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Diagnostic Accuracy of Early Secretory Antigenic Target-6–Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube

Diagnostic Accuracy of Early Secretory Antigenic Target-6–Free Interferon-gamma Release Assay... Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Clinical Infectious Diseases MAJOR ARTICLE Diagnostic Accuracy of Early Secretory Antigenic Target-6–Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube 1, 1 1 1 1 1 1 Elisa Nemes, Deborah Abrahams, Thomas J. Scriba, Frances Ratangee, Alana Keyser, Lebohang Makhethe, Mzwandile Erasmus, 1 1 1 1 1 2,a 3 2 Simbarashe Mabwe, Nicole Bilek, Virginie Rozot, Hennie Geldenhuys, Mark Hatherill, Maria D. Lempicki, Line Lindebo Holm, Leah Bogardus, 2,a 3 4 2 4 4 3 3 Ann M. Ginsberg, Thomas Blauenfeldt, Bronwyn Smith, Ruth D. Ellis, Andre G Loxton, Gerhard Walzl, Peter Andersen, and Morten Ruhwald 1 2 South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular Medicine & Division of Immunology, Department of Pathology, University of Cape Town; AERAS, 3 4 Rockville, Maryland; Statens Serum Institute, Copenhagen, Denmark; and South Africa Department of Science and Technology–National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town Background. Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) an- tigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6–free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6–containing vaccines. We aimed to qualify a recently developed ESAT-6–free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). Methods. Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6–free IGRA cuto, t ff est assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen- coated blood collection tubes. Results. ESAT-6–free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. e ESA Th T-6–free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6–free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74–0.90; McNemar test P = .48). ESAT-6–free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. Conclusions. e Th novel ESAT-6–free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection. Keywords. tuberculosis infection; diagnosis; IFN-γ release assay; qualification. Early secretory antigenic target-6 (ESAT-6) is an immunodominant 1 (ESX1) type VII secretion system [4] and are absent from bacille antigen expressed by Mycobacterium tuberculosis (M.tb) [1]. Calmette-Guerin (BCG), thereby, importantly, enabling distinc- ESAT-6 and its heterodimeric complex protein CFP-10 are the tion of M.tb infection from prior BCG vaccination [5]. principal components of immunodiagnostic tests for M.tb in- ESAT-6 is also increasingly recognized as an important fection, including the interferon-gamma (IFN-γ) release assay vaccine antigen with unique properties for infection contain- (IGRA) and novel specific skin tests [2, 3]. These small, highly ment and, potentially, for clearance [6–8]. As such, ESAT-6 immunodominant antigens are encoded in the region of differ- is contained in many candidate vaccines against tubercu- ence 1 of the M.tb genome, which comprises many important vir- losis (TB), including the subunit vaccine H56:IC31, which is ulence factors as components and substrates of the ESAT-6 system undergoing phase 2 clinical testing [6, 9]. Vaccine efficacy to prevent established M.tb infection, as a likely predictor of pre- vention of TB disease, has been proposed as an innovative and Received 4 October 2018; editorial decision 28 December 2018; accepted 16 January 2019; cost-effective endpoint to rationalize TB vaccine development published online January 21, 2019. a [10]. We recently showed that BCG revaccination or vaccina- Present address: International AIDS Vaccine Initiative, New York, NY, USA. Correspondence: M. Ruhwald, Center for Vaccine Research, Artillerivej 5 | 2300 København tion with a novel subunit vaccine candidate (H4:IC31) may S | Denmark (moru@ssi.dk and ssi.dk). oer p ff artial protection against sustained M.tb infection, meas- Clinical Infectious Diseases 2019;69(10):1724–30 ured by QuantiFERON-TB Gold In-tube (QFT) [11]. That trial © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons provided the first proof-of-concept efficacy signal for a subunit Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted vaccine candidate, which is very similar to H56:IC31 but does reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciz034 not contain ESAT-6. Such a clinical trial design, one that relies 1724 • cid 2019:69 (15 November) • Nemes et al Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 on IGRA to measure M.tb infection, cannot currently be used to sputum liquid culture and/or GeneXpert and/or smear micros- evaluate ESAT-6–containing vaccine candidates, since vaccine- copy. Patients with HIV infection, multidrug-resistant TB, or induced immune responses are cross-reactive with those meas- those who started TB treatment more than 3 days before enroll- ured by IGRA, rendering the test false positive. ment were excluded. Cohort 3: A subset of healthy adolescents Until recently, inclusion of ESAT-6 and CFP-10 has been screened for enrollment in a prevention-of-M.tb-infection trial considered essential for adequate immunodiagnostic sensi- at SATVI (HREC reference: 471/2013; clinicaltrials.gov identi- tivity. However, recent advances in antigen discovery tools and fier: NCT02075203) [11] were included in this study to evaluate M.tb antigen recognition studies in humans point to novel di- ESAT-6–free IGRA performance compared to QFT. agnostic antigens that could replace ESAT-6 [12–14]. Several Inclusion and exclusion criteria for cohort 3 have been studies explored the diagnostic potential of new candidates ei- described in detail elsewhere [16]. QFT results from cohort 2a, ther alone or in combination, and we recently established an 2b, and 3 have been published in a different form [16]. ESAT-6–free IGRA antigen cocktail aer s ft creening of several Assessment of Preselected Antigens candidates (TB7.7, EspJ, EspC, EspF, EccD1, PE35, and Rv2348) Venous blood was collected in lithium heparin–containing compared to ESAT-6 and CFP-10 [15]. vacutainers, and 1  mL per tube was dispensed in stimulation In this study, our aim was to confirm the recognition of an tubes (Sarstedt, Numbrecht, Germany) that contained (a) no an- ESAT-6–free IGRA antigen cocktail in adolescents from a high tigen, or pools of overlapping 15–20 mer peptides representing: TB burden area, define a diagnostic algorithm for the ESAT-6– (b) full length CFP-10, (c) a fragment of EspC (aa54-103), (d) free IGRA, evaluate ESAT-6–free IGRA diagnostic performance a fragment of EspF (aa9-44), (e) a fragment of Rv2348 (aa56- compared to QFT, and assess the variability of ESAT-6–free 108), or  (f) ESAT-6–free IGRA antigen cocktail (b+c+d+e, at IGRA blood collection tubes. a concentration of 5  μg per peptide/mL blood, as described [15]). Phytohemagglutinin (Dartford, Remel, United Kingdom) METHODS was used as the positive control (5  μg/mL), and a set of QFT Study Participants tubes (Nil, TB antigen, mitogen; Qiagen, Hilden, Germany) was All studies that involved human participants were conducted run in parallel as the reference. Within 2 hours of phlebotomy, according to the principles described in the Declaration of stimulated blood was incubated at 37°C for 18–22 hours and Helsinki and were approved by relevant institutional review then centrifuged. Plasma was harvested and cryopreserved at boards (Human Research Ethics Committee [HREC]). Written –80°C until batch analysis. informed assent and consent were obtained from participants and their legal guardians (for minors). QFT and ESAT-6–Free IGRA Assays We included the following 4 independent cohorts of patients Blood was collected directly in QFT tubes and in tubes that to address the study aims (Supplementary Figure 1A). Cohort contained lyophilized ESAT-6–free IGRA peptides, manu- 1: Healthy adolescents (cohort 1a) and adults (cohort 1b) were factured at SSI, as described [15], to define a cutoff for the enrolled at the South African Tuberculosis Vaccine Initiative ESAT-6–free IGRA assay, determine assay performance in (SATVI, South Africa, HREC reference: 126/2006) to confirm adolescents, and assess tube variability. Blood was processed ESAT-6–free IGRA antigen recognition and perform ESAT-6– following a standard operating procedure that was stricter free IGRA qualification, respectively. Individuals who had re- than that recommended by the manufacturer in order to re- ceived a tuberculin skin test (TST) in the last 12  months, had duce known sources of assay variability and improve reproduc- evidence of any acute or chronic disease or symptomatic infec- ibility, as described [16]. Briefly, 1  mL of blood was collected tion (including human immunodeficiency virus [HIV] positive and mixed by 10 manual