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There is an urgent need for new tools to combat the ongoing tuberculosis (TB) pandemic. Gene expression profiles based on blood signatures have proved useful in identifying genes that enable classification of TB patients, but have thus far been complex. Using real‐time PCR analysis, we evaluated the expression profiles from a large panel of genes in TB patients and healthy individuals in an Indian cohort. Classification models were built and validated for their capacity to discriminate samples from TB patients and controls within this cohort and on external independent gene expression datasets. A combination of only four genes distinguished TB patients from healthy individuals in both cross‐validations and on separate validation datasets with very high accuracy. An external validation on two distinct cohorts using a real‐time PCR setting confirmed the predictive power of this 4‐gene tool reaching sensitivity scores of 88% with a specificity of around 75%. Moreover, this gene signature demonstrated good classification power in HIV + populations and also between TB and several other pulmonary diseases. Here we present proof of concept that our 4‐gene signature and the top classifier genes from our models provide excellent candidates for the development of molecular point‐of‐care TB diagnosis in endemic areas.

BACKGROUND. Bacille Calmette-Guérin (BCG) vaccine is protective against Tuberculosis (TB) in children, but its efficacy wanes with age. Consequently, determining if BCG revaccination augments anti-TB immunity in young adults in TB endemic regions is vital. METHODS. Two hundred healthy adults, BCG vaccinated at birth, were tested for their IFN-γ release assay (IGRA) status. Of these, 28 IGRA + and 30 IGRAwere BCG revaccinated, and 24 IGRA + and 23 IGRAsubjects served as unvaccinated controls. T and innate cell responses to mycobacterial antigens were analyzed by 14-color flow cytometry over 34 weeks. RESULTS. IFN-γ and/or IL-2 Ag85A-and BCG-specific CD4 + and CD8 + T cell responses were boosted by revacciantion at 4 and 34 weeks, respectively, and were > 2-fold higher in IGRA + compared with IGRAvaccinees. Polyfunctional Ag85A, BCG, and mycobacterium tuberculosis (Mtb) latency Agspecific (LTAg-specific) CD4 + T cells expressing up to 8 cytokines were also significantly enhanced in both IGRA + and IGRAvaccinees relative to unvaccinated controls, most markedly in IGRA + vaccinees. A focused analysis of Th17 responses revealed expansion of Ag85A-, BCG-, and LTAgspecific total IL-17A + ,IL-17F + ,IL-22 + , and IL-10 + CD4 + T cell effectors in both IGRA + and IGRAsubjects. Also, innate IFN-γ + NK/γδ/NKT cell responses were higher in both IGRA + and IGRAvaccinees compared with controls. This is the first evidence to our knowledge that BCG revaccination significantly boosts antimycobacterial Th1/Th17 responses in IGRA + and IGRAsubjects. CONCLUSION. These data show that BCG revaccination is immunogenic in IGRAand IGRA + subjects, implying that Mtb preinfection in IGRA + subjects does not impact immunogenicity. This has implications for public health and vaccine development strategies.

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This study is the first to validate PCR-based assays for detection of microsporidial DNA in corneal scrapings. Pan microsporidian PCR can be a useful adjunct with smear examination in the diagnosis of microsporidial keratitis.