Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Introduction: Polyomavirus-associated nephropathy (BKPyVAN) is a common complication in kidney transplant recipients. The histological changes in the context of BKPyVAN and their association with the viral load and outcomes are still being investigated. Methods: This retrospective study involved 100 adult patients transplanted between 2000 and 2021, with available archived biopsy slides, aiming to analyze associations between viral load clearance in the blood (reduction in BKPyVAN-DNAemia below detection level) and histological features in biopsy-proven BKPyVAN. A kidney pathologist blinded to the clinical data reassessed the BANFF 2019 lesion scores in the BKPyVAN index biopsy. The primary endpoint was viral clearance three months after the diagnosis. Results: The presence of tubulointerstitial inflammation, peritubular capillaritis, and higher PVN Class at the diagnosis was linked to a reduced likelihood of viral clearance three months later (interstitial inflammation OR = 0.2, 95% CI [0.07–0.55], tubulitis OR = 0.39, 95% CI [0.21–0.73], peritubular capillaritis OR = 0.25, 95% CI [0.08–0.82], PVN Score OR = 0.1, 95% CI [0.03–0.4]), independently of other covariates. Combining the four lesions using the ROC analysis enhanced their capability to predict persistent BK viremia after 3 months with an AUC of 0.94. Conclusions: The presence of interstitial inflammation, tubulitis, and peritubular capillaritis, as well as the higher PVN Score, was associated with an up to 90% lower likelihood of viral load clearance three months post-diagnosis. These findings underscore the importance of histological evaluation as a surrogate of subsequent viral clearance and offer valuable insights for the management of BKPyVAN.

Peritubular capillaritis (ptc) is a hallmark lesion of antibody-mediated rejection (AMR), but the grading of its extent is historically based on arbitrary defined cut-offs. Molecular AMR diagnosis via intragraft gene expression measurements may provide evidence to challenge established ptc categories. We retrospectively included 38 renal allograft biopsies from clinical routine, performed because of suspicion of AMR. Biopsies were re-assessed by an experienced nephropathologist and intragraft gene expression was measured using the NanoString nCounter^® platform. Ptc categories were correlated with AMR gene expression to identify a ptc extent cut-off with optimal prediction of molecular diagnosis of AMR [gene expression levels above first quartile (AMR^Q>1)]. Finally, an independent validation cohort (n = 25, Erasmus MC, Rotterdam, The Netherlands) was included to reproduce the results. Re-assessment of biopsies revealed AMR in 26/68.4%, mixed rejection in 5/13.2%, and T-cell-mediated rejection in 3/7.9%. Biopsies with diffuse ptc had significantly higher AMR gene expression compared to biopsies with focal ptc and biopsies with no ptc (64.0/53.3–84.0 vs. 31.5/27.0–49.5, p = 0.023 and 27.0/14.3–31.8, p = 0.003, median/IQR). Sensitivity analysis revealed that a ≥35% ptc cut-off resulted in higher AUCs for predicting AMR^Q>1 compared to ptc^50% (AUC 0.78, 95% CI: 0.63–0.93, p = 0.009 versus AUC: 0.74, CI: 0.56–0.90, p = 0.03). In the validation cohort, only the ptc^35–, but not the ptc^50%, cut-off significantly predicted AMR^Q>1 (AUC 0.75, 95% CI: 0.54–0.96 p = 0.04 vs. AUC 0.69, CI: 0.46–0.93, p = 0.13). Using intragraft gene expression measurement, we identified a new ptc extent threshold with better prediction of molecular AMR. The newly proposed cut-off of ≥35% could potentially improve diagnostic evaluation and prognostication in cases with suspected or diagnosed AMR.

Background: In hemodialysis patients, precise hemoglobin (Hb) monitoring is essential for anemia management. Point-of-care blood gas analyzers (BGAs), such as the ABL800 Flex, offer rapid Hb determinations, but their accordance and comparability with central laboratory measurements remains to be assessed in the hemodialysis setting. Methods: We performed a retrospective analysis (April 2017–February 2024) of 10,802 paired Hb measurements from 291 hemodialysis patients. BGA and laboratory values within 90 min were compared using paired t-tests, non-inferiority testing (margin 0.5 g/dL), a Bland–Altman analysis, and linear regression. Results: The mean ± standard deviation Hb (g/dL) values were 10.14 ± 1.64 (BGA) versus 9.90 ± 1.55 (laboratory). The overall mean difference (BGA—laboratory) was 0.24 ± 0.49 g/dL (95% CI: 0.23–0.25), demonstrating non-inferiority (p < 0.0001). Measurement delay correlated with increasing analysis discrepancies (mean difference in g/dL: 0.22 at <30 min vs. 0.27 at 60–90 min; p < 0.001). We derived the equation of laboratory Hb = 0.90 × BGA Hb + 0.72; a simplified correction (BGA−0.3 g/dL) produced a mean absolute error (MAE) of 0.30 g/dL and root mean square error (RMSE) of 0.50 g/dL, and patient-level 10-fold cross-validation yielded MAE ≈ 0.30 and RMSE ≈ 0.49 g/dL. The Bland–Altman analysis confirmed a small systematic bias of 0.24 g/dL with 95% limits of agreement ranging from −0.73 to +1.21 g/dL. Conclusions: BGA Hb measurements via the ABL800 Flex are non-inferior to central laboratory values across clinical scenarios, with minimal bias. After regression correction, the estimated total error was ≈0.78 g/dL. If hemodialysis centers accept this level of total error and apply confirmatory testing near decision points, BGA could be used to guide anemia management.