Circulating cell-free RNA in blood as a host response biomarker for detection of tuberculosis.

Tuberculosis (TB) remains a leading cause of death from an infectious disease worldwide, partly due to a lack of effective strategies to screen and triage individuals with potential TB. Whole blood RNA signatures have been tested as biomarkers for TB, but have failed to meet the World Health Organization's (WHO) optimal target product profiles (TPP). Here, we use RNA sequencing and machine-learning to investigate the utility of plasma cell-free RNA (cfRNA) as a host-response biomarker for TB in cohorts from Uganda, Vietnam and Philippines. We report a 6-gene cfRNA signature, which differentiates TB-positive and TB-negative individuals with AUC = 0.95, 0.92, and 0.95 in test, training and validation, respectively. This signature meets WHO TPPs (sensitivity: 97.1% [95% CI: 80.9-100%], specificity: 85.2% [95% CI: 72.4-100%]) regardless of geographic location, sample collection method and HIV status. Overall, our results identify plasma cfRNA as a promising host response biomarker to diagnose TB.

Plasma Cell-free RNA Signatures of Inflammatory Syndromes in Children.

Inflammatory syndromes, including those caused by infection, are a major cause of hospital admissions among children and are often misdiagnosed because of a lack of advanced molecular diagnostic tools. In this study, we explored the utility of circulating cell-free RNA (cfRNA) in plasma as an analyte for the differential diagnosis and characterization of pediatric inflammatory syndromes. We profiled cfRNA in 370 plasma samples from pediatric patients with a range of inflammatory conditions, including Kawasaki disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), viral infections and bacterial infections. We developed machine learning models based on these cfRNA profiles, which effectively differentiated KD from MIS-C - two conditions presenting with overlapping symptoms - with high performance (Test Area Under the Curve (AUC) = 0.97). We further extended this methodology into a multiclass machine learning framework that achieved 81% accuracy in distinguishing among KD, MIS-C, viral, and bacterial infections. We further demonstrated that cfRNA profiles can be used to quantify injury to specific tissues and organs, including the liver, heart, endothelium, nervous system, and the upper respiratory tract. Overall, this study identified cfRNA as a versatile analyte for the differential diagnosis and characterization of a wide range of pediatric inflammatory syndromes.

Circulating Cell-Free RNA in Blood as a Host Response Biomarker for the Detection of Tuberculosis.

Tuberculosis (TB) remains a leading cause of death from an infectious disease worldwide. This is partly due to a lack of tools to effectively screen and triage individuals with potential TB. Whole blood RNA signatures have been extensively studied as potential biomarkers for TB, but they have failed to meet the World Health Organization's (WHOs) target product profiles (TPPs) for a non-sputum triage or diagnostic test. In this study, we investigated the utility of plasma cell-free RNA (cfRNA) as a host response biomarker for TB. We used RNA profiling by sequencing to analyze plasma samples from 182 individuals with a cough lasting at least two weeks, who were seen at outpatient clinics in Uganda, Vietnam, and the Philippines. Of these individuals, 100 were diagnosed with microbiologically-confirmed TB. Our analysis of the plasma cfRNA transcriptome revealed 541 differentially abundant genes, the top 150 of which were used to train 15 machine learning models. The highest performing model led to a 9-gene signature that had a diagnostic accuracy of 89.1% (95% CI: 83.6-93.4%) and an area under the curve of 0.934 (95% CI: 0.8674-1) for microbiologically-confirmed TB. This 9-gene signature exceeds the optimal WHO TPPs for a TB triage test (sensitivity: 96.2% [95% CI: 80.9-100%], specificity: 89.7% [95% CI: 72.4-100%]) and was robust to differences in sample collection, geographic location, and HIV status. Overall, our results demonstrate the utility of plasma cfRNA for the detection of TB and suggest the potential for a point-of-care, gene expression-based assay to aid in early detection of TB.

Nucleic acid biomarkers of immune response and cell and tissue damage in children with COVID-19 and MIS-C.

Differential host responses in coronavirus disease 2019 (COVID-19) and multisystem inflammatory syndrome in children (MIS-C) remain poorly characterized. Here, we use next-generation sequencing to longitudinally analyze blood samples from pediatric patients with COVID-19 or MIS-C across three hospitals. Profiling of plasma cell-free nucleic acids uncovers distinct signatures of cell injury and death between COVID-19 and MIS-C, with increased multiorgan involvement in MIS-C encompassing diverse cell types, including endothelial and neuronal cells, and an enrichment of pyroptosis-related genes. Whole-blood RNA profiling reveals upregulation of similar pro-inflammatory pathways in COVID-19 and MIS-C but also MIS-C-specific downregulation of T cell-associated pathways. Profiling of plasma cell-free RNA and whole-blood RNA in paired samples yields different but complementary signatures for each disease state. Our work provides a systems-level view of immune responses and tissue damage in COVID-19 and MIS-C and informs future development of new disease biomarkers.