Gang Huang

Background: As pivotal effector cells of innate immunity, macrophages may play a critical role in immune regulation and fibrotic remodeling in BK polyomavirus nephropathy (BKPyVN) following kidney transplantation. However, their subpopulation heterogeneity, molecular characteristics, and functional mechanisms remain poorly understood. Methods: We integrated single-cell RNA sequencing (scRNA-seq) data from BKPyVN and stable transplant kidney (STK) biopsies. Pseudotime trajectory, CellChat, and high-dimensional weighted gene co-expression network analysis (hdWGCNA) were used to characterize BKPyVN-associated macrophages (BKPyVN-Mac) and identify related gene modules. Characteristic genes from these modules were used to construct diagnostic models from an integrated renal allograft microarray dataset, and nine machine-learning algorithms were compared. Key genes were validated by RT-qPCR and immunohistochemistry in human biopsies and by time-course transcript and protein analyses in a persistent mouse polyomavirus (MPyV) infection model. Results: BKPyVN-Mac occupied a disease-associated macrophage state and may serve as a central communication hub within the allograft microenvironment. The support vector machine model exhibited the best diagnostic performance, with the highest area under the receiver operating characteristic curve (AUC = 0.923). ATF4 and MCL1 were identified as key feature genes, and their upregulation was confirmed in human BKPyVN biopsies and, through their mouse orthologs Atf4 and Mcl1, in the persistent MPyV infection model. Functional analysis suggested that these genes are associated with inflammation, fibrotic remodeling, and metabolic regulation. Conclusion: This study characterizes the molecular phenotype and regulatory network of a BKPyVN-Mac subpopulation, providing new insights into BKPyVN pathophysiology and identifying candidate biomarkers for clinical diagnosis.

BK polyomavirus (BKPyV) can establish persistent infections in 90% of the healthy adult population, and its reactivation is associated with graft failure in kidney transplant patients. Ischemic injury (IS) increases the risk of viral reactivation, making BKPyV a primary cause of allograft failure. However, the detailed molecular mechanisms of IS that promote BKPyV replication are not well characterized. The aim of the study was to identify the critical pathways and hub genes involved in IS's promotion of polyomavirus replication based on single‐cell RNA‐seq and bulk RNA‐seq data. We found that IS could activate the DNA damage response pathway to promote polyomavirus replication. Additionally, we identified CDK1 as a hub gene according to the conservation of gene expression pattern and key pathways in mice and humans. Subsequently, using siRNA experiments, we demonstrated that CDK1 knockdown inhibits BKPyV replication. Together, the present study found that IS could regulate the expression of CDK1 via the “DNA damage response” pathway to promote polyomavirus replication.

The clinical courses of BK polyomavirus nephropathy (BKPyVN) are variable. This study aimed to evaluate the usefulness of repeat donor‐derived cell‐free DNA (dd‐cfDNA) monitoring in identifying clinical courses. Repeat allograft biopsy and dd‐cfDNA monitoring were performed at 6–12 months post‐intervention or when renal allograft dysfunction occurred. Results demonstrated that recipients with rejection had a higher plasma dd‐cfDNA fraction (2.23% [1.17%, 5.52%]; n = 13) than those with resolved BKPyVN (0.80% [0.66%, 0.91%]; n = 30; p < 0.001) or persistent BKPyVN (0.82% [0.61%, 1.12%]; n = 10; p = 0.002). Compared to baseline at BKPyVN diagnosis, urine dd‐cfDNA concentration decreased in recipients with resolved BKPyVN post‐intervention (3.90 [1.35, 5.80] ng/mL vs. 7.88 [6.00, 15.37] ng/mL; p < 0.001), but not in persistent BKPyVN (9.02 [5.26, 10.35] ng/mL vs. 7.96 [6.43, 10.21] ng/mL; p = 0.463). Changes in urine dd‐cfDNA concentration were positively correlated with changes in the extent of SV40 LTag‐positive staining, while changes in plasma dd‐cfDNA fraction positively correlated with changes in glomerulitis score. A plasma dd‐cfDNA fraction > 1.12% following BKPyVN intervention could identify rejection (AUC = 0.924), and a urine dd‐cfDNA concentration > 5.95 ng/mL could identify persistent BKPyVN (AUC = 0.840). In summary, repeat dd‐cfDNA monitoring integrated with clinical data can serve as an adjunct to identify BKPyVN clinical courses.

