Rapid and accurate diagnosis is critical for the effective management of life threatening infections, such as pneumonia. Current culture based diagnosis has sub-optimal specificity and sensitivity and is too slow to impact on patient management. Shotgun metagenomics sequencing has the potential to change the way we diagnose pneumonia, combining rapidity with comprehensiveness beyond that of current methods. We have developed and optimised a nanopore metagenomics sequencing based pipeline for the diagnosis of bacterial pneumonia.
A diagnostics pipeline including human DNA depletion, DNA extraction (MagNA Pure Compact, Roche), library preparation (RLI001, RLB001) and MinION sequencing (R9.4/9.5 flowcells) was developed and optimised. Pneumonia samples (sputum, BAL and endotracheal aspirates) were collected from the Norfolk and Norwich University Hospital microbiology laboratory. Samples were sequenced individually using RLI001 library preparation kit or multiplexed in batches of six samples per flowcell using RLB001. Data was analysed in real-time using the Epi2Me Antibiotic Resistance pipeline. MinION results were compared to culture determined pathogens and antibiotic resistance.
MinION sequencing of 47 suspected bacterial pneumonia samples was performed. The turnaround time from sample to pathogen and acquired resistance gene identification was approximately 7 hours. The overall specificity and sensitivity of the method for pathogen identification compared to culture was approx. 99% and 98%, respectively (with the caveats that detection of additional pathogens in samples compared to culture were not considered false positives and detection of low numbers of Streptococcus pneumonia in samples reported as normal respiratory flora were not considered false positives). Analysis of resistance data is still underway, however, the mecA gene was detected in all samples positive for MRSA.
Real-time metagenomics has the potential to replace culture for the diagnosis of bacterial/fungal pneumonia and provides the rapid turnaround necessary for precision management of pneumonia patients.