Interferon-gamma release assays and childhood tuberculosis: systematic review and meta-analysis

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1 INT J TUBERC LUNG DIS 15(8): The Union doi: /ijtld Published online 8 June 2011 REVIEW ARTICLE Interferon-gamma release assays and childhood tuberculosis: systematic review and meta-analysis A. M. Mandalakas,* A. K. Detjen, A. C. Hesseling, A. Benedetti, # D. Menzies * Department of Pediatrics, Case Western Reserve University, Cleveland, Ohio, USA; Desmond Tutu TB Centre, Department of Paediatrics and Child Health, Stellenbosch University, Cape Town, South Africa; International Union Against Tuberculosis and Lung Disease, New York, New York, USA; Respiratory and Epidemiology Clinical Research Unit, Montreal Chest Institute, Montreal Department of Medicine, and # Department of Epidemiology, Biostatistics & Occupational Health, McGill University, Montreal, Quebec, Canada SUMMARY BACKGROUND: Children infected with Mycobacterium tuberculosis have significant risk of developing tuberculosis (TB) and can therefore benefit from preventive therapy. OBJECTIVE: To assess the value of interferon-gamma release assays (IGRAs) and the tuberculin skin test (TST) in the diagnosis of TB infection and disease in children. METHODS: Thirty-three studies were included, assessing commercial IGRAs (QuantiFERON -TB [QFT] and T-SPOT. TB) and TST. Reference standards for infection were incident TB or TB exposure. Test performance for disease diagnosis was evaluated in studies assessing children with confirmed and/or clinically diagnosed TB, compared to children where TB was excluded. RESULTS: Two small studies measured incident TB in children tested with QFT and found weak positive predictive value. Association of test response with exposure categorized dichotomously or as a gradient was similar for all tests. The sensitivity and specificity of all tests were similar in diagnosing the disease. Stratified analysis suggested lower sensitivity for all tests in young or human immunodeficiency virus infected children. CONCLUSIONS: Available data suggest that TST and IGRAs have similar accuracy for the detection of TB infection or the diagnosis of disease in children. Heterogeneous methodology limited the comparability of studies and the interpretation of results. A rigorous, standardized approach to evaluate TB diagnostic tests in children is needed. KEY WORDS: tuberculosis; pediatrics; TB infection; IGRAs; tuberculin skin test THE WORLD HEALTH ORGANIZATION (WHO) estimates that 2 billion people are infected with Mycobacterium tuberculosis, 1 creating a reservoir that leads to 9 million new tuberculosis (TB) cases and 2 million deaths annually. Children carry approximately 15% of the disease burden, 75% of which occurs in the 22 high-tb-burden countries. 2 TB exposure in young children frequently occurs in the household, and the risk of non-household exposure increases with age. Young children not identified for preventive therapy have a disproportionately high risk of early progression to disease and severe forms of TB. 3 The global TB epidemic is exacerbated by human immunodeficiency virus-1 (HIV) infection, especially in sub- Saharan Africa. HIV-infected children have an up to 24-fold higher risk of developing TB compared to HIV-negative children. 4 There is no gold standard for the detection of AMM and AKD contributed equally to this work. M. tuberculosis infection. The tuberculin skin test (TST) has known limitations in terms of sensitivity and specificity, 5 14 which may place children at risk of under- or over-treatment for latent infection. This could unnecessarily burden patients, families and r esource-limited health systems, while potentially failing to identify those children at highest risk of TB disease progression. The diagnosis of active TB in children is also difficult due to the paucibacillary nature of childhood TB, which makes bacteriological confirmation challenging. As a result, the diagnosis of active disease in this population often relies on a composite of contact history, clinical symptoms and radiological findings, as well as consideration of the TST reaction. 15 The TST and the recently developed interferongamma release assays (IGRAs) are immune-based diagnostic tests. IGRAs measure ex-vivo interferon-gamma (IFN-γ) production by circulating T-lymphocytes when incubated in the presence of highly specific Correspondence to: Anna Mandalakas, Global Health & Diseases, and Epidemiology & Biostatistics, Case Western Reserve University, Cleveland, Ohio 44106, USA. Tel: (+1) Fax: (+1) anna.mandalakas@ case.edu Article submitted 14 October Final version accepted 21 December 2010.

