Sequencing of Circulating Microbial Cell-Free DNA Can Identify Pathogens in Periprosthetic Joint Infections

Abstract

Background:Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing.Methods:In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results.Results:Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen.Conclusions:Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period.Level of Evidence:Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

Metagenomic next-generation sequencing in osteoarticular infections with a focus on pediatrics: current concepts and clinical applications

“…mNGS often involves obtaining tissue samples from the area of infection followed by extraction of nucleic acids from the cells within this tissue. cfNGS involves a peripheral blood draw that sequences cell-free DNA (cfDNA) present in the host bloodstream from already naturally lysed pathogenic cells ( 16 , 80 ). The cfDNA fragments have a short half-life in the circulating blood, so rapidly dividing oncologic and pathogenic cells produce increased and quantifiable amounts of cfDNA fragments valuable in the diagnosis and prognosis of pathogenic processes ( 16 , 20 , 80 , 81 ).…”

“…cfNGS involves a peripheral blood draw that sequences cell-free DNA (cfDNA) present in the host bloodstream from already naturally lysed pathogenic cells ( 16 , 80 ). The cfDNA fragments have a short half-life in the circulating blood, so rapidly dividing oncologic and pathogenic cells produce increased and quantifiable amounts of cfDNA fragments valuable in the diagnosis and prognosis of pathogenic processes ( 16 , 20 , 80 , 81 ). The mildly invasive nature and quantifiable measurements allow multiple samples to be obtained linearly in hospital and office settings to monitor disease processes.…”

“…They concluded that mNGS produced a higher diagnostic yield than invasive diagnostics, and 34 of those procedures could have been potentially avoided with cfNGS results alone. Echeverria et al ( 80 ) studied 53 patients with confirmed PJI who had cfNGS performed on peripheral blood prior to operative intervention. cfNGS detected a pathogen on average 3 days faster than other testing and identified a pathogen in 4/7 culture-negative cases.…”

Synovial fluid neutrophil extracellular traps could improve the diagnosis of periprosthetic joint infection

“…Presently, there are still many challenges in diagnosing PJI, especially culture-negative PJI. Microbial culture is regarded as the 'gold standard' for PJI diagnosis, however due to the presence of biofilms, viable but noncultural state organisms and fastidious bacteria, and the administration of antibiotics before sampling, the positive rates of microbial culture are far from satisfactory, varying from 47.1% to 80% in most centres according to previous reports, 4,5 and might be up to 90% after optimizing the culture methods in a few centres. [6][7][8] In the last few decades, the emergence of molecular diagnosis has initiated a new era for the microbial diagnosis of PJI, 9 especially next-generation sequencing (NGS), such as metagenomics next-generation sequencing (mNGS), which could detect all microorganisms in the sample at a time, including bacteria, fungi, viruses, and parasites, greatly improving the diagnosis of PJI.…”

Clinical impact of a metagenomic microbial plasma cell-free DNA next-generation sequencing assay on treatment decisions: a single-center retrospective study

“…The use of KT at our institution has been restricted to tertiary testing in scenarios where conventional assays do not provide a diagnosis or situations that require morbid invasive procedures. mcfDNA sequencing in general has been studied as a complimentary assay in the rapid diagnosis of sepsis, culture-negative endocarditis, pneumonia, invasive fungal infections, brain abscesses and more recently as an adjunct to conventional microbiology cultures in bloodstream infections and prosthetic joint infections [ 2 – 8 ]. Despite results of recent clinical trials and case reports demonstrating the potential value of mcfDNA testing as a diagnostic tool, the overall impact of mcfDNA testing on clinical care is less certain.…”