inversions and 5 minutes on a tube or TB symptoms), currently used immune-modifying drugs, rotator and incubated at 37°C within 2 hours of phlebotomy, had been enrolled in any experimental study, were pregnant or for 16–20 hours. lactating (women), weighed <50 kg, or those with raised suspi- To determine the ESAT-6–free IGRA assay cuto, b ff lood cion of anemia by recruiting officer were excluded. Cohort 2a: was collected and processed at SSI (M.tb unexposed controls, Healthy adults with no known exposure to M.tb were recruited cohort 2a) and IRG (TB patients, cohort 2b). Cryopreserved at the Statens Serum Institut (SSI) in Denmark (HREC ref- plasma was analyzed by QFT IFN-γ enzyme-linked immuno- erence: H-3-2012-008). Individuals with a medical history sorbent assay (ELISA) at SATVI. of M.tb exposure, travel, and/or risk-seeking behavior were To assess the performance of ESAT-6–free IGRA in South excluded. Cohort 2b: Adult patients with pulmonary TB di- African adolescents, samples were collected and processed at sease were recruited by the Immunology Research Group (IRG) SATVI. Blood was simultaneously collected and processed for at Stellenbosch University, South Africa (HREC reference: the QFT and ESAT-6–free IGRA. As results from QFT analysis N13/05/064). TB disease was microbiologically confirmed by were part of the screening procedures for real-time enrollment M.tb Infection Diagnosis by ESAT-6–Free IGRA • cid 2019:69 (15 November) • 1725 Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 in a clinical trial [11], fresh plasmas harvested from QFT tubes adolescents from a high TB burden area who represent a target were analyzed by QFT IFN-γ ELISA, while plasmas harvested population for prevention of M.tb infection trials (cohort 1a). from ESAT-6–free IGRA tubes were cryopreserved and analyzed e co Th ncentration of IFN-γ released in response to the in batches. We have previously shown that QFT results obtained ESAT-6–free IGRA antigen cocktail was significantly higher in from fresh vs frozen plasma are comparable [16]. participants with positive QFT status (QFT+; n = 35) compared to participants with negative QFT status (QFT–; n  =  25; ESAT-6–Free IGRA Tube Technical Qualification Supplementary Figure 2A), and the magnitude of the IFN-γ Blood was collected from participants enrolled in cohort 1b directly levels correlated with those induced by QFT (Supplementary in QFT and ESAT-6–free IGRA tubes (Supplementary Figure 1B) Figure 2B). The ESAT-6–free IGRA response was mostly driven and processed, as described [16]. Identical internal quality control by recognition of CFP-10 and EspC (Figure 1). samples were included in each plate to assess interassay variability. Definition of the ESAT-6–Free IGRA Cutoff IFN-γ ELISA The ESAT-6–free IGRA cutoff was determined by comparing IFNγ was quantified by QFT IFN-γ ELISA (Qiagen, Hilden, M.tb-unexposed participants enrolled in a low TB endemic Germany). All samples from the same participant were included setting (cohort 2a, n  =  50) to microbiologically confirmed in the same plate, and each sample was analyzed in triplicate TB patients recruited in South Africa (cohort 2b, n  =  51; or duplicate wells by a blinded operator. Results from replicate Supplementary Table 1). There was no significant differ- wells were averaged unless a technical error that affected 1 of ence in IFN-γ levels following whole blood incubation in the wells was recorded. QFT or ESAT-6–free IGRA Nil tubes (Figure 2A). Therefore, background IFN-γ values measured in Nil QFT tubes were Statistical Analyses subtracted from those measured in both QFT TB Ag and Statistical analyses were performed using SAS 9.2 (SAS Institute) ESAT-6–free IGRA TB Ag tubes in all subsequent experiments. and GraphPad Prism (V7, GraphPad Software Inc.). IFN-γ values In TB patients, the IFN-γ concentrations in the QFT assays (me- were compared using nonparametric methods across study dian, 6.79; interquartile range [IQR], 1.70–10.85 IU/mL) were groups (Mann-Whitney test) or assays (Wilcoxon matched-pairs higher than those measured by ESAT-6–free IGRA (median signed rank test or Spearman correlation). Diagnostic accuracy 4.27; IQR, 1.59–9.51 IU/mL; P  =  .005), while equivalent and and assay cutoff were determined by receiver operating charac- extremely low IFN-γ concentrations were measured in M.tb- teristic analysis. Assay sensitivity and specificity for a given cutoff unexposed individuals (Figure 2B). ESAT-6–free IGRA yielded are reported with Clopper-Pearson 95% confidence intervals diagnostic accuracy that was comparable to that of QFT for (CIs). Agreement and the marginal homogeneity between 2 classifying TB-diseased patients vs M.tb-unexposed individuals assays were calculated using Cohen’s kappa and the McNemar (Figure 2C). An ESAT-6–free IGRA assay cutoff (0.61 IU/mL) test, respectively. Assay variability and bias were calculated using was selected at the IFN-γ concentration that yielded no more Bland-Altman analysis. than a 5% lower specificity than QFT, as defined in a preset sta- tistical analysis plan. This cutoff resulted in a sensitivity of 82% RESULTS (Supplementary Figure 3). Recognition of ESAT-6–Free IGRA M.tb Antigens by South African Adolescents Validation of ESAT-6–Free IGRA Performance in South African Adolescents Since M.tb antigens included in the ESAT-6–free IGRA were Diagnostic accuracy of ESAT-6–free IGRA for M.tb infec- originally selected in participants from low and intermediate tion was benchmarked against QFT in an independent co- TB burden settings [15], we first confirmed their recognition in hort of 200 healthy adolescents from South Africa (cohort 3). Figure 1. Early secretory antigenic target-6–free IFN-γ release assay antigen recognition by South African adolescents. Heat map of log10(IFN-γ) showing recognition patterns of all antigens in each QFT-negative (left) and QFT-positive (right) participant. In the absence of a cutoff to define recognition of each E6FI antigen, log10(IFN-γ) are presented on a continuous quantitative scale. Abbreviations: IFN-γ, interferon-gamma; QFT, QuantiFERON-TB Gold In-tube. 1726 • cid 2019:69 (15 November) • Nemes et al Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Figure 2. ESAT-6–free IGRA diagnostic accuracy in M.tb-unexposed controls and TB patients. Blood was collected from M.tb-unexposed controls (n = 50, cohort 2a) and microbiologically confirmed TB patients (n = 51, cohort 2b) in QFT and ESAT-6 free IGRA (E6FI) tubes and stimulated for 16–20 hours at 37°C; the IFN-γ concentration was measured by QFT enzyme-linked immunosorbent assay. A, Paired analysis (Wilcoxon matched-pairs signed rank test) of IFN-γ values measured upon blood stimulation in QFT and E6FI Nil or TB Ag tubes in all participants. B, Paired analysis (Wilcoxon matched-pairs signed rank test) of IFN-γ values measured by QFT and E6FI (TB Ag – Nil) in M.tb-unexposed controls and TB patients. C, Receiver operating characteristic analysis of diagnostic accuracy of QFT (dashed line) and E6FI (solid line) assays. Area under the curve and 95% confidence interval are shown. Abbreviations: Ag, antigen; ESAT-6, early secretory antigenic target-6; IFN-γ, interferon-gamma; IGRA, interferon-gamma release assay; M.tb, Mycobacterium tuberculosis; QFT, QuantiFERON-TB Gold In-tube; TB, tuberculosis. Median participant age was 14 years (range, 12–18 years), 52% IU/mL; P  =  .015) in participants who tested QFT+, while no of participants were females, 46% of adolescents self-identified overall difference was observed for those who tested QFT– as black African, and 54% were from a mixed ethnic back- (Figure 3B). IFN-γ values obtained by ESAT-6–free IGRA ground (Cape mixed ancestry). All participants had received and QFT were highly correlated (r  =  0.828, P  <  .0001; Figure childhood BCG vaccination; none had received a TST within 3C). Overall, 91% of ESAT-6–free IGRA and QFT results were 3  months of recruitment, had previous TB disease nor were concordant (kappa  =  0.82; 95% CI, 0.74–0.90; McNemar test known to be exposed to patients with active TB at the time P = .48; Table 1). of blood collection; 4% were smokers. Figure 3A shows the An uncertainty zone to interpret QFT results close to the distribution of IFN-γ values in adolescents who tested QFT+ assay cuto, fa ff lling between 0.2 and 0.7 IU/mL, has been (n = 83) or QFT– (n = 117). ESAT-6–free IGRA yielded slightly proposed [16–19]. In this adolescent cohort, 5 of 18 (28%) QFT higher IFN-γ concentrations (median, 6.16; IQR, 2.97–10.91 and ESAT-6–free IGRA discordant results fell in the QFT un- IU/mL) compared to QFT (median, 5.70; IQR, 1.51–9.93 certainty zone (Supplementary Figure 4). Figure 3. ESAT-6–free IGRA diagnostic accuracy in healthy South African adolescents. Blood was collected from QFT-negative (n = 117, open symbols) and QFT-positive (n = 83, filled symbols) healthy South