1 March 2022
What Is Cell-Free DNA?
Human bodily fluids have been traditionally considered to be a completely sterile environment, such as blood with its morphotic (cell) elements suspended in surrounding plasma (water-based dispersant, rich in organic and inorganic compounds) as a supposed whole, the homeostatic composition of this liquid tissue. Until recent advancements in the field of biofluid microbiology, any confirmed presence of non-host elements, especially of bacteria, were treated as abnormal physiological states . Indeed, blood infectious diseases leading to sepsis are life-threatening emergencies, being the cause of one in five deaths worldwide. However, these conditions stem mainly from viable pathogen infections development due to weakened immune systems’ response of vulnerable populations (e.g. immunocapacitated, newborn, pregnant, elderly, or low-income setting individuals), not necessarily from colonization or contamination, which are one of the baseline strategies for the survival of naturally occurring blood microbiota (Fig. 1.)   .
Figure 1. Blood vessel cross-section graphic visualization with plasma-suspended red/white cells and platelets as the main tissue elements (MAG – magnification of plasma biocomponents). Besides the confirmation of blood bacteriome, virome, and fungome presence in both healthy and diseased individuals, other components have been proven to circulate in the bloodstream: live host cells, including cancerous ones, as well as undigested cell organelles and macromolecules, such as genetic information in form of genomic DNA (gDNA) parts, released to extracellular space from disintegrating biological agents, referred to as circulating cell-free DNA (cfDNA)4  .
Cell-free DNA is therefore an endo- or exogenous fragment of coding or non-coding deoxyribonucleic acid with an average size smaller than 300 bp, remaining in the biofluids after apoptosis, necrosis, or another breakdown pathway of cells or capsids  . Discovered in human blood samples as early as 1948, cfDNA has gained track only in recent decades as nucleic acid isolation and amplification techniques became more sensitive and efficient in detecting the nanogram per milliliter concentration of these biomolecules .
Why Is cfDNA Used for Diagnosis of Infections?
The makeup of human blood cfDNA profile is mostly composed of the genetic material coming from the organism’s own cells, constituting up to 99,99% of all free circulating deoxyribonucleic acids fragments, with as low as 0.00025% of the total cfDNA coming from the host’s malignant or fetal cells if present . The development of targeted molecular methods, including digital PCR (dPCR) and beads, emulsions, amplification, and magnetics (BEAMing), allowed for the detection of tumor cfDNA present in frequencies of 1% to 0.001% of the total cfDNA in vascular circulation. Based on these advancements, cfDNA sequencing has emerged as a novel laboratory technique for rapid and noninvasive diagnosis in cancer and prenatal screening as well as organ transplantation9  . The remaining part of extracellular DNA originates either from pathogenic or non-pathogenic components of microbiota such as bacteria (0.08%-4.85%), viruses (0.00%-0.16%), fungi (0.00%-0.01%) or protozoa. Collectively, the so-called microbial cell-free DNA (mcfDNA), have been proposed as an infectious disease biomarker for clinical diagnosis on the same principles as implemented in gynecology, oncology, or transplantology practice  .
Many DNA sequences, including cfDNA fragments, are species-specific and with the development of next-generation sequencing (NGS) technology, these molecules have been described as a promising, non-invasive tool for the early detection of several human diseases , including sepsis for which the latest research have reported significantly elevated fractions of cfDNA from retrotransposable elements in blood. Indeed, the use of cfDNA for the detection of infectious Epstein-Barr virus has been reported since the 20th century and more recently for the diagnosis of invasive fungal infection. Some more specific examples of infectious agents reported to be detected using cfDNA sequencing include Plasmodium, Trypanosoma, Leishmania, Schistosoma, Leptospira, and HIV. Microbial cfDNA can be also detected in biofluids of patients undergoing intensive antimicrobial treatments .
How cfDNA Is Used For Diagnosis of Infections?
mcfDNA-based next-generation sequencing (mcfDNA sequencing) is an emerging hypothesis-free test that detects mcfDNA shed into noninvasive samples (e.g. peripheral venous blood, urine) from sites of infection, increasingly promoted as a faster diagnostic method with higher sensitivity than standard blood cultures (BC) .
As of today, there is only one commercially available, however not FDA-approved test  for cfDNA-based medical disease diagnosis, developed by Karius, Inc. (USA) in 2018. The Karius® Test (KT) is an assay utilizing metagenomic NGS (mNGS) for microbial cfDNA detection in patient blood samples during infections diagnosis, based on a clinical-grade database limited to around 1,200 from more than 1,400 species of all known human pathogens . Following a set of custom-designed methods, including specific artificial intelligence algorithms, KT allows for efficient isolation, purification, sequencing, and taxonomic origin determination of identifiable, blood-circulating extracellular mcfDNA fragments, derived from non-viable microbes present in either healthy, diseased, or vulnerable individuals . Since its commercial introduction, the mcfDNA sequencing potential of KT has been also employed as a possible aid in the hard-to-detect and latent infections such as tuberculosis or some forms of sepsis, however with a varied success rate and the significance of Mycobacterium tuberculosis cfDNA detection in patients with pulmonary tuberculosis still remaining unclear  .
