Allograft biopsy remains the gold standard to diagnose rejection. New noninvasive biomarkers are needed to avoid unnecessary biopsies and to diagnose early rejection. We studied the performance of donor derived cell-free DNA (dd-cfDNA) to detect rejection in an unselected cohort of pediatric kidney transplant recipients (pKTRs) and determined whether dd-cfDNA could improve standard-of-care monitoring and detection of kidney allograft rejection in children.

We included 196 pKTRs, who underwent 367 biopsies with concomitant dd-cfDNA assessment. We assessed the association of dd-cfDNA with histological lesions and with rejection using a Cox regression model and compared the discrimination of 3 models: standard of care, dd-cfDNA alone, and combined.

We found a significant increase in dd-cfDNA levels with higher degree of inflammation on the biopsies. dd-cfDNA was strongly and independently associated with the presence of allograft rejection (odds ratio 1.89, 95% confidence interval [CI], 1.40-2.60). dd-cfDNA alone had a fair discrimination of 0.76 (95% CI, 0.69-0.81) to detect rejection and its addition to standard of care resulted in a significant increase in discrimination from 0.80 (95% CI, 0.71-0.85) to 0.84 (95% CI, 0.79-0.90), P = 0.01.

dd-cfDNA combined with standard of care improves the prediction of rejection in pKTRs. Further specific studies are needed to better define how to use this promising biomarker in various contexts of use in children.

Cell-free DNA (cfDNA) is a marker of organ injury and immune response. DNA methylation is an epigenetic regulator of gene expression. Here, we elucidate total plasma cfDNA methylation from kidney transplant recipients in presence versus absence of rejection. In doing so, we exploit cfDNA as a real-time biomarker to define molecular pathways of rejection. Twenty plasma cfDNA samples from pediatric kidney transplant recipients were collected at allograft biopsy. Differentially methylated cytosine residues (>20 % methylation difference, q-value <0.05) were identified in presence (N = 7) versus absence (N = 9) of acute rejection. Separate analyses were performed comparing borderline rejection (N = 4) to rejection and non-rejection. In rejection versus non-rejection, there were 1269 differentially methylated genes corresponding to 533 pathways. These numbers were 4-13× greater than comparisons against borderline samples. Enriched pathways between rejection and non-rejection samples were related to immune cell/inflammatory response, lipid metabolism, and tryptophan-kynurenine metabolism, suggesting differential methylation of these pathways contributes to rejection.

Alloimmune injury is a major cause of long-term kidney allograft failure whether due to functionally stable (subclinical) or overt clinical rejection. These episodes may be mediated by immune cells (cellular rejection) or alloantibody (antibody-mediated rejection). Early recognition of immune injury is needed for timely appropriate intervention to maintain graft functional viability. However, the conventional measure of kidney function (ie, serum creatinine) is insufficient for immune monitoring due to limited sensitivity and specificity for rejection. As a result, there is need for biomarkers that more sensitively detect the immune response to the kidney allograft. Recently, several biomarkers have been clinically implemented into the care of kidney transplant recipients. These biomarkers attempt to achieve multiple goals including (1) more sensitive detection of clinical and subclinical rejection, (2) predicting impending rejection, (3) monitoring for the adequacy of treatment response, and (4) facilitating personalized immunosuppression. In this review, we summarize the findings to date in commercially available biomarkers, along with biomarkers approaching clinical implementation. While we discuss the analytical and clinical validity of these biomarkers, we identify the challenges and limitations to widespread biomarker use, including the need for biomarker-guided prospective studies to establish evidence of clinical utility of these new assays.

The last decade has seen an explosion in clinical research focusing on the use of noninvasive biomarkers in kidney transplantation. Much of the published literature focuses on donor-derived cell-free DNA (dd-cfDNA). Although initially studied as a noninvasive means of identifying acute rejection, it is now clear that dd-cfDNA is more appropriately described as a marker of severe injury and irrespective of the etiology, elevated dd-cfDNA ≥0.5% portends worse graft outcomes. Blood gene expression profiling is also commercially available and has mostly been studied in the context of early identification of subclinical rejection, although additional data is needed to validate these findings. Torque teno virus, a ubiquitous DNA virus, has emerged as a biomarker of immunosuppression exposure as peripheral blood Torque teno virus copy numbers might mirror the intensity of host immunosuppression. Urinary chemokine tests including C-X-C motif chemokine ligand 9 and C-X-C motif chemokine ligand 10 have recently been assessed in large clinical trials and hold promising potential for early diagnosis of both subclinical and acute rejection, as well as, for long-term prognosis. Urinary cellular messenger RNA and exosome vesicular RNA based studies require additional validation. Although current data does not lend itself to conclusion, future studies on multimodality testing may reveal the utility of serial surveillance for individualization of immunosuppression and identify windows of opportunity to intervene early and before the irreversible allograft injury sets in.

The aim of this analysis was to evaluate early signals of the utility of gene expression profile (GEP) and donor-derived cell-free DNA (dd-cfDNA) for ruling out subclinical rejection in multiorgan transplant recipients.

This was a prospective, single-center, observational pilot study that began enrolling patients in September 2022. Participants were enrolled after providing informed consent and had biomarker samples drawn before surveillance or for-cause biopsy. GEP result of TX was considered negative and a dd-cfDNA of ≤0.69% was considered negative, regardless of a nonrenal organ.

There were 49 participants with 55 surveillance and/or for-cause biopsies. After exclusion of biopsies not paired with biomarkers, 51 biopsies were evaluated with at least 1 biomarker. Fifty-one biopsies had paired GEP results, whereas 47 biopsies had paired dd-cfDNA results. Overall, there were 12 biopsy-proven acute rejections (24%), 5 of which were T cell-mediated rejections (4-1A and 1-1B), 2 were antibody-mediated rejections, and 5 were borderline for T cell-mediated rejections. GEP by itself in 51 biopsies demonstrated a sensitivity of 17%, specificity of 74%, positive predictive value of 17%, negative predictive value of 74%, and balanced accuracy of 61%. Among 47 paired biopsies, dd-cfDNA demonstrated a sensitivity of 67% and specificity of 37%. Median dd-cfDNA was above the positivity threshold for both participants with rejection on biopsy and without (1.86% versus 1.35%, respectively). When evaluating GEP, specifically in surveillance biopsies and patients with liver transplants, diagnostic performance was maintained.

