The study, published in Frontiers in Cellular and Infection Microbiology, was conducted at Severance Hospital, a large tertiary teaching hospital affiliated with Yonsei University College of Medicine in Seoul. Between January 2024 and June 2025, the investigators enrolled 216 adult liver transplant recipients in a prospective diagnostic cohort. Twenty-three patients were later excluded because a cell-free DNA sample could not be obtained on either of the two scheduled sampling days, leaving 193 participants in the final analysis. Each recipient underwent plasma microbial cell-free DNA metagenomic next-generation sequencing, abbreviated mcfDNA mNGS, on postoperative days 7 and 14. The technique works by isolating the small fragments of DNA that microbes shed into the blood, sequencing them without any prior assumption about which organism might be present, and then matching the resulting reads against reference genomes to identify bacteria, viruses, and fungi at the species level.

The laboratory workflow behind the assay is technically demanding. Whole blood was collected in specialized tubes that stabilize cell-free DNA, then centrifuged twice to separate the plasma. Cell-free DNA was extracted with a magnetic reagent kit and quantified with a high-sensitivity fluorescence assay before libraries were prepared and sequenced on an Illumina NovaSeq 6000 system. The resulting reads were first aligned to the human genome to strip away host DNA; the unmapped remainder was then fed into Kraken2, a fast taxonomic classifier running against a custom database built from NCBI bacterial, viral, and fungal genomes. To make results comparable across samples sequenced at different depths, the team converted raw read counts into a normalized metric called reads per ten million, or RPTM, and applied a uniform positivity threshold of RPTM at or above 5. Known environmental and skin organisms such as Cutibacterium acnes were masked, and organisms appearing in high abundance only within specific processing batches were manually excluded as extraction or library-preparation contaminants.

Because fungal detection is particularly prone to database-driven false discoveries, the researchers built an unusual cross-validation layer into their pipeline. Every fungal call from Kraken2 was re-examined with two auxiliary classifiers: Kaiju, which matches translated protein sequences, and EukDetect, which aligns fungal marker genes. Supporting reads were then retrieved from the host-depleted sequencing files and mapped directly against a custom reference panel of 416 fungal genomes, with the human genome included as a decoy. A candidate fungal detection was considered confirmed only when at least ten reads with high mapping quality were distributed across at least three distinct one-kilobase windows. This adjudication process confirmed 92.9 percent of the primary pipeline’s fungal detections, but it also revealed a serious blind spot: the primary classifier achieved high precision for the fungi it called yet captured only a small fraction of the alignment-confirmed fungal detections in the cohort, missing organisms including Candida species, Nakaseomyces glabrata, and Aspergillus niger. All of the missed detections were recovered by Kaiju, and a consensus across multiple classifiers confirmed every detection when all three pipelines agreed.

The headline results concern agreement with conventional microbiology. Among the 193 recipients, mNGS returned positive results in 14.5 percent of patients on day 7 and 21.8 percent on day 14. When the sequencing results for bacterial pathogens were compared directly with blood cultures, concordance appeared modest: 35.0 percent on day 7 and just 14.3 percent on day 14. But that comparison understates the assay’s real performance. When the researchers widened the reference standard to include cultures from other clinically relevant specimens collected within 14 days, such as sputum, peritoneal drain fluid, and urine, overall concordance rose to 80.0 percent on day 7 and 71.4 percent on day 14, a level the authors describe as clinically acceptable. The low direct agreement with blood cultures likely reflects both the higher analytical sensitivity of sequencing and the fact that most patients were already receiving broad-spectrum empirical antibiotics, which suppress culture growth but leave microbial DNA fragments circulating in the plasma.

