Detection of uncultured organisms in paediatric bone and joint infections by a Multiplex real-time PCR panel
Abstract number: P1809
Ahmad N., Marsh P., Monk A., Pallett A., Saeed K., Clarke N.M.P., Faust S.N.
Objectives: The clinical features of osteoarticular infection (OAI) can vary considerably in children of different ages. Treatment is often started and continued empirically due to the low sensitivity of conventional blood and tissue culture in these patients. Better and more rapid microbiological diagnostic techniques are required to ensure more accurate therapy in order to reduce morbidity and overall (patient and financial) costs of treatment.
In a review of microbiology data at Southampton University Hospitals NHS Trust (SUHT), a defined pathogen was identified in only 8% of the 1200 samples received for culture and microscopy. Therefore our objectives were to develop a sensitive, specific and rapid multiplex PCR protocol for the detection of uncultured pathogens in paediatric OAI, and to investigate the level of infections left undetected by culture-based detection.
Method: A new real-time PCR diagnostic protocol was developed to identify the presence of the most common organisms known to cause bone and joint infections including the emergent paediatric pathogen Kingella kingae (identified from both the literature and basic local alignment search tool (BLAST) in samples taken from children with OAI at SUHT. A real-time PCR panel comprising of two triplexes was developed using three different reporter dyes (one for each target).
DNA extractions were carried out using an automated system, MagNA pure LC by Roche® using the MagNA Pure LC DNA isolation kit III (bacteria and fungi) Roche®..Real-time PCR was carried out using a Rotor-gene 6000 (Corbett).
Results: 95 paediatric bone and joint samples from SUHT were assayed from 56 patients (age range 270 days to 192 months) by PCR. Pathogenic bacteria were detected in 22 out of 56 patients where culture-based detection failed (including 6 K. Kingae, 7 Staphylococcus aureus and 3 Streptococcus pneumoniae) and 6/56 where culture confirmed the diagnosis.
Conclusions: We have developed a new, rapid (same day) method for detection of uncultured pathogens direct from paediatric bone and joint samples. Important OAI pathogens detected by PCR were missed by culture-based detection. Work is ongoing to establish the clinical value and cost-effectiveness of widespread introduction of this technique into routine clinical practice.
|Session name:||Abstracts 20th European Congress of Clinical Microbiology and Infectious Diseases|
|Location:||Vienna, Austria, 10 - 13 April 2010|
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