Detection of immune cell response to M. tuberculosis–specific antigens by quantitative polymerase chain reaction ☆ Ilona Bibova a,b, ⁎ , Irena Linhartova a , Ondrej Stanek a,b , Vendula Rusnakova c , Mikael Kubista c,d , Miloslav Suchanek e , Martina Vasakova f , Peter Sebo a,c a Institute of Microbiology AS CR, v.v.i., Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic b Department of Biochemistry and Microbiology, ICT Prague, 166 28 Prague 6, Czech Republic c Institute of Biotechnology AS CR, v.v.i., Academy of Sciences of the Czech Republic, 142 20 Prague 4, Czech Republic d TATAA Biocenter, 411 03 Odinsgatan 28, Göteborg, Sweden e Department of Analytical Chemistry, ICT Prague, 166 28 Prague 6, Czech Republic f Pneumological Clinic, Thomayer University Hospital with Policlinic, 140 59 Prague 4, Czech Republic Received 18 July 2011; accepted 23 September 2011 Abstract One third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb) and up to 10% of infected individuals develop active tuberculosis (TB) in their lifetime. Among the major challenges in the control of TB is the implementation of sensitive methods for detection of latent tuberculosis infection (LTBI). Currently, in vitro interferon gamma release assays, yielding single value readout, are used as an alternative to the traditional tuberculin skin test for the diagnosis of LTBI. More complex characterization of immune status of LTBI individuals, however, is desirable for indication of LTBI subjects for preventative chemotherapy. Here we describe a quantitative polymerase chain reaction (qPCR) for determination of expression levels of 14 genes, additional to interferon gamma, which was applied for comparison of the specific Mtb-antigen immune response of blood cells from healthy, latently infected, and TB individuals. With the use of principal component analysis and discriminant analysis, a pattern of mRNA levels of 6 genes was identified, allowing discrimination of healthy individuals from active TB and LTBI subjects. These results open the way to development of multimarker qPCR for the detection of LTBI. © 2011 Elsevier Inc. All rights reserved. Keywords: qPCR; Latent tuberculosis infection; Blood sample processing; Reference gene 1. Introduction Tuberculosis (TB) is a major public health problem that contributes considerably to morbidity and mortality from infectious diseases around the world. It is estimated that approximately one-third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), the causative agent of TB in humans (World Health Organization, 2010) and some 10% of those develop active TB (Kaufmann and McMichael, 2005), while 90% of the Mtb carriers are asymptomatic individuals with latent tuberculosis infection (LTBI). Of LTBI donors, the recent contacts of TB patients and immunosuppressed individuals (e.g., HIV-infected individuals, organ transplant patients, patients undergoing anti–TNF-α therapy, etc.) are at particular risk of developing active TB (Krejsek and Kopecky, 2004; Kroesen et al., 2003). Treatment of LTBI with chemoprophylaxis substantially reduces the risk that TB infection progresses to disease (Menzies et al., 2011). Therefore, targeting and treating LTBI subjects with high risk of disease progression are a key strategy for effective control of the spread of TB. Such efforts are, however, complicated by the lack of a gold standard diagnostic test for the detection of LTBI. Most often, identification of individuals infected with Mtb relies on the tuberculin skin Available online at www.sciencedirect.com Diagnostic Microbiology and Infectious Disease xx (2011) xxx – xxx www.elsevier.com/locate/diagmicrobio ☆ This work was supported by grants 2B06161 and KAN200520702 (to P.S.), grant IAA500970904 (to M.K.) and by the institutional research concept nos. AV0Z50200510 and AV0Z50520701 of the Academy of Sciences of the Czech Republic. ⁎ Corresponding author. Tel.: +42-420-24106-2016; fax: +42-420-241- 062-152. E-mail address: bibova@biomed.cas.cz (I. Bibova). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2011.09.024