifectio Table 3. Studies Comparig Iterfero- Release Assay (IGRA) with Tuberculi Ski Test (TST) agaist the Cliical Gold Stadard of Exposure Gradiets* Study, Year (Referece) Coutry Patiets with a History of BCG vacciatio, () Atiges for IGRA-Positive ad TST-Positive Type Results by Exposure Gradiet High Exposure Participats i Exposure Category, IGRA-Positive, TST-Positive, Elispot Lalvai et al., 2001 (31) Ewer et al., 2003 (41) Richeldi et al., 2004 (70) Hill et al., 2004 (71) Zellweger et al., 2005 (72) Shams et al., 2005 (73) Hill et al., 2006 (36) QuatiFERON Brock et al., 2004 (34) Kag et al., 2005 (30) Nakaoka et al., 2006 (51) Uited Kigdom 59 (most) ESAT-6 HEAF (PPD) Uited Kigdom 535 (most) ESAT ad CFP-10 HEAF (PPD) Italy 92 (10) ESAT-6 ad CFP-10 TST: 5TU-PPD Gambia 735 (45) ESAT-6 ad CFP-10 TST: 2TU-RT23 Switzerlad 91 (86) ESAT-6 ad CFP-10 TST: 2TU-RT23 Uited States 413 (49} ESAT-6 ad CFP-10 TST: 5TU-PPDS Gambia 718 (46) ESAT-6 ad CFP-10 TST: 2TU-RT23 Demark 85 (0) ESAT-6 ad CFP-10 TST: 2TU-RT23 Korea 273 (81) ESAT-6 ad CFP-10 TST: 2TU-RT23 Nigeria 207 (90) ESAT-6 ad CFP-10 TST: 5TU-PPDS * BCG bacille Calmette Guéri; PPD purified protei derivative; PPDS purified protei derivative stadard. The percetage is the proportio i that exposure category with a positive respose to IGRA or TST. This group represeted a radom sample from the geeral populatio. O the basis of historical data, the authors estimated that the prevalece of latet tuberculosis ifectio should have bee 30. O the basis of the average age of 25 years, the icidece of smear-positive tuberculosis (40/ ) (74), ad the Styblo equatio (75), the estimated prevalece of latet tuberculosis ifectio should have bee i 16 of treated persos i the third study (44). Of importace, i this last study, 7 of 25 (28) persos who were ot treated had reversio a pheomeo see i those who iitially had positive results o Elispot ad egative results o tuberculi ski test (44). Impact of Immue Suppressio To date, few studies have assessed IGRA i these highrisk populatios. I 2 studies (45, 46), resposes of IGRA were slightly reduced i immue-compromised patiets compared with those who were ot immue-compromised. I 3 studies with direct compariso, the prevalece of positive results o Elispot was substatially higher tha that of positive results o the tuberculi ski test (46 48), particularly i persos with greater immue suppressio (47). The oly study to evaluate QFT oted a low prevalece of positive results i asymptomatic HIV-ifected patiets ad that idetermiate results were more commo if the patiet s CD4 cout was less tha 100 cells/mm 3 (49). Results i Pediatric Populatios Studies of IGRA respose i pediatric samples are more heterogeeous because the populatios selected were suspected of havig active TB (23, 26, 50), had cotact with ifected persos (23, 36, 51), or were school childre (52). Some were healthy (36, 52, 53), whereas others were hospitalized (26, 50); some studies were coducted i lowicome coutries (26, 36, 51, 52), ad others were coducted i middle-icome (53) or high-icome (23, 50) coutries. Cofirmig or excludig active TB is also more difficult i childre. Hece, iformatio is curretly isufficiet to estimate sesitivity, specificity, ad reproducibility of IGRAs i childre, although the occurrece of idetermiate QFT (23, 50) ad failed phlebotomy (51, 52) may be importat potetial limitatios of IGRAs i this populatio. DISCUSSION Pricipal Fidigs Commercially available IGRAs have evolved rapidly over the past decade. The latest versios use more specific M. tuberculosis atiges ad are simpler to perform. These tests are promisig ad have bee successfully used by idepedet ivestigators i may settigs, icludig lowicome coutries. I our review, the sesitivity of the tuberculi ski test compared with QFT, usig active TB as a surrogate for latet TB ifectio, was low but similar for both tests, whereas the sesitivity of Elispot usig this sur March 2007 Aals of Iteral Medicie Volume 146 Number 5

