title: Development of a one-step probe based molecular assay for rapid immunodiagnosis of infection with M. tuberculosis using dried blood spots.
abstract: 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.
  
  
  OBJECTIVE
  To develop a robust IP-10 based molecular assay for the diagnosis of infection with M. tubercuolsis from whole blood and DBS.
  
  
  METHODS
  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.).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark; Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Blauenfeldt
authors.affiliation: Clinical Infectious Diseases, German Center for Infection Research (DZIF) Tuberculosis Unit, Research Center Borstel, Borstel, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Heyckendorf
authors.affiliation: Department of Pulmonary Medicine, Copenhagen University Hospital Gentofte, Copenhagen, Denmark.
authors.forename: Sidse
authors.initials: S
authors.lastname: Graff Jensen
authors.affiliation: 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.
authors.forename: Christoph
authors.initials: C
authors.lastname: Lange
authors.affiliation: Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
authors.forename: Camilla
authors.initials: C
authors.lastname: Drabe
authors.affiliation: International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Thomas S
authors.initials: TS
authors.lastname: Hermansen
authors.affiliation: Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark; Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
authors.forename: Lena
authors.initials: L
authors.lastname: de Thurah
authors.affiliation: International Reference Laboratory of Mycobacteriology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Troels
authors.initials: T
authors.lastname: Lillebaek
authors.affiliation: Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Eugen-Olsen
authors.affiliation: Department of Pulmonary Medicine, Copenhagen University Hospital Gentofte, Copenhagen, Denmark.
authors.forename: Niels
authors.initials: N
authors.lastname: Seersholm
authors.affiliation: Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Hoff
authors.affiliation: Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark; Department of Pathology, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
authors.forename: Jesper
authors.initials: J
authors.lastname: Bonde
authors.affiliation: Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark; Clinical Research Centre, Copenhagen University Hospital Hvidovre, Copenhagen, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Ruhwald
mesh_terms: D000328:Adult; D000368:Aged; D000942:Antigens, Bacterial / Q000097:blood / Q000276:immunology; D016022:Case-Control Studies; D054357:Chemokine CXCL10 / Q000097:blood / Q000276:immunology*; D059788:Dried Blood Spot Testing / Q000379:methods* / Q000592:standards; D005260:Female; D006801:Humans; D007118:Immunoassay / Q000379:methods* / Q000592:standards; D007371:Interferon-gamma / Q000097:blood / Q000276:immunology; D055985:Latent Tuberculosis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology; D008297:Male; D008875:Middle Aged; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D012333:RNA, Messenger / Q000097:blood / Q000276:immunology*; D012680:Sensitivity and Specificity; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000382:microbiology
chemical_list: D000942:Antigens, Bacterial; C114753:CXCL10 protein, human; D054357:Chemokine CXCL10; D012333:RNA, Messenger; D007371:Interferon-gamma