Diagnosis of <i>Mycoplasma pneumoniae</i> Pneumonia with Measurement of Specific Antibody-Secreting Cells

Mycoplasma pneumoniae (Mp) is reported to be the most common bacterial cause of community-acquired pneumonia (CAP) in hospitalized U.S. children (1). However, current diagnostic tests, including PCR of upper respiratory tract (URT) specimens and serology, do not differentiate between Mp infection and carriage (2). Mp carriage in the URT is found in up to 56% of healthy children (2, 3). A ≥4-fold increase in IgG levels is still used in most centers to confirm Mp infection but has low sensitivity (4) and is not helpful in acute clinical management (3). In the absence of an accurate diagnostic test, it is not surprising that studies and meta-analyses on the efficacy of antibiotics are inconclusive for Mp CAP in children (5, 6). Circulating antibody-secreting cell (ASC) responses have been demonstrated to be more rapid and shorter-lived than antibody responses (7). We hypothesized that Mp-IgM-ASCs circulate in peripheral blood only for a few days or weeks after Mp infection, whereas Mp-DNA in the URT and serum antibodies persist for months. We aimed to evaluate the measurement of Mp-IgM-ASCs by enzyme-linked immunospot (ELISpot) assay as a new test for diagnosing Mp CAP. Methods Pediatric patients with CAP (n = 152) and control subjects (n = 156) were enrolled from May 2016 to April 2017 after written informed consent. Inclusion criteria for patients with CAP were clinical diagnosis of pneumonia (fever &gt;38.5°C and tachypnea [8]) in previously healthy children aged 3–18 years. Children &lt;3 years were excluded because of a high probability of viral coexistence in the URT (8). Control individuals included healthy children (undergoing elective surgical procedures) and siblings of patients with CAP (with higher chance of being asymptomatic carriers) without recent (≤1 wk) respiratory tract infections. In all enrolled children, pharyngeal swabs were taken for Mp real-time PCR (9). If additional consent was given, blood samples also were collected in control individuals and patients with CAP (before antibiotic treatment) to test for the presence of Mp-IgM-ASCs by ELISpot assay (detailed in the legend of Figure 1) (10) and Mp-IgM, Mp-IgG, and Mp-IgA by ELISA (2). Finally, we only included children with fresh (isolated ≤4 h) peripheral blood mononuclear cells to avoid poor ELISpot assay performance resulting from decreased ASC viability (in case of isolation &gt;4 h after sampling) or reduced ASC recovery (after a freeze–thaw cycle) (10). Samples and clinical data (using a standardized questionnaire) were collected at follow-up visits at &lt;2 weeks, 2 weeks to 2 months, and 2–6 months.