Accepted Manuscript Prospective Head-to-Head Study Comparing Two Commercial Interferon-gamma Release Assays for the Diagnosis of Tuberculous Uveitis Marcus Ang, Wan Ling Wong, Sieh Yean Kiew, Xiang Li, Soon-Phaik Chee PII:
S0002-9394(14)00061-0
DOI:
10.1016/j.ajo.2014.01.031
Reference:
AJOPHT 8803
To appear in:
American Journal of Ophthalmology
Received Date: 29 August 2013 Revised Date:
24 January 2014
Accepted Date: 24 January 2014
Please cite this article as: Ang M, Wong WL, Kiew SY, Li X, Chee S-P, Prospective Head-to-Head Study Comparing Two Commercial Interferon-gamma Release Assays for the Diagnosis of Tuberculous Uveitis, American Journal of Ophthalmology (2014), doi: 10.1016/j.ajo.2014.01.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title Page 1. Title: Prospective Head-to-Head Study Comparing Two Commercial Interferongamma Release Assays for the Diagnosis of Tuberculous Uveitis
-
3
-
4
-
EP
5
-
SC
2
Singapore National Eye Centre 11 Third Hospital Avenue Singapore 168751. Singapore Eye Research Institute 11 Third Hospital Avenue Singapore 168751. Department of Ophthalmology, National University of Singapore Level 7 NUHS Tower block 1E Kent Ridge Road Singapore 119228. Department of Statistics and Applied Probability, National University of Singapore Block S16, Level 7, 6 Science Drive 2 Faculty of Science National University of Singapore Singapore 117546. Duke-National University of Singapore Graduate Medical School Singapore 8 College Road Singapore 169857.
M AN U
-
TE D
1
RI PT
2. Authors: Marcus Ang1,2 Wan Ling Wong2,3 Sieh Yean Kiew1 Xiang Li2,4 Soon-Phaik Chee1,2,3,5
3. Corresponding author:
AC C
Assoc. Professor Soon-Phaik Chee Singapore National Eye Centre 11 Third Hospital Avenue Singapore 168751 Tel: (65) 62277255 Fax: (65) 62277290 Email: [email protected] Short title: Interferon-gamma Release Assays for Tuberculous Uveitis
1
ACCEPTED MANUSCRIPT
Abstract
AC C
EP
TE D
M AN U
SC
RI PT
Purpose: To perform a head-to-head comparison of two commercially available interferon-gamma release assays, QuantiFERON-Gold-in-tube (Cellestis Inc, Australia) and T-SPOT.TB (Oxford Immunotech, UK), in the diagnosis of tuberculous uveitis. Design: Prospective cohort to study diagnostic accuracy Methods: We recruited consecutive new patients who presented with uveitis to a tertiary institution over a 2-year period. All patients underwent complete ocular examination and systemic evaluation, including T-SPOT.TB, QuantiFERON-Gold-in-tube and tuberculin skin test. Patients were followed-up for a minimum of 1 year after completion of antituberculous therapy where indicated. The main outcome measures were sensitivity, specificity and accuracy of each test estimated using Bayesian latent class analysis (presented with 95% Bayesian credible intervals, Crl). Prior information was obtained from published meta-analyses for diagnostic tests: QuantiFERON-Gold-in-tube sensitivity (0.64, 0.59-0.69) and specificity (0.99, 0.99-1.00); T-SPOT.TB sensitivity (0.50, 0.33-0.67) and specificity (0.91, 0.88-0.93). Results: From our study in patients with uveitis, QuantiFERON-Gold-in-tube was more specific but slightly less sensitive (sensitivity: 0.64, 0.60-0.69; specificity: 0.995, 0.9880.999) than T-SPOT.TB (sensitivity: 0.67, 0.60–0.74; specificity: 0.91, 0.88–0.93). However, QuantiFERON-Gold-in-tube was significantly more accurate in identifying ‘true positive tuberculous uveitis cases’ compared to T-SPOT.TB amongst discordant cases (QuantiFERON-Gold-in-tube positive 98% versus T-SPOT.TB positive 76%; ratio 1.28, 95%Crl: 1.11-1.72 i.e. 95%Crl > 1.0, statistically significant). Conclusion: Based on statistical decision theory, our head-to-head study suggests that QuantiFERON-Gold-in-tube is the first-line test that should be performed in preference to T-SPOT.TB (and the tuberculin skin test) for diagnosing tuberculous uveitis.
