Histological Patterns of Polyomavirus Nephropathy: Correlation with Graft Outcome and Viral Load

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1 American Journal of Transplantation 2004; 4: Blackwell Munksgaard Copyright C Blackwell Munksgaard 2004 doi: /j x Histological Patterns of Polyomavirus Nephropathy: Correlation with Graft Outcome and Viral Load Cinthia B. Drachenberg a, John C. Papadimitriou a,, Hans H. Hirsch d, Ravinder Wali b, Clinton Crowder a, Joseph Nogueira b, Charles B. Cangro b, Susan Mendley c,ayesa Mian c and Emilio Ramos b Departments of a Pathology, b Medicine and c Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, USA, d Department of Infectious Diseases, University of Basel, Basel, Switzerland Corresponding author: John C. Papadimitriou, jpapa001@umaryland.edu Polyomavirus-associated nephropathy (PVAN) is a significant cause of allograft loss. The diagnosis requires allograft biopsy, but the impact of the histological features on diagnosis and outcome has not been described. We studied the distribution and extent of PVAN in 90 patients. Viral cytopathic changes, tubular atrophy/fibrosis and inflammation were semiquantitatively scored and classified into histological patterns. The histological findings were correlated with viruria, viremia and graft survival. PVAN lesions were random, (multi-)focal and affected both cortex and medulla. Areas with PVAN coexisted with areas of unaffected parenchyma. In 36.5% (15/41) of biopsies with multiple tissue cores, discordant findings with PVAN-positive and -negative cores were observed. However, all patients with PVAN had decoy cells in urine as well as significant viruria and viremia (mean of and viral copies, respectively). Biopsies showing lesser degrees of renal scarring at the time of diagnosis were associated with, more likely, resolution of the infection, in response to decrease of immunosuppression (p = 0.001). More advanced tubulointerstitial atrophy, active inflammation and higher creatinine level at diagnosis correlated with worse graft outcome (p = , and ). Due to the focal nature of PVAN, correlation of biopsy results with viruria and viremia are required for diagnosis. Key words: BK virus, CD20, CD3, CD68, decoy cells, focal, Ki67, renal biopsy, urine cytology, viremia, viruria Received 1 April 2004, revised and accepted for publication 30 June 2004 Introduction The re-emergence of polyomavirus allograft nephropathy (PVAN) has been amply documented in many centers (1 15). The specific factors that influence outcome are not known, but graft loss has been attributed to a late diagnosis of PVAN in kidneys already showing irreversible tissue damage (scarring) (7). Several studies have shown that early modification of immunosuppression may favor stable graft function and viral clearance in a larger proportion of patients (14 18). In theory, early identification of patients at a time point prior to the occurrence of significant parenchymal damage would allow for a meaningful therapeutic intervention. PVAN is diagnosed by the histological demonstration of polyomavirus cytopathic changes in renal tubular epithelium (5,10,12,18 20). Performance of a renal biopsy is usually prompted by graft dysfunction, but more recently persistent PV viruria or viremia were suggested as appropriate indications (8,14,16). The histological diagnosis of PVAN is complicated by the wide spectrum of pathological changes ranging from limited to extensive parenchymal disease and the presence of inflammatory infiltrates similar to those of acute rejection (3,4,8,9,18). Given the importance of allograft biopsy for early diagnosis and intervention in PVAN, understanding of the histological features and consideration of potential tissue sampling errors are of paramount importance (21,22). In the current study, we sought to evaluate the morphological evolution of PVAN by the systematic evaluation of sequential histological samples from 90 patients, obtained over a period of 6 years. Specifically, we evaluated: (a) extent of parenchymal involvement at diagnosis and its impact on tissue sampling issues; (b) correlation between the histological pattern of PVAN in the initial biopsy and graft function, viruria, viremia and ultimate graft outcome and (c) histological features that could be useful for the diagnosis of PVAN and for the differentiation from rejection. Methods Between June 1997 and July 2003, the histological diagnosis of PVAN was made in 121 renal transplant patients. Patients included in this study were those who had complete medical records, continuous clinical care at 2082

