Pan-microbial pathogen detection in hours using metagenomic sequencing

Lower respiratory tract infections are the leading cause of sepsis-related deaths and the fourth leading cause of death worldwide¹. In cases of severe respiratory infection, starting effective treatment can be a race against time, as identifying the appropriate treatment is dependent on detecting the pathogenic microbe causing the infection. This could be any of a vast number of diverse pathogenic microbes, so fast, accurate, and comprehensive detection is essential. At the London Calling conference 2026, Luke Blagdon Snell and Adela Alcolea-Medina (NHS Respiratory Metagenomics Network, UK) shared how they have developed and validated a workflow using Oxford Nanopore sequencing that makes this possible.

Luke described how UK hospitals face multiple challenges when it comes to diagnosing respiratory infections¹. Antimicrobial resistance (AMR) and the risk of pandemic spillover are both on the rise, while biosecurity threats pose the possibility of novel pathogens. Plus, an increasing proportion of patients are immunocompromised, putting them at greater risk of infection by unusual pathogens that are not targeted by routine tests. Now, these researchers are utilising metagenomics to address all of these challenges.

Adela and Luke introduced their pan-microbial nanopore metagenomics workflow: an all-in-one, end-to-end assay that detects bacteria, DNA and RNA viruses, fungi, and parasitic pathogens. By making use of metagenomic sequencing to analyse all the nucleic acids in respiratory samples, their assay captures diverse microbial pathogens in a single workflow, whether common or rare. This is all possible from one sample, whereas standard-of-care diagnostic approaches require a sample to be split across multiple tests to pick up different microbes. Crucially, accessing this data from a single assay also avoids having to wait for these ‘very fragmented’, staggered processes to return results, after which the infecting pathogen may still remain undetected.

The team’s agnostic, whole-genome approach requires no prior assumptions about the pathogens that may be present. This contrasts with commonly used multiplexed PCR-based methods that narrow the search to a set number of targets, missing any microbes outside this list.

Building a rapid assay

In their rapid workflow, the team extracts DNA and RNA directly from respiratory research samples without the need for culturing, which would otherwise add considerable time and bias results toward only culturable microbes (Figure 1).

After converting RNA to cDNA, the team prepare the microbial nucleic acids for nanopore sequencing using the Rapid PCR Barcoding Kit, requiring only one hour plus PCR time. Then, they sequence the metagenomic libraries on a GridION, a benchtop device capable of sequencing on up to five independent flow cells. To analyse their data, they use an in-house bioinformatics pipeline, which provides automated reports at several time points across 24 hours. In just 6.5 hours, the researchers can generate the first ‘preliminary, actionable’ results.

Adela shared how the team validated their rapid workflow, comparing against the full standard-of-care diagnostic pathway in their clinical microbiology lab³. This in-house retrospective study demonstrated robust and acceptable performance for pathogen identification from an end-to-end nanopore sequencing workflow that generated actionable results in a matter of hours.

Going beyond identification

Nanopore reads of unrestricted length facilitate confident pathogen identification from metagenomic samples, as when mapping to microbial databases, long nanopore reads map unambiguously to microbial genomes. As well as this, genomic data goes beyond detection and into characterisation of these microbes, including critical AMR gene and virulence factor information with potential to inform effective treatments.

Next, the team conducted two pilot studies: the first using research samples from adult patients admitted to the intensive care unit (ICU) with respiratory failure⁵ and the second using research samples from paediatric ICU patients⁶. These showed similarly strong performance for pathogen identification, highlighting the workflow’s consistency. Crucially, their metagenomic approach detected additional pathogens in 30% and 24% of respiratory samples, respectively, that were not picked up by standard-of-care testing.

Detecting previously missed pathogens

Having validated their metagenomics workflow, the researchers expanded its reach. Just one year after Guy's and St Thomas' NHS Foundation Trust, London, sent the first samples to their laboratory, the assay is now deployed across multiple sites in the UK.

Luke emphasised the pattern across the sites using the workflow: in a rapid timeframe, they frequently identify additional pathogens that are not detected with routine diagnostic tests. Amongst these are microbes that cause pneumonia, which ‘in many cases ... would never have been detected by standard of care’, influenza C virus — which is not tested for via PCR-based methods — and rare zoonotic pathogens, such as avian paramyxovirus that caused severe respiratory failure in an immunocompromised individual. In one respiratory sample, the approach identified human immunodeficiency virus (HIV) in one day.

With this assay making it possible to reveal more pathogens, Luke was initially concerned that it could indicate a need for greater antimicrobial use than before. The result was the opposite: in two-thirds of cases, data that could suggest a change in treatment indicated de-escalation of antibiotic use. This is because of the ‘clarity’ offered by the approach, which can identify when a pathogen is resistant to prescribed antimicrobials and which treatment would be effective instead, reducing unnecessary antimicrobial use that contributes to rising resistance.

As well as demonstrating clinical potential, this information is highly valuable for monitoring outbreaks. As part of a network of National Health Service sites contributing anonymised data to the UK Health Security Agency Metagenomics Surveillance Collaboration and Analysis Programme (mSCAPE), the impact of the team’s workflow also expands to the public health scale. The data from their pipeline is shared with mSCAPE after 24 hours of sequencing, providing important insights for genomic pathogen surveillance to help identify potential outbreaks and inform control measures.

This assay, which consolidates what would otherwise require multiple tests across days or weeks, delivers comprehensive results in a day or less. The UK NHS Respiratory Metagenomics Network is also seeking ISO15189 clinical laboratory accreditations for the assay as it is rolled out. The group's work could mean significant changes in the way infectious disease is diagnosed, treated, and surveilled in the future.

Oxford Nanopore Technologies products are not intended for use for health assessment or to diagnose, treat, mitigate, cure, or prevent any disease or condition.

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