ACCEPTED MANUSCRIPT
SC
RI PT
Introduction Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide, with 8.7 million incident cases in 2011.1 Currently, the diagnosis of TB still depends on the centuryold Mantoux or tuberculin skin test.2 However, tuberculin skin testing has poor specificity due to false-positives in persons infected with non-tuberculous mycobacteria or vaccinated with Bacille Calmette–Guérin (BCG).3,4 Interferon-gamma release assays (IGRAs) are based on in-vitro detection of Interferon-gamma released by T-cells in response to antigens specific to Mycobacterium tuberculosis ;5 as opposed to tuberculin skin testing which uses a crude extract of proteins from Mycobacterium tuberculosis i.e. purified protein derivative (PPD).6, 7 Commercially available Interferon-gamma release assays s include the T-SPOT.TB (Oxford Immunotec, Oxford, United Kingdom) and QuantiFERON-TB Gold In-tube (Cellestis Incorporated, Carnegie, Australia).8
M AN U
The main advantage of Interferon-gamma release assays are that they provide an objective, reproducible test requiring only one visit.8 However, the main disadvantages are higher cost, logistical issues as the samples are time and temperature sensitive, and the need for trained personnel to analyze the results. Though similar, there are some key differences between TSPOT.TB and the QuantiFERON-TB Gold In-Tube. In T-SPOT.TB the number of Interferongamma producing T-cells are counted, after stimulating isolated peripheral blood mononuclear cells with early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), using an enzyme-linked immunospot assay.9 On the other hand, QuantiFERON-TB Gold In-Tube is a whole blood assay that quantifies interferon-gamma produced by T-cells in response to ESAT-6, CFP-10 and TB7.7 using an enzyme-linked immuno-sorbent assay.10
AC C
EP
TE D
Today, tuberculous uveitis is essentially a presumptive diagnosis. It is diagnosed when uveitis is present with a positive tuberculin skin test or Interferon-gamma release assay and chest x-ray (CXR) findings suggestive of pulmonary TB and/or evidence of associated systemic TB infection in the absence of other underlying disease.11 Few ocular biopsies3 are positive on culture, acid-fast bacilli smear or polymerase chain reaction (PCR) analysis.11 Thus, the role of Interferon-gamma release assays in the diagnosis of tuberculous uveitis has become increasingly important as these have implications for treatment and prognosis.12 While the role of Interferongamma release assays in diagnosing tubercular uveitis has been studied, to our knowledge,13-15 there is currently no head-to-head comparison between QuantiFERON-TB Gold In-Tube and TSPOT.TB specifically for the diagnosis of tuberculous uveitis. Thus, we conducted a prospective, direct comparative study to compare these two commercially available Interferon-gamma release assays to diagnose tuberculous uveitis in our population. Materials and Methods Overview of Management We conducted a prospective study of consecutive patients presenting with new onset of uveitis to the Singapore National Eye Centre Ocular Inflammation and Immunology Service over a 2-year period (1st January 2009- 31st December 2010). Ethical approval was obtained from our Singapore Health Services Centralized Institutional Review Board, and our study adhered to the tenets of the Declaration of Helsinki. After obtaining informed consent, all patients underwent a full systemic review, ocular examination, and standard baseline investigations as previously described.13 We included all patients who were undergoing systemic review for acute uveitis and
2
ACCEPTED MANUSCRIPT
gave informed consent to be enrolled in the study. We excluded patients who did not consent to the minimum follow-up period of 1 year after completion of ocular and/or systemic therapy.