Up to 10% of renal allograft failures are caused by BK polyomavirus nephropathy (BKPyVN). However, there is no specific antiviral agent for BKPyVN. The only treatment is to reduce the levels of immunosuppression, which is not always practical and increases the risk of rejection. Since targeting the microenvironment is a promising approach, we performed single-cell RNA sequencing (scRNA-seq) on BKPyVN samples and stable allografts to obtain BKPyVN microenvironmental atlases. Interestingly, we identified a novel subpopulation of proximal tubule cells (annotated as IGKC+ PT) with epithelial-immune dual features that may contribute to the progression of BKPyVN through T-cell exhaustion. Additionally, we determined that the IGKC+ PT subpopulation might serve as a non-invasive diagnostic marker through scRNA-seq of urine samples and co-immunofluorescence staining. These results improve our understanding of the BKPyVN microenvironment and may guide the development of new therapeutic and diagnostic approaches for a wide range of patients. IMPORTANCE: BKPyVN severely threatens kidney transplant recipients. Due to the lack of effective drugs against BK polyomavirus (BKPyV), reducing immunosuppressant therapy is the only treatment. Unfortunately, this approach is not always effective and increases the acute rejection risk. A growing body of research suggests that potential therapeutic targets may be identified by studying the disease microenvironment. However, traditional methods have not explained why the large number of infiltrating T cells in the BKPyVN microenvironment does not effectively clear BKPyV. Newly available large-scale scRNA-seq technology can be used to study gene expression at a single-cell resolution, offering a new way to investigate the BKPyVN microenvironment. By combining scRNA-seq with experimental analysis, we found a novel subpopulation of proximal tubule cells (annotated as IGKC+ PT) with epithelial-immune dual features that may contribute to the progression of BKPyVN through T-cell exhaustion.

This study systematically compared the diagnostic accuracy of seven assays for detecting the Mycobacterium tuberculosis complex, including metagenomic next-generation sequencing (mNGS), droplet digital polymerase chain reaction, real-time quantitative polymerase chain reaction, EasyNAT MTC, GeneXpert MTB/RIF, interferon-gamma release assay (IGRA), and acid‒fast staining (AFS). We try to select appropriate combinations of tuberculosis (TB) detection methods for regions with varying levels of medical resources, based on sensitivity, cost-effectiveness, and operational feasibility. A retrospective analysis was conducted on 141 samples collected from patients with suspected active TB at The First Affiliated Hospital of Sun Yat-sen University between April 2022 and April 2024. Among these samples, there were 100 cases assigned to the case group and 41 cases to the control group, based on the tuberculosis diagnostic criteria. Historical data for Xpert, IGRA, and AFS were collected, and parallel experiments using mNGS, droplet digital PCR (ddPCR), real-time quantitative polymerase chain reaction (RT-qPCR), and EasyNAT were conducted on all samples. Diagnostic performance was evaluated by comparing it with the final clinical diagnoses. Sensitivity, specificity, positive predictive value, negative predictive value, and receiver operating characteristic (ROC) curve analysis were conducted, along with DeLong tests for statistical comparison. Compared with the final clinical diagnosis, mNGS demonstrated the highest sensitivity (100%), followed by IGRA (79.2%), EasyNAT (79.1%), RT-qPCR (78.0%), ddPCR (75.8%), Xpert (75.3%), and AFS (16.7%). The specificity was 100% for both Xpert and AFS, followed by ddPCR (97.6%), RT-qPCR (95.1%), EasyNAT (92.7%), IGRA (72.7%), and mNGS (75.6%). ROC analysis revealed a significantly greater area under the ROC curve for mNGS (0.878) than for ddPCR (0.817, P = 0.031). DeLong tests revealed statistically significant differences in diagnostic performance between mNGS and ddPCR ( P < 0.05) and between IGRA and AFS ( P < 0.01). mNGS uniquely identified the pathogens involved in co-infection and quantified pathogen-specific sequencing reads. Through a comprehensive evaluation of the diagnostic efficacy, cost-effectiveness, and timeliness of tuberculosis detection methods, we propose corresponding combinations of TB testing approaches for regions with different healthcare resources. For undeveloped regions with limited resources, a combination of AFS +EasyNAT + chest X-ray is recommended. Primary care facilities may additionally employ IGRA + RT-qPCR. Intermediate-level hospitals can incorporate Xpert MTB/RIF for drug resistance testing, while tertiary hospitals or specialized centers should, on the basis of these fundamental tests, utilize mNGS for diagnosis and ddPCR for therapeutic monitoring in patients with complex mixed infections. IMPORTANCE: This study is the first to comprehensively evaluate the diagnostic efficacy, cost-effectiveness, and timeliness of seven TB detection methods in a single-center cohort. Our findings provide actionable solutions for optimizing TB diagnostics in diverse healthcare ecosystems, aligning with the WHO’s End TB Strategy to ensure equitable access to rapid diagnostics.

