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Nanopore Sequencing Shows Promise for Rapid Respiratory Infection Testing in ICU Patients


NEW YORK – Researchers from Guy’s and St Thomas’ NHS Foundation Trust (GSTT) in the UK have demonstrated the feasibility and utility of nanopore sequencing for rapid respiratory metagenomics analysis in critical care patients with suspected respiratory infections.

Based on results from their pilot project, conducted in collaboration with Oxford Nanopore Technologies and published last month in the American Journal of Respiratory and Critical Care Medicine, the GSTT researchers now plan to expand their program to multiple hospital sites across the UK after garnering £3 million ($3.8 million) in government funding in January.

"Respiratory infection is a huge challenge; [it is] probably the most common cause of sepsis worldwide," said Jonathan Edgeworth, director of the Centre for Clinical Infection and Diagnostics Research at GSTT and the lead researcher of the pilot study. "It's a major burden across society, particularly the intensive care unit."

Despite the significant impact of respiratory pathogens on the healthcare industry, Edgeworth said, the field has lacked a fast and comprehensive way to detect and analyze them. Standard-of-care microbiology tests can only provide partial information about a given pathogen over a few days and sometimes weeks, he said, often leading to overtreatment or undertreatment of patients.

To overcome this challenge, more than three years ago, Edgeworth and his team started to develop a rapid pathogen metagenomic analysis workflow leveraging nanopore sequencing.

"We were attracted by the potential for nanopore sequencing to provide results very quickly," he said, adding that the approach can not only identify pathogens but also unveil their "rich genomic details," such as strain typing and determinants of antimicrobial resistance and virulence.

After first evaluating the nanopore workflow in a research setting in the winter of 2022, the GSTT researchers kicked off a pilot project under hospital governance to assess the feasibility of implementing their assay as a same-day test for ICU patients with suspected respiratory infections. Edgeworth emphasized that the assay is currently not a diagnostic but is being evaluated as part of a research-use-only (RUO) workflow.

According to Rahul Batra, deputy director of GSTT’s Centre for Clinical Infection and Diagnostics Research, who also helped lead the pilot, the workflow the team has developed typically starts with depleting human DNA from the respiratory samples using centrifugation, bead beating, and degradation of host genetic material. This step ensures that the host DNA does not mask the pathogen reads during sequencing, he noted.

Because human DNA depletion is achieved through mechanical disruption rather than chemically, the workflow preserves both DNA and RNA from bacteria, fungi, and viruses, allowing for a pan-metagenomic analysis of the sample, he added.

After nucleic acid extraction, Batra said the samples undergo reverse transcription to generate cDNA for viral RNA, boosting the detection sensitivity. The samples are then processed using Oxford Nanopore's Rapid PCR Barcoding Kit before being loaded onto a GridIon platform for sequencing.

Currently, sequencing is optimized for the Oxford Nanopore R9 flow cell, but the team has also started to test out the newer R10 flow cell with the Kit 14 chemistry, which led to "a significant improvement" in sequencing quality, Batra said.

However, the issue with incorporating the new flow cell and chemistry into the assay is that their availability is "not fully there yet" for the Q-Line, Batra noted, referring to Oxford Nanopore's locked-down, standardized product line for validated assays. Once the R10 flow cell and Kit 14 chemistry are fully integrated into Q-Line products, the team will switch to using them "fairly soon," he added.

The GSTT team has also developed a real-time, automated analysis pipeline that can generate reports based on the sequencing data. Batra said the assay can typically generate actionable information after 30 minutes of sequencing and more details on antimicrobial resistance and speciation in about two hours. The team normally sequences samples for up to 24 hours, he added.

Typically, when the team receives patient samples at 8 a.m., Batra said, they will be able to return a report to the clinicians at around 3 p.m., followed by a more detailed report that includes additional information, such as antibiotic resistance, at about 5 p.m.

In their American Journal of Respiratory and Critical Care Medicine paper, the GSTT researchers described some results from their pilot, which included respiratory metagenomics sequencing on 128 samples from 87 patients with suspected lower respiratory tract infections in two general ICUs and one specialist respiratory ICU at the hospital.

During the first 15 weeks of the pilot, the nanopore assay provided same-day results for 110 samples, with a median turnaround time of 6.7 hours. Overall, the test was 93 percent sensitive and 81 percent specific for clinically relevant pathogens compared with routine testing, according to the study.

Additionally, almost half of the test results led to antimicrobial prescribing changes for the patients, with escalation based on speciation in 20 out of 24 cases and detection of acquired-resistance genes in four out of 24 cases, the study noted. The assay also picked up fastidious or unexpected organisms in 21 samples.

Bolstered by the pilot results, the GSTT team secured £3 million in new funding from NHS England and the Department for Science, Innovation, and Technology last month to expand the pilot project to about 10 other hospital sites across the UK over the next two years.

"The desire is to evaluate this workflow in a number of different sites [to see] if they get the same results and the same findings," Edgeworth said. "Then, there will be an opportunity to do a quality assurance scheme around that network, [and] then that will be a platform to go forward into clinical trials."

For the initial pilot project, the GSTT team signed a collaboration agreement with Oxford Nanopore in late 2022 that included resources and technical support for the clinical evaluation of the respiratory metagenomics workflow, Edgeworth said. He also signed a contract to work with Oxford Nanopore part-time in order to help "translate this technology into a more established setting."

The workflows developed by the GSTT researchers "are undergoing further refinement and evaluation that can now take place both at GSTT and other NHS hospital sites through the Network of Excellence program, with the commercial intention that solutions are globally applicable for healthcare and wider surveillance or public health needs," an Oxford Nanopore spokesperson said in an email. The company did not disclose further commercial plans for the test.

Besides the GSTT team, other researchers have also previously tried to achieve rapid respiratory infection testing using nanopore sequencing, including a group from the University of East Anglia's Quadram Institute Bioscience, for instance.

Moving forward, Edgeworth said the team plans to continue improving the sample processing workflow to include more automation, increasing volumes to between 20 and 30 samples per day to meet the critical care network’s demands.

With a streamlined sample preparation protocol and an "almost fully automated" analysis pipeline, Batra is optimistic that the metagenomics assay can be set up in local hospitals that have fewer sequencing and informatics resources.

Consumables costs for sample processing and sequencing is about £100 per sample, he said, with eight samples loaded per flow cell including controls.

"We can move out of the research and heavily supported settings into more standard healthcare settings," Batra said. "It does take a bit of time, but I would say, in the next three to five years, we would be looking to be able to roll out nanopore technology more widely in less supported healthcare settings."