8 Diagosis of Latet Tuberculosis Ifectio Article Table 3 Cotiued Results by Exposure Gradiet Moderate Exposure Low Exposure Very Low Exposure Participats i Exposure Category, IGRA-Positive, TST-Positive, Participats i Exposure Category, IGRA-Positive, TST-Positive, Participats i Exposure Category, IGRA-Positive, TST-Positive, rogate was somewhat superior. Whe a gradiet of exposure amog cotacts was used as the gold stadard, sesitivity of IGRA ad the tuberculi ski test were similar. Noe of these tests ca distiguish betwee latet ad active TB. Specificity of IGRA is excellet because either IGRA is affected by BCG vacciatio, although the effect of otuberculous mycobacteria o IGRA respose is poorly studied. However, BCG vacciatio has a greater effect o the specificity of the tuberculi ski test, particularly if BCG was give after ifacy. There is substatial discordace of IGRA with the tuberculi ski test i populatios with varyig likelihood of latet TB ifectio. Although some discordace may be explaied by superior specificity of IGRA, it would be overly simplistic to assume that the results of IGRA tests were always correct ad that those of tuberculi ski test were always icorrect. Some discordace may be explaied by 1 test beig close to the criteria for positive test results, but this pheomeo has ot bee aalyzed i most studies ad thus is ot well-uderstood. I 3 studies of immue-compromised patiets, sesitivity of Elispot was superior to that of the tuberculi ski test. Studies i pediatric populatios were too heterogeeous to draw firm coclusios. Studies of the effect of treatmet o IGRA respose are cotradictory. However, the true effect caot be ascertaied util the biological ad radom variability of IGRA respose has bee better characterized. I 2 studies, a substatial umber of persos with positive results o IGRA reverted to havig egative results without treatmet. This was associated with iitial egative results o the tuberculosis ski test (that is, discordat results) (39, 44) ad IGRA respose close to the cut-poit for positive test results (39). Limitatios The major limitatio of estimatio of sesitivity or specificity was the use of a cross-sectioal desig i all studies reviewed. With this desig, there is o gold stadard for latet TB ifectio. The oly certai measure that latet TB ifectio exists is whe the risk for active TB associated with a particular test result has bee defied. This requires large-scale cohort studies with log-term follow-up of utreated populatios with positive results at baselie. I additio to beig expesive ad complex, such studies are ethically impossible i most high-icome coutries, where the stadard of care is to offer treatmet to such persos. With cross-sectioal desigs, surrogates for true ifec- 6 March 2007 Aals of Iteral Medicie Volume 146 Number 5 347

9 Article Diagosis of Latet Tuberculosis Ifectio tio must be used. The most commoly used proxy is active TB, because persos with disease must be ifected. However, with active TB, the cell-mediated immue respose is ofte dimiished, particularly at the time of diagosis, i patiets with more advaced disease (54 56), malutritio (57), or older age (55, 58). Ituitively, oe would expect reduced performace of tests, such as the tuberculi ski test or IGRA, because the cell-mediated immuity they measure must have failed, to some extet, i ay perso with active disease. Of iterest, the greater sesitivity of Elispot i active TB is paralleled by fidigs i immue-suppressed patiets. We speculate that this may reflect the techical requiremet for stadardizatio of the umber of peripheral blood moouclear cells i each assay well. A additioal importat limitatio is the heterogeeity of the studies reviewed, which were coducted with differet timig of testig ad i differet settigs with samples that had markedly differet risks