Validation of the robust performance of novel transketolase epitopes for tuberculosis immunodiagnostics

Tuberculosis (TB) is a major global health concern, contributing to millions of deaths annually. Mycobacterium tuberculosis (M.tb) is the causative agent for TB. The dormancy/non-replicative phase of M.tb is characterized by a lower cell division and decreased metabolic rate. Transketolase (TKT) plays a crucial role in bacterial growth and M.tbTKT plays a key role in switch from the dormancy to proliferative phase. Despite advances in molecular TB-diagnostics, there is an unmet need for a point-of-care TB-diagnostics. Through immunoscreening of a novel T7 phage display library, we identified an antigen-clone with some homology to M.tb TKT (TKTμ). We designed two other antigenic peptides corresponding to M.tbTKT (M.tbTKT1 and M.tbTKT3). Utilizing a peptide-based ELISA against TKTμ, M.tbTKT1, M.tbTKT3, we tested 400 sera from TB (n = 100), Latent Tuberculosis Infection (LTBI n = 50), and non-infected individuals (n = 250). Receiver operating characteristic (ROC) by 10-fold cross-validation (CV) was applied to classify the performance of TKT epitopes for TB diagnosis. The TKTμ ROC achieved area under curve (AUC) of 0.98 ± 0.02. The 10-fold CV for TKTµ IgG-antibody yielded a mean sensitivity of 1, mean±SD specificity of 0.90 ± 0.03, accuracy of 0.93 ± 0.02, a positive predictive value (PPV) of 0.82 ± 0.12; and negative predictive value (NPV) of 1.00; TKT1 ROC curves yielded AUC of 0.88 ± 0.06, a mean sensitivity of 0.83 ± 0.13, specificity of 0.81 ± 0.1, accuracy of 0.82 ± 0.06, NPV of 0.91 ± 0.04 and PPV of 0.69 ± 0.15. TKT3 IgG-antibody reached ROC of 0.94 ± 0.02, a mean sensitivity of 0.88 ± 0.07, specificity of 0.84 ± 0.05, accuracy of 0.85 ± 0.04, NPV of 0.93 ± 0.03 and PPV of 0.72 ± 0.14. These results validate the robust performance of TKT-based TB immunodiagnostics.