In this pilot analysis, GEP maintained a high negative predictive value in a multiorgan cohort, regardless of the nonrenal organ. dd-cfDNA did not have good performance when using the kidney threshold cutoff, which was expected and driven by the liver component of multiorgan recipients. Further technological advances with dd-cfDNA to differentiate organs and multiple donors could be impactful. The results support the use of GEP for ruling out kidney rejection in a multiorgan population, including those with a liver transplant. Further evaluation is necessary to confirm the results.

There is a need for a noninvasive, affordable, sensitive, and specific biomarker to diagnose early acute rejection, to negate the need for frequent biopsies. Dd-cfDNA is a powerful adjunct yet there is limited data on the ethnic differences in its values. There is anecdotal evidence that dd-cfDNA values at rejection may be higher in Black as compared to non-Black recipients. This study aims to add to this literature while defining such variability and comparing it to previously validated cutoffs for dd-cfDNA of 0.5% or 1%.

This was a single-center retrospective observational study of patients who underwent graft biopsies with a preceding, paired, dd-cfDNA value. Recipients were separated into White, Black, and Hispanic racial and ethnic groups, and dd-cfDNA values at rejection versus nonrejection were compared.

With 0.5% and 1% cutoffs, false negative rates for rejection were 13% and 22%, respectively. The false positive rate was 38.4%. 12.2% of Black recipients, 11.8% of Hispanic recipients, and 44% of White recipients had rejection with a negative AlloSure®. Values >0.5% corresponded to histologic rejection in 61.5% of Black, 66.7% of White, and 56.3% of Hispanic recipients. Antibody-mediated rejection occurred in 65.5% of rejection cases in Black recipients, while exhibiting the lowest rate of T-cell-mediated rejection. Dd-cfDNA values gave an accurate diagnosis of rejection in 52.8% of recipients with AMR versus 19.3% in TCMR.

This study demonstrated that dd-cfDNA was applicable to Black recipients with a robust ability to detect antibody-mediated rejection, as compared to White and Hispanic recipients.

In the last two decades, many studies based on omics technologies have contributed to defining the clinical, immunological, and histological fingerprints of chronic antibody-mediated rejection (CAMR), the leading cause of long-term kidney allograft failure. However, the full biological machinery underlying CAMR has only been partially defined, likely due to the fact thatsingle-omics technologies capture only specific aspects of the biological system and fail to provide a comprehensive understanding of this clinical complication.

This study integrated mass spectrometry-based proteomic profiling of serum samples from 19 patients with clinical and histological evidence of CAMR and 26 kidney transplant recipients with normal graft function and histology (CTR) with transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) from an independent cohort of 10 CAMR and 8 CTR patients. Data analysis was conducted using unsupervised hierarchical clustering (multidimensional scaling with k-means) and Spearman's correlation test. Partial least squares discriminant analysis (PLS-DA) with the importance in projection (VIP) score identified key proteins differentiating CAMR from CTR. ELISA was used to validate the omics results.

Proteomic analysis identified 18 proteins that significantly differentiated CAMR from CTR (p < 0.01): five were more abundant (CHI3L1, LYZ, PRSS2, CPQ, IGLV3-32), while 13 were less abundant (SERPINA5, SERPING1, KNG1, CAMP, VNN1, BTD, WDR1, PON3, AHNAK2, MELTF, CA1, CD44, CUL1). Transcriptomic profiling revealed 6 downregulated and 33 upregulated genes in CAMR versus CTR (p < 0.01). Notably, only 2 biological elements were significantly deregulated in both omics analyses: chitinase-3-like protein 1 (CHI3L1) and plasma protease inhibitor C1 (SERPING1). CHI3L1, previously associated with the severity of tissue damage in kidney diseases, was up-regulated in CAMR in both transcriptomics and proteomics, while SERPING1, a serine esterase inhibitor that blocks the classical and lectin pathway of complement, was up-regulated in CAMR in transcriptomics but down-regulated in proteomics. ELISA validated the omics results, and the ROC curve showed that CHI3L1 has good discrimination power between CAMR and CTR (AUC of ROC curve of 0.81).

Our multi-omics data, although performed in a relatively small cohort of patients, revealed new systemic biological elements involved in the pathogenesis of CAMR and identified CHI3L1 as a new potential biomarker and/or therapeutic target for this important clinical complication. Future validation of these findings in larger patient cohorts should be conducted to better evaluate their clinical utility.

Kidney transplantation stands as the preferred treatment for end-stage kidney disease, significantly improving both the quality and longevity of life compared to dialysis. In recent years, the survival rates for patients and grafts have markedly increased thanks to innovative strategies in desensitization protocols for incompatible transplants and advancements in immunosuppressive therapies. For kidney transplant recipients, preventing allograft rejection is of paramount importance, necessitating the use of immunosuppressive medications. Regular follow-up appointments are essential, as monitoring the function of the kidney allograft is critical. Currently, established biomarkers such as serum creatinine, estimated Glomerular Filtration Rate (eGFR), proteinuria, and albuminuria are commonly employed to assess allograft function. However, these biomarkers have limitations, as elevated levels often indicate significant allograft damage only after it has occurred, thereby constraining treatment options and the potential for restoring graft function. Additionally, kidney biopsies, while considered the gold standard for diagnosing rejection, are invasive and carry associated risks. Consequently, the identification and development of new, sensitive, and specific biomarkers like dd-cfDNA, microRNAs (e.g., miR-21, miR-155), and sCD30 for allograft rejection are crucial. To tackle this challenge, intensive ongoing research employing cutting-edge technologies, including "omics" approaches, like genomic techniques, proteomics, or metabolomics, is uncovering a variety of promising new biomarkers.

