TY - JOUR
T1 - Assessment of an Antibody-in-Lymphocyte Supernatant Assay for the Etiological Diagnosis of Pneumococcal Pneumonia in Children
AU - PneumoNepal Study Group
AU - Carter, Michael J.
AU - Gurung, Pallavi
AU - Jones, Claire
AU - Rajkarnikar, Shristy
AU - Kandasamy, Rama
AU - Gurung, Meeru
AU - Thorson, Stephen
AU - Gautam, Madhav C.
AU - Prajapati, Krishna G.
AU - Khadka, Bibek
AU - Maharjan, Anju
AU - Knight, Julian C.
AU - Murdoch, David R.
AU - Darton, Thomas C.
AU - Voysey, Merryn
AU - Wahl, Brian
AU - O'Brien, Katherine L.
AU - Kelly, Sarah
AU - Ansari, Imran
AU - Shah, Ganesh
AU - Ekström, Nina
AU - Melin, Merit
AU - Pollard, Andrew J.
AU - Kelly, Dominic F.
AU - Shrestha, Shrijana
N1 - Funding Information:
This project was funded by the Wellcome Trust (Research Training Fellowship to MC, grant number 104439/Z/14/Z); Gavi, the Vaccine Alliance; and the European Union’s Horizon 2020 research and innovation programme under grant agreement number 668303.
Funding Information:
MC, CJ, DM, AP, DK, and SS designed the study and analysis. MC, PG, SR, MG, ST, SK, IA, GS, MCG, KP, BK, and AM performed the studies and collected sample material. MC, PG, SR, RK, NE, and MM performed the assays. MC, JK, MV, KO’B, BW, AP, and DK performed the analyses. MC, DK, and AP wrote the manuscript to which all authors had significant input. MC, DK, JK, and AP acquired the funding directly for this study. This study was also supported by the PneumoNepal Study Group.
Funding Information:
We thank all of the children, and their parents, who participated in this study; and the clinical teams at Patan Hospital who gave unstinting help. PhtD was kindly supplied by Sanofi Pasteur (Sanofi Pasteur S.A., Marcy L'Etoile, France), StkpC and PcsB by Dr. Andreas Meinke (Valneva, Vienna, Austria), and Ply and CbpA by Dr. Elaine Tuomanen (St. Jude Children's Research Hospital, Memphis, TN, USA). We thank Leena Saarinen and Camilla Virta for technical assistance, and Dr. Dafne Andrade and Dr. Igor Borges who set up and validated the FMIA for antibodies to pneumococcal proteins (all at THL). Dr. Timothy James (Biochemistry Laboratory, Oxford University Hospital NHS Foundation Trust, UK) kindly supported the measurement of CRP concentrations. Dr. Colin Fink and team (Micropathology Ltd, Warwick, UK), Prof. Mike Levin and team (Imperial College, London, UK), and the PERFORM consortium (https://www.perform2020.org/) supported PCR identification of NP viruses. We also thank reviewers for helpful comments. Funding. This project was funded by the Wellcome Trust (Research Training Fellowship to MC, grant number 104439/Z/14/Z); Gavi, the Vaccine Alliance; and the European Union's Horizon 2020 research and innovation programme under grant agreement number 668303.
Publisher Copyright:
© Copyright © 2020 Carter, Gurung, Jones, Rajkarnikar, Kandasamy, Gurung, Thorson, Gautam, Prajapati, Khadka, Maharjan, Knight, Murdoch, Darton, Voysey, Wahl, O'Brien, Kelly, Ansari, Shah, Ekström, Melin, Pollard, Kelly and Shrestha.
PY - 2020/1/17
Y1 - 2020/1/17
N2 - 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 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.
AB - 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 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.
KW - antibodies
KW - diagnostic test (MeSH)
KW - lymphocytes
KW - pneumococcus (Streptococcus pneumoniae)
KW - pneumonia
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U2 - 10.3389/fcimb.2019.00459
DO - 10.3389/fcimb.2019.00459
M3 - Article
C2 - 32039044
AN - SCOPUS:85078843141
SN - 2235-2988
VL - 9
JO - Frontiers in Cellular and Infection Microbiology
JF - Frontiers in Cellular and Infection Microbiology
M1 - 459
ER -