2 Histological Patterns of Polyomavirus Nephropathy our institution and a follow-up of at least 12 months post-transplantation. Thirty-one patients were excluded due to incomplete records (n = 6), clinical follow-up at another institution (n = 13) or insufficient follow-up (n = 12), leaving 90 patients for further study (mean age of 51 years, range 8 76, median 52). All renal biopsies available before (n = 58) and after (n = 100) the diagnosis of PVAN were evaluated and the main pathological findings and diagnoses were correlated with the first biopsy revealing PVAN (index biopsy). The biopsy results were correlated with urine cytology studies and, when available, with quantitative viremia and viruria performed on samples collected within 24 h of the biopsy. Quantitation of viremia and viruria were not performed on samples obtained before July Methods used for the cytological and quantitative molecular studies have been previously described (9,14). The morphological findings were correlated with the values of serum creatinine at the time of the biopsy and at the end of the study period (mean 36.5 months post-transplantation, range months). Ninety-eight percent of all the biopsies were performed because of acute increase in serum creatinine levels (20% from baseline). The remaining were intra-operative biopsies done at the time of laparotomy for various indications. Of the 90 patients, 20 had only one biopsy. The remaining 70 patients had 2 7 biopsies (mean 3.21, median 3). The diagnosis of PVAN was made at a mean of 14.4 months after transplantation (range 3 52 months, median 11). Three nephrectomy specimens from patients with ongoing polyomavirus replication were also available for evaluation. PVAN was defined by the typical viral cytopathic changes in the epithelium of tubules, glomeruli, and/or collecting ducts, which were confirmed by positive immunohistochemical nuclear staining for SV40 large T antigen as previously described (10). Viral cytopathic changes, interstitial inflammation and tubular atrophy/fibrosis were semi-quantitatively assessed and classified as follows: Pattern a consisted of viral cytopathic changes with no or negligible inflammation or tubular atrophy; Pattern b consisted of viral cytopathic changes with significant interstitial inflammation and atrophy of renal tubules (b1: features of Pattern b in less than 25% of the core and lack of involvement of the remaining tissue; b2: features of Pattern b in 25 50% of the core with residual areas of uninvolved tissue; b3: features of Pattern b involving the majority of the core >50%); Pattern c consisted of rare viral cytopathic changes in atrophic tubules, in a background of extensive tubular atrophy/fibrosis and chronic inflammation (end-stage PVAN). For statistical purposes, numerical values of 1 5 (histological score) were assigned to the histological Patterns a, b1, b2, b3 and c based on the premise that these represented increasing severity of parenchymal disease. Extent of viral cytopathic changes was recorded independently in the cortex and the medulla, and expressed as percentage of the tissue core affected. In addition, SV40-positive nuclei were quantitated in non-atrophic, non-inflamed versus atrophic and inflamed parenchyma at a magnification of 200. The degrees of lymphocytic interstitial and tubular inflammation (tubulitis) and grade of acute rejection were classified following the Banff 97 scheme (21). Unusual features of the infiltrates (i.e. large number of plasma cells, neutrophils or eosinophils) and the presence of interstitial edema and hemorrhage were also noted. Percentage of T cells, B cells and macrophages were determined on immunohistochemical stains for CD20, CD3 and CD68 in selected biopsies with significant mononuclear interstitial infiltrates (Banff i2, i3). The main diagnoses in these biopsies were PVAN (n = 15), acute rejection (n = 15) and chronic rejection/graft sclerosis (n = 15). The same biopsies were also stained for the Ki67 proliferation marker. Demographic information and clinical data including immunosuppression regimens and protocols for reduction in immunosuppression used in this group of patients have been previously described (7,8). Maintenance immunosuppression consisted of tacrolimus, MMF (mycophenolate mofetil) and prednisone in 85 patients. Cyclosporine A, MMF and prednisone was used in four patients, and azathioprine, tacrolimus and prednisone in the remaining patient. Immunosuppression was reduced in all patients: within 6 weeks of the histological diagnosis of PVAN in 82 patients and within 10 weeks of diagnosis in 8 patients. Initial immunosuppression reduction