TY - JOUR T1 - An mRNA based IP-10 release assay for LTBI combining RT-qPCR and dried blood spots JF - European Respiratory Journal JO - Eur Respir J VL - 44 IS - Suppl 58 SP - 3249 AU - Thomas Blauenfeldt AU - Jesper Bonde AU - Sidse Graff AU - Niels Seersholm AU - Christoph Lange AU - Jan Heyckendorf AU - Morten Ruhwald Y1 - 2014/09/01 UR - http://erj.ersjournals.com/content/44/Suppl_58/3249.abstract N2 - Introduction:IP-10 is an immunodiagnostic biomarker for latent TB (LTBI) expressed at levels 100 fold higher than IFN-γ. We compared mRNA and protein expression patterns of IP-10 and IFN-γ in whole blood stimulated with M.Tb specific antigens, and show proof-of-principle detecting LTBI using an mRNA based IP-10 release assay on dried blood spots (DBS).Method:Blood from 27 TB patients, 9 healthy IGRA positive and 96 controls was stimulated in QuantiFERON tubes. mRNA was detected at 8h and protein levels at 20h. In a subgroup of 5 patients, both levels were determined at 2 hour intervals for 48h. mRNA expression was assessed using RT-qPCR and protein levels using ELISA.Results:IP-10 mRNA is stable and readily detected from whole blood as well as DBS. Assay reproducibility was very high (CV% <1). The kinetics of IP-10 and IFN-γ expression were comparable, with IP-10 levels substantially higher than IFN-γ. IP-10 mRNA levels were detectable at 4h and peaked at 8h, protein levels plateaued at 18h. IP-10 mRNA and protein release were comparable for LTBI and TB. The diagnostic accuracy was high (area under ROC curve; 0.93) and both mRNA and protein based methods yielded sensitivity of 86% (31/36) with a specificity of 96% (92/96).Discussion:Given the shorter incubation time, mRNA based IP-10 release assays enables a fast workflow. Compared to protein-based assays, molecular detection is more accurate and optimal for automation, suggesting that next generation IGRA assays could be developed from this blueprint.This study is ongoing and for the conference, we will present a larger cohort. ER -