Feasibility of using recombinase polymerase amplification for point of care diagnosis of 'Mycoplasma pneumoniae', a major cause of community acquired pneumonia

'Mycoplasma pneumoniae' is a major cause of community acquire pneumonia (CAP), causing up to 40% of all CAP cases. 'M. pneumoniae' is a small fastidious bacterium which is difficult to diagnose, it lacks a cell wall, making it resistant to commonly-used antibiotics that act on cell walls. Current methods for 'M. pneumoniae' detection rely on culture methods, serology, PCRand real-time PCR,the current "gold standard". These techniques can be slow, insensitive and expensive. Recombinase polymerase amplification (RPA), developed at TwistDx, Cambridge, UK, is a novel, rapid, isothermal nucleic acid amplification method that is sensitive, specific and robust to temperature fluctuations and sample impurities, which would be ideal for point-of-care (POC) use. This study aimed to deliver a prototype RPA assay by developing RPA primers and probes for fluorescent detection and optimising methods for sample preparation. The feasibility of the RPA test for 'M. pneumoniae' detection was evaluated using a spiked throat swab model which imitated a real clinical samples. A saliva model was also developed, to test RPA's sensitivity. Real and artificial saliva were used in this study. Findings show that the assay sensitivity, specificity and robustness was similar to Realtime PCR. A direct comparison of both methods was achieved by analysing the same samples with the two different methods. Comparison of both methods also showed similar limits of detection as well as sensitivity. This study successfully demonstrates that the RPA assay has the potential to establish itself as a widely used diagnostic POC test for 'M. pneumoniae' detection.