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Background RISK6, a human-blood transcriptomic signature, has shown promise as a non-sputum-based tool for tuberculosis (TB) screening, triage and treatment monitoring among immunocompetent adults in high TB burden settings. 1 The TB-HIRA assay is a one-step multiplex qRT-PCR reaction that has been developed to streamline and accelerate the original multi-step singleplex protocol for measuring signature expression.Aim To optimise the TB-HIRA qRT-PCR assay, determine concordance with the original RISK6 singleplex assays and determine performance of RISK6 scores using both methods to discriminate between active pulmonary TB (PTB), QuantiFERON-TB Gold (QFT) positive/latent TB (LTBI) and healthy (QFT negative) controls.Method Venous blood samples stored in TempusTM RNA tubes from 20 individuals with active PTB, 10 with LTBI and 18 healthy controls were evaluated. Specimens were blinded and randomised before RNA extraction and throughout analysis. Both TB-HIRA and RISK6 assays were run in RT-PCR/PCR triplicates per sample. RISK6 scores were calculated in R using the risk6.R script 2 and expanded ROC analysis was performed in STATA to determine optimal cut-off thresholds.Results There was excellent correlation between the original RISK6 score and the TB-HIRA assay (Spearman correlation coefficient 0.98. TB-HIRA systematically yielded higher RISK6 scores than the original singleplex assays. For both protocols, the RISK6 scores were significantly higher for active PTB compared with LTBI/healthy control groups with the optimal cut-off threshold distinguishing between them with 95% sensitivity and 96% specificity (AUC>0.99). There was no significant difference in scores between the healthy and LTBI groups ( figure 1).Abstract M35 Figure 1Comparison of RISK6 score generated from the TB-HIRA vs singleplex assaysConclusion In this proof-of-concept pilot study, the RISK6 signature offers robust discrimination of active PTB from LTBI and healthy subgroups, supporting potential utility as a non-sputum-based TB diagnostic test in a low TB burden setting. Strong concordance between the TB-HIRA multiplex assay and original singleplex assays, offers scope to develop a rapid near-patient TB triage test.References Bayaa R, et al. Multi-country evaluation of RISK6, a 6-gene blood transcriptomic signature, for tuberculosis diagnosis and treatment monitoring. Scientific Reports 2021;11(1).Penn-Nicholson A, et al. RISK6, a 6-gene transcriptomic signature of TB disease risk, diagnosis and treatment response. Scientific Reports 2020;10(1).