consisted of a decrease in the target level of tacrolimus from ng/ml to 6 8 ng/ml and cyclosporine A from mg/ml to mg/ml. Tacrolimus was changed to low-dose cyclosporine A in 11 patients. Sirolimus was used instead of the calcineurin inhibitor in 11 patients. Dose of MMF was reduced by 50%. Mycophenolate mofetil was eventually discontinued in 58 patients. All patients continued to receive prednisone. In addition, cidofovir was used in two patients at a dose of mg/kg. The patients were treated for 5 6 months starting at 4 and 15 months after the diagnosis of PVAN, respectively. Clearance of viruria and viremia occurred 8 months after cidofovir therapy with stabilization of renal function in the first patient. The second patient had slow and progressive deterioration of graft function and never achieved clearance of viremia and viruria. Followbiopsies were only available in the second patient (see Table 1, group 4). The study was approved by the University of Maryland Institutional Review Board. Results Histological pattern of PVAN at diagnosis (index biopsies), allograft function and outcome The histological pattern of PVAN in the index biopsies corresponded to patterns a, b(1 3) and c in 31, 56 and 3 patients, respectively (Figure 1). The pattern of PVAN in the index biopsy correlated with the ultimate fate of the graft, and patients that lost graft function had more severe disease in the index biopsy (p = ) (Figure 1). Graft outcome also correlated with the serum creatinine level at the time of the index biopsy. Patients that lost graft function had a mean serum creatinine of 3 mg/dl at the time of the index biopsy in comparison to a mean serum creatinine of 2.4 mg/dl in patients that maintained function (p = ). A lower histological score in the index biopsy correlated with resolution of the viral infection (negative biopsy, viremia and decoy cells in urine) at the time of follow-up in comparison to patients that continued to have PVAN in biopsy, viremia and decoy cells in urine (p = 0.001). Discordance of PVAN diagnosis in simultaneously procured biopsy tissue cores Two or more tissue cores were available in 41 biopsies with PVAN (four cores: n = 2; three cores: n = 7; two cores: n = 32). In 15 biopsies (36.5%), PVAN was absent in at least one of the tissue cores by light microscopy and immunohistochemistry (Figure 2A,B). Concordance of histological findings in simultaneously obtained tissue cores was seen in the remaining 26 biopsies (63.5%). Biopsies with concordant PVAN findings had significantly more extensive involvement of the tissue cores than biopsies with discordant findings (mean of 52.8% vs. 18%, p = 0.02). In the tissue cores that lacked polyomavirus cytopathic changes, focal, mononuclear interstitial inflammation predominantly consisting of lymphocytes and plasma cells were seen in 11 of the 15 cases. Also, in 12 of the 15 cases the negative cores showed evidence of tubular injury consisting of mild American Journal of Transplantation 2004; 4:

3 Drachenberg et al. HISTOLOGICAL PATTERN IN INDEX BIOPSY CORRELATION WITH GRAFT LOSS Number of Cases Percentage graft loss 10 0 a b1 b2 b3 c Number of patients % graft loss Figure 1: Histological pattern in index biopsy/correlation with graft loss. Pattern a: focal viral cytopathic effect with negligible inflammation or tubular atrophy interstitial fibrosis. Pattern b: viral cytopathic changes with significant interstitial inflammation and atrophy of renal tubules/fibrosis (b1: <25% of the biopsy core, b2: 25 50% of the biopsy core, b3: >50%, majority of the core). Pattern c: viral cytopathic changes in atrophic tubules, in a background of extensive tubular atrophy/fibrosis and chronic inflammation (end-stage PVAN). tubular dilatation, apical tubular cell blebbing/vacuolization and few microcalcifications. Distribution of PVAN in index biopsies Of the 90 index biopsies, 53 contained areas representing both cortex and medulla. In 37 (69.8%) biopsies, both areas were involved by PVAN. Viral changes limited to the cortex or to the medulla were seen in 11 (20.7%) and 5 (9.43%) biopsies, respectively. In 13 patients (14.4%), sequential biopsies obtained over a period of 2 12 weeks showed change from cortical to medullary involvement, or vice versa. In five patients, urothelial lining was also present in the biopsy cores and showed polyomavirus cytopathic changes in all cases regardless of solely medullary or solely cortical involvement. The percentage of tissue core with viral cytopathic changes in the index biopsy ranged from 2% to 100% (mean 33%, median 45%) and did not correlate with graft survival (p = 