African adolescents (cohort 3) in QFT and ESAT-6–free IGRA (E6FI) tubes and stimulated for 16–20 hours at 37°C; the IFN-γ concentra- tion was measured by QFT enzyme-linked immunosorbent assay. A, Distribution of IFN-γ values measured by QFT and E6FI in all participants. The dotted line denotes QFT cutoff. B, Paired analysis (Wilcoxon matched-pairs signed rank test) of IFN-γ values measured by QFT and E6FI (TB Ag – Nil) in QFT-negative and QFT-positive adolescents. C, Correlation between IFN-γ values obtained by QFT and E6FI in all participants was assessed using the Spearman test. The dotted lines denote assay cutoffs: 0.35 IU/mL for QFT (vertical line) and 0.61 IU/mL for E6FI (horizontal line). Abbreviations: Ag, antigen; ESAT-6, early secretory antigenic target; IFN-γ, interferon-gamma; IGRA, interferon- gamma release assay; QFT, QuantiFERON-TB Gold In-tube; TB, tuberculosis. M.tb Infection Diagnosis by ESAT-6–Free IGRA • cid 2019:69 (15 November) • 1727 Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Table 1. Concordance of ESAT-6–Free IGRA and Quantiferon-TB Gold in Analysis of blood collected in 3 ESAT-6–free IGRA tubes Healthy South African Adolescents from lot 2 yielded consistent quantitative and qualitative results, with 1 exception (Figure 4B). Variability observed across rep- ESAT-6–Free IGRA licate ESAT-6–free IGRA assays appeared substantially higher Negative (n) Positive (n) Total (N) than that due to IFN-γ ELISA intra- and interassay variability Quantiferon-TB Gold Negative (n) 106 11 117 (Supplementary Figure 5C). Positive (n) 7 76 83 Storage of ESAT-6–free IGRA blood collection tubes for 2 Total (n) 113 87 200 weeks at 4, 20, 37, or 50°C prior to long-term storage at 4°C Abbreviations: ESAT-6, early secretory antigenic target; IGRA, interferon-gamma release did not ae ff ct quantitative and qualitative results (Figure 4C) assay. nor did it introduce a systematic bias (Supplementary Figure 5D), although high variability was observed in some samples (Supplementary Figure 5E). ESAT-6 Free IGRA Variability To complete the qualification of the ESAT-6–free IGRA assay, we DISCUSSION assessed lot-to-lot variation, precision, and stability of ESAT-6– free IGRA blood collection tubes (Supplementary Figure 1B). Our aim in this study was to qualify the recently developed Experiments were conducted concurrently on blood collected ESAT-6–free IGRA, a diagnostic tool for the detection of M.tb from 12 healthy donors (3 who tested QFT– and 9 who tested infection in individuals vaccinated with ESAT-6–containing QFT+; cohort 1b). TB vaccines. We demonstrate that the 3 antigens substituting No significant difference was observed in IFN-γ values ESAT-6 are specific and recognized in healthy individuals with obtained by QFT, ESAT-6–free IGRA lot 1, and ESAT-6–free a QFT-positive test. Using a cross-validation design, we defined IGRA lot 2 (P > .5 for all comparisons; Figure 4A). Qualitatively, a cutoff for positive ESAT-6–free IGRA, which is essential to the 1 discordant result was observed when QFT was compared to development of a diagnostic algorithm for test interpretation, ESAT-6–free IGRA lot 1 and when ESAT-6–free IGRA lot 1 was and demonstrated that the ESAT-6–free IGRA had diagnostic compared to ESAT-6–free IGRA lot 2, while 2 discordant results performance that was comparable to that of QFT in South were observed when QFT was compared to ESAT-6–free IGRA African adolescents. Also, we explored the variability, precision, lot 2 (Figure 4A). Bland-Altman analysis of ESAT-6–free IGRA and stability of the ESAT-6–free IGRA test and demonstrated lot 1 and lot 2 tubes did not show lot-associated systematic bias that the novel test is sufficiently robust for its intended use in (bias  =  –10%; 95% limits of agreement, –123% to 101%), al- clinical research. though a high percent difference between lots was observed in e s Th trength of this analysis was the cross-validation de- some instances (Supplementary Figure 5A), resulting in a con- sign; this was achieved by determining the cutoff in a popu- cordant correlation coefficient (ρc) = 0.89 (95% CI, 0.68–0.97; lation with stringently defined M.tb-unexposed controls and Supplementary Figure 5B). TB cases and validating it in a separate cohort, representative of Figure 4. ESAT-6–free IGRA tube variability. Blood was collected from healthy donors (n = 12, cohort 1b) and analyzed as described in Supplementary Figure 1B. IFN-γ values were measured by QFT enzyme-linked immunosorbent assay upon blood stimulation in (A) QFT and 2 different lots of ESAT-6–free IGRA (E6FI) tubes (lot-to-lot vari- ability), (B) 3 E6FI tubes from lot 2 (precision), and (C) 4 E6FI tubes stored at the indicated temperatures for 2 weeks prior to long-term storage at 4°C (stability). Descriptive statistics are shown in Supplementary Figure 5. In all figures, the dotted line denotes the E6FI assay cutoff. Abbreviations: Ag, antigen; ESAT-6, early secretory antigenic target; IFN-γ, interferon-gamma; IGRA, interferon-gamma release assay; QFT, QuantiFERON-TB Gold In-tube; TB, tuberculosis. 1728 • cid 2019:69 (15 November) • Nemes et al Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 the target population for the test. In this study, QFT performed Another limitation in the study was the reliance on active as expected both in terms of magnitude of IFN-γ release in TB cases and uninfected controls to determine the cutoff for a samples from TB patients and controls [20] and in terms of positive test. Although this approach has typically been used to positivity rates in the validation cohort. The validation cohort define the IGRA cutoff [20, 24], ideally, endpoints should have was recruited in a region with extremely high exposure to TB, been derived from ESAT-6–free IGRA-positive participants resulting in about 40% prevalence of QFT+ individuals among who develop active TB in a prospective trial. However, the scale adolescents aged 14  years, which is consistent with previous and time required for this endeavor would vastly outweigh the observations in the same community [21]. potential benefit to be gained. Further, in the absence of an We observed 9% discordance between ESAT-6–free IGRA alternative gold standard to measure M.tb exposure, it is not and QFT. This finding was expected, as the antigens used in possible to identify M.tb-unexposed controls in the same high the 2 tests differ and are differentially recognized in different endemic population. persons [15] (Figure 1). Our findings confirm that EspC is an A challenge that limited the generalizability of the blood col- immunodominant and M.tb-specific antigen and the key sup- lection tube variability and stability analysis is that we did not port to CFP-10 in the ESAT-6–free IGRA antigen cocktail [13, include a direct experimental comparison to QFT. This would 15]. Our initial study identified EspF and Rv2348 as impor - allow better referencing to state-of-art  assay; however, when tant supplement antigens that augment the level of IFN-γ re- compared to the literature [22], the ESAT-6–free IGRA tubes lease and extend diagnostic coverage, especially in individuals appear robust and field applicable. with latent infection [15]. However, in the population included In conclusion, we demonstrated that the novel ESAT-6–free in this study, these antigens were less frequently recognized. IGRA yields a diagnostic accuracy for M.tb infection that is Of note, one third of discordant results fell in the QFT uncer- comparable to that of QFT in South African adolescents. The tainty zone (IFN-γ values between 0.2 and 0.7 IU/mL) [16]. Our ESAT-6–free IGRA is thus suitable to measure M.tb infection in results suggest that the ESAT-6–free IGRA is subject to biolog- clinical trials of novel TB vaccine candidates that contain ESAT- ical and analytical variability that is similar to that of QFT [16, 6, targeting young, healthy adults. 