Evaluation of Pathogenesis and Diagnosis of Mycoplasma Pneumoniae Community-acquired Pneumonia (CAP)

Study Overview
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Brief Summary
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To investigate the Mycoplasma pneumoniae-specific circulating antibody-secreting cell (ASC) response and Mycoplasma pneumoniae-specific interferon (INF)-γ-secreting T cell response, along with polymerase chain reaction (PCR) and serology, in a cohort of children with community-acquired pneumonia (CAP) and controls.

Official Title
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The Role of Adaptive Immune Responses to Mycoplasma Pneumoniae in Pathogenesis and Diagnosis of Community-acquired Pneumonia (CAP) in Children: an Observational Single-center Study (myCAP Study)

Conditions
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Childhood Pneumonia, Mycoplasma Pneumonia, Antibody-secreting Cells, Enzyme-linked Immunospot (ELISpot), Diagnosis

Intervention / Treatment
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* Diagnostic Test: Enzyme-linked immunospot (ELISpot) assay [Blood]


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: CAP cohort: Children of age 3 to 18 years; In- and outpatients; Clinically diagnosed community-acquired pneumonia (CAP); Healthy control cohort: - Healthy asymptomatic children of age 3 to 18 years undergoing an elective surgical procedure; Family control cohort: - Family members of index CAP patients. Exclusion Criteria: Hospital-acquired pneumonia; Immunodeficiencies; Chronic lung disorders.

Ages Eligible for Study
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Minimum Age: 3 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| CAP cohort<br>Children of age 3 to 16 years; In- and outpatients; Clinically diagnosed community-acquired pneumonia (CAP). | Diagnostic Test: Enzyme-linked immunospot (ELISpot) assay [Blood]<br>* The ASC ELISpot will be developed based on the improved methods recently described [Nat Protoc 2013;8:1073-87]. This protocol allows rapid (6-8 h) detection of specific ASCs in small volumes (1-2 ml) of blood. M. pneumoniae protein P1 (50 μl/ml) will be used as antigen. The optimal concentration of coating antigen will be assessed in advance in two-fold serial dilutions for clear spot definition. The M. pneumoniae-specific T cell ELISpot will be developed based on methods recently described [Nat Protoc 2009;4:461-9].<br>* Other names: Enzyme-linked immunosorbent assay (ELISA) [Serum];|
| Healthy control cohort<br>Healthy asymptomatic children of age 3 to 16 years; undergoing an elective surgical procedure. | Diagnostic Test: Enzyme-linked immunospot (ELISpot) assay [Blood]<br>* The ASC ELISpot will be developed based on the improved methods recently described [Nat Protoc 2013;8:1073-87]. This protocol allows rapid (6-8 h) detection of specific ASCs in small volumes (1-2 ml) of blood. M. pneumoniae protein P1 (50 μl/ml) will be used as antigen. The optimal concentration of coating antigen will be assessed in advance in two-fold serial dilutions for clear spot definition. The M. pneumoniae-specific T cell ELISpot will be developed based on methods recently described [Nat Protoc 2009;4:461-9].<br>* Other names: Enzyme-linked immunosorbent assay (ELISA) [Serum];|
| Family control cohort<br>- Family members of index CAP patients. | Diagnostic Test: Enzyme-linked immunospot (ELISpot) assay [Blood]<br>* The ASC ELISpot will be developed based on the improved methods recently described [Nat Protoc 2013;8:1073-87]. This protocol allows rapid (6-8 h) detection of specific ASCs in small volumes (1-2 ml) of blood. M. pneumoniae protein P1 (50 μl/ml) will be used as antigen. The optimal concentration of coating antigen will be assessed in advance in two-fold serial dilutions for clear spot definition. The M. pneumoniae-specific T cell ELISpot will be developed based on methods recently described [Nat Protoc 2009;4:461-9].<br>* Other names: Enzyme-linked immunosorbent assay (ELISA) [Serum];|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in numbers of M. pneumoniae-specific ASCs and M. pneumoniae-specific INF-γ-secreting T cells in blood from inclusion (day 0) to 1-month follow-up (day 28) | Enzyme-linked immunospot (ELISpot) assay and flow cytometry | At day 0 (inclusion, disease presentation) and at day 28 (follow-up, disease resolution) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Change in M. pneumoniae DNA levels in respiratory samples from inclusion (day 0) to 1-month follow-up (day 28) | PCR | At day 0 (inclusion, disease presentation) and at day 28 (follow-up, disease resolution) | 
| Change in total and M. pneumoniae-specific antibody levels (immunoglobulin (Ig)G, IgM, IgA) from inclusion (day 0) to 1-month follow-up (day 28) | Enzyme-linked immunosorbent assay (ELISA) | At day 0 (inclusion, disease presentation) and at day 28 (follow-up, disease resolution) | 
| Outcome of community-acquired pneumonia (CAP) assessed by clinical assessment of body temperature (°C) and respiratory rate (per minute) at 1-month follow-up (day 28) | Clinical assessment of body temperature (°C) and respiratory rate (per minute), with worse outcome defined as body temperature more than 38.5°C and respiratory rate according to age more than 40/min for 3 years, more than 34/min for 4-5 years, more than 30/min for 6-12 years, and more than 16/min for 13-18 years. | At day 28 (follow-up) |