Transplant rejection remains a significant barrier to the long-term success of organ transplantation. Biopsy, although considered the gold standard, is invasive, costly, and unsuitable for routine monitoring. Traditional biomarkers, such as creatinine and troponin, offer limited predictive value owing to their low specificity, and conventional imaging techniques often fail to detect early organ damage, increasing the risk of undiagnosed rejection episodes. Considering these limitations, emerging noninvasive biomarkers and molecular imaging techniques hold promise for the early and accurate detection of transplant rejection, enabling personalized management strategies. This review highlights noninvasive biomarkers that predict, diagnose, and assess transplant prognosis by reflecting graft injury, inflammation, and immune responses. For example, donor-derived cell-free DNA (dd-cfDNA) is highly sensitive in detecting early graft injury, whereas gene expression profiling effectively excludes moderate-to-severe acute rejection (AR). Additionally, microRNA (miRNA) profiling enhances the diagnostic specificity for precise AR detection. Advanced molecular imaging techniques further augment the monitoring of rejection. Fluorescence imaging provides a high spatiotemporal resolution for AR grading, ultrasound offers real-time and portable monitoring, and magnetic resonance delivers high tissue contrast for anatomical assessments. Nuclear imaging modalities such as single photon emission computed tomography and positron emission tomography, enable dynamic visualization of immune responses within transplanted organs. Notably, dd-cfDNA and nuclear medicine imaging have already been integrated into clinical practice, thereby demonstrating the translational potential of these techniques. Unlike previous reviews, this work uniquely synthesizes advancements in both noninvasive biomarkers and molecular imaging, emphasizing their complementary strengths. Biomarkers deliver molecular-level insights, whereas imaging provides spatial and temporal resolution. Together, they create a synergistic framework for comprehensive and precise transplant monitoring. By bridging these domains, this review underscores their individual contributions and collective potential to enhance diagnostic accuracy, improve patient outcomes, and guide future research and clinical applications in transplant medicine.

The use of noninvasive biomarkers may reduce the need for biopsy and guide immunosuppression adjustments during transplantation. The scientific community in solid organ transplantation currently considers that chemokines, T- and B-cell immunophenotypes, and gene expression, among other molecular biomarkers, have great potential as diagnostic and predictive biomarkers for graft evolution; however, in clinical practice, few valid early biomarkers have emerged. This review focuses on the most relevant scientific advances in this field in the last 5 years regarding the role of 3 biomarkers: miRNAs, chemokines, and ddcf-DNA, in both adult and pediatric populations. An update was provided on the scores based on the combination of these biomarkers. The most-featured articles were identified through a literature search of the PubMed database. This review provides a comprehensive analysis of the potential clinical applications of these biomarkers in the diagnosis and prediction of graft outcomes and discusses the reasons why none have been implemented in clinical practice to date. Translating these biomarkers into routine clinical practice and combining them with pharmacogenetics and pharmacokinetic monitoring is challenging; however, it is the key to present/future individualized immunosuppressive therapies. It is essential that they be shown to be applicable and robust in real-life patient conditions and properly evaluate their added value when combined with the standard-of-care factor monitoring for graft clinical assessment. Partnership strategies among scientists, academic institutions, consortia, including expert working groups and scientific societies, and pharmaceutical and/or biotechnology companies should promote the development of prospective, randomized, multicenter intervention studies for adequate clinical validation of these biomarkers and their monitoring frequency, and their commercialization to make them available to transplant physicians.

Background: This study aimed to assess the diagnostic accuracy of combining donor-derived cell-free DNA (dd-cfDNA) fraction and absolute quantification in detecting kidney allograft rejection. Methods: A prospective study was conducted from December 2019 to April 2021 at the First Affiliated Hospital of Sun Yat-sen University. Kidney transplant recipients who underwent biopsy, including cases of T-cell-mediated rejection (TCMR), antibody-mediated rejection (ABMR), and borderline rejection, were included. dd-cfDNA fraction and absolute concentrations were measured, and diagnostic efficacy was evaluated using receiver operating characteristic (ROC) analysis. The double-positive and double-negative methods were applied to assess performance. Results: A total of 50 kidney transplant recipients were included. The dd-cfDNA fraction cutoff of 1.08% achieved 93.33% sensitivity and 91.43% specificity (AUC = 0.95), with an NPV of 96.97% and a PPV of 82.35%. The absolute dd-cfDNA threshold of 32 cp/mL yielded 80.00% sensitivity and 71.43% specificity (AUC = 0.78), with an NPV of 89.29% and a PPV of 54.55%. The double-positive method provided superior accuracy, with a PPV of 91.67% and an NPV of 89.47%, demonstrating 73.33% sensitivity and 97.14% specificity. The double-negative method achieved 100% NPV and 100% sensitivity. Conclusions: Combining dd-cfDNA fraction and absolute quantification improves diagnostic accuracy for kidney transplant rejection, especially ABMR. The double-positive and double-negative approaches show high predictive value, offering potential clinical value for monitoring kidney transplant recipients.

Chronic kidney disease (CKD) is associated with chronic low-grade inflammation, but the primary factors triggering this inflammation remain unclear. Extracellular or cell-free DNA (exDNA) originates from virtually all tissues, being released during cell death, and stimulates the innate immune system. Our study was designed as an observational, cross-sectional cohort study of children with CKD (both before and after kidney transplantation) and controls to analyze associations between exDNA, markers of inflammation, and cardiovascular health. Extracellular DNA (total, nuclear, and mitochondrial) was analyzed in plasma using fluorometry and real-time PCR. We found that children with CKD after kidney transplantation had higher concentrations of total and nuclear extracellular DNA (total exDNA and nc_exDNA) in plasma compared to controls. In univariate analysis, levels of interleukin-6 (IL-6), antimicrobial peptide cathelicidin (LL-37), soluble vascular cell adhesion molecule-1 (VCAM-1) and left ventricular mass index (LVMI) were positively correlated with total exDNA and nc_exDNA concentrations. Multivariate analysis revealed LVMI as the only independent variable associated with high levels of both total exDNA and nc_exDNA. We believe that our results contribute new knowledge to the pathogenesis of CKD and its complications and may help identify new treatment targets.

