Host Response to Infection by Direct Analysis of Leukocyte Single Cell-type Gene Expression/transcript Abundance, Direct LS-TA

Peripheral Blood Single Cell-type Expression Profile of Interferon-stimulated and Other Biomarker Genes for Triage of Febrile Patients

Febrile illness is a common condition, particularly among young patients and it is crucial to have an early triage of patients according to various aetiologies to enable appropriate treatment. Most diagnostic tests are targeted towards the detection of pathogens while other assays are mostly related to serum proteins. Blood cells transcriptome has been explored to differentiate bacterial and viral infections.

Here, we propose to develop a rapid test using the host responses in terms of gene expressions of single-cell populations of peripheral leukocytes (monocytes and granulocytes) 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 (e.g. monocytes and granulocytes) among various leukocyte cell populations directly in a peripheral blood sample. Such results signify the nature of host response and can be used to indicate the type of infection (viral, bacterial or active tuberculosis).

Study Overview

• Status: Active, not recruiting
• Conditions: Bacterial Infections; Infection; Active Tuberculosis; Gene Expression Profiling; Gene Expression; Viral Diseases
• Intervention / Treatment: Other: No intervention

Detailed Description

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 these retrospective samples to evaluate and compare their diagnostic (triage) performance of febrile patients into different pathogen etiologies.

• Study Type: Observational
• Enrollment (Actual): 192

Contacts and Locations

Study Locations

• Hong Kong
  • Hong Kong, Hong Kong
    • Dept of Chemical Pathology, Chinese University of Hong Kong

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: Yes

• Sampling Method: Non-Probability Sample

Study Population

Patients with Infections were patients admitted to ward for treatment of infection.

Description

Inclusion Criteria:

• Bacterial infection sample: positive isolation of organism in blood culture (Bacterial infection sample group)
• Clinical diagnosed active TB (Tuberculosis infection group)

Exclusion Criteria:

• End stage renal failure
• Pregnancy
• Infections for which long term antibiotic treatment is strongly recommended (including infective endocarditis, osteoarticular infections, cerebral or hepatic or lung abscesses, tuberculosis or nontuberculous mycobacterial infections)

Study Plan

How is the study designed?

Number of groups / cohorts

3

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Vaccination Controls; Up to 200 adult samples collected as the baseline of this study will be used to establish the reference ranges of the ratio-based biomarkers by quantitative PCR or digital PCR.	Other: No intervention; No intervention for this retrospective study
Bacterial infection samples; A retrospective sample of adult patients with positive bacterial blood culture.	Other: No intervention; No intervention for this retrospective study
Tuberculosis samples; A retrospective sample of adult patients who were diagnosed to have active tuberculosis disease.	Other: No intervention; No intervention for this retrospective study

