Detection of uncultured organisms in paediatric bone and joint infections by a multiplex real-time PCR panel

Background and aims: In paediatric osteoarticular infections (OAI), treatment is often started and continued empirically due to the low sensitivity of conventional blood and tissue culture in these patients. Complex cases are more likely to undergo surgery. Better and more rapid microbiological diagnostic techniques could ensure more accurate therapy in order to reduce morbidity and overall (patient and financial) costs of treatment. This study investigated the use of real-time multiplex polymerase chain reaction (PCR) in paediatric OAI to increase the speed and accuracy of diagnosis and to demonstrate the potential clinical impact of the new test. Methods: A new real-time PCR diagnostic protocol was developed to identify the presence of the most common organisms known to cause paediatric OAI (identified from both the literature and local microbiology database). A real-time PCR panel (comprising two triplexes+one single PCR) was developed using three different reporter dyes (one for each target). DNA extraction was carried out using the MagNA Pure platform (Roche). Real-time PCR was carried out using a Rotor-gene 6000 (Corbett). The detection limit was found to be 103–102 CFU/ml (assay is detecting five genomic copies per PCR reaction as microlitre samples are being tested). Research Ethics Committee approval was obtained to link clinical and laboratory data. Results: 55 paediatric bone and joint samples were assayed from 32 patients (age range 270 days to 192 months). 18.2% of PCR samples were positive for pathogenic bacteria on day 1 (21.8% by day 4 of culture). Pathogenic bacteria detected by PCR were six Staphylococcus aureus, two Kingella kingae, one Streptococcus pneumoniae, one Group A Streptococcus and three Group B Streptococcus). Three samples were PCR and conventional culture +ve, 20 PCR +ve but culture –ve, and 30 PCR and culture –ve. No samples were culture positive for organisms tested by PCR. The diagnostic yield of pathogenic bacteria by culture alone was two of 32 patients (6%) increasing to 13 of 32 patients (41%) using PCR. The potential clinical impact is demonstrated by collated case examples (data not shown). Conclusions: We have developed a new, rapid (same day) method for detecting uncultured pathogens direct from paediatric bone and joint samples. Case examples demonstrate the clinical benefit of the technique. Work is ongoing to establish the clinical value and cost-effectiveness of the technique prior to introduction into routine clinical practice.