Donor-derived cell-free DNA (dd-cfDNA) is a promising non-invasive biomarker for detecting graft injury in solid organ transplant recipients. Elevated dd-cfDNA levels are strongly associated with rejection and graft injury, especially antibody-mediated rejection (ABMR). While de novo donor-specific antibodies (dnDSA) are crucial in ABMR, the relationship between dd-cfDNA levels and dnDSA features, such as DSA category, MFI and HLA target loci, remains unclear.

We analyzed dd-cfDNA levels in 75 kidney transplant recipients who developed dnDSA post-transplant. dnDSA were categorized as "true", "possible", or "false" based on bead reactivity patterns and HLA typing. dd-cfDNA was assessed alongside dnDSA detection and sequential follow-up samples in a subgroup.

"True" dnDSA showed significantly higher dd-cfDNA levels compared to "possible" and "false" groups. None of the dd-cfDNA values in the "false" group exceeded 0.6%, and only a small fraction of the "possible" group had values slightly above 0.6%. dd-cfDNA levels were not significantly affected by dnDSA target loci or number. A strong correlation between cumulative dnDSA MFI and dd-cfDNA levels was observed, especially in patients with "true" HLA-DQ-directed dnDSA. Sequential dd-cfDNA analysis showed dynamic changes in 25% of patients, all from the "true" dnDSA group, which tended to align with shifts in cumulative MFI over time.

These findings highlight the correlation between cumulative dnDSA MFI and dd-cfDNA levels, particularly in HLA-DQ-directed dnDSA, and suggest graft injury is dynamic in dnDSA-positive patients. Integrated monitoring of dnDSA and dd-cfDNA offers a promising non-invasive approach for assessing graft injury and alloimmunity, potentially enhancing post-transplant care.

To investigate whether the methylation of circulating cell-free DNA (cfDNA) differentiates active tuberculosis (TB) from latent TB infection (LTBI).

Patients with pulmonary TB, contacts with LTBI, and healthy controls were enrolled (2018-2021). Plasma cfDNA was extracted, and using a 5-methylcytosine (5mC) DNA ELISA kit, the global methylation of cfDNA (5mC-cfDNA) was measured.

59 TB, 63 LTBI, 39 healthy controls were included. The 5mC-cfDNA level was higher in TB (6.4 %) than LTBI (4.1 %) and healthy controls (4.9 %) (both p<0.05). Independent TB factors were 5mC-cfDNA ≥6.6 % and CRP ≥0.32 mg/dL (adjusted odds ratio (aOR) 4.594 [95 % CI:1.628-12.965], p=0.004 and 5.338 [1.659-17.176], p=0.005). Having one or both factors increased TB odds 8- and 16-fold (aOR 8.688 [3.229-23.378], p <0.001 and 16.080 [3.092-83.632], p =0.001).

The global cfDNA methylation level was higher in TB than contacts without TB and helped differentiate patients with TB from contacts with LTBI.

Dual heart - kidney transplantation (HKT) is an increasingly utilized option for patients with end-stage heart failure and impaired renal function. In 2023, the United Network for Organ Sharing (UNOS) formalized indications for simultaneous heart - kidney transplantation (SHKT) and kidney-after-heart transplantation (KAHT). In light of this landmark effort, we review the evolution in HKT indications, outcomes after SHKT and KAHT in various patient populations, novel considerations in pre and posttransplant management, and areas for future research.

The UNOS policy was borne out of retrospective outcome studies suggesting that SHKT is superior to single-organ heart transplantation (HT) in select patients with sufficiently severe renal impairment. KAHT utilization continues to evolve, providing a helpful safety net for HT recipients with irreversible worsening of renal function. Time-limited pretransplant device trials are useful for identifying optimal candidates for HKT, and calcineurin inhibitor-sparing regimens can preserve posttransplant renal function. Explorations into artificial intelligence algorithms and biomarker technology represent interesting future opportunities.

Recent studies have refined selection and management of dual HKT recipients. The new UNOS policies now provide helpful standardization to the field. Rapid innovation to date has made dual HKT an exciting therapeutic strategy for patients with few options left.

Pregnancy significantly alters the maternal immune system, affecting fetal development. The collection of tissues from the human placenta and fetus is not ethically or practically feasible at various gestational stages, thus limiting the study of gene expression in the fetus and placenta. Recent studies have shown that plasma cell-free DNA (cfDNA) nucleosome patterns can predict gene expression in the source tissue, offering insights into an individual's health status. This study aimed to identify pregnancy-related gene expression changes across gestational periods using cfDNA nucleosome distribution to understand fetal development and maternal immune changes.

Plasma samples were collected from 150 healthy pregnant women in different trimesters (early, mid, and late) and 32 healthy nonpregnant women. The correlation between gene expression and physiological changes during pregnancy was evaluated by inferring differential expression profiles around the transcription start site (TSS) using cfDNA nucleosome distribution patterns obtained through whole-genome sequencing. We utilized Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses to annotate differentially expressed genes with the mother and fetus.

We identified gene expression changes that support the regulation of fetal development and immune system function during pregnancy. Differential coverage genes were mainly enriched in pathways related to transcription and translation, organic compound metabolism, and immune regulation. In addition, differentially expressed genes with significant temporal trends were identified. Among them, the upregulated differential genes were mainly related to development, whereas those with downregulated trends were mainly related to the immune system response. This indicates that differential changes of the placenta and maternal are significantly correlated with the pregnancy status.

This study demonstrated the differential gene expression represented by the characteristic distribution of cfDNA nucleosome in maternal peripheral blood can effectively capture significant changes in maternal immunity and fetal development throughout pregnancy stages. It may help identify abnormal gene expression patterns associated with complications in pregnancy and childbirth, enhancing the quality of life and safety for both mother and fetus.

