• Plos One · Jan 2014

    Development of a one-step probe based molecular assay for rapid immunodiagnosis of infection with M. tuberculosis using dried blood spots.

    • Thomas Blauenfeldt, Jan Heyckendorf, Graff JensenSidseSDepartment of Pulmonary Medicine, Copenhagen University Hospital Gentofte, Copenhagen, Denmark., Christoph Lange, Camilla Drabe, Thomas S Hermansen, Lena de Thurah, Troels Lillebaek, Jesper Eugen-Olsen, Niels Seersholm, Søren Hoff, Jesper Bonde, and Morten Ruhwald.
    • Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark; Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
    • Plos One. 2014 Jan 1; 9 (9): e105628.

    BackgroundAntigen specific release of IP-10 is the most promising alternative marker to IFN-γ for infection with M. tuberculosis. Compared to Interferon-γ release assays (IGRA), IP-10 is released in high levels enabling novel approaches such as field friendly dried blood spots (DBS) and molecular detection.AimTo develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS.MethodWe developed a one-step probe based multiplex RT-qPCR assay for detecting IP-10 and IFN-γ mRNA expression from whole blood and DBS samples. The assay was validated and applied for the diagnosis of M. tuberculosis infection in DBS samples from 43 patients with confirmed TB, 13 patients with latent TB and 96 presumed uninfected controls. In parallel, IP-10 and INF-γ levels were measured in Quantiferon (QFT-TB) plasma supernatants.ResultsIP-10 mRNA upregulation was detectable at 4 hours after stimulation (6 fold upregulation) peaking at 8 hours (108 fold upregulation). IFN-γ expression occurred in concert but levels were lower (peak 6.7 fold upregulation). IP-10 gene expression level was significantly higher in patients with tuberculosis (median 31.2, IQR 10.7-67.0) and persons with latent tuberculosis infection (LTBI) (41.2, IQR 9.8-64.9) compared to healthy controls (1.6, IQR 1.1-2.4; p<0.0001). The IP-10 mRNA and protein based tests had comparable diagnostic accuracy to QFT-TB, sensitivity (85% and 88% vs 85%) and specificity (96% and 96% vs 97%, p = ns.).ConclusionWe developed a rapid, robust and accurate molecular immunodiagnostic test for M. tuberculosis infection. By combining DBS based sample acquisition, mail or currier based sample transport with centralized molecular detection, this immunodiagnostic test concept can reduce the local technological requirements everywhere and make it possible to offer highly accurate immunodiagnostic tests in low resource settings.

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