for exposure ad ifectio. Because of these methodological differeces, poolig of estimates could be made oly for results of sesitivity amog patiets with active TB (as a surrogate) ad of specificity i low-risk populatios. The estimatio of the sesitivity of these tests amog cotacts who were categorized ito gradiets of exposure was limited by the differeces i degree ad categorizatio of exposure. Thus, differeces i results may have reflected differeces i exposure or exposure defiitio rather tha differeces i test performace. There were too few studies that evaluated IGRAs i immue-compromised persos ad i pediatric patiets to make firm coclusios. To date, most studies of the ew IGRA have icluded the ivestigators who developed these assays. This is ievitable because of their recet itroductio, but could result i 2 problems. First, highly complex tests could achieve iitially excellet results i research laboratories, but test performace may be reduced i the hads of idepedet ivestigators who lack the specialized expertise to overcome Table 4. Cocordace of QuatiFERON ad Tuberculi Ski Test (TST) i Healthy Populatios with Varyig Risk for Latet Tuberculosis Ifectio* Study, Year (Referece) Brock et al., 2004 (34) Pai et al., 2005 (76) Kag et al., 2005 (30) Porsa et al., 2006 (35) Harada et al., 2006 (32) Ferrara et al., 2006 (23) Dogra et al., 2006 (26) Mahomed, 2006 (80) Tsiouris et al., 2006 (52) Lee et al., 2006 (22) Nakaoka et al., 2006 (51) Coell et al., 2006 (50) Total QuatiFERON Coutry Risk Group Total Participats, BCG, Age at BCG Vacciatio Cocordat Results TST- Positive ad IGRA- Positive, () TSTad Negative IGRA- Negative, () Discordat Results TST- Negative ad IGRA- Positive, () TST- Positive ad IGRA- Negative, () Demark Cotacts of persos with tuberculosis (51) 19 (42) 1 (2) 2 (4) Idia Health care Ifacy 223 (31) 360 (50) 65 (9) 72 (10) workers Korea Close ad casual Older 24 (20) 40 (33) 4 (3) 53 (44) cotacts of persos with tuberculosis U.S. Prisoers 409 All ages 8 (2) 360 (88) 12 (3) 29 (7) Japa Health care workers Older 24 (8) 18 (6) 1 (1) 261 (86) Italy Hospitalized All ages 67 (23) 143 (50) 21 (7) 55 (19) adults Idia Hospitalized Ifacy 3 (3) 89 (92) 3 (3) 2 (2) childre South Healthy adults Ifacy 189 (53) 57 (16) 12 (3) 100 (28) Africa South Pediatric Ifacy 51 (28) 94 (51) 10 (5) 29 (16) Africa cotacts Korea Healthy studets Older 3 (2) 95 (73) 8 (6) 25 (19) Nigeria Australia Pediatric cotacts Pediatric cotacts Ifacy 40 (22) 106 (59) 19 (11) 14 (8) All ages 10 (13) 38 (51) 4 (5) 23 (31) 3216 (100) 1890 (59) 693 (21.5) 1586 (49.3) 163 (5.1) 774 (24.1) * BCG bacille Calmette Guéri; IGRA iterfero- release assay. I these 3 studies, the participats who had received BCG vacciatio were immigrats from may coutries i which BCG vacciatio policy differed. Study participats had previous multiple BCG vacciatios ad TSTs. Eight participats with active tuberculosis were excluded from this aalysis. Results from QuatiFERON-Gold I-Tube (ESAT-6/CFP-10/TB7.7) are show. Results with QuatiFERON-Gold (ESAT-6/CFP-10) were less cocordat with TST March 2007 Aals of Iteral Medicie Volume 146 Number 5

10 Diagosis of Latet Tuberculosis Ifectio Article Table 5. Cocordace of Elispot or T-SPOT.TB with Tuberculi Ski Test (TST) i Healthy Populatios with Varyig Risk for Latet Tuberculosis Ifectio* Study, Year (Referece) Chapma et al., 2002 (46) Ewer et al., 2003 (41) Richeldi et al., 2004 (66) Hill et al., 2004 (71) Shams et al., 2005 (73) Zellweger et al., 2005 (72) Ferrara et al., 2006 (23) Hill et al., 2006 (36) Total ELISPOT, () Coutry Risk Group Participats, Zambia Uited Kigdom Italy Adults (29 were HIV ifected) Cotacts i high school outbreak Nosocomial cotacts BCG Vacciatio, Age at BCG Vacciatio Cocordat Results TST- Positive ad IGRA- Positive, () TSTad Negative IGRA- Negative, () Discordat Results TST- Positive ad IGRA- Negative, () TST- Negative ad