BK polyomavirus (BKPyV) can establish persistent infections in 90% of the healthy adult population, and its reactivation is associated with graft failure in kidney transplant patients. Ischemic injury (IS) increases the risk of viral reactivation, making BKPyV a primary cause of allograft failure. However, the detailed molecular mechanisms of IS that promote BKPyV replication are not well characterized. The aim of the study was to identify the critical pathways and hub genes involved in IS’s promotion of polyomavirus replication based on single-cell RNA-seq and bulk RNA-seq data. We found that IS could induce reactive oxygen species (ROS) generation and then activate the DNA damage response pathway, which could promote polyomavirus replication. Additionally, we identified CDK1 as a hub gene according to the conservation of gene expression pattern and key pathways in mice and humans. Subsequently, we screened the potential drug repurposing targeted CDK1 by docking-based virtual screening to help improve the outcome of BKPyV-associated nephropathy (BKPyVN). Together, the present study found that IS could induce ROS to regulate the expression of CDK1 via the ‘DNA damage response’ pathway to promote polyomavirus replication. Keywords: BKPyV; ischemic injury; single-cell sequencing; pathway enrichment

The objective of this study was to establish a more sensitive and specific diagnostic method for detecting plasma BK polyomavirus (BKPyV) DNA load in patients after renal transplantation using droplet digital polymerase chain reaction (ddPCR) and to validate the methodology. The linear range, lower limit of detection, accuracy, precision, and specificity of the detection system were evaluated by using the WHO BKPyV standard (7.2 log ₁₀ IU/mL) as a reference, in accordance with the relevant documents of the Clinical and Laboratory Standards Institute. Plasma samples were collected from 74 renal transplantation patients with urinary BKPyV-DNA levels exceeding 7 log ₁₀ copies/mL. Quantitative PCR (qPCR) and ddPCR were performed, and their diagnostic efficacy for BKPyV-DNA in the diagnosis of BK polyomavirus-associated nephropathy was evaluated using a receiver operating characteristic (ROC) curve. The coefficients of variation for the repeated detection of BKPyV standard DNA were 2.55 and 4.71 at concentrations of 6.2 and 3.2 log ₁₀ IU/mL, respectively. The linear range was 2.2–6.2 log ₁₀ IU/mL, and the lowest detection limit was 100 IU/mL. By utilizing histopathological examination of renal biopsy as the gold standard for BKPyV diagnosis, the area under the ROC curve of 74 post-transplantation plasma samples detected by the ddPCR system was found to be 0.875 (95% CI: 0.797–0.953, P < 0.01). The optimal threshold was 512.86 copies/mL, with a sensitivity of 90.0% and a specificity of 67.6%. In comparison, the area under the ROC curve for qPCR was 0.668 (95% CI: 0.583–0.752, P < 0.01), with an optimal threshold of 11,481.54 copies/mL, a sensitivity of 35.0%, and a specificity of 100.0%. Pairwise comparison (Delong test) of the ROC curves of the two systems showed a significant difference in the area under the curve, with a difference of 0.207 and a P -value <0.01. The BKPyV nucleic acid detection system, based on ddPCR, is appropriate for the regular monitoring of the BK polyomavirus, specifically in plasma samples containing low viral DNA loads while it provides the benefits of both absolute quantification and high sensitivity. IMPORTANCE: It was previously believed that droplet digital polymerase chain reaction had limitations, including high cost, limited throughput, and cumbersome operation, which hindered its widespread application in clinical practice. However, the current fully automated digital PCR platform, combined with streamlined operations, can detect 96 samples at once, and the entire process can be completed within an hour, laying a solid foundation for its extensive use.