2 IGRAs and childhood TB: systematic review 1019 M. tuberculosis antigens (early secreted antigenic target 6 [ESAT-6], culture filtrate protein 10 [CFP-10] ± TB7.7). There are two commercially available IGRAs: QuantiFERON -TB (QFT; QuantiFERON -TB Gold [QFT-G] and QuantiFERON -TB Gold In-Tube [QFT-GIT], Cellestis, Carnegie, VIC, Australia) and T-SPOT. TB (Oxford Immunotec, Oxford, UK). The QFT test incubates whole blood and measures IFN-γ production with an enzyme-linked immunosorbent assay (ELISA), while T-SPOT.TB measures the number of IFN-γ producing peripheral mononuclear cells (PBMCs). Like the TST, IGRAs cannot differentiate between M. tuberculosis infection and active TB. A growing number of studies have compared the TST and IGRAs in the detection of M. tuberculosis infection and active TB in children. In the absence of a gold standard for infection, some studies have measured sensitivity in populations with active TB as a surrogate for M. tuberculosis-infected persons, while others have used M. tuberculosis exposure as a surrogate for infection. 16,17 The value of IGRAs for the diagnosis of active TB and M. tuberculosis infection in children remains unclear. We systematically reviewed the existing evidence on the accuracy of IGRAs for the detection of M. tuberculosis infection and diagnosis of active TB in children in settings with varying incidence of TB. This review was presented at the July 2010 WHO expert meeting where recommendations were drafted for the use of IGRAs in high-burden settings. METHODS In collaboration with an experienced librarian at Case Western Reserve University, we systematically searched Medline and Web of Science for articles published in English, French or Spanish from 1998 until January Search terms included tuber culosis infection, or tuberculosis disease, AND P e diatrics or child*, AND QuantiFERON, or ELISpot, or interferon-gamma assays, or interferon-gamma release assays, or T-cell assays, AND ESAT-6, or CFP10, or RD1 antigens. We searched reference lists of all articles selected, existing systematic reviews and an existing IGRA database (courtesy of M Pai, McGill University, Montreal, QC, Canada). We also captured new publications identified until 1 June 2010 (Figure 1). We considered published peer-reviewed studies that included at least 20 children aged <18 years, assessing commercially available IGRAs (QFT-G, QFT- GIT and T-SPOT.TB). Studies using in-house assays or pre-commercial IGRA versions were excluded. Case series and case reports were not considered. Selection of articles for inclusion in this review was performed in two stages. Two reviewers (AM and AD) first screened titles and abstracts, and then performed a full text review to determine eligibility. Figure 1 Flow of study selection. * See Table 1. ISI = Institute for Scientific Information; IGRA = interferon-gamma release assay. Disagreements regarding inclusion were resolved by consensus, or with input from a third reviewer (DM) when consensus could not be reached. Information was extracted independently from all selected publications by the same two reviewers using a standardized data extraction form, developed, piloted and used specifically for this review. Discrepancies in extracted data were resolved by consensus. All reference standards were defined a priori: the primary reference standard for M. tuberculosis infection was incident TB in cohorts who were exposed but disease-free and tested for infection at study enrollment, then followed prospectively with active case finding to identify children who subsequently developed active TB. The secondary reference standard for M. tuberculosis infection was TB exposure, defined dichotomously (exposed or not) or as a gradient, based on index case microbiologic indicators (sputum smear) or proximity or duration of contact.

3 1020 The International Journal of Tuberculosis and Lung Disease For the assessment of sensitivity for the diagnosis of active TB, two definitions of disease were accepted: definite (confirmed) and probable TB. Definite TB was defined as the presence of at least one clinical specimen positive for M. tuberculosis on culture, or positive acid-fast bacilli smear microscopy, or one histology sample positive for necrotizing granulomas, or nucleic acid amplification test positive for M. tuberculosis. Probable TB was defined as the presence of three or more of the following: 1) chest radiologic findings consistent with active TB; 2) typical symptoms such as cough and weight loss; 3) other radiological evidence of active TB, including extra-pulmonary TB (e.g., computed tomography/magnetic resonance imaging findings consistent with TB meningitis) in conjunction with symptoms; 4) exposure to a case with active infectious TB; and 5) response to appropriate anti-tuberculosis therapy. TST and IGRA could not be included in either case definition. For assessment of specificity in the diagnosis of active TB, we used data from groups of children in whom active TB was systematically excluded: 1) TB suspects with symptoms suggestive of active TB, or 2) children with exposure to a case with active TB. We did not include data from groups if they did not have clear risk factors for active TB or if it was unclear whether active TB was systematically excluded in all subjects. Data were extracted on the number of positive, negative and indeterminate results for each of the reference standards and tests assessed. Data were also extracted to support stratified and subgroup analysis. We used 13 QUADAS (Quality Assessment of Diagnostic Accuracy Studies) items for the assessment of study quality and added an additional item on industry involvement. 