IGRA- Positive, () Ifacy 22 (45) 8 (16) 11 (22) 8 (16) Older 118 (22) 353 (66) 32 (6) 27 (5) Mixed 2 (2) 73 (79) 2 (2) 15 (16) Gambia Cotacts Ifacy 162 (22) 382 (52) 139 (19) 58 (8) Uited Cotacts Mixed 132 (32) 174 (42) 74 (18) 29 (7) States Switzerlad Cotacts Mixed 11 (12) 48 (53) 27 (32) 3 (3) Italy Hospitalized Mixed 90 (29) 146 (47) 42 (14) 30 (10) patiets Gambia Cotacts Ifacy 165 (24) 413 (60) 60 (9) 55 (8) 2916 (100) 1493 (51) 702 (24.1) 1597 (54.8) 387 (13.3) 225 (7.7) * BCG bacille Calmette Guéri; IGRA iterfero- release assay. The participats who had received BCG vacciatio were immigrats from may coutries where BCG vacciatio policy differed. techical challeges. Secod, ivestigators could have a coflict of iterest if they retaied fiacial iterest i the assays. The stregths of this review iclude the focus o commercially available versios of the ew IGRAs or i-house versios that used idetical techiques, makig the results more relevat to practicig cliicias. I additio, most of the studies we icluded were published i the past 2 years, makig this review as up-to-date as possible. However, because this is a active field of ivestigatio, estimated sesitivity, specificity, ad reproducibility may chage with additioal publicatios, particularly where evidece is limited. Implicatios The most cosistet fidig i this review is the high specificity of IGRA, which was uaffected by BCG vacciatio, i all populatios. This reflects the atiges used i these assays, which are ot preset i BCG or i certai otuberculous mycobacteria (59). I the studies reviewed, the effect of BCG vacciatio o the results of the tuberculi ski test varied, which may reflect differeces i patiets ages whe the vacciatio was admiistered. I a earlier review of 24 studies that ivolved persos who had bee vacciated with BCG i ifacy ad a similar umber of persos who were ot vacciated, falsepositive results o tuberculi ski tests ( 10 mm) attributable to BCG vacciatio occurred i 6.3 persos overall ad oly i 1 of those who were tested after more tha 10 years (60). I the same review, i the 12 studies ivolvig persos who were vacciated with BCG at 2 years of age or older, BCG was resposible for falsepositive results o tuberculi ski tests i 40 of all persos ad i 20 after 10 or more years (60). It is clear that IGRA will be most useful i BCG-vacciated populatios, particularly i those that received BCG vacciatio repeatedly or after ifacy. This practice was commo i Europe ad Lati America for may years, but ow global vacciatio policy is to vacciate ifats oly (61). I populatios that received BCG vacciatios as ifats, the IGRA will be slightly more specific i childhood but o more specific i adolescece or i adult life (60). Noe of the reviewed studies assessed the effect of otuberculous mycobacteria o IGRA respose. Persos with disease caused by M. marium ad M. kasasei ca have positive results o RD1-IGRA (62), which is of iterest because the geomes of these otuberculous mycobacteria orgaisms cotai the RD1 regio (59). It would be iterestig to perform RD1 o the basis of IGRA ad dual ski testig with purified protei derivative (or RT23) plus otuberculous mycobacteria atiges, such as M. 6 March 2007 Aals of Iteral Medicie Volume 146 Number 5 349

11 Article Diagosis of Latet Tuberculosis Ifectio Table 6. Cocordace of T-SPOT.TB ad QuatiFERON* Study, Year (Referece) Coutry Risk Group Participats, BCG Vacciatio, Age at BCG Vacciatio Cocordat Reactios T- SPOT.TB Positive ad QFT-G Positive, () T- SPOT.TB Negative ad QFT-G Negative, () Discordat Reactios T- SPOT.TB Positive ad QFT-G Negative, () Ferrara et al., Italy Persos with All ages 94 (25) 233 (61) 50 (13) 6 (2) 2006 (23) suspected TB Lee et al., Korea Persos with All ages 62 (28) 104 (48) 34 (16) 10 (5) 2006 (22) active TB ad those at low risk for TB Goletti et al., Italy Persos with All ages 12 (37) 18 (56) 1 (3) 1 (3) 2006 (24) suspected TB Total (27) 