Challenges in the Development of an Immunochromatographic Interferon-Gamma Test for Diagnosis of Pleural Tuberculosis

Claudia M Denkinger,Madhukar Pai,Yatiraj Kalantri,Devasahayam J Christopher,Nira R Pollock,Natarajan Sriram,Robert Luo,Samuel G Schumacher,Arvind Saxena,Thangakunam Balamugesh,Joy S Michael,Deepa Shankar

doi:10.1371/journal.pone.0085447

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 300pg/ml; 94% sensitivity and 93% specificity). The ICT was then designed; however, its achievable limit of detection (5000pg/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%; 1out 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.

Highlights

Extrapulmonary TB (EPTB) accounts for approximately 25% of all TB, and poses major diagnostic challenges
We considered the diagnosis of pleural TB confirmed if smear, culture or Xpert on pleural tissue or fluid was positive for Mycobacterium tuberculosis (MTB), histopathology of pleural tissue identified granulomas, or MTB was present in any other respiratory sample
Initial evaluation of 1st prototype Initial results under optimized conditions at Tulip Diagnostics, using aliquots of the same samples that were used to define the cut-off for the enzyme-linked immunosorbent assay (ELISA), showed a sensitivity of 69% (95% confidence interval (CI): 41-89) and specificity of 100% in comparison to the ELISA

Summary

Introduction

Extrapulmonary TB (EPTB) accounts for approximately 25% of all TB, and poses major diagnostic challenges. Pleural TB is the second most common manifestation of EPTB after lymph node TB [1,2]. A pleural fluid aspiration rarely yields a definite diagnosis. A biopsy of the pleural tissue for histology and culture is considered the diagnostic gold standard, but may still be falsely negative in 10% to 20% of cases [3,4]. Nucleic acid amplification tests (NAATs) for evaluation of TB in pleural effusions appear to have high specificity (98%) but relatively low sensitivity (62%) [5]. Xpert MTB/RIF (Cepheid Inc., Sunnyvale, CA), a recently developed NAAT, had low sensitivity (25-50%) across a number of studies on pleural fluid (and one study on pleural biopsy), with consistently high specificity [6,7,8]

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