22]; therefore, an uncertainty zone should also be established Further evaluation of the assay needs to be carried out to as- for the ESAT-6–free IGRA. sess its diagnostic potential among specific subpopulations such e de Th velopment of the ESAT-6–free IGRA arose from the as immunocompromised patients and children. need to detect M.tb infection aer vaccin ft ation with ESAT-6– Supplementary Data containing vaccines. ESAT-6 seems to possess unique properties Supplementary materials are available at Clinical Infectious Diseases online. that drive protective immune responses and has become a cen- Consisting of data provided by the authors to benefit the reader, the posted tral antigen in several vaccine candidates that are in develop- materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. ment [23]. However, clinical efficacy trials are required before even the most advanced and promising candidates will be ready Notes for implementation in the field. A robust diagnostic assay to de- Acknowledgments. The authors thank all study participants; the South tect M.tb infection aer vaccin ft ation is critical for the evaluation African Tuberculosis Vaccine Initiative, Immunology Research Group, of the efficacy in preventing M.tb infection in these trials [10], and Statens Serum Institut (SSI) clinical and laboratory teams; Kathryn Rutkowski (AERAS) for statistical input; Marcella Sarzotti-Kelsoe and which we showed is feasible in countries with high TB burden Daniel Akio Ozaki (QualCon, the Quality Consultants) and Peggy Johnston and can provide proof of concept to inform further clinical de- (the Bill & Melinda Gates Foundation) for input on the assay qualification velopment of vaccine candidates [11]. plan. Elisa Nemes is an International Society for Advancement of Cytometry Marylou Ingram Scholar. In TB patients, the magnitude of IFN-γ release was signifi- Financial support. This work was supported by AERAS and SSI. The val- cantly lower in the ESAT-6–free IGRA compared to QFT. While idation cohort was recruited as a substudy of a trial cofunded by AERAS this might have ae ff cted the assay cutoff determination, it did and Sanofi Pasteur. M. R., T. J. S., and G. W. receive funding from European not translate into a lower diagnostic sensitivity, determined as Commission H2020 program (grant TBVAC2020 643381). M.  R.  receives funding from the Research Council Norway (GLOBVAC 248042/H10). B. S., positivity rate in the validation cohort. A. G. L., and G. W. receive funding from the National Institutes of Health Regardless, these observations, along with tube-associated (grant 1U01AI115619) and a Strategic Health Innovation Partnership grant variability, suggest a need for improvement of the blood collec- from the South African Department of Science and Technology and South African Medical Research Council. tion tubes. This was addressed through the development of an Potential conifl cts of interest. E.  N., T.  J. S., and M.  H.  report funding improved freeze-drying procedure that enhanced peptide solu- from AERAS during the conduct of this study. P.  A.  has a patent bility. Tubes manufactured through this improved process in- (WO2015090322 A8) pending to the SSI. M. R. and L. L. H. are employed by the SSI, a governmental not-for-prot r fi esearch organization that holds duced IFN-γ levels equivalent to QFT in individuals who tested intellectual property rights on several of the antigens explored in this work QFT+ and lower levels of IFN-γ in controls and will be used for vaccine and diagnostic purposes. All rights are assigned to SSI. A. M. G., in future efficacy studies (Kidola and Ruhwald, manuscript in R. D. E., and M. D. L. report grants to AERAS from the Bill and Melinda preparation). Gates Foundation and the United Kingdom Department for International M.tb Infection Diagnosis by ESAT-6–Free IGRA • cid 2019:69 (15 November) • 1729 Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Development that supported the conduct of the study and shared clinical 12. Arlehamn  CS, Sidney  J, Henderson  R, et  al. Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, trial costs with Sanofi Pasteur and GlaxoSmithKline outside the submitted Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ). J Immunol 2012; work. All other authors: No reported conflicts. All authors have submitted 188:5020–31. the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts 13. Millington KA, Fortune SM, Low J, et al. Rv3615c is a highly immunodominant that the editors consider relevant to the content of the manuscript have been RD1 (region of difference 1)-dependent secreted antigen specific for disclosed. Mycobacterium tuberculosis infection. Proc Natl Acad Sci U S A 2011; 108:5730–5. 14. Coppola M, van Meijgaarden KE, Franken KL, et al. New genome-wide algorithm References identifies novel in-vivo expressed Mycobacterium tuberculosis antigens inducing human T-cell responses with classical and unconventional cytokine profiles. Sci 1. Carpenter C, Sidney J, Kolla R, et al. A side-by-side comparison of T cell reactivity Rep 2016; 6:37793. to fifty-nine Mycobacterium tuberculosis antigens in diverse populations from five 15. Ruhwald M, de Thurah L, Kuchaka D, et al. Introducing the ESAT-6 free IGRA, a continents. Tuberculosis (Edinb) 2015; 95:713–21. companion diagnostic for TB vaccines based on ESAT-6. Sci Rep 2017; 7:45969. 2. Pai M, Denkinger CM, Kik SV, et al. Gamma interferon release assays for detec- 16. Nemes E, Rozot V, Geldenhuys H, et al; C-040-404 Study Team and the Adolescent tion of Mycobacterium tuberculosis infection. Clin Microbiol Rev 2014; 27:3–20. Cohort Study Team. Optimization and interpretation of serial QuantiFERON 3. Ruhwald M, Aggerbeck H, Gallardo RV, et al; TESEC Working Group. Safety and testing to measure acquisition of Mycobacterium tuberculosis infection. Am J efficacy of the C-Tb skin test to diagnose Mycobacterium tuberculosis infection, Respir Crit Care Med 2017; 196:638–48. compared with an interferon γ release assay and the tuberculin skin test: a phase 17. Pai M, Joshi R, Dogra S, et al. Serial testing of health care workers for tuberculosis 3, double-blind, randomised, controlled trial. Lancet Respir Med 2017; 5:259–68. using interferon-gamma assay. Am J Respir Crit Care Med 2006; 174:349–55. 4. Gröschel  MI, Sayes  F, Simeone  R, Majlessi  L, Brosch  R. ESX secretion systems: 18. van  Zyl-Smit  RN, Zwerling  A, Dheda  K, Pai  M. Within-subject variability of mycobacterial evolution to counter host immunity. Nat Rev Microbiol 2016; interferon-g assay results for tuberculosis and boosting effect of tuberculin skin 14:677–91. testing: a systematic review. PLoS One 2009; 4:e8517. 5. Brosch R, Gordon SV, Garnier T, et al. Genome plasticity of BCG and impact on 19. Zwerling  A, Joshi  R, Kalantri  SP, et  al. Trajectories of tuberculosis-specific vaccine efficacy. Proc Natl Acad Sci U S A 2007; 104:5596–601. interferon-gamma release assay responses among medical and nursing students 6. Aagaard  C, Hoang  T, Dietrich  J, et  al. A multistage tuberculosis vaccine that in rural India. J Epidemiol Glob Health 2013; 3:105–17. confers efficient protection before and after exposure. Nat Med 2011; 17:189–94. 20. Ruhwald M, Bodmer T, Maier C, et al; TBNET. Evaluating the potential of IP-10 7. Lin PL, Dietrich J, Tan E, et al. The multistage vaccine H56 boosts the effects of and MCP-2 as biomarkers for the diagnosis of tuberculosis. Eur Respir J 2008; BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection. J Clin Invest 2012; 122:303–14. 32:1607–15. 8. Hoang T, Aagaard C, Dietrich J, et al. ESAT-6 (EsxA) and TB10.4 (EsxH) based 21. Mahomed  H, Hawkridge  T, Verver  S, et  al; SATVI Adolescent Study Team. vaccines for pre- and post-exposure tuberculosis vaccination. PLoS One 2013; Predictive factors for latent tuberculosis infection among adolescents in a high- 8:e80579. burden area in South Africa. Int J Tuberc Lung Dis 2011; 15:331–6. 9. Luabeya  AK, Kagina  BM, Tameris  MD, et  al; H56-032 Trial Study Group. 22. Tagmouti S, Slater M, Benedetti A, et al. Reproducibility of interferon gamma First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in (IFN-γ) release assays. A  systematic review. Ann Am Thorac Soc 2014; Mycobacterium tuberculosis infected and non-infected healthy adults. Vaccine 11:1267–76. 2015; 33:4130–40. 23. Suliman S, Luabeya AKK, Geldenhuys H, et al. Dose optimization of H56:IC31 10. Ellis RD, Hatherill M, Tait D, et al. Innovative clinical trial designs to rationalize vaccine for TB endemic populations: a double-blind, placebo-controlled, dose- TB vaccine development. Tuberculosis (Edinb) 2015; 95:352–7. selection trial. Am J Respir Crit Care Med 2019; 199:220–31. 11. Nemes  E, Geldenhuys  H, Rozot  V, et  al; C-040-404 Study Team. Prevention of 24. Mori T, Sakatani M, Yamagishi F, et al. Specific detection of tuberculosis infection: M.  tuberculosis infection with H4:IC31 vaccine or BCG revaccination. N Engl J an interferon-gamma-based assay using new antigens. 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Diagnostic Accuracy of Early Secretory Antigenic Target-6–Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube

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Oxford University Press
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Copyright © 2022 Infectious Diseases Society of America
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10.1093/cid/ciz034
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Abstract

Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Clinical Infectious Diseases MAJOR ARTICLE Diagnostic Accuracy of Early Secretory Antigenic Target-6–Free Interferon-gamma Release Assay Compared to QuantiFERON-TB Gold In-tube 1, 1 1 1 1 1 1 Elisa Nemes, Deborah Abrahams, Thomas J. Scriba, Frances Ratangee, Alana Keyser, Lebohang Makhethe, Mzwandile Erasmus, 1 1 1 1 1 2,a 3 2 Simbarashe Mabwe, Nicole Bilek, Virginie Rozot, Hennie Geldenhuys, Mark Hatherill, Maria D. Lempicki, Line Lindebo Holm, Leah Bogardus, 2,a 3 4 2 4 4 3 3 Ann M. Ginsberg, Thomas Blauenfeldt, Bronwyn Smith, Ruth D. Ellis, Andre G Loxton, Gerhard Walzl, Peter Andersen, and Morten Ruhwald 1 2 South African Tuberculosis Vaccine Initiative, Institute of Infectious Disease and Molecular Medicine & Division of Immunology, Department of Pathology, University of Cape Town; AERAS, 3 4 Rockville, Maryland; Statens Serum Institute, Copenhagen, Denmark; and South Africa Department of Science and Technology–National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town Background. Early secretory antigenic target-6 (ESAT-6) is an immunodominant Mycobacterium tuberculosis (M.tb) an- tigen included in novel vaccines against tuberculosis (TB) and in interferon-gamma (IFN-γ) release assays (IGRAs). Therefore, the availability of an ESAT-6–free IGRA is essential to determine M.tb infection status following vaccination with ESAT-6–containing vaccines. We aimed to qualify a recently developed ESAT-6–free IGRA and to assess its diagnostic performance in comparison to QuantiFERON-TB Gold In-tube (QFT). Methods. Participants with different levels of M.tb exposure and TB disease were enrolled to determine the ESAT-6–free IGRA cuto, t ff est assay performance in independent cohorts compared to standard QFT, and perform a technical qualification of antigen- coated blood collection tubes. Results. ESAT-6–free IGRA antigen recognition was evaluated in QFT-positive and QFT-negative South African adolescents. e ESA Th T-6–free IGRA cutoff was established at 0.61 IU/mL, based on receiver operating characteristic analysis in M.tb-unexposed controls and microbiologically confirmed pulmonary TB patients. In an independent cohort of healthy adolescents, levels of IFN-γ released in QFT and ESAT-6–free IGRA were highly correlated (P < .0001, r = 0.83) and yielded comparable positivity rates, 41.5% and 43.5%, respectively, with 91% concordance between the tests (kappa = 0.82; 95% confidence interval, 0.74–0.90; McNemar test P = .48). ESAT-6–free IGRA blood collection tubes had acceptable lot-to-lot variability, precision, and stability. Conclusions. e Th novel ESAT-6–free IGRA had diagnostic accuracy comparable to QFT and is suitable for use in clinical trials to assess efficacy of candidate TB vaccines to prevent established M.tb infection. Keywords. tuberculosis infection; diagnosis; IFN-γ release assay; qualification. Early secretory antigenic target-6 (ESAT-6) is an immunodominant 1 (ESX1) type VII secretion system [4] and are absent from bacille antigen expressed by Mycobacterium tuberculosis (M.tb) [1]. Calmette-Guerin (BCG), thereby, importantly, enabling distinc- ESAT-6 and its heterodimeric complex protein CFP-10 are the tion of M.tb infection from prior BCG vaccination [5]. principal components of immunodiagnostic tests for M.tb in- ESAT-6 is also increasingly recognized as an important fection, including the interferon-gamma (IFN-γ) release assay vaccine antigen with unique properties for infection contain- (IGRA) and novel specific skin tests [2, 3]. These small, highly ment and, potentially, for clearance [6–8]. As such, ESAT-6 immunodominant antigens are encoded in the region of differ- is contained in many candidate vaccines against tubercu- ence 1 of the M.tb genome, which comprises many important vir- losis (TB), including the subunit vaccine H56:IC31, which is ulence factors as components and substrates of the ESAT-6 system undergoing phase 2 clinical testing [6, 9]. Vaccine efficacy to prevent established M.tb infection, as a likely predictor of pre- vention of TB disease, has been proposed as an innovative and Received 4 October 2018; editorial decision 28 December 2018; accepted 16 January 2019; cost-effective endpoint to rationalize TB vaccine development published online January 21, 2019. a [10]. We recently showed that BCG revaccination or vaccina- Present address: International AIDS Vaccine Initiative, New York, NY, USA. Correspondence: M. Ruhwald, Center for Vaccine Research, Artillerivej 5 | 2300 København tion with a novel subunit vaccine candidate (H4:IC31) may S | Denmark (moru@ssi.dk and ssi.dk). oer p ff artial protection against sustained M.tb infection, meas- Clinical Infectious Diseases 2019;69(10):1724–30 ured by QuantiFERON-TB Gold In-tube (QFT) [11]. That trial © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons provided the first proof-of-concept efficacy signal for a subunit Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted vaccine candidate, which is very similar to H56:IC31 but does reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciz034 not contain ESAT-6. Such a clinical trial design, one that relies 1724 • cid 2019:69 (15 November) • Nemes et al Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 on IGRA to measure M.tb infection, cannot currently be used to sputum liquid culture and/or GeneXpert and/or smear micros- evaluate ESAT-6–containing vaccine candidates, since vaccine- copy. Patients with HIV infection, multidrug-resistant TB, or induced immune responses are cross-reactive with those meas- those who started TB treatment more than 3 days before enroll- ured by IGRA, rendering the test false positive. ment were excluded. Cohort 3: A subset of healthy adolescents Until recently, inclusion of ESAT-6 and CFP-10 has been screened for enrollment in a prevention-of-M.tb-infection trial considered essential for adequate immunodiagnostic sensi- at SATVI (HREC reference: 471/2013; clinicaltrials.gov identi- tivity. However, recent advances in antigen discovery tools and fier: NCT02075203) [11] were included in this study to evaluate M.tb antigen recognition studies in humans point to novel di- ESAT-6–free IGRA performance compared to QFT. agnostic antigens that could replace ESAT-6 [12–14]. Several Inclusion and exclusion criteria for cohort 3 have been studies explored the diagnostic potential of new candidates ei- described in detail elsewhere [16]. QFT results from cohort 2a, ther alone or in combination, and we recently established an 2b, and 3 have been published in a different form [16]. ESAT-6–free IGRA antigen cocktail aer s ft creening of several Assessment of Preselected Antigens candidates (TB7.7, EspJ, EspC, EspF, EccD1, PE35, and Rv2348) Venous blood was collected in lithium heparin–containing compared to ESAT-6 and CFP-10 [15]. vacutainers, and 1  mL per tube was dispensed in stimulation In this study, our aim was to confirm the recognition of an tubes (Sarstedt, Numbrecht, Germany) that contained (a) no an- ESAT-6–free IGRA antigen cocktail in adolescents from a high tigen, or pools of overlapping 15–20 mer peptides representing: TB burden area, define a diagnostic algorithm for the ESAT-6– (b) full length CFP-10, (c) a fragment of EspC (aa54-103), (d) free IGRA, evaluate ESAT-6–free IGRA diagnostic performance a fragment of EspF (aa9-44), (e) a fragment of Rv2348 (aa56- compared to QFT, and assess the variability of ESAT-6–free 108), or  (f) ESAT-6–free IGRA antigen cocktail (b+c+d+e, at IGRA blood collection tubes. a concentration of 5  μg per peptide/mL blood, as described [15]). Phytohemagglutinin (Dartford, Remel, United Kingdom) METHODS was used as the positive control (5  μg/mL), and a set of QFT Study Participants tubes (Nil, TB antigen, mitogen; Qiagen, Hilden, Germany) was All studies that involved human participants were conducted run in parallel as the reference. Within 2 hours of phlebotomy, according to the principles described in the Declaration of stimulated blood was incubated at 37°C for 18–22 hours and Helsinki and were approved by relevant institutional review then centrifuged. Plasma was harvested and cryopreserved at boards (Human Research Ethics Committee [HREC]). Written –80°C until batch analysis. informed assent and consent were obtained from participants and their legal guardians (for minors). QFT and ESAT-6–Free IGRA Assays We included the following 4 independent cohorts of patients Blood was collected directly in QFT tubes and in tubes that to address the study aims (Supplementary Figure 1A). Cohort contained lyophilized ESAT-6–free IGRA peptides, manu- 1: Healthy adolescents (cohort 1a) and adults (cohort 1b) were factured at SSI, as described [15], to define a cutoff for the enrolled at the South African Tuberculosis Vaccine Initiative ESAT-6–free IGRA assay, determine assay performance in (SATVI, South Africa, HREC reference: 126/2006) to confirm adolescents, and assess tube variability. Blood was processed ESAT-6–free IGRA antigen recognition and perform ESAT-6– following a standard operating procedure that was stricter free IGRA qualification, respectively. Individuals who had re- than that recommended by the manufacturer in order to re- ceived a tuberculin skin test (TST) in