title: Challenges in the development of an immunochromatographic interferon-gamma test for diagnosis of pleural tuberculosis.
abstract: Existing diagnostic tests for pleural tuberculosis (TB) have inadequate accuracy and/or turnaround time. Interferon-gamma (IFNg) has been identified in many studies as a biomarker for pleural TB. Our objective was to develop a lateral flow, immunochromatographic test (ICT) based on this biomarker and to evaluate the test in a clinical cohort. Because IFNg is commonly present in non-TB pleural effusions in low amounts, a diagnostic IFNg-threshold was first defined with an enzyme-linked immunosorbent assay (ELISA) for IFNg in samples from 38 patients with a confirmed clinical diagnosis (cut-off of 300 pg/ml; 94% sensitivity and 93% specificity). The ICT was then designed; however, its achievable limit of detection (5000 pg/ml) was over 10-fold higher than that of the ELISA. After several iterations in development, the prototype ICT assay for IFNg had a sensitivity of 69% (95% confidence interval (CI): 50-83) and a specificity of 94% (95% CI: 81-99%) compared to ELISA on frozen samples. Evaluation of the prototype in a prospective clinical cohort (72 patients) on fresh pleural fluid samples, in comparison to a composite reference standard (including histopathological and microbiologic test results), showed that the prototype had 65% sensitivity (95% CI: 44-83) and 89% specificity (95% CI: 74-97). Discordant results were observed in 15% of samples if testing was repeated after one freezing and thawing step. Inter-rater variability was limited (3%; 1 out of 32). In conclusion, despite an iterative development and optimization process, the performance of the IFNg ICT remained lower than what could be expected from the published literature on IFNg as a biomarker in pleural fluid. Further improvements in the limit of detection of an ICT for IFNg, and possibly combination of IFNg with other biomarkers such as adenosine deaminase, are necessary for such a test to be of value in the evaluation of pleural tuberculosis.
authors.affiliation: Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America ; McGill International TB Centre & Department of Epidemiology, Biostatistics, and Occupational Health, McGill University, Montreal, Quebec, Canada.
authors.forename: Claudia M
authors.initials: CM
authors.lastname: Denkinger
authors.affiliation: Tulip Diagnostics, Goa, India.
authors.forename: Yatiraj
authors.initials: Y
authors.lastname: Kalantri
authors.affiliation: McGill International TB Centre & Department of Epidemiology, Biostatistics, and Occupational Health, McGill University, Montreal, Quebec, Canada.
authors.forename: Samuel G
authors.initials: SG
authors.lastname: Schumacher
authors.affiliation: Department of Microbiology, Christian Medical College, Vellore, India.
authors.forename: Joy S
authors.initials: JS
authors.lastname: Michael
authors.affiliation: Department of Pulmonary Medicine, Christian Medical College, Vellore, India.
authors.forename: Deepa
authors.initials: D
authors.lastname: Shankar
authors.affiliation: Tulip Diagnostics, Goa, India.
authors.forename: Arvind
authors.initials: A
authors.lastname: Saxena
authors.affiliation: Tulip Diagnostics, Goa, India.
authors.forename: Natarajan
authors.initials: N
authors.lastname: Sriram
authors.affiliation: Department of Pulmonary Medicine, Christian Medical College, Vellore, India.
authors.forename: Thangakunam
authors.initials: T
authors.lastname: Balamugesh
authors.affiliation: Department of Pathology, Stanford University, Stanford, California, United States of America.
authors.forename: Robert
authors.initials: R
authors.lastname: Luo
authors.affiliation: Division of Infectious Diseases, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States of America ; Department of Laboratory Medicine, Boston Children's Hospital, Boston, Massachusetts, United States of America.
authors.forename: Nira R
authors.initials: NR
authors.lastname: Pollock
authors.affiliation: McGill International TB Centre & Department of Epidemiology, Biostatistics, and Occupational Health, McGill University, Montreal, Quebec, Canada.
authors.forename: Madhukar
authors.initials: M
authors.lastname: Pai
authors.affiliation: Department of Pulmonary Medicine, Christian Medical College, Vellore, India.
authors.forename: Devasahayam J
authors.initials: DJ
authors.lastname: Christopher
mesh_terms: D000293:Adolescent; D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D015331:Cohort Studies; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D014396:Tuberculosis, Pleural / Q000175:diagnosis* / Q000276:immunology
chemical_list: D015415:Biomarkers; D007371:Interferon-gamma