355 (56) 85 (13) 17 (3) * BCG bacille Calmette Guéri; TB tuberculosis. T- SPOT.TB Negative ad QFT-G Positive, () marium, M. kasasei, M. szulgai, or M. flavesces, from orgaisms cotaiig the RD1 regio. I our earlier review, we foud that for every 100 persos with otuberculous mycobacteria sesitivity, oly 2 (2) would have false-positive results, measurig 10 to 14 mm, o tuberculi ski tests. This effect was reasoably cosistet i all coutries where dual testig was performed, despite the wide variatio i prevalece i otuberculous mycobacteria sesitivity (60). This meas that eve i populatios i which every perso was sesitized to otuberculous mycobacteria, tuberculi ski test specificity would still be 98. This modest effect will be cliically importat oly i populatios with a high prevalece of otuberculous mycobacterial sesitivity ad a very low prevalece of true TB ifectio (such as i the souther Uited States [63, 64]). For the diagosis of latet TB ifectio, the sesitivity of IGRA is less clear, although i study samples with a gradiet of exposure, the prevalece of positive test results was similar i the most-exposed categories. The biggest problem i estimatig sesitivity is the lack of a proper gold stadard for latet TB ifectio. I cross-sectioal studies, the most commoly used surrogate of latet TB ifectio has bee ewly diagosed, active TB. However, this is a poor surrogate because of the kow reductio i cell-mediated respose i such patiets. The ideal gold stadard i cross-sectioal studies would be healthy persos kow to have TB ifectio. The oly patiets who meet these criteria are those beig treated for active TB, who have cliically recovered. I a older multiceter study of more tha 1000 such patiets, tuberculi ski test reactios were remarkably similar i all populatios, ad overall sesitivity exceeded 93 (29). I 3 earlier studies of such patiets, sesitivity of purified protei derivative based IGRA raged from 59 to 71; however, tuberculi ski test sesitivity was 95 (65). Does this mea that the IGRA is less sesitive or that treatmet chages the immue respose to the IGRA measure? The results of our review idicate that IGRA respose geerally decreased with treatmet i patiets who had active TB, although this pheomeo was ot see i patiets with latet TB ifectio. But, why ad how would patiets with active TB differ from those with utreated latet TB ifectio after 3 to 6 moths of treatmet? This issue is uresolved. Ufortuately, there are o recet studies usig RD1-based IGRA i patiets who have recovered from active TB, although these would be of great iterest. The effect of treatmet o IGRA respose also caot be ascertaied without uderstadig the iheret biological variability i IGRA respose over moths or years. Short-term variability was approximately 9 i 2 studies (37, 38). However, i the oly 2 studies with follow-up of 1 to 2 years, spotaeous reversio of IGRA was documeted i 24 to 28 of persos ad was associated with discordat ad weaker iitial IGRA resposes (39, 44). Util the variability of IGRA respose has bee studied, the effect of treatmet o IGRA respose caot be ascertaied. Without this iformatio, the accuracy of evaluatio of IGRA sesitivity will ot be kow i patiets with active TB who have recovered cliically. The umerous studies that have used active TB as a surrogate for latet ifectio may give rise to the miscoceptio that these tests are useful for diagosig active TB. However, they do ot have optimal sesitivity, ad more importat, caot distiguish active from latet TB, which severely reduces their specificity for active TB. The oly exceptio is diagosis of active TB i childre, because tuberculi ski testig is commoly used for this idicatio. Results from our review idicate that sesitivity of QFT ad Elispot are somewhat lower i childre tha i adults. There are too few studies to make defiitive statemets, but the results of this review support the urget March 2007 Aals of Iteral Medicie Volume 146 Number 5