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Viral host response Direct LS-TA in monocytes (Type I interferon response)	Direct LS-TA is a kind of RBB that can be readily quantified by qPCR or dPCR of a pair of genes, the RBB numerator gene and the RBB denominator gene. The biomarker is the ratio of the 2 genes. Single cell-type: Monocyte. RBB numerator gene: IFI27 or IFI44L. RBB denominator gene: PSAP, CTSS or CPVL. The various RBB (e.g. IFI27/PSAP and IFI44L/PSAP ratios) will be compared in terms of their sensitivity/specificity of diagnostic performance in the vaccination control samples and samples from infection groups. AUC of ROC analysis will be performed where appropriate.	1 day (first collected sample after admission)
Viral host response Direct LS-TA in granulocytes (Type I interferon response)	Direct LS-TA is a kind of RBB that can be readily quantified by qPCR or dPCR of a pair of genes, the RBB numerator gene and the RBB denominator gene. The biomarker is the ratio of the 2 genes. Single cell-type: Granulocyte. RBB numerator gene: RSAD2 or IFIT1. RBB denominator gene: SAT1 or SRGN. The various RBB will be compared in terms of their sensitivity/specificity of diagnostic performance in the vaccination control samples and samples from infection groups. AUC of ROC analysis will be performed where appropriate.	1 day (first collected sample after admission)
Bacterial infection host response Direct LS-TA in monocytes (pro-inflammatory response)	Direct LS-TA is a kind of RBB that can be readily quantified by qPCR or dPCR of a pair of genes, the RBB numerator gene and the RBB denominator gene. The biomarker is the ratio of the 2 genes. Single cell-type: Monocyte. RBB numerator gene: VNN1, or NLRC4. RBB denominator gene: PSAP, CTSS or CPVL. The various RBBs will be compared in terms of their sensitivity/specificity of diagnostic performance in the vaccination control samples and samples from infection groups. AUC of ROC analysis will be performed where appropriate.	1 day (first collected sample after admission)
Bacterial infection host response Direct LS-TA in granulocytes (pro-inflammatory response)	Direct LS-TA is a kind of RBB that can be readily quantified by qPCR or dPCR of a pair of genes, the RBB numerator gene and the RBB denominator gene. The biomarker is the ratio of the 2 genes. Single cell-type: Granulocyte. RBB numerator gene: ALPL, ARG1, or ANXA3. RBB denominator gene: SAT1 or SRGN. The various RBB will be compared in terms of their sensitivity/specificity of diagnostic performance in the vaccination control samples and samples from infection groups. AUC of ROC analysis will be performed where appropriate.	1 day (first collected sample after admission)
Active TB host response Direct LS-TA in monocytes (Type II interferon response)	Direct LS-TA is a kind of RBB that can be readily quantified by qPCR or dPCR of a pair of genes, the RBB numerator gene and the RBB denominator gene. The biomarker is the ratio of the 2 genes. Single cell-type: Monocyte. RBB numerator gene: WARS1, ATF3 or CALHM6. RBB denominator gene: PSAP, CTSS or CPVL. The various RBB will be compared in terms of their sensitivity/specificity of diagnostic performance in the vaccination control samples and samples from infection groups. AUC of ROC analysis will be performed where appropriate.	1 day (first collected sample after admission)
Active TB host response Direct LS-TA in granulocytes (Type II interferon response)	Direct LS-TA is a kind of RBB that can be readily quantified by qPCR or dPCR of a pair of genes, the RBB numerator gene and the RBB denominator gene. The biomarker is the ratio of the 2 genes. Single cell-type: granulocyte. RBB numerator gene: ANKRD22, BATF2, CD274, FCGR1A or ETV7. RBB denominator gene: SAT1 or SRGN. The various RBB will be compared in terms of their sensitivity/specificity of diagnostic performance in the vaccination control samples and samples from infection groups. AUC of ROC analysis will be performed where appropriate.	1 day (first collected sample after admission)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Reference intervals and median of these DIRECT LS-TA RBB in the control group	The median value and the central 95% range of each RBB will be determined.	1 day (first collected sample after admission)

Collaborators and Investigators

• Sponsor: Chinese University of Hong Kong

Publications and helpful links

General Publications

• Huang D, Liu AYN, Leung KS, Tang NLS. Direct Measurement of B Lymphocyte Gene Expression Biomarkers in Peripheral Blood Transcriptomics Enables Early Prediction of Vaccine Seroconversion. Genes (Basel). 2021 Jun 25;12(7):971. doi: 10.3390/genes12070971.
• Huang B, Huang J, Chiang NH, Chen Z, Lui G, Ling L, Kwan MYW, Wong JSC, Mak PQ, Ling JWH, Lam ICS, Ng RWY, Wang X, Gao R, Hui DS, Ma SL, Chan PKS, Tang NLS. Interferon response and profiling of interferon response genes in peripheral blood of vaccine-naive COVID-19 patients. Front Immunol. 2024 Jan 10;14:1315602. doi: 10.3389/fimmu.2023.1315602. eCollection 2023.
• Direct single cell-type gene expression analysis in Whole blood: Novel ratio-based gene expression biomarkers using 2 novel monocyte reference genes (PSAP and CTSS) for monitoring bacterial infection Nelson LS Tang, Tsz-Ki Kwan, Dan Huang, Suk Ling Ma, K.S. Leung bioRxiv 2025.01.27.634977; doi: https://doi.org/10.1101/2025.01.27.634977

Helpful Links

• Patent about the model to obtain single cell-type gene expression in peripheral blood
• Patent about the model to obtain single cell-type gene expression in peripheral blood

Study record dates

Study Major Dates

• Study Start (Actual): January 1, 2018
• Primary Completion (Estimated): January 1, 2026
• Study Completion (Estimated): January 1, 2026

Study Registration Dates

• First Submitted: February 17, 2025
• First Submitted That Met QC Criteria: February 20, 2025
• First Posted (Actual): March 25, 2025

Study Record Updates

• Last Update Posted (Actual): March 25, 2025
• Last Update Submitted That Met QC Criteria: February 27, 2025
• Last Verified: February 1, 2025

More Information

Terms related to this study

• Keywords: fever; triage; host response; transcriptome; Single cell-type gene expression; febrile patients
• Additional Relevant MeSH Terms: Pathologic Processes; Disease Attributes; Gram-Positive Bacterial Infections; Bacterial Infections and Mycoses; Actinomycetales Infections; Mycobacterium Infections; Infections; Communicable Diseases; Virus Diseases; Tuberculosis; Bacterial Infections
• Other Study ID Numbers: Direct LS-TA retrospective

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED
• IPD Plan Description: Descriptive statistics of each RBB (DIRECT LS-TA) will be provided on a reasonable request.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No