Introduction: Donor-derived cell-free DNA (dd-cfDNA) in the peripheral blood of allograft recipients has shown to early identify allograft injury. In this study, we assessed the factors that influence the amount of circulating dd-cfDNA during the first month postkidney transplant as well as its longitudinal trend. Materials and Methods: A consecutive series of 98 adult kidney transplant recipients at a single center between July 2018 and January 2020 were included in this study. All demographic and operative details were collected for donors and recipients of the organ transplant. Median eGFR, dd-cfDNA, and serum creatinine were calculated at 1, 2, 3, 6, and 12 month posttransplant. Descriptive statistics were used for patient demographics. Nonparametric comparisons of dd-cfDNA cumulative distributions between dichotomized groupings were evaluated using Kruskal-Wallis or Mann-Whitney U tests. Results: The median age of recipients was 54.5 years (IQR: 42.7-62.2). The cause of ESRD among recipients was hypertension (43%) and Type II diabetes mellitus (29%). Eighty-two percentage of patients received a deceased donor allograft, 14% received a living unrelated allograft, and 4% received a living related allograft. Sixteen percentage of recipients experienced delayed graft function (DGF). Median creatinine level at 1 month posttransplant was 1.75 mg/dL (IQR: 1.34-2.26) and median eGFR at 1 month posttransplant was 49.6 mL/min/1.73 m2 (IQR: 35-65). The median dd-cfDNA score at 1 month posttransplant for all recipients was 0.4% (IQR: 0.15-5.3). Donor sex was a statistically significant differential for dd-cfDNA score. Recipients from male donors had a significantly higher median dd-cf DNA score at 1 month posttransplant versus those who received a female kidney (0.57% vs. 0.28%, p < 0.01). Highest median score was recorded at the first month posttransplant (0.4%, IQR: 0.26-0.74), and a sustained downward trend was observed through Month 2 (0.19%, IQR: 0.17-0.31) and Month 3 (0.19%, IQR: 0.15-0.26). Correlation between 1-, 2-, 3-, 6-, and 12-month posttransplant median dd-cfDNA scores between deceased donor and living donor (LRD and LURD) cohorts was not statistically significant. Conclusion: This study provides further insight into donor and recipient variables' effects on dd-cfDNA in the early posttransplant phase by analyzing a more diverse cohort of patients and adds to the knowledge around interpreting dd-cfDNA scores with clinical correlation for posttransplant management.

Traditional monitoring of kidney transplant recipients for allograft dysfunction caused by rejection involves serial checks of serum creatinine with biopsy of the renal allograft if dysfunction is suspected. This approach is labor-intensive, invasive and costly. In addition, because this approach relies on a rise in serum creatinine above historical baselines, injury to the allograft can be extensive before this rise occurs. In an effort to address this, donor-derived cell-free DNA (dd-cf DNA) is being used with increasing frequency in the clinical setting as a means of diagnosing a rejection of the renal allograft early in the course. This can potentially allow for early intervention to minimize not only injury, but the intensity of antirejection therapy needed and the avoidance of side effects. Here, we will review the available methodology for the determination and quantification of dd-cf DNA, the data supporting its use in clinical practice and the limitations of this technology.

Donor-derived cell-free DNA (dd-cfDNA) is an emerging non-invasive biomarker for allograft injury detection. This study aimed to evaluate a new, decentralized dd-cfDNA testing kit against a centralized dd-cfDNA testing service broadly utilized in the United States. Kidney transplant recipients with decentralized and centralized dd-cfDNA measurements and concomitant kidney allograft biopsies were included in the study. 580 kidney allograft recipients from 3 referral centers were included for 603 total evaluations. Correlation between assays was evaluated using r-squared (r 2) and Spearman's rank correlation test, and associations with rejection using logistic regression analyses and discrimination using area under the curve. Mean dd-cfDNA levels from decentralized and centralized tests were 0.51% ± 0.81% and 0.43% ± 0.78%, respectively. The assays were highly correlated, with r 2 = 0.95 and Spearman's rank correlation 0.88 (p < 0.0001). Both tests showed significant association with allograft rejection (p < 0.0001) and good and similar discriminations to predict rejection (AUC: 0.758 for the decentralized and AUC: 0.760 for the centralized dd-cfDNA; p = 0.8466). Consistency between the assays was also confirmed across clinical scenarios including post-transplant timepoint, allograft stability, and allograft rejection subcategories. This decentralized dd-cfDNA assessment demonstrates high accuracy and value to non-invasively monitor kidney recipients.

Model-informed precision dosing (MIPD) stands as a significant development in personalized medicine to tailor drug dosing to individual patient characteristics. MIPD moves beyond traditional therapeutic drug monitoring (TDM) by integrating mathematical predictions of dosing and considering patient-specific factors (patient characteristics, drug measurements) as well as different sources of variability. For this purpose, rigorous model qualification is required for the application of MIPD in patients. This review delves into new methods in model selection and validation, also highlighting the role of machine learning in improving MIPD, the utilization of biosensors for real-time monitoring, as well as the potential of models integrating biomarkers for efficacy or toxicity for precision dosing. The clinical evidence of TDM and MIPD is discussed for various medical fields including infection medicine, oncology, transplant medicine, and inflammatory bowel diseases, thereby underscoring the role of pharmacokinetics/pharmacodynamics and specific biomarkers. Further research, particularly randomized clinical trials, is warranted to corroborate the value of MIPD in enhancing patient outcomes and advancing personalized medicine.