12 Diagosis of Latet Tuberculosis Ifectio Article eed for further evaluatio of IGRA i the diagosis of TB i pediatric patiets. We hope that i the ear future, the gold stadard will be logitudial studies that determie the icidece of active TB i persos with positive ad egative results o IGRA ad tuberculi ski tests. Results from these studies will establish the risk for TB i persos with positive results o IGRA particularly those with discordat egative results o tuberculi ski testig. As oted i our review, these discordat reactios are commo ad poorly uderstood at preset, rederig cliical maagemet of persos with such reactios very difficult. Some discordace may be explaied by radom variatio with 1 or both tests beig close to the cut-poit for positivity (66, 67). Oe importat use of tuberculi ski testig is serial testig of exposed populatios to detect ew TB ifectio. The variability of tuberculi ski testig has bee well established, but the variatio i IGRA respose has ot. Two studies have show that similar to the tuberculi ski test, IGRA shows ospecific variatios ad reversios from positive to egative results durig serial testig (39, 44). Util iformatio is available to defie IGRA coversio, results of serial IGRA testig will be largely uiterpretable. Recommedatios for Research O the basis of the data preseted, we recommed further research i the followig areas. Idepedet studies of the reproducibility of IGRA should be doe. To estimate test variability, repeated assays should be performed o the same samples by the same techicia, by differet techicias i the same laboratory, or i differet laboratories. The effect of mior modificatios i the field testig protocol, such as time from phlebotomy to icubatio, time of icubatio, or temperature of storage, should be tested i the same way. To estimate biological variability, repeated tests should be repeated i uexposed ad u- Table 7. Effect of Treatmet o Iterfero- Release Assay (IGRA) Respose i All Cohort Studies Usig RD1 Atiges ad Overight Assays Oly* Study, Year (Referece) Patha et al., 2001 (40) Carrara et al., 2004 (78) Nicol et al., 2005 (42) Aike et al., 2006 (38) Ewer et al., 2006 (44) Wilkiso et al., 2006 (79) Pai et al., 2006 (43) Coutry Test Type (Icubatio Time) Participats Uited Kigdom Elispot (14 h) Persos with active TB: 12 tested before, durig, ad after treatmet Italy Elispot Persos with active TB: 18 were treated South Africa Elispot (18 h) Pediatric patiets with active TB: 15 had probable or possible disease Gambia Elispot (6 14 h) Persos with active TB: 82 tested before ad after treatmet Uited Kigdom Elispot Persos with latet TB ifectio: 38 with positive TST ad IGRA results were treated; 11 with positive TST ad IGRA results were ot treated; 14 with egative TST results ad positive IGRA results were ot treated Uited Kigdom Elispot (14 h) Persos with latet TB ifectio: 33 received INH/RIF; 8 received o treatmet Idia QFT-IT (16 20 h) Persos with latet TB ifectio: 10 received INH Days Whe Tested 0 ad Chage or Differece Decrease Details 62 had a decrease i mea levels over a average of 19 wk 0, 90, ad 180 Decrease 13 of 18 had reversio to egative results 0 ad 30 Icrease the Mea levels icreased decrease by 45 after 1 mo of therapy compared with before therapy 0 ad 365 Decrease 82 had positive results before treatmet, ad 46 had positive results 6 mo after treatmet 0, 180, 365, ad 640 0, 26, ad 82 0, 365, ad 640 Decrease, o chage, ad decrease 6 of 38 (16) had reversio after treatmet; 0 of 11 (0) had reversio who were ot treated; ad 7 of 14 (50) had reversio without treatmet No chage Mea levels icreased durig treatmet but decreased at the ed of treatmet; o chage was see i utreated persos No chage Media levels 10 U 3 5U3 7.9 U; percetage positive ( 0.35): * I this study, differeces betwee group meas were less marked ad less sigificat after 18 hours tha after 6 days icubatio, but treds were similar. INH isoiazid; RIF rifampi; QFT-IT QuatiFERON-Gold I-Tube; TB tuberculosis; TST tuberculi ski test. Chage or differece comparig results o or after treatmet relative to before treatmet. 6 March 2007 Aals of Iteral Medicie Volume 146 Number 5 351

