1 Pulmonary Medicine, Herlev and Gentofte Hospital, The Capital Region of Denmark2 Clinical Research Centre, Amager and Hvidovre Hospital, The Capital Region of Denmark3 Department of Pathology, Amager and Hvidovre Hospital, The Capital Region of Denmark4 Clinical Infectious Diseases, German Center for Infection Research (DZIF) Tuberculosis Unit, Research Center Borstel, Borstel, Germany.5 Department of Thoracic Medicine and Infectious Disease, Nordsjællands Hospital, The Capital Region of Denmark6 Clinical Infectious Diseases, German Center for Infection Research (DZIF) Tuberculosis Unit, Research Center Borstel, Borstel, Germany; Department of Medicine, University of Namibia School of Medicine, Windhoek, Namibia.7 Department of Radiology, Centre for Functional and Diagnostic Imaging and research, Amager and Hvidovre Hospital, The Capital Region of Denmark8 Statens Serum Institut
BACKGROUND: Antigen 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. AIM: To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS. METHOD: We 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. RESULTS: IP-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.). CONCLUSION: We 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.