18,19 Analysis Data were analyzed using SAS version 9.2 (SAS Institute Inc, Cary, NC, USA) and STATA version 11 (Stata Corporation, College Station, TX, USA). Several analytic approaches were used to evaluate test performance for the detection of M. tuberculosis infection. In studies that measured exposure dichotomously, we assessed the association between test result and TB exposure as an odds ratio (OR). These results were pooled using both a fixed- and a randomeffects approach. 20 In studies that expressed exposure via a gradient, we measured the correlation between exposure gradients and prevalence of positive tests. We estimated the Spearman correlation between the categorical test result and the outcome and calculated a pooled correlation coefficient for each test with both a fixed- and a random-effects approach. 21 Next, we used the OR as the measure of effect to assess performance by calculating the OR for each level of exposure relative to the reference group, and then an overall OR for increasing exposure category. We again used both fixed- and random-effects approaches to estimate a pooled exposure effect across studies. 22,23 We estimated inter-study heterogeneity via I 2 statistics. 20 To evaluate test performance for the diagnosis of active TB, we used data from studies that measured IGRAs in children with active TB to estimate test sensitivity and data from studies that included an appropriate group in whom active TB was excluded to estimate test specificity. We used a random effects meta-analysis to estimate the overall pooled estimates of sensitivity, specificity and 95% confidence interval (95%CI; Proc Nlmixed in SAS), and used the exact binomial likelihood approach to approximate the distribution of the outcome of interest. 24 We assessed heterogeneity by estimating the I 2 statistic and associated 95%CIs. 25 To calculate the I 2, zero cells were corrected by 0.5. To explore sources of heterogeneity, we performed sub-group analyses stratified by predefined covariates of interest. These included TB incidence, age, World Bank income, bacille Calmette- Guérin (BCG) vaccination rate, HIV prevalence, TST cut-off point and QFT test type (QFT-G or QFT-GIT). To assess the impact of indeterminate IGRA results, sensitivity estimates were calculated with indeterminates considered as false-negatives; the relationship between the frequency of IGRA indeterminate results and important covariates was described. RESULTS Selection and quality of studies Our search strategy identified 240 studies. From these, 67 articles were selected for full text review, of which 31 articles describing 32 studies (one article describing two studies conducted in two countries was included; Figure 1, Table 1) Thirtysix studies that completed full text review were excluded for a number of reasons, including data not available; 27,42,47,48,61,62,64,80,81,83,91 no exposure gradient used; 31,34,71,74,86,89,90 pre-commercial IGRAs used; 29,37,40,66,76,85 fewer than 20 children included; 28,60,65,70,77 IGRA testing completed in TSTpositive children only, precluding a comparison group; 46,56,78,93 study population overlapped with an included study; 39,84 and children with positive TST referred preferentially to the study, creating incorporation bias. 63 Studies were performed in 18 countries; the incidence of smear-positive TB was >25 per population in 10. Nineteen studies (59%) were performed in high-income countries; these included 11% to 100% immigrant children. The 32 studies described results in 5525 children; our analysis included 4122 of these children, as some sub-groups did not meet the criteria for any reference standard. The mean age of the children was 7.6 years (range ); BCG coverage ranged between 8% and 100%. Six studies included HIV-infected children (range

4 IGRAs and childhood TB: systematic review 1021 Table 1 Characteristics of included studies sorted by World Bank income index Author, year, reference Country World Bank income index TB incidence* Included for analysis/ total study n/n Mean or median age, years BCG Immigrants % % HIVinfected % Immune suppressed % Adetifa, The Gambia LIC / NR 1 NR Okada, Cambodia LIC / NR NR NR Petrucci, Nepal LIC / NR NR NR Dogra, India LMIC / NR 1 57 Nakaoka, Nigeria LMIC / NR NR NR Warier, India LMIC / NR NR NR NR Hansted, Lithuania UMIC / NR 0 NR Hesseling, South Africa UMIC / NR 0 0 Mandalakas, South Africa UMIC / NR Nicol, South Africa UMIC / NR 0.5 NR Petrucci, Brazil UMIC / NR NR NR Stavri, Romania UMIC 51 36/ NR Stefan, South Africa UMIC /34 7 NR NR Bamford, UK HIC / NR NR Bergamini, Italy HIC / Bianchi, Italy HIC / Chun, Korea HIC / NR 0 NR Connell, Australia HIC 2.8 9/ NR NR Connell, Australia HIC 2.8 9/ NR NR Detjen, Germany HIC / Diel, Germany HIC / Dominguez, Spain HIC / Girardi, Italy HIC 3.3 9/ NR NR Grare, France HIC 6.2 7/ NR NR Haustein, UK HIC / NR Herrmann, France HIC / Higuchi, Japan HIC / NR NR NR Higuchi, Japan HIC / NR NR NR Higuchi, Japan HIC / NR NR NR Kampmann, UK HIC / NR Lighter, USA HIC / Lucas, Australia HIC / NR NR * Incidence of smear-positive TB per population reported by the WHO in These data indicate the proportion of BCG-vaccinated/immigrants/HIV-infected or immune-suppressed children within the sub-group used for analysis. If the subgroup data were not available, data for the whole group were used. Author contacted to ensure that there was no data overlap with other publications or to provide clarification/re-analysis of data. Data provided in the published manuscript were analyzed as two independent studies. Median age used as mean age was not reported. TB = tuberculosis; BCG = bacille Calmette-Guérin; HIV = human immunodeficiency virus; LIC = low-income country; NR = not reported; LMIC = low- and middle-income countries; UMIC = upper-middle-income country; HIC = high-income country; WHO = World Health Organization ), 26,44,52,69,75,87 and three studies included children with non-hiv-related immune suppression. 