Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells.

Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6-32% of VP1+ cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68-94% of them losing parts of their DNA and exhibiting membrane damage, appearing as "ghost cells" but still VP1+. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.

The infection process of BK virus in transplant recipients after renal transplantation / 中华泌尿外科杂志

Objective To investigate the characteristics and manifestations of the different stages of BK virus infection in the recipients after renal transplantation.Methods A retrospective survey from January 2015 to December 2016 was done in our hospital.A total 135 recipients were included and accepted BK virus detection in 1,3,6,9,12,15 months respectively after renal transplantation.The prevalence of decoy cell,BK virus DNA load in urine and BK virus DNA load in blood was 56 cases (41.5％),9 cases (43.7％) and 30 cases (22.2％),5 cases of BK vims nephropathy confirmed by pathological biopsy (3.7％).At the same time,51 cases (37.8％) were combined with decoy cells and virus DNA load in urine.Positive decoy cells and negative BK virus DNA load in urine was 5 cases,and Positive BK virus DNA load in urine and negative decoy cells was 8 cases.The recipients were divided into positive group of urine decoy cell,positive group of urinary BK virus DNA load,and positive group of blood BK virus DNA load.Statistical correlation analysis was conducted on the laboratory test results of the 3 groups.Results The positive group of blood BK virus DNA load were detected the high level urine decoy cell count [median of 23/10HPF(2-48/10HPF)] and high level of urinary BK virus DNA load [4.52 × 106 copies/ml (6.51 × 103-7.89 × 109 copies/ml)],significantly higher than the positive group of decoy cells [8/10HPF(2-40/10HPF)] and the positive group of urine BK virus DNA load [4.56 × 105 copies/ml(5.62 × 103-7.89 ×109 copies/ml)] (P ＜ 0.05).The decoy cell count and urine DNA load has a significant linear correlation in viruria recipients,and the urinary BK DNA load and blood BK virus DNA load has the same significant 0.939 and 0.702 in 3 months,0.969 and 0.910 in 6 months,0.782 and 0.766 in 9 months,0.898 and 0.615 in 12 months after renal transplantation.Conclusions There is a linear correlation between decoy cell in urine,viruria and viremia,suggesting that the infection of BK virus in kidney transplant recipients is a continuous process.linear correlation in viremia recipients(P ＜ 0.05).The correlation coefficients at different time points were

BK polyomavirus nephropathy in two kidney transplant patients with distinct diagnostic strategies for BK virus and similar clinical outcomes: two case reports.

BACKGROUND: BK polyomavirus-associated nephropathy is an important cause of post-transplantation renal failure. We present two cases of BK polyomavirus-associated nephropathy who were submitted to contrasting strategies of clinical follow-up to BK polyomavirus reactivation, but progressed to a similar final outcome. CASE PRESENTATION: Case 1 is a 37-year-old white man whose graft had never presented a good glomerular filtration rate function, with episodes of tacrolimus nephrotoxicity, and no urinary monitoring for BK polyomavirus; stage B BK polyomavirus-associated nephropathy was diagnosed by biopsy at 14 months post-transplant. Despite clinical treatment (dosage decrease and immunosuppressive drug change), he progressed to stage C BK polyomavirus-associated nephropathy and loss of graft function 30 months post-transplant. Case 2 is a 49-year-old mulatto man in his second renal transplantation who was submitted to cytological urinary monitoring for BK polyomavirus; he presented early, persistent, and massive urinary decoy cell shedding and concomitant tacrolimus nephrotoxicity. Even with decreasing immunosuppression, he developed BK polyomavirus-associated nephropathy 1-year post-transplant. Loss of graft function occurred 15 months post-transplant. CONCLUSIONS: Cytological urinary monitoring was an efficient strategy for monitoring BK virus reactivation. Decoy cell shedding may be related to BK polyomavirus-associated nephropathy when extensive and persistent. The presence of associated tacrolimus nephrotoxicity may be a confounding factor for the clinical diagnosis of BK polyomavirus-associated nephropathy.

Urinary decoy cell grading and its clinical implications.

