Abstract
Pneumonia is one of the most common and severe diseases associated with Streptococcus pneumoniae infections in children and adults. Etiological diagnosis of pneumococcal pneumonia in children is generally challenging because of limitations of diagnostic tests and interference with nasopharyngeal colonizing strains. Serological assays have recently gained interest to overcome some problems found with current diagnostic tests in pediatric pneumococcal pneumonia. To provide insight into this field, we have developed a protein array to screen the antibody response to many antigens simultaneously. Proteins were selected by experimental identification from a collection of 24 highly prevalent pediatric clinical isolates in Spain, using a proteomics approach consisting of "shaving" the cell surface with proteases and further LC/MS/MS analysis. Ninety-five proteins were recombinantly produced and printed on an array. We probed it with a collection of sera from children with pneumococcal pneumonia. From the set of the most seroprevalent antigens, we obtained a clear discriminant response for a group of three proteins (PblB, PulA, and PrtA) in children under 4 years old. We validated the results by ELISA and an immunostrip assay showed the translation to easy-to-use, affordable tests. Thus, the protein array here developed presents a tool for broad use in serodiagnostics.
Highlights
We based the design of our pneumococcal protein array on antigens experimentally identified on the surface of a collection of pediatric clinical isolates, as surface proteins are those with the highest probabilities to raise an effective humoral immune response
We analyzed 24 clinical isolates collected from children with invasive pneumococcal disease (IPD), which corresponded to 12 different serotypes
We found 20 different clonal sequence types (ST), including several major global clones recognized by the PMEN as shown in supplemental Table S1
Summary
The amplification and quantification of pneumococcal genes (namely spn9802, ply or pcpA) by PCR has been used, but with lower sensitivity than culture in blood samples in adults and inability to discriminate between carriage and disease in nasopharyngeal and sputum samples (9 –11). To better discriminate between diseased and healthy people, a combination of antigens would be desirable. To this regard, proteomics offers an excellent platform to develop the necessary more sensitive and specific. Three proteins were proven to discriminate with optimal sensitivity, specificity, and accuracy between nonpneumococcal disease and disease status for Ͻ4-year-old children. As a proof-of-concept, we have developed an immunostrip assay with such proteins, obtaining the same sensitivity, specificity, and accuracy, demonstrating the power of high-throughput technologies for discovering diagnostic biomarkers of infection and its possible translation to an easy-to-use clinical tool
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