52,87,88 Two studies reported incident TB in cohorts, 18 described the association of tests with exposure (Table 2), 21 assessed test sensitivity in children with active TB, and nine provided data that could be used to estimate test specificity for active TB. Studies evaluated one or more index tests, including T-SPOT.TB (n = 15), QFT-G (n = 10) and QFT-GIT (n = 21). Thirty studies provided TST data that could be used for analysis (Table 1). Assessment of the study quality using QUADAS showed that only a minority of studies clearly reported on sampling methods or included a spectrum of subjects representative of patients in whom the tests might be used clinically. Blinding of clinicians to IGRA results was reported in 29% of the studies assessing active TB. Of 32 studies, 11 (33%) were supported by either or both IGRA manufacturers, mainly through donation of test kits. In 43% of the studies assessing active TB, it remained unclear whether the reference standard was applied to all subjects included (i.e., whether active TB was excluded in all subjects). Seventeen of 21 studies (81%) described their definition of the reference standard in detail sufficient to support replication. However, there was still wide variation among studies regarding the criteria used and data provided on the definition of confirmed or probable TB. Test failure was infrequently reported. IGRA test failure was defined as technical errors, failed phlebotomy or insufficient peripheral blood mononuclear cells (T-SPOT.TB). For the TST, failure was defined as unread tests. Failure rates ranged from 0% to 7% for QFT-GIT (n = 19), 0% for QFT-G (n = 6), 0% to 21% for T-SPOT.TB (n = 15) and 0% to 11% for TST (n = 20). Average rates of indeterminate results across all studies were respectively 6.5% for QFT-GIT, 6.4% for QFT-G and 3.5% for T-SPOT.TB. In several studies, indeterminate rates >10% were associated with multiple factors such as young age, helminth coi nfection and immune suppression. 32,50,54,68,36,52,87,88,92 In our analysis, a significant correlation of indeterminate results with specific risk factors could not be

5 1022 The International Journal of Tuberculosis and Lung Disease Table 2 Characteristics of dichotomous and graded expression of M. tuberculosis exposure, sorted by World Bank income index Author, year, reference Dichotomous comparison groups Description exposed Description unexposed LMIC Hansted, Household or school contact No TB contact, no symptoms, chest radiography normal Hesseling, Known TB contact No known TB contact Mandalakas, Known household contact No known household contact Stefan, Known TB contact No known TB contact HIC Bianchi, TB contacts, Italian and immigrant Immigrants without TB contact Chun, Household contacts Contact outside household Dominguez, Children from contact investigations TST-positive children detected during routine screening* Higuchi, Same class as index case (contact 90 h) Same school, different classes from index case (contact <18 h) Lighter, Close contact to TB index case No risk factors for TB exposure Lucas, Immigrants with household contact Immigrants without household contact Graded exposure comparison groups (characteristics of exposure) Grade 0 Grade 1 Grade 2 Grade 3 LMIC Adetifa, Different house Different room Same room Nakaoka, Community controls Smear TB Smear+ TB Okada, Smear TB Smear+ TB Smear++ TB Smear+++ TB Petrucci, Scanty TB Smear+ TB Smear++ TB Smear+++ TB HIC Bergamini, Probable TB Smear/culture+ TB Smear+ TB Diel, h exposure h exposure h exposure 200 h exposure Girardi, Other students Activities with index case Attending class with index case * TST was not used for analysis due to incorporation bias LMIC = low- and middle-income countries; TB = tuberculosis; HIC = high-income countries; = negative; + = positive; TST = tuberculin skin test. Table 3 Concordance of tests for M. tuberculosis infection with dichotomized exposure to tuberculosis Test Author, year, reference Exposed positive/ total Unexposed positive/ total OR* Pooled effects OR (95%CI) Fixed Random TST 5 mm Bianchi, /38 154/ ( ) 1.3 ( ) Chun, /42 16/ Hansted, /45 36/ Higuchi, /38 186/ Lucas, /26 83/ Mandalakas, /6 4/ Stefan, /4 4/ TST 10 mm Bianchi, /38 31/ ( ) 1.9 ( ) Chun, /42 7/ Hansted, /45 34/ Hesseling, /26 1/2 1.2 Higuchi, /38 77/ Lighter, /13 8/ Lucas, /26 48/ Stefan, /4 4/ TST 15 mm Bianchi, /38 16/ ( ) 1.8 ( ) Hansted, /45 21/ Lucas, /26 18/ QuantiFERON -TB Bianchi, /38 35/ ( ) 3.5 ( ) Chun, /42 2/ Dominguez, /64 16/ Hesseling, /16 0/2 5.0 Higuchi, /41 3/ Lighter, /13 0/30 94 Lucas, /33 38/ Mandalakas, /3 2/9 0.4 Stefan, /3 3/ T-SPOT.TB Dominguez, /63 13/ ( ) 1.3 ( ) Hansted, /45 5/ Hesseling, /25 1/ Lucas, /30 32/ Mandalakas, /6 4/ Stefan, /3 5/ * In the calculation of ORs, a value of 0.5 was added to all cells if one cell had a 0 value. Indicates country classified as low-, low and middle or upper middle-income country according to the World Bank. Three exposure groups defined by Lighter et al. However, results from the intermediate group were excluded from analysis, as their exposure was judged too heterogeneous. OR = odds ratio; CI = confidence interval; TST = tuberculin skin test.