BACKGROUND: Examination of urine for decoy cells (DCs) is a useful screening test for polyomavirus (PV) activation. We explored the significance of the amount of DCs in persistent shedding, PV nephropathy and acute rejection. METHODS: A case-controlled study was performed in 88 renal allograft patients who had DCs detected at least once in four or more urine samples. RESULTS: Fifty one patients were classified into the high-grade shedding group (HG) and 37 patients into the low-grade shedding group (LG) according to DC shedding (≥10 or <10 DCs/10 high power field [HPF]). DC shedding of more than three consecutive months was significantly more prevalent in the HG as compared with their LG counterparts (p<0.0001). Urinary DCs were present for more than one year in 29.4% of the HG and 8.1% of the LG. Real-time polymerase chain reaction for PV was higher in both urine (51.4% vs. 11.1%) and plasma (9.1% vs. 0%) of the HG than the LG. The prevalence of PV nephropathy was higher in the HG than the LG (p=0.019). However, there was no significant difference in the prevalence of acute rejection. CONCLUSIONS: Shedding of ≥10 DCs/10 HPF is associated with sustained shedding, polymerase chain reaction positivity and PV nephropathy, but not a predictor of acute rejection.

Urinary Decoy Cell Grading and Its Clinical Implications

BACKGROUND: Examination of urine for decoy cells (DCs) is a useful screening test for polyomavirus (PV) activation. We explored the significance of the amount of DCs in persistent shedding, PV nephropathy and acute rejection. METHODS: A case-controlled study was performed in 88 renal allograft patients who had DCs detected at least once in four or more urine samples. RESULTS: Fifty one patients were classified into the high-grade shedding group (HG) and 37 patients into the low-grade shedding group (LG) according to DC shedding (> or =10 or or =10 DCs/10 HPF is associated with sustained shedding, polymerase chain reaction positivity and PV nephropathy, but not a predictor of acute rejection.

Urinary Decoy Cell Grading and Its Clinical Implications

BACKGROUND: Examination of urine for decoy cells (DCs) is a useful screening test for polyomavirus (PV) activation. We explored the significance of the amount of DCs in persistent shedding, PV nephropathy and acute rejection. METHODS: A case-controlled study was performed in 88 renal allograft patients who had DCs detected at least once in four or more urine samples. RESULTS: Fifty one patients were classified into the high-grade shedding group (HG) and 37 patients into the low-grade shedding group (LG) according to DC shedding (> or =10 or or =10 DCs/10 HPF is associated with sustained shedding, polymerase chain reaction positivity and PV nephropathy, but not a predictor of acute rejection.

BK Virus Associated Late Onset Haemorrhagic Cystitis After Allogeneic Peripheral Blood Stem Cell Transplant.

Haemorrhagic cystitis (HC) after allogeneic stem cell transplant is a clinical disorder with multiple etiologies. There is an inflammation of the bladder mucosa leading to painful haematuria. Early onset HC is mainly attributed to conditioning chemotherapy consisting of cyclophosphamide. Late onset HC is now being related to reactivation of latent polyoma BK virus (BKV). We describe the case of a 37 year old male with acute myeloid leukemia presenting with haematuria after allogeneic stem cell transplant.

BK virus infection in renal transplant recipients: Diagnosis and risk factors analysis / 第二军医大学学报

Objective: To investigate the incidence of BK virus infection in renal transplant recipients and to analyze the risk factors of BKV infection. Methods: The urine samples and peripheral blood samples of 63 renal transplant recipients were collected at 1, 2, 3, 4, 6 and 8 months after transplantation. A real-time fluorescent PCR procedure was used to detect BK virus DNA in the samples and the samples were divided into the following 3 groups according to the detection outcomes: UV+PV+ group (BKV DNA positive in both urine and blood samples), UV- PV- group (BKV DNA positive in urine but negative in blood samples), and UV- PV- group (BKV DNA negative in both urine and blood samples). Urinary sediment smears of patients were checked for decoy cells and the decoy cells were subjected to cytological immunostaining. Renal graft biopsy was performed when a sample was highly suspected of BKVAN by its clinical manifestations. The age, cold ischemia time, hematodialysis duration, immunosuppressive agents, panel reactive antibody, and other clinical parameters were compared between the 3 groups and a Logistic regression was performed to analyze the risk factors of BK virus infection. Results: There were 19 (30.1%) patients in UV+ PV- group, 9(14.3%)in UV-PV+ group,and 35 in UV-PV- group. The median time for the first detection of BK virus was 4 months in UV-PV- group and 3 months in UV+ PV+ group. Decoy cells were detected in 39.7% of the 63 patients and the positive rate of decoy cell immunostaining was 54.3%. One patient showed no BDVAN manifestation in renal biopsy. Logistic regression found that the cold ischemia time was significantly related to the BKV DNA infection (P=0.048; OR=1.151), but not to other parameters (P=0.069). Conclusion: Real-time fluorescent quantitative PCR is a good way for detection of BKV infection after renal transplantation. The peak time for BKV shedding is 3-4 months after transplantation. Cold ischemia time may be one of the risk factors of BKV infection, and immunosupressive regiment may has no obvious influence on BK virus infection.