0.62) or with post-transplant time to PVAN diagnosis (p = 0.58). There was no correlation of the time post-transplantation with cortical or medullary or simultaneous involvement. The random multi-focal distribution of PVAN was also evident in the three nephrectomies. These showed residual areas of uninfected parenchyma representing up to 40% of the cut surface. Active viral replication, as highlighted by the SV40 immunohistochemical stain, varied markedly from area to area and the number of infected cells was inversely proportional to the tubular atrophy and inflammation (Figure 3A,B). Nonatrophic/non-inflamed infected areas had a mean of 13.6 SV40-positive tubular nuclei per medium power histological field (200 ) in comparison to 6.1 in atrophic/inflamed areas (p = ). The periphery of a typical PVAN lesion showed active viral replication, whereas the central areas contained scarred parenchyma with minimal viral replication. Characteristics and significance of inflammation in the index biopsy In addition to the viral cytopathic changes, interstitial inflammation with moderate-to-marked tubulitis (>4 lymphocytes per tubular cross-section) was seen in 46.6% of index biopsies. The tubulitis was irregularly distributed and involved 5 30% of tubules in the affected areas. When present, tubulitis was found in tubules with and without viral cytopathic changes. Moderate-to-marked tubulitis in the index biopsy (Banff t2, t3), correlated with ultimate graft loss (p = ) and was seen in 78% of index biopsies in patients that lost graft function in comparison to 24% in patients that still 2084 American Journal of Transplantation 2004; 4:

4 Histological Patterns of Polyomavirus Nephropathy Figure 2: (A) Area of essentially normal renal cortex. There is no evidence of PVAN, inflammation or atrophy in contrast to Figure 1B. (B) Same specimen as Figure 1A. Area of end-stage PVAN. There is extensive cortical atrophy with scattered mononuclear inflammatory infiltrates. had graft function after a mean follow-up of 34 months (range months). The type of inflammation in PVAN was almost purely mononuclear, consisting of lymphocytes, plasma cells and macrophages (Figure 4A). Focal or diffuse interstitial aggregates of plasma cells between tubules with and without viral changes were seen in 51% of index biopsies. CD20 stains identified a mean of 5% of B cells (range 1 15%) in biopsies with acute rejection. This was in contrast to biopsies with PVAN and biopsies with chronic rejection/graft sclerosis, which showed a much larger proportion of B cells (mean 32%, range 5 75% versus mean 2.5%, range 10 60%, p = 0.02). In biopsies with PVAN pattern b, and in biopsies with chronic rejection, the B cells tended to form aggregates in a background of T cells. Macrophages as determined by the CD68 stain represented a minor proportion of the mononuclear infiltrates with a similar pattern of distribution in all three biopsy groups (mean 2 3%, range 1 5% of infiltrates). Macrophages accumulated predominantly in areas showing tubular atrophy and interstitial fibrosis. They also clustered around, and permeated tubules showing tubular cell injury, independently of the presence of viral cytopathic changes. Clusters of neutrophils and or significant interstitial edema were seen in less than 10% of biopsies, in association with severe virally induced tubular injury. Significant interstitial hemorrhage and significant numbers of eosinophils were not seen in any of the index biopsies. Stains for Ki67 failed to identify a distinctive pattern of epithelial proliferation in biopsies with PVAN in American Journal of Transplantation 2004; 4:

5 Drachenberg et al. Figure 2: Continued. comparison to biopsies showing acute or chronic rejection. The tubular cell proliferation fraction ranged from <1% to 7%. Proliferation of inflammatory cells (lymphoid cells) was prominent in some cases of acute rejection (up to 8% in lymphoid aggregates), but this finding was not consistent. Significance of interstitial inflammation/tubulitis and persistence of viral changes in follow-up biopsies Of the 90 patients 56 had one to four follow-up biopsies (total n = 100) performed 1 week to 60 months after the diagnosis of PVAN (mean 12.4 months, median 9 months). Based on the presence or absence of PVAN and active tubulitis (Banff t2, t3) in the follow-up biopsies, the patients fell into one of four groups as summarized in Table 1. When available, surrogate markers of PVAN (viremia, viruria and urine cytology) were correlated with the biopsy findings. Group 1 Persistence of PVAN with active tubulointerstitial inflammation and prominent tubulitis: This pattern was seen in 21 patients (37.5%), all of whom lost graft function 4 35 months after the diagnosis of PVAN (mean 15.7 months, median 12). Concurrent urine cytologies were positive for decoy cells in all 21 patients at the time of the follow-up biopsies. Measurements of viremia available in 17 of these patients showed evidence of active disease at the time of the biopsy (range BK viral copies/ml of plasma). Group 2 No evidence of PVAN by light microscopy and immunohistochemistry, but active tubulointerstitial inflammation with prominent tubulitis: These 2086 American Journal of Transplantation 2004; 4:

