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

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Michael James Carter, Pallavi Gurung, Claire Jones, Shristy Rajkarnikar, Rama Kandasamy, Meeru Gurung, Stephen Thorson, Madhav Gautam, Krishna Prajapati, Bibek Khadka, Anju Maharjan, Julian C Knight, David Murdoch, Thomas C Darton, Merryn Voysey, Brian Wahl, Katherine L O'Brien, Sarah Kelly, Imran Ansari, Ganesh Shah & 5 more Nina Ekström, Merit Melin, Andrew J Pollard, Dominic F Kelly, Shrijana Shrestha

Original languageEnglish
JournalFrontiers in Cellular and Infection Microbiology
Accepted/In press16 Dec 2019
Published17 Jan 2020


  • Carter2020ALSpneumonia

    Carter2020ALSpneumonia.pdf, 2.79 MB, application/pdf

    Uploaded date:19 Dec 2019

    Version:Accepted author manuscript

    Licence:CC BY

King's Authors


New diagnostic tests for the aetiology 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 aetiological 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 (6 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 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 years 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.

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