Immunologic Control for Polyomavirus Infection after Kidney Transplantation / 대한이식학회지

Purpose: The purposes of this study were to compare the relative efficacy of urine decoy cell (UDC) and polymerase chain reaction (PCR) for the polyomavirus infection (PVI), and to search the efficacy of preemptive immunologic control for PVI in earlier stage before irreversible graft injury. Methods: Between Mar. 2003 to Sep. 2005, 265 patients were monitored for the PVI after kidney transplantation. Of the 265 patients, the results of preemptive immunologic modifications were searched among 222 recipients who had the complete data. Results: Of the total 222 patients, 75 patients (33.8%) were positive for UDCs in at least one examination. Overall cumulative incidence of PVI was 32.9%. According to the episode of acute rejection, the one year incidences of PVI were 51.4% and 29.5% in recipients with and without the episode of acute rejection, respectively (P=0.0047). Using decoy cells as a marker of PV viruria, cytology has a sensitivity of 57.1% and negative predictive value of 74.1%. The specificity and positive predictive value for viruria (not viral nephropathy) are 67.2% and 48.8%. False-negative results occurred in samples with suboptimal cellularity, and a low viral load. Three cases of PV nephropathy (PVN) were documented. From January 2001 to December 2002, when we did not prospectively monitor UDCs, 7 cases of PVN were documented among the 116 recipients. Conclusion: The combination test of UDC and PV PCR should be considered as screening test for PVI due to low positive predictive value of UDC. The modulation of net immunosuppression based on UDC values and PV viral loads may reduce the development of PVN.

Prospective Monitoring of Urine Decoy Cell after Kidney Transplantation / 대한이식학회지

PURPOSE: To find the incidence and risk factors for polyomavirus (PV) infection, we monitored urine decoy cell (UDC) after renal transplantation. METHODS: From March 2003 to September 2004, 142 de novo renal recipients were prospectively monitored for UDC at post-transplant 1, 3, 6, 9, 12 months. According to the number of UDC in Cytospin, patients were divided into 3 groups: A (0), B (1~9) and C (> or =10). We decreased immunosuppression (IS) when group C status persisted for more than 1 month or more than 4 UDC was continuously detected for more than 3 months. Differences in demographics and clinical characteristics among the groups were compared. RESULTS: Forty four (31%) patients were found to have positive UDC at least at one examination (30 in group B and 14 in C). The number of patients with positive UDC at postoperative 1, 3, 6, 9, 12 months were 10 (22.7%), 14 (31.8%), 17 (38.6%), 27 (61.3%), 20 (45.4%) respectively with a highest at 9 months. One PV nephropathy was documented by renal biopsy. During the period from January 2001 to December 2002 when we did not prospectively monitor UDC, 7 PV nephropathy cases were documented among 116 recipients. Tacrolimus (Tac) and episode of acute rejection (AR) were significant risk factor for positive UDC (P=0.036, 0.010, respectively). Cumulative incidence of PV infection was significantly different by the use of Tac and episode of AR (P=0.03, 0.013, respectively). CONCLUSION: Use of Tac and episode of AR were risk factor for positive UDC and PV infection. Modulation of IS by the result of UDC monitoring could decrease the development of PV nephropathy after renal transplantation.