Infection and rejection are major complications that impact transplant longevity and recipient survival. Balancing their risks is a significant challenge for clinicians. Current strategies aimed at interrogating the degree of immune deficiency or activation and their attendant risks of infection and rejection are imprecise. These include immune (cell counts, function and subsets, immunoglobulin levels) and non-immune (drug levels, viral loads) markers. The shared risk factors between infection and rejection and the bidirectional and intricate relationship between both entities further complicate transplant recipient care and decision-making. Understanding the dynamic changes in the underlying net state of immunity and the overall risk of both complications in parallel is key to optimizing outcomes. The allograft biopsy is the current gold standard for the diagnosis of rejection but is associated with inherent risks that warrant careful consideration. Several biomarkers, in particular, donor derived cell-free-DNA and urinary chemokines (CXCL9 and CXCL10), show significant promise in improving subclinical and clinical rejection risk prediction, which may reduce the need for allograft biopsies in some situations. Integrating conventional and emerging risk assessment tools can help stratify the individual's short- and longer-term infection and rejection risks in parallel. Individuals identified as having a low risk of rejection may tolerate immunosuppression wean to reduce medication-related toxicity. Serial monitoring following immunosuppression reduction or escalation with minimally invasive tools can help mitigate infection and rejection risks and allow for timely diagnosis and treatment of these complications, ultimately improving allograft and patient outcomes.

(1) Background: donor-derived circulating free DNA (dd-cfDNA), an innovative biomarker with great potential for the early identification and prevention of graft damage. (2) Methods: Samples were collected prospectively and the study was performed retrospectively to analyze dd-cfDNA plasma levels in 30 kidney transplant patients during their post-transplant follow-up (15 days, 3, 6, and 9 months), to determine if the result could be of interest in the identification of possible adverse events, especially rejection. The aim was to verify whether the data on sensitivity, specificity, NPV, and PPV compare with reference values and creatinine values. (3) Results: We observed levels of dd cfDNA > 1% in six of nine patients with active rejection (ABMR or TCMR) and elevated values (>0.5%) in two other patients in this rejection group. Our results show low values of sensitivity = 50%, specificity = 61.11%, rejection NPV = 64.71%, and rejection PPV = 46.13% of the technique compared to reference values previously published. With respect to creatinine, only for TCRM, we observed better results for dd-cfDNA in these parameters than in creatinine. Also, our data suggest that dd-cfDNA could help to differentiate those patients with dnDSAs that are going to through rejection better than creatinine, specially at 15 d post transplant. In this study, this appears to have no positive predictive value for borderline rejection (BR) or TCMR IA. (4) Conclusions: plasma levels of dd-cfDNA could be considered an additional or alternative biomarker for graft rejection monitoring in early post-kidney transplant up to several months before its clinical presentation, especially for patients with suspected TCMR or ABMR.

Serial monitoring of dd-cfDNA and change from baseline can provide meaningful information beyond absolute thresholds. We describe dd-cfDNA trajectories from the baseline before and after acute rejection (AR) and de novo donor-specific antibodies (dnDSA) detection in kidney transplant recipients (KTRs).

We included KTR from 02/2019 to 03/2022 with serial dd-cfDNA. The primary analysis compared the time-varying change in dd-cfDNA from baseline in KTR first AR on biopsy [AR] to patients with no-AR on biopsy [no-AR].

151 KTR were analyzed (AR = 56 KTR, no-AR = 95 KTRs). In the AR group, dd-cfDNA rose ahead of diagnosis: median rise from baseline was 75% at -3 months, 32% at -2 months, and 325% at -1 month before biopsy. At the time of biopsy, the median rise in dd-cfDNA from baseline was 291% (IQR [interquartile range] 88%-1081%) in AR and 17% (IQR 0%- 194%) in no-AR (p < 0.0001). Following treatment, dd-cfDNA values fell in the AR group with a median change from baseline of 94.7% at +1 month, 10.5% at +2 months, and 0% at +3 months. These trajectories were not observed in the no-AR group. Similarly, there were no significant differences in eGFR (estimated glomerular filtration rate) trajectories between the two groups. The median change from baseline to dnDSA detection was 141% (IQR 112%-574%). In KTRs with persistent rejection, median dd-cfDNA was 0.95% (IQR 0.44-1.8) compared to 0.19% (IQR 0.12-0.31) in subjects with no rejection on follow-up (p < 0.001).

The significant changes from baseline observed before and after AR show how serial monitoring enhances dd-cfDNA utility and allows for earlier identification of evolving injury and monitoring treatment response.

Immune checkpoint inhibitor (ICI) therapy has enabled a paradigm shift in Oncology, with the treatment of metastatic cancer in certain tumor types becoming akin to the treatment of chronic disease. Kidney transplant recipients (KTR) are at increased risk of developing cancer compared to the general population. Historically, KTR were excluded from ICI clinical trials due to concern for allograft rejection and decreased anti-tumor efficacy. While early post-marketing data revealed an allograft rejection risk of 40%-50%, 2 recent small prospective trials have demonstrated lower rates of rejection of 0%-12%, suggesting that maintenance immunosuppression modification prior to ICI start modulates rejection risk. Moreover, objective response rates induced by ICI for the treatment of advanced or metastatic skin cancer, the most common malignancy in KTR, have been comparable to those achieved by immune intact patients. Non-invasive biomarkers may have a role in risk-stratifying patients before starting ICI, and monitoring for rejection, though allograft biopsy is required to confirm diagnosis. This clinically focused review summarizes current knowledge on complications of ICI use in KTR, including their mechanism, risk mitigation strategies, non-invasive biomarker use, approaches to treatment of rejection, and suggestions for future directions in research.

There is a critical need for a rapid and sensitive pathogen detection method for septic patients. This study aimed to investigate the diagnostic efficacy of Digital droplet polymerase chain reaction (ddPCR) in identifying pathogens among suspected septic patients.

We conducted a prospective pilot diagnostic study to clinically validate the multiplex ddPCR panel in diagnosing suspected septic patients. A total of 100 sepsis episodes of 89 patients were included in the study.

In comparison to blood culture, the ddPCR panel exhibited an overall sensitivity of 75.0% and a specificity of 69.7%, ddPCR yielded an additional detection rate of 17.0% for sepsis cases overall, with a turnaround time of 2.5 h. The sensitivity of ddPCR in the empirical antibiotic treatment and the non-empirical antibiotic treatment group were 78.6% versus 80.0% (p > 0.05). Antimicrobial resistance genes were identified in a total of 13 samples. Whenever ddPCR detected the genes beta-lactamase-Klebsiella pneumoniae carbapenemase (blaKPC) or beta-lactamase-New Delhi metallo (blaNDM), these findings corresponded to the cultivation of carbapenem-resistant gram-negative bacteria. Dynamic ddPCR monitoring revealed a consistent alignment between the quantitative ddPCR results and the trends observed in C-reactive protein and procalcitonin levels.

