This article has been updated to include additional information from Kai Sohn.
NEW YORK – Researchers in Germany have developed a nanopore sequencing assay that uses cell-free DNA from plasma to detect sepsis-causing pathogens.
"We can significantly reduce time to diagnosis," said Kai Sohn, senior author of a paper describing the method that was published Thursday in the Journal of Molecular Diagnostics. "From having the sample in the lab to the first result, we can reduce this time to six hours for most samples."
The proof-of-concept study used Oxford Nanopore Technologies' MinIon device to analyze eight samples from four patients that were determined to have sepsis in a previous study, as well as samples from three healthy controls. It used an optimized sample preparation method for cell-free DNA and compared the sequence reads to a database of pathogens associated with the sepsis response. For seven of the sepsis samples, the nanopore method detected the pathogen and for all seven samples, some pathogen sequence hits were found within one hour; 16 of the 25 total hits were found within two hours.
An hour of sequencing produced approximately 50,000 reads, or about 100 Mb of data, Sohn said. The cost per sample, including cell-free DNA isolation and library preparation, was approximately $1,100, the researchers said, compared with approximately $300 per sample for Illumina sequencing.
According to an accompanying commentary, the study is "the first description of the use of MinIon for detecting pathogens directly from blood samples from septic patients."
"It's a promising technology," said Justin O'Grady, a researcher at the UK's Quadram Institute who is developing his own nanopore sequencing-based sepsis test, which analyzes cellular DNA. "They've now shown they can do it with real-time sequencing, which is an advance. And they do detect the correct pathogens."
But O'Grady, who has been critical of the specificity of other cell-free DNA sepsis tests, such as the test from Stanford University spinout Karius, said he remains unconvinced it's a good approach for the condition because cell-free DNA from the human microbiome can make its way into the blood. "Sometimes you find the pathogen causing the infection, but sometimes you're detecting background levels [of bacterial DNA]" he said. In addition, it was unclear whether or not the new study detected bacterial DNA in the control samples, O'Grady said.
Still, cell-free DNA testing offers the opportunity to "dramatically improve diagnostic yield for patients with sepsis," Karius CSO and Cofounder Tim Blauwkamp said. "The beauty of it is, [these tests] don't require a sample of the microbe itself," eliminating the need for invasive procedures intended to find the specific infection that is eliciting sepsis.
The new study, conducted by researchers at the Fraunhofer Institute for Interfacial Engineering and Biotechnology, Heidelberg University Hospital, and Noscendo — a German diagnostics firm that Sohn cofounded, as well as Westpfalz-Klinikum hospital, built on previous research that used Illumina sequencing to diagnose sepsis with cell-free DNA.
"We were originally motivated to find a diagnosis workflow that was the most reliable," Sohn said. An NGS-based method provided precise analysis of biomarkers that provided good sensitivity, but took a day or more. That's faster than blood culture, considered the standard of care for sepsis testing, "but still quite long," he said. They turned to Oxford Nanopore's MinIon to make the method even faster, as the mortality risk increases with every hour that a sepsis patient doesn't receive the proper treatment.
"Nanopore sequencing has features that when used in the right context, can provide some advantages, such as ability to get real-time data," noted Blauwkamp. Karius' own test, which the company says can be used for a broad range of infection-related issues, uses Illumina sequencing, but he said the firm has considered other platforms. The German team also modified the Oxford Nanopore sample preparation steps to save time and increase yields, cutting 1.5 hours off the library preparation.
But nanopore technology also has disadvantages in terms of the quality of sequence information, Blauwkamp said. In the paper, the authors provided data on their method's "discriminative power." While they were able to identify pathogens also seen with their Illumina-based method, there were some pathogens found with MinIon that weren't seen with other methods — likely false positives, the authors said —and others that the nanopore method missed.
Moreover, O'Grady said it was unclear, based on the data presented, whether the MinIon reads were identifying pathogens in the control samples. "If they detect pathogen reads in healthy patients, then that’s another issue of specificity," he said. Such results, if acted on by a healthcare provider, could lead to treating a patient for microbes that weren't actually associated with sepsis. Sohn said that MinIon sequencing did identify microbes in the three controls samples, but that they were species that other sequencing studies either did not identify or had considered not relevant to sepsis. "A relevance score for pathogen classification based on MinIon sequencing is not established yet," Sohn noted.
Both O'Grady and Blauwkamp noted that the sample size in the new study was small, so sensitivity and specificity cannot be calculated. But Sohn said he would apply the method to a greater number of patients soon: His lab is involved in a multi-center study that is recruiting up to 500 patients to test their Illumina-based method of sepsis diagnosis. He "definitely" plans to test some of those samples with the MinIon workflow, as well, he said.
This nanopore-based method is certainly not ready as a diagnostic tool, Sohn said, but he could see sequencing-based sepsis tests, in general, following a similar path to the clinic as NGS-based noninvasive prenatal testing.
Noscendo is already offering a sequencing-based infection testing service in Germany that doesn't receive insurance reimbursement at the moment, but NIPT didn't initially get any, either, he noted. A decade later, Germany is about to get reimbursement for NIPT. "Because it's similar technology, I wouldn't be too surprised if this is on the same route," he said.
For now, Sohn said his team continues to optimize its workflow for lower inputs and smaller fragments. He and his colleagues are also looking into adapting the method for other Oxford Nanopore devices, such as Flongle, which could reduce costs, and to use newer sequencing chemistries.
"This is really on the move, these kinds of protocols, it's not fixed yet," he said. "I think it shows a promising starting point to dig a bit deeper."