6 IGRAs and childhood TB: systematic review 1023 shown; there was no difference in the frequency of IGRA indeterminate results in stratified analysis. Results of review: latent TB infection Two longitudinal studies assessed incident active TB. 43,57 A school outbreak investigation in Japan assessed 313 children with TST and QFT-G tests. 57 QFT-G-positive children and QFT-G-indeterminate/ TST-positive children received preventive chemotherapy. One year after the index case was reported, all children underwent chest radiography; no child developed active TB during the 3-year follow-up (positive predictive value [PPV] 0%, 95%CI 0 35, negative predictive value [NPV] 100%, 95%CI 0 1.5). German contact investigations assessed 168 children with QFT-GIT and completed approximately 2 years of follow-up. 43 Three of seven QFT-GIT-positive children developed probable TB (PPV 43%, 95%CI 16 75), whereas none of the 161 QFT-GIT-negative children developed active TB (NPV 100%, 95%CI 0 3). As seen in Table 3, the pooled ORs (random effects model) for the association of positive TST using respectively 5, 10 or 15 mm cut-offs, with dichotomous exposure, was 1.34 (95%CI ), 1.93 (95%CI ) and 1.83 (95%CI ). For QFT-G/QFT-GIT, the pooled OR was 3.51 (95%CI ) and for T-SPOT.TB it was 1.31 (95%CI ; Table 3). When the analysis was restricted to low- and middle-income countries (LMICs), QFT and T-SPOT.TB results were positively correlated with exposure (OR 1.30 and 2.24, respectively), but TST was not (OR 1.04 [95%CI ], 0.81 [95%CI ] and 0.48 [only one study] for cut-offs of 5, 10 and 15 mm, respectively). The pooled correlation coefficient between QFT-G/ QFT-GIT results and TB exposure expressed as a gradient (random-effects model) was 0.19 (95%CI ). The pooled correlation coefficient for T-SPOT.TB was 0.13 (95%CI ) and for TST respectively 0.17 (95% CI ), 0.18 (95% CI ) and 0.09 (95%CI ) when 5, 10 and 15 mm were used as the cut-off (Table 4). Findings were similar when the analysis was restricted to studies from LMIC. We also estimated slopes of the effect of TB exposure by regressing the log-or between each successive higher exposure category. Using either fixed- or random-effects models, all slope estimates were similar and associated with wide CIs, such that no test could be declared superior (Figure 2). Regression slopes for studies from LMICs showed a similar trend (Figure 2). Sensitivity and specificity in active TB The sensitivity and specificity among all children with confirmed and/or probable active TB was slightly Table 4 Correlation of tests for M. tuberculosis infection with graded exposure to tuberculosis Test Author, year, reference Exposure category, n positive/total tested Pooled correlation (95%CI) 0 least most* Fixed Random I 2 TST 5 mm Bergamini, /99 10/93 16/ ( ) 0.17 ( ) 0.67 Diel, /55 10/35 3/62 13/62 Nakaoka, /41 23/80 49/78 Petrucci, /9 24/36 21/40 35/60 Petrucci, /10 9/40 17/49 8/14 TST 10 mm Adetifa, /72 34/126 16/ ( ) 0.18 ( ) 0.71 Bergamini, /99 2/93 9/69 Diel, /55 3/35 0/16 1/62 Nakaoka, /41 13/80 38/72 Okada, /54 14/64 13/49 12/28 Petrucci, /9 22/36 17/40 33/60 Petrucci, /10 8/40 16/49 8/14 TST 15 mm Bergamini, /99 1/93 5/ ( ) 0.09 ( ) 0.75 Diel, /55 1/35 0/16 0/62 QFT-G/QFT-GIT Adetifa, /70 42/26 15/ ( ) 0.19 ( ) 0.88 Bergamini, /71 5/47 1/36 Diel, /55 3/35 0/16 2/62 Girardi, /1 4/7 0/1 Nakaoka, /39 8/81 53/72 Okada, /54 12/64 9/49 9/38 Petrucci, /9 17/35 19/38 30/59 Petrucci, /10 10/39 23/49 10/14 T-SPOT.TB Adetifa, /72 40/126 14/ ( ) 0.13 ( ) 0.20 Bergamini, /14 0/56 4/40 Girardi, /1 4/7 0/1 * In four studies there were only three exposure categories. In Bergamini, one un-exposed group had significant prior risk of exposure, and was excluded from analysis. Indicates country classified as low-, low and middle- or upper middle-income according to the World Bank. Petrucci 2008 study conducted in two countries. Results shown separately for each country. CI = confidence interval; TST = tuberculin skin test; QFT-G = QuantiFERON -TB Gold; QFT-GIT = QuantiFERON -TB Gold In-Tube.

7 1024 The International Journal of Tuberculosis and Lung Disease higher for both IGRAs than for the TST, but the CIs were overlapping. Forest plots show study-specific and pooled estimates of sensitivity and specificity by World Bank income index (Figures 3 and 4). There was significant heterogeneity among studies, suggesting that pooled results be interpreted cautiously (Table 5). Stratified analysis suggested reduced sensitivity of TST, QFT-G/QFT-GIT and T-SPOT.TB in studies conducted in LMICs or when the average age was <5 years, all children were HIV-infected or >50% were BCG-vaccinated. The inclusion of indeterminate results as false-negatives had a modest effect on the sensitivity of both IGRAs (Table 6). No studies systematically assessed operational aspects of IGRAs such as cost, reproducibility, transport time, time to results or impact on treatment or feasibility. Several studies briefly addressed operational challenges such as phlebotomy in young children or the relatively high costs of IGRAs for lowincome settings. 44,50,73,79,87 Figure 2 Regression slopes for exposure gradients. The slopes are estimated from the regression of the logs of ORs of each successive higher exposure compared to the least exposed group. Hence, a steeper slope represents a greater change in the log ORs as exposure increases. A greater change in the log OR in response to increasing exposure (i.e., steeper slope) suggests that a test is better able to detect infection. A random effects model was used to calculate regression slopes for TST 10 mm and QFT. A fixed effects model was used to calculate the slopes for TST 5 mm, 15 mm and T-SPOT.TB. HIC = highincome country; LMIC = low- and middle-income country; TST = tuberculin skin test; QFT-G = QuantiFERON Gold; QFT-GIT = QuantiFERON Gold In-Tube; OR = odds ratio. DISCUSSION Commercial IGRAs are increasingly used and recommended for the diagnosis of M. tuberculosis infection, mainly in high-income countries with a low incidence of active TB The value of IGRAs in TB intermediate and high-burden settings is less clear and depends not only on test performance but also on the availability of preventive TB therapy, health systems capacity, laboratory infrastructure and associated health system costs. Figure 3 Pooled sensitivity in active TB, stratified by World Bank income index. (continued )

8 IGRAs and childhood TB: systematic review 1025 Figure 3 (Continued ) Various systematic reviews on IGRAs have been published, mainly focusing on adults. 97,98 Unique considerations in children include their developing immune system, high risk of disease progression following recent exposure and substantial benefits from preventive therapy. Current recommendations in highburden settings are to provide preventive therapy to children based on risk factors if the TST is not available. 99 An accurate test for the identification of M. tuberculosis-infected children could support more focused programmatic delivery of preventive therapy to at-risk children. As there is no gold standard for the diagnosis of M. tuberculosis infection, surrogate measures for infection are considered reference standards in diagnostic studies. Existing systematic reviews on the value of IGRAs for M. tuberculosis infection have mainly used active TB as a surrogate reference standard. 97,98 This approach assumes that estimates of IGRA performance in populations with active TB reflect performance in

9 1026 The International Journal of Tuberculosis and Lung Disease Figure 4 Pooled specificity in active TB, stratified by World Bank income index.

