Liquid biopsy for infectious diseases: Sequencing of cell-free plasma to detect pathogen DNA in patients with invasive fungal disease

Abstract

Diagnosis of life-threatening deep-seated infections currently requires invasive sampling of the infectedtissue to provide a microbiologic diagnosis. These procedures can lead to high morbidity in patients and add to healthcare costs. Here we describe a novel next-generation sequencing assay that was used to detect pathogen-derived cell-free DNA in peripheral blood of patients with biopsy-proven invasive fungal infections. The non-invasive nature of this approach could provide rapid, actionable treatment information for invasive fungal infections when a biopsy is not possible.

Cell‐free DNA next‐generation sequencing successfully detects infectious pathogens in pediatric oncology and hematopoietic stem cell transplant patients at risk for invasive fungal disease

“…Sequencing reads identified as human were removed, and remaining sequences were aligned to a curated pathogen database. Any of over 1000 organisms in the Karius clinical reportable range found to be present above a predefined statistical threshold were reported as previously described . Those interpreting the NGS results were blinded to clinical information.…”

“…Next‐generation sequencing (NGS) technologies are being investigated for noninvasive diagnosis and monitoring of infectious diseases, including fungal pathogens . Sequencing of cell‐free DNA (cfDNA) in the bloodstream has previously demonstrated clinical utility in the detection of fetal abnormalities, transplanted organ rejection, and malignant tumors,although results are disappointing for several major tumor types .…”

“…Similar approaches have been used to sequence circulating cfDNA, identify nonhuman sequences, and compare them with known genomic databases of bacterial, viral, and fungal pathogens . While NGS technology is newer with a smaller number of samples studied, inhibiting comprehensive evaluation of its performance, this method has been effective for diagnosing bacterial infections in a real‐time setting as well as noninvasively identifying molds in patients with invasive fungal infection . In this pilot study (ClinicalTrials.gov identifier: NCT03262584), we hypothesized that cfDNA NGS could accurately and noninvasively identify fungal pathogens in pediatric hematology, oncology, and HSCT patients at risk for new IFD.…”

Clinical metagenomics

“…Moreover, defining specific microbial profiles that are diagnostic or predictive of disease development can be difficult, especially from nonsterile sites that harbour a complex microbiome, such as respiratory secretions or stool 6 . Nevertheless, several groups have successfully validated mNGS in Clinical Laboratory Improvement Amendments (CLIA)certified clinical laboratories for the diagnosis of infections, including meningitis or encephalitis 36,37 , sepsis 33,57 and pneumonia 58 , and these assays are now available for clinical reference testing of patients.…”

Community-acquired pneumonia in children: cell-free plasma sequencing for diagnosis and management

“…DNA sequencing of cell-free plasma (De Vlaminck et al, 2015;Long et al, 2016;Hong et al, 2018) allows for detection of pathogen DNA derived from both bloodstream infections and deeper body sites, including cases where there has been antibiotic pretreatment prior to cultures and in those with fastidious, difficult-to-culture organisms (Abril et al, 2016). This case series reflects an institutional experience applying…”