What Are cfDNA Limitations and Possible Alternatives?
With continued advancements in molecular microbiology techniques, a wide number of sequencing-based diagnostics tests for an array of pathogens are emerging, but these tests often require invasive biopsy samples taken from infected tissue, which is not advised during some medical conditions or emergencies, therefore prolonging and hindering the determination of a proper diagnosis. Based on the fact that cfDNA from specific tissues or sites circulates freely around the bloodstream, mcfDNA has been proposed as an alternative method of noninvasive blood biopsies for rapid infectious diseases diagnosis ongoing throughout the body. Nevertheless, recent research indicates the distribution of circulating cfDNA coverage over the respective reference genome is highly non-uniform increasing the risk of missing relevant fragments, especially given the small volume of biofluid analyzed, low amounts of mcfDNA in relation to interfering human cfDNA, even smaller if taken into account overall cfDNA concentration in the blood varies significantly, ranging between 0–5 and >1000 ng per ml in patients with cancer and between 0 and 100 ng per ml in healthy subjects    .
The overall value of each infectious agent’s detection method for clinical practice is determined by a wide spectrum of features including their accuracy, safety, technical, or manufacturing parameters. From the patient’s healthcare point of view, the diagnosis speed and effectiveness of subsequent therapy are influenced to the highest degree by the test’s specificity, sensitivity, turnaround time, costs, and the degree with which pathogen characteristics are determined (e.g. species, strains, and specific traits). The summary of these frequently addressed tests’ features for clinical application indicates the mcfDNA mNGS technique indeed has improved sensitivity as compared with most widely used BC, yet its specificity is decreased in relation to both BC and gDNA sequencing of whole blood pathogen samples (Tabl. 1.). As with every DNA sequencing technology, both gDNA and mcfDNA rapid diagnostics are less available and more expensive (with the former still cheaper than the latter) than pathogen ID based on microbial culturing, but at the same time constitute much faster and more sensitive methods. Although KT could diagnose opportunistic pathogens otherwise missed by standard microbiological testing, it also yields polymicrobial detections and organisms of uncertain clinical significance and actionability. Recent empirical and theoretical research points out the KT detection method is not uniform and the standardization process is lacking, with the main obstacles of mcfDNA use in rapid diagnostics of infectious diseases (e.g. low abundance, high rate of non-microbial impurities, or incorrect sequence attribution) overcomed on a basis of custom solutions. A significant drawback of the mcfDNA mNGS is definitely seen in the inability to determine specific strains and traits of detected species, as most of the genetic information needed for such ID is fragmented and the diversity of genes encoding the clinically relevant features such as antimicrobial resistance, is defined in hundreds of sequences among single species. The quantification of viable microbiome presence and unconstricted range of reference genomes during pathogen gDNA sequencing allowing for the NGS-based identification of pathogenic and non-pathogenic microbes in whole blood samples with a sensitivity of 102 genome copies per ml, is unavailable while using mcfDNA for diagnostic purposes, resulting in the knowledge gaps about the nature of detected microbiota components (e.g. infection, colonization, contamination could not be distinguished)           .
Table 1. Comparison of different methods in clinical microbiology for diagnosis of human infectious diseases from blood.
|Diagnostic Method||Blood Culture||gDNA mNGS||mcfDNA mNGS|
|Agents Identified||Viable Pathogen Colonies||Viable Pathogen’s Whole gDNA||Non-Viable Pathogen’s cfDNA|
|Species ID Range||Medium||High||Medium|
|Strain/Variant ID Range||Medium||High||Low|
|Antimicrobial Resis. ID||Yes||Yes||No|
|Turnaround Time||≥ 2 days||1 day||2 days|
|Commercial Tests Examples||WORKSAFE™ BC Kits (bioMérieux SA, France)||PaRTI-Seq® (Micronbrane Medical Co., Taiwan)||Karius® Test (Karius Inc., USA)|
All in all, the mcfDNA sequencing could in the future become a valuable resource for clinical microbiology practice, especially useful during difficult or extraordinary cases, however as an additional and not the only or definitive source of medical diagnoses during infectious diseases. KT results should be examined with caution, by physicians with solid ID expertise, familiarity with the technology and result interpretation, preferably as a supplemental verification of BC or whole blood gDNA sequencing interpretations .
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