the last 12  months, had duce known sources of assay variability and improve reproduc- evidence of any acute or chronic disease or symptomatic infec- ibility, as described [16]. Briefly, 1  mL of blood was collected tion (including human immunodeficiency virus [HIV] positive and mixed by 10 manual inversions and 5 minutes on a tube or TB symptoms), currently used immune-modifying drugs, rotator and incubated at 37°C within 2 hours of phlebotomy, had been enrolled in any experimental study, were pregnant or for 16–20 hours. lactating (women), weighed <50 kg, or those with raised suspi- To determine the ESAT-6–free IGRA assay cuto, b ff lood cion of anemia by recruiting officer were excluded. Cohort 2a: was collected and processed at SSI (M.tb unexposed controls, Healthy adults with no known exposure to M.tb were recruited cohort 2a) and IRG (TB patients, cohort 2b). Cryopreserved at the Statens Serum Institut (SSI) in Denmark (HREC ref- plasma was analyzed by QFT IFN-γ enzyme-linked immuno- erence: H-3-2012-008). Individuals with a medical history sorbent assay (ELISA) at SATVI. of M.tb exposure, travel, and/or risk-seeking behavior were To assess the performance of ESAT-6–free IGRA in South excluded. Cohort 2b: Adult patients with pulmonary TB di- African adolescents, samples were collected and processed at sease were recruited by the Immunology Research Group (IRG) SATVI. Blood was simultaneously collected and processed for at Stellenbosch University, South Africa (HREC reference: the QFT and ESAT-6–free IGRA. As results from QFT analysis N13/05/064). TB disease was microbiologically confirmed by were part of the screening procedures for real-time enrollment M.tb Infection Diagnosis by ESAT-6–Free IGRA • cid 2019:69 (15 November) • 1725 Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 in a clinical trial [11], fresh plasmas harvested from QFT tubes adolescents from a high TB burden area who represent a target were analyzed by QFT IFN-γ ELISA, while plasmas harvested population for prevention of M.tb infection trials (cohort 1a). from ESAT-6–free IGRA tubes were cryopreserved and analyzed e co Th ncentration of IFN-γ released in response to the in batches. We have previously shown that QFT results obtained ESAT-6–free IGRA antigen cocktail was significantly higher in from fresh vs frozen plasma are comparable [16]. participants with positive QFT status (QFT+; n = 35) compared to participants with negative QFT status (QFT–; n  =  25; ESAT-6–Free IGRA Tube Technical Qualification Supplementary Figure 2A), and the magnitude of the IFN-γ Blood was collected from participants enrolled in cohort 1b directly levels correlated with those induced by QFT (Supplementary in QFT and ESAT-6–free IGRA tubes (Supplementary Figure 1B) Figure 2B). The ESAT-6–free IGRA response was mostly driven and processed, as described [16]. Identical internal quality control by recognition of CFP-10 and EspC (Figure 1). samples were included in each plate to assess interassay variability. Definition of the ESAT-6–Free IGRA Cutoff IFN-γ ELISA The ESAT-6–free IGRA cutoff was determined by comparing IFNγ was quantified by QFT IFN-γ ELISA (Qiagen, Hilden, M.tb-unexposed participants enrolled in a low TB endemic Germany). All samples from the same participant were included setting (cohort 2a, n  =  50) to microbiologically confirmed in the same plate, and each sample was analyzed in triplicate TB patients recruited in South Africa (cohort 2b, n  =  51; or duplicate wells by a blinded operator. Results from replicate Supplementary Table 1). There was no significant differ- wells were averaged unless a technical error that affected 1 of ence in IFN-γ levels following whole blood incubation in the wells was recorded. QFT or ESAT-6–free IGRA Nil tubes (Figure 2A). Therefore, background IFN-γ values measured in Nil QFT tubes were Statistical Analyses subtracted from those measured in both QFT TB Ag and Statistical analyses were performed using SAS 9.2 (SAS Institute) ESAT-6–free IGRA TB Ag tubes in all subsequent experiments. and GraphPad Prism (V7, GraphPad Software Inc.). IFN-γ values In TB patients, the IFN-γ concentrations in the QFT assays (me- were compared using nonparametric methods across study dian, 6.79; interquartile range [IQR], 1.70–10.85 IU/mL) were groups (Mann-Whitney test) or assays (Wilcoxon matched-pairs higher than those measured by ESAT-6–free IGRA (median signed rank test or Spearman correlation). Diagnostic accuracy 4.27; IQR, 1.59–9.51 IU/mL; P  =  .005), while equivalent and and assay cutoff were determined by receiver operating charac- extremely low IFN-γ concentrations were measured in M.tb- teristic analysis. Assay sensitivity and specificity for a given cutoff unexposed individuals (Figure 2B). ESAT-6–free IGRA yielded are reported with Clopper-Pearson 95% confidence intervals diagnostic accuracy that was comparable to that of QFT for (CIs). Agreement and the marginal homogeneity between 2 classifying TB-diseased patients vs M.tb-unexposed individuals assays were calculated using Cohen’s kappa and the McNemar (Figure 2C). An ESAT-6–free IGRA assay cutoff (0.61 IU/mL) test, respectively. Assay variability and bias were calculated using was selected at the IFN-γ concentration that yielded no more Bland-Altman analysis. than a 5% lower specificity than QFT, as defined in a preset sta- tistical analysis plan. This cutoff resulted in a sensitivity of 82% RESULTS (Supplementary Figure 3). Recognition of ESAT-6–Free IGRA M.tb Antigens by South African Adolescents Validation of ESAT-6–Free IGRA Performance in South African Adolescents Since M.tb antigens included in the ESAT-6–free IGRA were Diagnostic accuracy of ESAT-6–free IGRA for M.tb infec- originally selected in participants from low and intermediate tion was benchmarked against QFT in an independent co- TB burden settings [15], we first confirmed their recognition in hort of 200 healthy adolescents from South Africa (cohort 3). Figure 1. Early secretory antigenic target-6–free IFN-γ release assay antigen recognition by South African adolescents. Heat map of log10(IFN-γ) showing recognition patterns of all antigens in each QFT-negative (left) and QFT-positive (right) participant. In the absence of a cutoff to define recognition of each E6FI antigen, log10(IFN-γ) are presented on a continuous quantitative scale. Abbreviations: IFN-γ, interferon-gamma; QFT, QuantiFERON-TB Gold In-tube. 1726 • cid 2019:69 (15 November) • Nemes et al Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Figure 2. ESAT-6–free IGRA diagnostic accuracy in M.tb-unexposed controls and TB patients. Blood was collected from M.tb-unexposed controls (n = 50, cohort 2a) and microbiologically confirmed TB patients (n = 51, cohort 2b) in QFT and ESAT-6 free IGRA (E6FI) tubes and stimulated for 16–20 hours at 37°C; the IFN-γ concentration was measured by QFT enzyme-linked immunosorbent assay. A, Paired analysis (Wilcoxon matched-pairs signed rank test) of IFN-γ values measured upon blood stimulation in QFT and E6FI Nil or TB Ag tubes in all participants. B, Paired analysis (Wilcoxon matched-pairs signed rank test) of IFN-γ values measured by QFT and E6FI (TB Ag – Nil) in M.tb-unexposed controls and TB patients. C, Receiver operating characteristic analysis of diagnostic accuracy of QFT (dashed line) and E6FI (solid line) assays. Area under the curve and 95% confidence interval are shown. Abbreviations: Ag, antigen; ESAT-6, early secretory antigenic target-6; IFN-γ, interferon-gamma; IGRA, interferon-gamma release assay; M.tb, Mycobacterium tuberculosis; QFT, QuantiFERON-TB Gold In-tube; TB, tuberculosis. Median participant age was 14 years (range, 12–18 years), 52% IU/mL; P  =  .015) in participants who tested QFT+, while no of participants were females, 46% of adolescents self-identified overall difference was observed for those who tested QFT– as black African, and 54% were from a mixed ethnic back- (Figure 3B). IFN-γ values obtained by ESAT-6–free IGRA ground (Cape mixed ancestry). All participants had received and QFT were highly correlated (r  =  0.828, P  <  .0001; Figure childhood BCG vaccination; none had received a TST within 3C). Overall, 91% of ESAT-6–free IGRA and QFT results were 3  months of recruitment, had previous TB disease nor were concordant (kappa  =  0.82; 95% CI, 0.74–0.90; McNemar test known to be exposed to patients with active TB at the time P = .48; Table 1). of blood collection; 4% were smokers. Figure 3A shows the An uncertainty zone to interpret QFT results close to the distribution of IFN-γ values in adolescents who tested QFT+ assay cuto, fa ff lling between 0.2 and 0.7 IU/mL, has been (n = 83) or QFT– (n = 117). ESAT-6–free IGRA yielded slightly proposed [16–19]. In this adolescent cohort, 5 of 18 (28%) QFT higher IFN-γ concentrations (median, 6.16; IQR, 2.97–10.91 and ESAT-6–free IGRA discordant results fell in the QFT