Compared to blood culture, ddPCR exhibited higher sensitivity for pathogen diagnosis in suspected septic patients, and it provided pathogen and drug resistance information in a shorter time. The quantitative results of ddPCR generally aligned with the trends seen in C-reactive protein and procalcitonin levels, indicating that ddPCR can serve as a dynamic monitoring tool for pathogen load in septic patients.

Monitoring the status of grafts and the occurrence of postoperative complications, such as rejection, is crucial for ensuring the success and long-term survival of organ transplants. Traditional histopathological examination, though effective, is an invasive procedure and poses risks of complications, making frequent use impractical. In recent years, graft-derived cell-free DNA (gd-cfDNA) has emerged as a promising non-invasive biomarker. It not only provides early warnings of rejection and other types of graft injury but also offers important information about the effectiveness of immunosuppressive therapy and prognosis. gd-cfDNA shows potential in the monitoring of organ transplants. The early, real-time information on graft injury provided by gd-cfDNA facilitates timely individualized treatment and improves patient outcomes. However, the progress of research on gd-cfDNA varies across different organs. Therefore, this article will comprehensively review the application and findings of gd-cfDNA in monitoring various solid organs, discussing the advantages, limitations, and some future research directions to aid in its clinical application.

The donor-derived cell-free DNA (ddcfDNA) is found in the plasma and urine of kidney transplant recipients and displays notable potential in diagnosing rejection, specifically antibody-mediated rejection (ABMR). Nonetheless, the quantitative methods of ddcfDNA lacking standardization and diverse detection techniques can impact the test outcomes. Besides, both the fraction and absolute values of ddcfDNA have been reported as valuable markers for rejection diagnosis, but they carry distinct meanings and are special in various pathological conditions. Additionally, ddcfDNA is highly sensitive to kidney transplant injury. The various sampling times and combination with other diseases can indeed impact ddcfDNA detection values. This review comprehensively analyses the various factors affecting ddcfDNA detection in kidney transplantation, including the number of SNPs and sequencing depths. Furthermore, different pathological conditions, distinct sampling time points, and the presence of complex heterologous signals can influence ddcfDNA testing results in kidney transplantation. The review also provides insights into ddcfDNA testing on different platforms along with key considerations.

The successes in the field of pediatric kidney transplantation over the past 60 years have been extraordinary. Year over year, there have been significant improvements in short-term graft survival. However, improvements in longer-term outcomes have been much less apparent. One important contributor has been the phenomenon of low-level rejection in the absence of clinical manifestations-so-called subclinical rejection (SCR).

Traditionally, rejection has been diagnosed by changes in clinical parameters, including but not limited to serum creatinine and proteinuria. This review examines the shortcomings of this approach, the effects of SCR on kidney allograft outcome, the benefits and drawbacks of surveillance biopsies to identify SCR, and new urine and blood biomarkers that define the presence or absence of SCR.

Serum creatinine is an unreliable index of SCR. Surveillance biopsies are the method most utilized to detect SCR. However, these have significant drawbacks. New biomarkers show promise. These biomarkers include blood gene expression profiles and donor derived-cell free DNA; urine gene expression profiles; urinary cytokines, chemokines, and metabolomics; and other promising blood and urine tests.

Specific emphasis is placed on studies carried out in pediatric kidney transplant recipients.

ClinicalTrials.gov: NCT03719339.

Currently in the United States, there are more than 250,000 patients with a functioning kidney allograft and over 100,000 waitlisted patients awaiting kidney transplant, with a burgeoning number added to the kidney transplant wait list every year. Although early post-transplant care is delivered at the transplant center, the increasing number of kidney transplant recipients requires general nephrologists to actively participate in the long-term care of these patients. Serum creatinine and proteinuria are imperfect traditional biomarkers of allograft dysfunction and lag behind subclinical allograft injury. This manuscript reviews the various clinically available biomarkers in the field of kidney transplantation for a general nephrologist with a focus on the utility of donor-derived cell-free DNA, as a marker of early allograft injury. Blood gene expression profiling, initially studied in the context of early identification of subclinical rejection, awaits validation in larger multicentric trials. Urinary cellular messenger ribonucleic acid and chemokine CXCL10 hold promising potential for early diagnosis of both subclinical and acute rejection. Torque tenovirus, a ubiquitous DNA virus is emerging as a biomarker of immunosuppression exposure as peripheral blood torque tenovirus copy numbers might mirror the intensity of host immunosuppression. Although high-quality evidence is still being generated, evidence and recommendations are provided to aid the general nephrologist in implementation of novel biomarkers in their clinical practice.

Standard-of-care biomarkers for renal allograft rejection are lagging indicators, signaling existing organ injury. This precludes early intervention, when immunological cascades leading to rejection are most susceptible. Donor-derived cell-free DNA (dd-cfDNA) shows promise as an early indicator of rejection, allowing earlier and possibly more effective treatment. This analysis was designed to assess this promise using real-world dd-cfDNA testing evidence.

This retrospective analysis of the prospective, observational ProActive registry study (NCT04091984) assessed dd-cfDNA and serum creatinine levels before biopsy in 424 patients with ≥1 dd-cfDNA test (n = 1013) in the 6 mo before biopsy.

Of 4667 enrolled patients, 1631 patients had ≥18 mo of follow-up data, of which 424 had a biopsy and were included in this analysis. Twenty-six biopsies showed antibody-mediated rejection (ABMR), 62 showed T cell-mediated rejection, and 336 showed nonrejection; each from a unique patient. dd-cfDNA fractions were significantly elevated 5 mo before ABMR biopsies, and 2 mo before T cell-mediated rejection biopsies, compared with nonrejection biopsies. In contrast, serum creatinine did not discriminate between rejection and nonrejection in advance, or concurrent with biopsy. Among patients with nonrejection biopsies, estimated glomerular filtration rate was significantly lower in cases with ≥2 increased dd-cfDNA results (≥1%), compared with those with 0 or 1 increased dd-cfDNA result.

