Abstract
Osteoarticular infections (OAI) pose a significant challenge to children in New Zealand with reported OAI rates much higher than in other OECD countries. Osteoarticular infection is an umbrella term for infections of the bone or joint tissue, requiring targeted and rapid antimicrobial treatment to avoid morbidity and long-term sequelae.
With the introduction of highly sensitive molecular diagnostics, Kingella kingae is now considered the leading cause of OAI in young children. Early identification of the disease-causing microorganism allows for targeted treatment which can reduce treatment duration and improve patient prognoses. Unfortunately, the current gold standard diagnostic techniques, polymerase chain reaction (PCR) and culture, are not very sensitive and one third of suspected OAI cases have no identified pathogen.
Blood cultures are insensitive but routinely done, aspirates of joints, invasive surgery and biopsies are more sensitive but not routinely carried out. Due to these diagnostic difficulties the microbiological aetiology of some cases cannot be determined. Short segments of cell free DNA (cfDNA) released by the invading pathogen may pass into patient’s blood and urine and detection of these segments by PCR has the potential to improve the rate of microbiological diagnosis. The sensitivity and specificity of this technique for K. kingae is unknown. K. kingae cfDNA can be concentrated from urine using K. kingae specific probes coupled to magnetic beads. The captured cfDNA can then be amplified and quantified with quantitative PCR (qPCR). This project aimed to develop specific and sensitive K. kingae primers for use in qPCR.
Nine different primer combinations were tested for their performance and K. kingae specificity. All six natural DNA primers formed primer-dimers. The melting temperatures for the target DNA were similar to those of primer-dimers (primers 1, 3) or those of other bacterial species tested (primers 2,4,5,6). This led to designing locked nucleic acid primers (LNA), with two primers that were free of primer-dimers. During the specificity testing primer 5LNA performed the best and only cross-reacted with a closely related species, K. denitrificans.
In summary, this study developed and optimised a qPCR assay for the detection of K. kingae with a short amplicon sequence suitable for cfDNA. Although we were unable to design a primer that is specific to K. kingae alone in the given timeframe, we have established a strong foundation for future primer design, which can include modifying the number and positioning of LNAs. The amplicon sequence will also serve as template for the probes that are coupled to magnetic beads used to extract the bacterial cfDNA from urine samples. Lastly, this platform has the potential to be a foundation for the detection of other infectious diseases, following development of specific primers and probes for different pathogenic microorganisms.