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Rapid identification of pathogens involved in pediatric osteoarticular infections by multiplex PCR
Chi Gan1, Jinfeng Hu1, Qing Cao2
1The Laboratory of Pediatric Infectious Diseases, Pediatric Translational Medicine Institute, Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.
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 orpI 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 1×103 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.
Insights
A new multiplex PCR assay accurately detects key pathogens in pediatric osteoarticular infections (OAIs), including Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa. This rapid diagnostic tool aids in timely treatment and reduces long-term complications in children.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Pediatric Infectious Diseases
Background:
- Pediatric osteoarticular infections (OAIs) can lead to severe complications if not diagnosed promptly.
- Accurate and rapid pathogen identification is crucial for effective treatment of pediatric OAIs.
Purpose of the Study:
- To develop a multiplex PCR assay for the simultaneous detection of major pathogens in pediatric OAIs.
- To improve the speed and accuracy of diagnosing infections caused by Staphylococcus aureus, Streptococcus pyogenes, and Pseudomonas aeruginosa in children.
Main Methods:
- Designed gene-specific primers for methicillin-sensitive and resistant Staphylococcus aureus (nuc, mecA), Streptococcus pyogenes (spyM), and Pseudomonas aeruginosa (oprI).
- Developed a one-tube multiplex PCR assay.
- Evaluated the assay using 39 isolated clinical strains and 41 clinical specimens from patients with suspected OAIs.
Main Results:
- Successfully amplified target genes simultaneously with specific product sizes.
- Achieved 100% sensitivity and 100% specificity in assay evaluation.
- Established a limit of detection of approximately 1x10^3 CFU/mL at the bacterial cell level.
Conclusions:
- The developed multiplex PCR assay provides rapid and accurate diagnosis of common bacterial pathogens in pediatric OAIs.
- This assay can serve as a valuable tool for urgent pathogen determination, potentially improving patient outcomes.
- The assay offers a non-sequencing based diagnostic approach for pediatric osteoarticular infections.
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