un- IU/mL) compared to QFT (median, 5.70; IQR, 1.51–9.93 certainty zone (Supplementary Figure 4). Figure 3. ESAT-6–free IGRA diagnostic accuracy in healthy South African adolescents. Blood was collected from QFT-negative (n = 117, open symbols) and QFT-positive (n = 83, filled symbols) healthy South African adolescents (cohort 3) in QFT and ESAT-6–free IGRA (E6FI) tubes and stimulated for 16–20 hours at 37°C; the IFN-γ concentra- tion was measured by QFT enzyme-linked immunosorbent assay. A, Distribution of IFN-γ values measured by QFT and E6FI in all participants. The dotted line denotes QFT cutoff. B, Paired analysis (Wilcoxon matched-pairs signed rank test) of IFN-γ values measured by QFT and E6FI (TB Ag – Nil) in QFT-negative and QFT-positive adolescents. C, Correlation between IFN-γ values obtained by QFT and E6FI in all participants was assessed using the Spearman test. The dotted lines denote assay cutoffs: 0.35 IU/mL for QFT (vertical line) and 0.61 IU/mL for E6FI (horizontal line). Abbreviations: Ag, antigen; ESAT-6, early secretory antigenic target; IFN-γ, interferon-gamma; IGRA, interferon- gamma release assay; QFT, QuantiFERON-TB Gold In-tube; TB, tuberculosis. M.tb Infection Diagnosis by ESAT-6–Free IGRA • cid 2019:69 (15 November) • 1727 Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Table 1. Concordance of ESAT-6–Free IGRA and Quantiferon-TB Gold in Analysis of blood collected in 3 ESAT-6–free IGRA tubes Healthy South African Adolescents from lot 2 yielded consistent quantitative and qualitative results, with 1 exception (Figure 4B). Variability observed across rep- ESAT-6–Free IGRA licate ESAT-6–free IGRA assays appeared substantially higher Negative (n) Positive (n) Total (N) than that due to IFN-γ ELISA intra- and interassay variability Quantiferon-TB Gold Negative (n) 106 11 117 (Supplementary Figure 5C). Positive (n) 7 76 83 Storage of ESAT-6–free IGRA blood collection tubes for 2 Total (n) 113 87 200 weeks at 4, 20, 37, or 50°C prior to long-term storage at 4°C Abbreviations: ESAT-6, early secretory antigenic target; IGRA, interferon-gamma release did not ae ff ct quantitative and qualitative results (Figure 4C) assay. nor did it introduce a systematic bias (Supplementary Figure 5D), although high variability was observed in some samples (Supplementary Figure 5E). ESAT-6 Free IGRA Variability To complete the qualification of the ESAT-6–free IGRA assay, we DISCUSSION assessed lot-to-lot variation, precision, and stability of ESAT-6– free IGRA blood collection tubes (Supplementary Figure 1B). Our aim in this study was to qualify the recently developed Experiments were conducted concurrently on blood collected ESAT-6–free IGRA, a diagnostic tool for the detection of M.tb from 12 healthy donors (3 who tested QFT– and 9 who tested infection in individuals vaccinated with ESAT-6–containing QFT+; cohort 1b). TB vaccines. We demonstrate that the 3 antigens substituting No significant difference was observed in IFN-γ values ESAT-6 are specific and recognized in healthy individuals with obtained by QFT, ESAT-6–free IGRA lot 1, and ESAT-6–free a QFT-positive test. Using a cross-validation design, we defined IGRA lot 2 (P > .5 for all comparisons; Figure 4A). Qualitatively, a cutoff for positive ESAT-6–free IGRA, which is essential to the 1 discordant result was observed when QFT was compared to development of a diagnostic algorithm for test interpretation, ESAT-6–free IGRA lot 1 and when ESAT-6–free IGRA lot 1 was and demonstrated that the ESAT-6–free IGRA had diagnostic compared to ESAT-6–free IGRA lot 2, while 2 discordant results performance that was comparable to that of QFT in South were observed when QFT was compared to ESAT-6–free IGRA African adolescents. Also, we explored the variability, precision, lot 2 (Figure 4A). Bland-Altman analysis of ESAT-6–free IGRA and stability of the ESAT-6–free IGRA test and demonstrated lot 1 and lot 2 tubes did not show lot-associated systematic bias that the novel test is sufficiently robust for its intended use in (bias  =  –10%; 95% limits of agreement, –123% to 101%), al- clinical research. though a high percent difference between lots was observed in e s Th trength of this analysis was the cross-validation de- some instances (Supplementary Figure 5A), resulting in a con- sign; this was achieved by determining the cutoff in a popu- cordant correlation coefficient (ρc) = 0.89 (95% CI, 0.68–0.97; lation with stringently defined M.tb-unexposed controls and Supplementary Figure 5B). TB cases and validating it in a separate cohort, representative of Figure 4. ESAT-6–free IGRA tube variability. Blood was collected from healthy donors (n = 12, cohort 1b) and analyzed as described in Supplementary Figure 1B. IFN-γ values were measured by QFT enzyme-linked immunosorbent assay upon blood stimulation in (A) QFT and 2 different lots of ESAT-6–free IGRA (E6FI) tubes (lot-to-lot vari- ability), (B) 3 E6FI tubes from lot 2 (precision), and (C) 4 E6FI tubes stored at the indicated temperatures for 2 weeks prior to long-term storage at 4°C (stability). Descriptive statistics are shown in Supplementary Figure 5. In all figures, the dotted line denotes the E6FI assay cutoff. Abbreviations: Ag, antigen; ESAT-6, early secretory antigenic target; IFN-γ, interferon-gamma; IGRA, interferon-gamma release assay; QFT, QuantiFERON-TB Gold In-tube; TB, tuberculosis. 1728 • cid 2019:69 (15 November) • Nemes et al Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 the target population for the test. In this study, QFT performed Another limitation in the study was the reliance on active as expected both in terms of magnitude of IFN-γ release in TB cases and uninfected controls to determine the cutoff for a samples from TB patients and controls [20] and in terms of positive test. Although this approach has typically been used to positivity rates in the validation cohort. The validation cohort define the IGRA cutoff [20, 24], ideally, endpoints should have was recruited in a region with extremely high exposure to TB, been derived from ESAT-6–free IGRA-positive participants resulting in about 40% prevalence of QFT+ individuals among who develop active TB in a prospective trial. However, the scale adolescents aged 14  years, which is consistent with previous and time required for this endeavor would vastly outweigh the observations in the same community [21]. potential benefit to be gained. Further, in the absence of an We observed 9% discordance between ESAT-6–free IGRA alternative gold standard to measure M.tb exposure, it is not and QFT. This finding was expected, as the antigens used in possible to identify M.tb-unexposed controls in the same high the 2 tests differ and are differentially recognized in different endemic population. persons [15] (Figure 1). Our findings confirm that EspC is an A challenge that limited the generalizability of the blood col- immunodominant and M.tb-specific antigen and the key sup- lection tube variability and stability analysis is that we did not port to CFP-10 in the ESAT-6–free IGRA antigen cocktail [13, include a direct experimental comparison to QFT. This would 15]. Our initial study identified EspF and Rv2348 as impor - allow better referencing to state-of-art  assay; however, when tant supplement antigens that augment the level of IFN-γ re- compared to the literature [22], the ESAT-6–free IGRA tubes lease and extend diagnostic coverage, especially in individuals appear robust and field applicable. with latent infection [15]. However, in the population included In conclusion, we demonstrated that the novel ESAT-6–free in this study, these antigens were less frequently recognized. IGRA yields a diagnostic accuracy for M.tb infection that is Of note, one third of discordant results fell in the QFT uncer- comparable to that of QFT in South African adolescents. The tainty zone (IFN-γ values between 0.2 and 0.7 IU/mL) [16]. Our ESAT-6–free IGRA is thus suitable to measure M.tb infection in results suggest that the ESAT-6–free IGRA is subject to biolog- clinical trials of novel TB vaccine candidates that contain ESAT- ical and analytical variability that is similar to that of QFT [16, 6, targeting young, healthy adults. 