M AN U
SC
RI PT
Investigations At presentation, all patients were tested with a standard panel of investigations as described,13 essentially: a complete blood count, erythrocyte sedimentation rate analysis, liver enzyme panel analysis, and infectious disease screening, which included a venereal disease research laboratory (VDRL) test for syphilis, tuberculin skin test, urine microscopy, and a CXR. Other tests such as HLA-B27 screen, an acid and alcohol fast bacilli smear from throat swabs, and PCR assays for TB DNA from ocular samples were performed if the patient consented to the procedure. Blood was taken for QuantiFERON-TB Gold In-Tube and T-SPOT.TB testing before the tuberculin skin test was performedto avoid any boosting effect (although it has been shown that this is unlikely to be significant).16, 17 The tuberculin skin test was performed using the standard Mantoux method: intradermal injection of 0.1 ml (2 tuberculin units) purified protein derivative (PPD) (RT23 SSI – 2T.U./0.1 ml Statens Serum Institut, Copenhagen, Denmark).18 Induration was measured at 72 hours with a ruler and considered positive if it was more than or equal to 15 mm (as validated in our population).13
EP
TE D
T-SPOT.TB was performed according to the manufacturer's instructions.19 For each patient, 8 ml of blood was collected in Lithium Heparin tubes and processed within 8 hours of sampling. Peripheral blood mononuclear cells were prepared by density gradient centrifugation over Ficoll Paque™Plus (GE Healthcare). 250 000 cells were seeded in each of four wells of the assay plate. The cells were stimulated for 16–20 h (under 5% carbon dioxide at 37°C) with GIBCO AIMV™ medium (nil control), phytohaemagglutinin (mitogen-positive control) or the TB-specific peptide antigens (peptide pools for ESAT-6 and CFP-10 in separate wells) in a total volume of 150 µL per well. Two readers quantified the number of Interferon-gamma spot-forming T-cells visually, and a third reader was consulted if the results were disparate. The T-SPOT.TB test was considered positive if there were >8 spots compared to the negative control well; negative if there were 10 spots and/or
S0002-9394(14)00061-0
DOI:
10.1016/j.ajo.2014.01.031
Reference:
AJOPHT 8803
To appear in:
American Journal of Ophthalmology
Received Date: 29 August 2013 Revised Date:
24 January 2014
Accepted Date: 24 January 2014
Please cite this article as: Ang M, Wong WL, Kiew SY, Li X, Chee S-P, Prospective Head-to-Head Study Comparing Two Commercial Interferon-gamma Release Assays for the Diagnosis of Tuberculous Uveitis, American Journal of Ophthalmology (2014), doi: 10.1016/j.ajo.2014.01.031. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Title Page 1. Title: Prospective Head-to-Head Study Comparing Two Commercial Interferongamma Release Assays for the Diagnosis of Tuberculous Uveitis
-
3
-
4
-
EP
5
-
SC
2
Singapore National Eye Centre 11 Third Hospital Avenue Singapore 168751. Singapore Eye Research Institute 11 Third Hospital Avenue Singapore 168751. Department of Ophthalmology, National University of Singapore Level 7 NUHS Tower block 1E Kent Ridge Road Singapore 119228. Department of Statistics and Applied Probability, National University of Singapore Block S16, Level 7, 6 Science Drive 2 Faculty of Science National University of Singapore Singapore 117546. Duke-National University of Singapore Graduate Medical School Singapore 8 College Road Singapore 169857.
