Host Response to Infection by Direct Analysis of Leukocyte Single Cell-type Gene Expression/transcript Abundance, Direct LS-TA. a Prospective Study Will Evaluate the Performance of Direct LS-TA in Triage Febrile Patients Into Major Categories of Infections: Viral, Bacterial or Active Tuberculosis.

Active Tuberculosis, Bacterial Diseases, Gene Expression, Gene Expression Profiling, Host Defense Mechanisms, Host Microbial Interactions, Infection, Single Cell Sequencing Technology, Transcriptome, Viral Diseases

host response, transcriptome, single cell-type gene expression, single cell RNA, triage, fever, febrile patient, Direct LS-TA

Febrile illness is a common condition and it is crucial to have an early triage of patients according to various aetiologies to enable appropriate treatment. Currently, most screening/diagnostic tests target the detection of pathogens, while only a few assays aim to understand the host response, and they are mostly based on a measurement of serum proteins (e.g. CRP or procalcitonin). Recently, blood transcriptome has been explored to differentiate bacterial and viral infections. However, gene expression in blood represents a composite score of gene expression of all the component cell-types present in the sample. Here, we propose to develop a rapid test that can determine gene expressions of a specified single cell type in peripheral blood (e.g., monocytes or granulocytes) as a host response biomarker to differentiate three major categories of infections that are bacterial, viral, and tuberculosis The assay is called Direct Leukocyte Single cell-type transcript abundance (TA) assay (DIRECT LS-TA) as it can directly determine the gene expression of a specified single cell-type among various other leukocyte populations directly in a peripheral blood sample. Such results signify the nature of host response according to 3 or more axes (Type I or Type II interferon signaling response or pro-inflammatory cytokine signaling) And it can be used to indicate the type of underlying infection (viral, bacterial, or active tuberculosis).

DIRECT LS-TA is a ratio-based biomarker (RBB) for blood gene expression analysis which can be performed in commonly available equipments (e.g. qPCR or digital PCR machines). Using the ratio of TA of prior defined numerator gene and denominator gene, this RBB can quantify gene expression of the specified constitutional single cell-type (e.g. monocytes and granulocytes) inside a cell-mixture sample of Whole blood. DIRECT LS-TA was a method pioneered by the PI \[Tang 2017, https://patents.google.com/patent/US9589099B2/\]. And it has been developed for quantification of early B cell response after vaccination \[DOI: 10.3390/genes12070971\]. Recently, the method is used to develop host response biomarkers after infection to differentiate the type of pathogens (such as viral, bacterial or active tuberculosis). Numerator and denominator genes have been identified by using public gene expression datasets for monocytes and granulocytes. Diagnostic performance was good using these public data. Therefore, these RBBs will be applied in the prospective study to evaluate and compare their diagnostic (triage) performance of febrile patients into different pathogen etiologies. Comparing to other methods to obtain single cell-type gene expression data, DIRECT LS-TA has many advantages The conventional (gold standard) approach is to quantify gene expression in a sample of purified single cell-type from peripheral blood but it is very labour intensive procedure. Recent single-cell RNA sequencing can also provide gene expression data for every individual cell in a sample, but it is slow and very costly. DIRECT LS-TA provides the unique technique to quantify single cell-type gene expression in blood samples without the need to isolate the targeted cell-type. It has been used to differentiate the type of host response in fever patients into 3 major axes (Type I or Type II interferon signaling response or pro-inflammatory cytokine signaling) which correspond to the nature of underlying infection aetiologies (viral infection, active tuberculosis and bacterial infection). See preprint DOI: 10.1101/2025.01.27.634977. and patents in B cells : https://patents.google.com/patent/WO2022089426A1/ Monocytes: https://patents.google.com/patent/WO2023109365A1/ Granulocytes : https://patents.google.com/patent/GB202400180D0/

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