Application of Flow Cytometry in the Diagnostics Pipeline of Primary Immunodeficiencies Underlying Disseminated
Pamela P Lee1,2, Mongkol Lao-Araya3, Jing Yang1
1Department of Paediatrics and Adolescent Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
Abstract:
Talaromyces (Penicillium) marneffei is an AIDS-defining infection in Southeast Asia and is associated with high mortality. It is rare in non-immunosuppressed individuals, especially children. Little is known about host immune response and genetic susceptibility to this endemic fungus. Genetic defects in the interferon-gamma (IFN-γ)/STAT1 signaling pathway, CD40/CD40 ligand- and IL12/IL12-receptor-mediated crosstalk between phagocytes and T-cells, and STAT3-mediated Th17 differentiation have been reported in HIV-negative children with talaromycosis and other endemic mycoses such as histoplasmosis, coccidioidomycosis, and paracoccidioidomycosis. There is a need to design a diagnostic algorithm to evaluate such patients. In this article, we review a cohort of pediatric patients with disseminated talaromycosis referred to the Asian Primary Immunodeficiency Network for genetic diagnosis of PID. Using these illustrative cases, we propose a diagnostics pipeline that begins with immunoglobulin pattern (IgG, IgA, IgM, and IgE) and enumeration of lymphocyte subpopulations (T-, B-, and NK-cells). The former could provide clues for hyper-IgM syndrome and hyper-IgE syndrome. Flow cytometric evaluation of CD40L expression should be performed for patients suspected to have X-linked hyper-IgM syndrome. Defects in interferon-mediated JAK-STAT signaling are evaluated by STAT1 phosphorylation studies by flow cytometry. STAT1 hyperphosphorylation in response to IFN-α or IFN-γ and delayed dephosphorylation is diagnostic for gain-of-function STAT1 disorder, while absent STAT1 phosphorylation in response to IFN-γ but normal response to IFN-α is suggestive of IFN-γ receptor deficiency. This simple and rapid diagnostic algorithm will be useful in guiding genetic studies for patients with disseminated talaromycosis requiring immunological investigations.
Insights
Talaromyces marneffei infections in children can indicate primary immunodeficiency. A new diagnostic algorithm using immunoglobulin levels and lymphocyte analysis helps identify genetic defects, guiding further immunological investigations for better patient outcomes.
Area of Science:
- Immunology
- Medical Mycology
- Genetics
Background:
- Talaromyces marneffei is an AIDS-defining fungal infection prevalent in Southeast Asia, causing high mortality, particularly in immunocompromised individuals.
- Understanding the host immune response and genetic susceptibility in non-immunosuppressed children with talaromycosis is crucial.
- Previous studies identified genetic defects in IFN-γ/STAT1, CD40/CD40L, IL12/IL12R, and STAT3 pathways in children with endemic mycoses.
Purpose of the Study:
- To review a cohort of pediatric patients with disseminated talaromycosis evaluated for primary immunodeficiency (PID).
- To propose a diagnostic algorithm for evaluating pediatric talaromycosis patients for underlying genetic immune defects.
- To guide genetic studies by outlining a rapid immunological investigation pipeline.
Main Methods:
- Review of a pediatric patient cohort with disseminated talaromycosis referred for PID genetic diagnosis.
- Proposed diagnostic pipeline starting with immunoglobulin (IgG, IgA, IgM, IgE) assessment and lymphocyte subset enumeration (T, B, NK cells).
- Flow cytometry for CD40L expression and STAT1 phosphorylation studies in response to interferons (IFN-α, IFN-γ).
Main Results:
- Immunoglobulin patterns can suggest hyper-IgM or hyper-IgE syndromes.
- CD40L expression analysis aids in diagnosing X-linked hyper-IgM syndrome.
- STAT1 phosphorylation studies differentiate between STAT1 gain-of-function disorders and IFN-γ receptor deficiency.
Conclusions:
- A simple, rapid diagnostic algorithm can be established for pediatric disseminated talaromycosis.
- This algorithm aids in identifying primary immunodeficiencies by evaluating immunoglobulin patterns, lymphocyte subsets, and interferon-mediated signaling pathways.
- The proposed pipeline is valuable for guiding genetic investigations in affected children.
