Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children

Abstract

New diagnostic tests for the etiology of childhood pneumonia are needed. We evaluated the antibody-in-lymphocyte supernatant (ALS) assay to detect immunoglobulin (Ig) G secretion from ex vivo peripheral blood mononuclear cell (PBMC) culture, as a potential diagnostic test for pneumococcal pneumonia. We enrolled 348 children with pneumonia admitted to Patan Hospital, Kathmandu, Nepal between December 2015 and September 2016. PBMCs sampled from participants were incubated for 48 h before harvesting of cell culture supernatant (ALS). We used a fluorescence-based multiplexed immunoassay to measure the concentration of IgG in ALS against five conserved pneumococcal protein antigens. Of children with pneumonia, 68 had a confirmed etiological diagnosis: 12 children had pneumococcal pneumonia (defined as blood or pleural fluid culture-confirmed; or plasma CRP concentration ≥60 mg/l and nasopharyngeal carriage of serotype 1 pneumococci), and 56 children had non-pneumococcal pneumonia. Children with non-pneumococcal pneumonia had either a bacterial pathogen isolated from blood (six children); or C-reactive protein <60 mg/l, absence of radiographic consolidation and detection of a pathogenic virus by multiplex PCR (respiratory syncytial virus, influenza viruses, or parainfluenza viruses; 23 children). Concentrations of ALS IgG to all five pneumococcal proteins were significantly higher in children with pneumococcal pneumonia than in children with non-pneumococcal pneumonia. The concentration of IgG in ALS to the best-performing antigen discriminated between children with pneumococcal and non-pneumococcal pneumonia with a sensitivity of 1.0 (95% CI Carter et al.Antibody-in-Lymphocyte Supernatant for Pneumococcal Pneumonia 0.73-1.0), specificity of 0.66 (95% CI 0.52-0.78) and area under the receiver-operating characteristic curve (AUROCC) 0.85 (95% CI 0.75-0.94). Children with pneumococcal pneumonia were older than children with non-pneumococcal pneumonia (median 5.6 and 2.0 years, respectively, p < 0.001). When the analysis was limited to children ≥2 years of age, assay of IgG ALS to pneumococcal proteins was unable to discriminate between children with pneumococcal pneumonia and non-pneumococcal pneumonia (AUROCC 0.67, 95% CI 0.47-0.88). This method detected spontaneous secretion of IgG to pneumococcal protein antigens from cultured PBMCs. However, when stratified by age group, assay of IgG in ALS to pneumococcal proteins showed limited utility as a test to discriminate between pneumococcal and non-pneumococcal pneumonia in children.

Antibiotic Use and Treatment Outcomes among Children with Community-Acquired Pneumonia Admitted to a Tertiary Care Public Hospital in Nepal

“…Given the presence of wheezing, low yield of blood culture, and normal chest radiography in the majority of children, we hypothesize that many of the pneumonia cases had viral etiology. This hypothesis is supported by the findings of a previous study from the same hospital [ 5 ]. Given the lack of diagnostic capacity in the hospital to confirm viral etiology of pneumonia, the treating pediatricians presumed bacterial etiology and treated every child with antibiotics.…”

“…In Nepal, pneumonia accounted for 10% of all childhood acute respiratory infections (ARI) in 2017 with an annual incidence of 66 cases per 1000 under-five children [ 4 ]. Previous studies from tertiary care settings in Nepal show that most of the pneumonia in under-five children were viral in origin and those with bacterial etiology were sensitive to the penicillin group of antibiotics [ 5 , 6 ].…”

Evaluation of Acute and Convalescent Antibody Concentration Against Pneumococcal Capsular Polysaccharides for the Diagnosis of Pneumococcal Infection in Children with Community-Acquired Pneumonia

“…However, the use of controls from a different population and disease may have confounded these results. We previously extended this work by examining the production of IgG to 5 conserved pneumococcal proteins in the antibody in lymphocyte supernatant assay in Nepali children, 8 finding that lymphocyte production of IgG to pneumococcal proteins discriminated between pneumococcal and nonpneumococcal pneumonia with areas under the curve of 0.60–0.85. However, when stratified into children ≥2 years of age, there were no significant differences in protein-specific IgG production between pneumococcal and nonpneumococcal pneumonia.…”