Diagnostic performance of transcriptomic signatures for the evaluation of suspected tuberculosis in routine clinical practice

Background: Blood transcriptomic signatures for diagnosis of tuberculosis (TB) have shown promise in case-control studies but their diagnostic accuracy has not been prospectively validated in adults presenting with the full clinical spectrum of suspected TB, including extra-pulmonary TB, and its associated differential diagnoses with concomitant latent TB infection. Methods: Our study was nested within a prospective multi-center cohort study in secondary care in England. Patients had whole-blood taken for microarray to measure abundance of 47,275 transcripts and interferon-gamma release assays (IGRAs) at clinical presentation with suspected TB and were followed up for 6-12 months’ to determine final diagnoses based on pre-defined diagnostic criteria. The diagnostic accuracy of published transcriptomic signatures and a novel cohort-derived signature were assessed to generate area under the receiver-operating characteristic curves (AUC), sensitivities and specificities. Three millilitres of whole blood were collected into each of two Tempus tubes (Applied Biosystems/Ambion) by trained research staff following a standard phlebotomy protocol. Blood was vigorously mixed immediately following collection and stored at -80°C before RNA extraction. For each patient, the contents of one tube were used for analysis and the other tube was retained in case of assay failure. RNA was isolated using 1.5 ml whole blood and the MagMAX-96 Blood RNA Isolation Kit (Applied Biosystems/Ambion), as per the manufacturer’s instructions. 250 g of isolated total RNA was globin-reduced using the GLOBINclear 96-well format kit (Applied Biosystems/Ambion) according to the manufacturer’s instructions. Total and globin-reduced RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA yield was assessed using a NanoDrop8000 spectrophotometer (NanoDrop Products, Thermo Fisher Scientific). High-quality (>6.5 RIN) whole blood RNA was successfully obtained and processed by microarray in all cases. Biotinylated, amplified antisense complementary RNA (cRNA) targets were prepared from 200-250 ng of globin-reduced RNA using the Illumina CustomPrep RNA amplification kit (Applied Biosystems/Ambion). For each sample, seven hundred and fifty nanograms of labelled cRNA were hybridised overnight to Illumina Human HT12 V4 BeadChip arrays (Illumina), which contained greater than 47,000 probes. The arrays were washed, blocked, stained and scanned on an Illumina iScan, as per the manufacturer’s instructions. GenomeStudio (Illumina) was used to perform quality control and generate signal intensity values.