The sequencing assay also uncovered pathogens that routine testing missed entirely. Bacteria were detected exclusively by mNGS in 59.3 percent of cases on day 7 and 74.2 percent on day 14. Fungal pathogens appeared in five and four cases on the two sampling days, with Aspergillus species identified only by sequencing on day 7. Viral detections were especially revealing: nine viral pathogens were found on day 7 and seventeen on day 14, including cytomegalovirus, herpes simplex virus, varicella-zoster virus, and roseolovirus. Cytomegalovirus detections were confirmed by quantitative PCR, but the other viruses are not routinely tested after liver transplant, and prophylactic strategies for them vary widely across countries. A retrospective chart review found that most patients with these unexpected viral detections had mucocutaneous manifestations suggestive of active infection, even though no diagnostic testing had been ordered and many had not received antiviral treatment. The authors suggest that these infections may be under-recognized under immunosuppression, and that sequencing could serve as an adjunctive diagnostic tool, while cautioning that some detected viruses might represent bystanders rather than true pathogens.

Discordant cases, where blood culture was positive but sequencing was negative, received detailed scrutiny. Thirteen such cases were identified. Two, or 15.4 percent, were classified as contamination or colonization, involving skin flora such as coagulase-negative staphylococci isolated from only one bottle of a blood culture pair or from a catheter specimen alone. Five, or 38.5 percent, were interpreted as weak positives, meaning the organism was present in the sequencing data but below the predefined RPTM threshold. The remaining six, or 46.2 percent, were classified as not detected; four involved Candida species, one Enterococcus faecium, and one Achromobacter species, and several of these were identified only from catheter blood cultures rather than peripheral blood. The Candida findings align with the pipeline’s documented limited sensitivity for fungi, reinforcing the authors’ argument that a single taxonomic classifier is insufficient for fungal surveillance and that negative sequencing results cannot reliably exclude fungal infection.

Sequencing positivity also tracked closely with clinical severity and survival. On day 7, patients with positive mNGS results had significantly higher plasma cell-free DNA concentrations, higher total bilirubin, higher C-reactive protein levels, and more impaired pre-transplant liver function than those with negative results. Their 28-day mortality was 26.9 percent versus zero percent among mNGS-negative patients, and their 90-day mortality was 30.8 percent versus 3.9 percent. On day 14, the 90-day mortality rate remained significantly higher among mNGS-positive patients. Among the 160 patients sampled at both time points, 15 of the 22 who were positive on day 7 remained positive on day 14, and the same organism persisted in 86.7 percent of those cases, indicating good reproducibility of the assay. Persistent positivity was associated with higher Sequential Organ Failure Assessment scores and elevated bilirubin and C-reactive protein levels, suggesting that the sequencing results mirror the recipient’s evolving clinical status rather than floating free of it.

The authors are careful to frame the technique as an adjunct rather than a replacement. Because mNGS is hypothesis-free and highly sensitive, it can detect DNA from dead or dying organisms, complicating interpretation and potentially prompting unnecessary antimicrobial use. Testing is also expensive, and the positivity rate in this unselected cohort was low, which the researchers argue supports selective deployment in situations where the expected diagnostic benefit is high, such as clinically ambiguous cases or patients already on broad empirical therapy in whom sequencing might facilitate early antibiotic de-escalation. Notably, most mNGS-positive patients in the study were already receiving active antibiotics against the detected pathogens, which limited the potential for the assay to trigger unnecessary new treatment. The study has limitations: sequencing was performed regardless of clinical suspicion, so positivity rates might be higher in suspected-infection settings; the comparison with blood cultures may have been skewed by prior antibiotic exposure; and the retrospective elements prevented assessment of whether sequencing-guided treatment improved outcomes. Cost-effectiveness, turnaround time, and antimicrobial resistance gene detection were not evaluated, and the authors suggest that differentiated thresholds, such as RPTM at or above 3 for viruses and at or above 8 for bacteria and fungi, might better balance sensitivity and specificity. Whether sequencing-guided management can improve clinical outcomes after liver transplantation remains the central question for future studies, but the evidence here indicates that plasma microbial cell-free DNA sequencing agrees substantially with conventional microbiology when clinically relevant specimens are considered, and that it reveals pathogens, particularly viruses and antibiotic-suppressed bacteria, that routine diagnostics would otherwise leave undetected during the fragile early weeks after surgery.

