Clinical metagenomics by nanopore sequencing to improve the success rate of respiratory pathogen identification

Abstract

Background Respiratory infections can be caused by viruses, bacteria and fungi affecting the upper or lower respiratory tract. They are associated with high morbidity, mortality, and prolonged hospitalisation. Culture-based identification of infection agents is time-intensive (48-72 hours) and contingent on microorganism viability in the samples. As a result, empirical antibiotic use affects the microbiota and increases resistance. Metagenomics, on the other hand, analyses all genetic material and can identify most microorganisms in less than 24 hours. The aims of this work were to evaluate the success rate of culture-based identification of causative agents of respiratory infection, at Centro Hospitalar Baixo Vouga (Aveiro, Portugal), between 2017 and 2022 and to compare the efficacy of nanopore sequencing with the culture method, in identifying aetiological infectious agents. Materials In a retrospective analysis, infection-causing agents were identified in respiratory samples from emergency departments, hospital admissions, and specialist consultations. This study encompassed 7455 culture results from sputum, endotracheal aspirates, bronchoalveolar aspirates, and lavages. Samples from the lower respiratory tract were examined through metagenomic analyses: six with negative or polymicrobial culture results and six with culture identification. The traditional culture results were compared with the nanopore sequencing using MinION® technology and analysed on the EPI2ME platform. Results The success rate of pathogen identification, retrieved from culture, was consistently less than 50%, regardless of sample type. Endotracheal aspirate had the highest and bronchoalveolar lavage the lowest success rate, with 40,1% and 15,3%, respectively. Pseudomonas aeruginosa was the most commonly isolated microorganism, identified in 22,7% of positive samples. Comparing the two methodologies, from six samples with positive culture results, five nanopore sequencing accurately matched the culture findings. Nanopore sequencing successfully identified microorganisms in four out of six samples whereas culture-based identification was unsuccessful. Two hours of sequencing proved to be effective in the identification of microorganisms and resistance genes.

Comparing the two methodologies, within six samples with positive culture results, five were a match.

Conclusions Poor culture results highlight the need for an alternative and faster infection aetiological agent identification method. Clinical metagenomics can be an alternative to improve success rates and response times, leading to a targeted and appropriate therapy. Future studies will refine this approach and evaluate its cost-effectiveness and impact on antimicrobial resistance prevention.