Additionally, in the preliminary experiment, it was observed that 4 of 5 sera from individuals with culture-positive active infections contained measurable HspX

Additionally, in the preliminary experiment, it was observed that 4 of 5 sera from individuals with culture-positive active infections contained measurable HspX. this study, using standard, and Luminex xMAP? bead capture ELISA, respectively, we report on detection of anti-HspX IgG and IgM antibodies and HspX protein in sera from acute and latent TB patients. For the antibody screen, levels of IgG and IgM antibodies were similar between non-infected and active TB patients; however, individuals classified into the group with latent TB showed higher values of anti-HspX IgM (p = 0.003) compared to active TB patients. Using the bead capture antigen detection assay, HspX protein was detected in sera from 56.5% of putative latent cases (p< 0.050) compared to the background median with an average of 9,900 pg/ml and a range of 1 1,000 to 36,000 pg/ml. Thus, presence of anti-HspX IgM antibodies and HspX protein in sera may be markers of latent TB. Introduction Tuberculosis (TB) is a bacterial disease primarily caused by infection with antigens without evidence of clinically manifested active TB [2, 3]. Persons with LTBI have negative bacteriological tests and the presumptive diagnosis is based on a positive result of either a skin (tuberculin skin test, TST) or blood (interferon-gamma release assay, IGRA) test indicating an immune response to infection. There are treatment regimens that can reduce but not eliminate the risk of reactivation disease in individuals with LTBI, but such prophylactic therapy involves taking antibiotics for up to 9 months, and carries a small risk of serious, or even fatal, side-effects in these TST/IGRA-positive individuals only suspected of having LTBI. At present, a direct measurement tool for LTBI in humans is currently unavailable [2, 3], and the inability to identify the population of individuals with LTBI constitutes a major impediment to TB control efforts [4]. An Institute of Medicine report concluded that the development of new diagnostics that can distinguish the truly infected individuals from those who are exposed but self-cured/false positive will be the key to ultimately eliminating TB [5]. Proteins expressed under stress are ubiquitous PX-866 (Sonolisib) in nature and they play an important role in helping bacterial cells to survive under extreme conditions. It is hypothesized that the primary location for latent bacilli is the granuloma, and within these structures, the bacteria are exposed to adverse conditions including hypoxic stress, low pH, hydrolytic enzymes, toxic fatty acids and reactive oxygen radicals. To survive, bacilli upregulate stress-response proteins including chaperones and transport proteins associated with pH control, metabolite movement, and lipid metabolism [6C9]. The gene encodes HspX (also known as -crystallin, Acr, or the 16-kDa antigen) which is PX-866 (Sonolisib) expressed during stationary growth, under hypoxia, and low pH, similar to conditions present within areas of some granulomas [10]. HspX may be an important element of replication control in the granuloma since its over-expression inhibits growth [11C13]. The use of a serodiagnostic test for TB was analyzed by Limongi et al. [14], who reported that specific anti-HspX IgA in pleural fluid samples was useful to discriminate between pleural effusion due to TB and other pulmonary disease. Similarly, detection of IgG, IgA, and IgM antibodies against the 38-kDa and 16-kDa antigens, but does not include the detection of IgM. Alternatively, two groups have reported that they are examining direct detection of antigens released into body fluids, however, those tests target abundant non-specific mycobacterial cell wall components: lipoarabinomannan (LAM) in urine [17, 18]. The relative reluctance PX-866 (Sonolisib) of investigators to employ the detection of a low abundance secreted protein as a diagnostic is due to uncertainty as to what specific antigens are most appropriate, and the expectation that these factors may only be present in very low amounts in serum, especially in the case of LTBI. As described, HspX is produced exclusively during periods of Sox2 bacterial stress, including survival within granulomas [19]. In that study, naphthol red-tagged anti-HspX monoclonal antibody highlighted the presence of HspX protein on bacterial outer membranes within guinea pig early granulomas (3 weeks after infection) and later granulomas (10 weeks post-infection). Based on these and other unpublished studies, it was expected that this protein would be present in patient sera in picogram/ml to femtogram/ml concentrations; theoretically detectable amounts by ultra-sensitive detection systems. In this study, we report on successful detection of anti-HspX antibodies and HspX protein in LTBI patient sera, and statistically determine diagnostic potential. Materials and methods Ethics statement The present study and the consent procedures were specifically approved by the Review Board of the National Research Commission of Instituto Mexicano del Seguro Social with approval number: R-2011-1906-41 in Mexico and The University of Georgia,.