10 IGRAs and childhood TB: systematic review 1027 Table 5 Characteristic of comparison groups for active TB, sorted by World Bank income index Author, year, reference Active TB category* No TB category LMIC Dogra, Definite/probable TB combined Hospitalized children with clinical suspicion of TB or TB contact, TB disease was ruled out Warier, Definite TB, probable TB Hospitalized children with other diagnosis, no TB contact Hansted, Definite TB Reported group not used Nicol, Definite/probable TB combined Children admitted for either clinically suspected TB or TB contact, active TB ruled out by CXR and culture Stavri, Definite TB No group reported HIC Bamford, Definite TB, probable TB No group reported Bergamini, Definite/probable TB combined Reported group not used Bianchi, Definite/probable TB combined Reported group not used Chun, Probable TB TB ruled out, other diagnosis Connell, Definite/probable TB combined Reported group not used Connell, Probable TB Reported group not used Detjen, Definite TB Children with other respiratory illness, low risk for TB Dominguez, Definite/probable TB combined Reported group not used Grare, Definite/probable TB combined Reported group not used Haustein, Definite TB, probable TB Reported group not used Herrmann, Definite/probable TB combined Children hospitalized for any other disease, no TB contact Higuchi, Definite/probable TB combined School outbreak investigation, active TB excluded by CXR (detected in school outbreak) Higuchi, Definite/probable TB combined Reported group not used Kampmann, Definite TB, probable TB Children with risk factors for TB but disease ruled out, other diagnosis made Lighter, Definite/probable TB combined Reported group not used * Definite TB = culture-confirmed disease, probable TB = diagnosis made on the basis of symptoms and radiologic findings, no culture result. Definite/probable combined = some cases confirmed but others diagnosed on clinical and radiological criteria only, and results not stratified by method of diagnosis. No TB = active TB systematically excluded in either TB suspects with symptoms suggestive of active TB, or TB contacts. No group reported = study assessed active TB group only. Reported group not used = reported control group did not meet review criteria for an appropriate control group. TB = tuberculosis; LMIC = low- and middle-income countries; CXR = chest radiography; HIC = high-income countries. populations with infection. Not only does the immune status of diseased persons differ from that of infected persons, but populations evaluated for active TB differ greatly from populations evaluated for infection. For M. tuberculosis i nfection, this review therefore used two reference standards, incident TB and TB exposure, which were considered superior surrogate measures of infection. A test identifying individuals at highest risk for disease progression would greatly aid in the selection of individuals who would most benefit from preventive therapy. Longitudinal studies using incident TB as the reference standard are therefore ideal. We identified two studies completed in high-income countries that employed this reference standard using commercial IGRAs. These studies, which included very small sample sizes of QFT-positive children, showed a weak association between positive QFT assays and subsequent active TB, suggesting low PPVs. 43,57 Noncommercial ELISpot assays (not included in this review) have also shown performance similar to the TST in longitudinal studies assessing incident TB. 29,100 Exposure to M. tuberculosis increases the likelihood of infection. A strong correlation of the index test with exposure therefore suggests that the index test can identify people most likely to benefit from preventive therapy. We compared the performance of the IGRAs and the TST in exposed vs. unexposed individuals as well as across a gradient of exposure and found comparable performance between all tests using both methodological approaches. Our analysis compared differences in exposure gradients (our gold standard comparison) rather than absolute exposure. Although studies used different selection methods and measure of exposure, we analyzed studies collectively by defining study-specific gradients that progressed from the least to highest