Subject of Research: Plasma microbial cell-free DNA metagenomic sequencing for early infection detection after liver transplantation

Article Title: Plasma microbial cell-free DNA metagenomic sequencing for early detection of infection after liver transplantation: a prospective cohort study

Article References: Lee, Y., Min, E.-K., Park, Y. J., Kim, D.-G., Joo, D. J., Kim, M. S., Shin, S., Lee, S.-T., Lee, J. G., & Jeong, S. J. (2026). Plasma microbial cell-free DNA metagenomic sequencing for early detection of infection after liver transplantation: a prospective cohort study. Frontiers in Cellular and Infection Microbiology, 16, Article 1930701. https://doi.org/10.3389/fcimb.2026.1930701

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1930701

Keywords: liver transplantation, metagenomic next-generation sequencing, microbial cell-free DNA, infection diagnosis, bloodstream infection, organ transplant recipients, opportunistic pathogens, diagnostic microbiology, viral detection, fungal surveillance, prospective cohort study, antibiotic de-escalation

The research team, led by corresponding author Ying Xu of the Department of Clinical Laboratory, enrolled febrile patients admitted to the hospital between 2021 and 2023. From this population, 457 eligible patients were classified into three etiological groups—bacterial, viral, or fungal infection—based on final clinical diagnoses corroborated by microbiological results. Rather than hunting for the pathogen itself, the investigators turned the diagnostic lens inward, asking whether the patient’s own immune response could reveal the identity of the invader. The rationale rests on a well-established biological principle: different classes of pathogens trigger distinct patterns of cytokine signaling, the chemical messengers through which immune cells coordinate their defense.

The biomarker panel the team evaluated combined six cytokines—interleukin-2 receptor (IL2R), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β)—with three routine inflammatory markers: procalcitonin (PCT), C-reactive protein (CRP), and the neutrophil-to-lymphocyte ratio (NLR). Each component carries distinct immunological meaning. IL-6 is an early-response cytokine that drives the acute-phase reaction and is often markedly elevated in bacterial infections. IL-10, an anti-inflammatory cytokine, rises when the immune system attempts to restrain excessive inflammation, and the balance between the two—captured by the IL-6/IL-10 ratio—reflects the net inflammatory tone of the host. IL-8 is a chemokine that recruits neutrophils to sites of infection, while PCT, a prohormone of calcitonin, is released by extra-thyroid tissues under bacterial endotoxin stimulation and has long been regarded as one of the more specific markers of bacterial insult. The NLR, a simple calculation from a standard complete blood count, indexes the relative dominance of innate, neutrophil-driven immunity over adaptive, lymphocyte-driven immunity.

To translate these measurements into clinically usable decision rules, the researchers performed receiver operating characteristic (ROC) curve analysis, a statistical technique that plots diagnostic sensitivity against the false-positive rate across all possible cut-off values. The area under the ROC curve (AUC) quantifies overall discriminative ability, with 0.5 indicating performance no better than chance and 1.0 representing perfect classification. From these analyses, the team derived optimal threshold values for each marker and then combined them into multivariable models tailored to specific diagnostic questions. The result is not a single test but a sequential pathway: first determine whether the infection is bacterial or viral, then, if bacterial, determine whether the pathogen is Gram-negative or Gram-positive, and finally assess whether the bacterial infection has progressed to sepsis.

The first step of this hierarchy addresses the most common and consequential dilemma in febrile patients—separating bacterial from viral disease. A model combining an IL-6/IL-10 ratio greater than 1.13, IL-8 above 28.70 picograms per milliliter, and an NLR above 2.96 achieved an AUC of 0.80 (95% confidence interval: 0.74–0.87) for this distinction. In practical terms, an AUC of 0.80 indicates good discriminative ability, meaning the model correctly ranks a randomly chosen bacterial case above a randomly chosen viral case about four times out of five. The logic behind the chosen variables is coherent: a high IL-6/IL-10 ratio signals unopposed pro-inflammatory activity typical of bacterial stimulation, elevated IL-8 reflects intense neutrophil recruitment, and a high NLR confirms the neutrophil-dominant blood picture that accompanies acute bacterial infection. Viral infections, by contrast, tend to provoke lymphocyte-dominated responses with comparatively restrained IL-6 and IL-8 production.