M AN U
-
TE D
1
RI PT
2. Authors: Marcus Ang1,2 Wan Ling Wong2,3 Sieh Yean Kiew1 Xiang Li2,4 Soon-Phaik Chee1,2,3,5
3. Corresponding author:
AC C
Assoc. Professor Soon-Phaik Chee Singapore National Eye Centre 11 Third Hospital Avenue Singapore 168751 Tel: (65) 62277255 Fax: (65) 62277290 Email: [email protected] Short title: Interferon-gamma Release Assays for Tuberculous Uveitis
1
ACCEPTED MANUSCRIPT
Abstract
AC C
EP
TE D
M AN U
SC
RI PT
Purpose: To perform a head-to-head comparison of two commercially available interferon-gamma release assays, QuantiFERON-Gold-in-tube (Cellestis Inc, Australia) and T-SPOT.TB (Oxford Immunotech, UK), in the diagnosis of tuberculous uveitis. Design: Prospective cohort to study diagnostic accuracy Methods: We recruited consecutive new patients who presented with uveitis to a tertiary institution over a 2-year period. All patients underwent complete ocular examination and systemic evaluation, including T-SPOT.TB, QuantiFERON-Gold-in-tube and tuberculin skin test. Patients were followed-up for a minimum of 1 year after completion of antituberculous therapy where indicated. The main outcome measures were sensitivity, specificity and accuracy of each test estimated using Bayesian latent class analysis (presented with 95% Bayesian credible intervals, Crl). Prior information was obtained from published meta-analyses for diagnostic tests: QuantiFERON-Gold-in-tube sensitivity (0.64, 0.59-0.69) and specificity (0.99, 0.99-1.00); T-SPOT.TB sensitivity (0.50, 0.33-0.67) and specificity (0.91, 0.88-0.93). Results: From our study in patients with uveitis, QuantiFERON-Gold-in-tube was more specific but slightly less sensitive (sensitivity: 0.64, 0.60-0.69; specificity: 0.995, 0.9880.999) than T-SPOT.TB (sensitivity: 0.67, 0.60–0.74; specificity: 0.91, 0.88–0.93). However, QuantiFERON-Gold-in-tube was significantly more accurate in identifying ‘true positive tuberculous uveitis cases’ compared to T-SPOT.TB amongst discordant cases (QuantiFERON-Gold-in-tube positive 98% versus T-SPOT.TB positive 76%; ratio 1.28, 95%Crl: 1.11-1.72 i.e. 95%Crl > 1.0, statistically significant). Conclusion: Based on statistical decision theory, our head-to-head study suggests that QuantiFERON-Gold-in-tube is the first-line test that should be performed in preference to T-SPOT.TB (and the tuberculin skin test) for diagnosing tuberculous uveitis.
ACCEPTED MANUSCRIPT
SC
RI PT
Introduction Tuberculosis (TB) remains one of the leading causes of morbidity and mortality worldwide, with 8.7 million incident cases in 2011.1 Currently, the diagnosis of TB still depends on the centuryold Mantoux or tuberculin skin test.2 However, tuberculin skin testing has poor specificity due to false-positives in persons infected with non-tuberculous mycobacteria or vaccinated with Bacille Calmette–Guérin (BCG).3,4 Interferon-gamma release assays (IGRAs) are based on in-vitro detection of Interferon-gamma released by T-cells in response to antigens specific to Mycobacterium tuberculosis ;5 as opposed to tuberculin skin testing which uses a crude extract of proteins from Mycobacterium tuberculosis i.e. purified protein derivative (PPD).6, 7 Commercially available Interferon-gamma release assays s include the T-SPOT.TB (Oxford Immunotec, Oxford, United Kingdom) and QuantiFERON-TB Gold In-tube (Cellestis Incorporated, Carnegie, Australia).8
M AN U
The main advantage of Interferon-gamma release assays are that they provide an objective, reproducible test requiring only one visit.8 However, the main disadvantages are higher cost, logistical issues as the samples are time and temperature sensitive, and the need for trained personnel to analyze the results. Though similar, there are some key differences between TSPOT.TB and the QuantiFERON-TB Gold In-Tube. In T-SPOT.TB the number of Interferongamma producing T-cells are counted, after stimulating isolated peripheral blood mononuclear cells with early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10), using an enzyme-linked immunospot assay.9 On the other hand, QuantiFERON-TB Gold In-Tube is a whole blood assay that quantifies interferon-gamma produced by T-cells in response to ESAT-6, CFP-10 and TB7.7 using an enzyme-linked immuno-sorbent assay.10