13 Article Diagosis of Latet Tuberculosis Ifectio treated persos at itervals ragig from days to years. This iformatio is crucial to distiguish radom variatio from coversios attributable to ew TB ifectio ad to study the effect of treatmet o IGRA respose. More studies are eeded that compare the sesitivity of the tuberculi ski test with IGRA i HIV-ifected ad other immue-compromised groups, itraveous drug users, ad pediatric ad elderly populatios. More data are eeded to uderstad discordat tuberculi ski test ad IGRA reactios, icludig the effect of chages i cut-poits, the role of otuberculous mycobacteria, ad time to coversio after exposure ad ifectio. Now that both IGRAs are widely commercially available, idepedet field studies ca evaluate the feasibility, utility, ad costs of these tests i differet populatios ad uder differet coditios. Such studies should report o the actual completio of tests ad the subsequet evaluatio ad treatmet for patiets with positive test results. Much of the value of a test is lost if persos who are tested do ot retur to lear the sigificace of their test result or if those with positive results are ot evaluated further. With these results, the cost-effectiveess of IGRA ad the tuberculi ski test ca be compared i real-world settigs. Fially, large-scale cohort studies are eeded that estimate risk for progressio to active disease i persos who have had tuberculi ski test ad IGRA. Of particular iterest is the risk for disease i persos with discordat reactios. From McGill Uiversity, Motréal, Québec, Caada, ad Johs Hopkis Uiversity, Baltimore, Marylad. Ackowledgmets: The authors thak Drs. Jaice Pogoda, Peter Bares, Philip Hill, Thomas Meier, Peter Aderse, ad Delia Goletti for providig additioal iformatio. Grat Support: Noe. Potetial Fiacial Coflicts of Iterest: Noe disclosed. Requests for Sigle Reprits: Dick Mezies, MD, MSc, Respiratory Epidemiology ad Cliical Research Uit, Motréal Chest Istitute, 3650 St-Urbai, Room K1.24, Motréal, Québec H2X 2P4, Caada; , dick.mezies@mcgill.ca. Curret author addresses ad author cotributios are available at Refereces 1. Matoux MC. La voie itradermique e tuberculiothérapie. Presse Med. 1912;20: World Health Orgaizatio. The WHO stadard tuberculi test. Geeva: World Health Orgaizatio; Bearma JE, Kleima H, Glyer VV, Lacroix OM. A study of variablilty i tuberculi test readig. Am Rev Respir Dis. 1964;90: [PMID: ] 4. Perez-Stable EJ, Slutki G. A demostratio of lack of variability amog six tuberculi ski test readers. Am J Public Health. 1985;75: [PMID: ] 5. Erdtma FJ, Dixo KE, Llewelly CH. Ski testig for tuberculosis. Atige ad observer variability. JAMA. 1974;228: [PMID: ] 6. Pouchot J, Graslad A, Collet C, Coste J, Esdaile JM, Viceeux P. Reliability of tuberculi ski test measuremet. A Iter Med. 1997;126: [PMID: ] 7. 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Goletti D, Carrara S, Viceti D, Saltii C, Rizzi EB, Schiià V,etal. Accuracy of a immue diagostic assay based o RD1 selected epitopes for active tuberculosis i a cliical settig: a pilot study. Cli Microbiol Ifect. 2006; 12: [PMID: ] 25. A Q, Buxto D, Hedricks A, Robiso L, Shah J, Lu L, et al. Compariso of amplified Q beta replicase ad PCR assays for detectio of Mycobacterium tuberculosis. J Cli Microbiol. 1995;33: [PMID: ] 26. Dogra S, Narag P, Mediratta DK, Chaturvedi P, Reigold AL, Colford JM Jr, et al. Compariso of a whole blood iterfero-gamma assay with tuberculi ski testig for the detectio of tuberculosis ifectio i hospitalized childre i rural Idia. J Ifect DOI: /J.Jif (electroic March 2007 Aals of Iteral Medicie Volume 146 Number 5