BARCELONA, Spain – A group of German researchers from the Fraunhofer Institute for Interfacial Engineering and Biotechnology have developed a method that combines multiplex suppression PCR and nanopore sequencing to detect cell-free DNA from bacterial pathogens and antimicrobial resistance in sepsis patients.
In a presentation at the European Society of Clinical Microbiology and Infectious Diseases global congress (ESCMID Global) here on Sunday and in a preprint published last week, the team described its Suppression PCR-based selective target sequencing (SUPSETS) approach to identify the most common sepsis-causing pathogens and instances of antimicrobial resistance.
Mirko Sonntag, one of the researchers who developed the method, noted in his ESCMID Global presentation that cell-free DNA is "permanently secreted" by both the host and the pathogen, resulting in microbial cfDNA remaining present inside the bloodstream of sepsis patients. It "delivers a precise snapshot of the current state of the patient," but it is highly and randomly fragmented, short, and has no defined beginning or end, he added. However, because of its sensitivity and abundance, it is a "highly suitable" biomarker for sepsis-related diagnostics.
The team's SUPSETS approach starts with the isolation of cell-free DNA, using bioinformatics to select its target regions and amplifying those regions with megaplex suppression PCR followed by nanopore real-time sequencing. The team then uses a simplified bioinformatic analysis for simultaneous pathogen and AMR detection with results deliverable within one workday, Sonntag said.
To conduct the suppression PCR, the researchers ligate suppression adaptors to the ends of the isolated cfDNA, regardless of whether it is human or microbial DNA. The ligation forces self-annealing that causes a stem-loop formation upon denaturation, which "diminishes unwanted amplification," Sonntag said. The team then uses an F-primer to elongate the cfDNA fragments, which consists of a target-specific sequence at the 3' end and a universal sequence at the 5' end. Per target, the team only uses one F-primer, which "allows us to [do] higher multiplexing against dozens of targets at the current state," he added.
With the F-primer, the team then creates a detection template that defines the endings of the sites that can be used for amplification. The amplicons are then sequenced using nanopore sequencing.
The researchers first technically validated their method on synthetic pathogen DNA samples spiked with human background DNA to simulate real samples. The complete target-primer panel was applied against one type of pathogen DNA, and the team saw clear signals for the respective primers with little to no background signals on the target regions, Sonntag said.
The team also tested its panel on samples spiked with all pathogens and AMR plasmids and received "comparable results with resolvable signals for all amplicons in positive" controls, he added.
After its technical validation, the team conducted a proof-of-concept clinical validation study, which was posted as a preprint last week. The SUPSETS approach was compared to standard blood culture and clinical metagenomics on both a MinIon flow cell from Oxford Nanopore Technologies and the firm's MinIon Flongle to see if costs could be decreased, Sonntag said. The method detected 11 out of 15 pathogens correctly, while blood culture detected only one pathogen. Overall, the test was able to detect 50 out of 53 negative samples, leading to a specificity of around 95 percent, he added.
The team also did a retrospective analysis of samples, finding that their method detected reads for two pathogens and antimicrobial resistance present in a sample within 18 minutes.
According to the preprint, the cost of using SUPSETS was between about €160 ($172) and €225 ($241) per sample.
The team also noted the limitations of the targeted approach, writing "since only selected targets are addressed, pathogens not included into the panel cannot be detected." However, "since only species-specific primers are required, the tested panel can be quickly expanded and adapted to other targets." The researchers added that other pathogen and AMR targets should be reevaluated and included to increase clinical value.
With further improvement of the panel, Sonntag noted, the team may be able to apply its method directly to blood samples, diminishing the isolation steps and speeding up its process.