Development and Validation of a Multiplex Real-Time PCR Assay for Detection and Quantification ofStreptococcus pneumoniaein Pediatric Respiratory Samples

Abstract

AbstractStreptococcus pneumoniae(Spn) is a bacterial pathogen that causes a range of disease manifestations in children, from acute otitis media to pneumonia, septicemia, and meningitis. PrimarySpnlaboratory diagnostic identification methods include culture, antigen testing, single-plex real-time PCR, and syndromic PCR panels. However, each method lacks sensitivity, specificity, and/or cost efficiency. We developed and validated a quantitative, multiplex PCR assay that uses threeSpngenomic targets (lytA, piaB, and SP2020) for improved sensitivity and specificity to detectSpnin pleural fluid (PF), bronchoalveolar lavage (BAL), tracheal aspirate (TA), and upper respiratory (UR, research only) samples. Validation testing included analytical sensitivity (limit of detection), specimen storage, analytical specificity (cross-reactivity), and accuracy studies. Limit of detection is 500 genome copies/mL in lower respiratory samples and 100 copies/mL in upper respiratory specimens, with quantification range of 1,000 to 10,000,000 copies/mL. Specimens can be stored frozen at least 60 days andSpnDNA is stable through 3 freeze-thaw cycles. No cross-reactivity was observed against 20 closely related microorganisms and/or microorganisms that can be detected in similar sample types, includingStreptococcus pseudopneumoniae. In reference range testing,Spnwas detected in 5 of 23 (21.7%) PF, 2 of 19 (10.5%) BAL, 1 of 20 (5.0%) TA, and 44 of 178 (24.7%) UR residual specimens. For accuracy studies, 98 specimens were tested and overall percent agreement with a qualitative,lytA-based comparator assay was 96.9% across all sample types. This multiplex, quantitative PCR assay is a sensitive and specific method forSpndetection in pediatric respiratory samples.