Diagnosis of lower respiratory tract infections using nanopore sequencing

Background: According to WHO, lower respiratory tract infections (LRTI) are the deadliest communicable disease globally, causing 3.2 million deaths annually. Culture is the gold-standard diagnostic, but produces sub-optimal results and takes a minimum of 48hr. Poor diagnostics impact on patient management and treatment. Shotgun-metagenomics can overcome these issues, by reducing turnaround time and improving diagnostic accuracy.

Aim: To develop a rapid metagenomics sequencing pipeline for the diagnosis of bacterial LRTIs directly from respiratory samples.

Methods: Respiratory samples (sputum and aspirates) from patients with suspected bacterial LRTIs were used to develop and optimise a metagenomics pipeline which included: novel human DNA depletion, pathogen DNA extraction, library preparation and MinION sequencing. Pathogens and antibiotic resistance genes were identified in real-time by MinION sequencing and Epi2ME analysis (Antimicrobial Resistance pipeline) and were compared to clinical microbiology results.

Results: The initial sample set (n=42) was 89% concordant with culture for pathogen detection. After optimisation (improving turnaround and sensitivity) the pipeline was tested on a second sample set (n=13) and was 100% concordant with culture. The turnaround time was 6hrs from sample to pathogen and acquired-resistance gene identification. Routine microbiology identified 2 MRSA positive samples, both of which were mecA gene positive using the developed metagenomics sequencing pipeline.

Conclusion: LRTI pathogens and antibiotic resistance genes were identified within 6hrs with our pipeline, demonstrating that metagenomic sequencing has the potential to replace culture.