exposure. In theory, a steeper slope in the regression analysis is associated with a greater change in odds across exposure categories, indicating increased ability of the test to distinguish infection across exposure categories. Of note, differences between exposure grades were sometimes subtle (e.g., exposure to smear+ vs. smear++ TB); the expected magnitude of the test effect is therefore unclear. Despite the methodological heterogeneity of study groups, we found a positive and increasing correlation between each test and each progressive grade of exposure. This correlation would be stronger if studies used a standardized tool to measure TB exposure. However, despite the heterogeneous nature of the studies, our analysis clearly illustrates that both the IGRAs and the TST have the ability to detect M. tuberculosis infection. In studies reviewed for dichotomous exposure, the overall odds of a positive QFT-G or QFT-GIT was higher in exposed than unexposed children (OR 3.51, 95%CI ). QFT may differentiate between these two groups more accurately than the TST (10 mm cut-off) and T-SPOT.TB (OR 1.93, 95%CI and OR 1.31, 95%CI , respectively). Nevertheless, the wide and overlapping CIs preclude identification of a superior test and highlight the significant heterogeneity among studies. Studies were performed in countries and populations

11 Table 6 Diagnostic accuracy of TST, QFT and T-SPOT.TB for definite and/or probable active TB stratified by key variables n Sensitivity (true-positives) Positive/ tested n/n Sensitivity % (95%CI) n Specificity (false-positives) Positive/ tested n/n Specificity % (95%CI) TST Overall* / (70 90) 6 105/ (63 100) TST, mm / (84 98) 4 104/ (17 100) / (75 93) 5 86/ (62 100) / (50 83) 3 44/ (71 100) World Bank income index HIC / (73 90) 4 94/ (39 100) LMIC 4 74/ (45 94) 2 11/ (77 100) TB incidence < / (73 93) 3 75/ (36 100) / (39 84) 3 30/ (71 100) BCG vaccination, % < /66 85 (70 100) 1 0/ (85 100) / (65 90) 5 105/ (50 100) Age, years <5 5 77/ (57 98) 3 27/ (75 100) / (69 93) 3 78/ (35 100) HIV prevalence, % < / (73 90) 6 105/ (63 100) /36 39 (0 92) 0 QFT (QFT-G and QFT-GIT unless otherwise specified) Overall / (75 92) 6 71/ (78 100) Overall, indeterminates / (71 92) QFT test type QFT-G 5 63/74 92 (82 100) 1 53/82 35 (0 80) QFT-GIT / (65 88) 6 66/ (86 100) World Bank income index HIC / (78 94) 5 65/ (74 100) LMIC 2 22/37 58 (28 87) 1 6/95 94 (71 100) TB incidence < / (78 93) 4 64/ (59 100) /42 68 (34 78) 2 6/ (19 100) BCG vaccination, % < /62 93 (85 100) 1 0/ (84 100) / (68 87) 5 71/ (69 100) Age, years <5 4 54/58 94 (86 100) 2 1/92 99 (57 100) / (69 92) 4 70/ (66 100) HIV prevalence, % < / (76 92) 6 71/ (78 100) /27 63 (16 100) 0 0 T-SPOT. TB Overall 9 194/ (63 100) 4 12/ (87 100) Overall, indeterminates 9 194/ (59 100) World Bank income index HIC 6 126/ (67 100) 2 3/46 95 (84 100) LMIC 3 68/ (23 100) 2 9/97 93 (83 100) TB incidence < / (68 100) 2 3/46 95 (86 100) / (29 100) 2 9/97 93 (83 100) BCG vaccination, % < /44 97 (92 100) 1 0/ (84 100) / (45 93) 3 11/75 87 (53 100) Age, years <5 2 49/86 74 (23 100) 2 8/71 92 (80 100) / (65 100) 2 4/72 95 (88 100) HIV prevalence, % < / (63 100) 4 12/ (87 100) * For overall and stratified analysis, a TST cut-off of 10 mm was preferentially used; for two studies, TST 5 mm data were used and for one study, TST 15 mm data were used as these were the only data available; one of these studies was conducted in HIV-infected children. Incidence of smear-positive TB per population reported by the WHO in These data indicate the proportion of BCG-vaccinated within the subgroup used for analysis. BCG vaccination information not provided in one study; study was categorized as >50% BCG vaccinated as national guidelines recommend neonatal. Indeterminate results were included as false-negative in this analysis of the all TB group. Indeterminates were excluded for all other analysis. HIV prevalence in study population. If HIV status of subjects not reported, study was categorized as having <15% of subjects HIV-infected. TST = tuberculin skin test; QFT = QuantiFERON -TB; TB = tuberculosis; CI = confidence interval; BCG = bacille-calmette Guerin; HIV = human immunodeficiency virus; QFT-G = QuantiFERON -TB Gold; QFT-GIT = QuantiFERON -TB Gold In-Tube; WHO = World Health Organization.