Background: The purpose of the study was to evaluate renal quality and predict posttransplant graft function using ex vivo sound touch elastography (STE). Methods: In this prospective study, 106 donor kidneys underwent ex vivo STE examination and biopsy from March 2022 to August 2023. The mean stiffness of the superficial cortex (STE_sc), deep cortex (STE_dc), and medulla (STE_me) was obtained and synthesized into one index (STE) through the factor analysis method. Additionally, 100 recipients were followed up for 6 months. A random forest algorithm was employed to explore significant predictive factors associated with the Remuzzi score and allograft function. The performance of parameters was evaluated by using the area under the receiver operating characteristic curve (AUC). Results: STE had AUC values of 0.803 for diagnosing low Remuzzi and 0.943 for diagnosing high Remuzzi. Meanwhile, STE had an AUC of 0.723 for diagnosing moderate to severe ATI. Random forest algorithm identified STE and Remuzzi score as significant predictors for 6-month renal function. The AUC for STE in predicting postoperative allograft function was 0.717, which was comparable with that of the Remuzzi score (AUC = 0.756). Nevertheless, the specificity of STE was significantly higher than that of Remuzzi (0.913 vs 0.652, p < 0.001). Given these promising results, donor kidneys can be transplanted directly without the need for biopsy when STE ≤ 11.741. Conclusions: The assessment of kidney quality using ex vivo STE demonstrated significant predictive value for the Remuzzi score and allograft function, which could help avoid unnecessary biopsy. Critical relevance statement: Pre-transplant kidney quality measured with ex vivo STE can be used to assess donor kidney quality and avoid unnecessary biopsy. Key Points: STE has significant value for diagnosing low Remuzzi and high Remuzzi scores. STE achieved good performance in predicting posttransplant allograft function. Assessment of kidney quality using ex vivo STE could avoid unnecessary biopsies. Graphical

Background.: Co-infection of JC polyomavirus (JCPyV) and BK polyomavirus (BKPyV) is uncommon in kidney transplant recipients, and the prognosis is unclear. This study aimed to investigate the effect of concurrent JCPyV-DNAemia on graft outcomes in BKPyV-infected kidney transplant recipients with polyomavirus-associated nephropathy (PyVAN). Methods.: A total of 140 kidney transplant recipients with BKPyV replication and PyVAN, 122 without concurrent JCPyV-DNAemia and 18 with JCPyV-DNAemia were included in the analysis. Least absolute shrinkage and selection operator regression analysis and multivariate Cox regression analysis were used to identify prognostic factors for graft survival. A nomogram for predicting graft survival was created and evaluated. Results.: The median tubulitis score in the JCPyV-DNAemia-positive group was higher than in JCPyV-DNAemia-negative group (P = 0.048). At last follow-up, the graft loss rate in the JCPyV-DNAemia-positive group was higher than in the JCPyV-DNAemia-negative group (50% versus 25.4%; P = 0.031). Kaplan–Meier analysis showed that the graft survival rate in the JCPyV-DNAemia-positive group was lower than in the JCPyV-DNAemia-negative group (P = 0.003). Least absolute shrinkage and selection operator regression and multivariate Cox regression analysis demonstrated that concurrent JCPyV-DNAemia was an independent risk factor for graft survival (hazard ratio = 4.808; 95% confidence interval: 2.096-11.03; P < 0.001). The nomogram displayed favorable discrimination (C-index = 0.839), concordance, and clinical applicability in predicting graft survival. Conclusions.: Concurrent JCPyV-DNAemia is associated with a worse graft outcome in BKPyV-infected kidney transplant recipients with PyVAN.