22]; therefore, an uncertainty zone should also be established Further evaluation of the assay needs to be carried out to as- for the ESAT-6–free IGRA. sess its diagnostic potential among specific subpopulations such e de Th velopment of the ESAT-6–free IGRA arose from the as immunocompromised patients and children. need to detect M.tb infection aer vaccin ft ation with ESAT-6– Supplementary Data containing vaccines. ESAT-6 seems to possess unique properties Supplementary materials are available at Clinical Infectious Diseases online. that drive protective immune responses and has become a cen- Consisting of data provided by the authors to benefit the reader, the posted tral antigen in several vaccine candidates that are in develop- materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. ment [23]. However, clinical efficacy trials are required before even the most advanced and promising candidates will be ready Notes for implementation in the field. A robust diagnostic assay to de- Acknowledgments. The authors thank all study participants; the South tect M.tb infection aer vaccin ft ation is critical for the evaluation African Tuberculosis Vaccine Initiative, Immunology Research Group, of the efficacy in preventing M.tb infection in these trials [10], and Statens Serum Institut (SSI) clinical and laboratory teams; Kathryn Rutkowski (AERAS) for statistical input; Marcella Sarzotti-Kelsoe and which we showed is feasible in countries with high TB burden Daniel Akio Ozaki (QualCon, the Quality Consultants) and Peggy Johnston and can provide proof of concept to inform further clinical de- (the Bill & Melinda Gates Foundation) for input on the assay qualification velopment of vaccine candidates [11]. plan. Elisa Nemes is an International Society for Advancement of Cytometry Marylou Ingram Scholar. In TB patients, the magnitude of IFN-γ release was signifi- Financial support. This work was supported by AERAS and SSI. The val- cantly lower in the ESAT-6–free IGRA compared to QFT. While idation cohort was recruited as a substudy of a trial cofunded by AERAS this might have ae ff cted the assay cutoff determination, it did and Sanofi Pasteur. M. R., T. J. S., and G. W. receive funding from European not translate into a lower diagnostic sensitivity, determined as Commission H2020 program (grant TBVAC2020 643381). M.  R.  receives funding from the Research Council Norway (GLOBVAC 248042/H10). B. S., positivity rate in the validation cohort. A. G. L., and G. W. receive funding from the National Institutes of Health Regardless, these observations, along with tube-associated (grant 1U01AI115619) and a Strategic Health Innovation Partnership grant variability, suggest a need for improvement of the blood collec- from the South African Department of Science and Technology and South African Medical Research Council. tion tubes. This was addressed through the development of an Potential conifl cts of interest. E.  N., T.  J. S., and M.  H.  report funding improved freeze-drying procedure that enhanced peptide solu- from AERAS during the conduct of this study. P.  A.  has a patent bility. Tubes manufactured through this improved process in- (WO2015090322 A8) pending to the SSI. M. R. and L. L. H. are employed by the SSI, a governmental not-for-prot r fi esearch organization that holds duced IFN-γ levels equivalent to QFT in individuals who tested intellectual property rights on several of the antigens explored in this work QFT+ and lower levels of IFN-γ in controls and will be used for vaccine and diagnostic purposes. All rights are assigned to SSI. A. M. G., in future efficacy studies (Kidola and Ruhwald, manuscript in R. D. E., and M. D. L. report grants to AERAS from the Bill and Melinda preparation). Gates Foundation and the United Kingdom Department for International M.tb Infection Diagnosis by ESAT-6–Free IGRA • cid 2019:69 (15 November) • 1729 Downloaded from https://academic.oup.com/cid/article/69/10/1724/5298338 by DeepDyve user on 19 July 2022 Development that supported the conduct of the study and shared clinical 12. Arlehamn  CS, Sidney  J, Henderson  R, et  al. Dissecting mechanisms of immunodominance to the common tuberculosis antigens ESAT-6, CFP10, trial costs with Sanofi Pasteur and GlaxoSmithKline outside the submitted Rv2031c (hspX), Rv2654c (TB7.7), and Rv1038c (EsxJ). J Immunol 2012; work. All other authors: No reported conflicts. All authors have submitted 188:5020–31. the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts 13. Millington KA, Fortune SM, Low J, et al. Rv3615c is a highly immunodominant that the editors consider relevant to the content of the manuscript have been RD1 (region of difference 1)-dependent secreted antigen specific for disclosed. Mycobacterium tuberculosis infection. Proc Natl Acad Sci U S A 2011; 108:5730–5. 14. Coppola M, van Meijgaarden KE, Franken KL, et al. New genome-wide algorithm References identifies novel in-vivo expressed Mycobacterium tuberculosis antigens inducing human T-cell responses with classical and unconventional cytokine profiles. Sci 1. Carpenter C, Sidney J, Kolla R, et al. A side-by-side comparison of T cell reactivity Rep 2016; 6:37793. to fifty-nine Mycobacterium tuberculosis antigens in diverse populations from five 15. Ruhwald M, de Thurah L, Kuchaka D, et al. Introducing the ESAT-6 free IGRA, a continents. Tuberculosis (Edinb) 2015; 95:713–21. companion diagnostic for TB vaccines based on ESAT-6. Sci Rep 2017; 7:45969. 2. Pai M, Denkinger CM, Kik SV, et al. Gamma interferon release assays for detec- 16. Nemes E, Rozot V, Geldenhuys H, et al; C-040-404 Study Team and the Adolescent tion of Mycobacterium tuberculosis infection. Clin Microbiol Rev 2014; 27:3–20. Cohort Study Team. Optimization and interpretation of serial QuantiFERON 3. Ruhwald M, Aggerbeck H, Gallardo RV, et al; TESEC Working Group. Safety and testing to measure acquisition of Mycobacterium tuberculosis infection. Am J efficacy of the C-Tb skin test to diagnose Mycobacterium tuberculosis infection, Respir Crit Care Med 2017; 196:638–48. compared with an interferon γ release assay and the tuberculin skin test: a phase 17. Pai M, Joshi R, Dogra S, et al. Serial testing of health care workers for tuberculosis 3, double-blind, randomised, controlled trial. Lancet Respir Med 2017; 5:259–68. using interferon-gamma assay. Am J Respir Crit Care Med 2006; 174:349–55. 4. Gröschel  MI, Sayes  F, Simeone  R, Majlessi  L, Brosch  R. ESX secretion systems: 18. van  Zyl-Smit  RN, Zwerling  A, Dheda  K, Pai  M. Within-subject variability of mycobacterial evolution to counter host immunity. Nat Rev Microbiol 2016; interferon-g assay results for tuberculosis and boosting effect of tuberculin skin 14:677–91. testing: a systematic review. PLoS One 2009; 4:e8517. 5. Brosch R, Gordon SV, Garnier T, et al. Genome plasticity of BCG and impact on 19. Zwerling  A, Joshi  R, Kalantri  SP, et  al. Trajectories of tuberculosis-specific vaccine efficacy. Proc Natl Acad Sci U S A 2007; 104:5596–601. interferon-gamma release assay responses among medical and nursing students 6. Aagaard  C, Hoang  T, Dietrich  J, et  al. A multistage tuberculosis vaccine that in rural India. J Epidemiol Glob Health 2013; 3:105–17. confers efficient protection before and after exposure. Nat Med 2011; 17:189–94. 20. Ruhwald M, Bodmer T, Maier C, et al; TBNET. Evaluating the potential of IP-10 7. Lin PL, Dietrich J, Tan E, et al. The multistage vaccine H56 boosts the effects of and MCP-2 as biomarkers for the diagnosis of tuberculosis. Eur Respir J 2008; BCG to protect cynomolgus macaques against active tuberculosis and reactivation of latent Mycobacterium tuberculosis infection. J Clin Invest 2012; 122:303–14. 32:1607–15. 8. Hoang T, Aagaard C, Dietrich J, et al. ESAT-6 (EsxA) and TB10.4 (EsxH) based 21. Mahomed  H, Hawkridge  T, Verver  S, et  al; SATVI Adolescent Study Team. vaccines for pre- and post-exposure tuberculosis vaccination. PLoS One 2013; Predictive factors for latent tuberculosis infection among adolescents in a high- 8:e80579. burden area in South Africa. Int J Tuberc Lung Dis 2011; 15:331–6. 9. Luabeya  AK, Kagina  BM, Tameris  MD, et  al; H56-032 Trial Study Group. 22. Tagmouti S, Slater M, Benedetti A, et al. Reproducibility of interferon gamma First-in-human trial of the post-exposure tuberculosis vaccine H56:IC31 in (IFN-γ) release assays. A  systematic review. Ann Am Thorac Soc 2014; Mycobacterium tuberculosis infected and non-infected healthy adults. Vaccine 11:1267–76. 2015; 33:4130–40. 23. Suliman S, Luabeya AKK, Geldenhuys H, et al. Dose optimization of H56:IC31 10. Ellis RD, Hatherill M, Tait D, et al. Innovative clinical trial designs to rationalize vaccine for TB endemic populations: a double-blind, placebo-controlled, dose- TB vaccine development. Tuberculosis (Edinb) 2015; 95:352–7. selection trial. Am J Respir Crit Care Med 2019; 199:220–31. 11. Nemes  E, Geldenhuys  H, Rozot  V, et  al; C-040-404 Study Team. Prevention of 24. Mori T, Sakatani M, Yamagishi F, et al. Specific detection of tuberculosis infection: M.  tuberculosis infection with H4:IC31 vaccine or BCG revaccination. N Engl J an interferon-gamma-based assay using new antigens. 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Journal

Clinical Infectious Diseases – Oxford University Press

Published: Oct 30, 2019

Keywords: adolescent; antigens; interferon type ii; tuberculosis; diagnosis; quantiferon-tb gold test; interferon gamma release assay; infections; tuberculosis vaccines; mycobacterium tuberculosis

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