These data indicate that dd-cfDNA is an early indicator of biopsy-proven rejection, especially ABMR, suggesting a greater role for dd-cfDNA in surveillance to identify patients at high risk of ongoing or future rejection, thus requiring closer monitoring, biopsy, or other management changes.

Antibody-mediated rejection (AMR) is a major cause of graft failure limiting long-term graft survival after kidney transplantation. Current diagnostic strategy to detect AMR is suboptimal and requires further improvement. Previously suggested treatment regimens for AMR could not demonstrate efficacy, however novel therapeutic agents are currently under investigation. Donor-derived cell-free DNA (dd-cfDNA) is a novel non-invasive biomarker for allograft injury, that has been mainly studied in the context of rejection. Its short-half-life in circulation and injury-dependent release are its key advantages that contribute to its superior diagnostic accuracy, compared to traditional biomarkers. Moreover, previous studies showed that dd-cfDNA-release is well-linked to histological and molecular features of AMR, and thus able to reflect real-time injury. Further observations suggest that dd-cfDNA can be used as a suitable screening tool for early detection of AMR in patients with donor-specific-anti-HLA-antibodies (DSA), as well as for monitoring AMR activity after anti-rejection treatment. The weight of evidence suggests that the integration of dd-cfDNA in the graft surveillance of patients with AMR, or those suspicious of AMR (e.g., due to the presence of donor-specific anti-HLA-antibodies) has an added value and might have a positive impact on outcomes in this specific cohort.

Little is known about the urinary virome and how it interacts with the host, particularly in renal transplant diseases. Using metagenomic sequencing, we characterized the urinary virome of 23 kidney transplant recipients longitudinally (11 BKV+ patients and 12 BKV- patients). We applied linear mixed effects models, PERMANOVA, k-means clustering, and MaAsLin2 algorithms to determine virome signatures associated with post-transplant time, BK viremia status, and patient sex. We found that the richness and alpha diversity of urinary virome were significantly different in renal transplant recipients with BKV+ over time in comparison to BKV- (richness P = 0.012, alpha P < 0.0001). Female BKV- patients had significantly higher virome richness than males (P = 0.0063). Virome beta diversity was significantly different between patients by BKV status (P < 0.001). Additionally, we identified underlying interactions between patient sex and BKV status, in terms of virome beta diversity (P = 0.008). BK polyomavirus infections were primarily of subtypes IA, IB1, and IB2. The non-BK dominant samples clustered into six urinary virome community states. BKV- samples had more anelloviruses than BKV+ samples though this difference was not statistically significant. Lastly, we identified specific viruses, associated with BKV+ and time in our samples. Our results indicate that dynamic alterations in the urinary virome over the post-transplant period in kidney transplant recipients can be shaped by BK viremia and patient sex. These findings advance our fundamental understanding of the urinary virome and support a new line of investigation in renal disease and transplantation.

The urinary microbiome is increasingly implicated in renal health and disease. While most research focuses on bacteria communities of the microbiome, factors that influence the urinary virome are not understood. Here, we investigated the urinary virome of 23 adult kidney transplant recipients longitudinally over 14 weeks post-transplant. We show that alterations in the urinary virome are associated with kidney transplant recipients with BK polyomavirus viremia that can lead to BK nephropathy and allograft rejection. By modeling the temporal dynamics post-transplant, we delineated specific profiles of the urinary virome associated with patient sex and urinary community states. These findings reveal fundamental aspects of the urinary virome that can be leveraged to better manage kidney diseases.

Donor-derived cell-free DNA (dd-cfDNA) is an emerging noninvasive biomarker that has the potential to detect allograft injury. The capacity of dd-cfDNA to detect kidney allograft rejection and its added clinical value beyond standard of care patient monitoring is unclear. We enrolled 2,882 kidney allograft recipients from 14 transplantation centers in Europe and the United States in an observational population-based study. The primary analysis included 1,134 patients. Donor-derived cell-free DNA levels strongly correlated with allograft rejection, including antibody-mediated rejection (P < 0.0001), T cell-mediated rejection (P < 0.0001) and mixed rejection (P < 0.0001). In multivariable analysis, circulating dd-cfDNA was significantly associated with allograft rejection (odds ratio 2.275; 95% confidence interval (CI) 1.902-2.739; P < 0.0001) independently of standard of care patient monitoring parameters. The inclusion of dd-cfDNA to a standard of care prediction model showed improved discrimination (area under the curve 0.777 (95% CI 0.741-0.811) to 0.821 (95% CI 0.784-0.852); P = 0.0011) and calibration. These results were confirmed in the external validation cohorts (n = 1,748) including a cohort of African American patients (n = 439). Finally, dd-cfDNA showed high predictive value to detect subclinical rejection in stable patients. Our study provides insights on the potential value of assessing dd-cfDNA, in addition to standard of care monitoring, to improve the detection of allograft rejection. ClinicalTrials.gov registration: NCT05995379 .

During the last few years, cell-free DNA (cfDNA) has emerged as a possible non-invasive biomarker for prediction of complications after lung transplantation. We previously published a proof-of-concept study using a digital droplet polymerase chain reaction (ddPCR)-based method for detection of cfDNA. In the current study, we aimed to further evaluate the potential clinical usefulness of detecting chronic lung allograft dysfunction (CLAD) using three different ddPCR applications measuring and calculating the donor fraction (DF) of cfDNA as well as one method using the absolute amount of donor-derived cfDNA. We analyzed 246 serum samples collected from 26 lung transplant recipients. Nine of the patients had ongoing CLAD at some point during follow-up. All four methods showed statistically significant elevation of the measured variable in the CLAD samples compared to the non-CLAD samples. The results support the use of ddPCR-detected cfDNA as a potential biomarker for prediction of CLAD. These findings need to be validated in a subsequent prospective study.