The goal of this observational study is to identify biomarkers and host factors associated with dengue severity in patients from dengue-endemic regions. The main question it aims to answer is:

Can multi-omics approaches predict disease severity and identify key factors contributing to severe dengue infections?

Participants will include individuals with dengue infection, and the study will analyze their blood samples using multi-omics techniques to uncover host immune responses, disease pathogenesis, and cellular pathway interactions. The study will also assess the role of primary and secondary infections, virus serotypes, and immunological factors in disease progression. Findings aim to support disease management, improve severity prediction, and identify potential therapeutic targets to prevent severe outcomes.

Background: Many ongoing dengue clinical trials aim to evaluate both therapeutic compounds and potential vaccine candidates. The therapeutic compounds aim to target various stages of dengue virus replication, such as viral entry, by inhibiting virus fusion, NS5 polymerase inhibitor, and NS1 antigenemia reduction, to mention a few. However, an omics-based approach to identifying the metabolic pathways that dengue utilizes to subvert the host system to derive energy from and subsequently deploy viral immune evasion strategies has yet to be established. This study will focus on exploring and understanding the pathways that the dengue virus exploits and investigate if blocking the identified pathway can cause the virus to 'starve.'

• The study will offer insight into the diverse system-level range of dengue infection markers and the dysregulated molecules involved in cellular and metabolic pathways during dengue infection.
• The identification of novel pathways affected by molecule dysregulation will unveil the association of the biomarkers with immune response and disease severity, thereby helping to improve patient triage and treatment.
• The identified biomarkers will be significant in monitoring patients, creating a platform for early intervention to mitigate severe dengue through novel therapeutic targets and management strategies.

The primary objectives of the proposed study are:

• Case-wise viral serotyping and investigation of host cytokine profile.
• System-wide blood-transcriptomics and proteomics during dengue infection and its relationship with disease severity.
• Identification of pathways influencing severity by in vitro mechanistic studies.

Study Cohorts:

Clinical material from two endemic regions, one from Central America (Guatemala) and Southeast Asia (India) will be used. Guatemala is an exemplary location for studying the DENV infection, owing to its distinctive combination of climatic diversity and endemic and dengue presence. Two blood samples (on the day of hospitalization and the day of discharge) from the patients will be collected as per the WHO revised 2009 case classification: 1) dengue without warning signs (DwoWS, n=200), 2) dengue with warning signs (DWS, n=200), 3) severe dengue (SD, n=200). Additionally, non-dengue samples will be collected from healthy controls (n=200). This cohort will act as the DISCOVERY cohort. We will collect whole blood in a TempusTM tube (for RNA sequencing) and serum (for proteomics and metabolomics).

Under the India-EU cooperation on research & innovation (R&I) and co-funding partnership under the EU framework program on R&I 'Horizon Europe, samples will be collected from India, from Artemis Hospital, New Delhi, Max Health Care, New Delhi (a unit of Devki Devi Foundation) and Kasturba Medical College Hospital, Mangalore. Those are the two epicentres of the dengue endemic in India. 50 samples from each dengue severity classification (total samples 150 in each category) and DENV-negative healthy control (total 150: 50 in each site) will be collected. The Indian cohorts will act as the VALIDATION cohorts.

Recruitment of controls Healthy participants who have tested negative for dengue infection in the past three months will not be recruited to the study, which will be further age-and gender-matched with the cases. These samples will be tested for anti-dengue IgM and NS1 ELISA to rule out ongoing asymptomatic infection. They will also be subjected to anti-dengue IgG ELISA to check for past dengue infection. Each of the control participants will also be asked to provide a detailed medical history

Inclusion Criteria:

Group 1: Dengue without warning signs Patients presenting with the following symptoms confirmed positive for dengue infection by lab diagnosis by anti-dengue IgM or NS1 ELISA will be recruited for the study under this cohort.

Symptoms will be fever along with at least two of the following -

• Myalgia, arthralgia
• Nausea and/or vomiting
• Rashes
• Leukopenia Group 2: DENV with warning signs This cohort will include patients with the above symptoms and any of the following clinical signs.
• Abdominal pain with tenderness
• Persistent vomiting
• Fluid accumulation
• Mucosal bleeding
• Restlessness, lethargy
• Liver enlargement >2cm
• Laboratory findings: An increase in hematocrit levels and a rapid decrease in platelet count Group 3: Severe dengue
• Severe plasma leakage leads to hypovolemic shock (dengue shock syndrome) and fluid accumulation with respiratory distress.
• Severe bleeding, such as epistaxis, internal bleeding, etc., is evaluated by a clinician.
• Severe organ involvement with any of the following symptoms
• Liver AST or ALT ≥ 1000
• CNS: impaired consciousness
• Heart and other organs failure

Exclusion Criteria:

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• Patients who are pregnant, immunocompromised, or undergoing chemotherapy, on steroids or immunosuppressive drugs in the last year, liver cirrhosis, or coinfection with a non-dengue pathogen.
• Cases with a lab-confirmed dengue infection within the past three months will excluded from the case cohort.

Annonymised and delinked IPD can be shared after the completion of the study after getting approval from the steering board and obtaining the ethical clearance of the requesting party.