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

<div><p>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.</p> </div

Prototype of the IFNg lateral flow test (named Gammacheck).

<p>Prototype of the IFNg lateral flow test (named Gammacheck).</p

IFNg lateral flow test.

<p>(A) Initial results at Tulip with clearly defined lines indicating a positive (top) and negative (bottom) test. (B) Initial results at clinical evaluation site (CMC) with smearing of sample and incomplete advancement.</p

Study flow and location of testing.

<p>Legend: CMC=Christian Medical College; TB=tuberculosis; ELISA=enzyme-linked immunosorbent assay; ICT= lateral flow, immunochromatographic test; IFNg=interferon gamma; ADA=adenosine deaminase.</p

Scatter plots of relations of implicit (IAT) and explicit weight bias with BMI at the individual level.

<p>Note. Each point in the plots represents the average preference of participants as a function of their BMI. The weight bias scores ranges from +2 to -2 for the IAT and from +3 to -3 for the explicit, with 0 indicating no relative preference between thin people over overweight people. More positive scores indicate a preference for thin people over overweight people, while more negative scores indicate a preference for overweight people over thin people. Vertical bars signify standard error. Data for participants with BMI greater than 60 were not included in the plot (0.15%). The regression line was computed on the original and not on the average data.</p

Scatter plots of relations of implicit (IAT) and explicit weight bias as a function of three weight indicators (BMI, percentage of overweight and underweight people) at the national level.

<p>Note. The weight bias scores ranges from +2 to -2 for the IAT and from +3 to -3 for the explicit, with 0 indicating no relative preference between thin people over overweight people. More positive scores indicate a preference for thin people over overweight people, while more negative scores indicate a preference for overweight people over thin people.</p