Rapid identification of pathogens involved in pediatric osteoarticular infections by multiplex PCR

Background: Delays in the diagnosis of pediatric osteoarticular infections (OAIs) can cause associated acute complications or long-term morbidity. This study attempts to develop a multiplex PCR-based assay that can rapidly and accurately detect the main pathogens involved in pediatric OAIs, namely, methicillin-sensitive or methicillin-resistant Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa. Methods: A set of four gene-specific primers suitable for use in a one-tube PCR assay was designed to detect four common pathogens involved in pediatric OAIs, namely, nuc for methicillin-sensitive Staphylococcus aureus, nuc and mecA for methicillin-resistant Staphylococcus aureus, spyM for Streptococcus pyogenes and orpl for Pseudomonas aeruginosa. The multiplex PCR was first evaluated with 39 isolated clinical strains and further with 41 specimens collected from patients suspected of having OAIs. Results: Specific primer pairs were successfully designed, and the targeted genes were simultaneously amplified. The product sizes in the assay for nuc, mecA, spyM and opri were 233, 158, 336 and 109 bp, respectively. Evaluation of the multiplex PCR with 39 isolated clinical strains and 41 specimens revealed 100% sensitivity and 100% specificity. The limit of detection of the multiplex PCR assay was approximately 1 x10(3) CFU at the bacterial cell level. Conclusions: This newly developed multiplex PCR assay, without sequencing, enables a rapid and accurate diagnosis of the major bacterial species in children with OAIs and might serve as an additional diagnostic approach for urgent pathogen determination.