12 IGRAs and childhood TB: systematic review 1029 with different annual risk of TB infection and other factors modifying the risk of infection, such as diagnostic delay and strain type. The categories unexposed and exposed therefore describe very different populations in each study. Sub-group analysis, including World Bank income status, did not suggest possible explanations for differences in estimates of test performance. By comparing studies with similar methodological approaches and comparable study populations, the correlation of either test with exposure would be stronger and differences between tests might become apparent. For the diagnosis of active TB, the overall sensitivity of both IGRAs and the TST was similar when assessed in children with all categories of active TB combined. Our results suggest that TST and QFT sensitivity may be higher in children with definite TB compared to children with definite and probable TB. Although estimates of test sensitivity are most certain in the definite TB group, study-specific case definitions of probable TB varied considerably and might have introduced differential bias among studies. The assessment of test performance in children with all categories of active TB is thus most representative of clinical practice and provides useful insight upon which to base clinical guidelines. Overall, the ability of either TST or IGRAs was suboptimal to rule in or rule out active TB, reinforcing the appropriate use of these tests as adjuncts in the clinical diagnosis of active TB. Young children have an increased risk of developing active TB after infection, which may indicate differences in the pediatric immune system compared to adults. Individual studies have suggested poorer sensitivity and higher rates of indeterminate results in children aged <5 years, who could most benefit from accurate detection of M. tuberculosis infection. 32,50,54,59 Similarly, our stratified analysis found a trend towards lower sensitivity of all tests in studies assessing children younger than vs. older than 5 years. Nevertheless, the small number of studies completed in younger children and the lack of data reported by age strata limited our ability to demonstrate a statistically significant difference. Our review clearly highlights the need for more data on test performance in young children. Trends in subgroup and stratified analysis suggest that the sensitivity of all tests for the diagnosis of disease may be dependent on the study setting and population. The sensitivity of all tests may be higher in high-income countries, and T-SPOT.TB could be the most sensitive in LMICs, although more robust data are needed. In contrast, all tests may have lower sensitivity in HIV-infected children. Indeterminate results may lower the sensitivity of IGRAs slightly. Most interesting was a tendency towards lower sensitivity of all tests in study populations with >50% BCG coverage. Although BCG vaccination can explain differences in specificity between TST and IGRAs, BCG is not expected to influence sensitivity. We note that BCG coverage is high in countries with higher TB burden or among immigrants in low-burden settings. Populations with higher BCG coverage rates may also have higher rates of underlying conditions that may impair test accuracy, such as co-infections with helminths and malnutrition. 68 Although small subgroup sample sizes limited our ability to demonstrate statistically significant differences, trends in sub-group analysis highlight important factors to consider when choosing a preferred testing strategy in different settings or patient groups or targeting research areas. Operational aspects and feasibility are important considerations when contemplating the programmatic implementation of a diagnostic test, particularly in resource-constrained settings. We aimed to assess some important aspects such as cost, transport times, reproducibility and feasibility in this systematic review, but studies rarely addressed operational aspects of study implementation or test performance. Future studies should assess operational aspects and feasibility of IGRAs to comprehensively inform guidelines regarding their use in children. Our review had limitations. In addition to those previously discussed, the studies included assessed very different populations in diverse settings. Sample sizes were less than 10 in some subgroup analyses, limiting our ability to generalize these subgroup results. The tremendous variation in methodological approaches and the use of non-uniform reference standards led to significant analytic heterogeneity. Nevertheless, we used a variety of analytic approaches to improve the robustness of our findings. A limited number of studies employed an acceptable definition of the non-diseased group, limiting our power to estimate specificity. Although additional data in children exist on inhouse assays and pre-commercial versions of current IGRAs, this review focused on two standardized commercial IGRAs, which are the most widely used and have recently been considered for policy guidance and programmatic implementation. This review found that TST, QFT and T-SPOT.TB had similar accuracy for the diagnosis of M. tuberculosis infection and active TB in children. Diverse study methodologies limited the comparability of studies and interpretation of results, emphasizing the importance of rigorous, standardized approaches for the evaluation of TB diagnostic tests in children. Welldefined exposure gradients serve as a good surrogate reference standard for M. tuberculosis infection and patient-important outcomes. Based on the evidence generated by this review, the WHO s Strategic and Technical Advisory Group for TB (STAG-TB) has discouraged the use of IGRAs for the diagnosis of active TB and M. tuberculosis infection in children living in LMICs. 101

13 1030 The International Journal of Tuberculosis and Lung Disease There is a clear need for more coordinated research on the value of IGRAs in different pediatric populations in settings with high burden of TB and HIV. Although we were not able to determine a superior test, our analysis confirms that the TST and both IGRAs are able to identify children with M. tuberculosis infection. In settings with high burdens of TB, accurate identification of children with M. tuberculosis infection may help guide the targeted delivery of isoniazid preventive therapy (IPT), and might improve the cost-effectiveness of IPT. Children of all ages, but particularly very young and HIV-infected children with the highest risk of disease progression, stand to benefit most from TB preventive therapy. Although the limited available data did not support a meaningful comparison of test performance for the TST and the commercial IGRAs in these subgroups of vulnerable children, more high-quality diagnostic studies and cost-effectiveness analyses are needed to inform recommendations regarding IGRA use in these high-risk groups. There was sufficient data to assess test performance in the diagnosis of active TB. Our analysis demonstrated that neither the TST nor the IGRAs perform sufficiently to rule in or rule out active TB as a single test. Our findings reinforce the acceptable use of these tests as adjuncts in the clinical diagnosis of active TB where resources are sufficient to support the use of a test for infection. Acknowledgements The authors thank the studies authors for kindly providing additional information upon request, including I Adetifa, S Arend, P Beffa, A Bose, T Connell, R Diel, G Dixon, J Dominguez, M Grare, K Higuchi, Y Kang, Y Kobashi, B Kampman, Y Lee, W Lew, M Losi, M Lucas, P Mantegani, B McKinnon, A Nienhaus, R Petrucci, H Pollack and E Tavast. The authors thank M P McGraw, Case Western Reserve University, for support to complete their electronic literature search and K Steingart for helpful discussions on QUADAS. This systematic review was made possible by the generous support of the American people through the United States Agency for International Development (USAID) under Cooperative Agreement GHN-A The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government. DM and AB receive salary support from Fond de la recherche en Santé de Québec. 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