AC C
EP
TE D
Today, tuberculous uveitis is essentially a presumptive diagnosis. It is diagnosed when uveitis is present with a positive tuberculin skin test or Interferon-gamma release assay and chest x-ray (CXR) findings suggestive of pulmonary TB and/or evidence of associated systemic TB infection in the absence of other underlying disease.11 Few ocular biopsies3 are positive on culture, acid-fast bacilli smear or polymerase chain reaction (PCR) analysis.11 Thus, the role of Interferon-gamma release assays in the diagnosis of tuberculous uveitis has become increasingly important as these have implications for treatment and prognosis.12 While the role of Interferongamma release assays in diagnosing tubercular uveitis has been studied, to our knowledge,13-15 there is currently no head-to-head comparison between QuantiFERON-TB Gold In-Tube and TSPOT.TB specifically for the diagnosis of tuberculous uveitis. Thus, we conducted a prospective, direct comparative study to compare these two commercially available Interferon-gamma release assays to diagnose tuberculous uveitis in our population. Materials and Methods Overview of Management We conducted a prospective study of consecutive patients presenting with new onset of uveitis to the Singapore National Eye Centre Ocular Inflammation and Immunology Service over a 2-year period (1st January 2009- 31st December 2010). Ethical approval was obtained from our Singapore Health Services Centralized Institutional Review Board, and our study adhered to the tenets of the Declaration of Helsinki. After obtaining informed consent, all patients underwent a full systemic review, ocular examination, and standard baseline investigations as previously described.13 We included all patients who were undergoing systemic review for acute uveitis and
2
ACCEPTED MANUSCRIPT
gave informed consent to be enrolled in the study. We excluded patients who did not consent to the minimum follow-up period of 1 year after completion of ocular and/or systemic therapy.
M AN U
SC
RI PT
Investigations At presentation, all patients were tested with a standard panel of investigations as described,13 essentially: a complete blood count, erythrocyte sedimentation rate analysis, liver enzyme panel analysis, and infectious disease screening, which included a venereal disease research laboratory (VDRL) test for syphilis, tuberculin skin test, urine microscopy, and a CXR. Other tests such as HLA-B27 screen, an acid and alcohol fast bacilli smear from throat swabs, and PCR assays for TB DNA from ocular samples were performed if the patient consented to the procedure. Blood was taken for QuantiFERON-TB Gold In-Tube and T-SPOT.TB testing before the tuberculin skin test was performedto avoid any boosting effect (although it has been shown that this is unlikely to be significant).16, 17 The tuberculin skin test was performed using the standard Mantoux method: intradermal injection of 0.1 ml (2 tuberculin units) purified protein derivative (PPD) (RT23 SSI – 2T.U./0.1 ml Statens Serum Institut, Copenhagen, Denmark).18 Induration was measured at 72 hours with a ruler and considered positive if it was more than or equal to 15 mm (as validated in our population).13
EP
TE D
T-SPOT.TB was performed according to the manufacturer's instructions.19 For each patient, 8 ml of blood was collected in Lithium Heparin tubes and processed within 8 hours of sampling. Peripheral blood mononuclear cells were prepared by density gradient centrifugation over Ficoll Paque™Plus (GE Healthcare). 250 000 cells were seeded in each of four wells of the assay plate. The cells were stimulated for 16–20 h (under 5% carbon dioxide at 37°C) with GIBCO AIMV™ medium (nil control), phytohaemagglutinin (mitogen-positive control) or the TB-specific peptide antigens (peptide pools for ESAT-6 and CFP-10 in separate wells) in a total volume of 150 µL per well. Two readers quantified the number of Interferon-gamma spot-forming T-cells visually, and a third reader was consulted if the results were disparate. The T-SPOT.TB test was considered positive if there were >8 spots compared to the negative control well; negative if there were 10 spots and/or