The second step of the pathway tackles a subtler question: once a bacterial infection is confirmed, is the culprit Gram-negative or Gram-positive? This distinction matters because the two bacterial classes differ in cell-wall structure, antibiotic susceptibility profiles, and clinical behavior. The study found that no single universal model sufficed; instead, site-specific models were required, reflecting the fact that the immune signature of a bacterial infection is shaped by both the pathogen’s Gram status and the anatomical location of the infection. Notably, in bloodstream infections, a model incorporating IL-6 above 44.91 picograms per milliliter and PCT above 2.30 nanograms per milliliter yielded an AUC of 0.82 (95% confidence interval: 0.70–0.93). The pairing is immunologically sensible—Gram-negative organisms release endotoxin, a potent inducer of both IL-6 and PCT, so elevated values of these two markers together point toward a Gram-negative etiology.

The third and final step addresses severity stratification: among patients with confirmed bacterial infection, which ones have developed sepsis, the life-threatening dysregulated response that demands immediate aggressive intervention? Here the combination of IL-8 above 72.45 picograms per milliliter and PCT above 1.60 nanograms per milliliter achieved an AUC of 0.80 (95% confidence interval: 0.72–0.89). Very high IL-8 concentrations likely reflect the massive neutrophil activation and endothelial inflammation that characterize septic physiology, while elevated PCT tracks the systemic bacterial burden. Together, the three steps of the pathway achieved AUC values of 0.80 to 0.82 across the key diagnostic decisions—a consistent level of performance that, while not replacing microbiological confirmation, is sufficient to guide early therapeutic choices in the critical first hours of illness.

The practical appeal of this strategy lies in its accessibility. Every marker in the panel—cytokines, PCT, CRP, and the NLR—can be measured with routine laboratory instrumentation, and many are already part of standard workups for febrile patients. The study’s authors emphasize that the approach constitutes a cost-effective, rapid decision-support tool designed to facilitate early clinical decisions and antibiotic stewardship, with particular relevance for medical institutions that lack access to expensive molecular diagnostics such as multiplex PCR panels or next-generation sequencing. In resource-limited settings, where blood cultures may take days or be unavailable altogether and where antibiotic misuse is often most severe, a tiered pathway built from existing assays could meaningfully shorten the time to targeted therapy. The work was supported by the National Natural Science Foundation of China and by Science and Technology Projects funding from Guangzhou.

As with any biomarker study, the findings come with caveats that will shape future validation. The analysis was retrospective, drawing on existing medical records from a single center, and the classification of patients depended on final clinical diagnoses, which themselves carry inherent uncertainty. Cut-off values derived from one population may require recalibration in others, and the confidence intervals around some models—particularly the Gram-negative versus Gram-positive bloodstream infection model—indicate a degree of statistical uncertainty that larger cohorts could narrow. Nonetheless, the study demonstrates a compelling concept: that the immune system’s own chemical conversation, read through a carefully chosen panel of cytokines and routine markers, can be decoded into a structured diagnostic pathway. By sequencing the questions—etiology, pathogen subtype, severity—the strategy converts a scatter of laboratory values into an actionable clinical narrative, offering a template for how host-response diagnostics might complement, and in some cases temporarily stand in for, the slower tools of classical microbiology.

Subject of Research: Host immune response biomarkers for rapid differentiation of bacterial, viral, and fungal infections

Article Title: A rapid diagnostic strategy of pathogen infection using host markers of immune response

Article References: A rapid diagnostic strategy of pathogen infection using host markers of immune response. (n.d.). https://doi.org/10.1186/s12879-026-14518-6

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14518-6

Keywords: biomarkers, cytokines, interleukin-6, interleukin-8, procalcitonin, neutrophil-to-lymphocyte ratio, bacterial infection, viral infection, sepsis, antibiotic stewardship, diagnostic markers, ROC analysis