6 Histological Patterns of Polyomavirus Nephropathy Figure 3: (A) Area of PVAN with active viral replication and no significant inflammation or tubular atrophy (pattern a). SV40 stain is positive in scattered infected tubular cell nuclei and in some casts. (B) Area of PVAN with extensive tubular atrophy and chronic inflammation (pattern c). SV40 stain is positive in rare nuclei of atrophic tubular cells. patients had negative urine cytology and negative viremia at the time of the biopsy, therefore, the histological findings were considered most consistent with acute cellular rejection. This pattern was seen in eight patients (14.8%), seven of whom continued to have graft function after a mean of 23.8 months after the index biopsy (range months, median 28, mean creatinine 2.48 mg/dl). Group 3 Persistent viral cytopathic changes with minimal or inactive mononuclear inflammation (no tubulitis): Mild, bland fibrosis (Banff c1-2) similar to that seen in non-specific graft sclerosis/chronic allograft nephropathy was present. Sixteen patients (17.7%) presented with these features, six of which lost graft function 2 23 months after the diagnosis of PVAN (mean 11.1 months, median 10 months). The remaining 10 patients continued to have graft function for a mean of 34 months after the diagnosis of PVAN (range months, median 40.5 months) and had a mean creatinine of 3.1 mg/dl (range ). Concurrent urine cytologies showed decoy cells and concurrent viremia (available in 9 of 10 patients) ranged from 3000 to BK copies/ml plasma. Group 4 No evidence of PVAN by light microscopy and immunohistochemistry, no tubulitis: These biopsies showed interstitial fibrosis and tubular atrophy, often in stripped pattern and patchy interstitial inflammation (Banff c1-2), without active inflammation (Figure 4B). These biopsies showed features indistinguishable from non-specific graft sclerosis or chronic allograft nephropathy. Eleven patients (19.6%) had follow-up biopsies showing these features. Concurrent urine cytologies and quantitation of viremia in this group were variable. Of the 11 patients, 7 had negative urine cytologies and absence of viremia. The remaining four patients had urine samples positive for decoy cells and mean viremia of BK viral copies/ml (range ). The cytology findings and positive viremia were consistent with persistently active PVAN despite the negative biopsy. Of the 11 patients in this group, 2 lost graft function 10 and 23 months after the diagnosis of PVAN. These patients had American Journal of Transplantation 2004; 4:

7 Drachenberg et al. Figure 3: Continued. increasing viremia ( and BK virus copies/ml, at the time of the biopsy, respectively) and persistence of decoy cells in urine. The remaining nine patients continued to have graft function for a mean of 34.2 months (range months, median 37 months, mean creatinine 2.65 mg/dl with a range of mg/dl). The patients in group 1 (persistent PVAN and active tubulitis) had significantly worse outcome (graft loss) in comparison to the three other groups (p = ). Patients in group 1 had a more advanced histological pattern (histological score) in the index biopsy in comparison to patients in groups 2, 3 and 4 (p = , 0.03 and 0.001, respectively) (see Table 1). The post-transplant time at diagnosis and the follow-up time were similar in all groups. The level of renal function and the graft loss were not significantly different in groups 2, 3 and 4 at the end of the follow-up period. The protocols for immunosuppression reduction were not significantly different in groups 1, 2, 3 and 4. In patients that had overcome the infection (five patients in group 4) and had maintained adequate allograft function for more than 12 months, follow-up biopsies showed features overlapping with those described in non-specific graft sclerosis/chronic allograft nephropathy. The distribution of healed PVAN lesions followed the pattern of the active infection (random, multi-focal, stripped fibrosis). Correlation of tissue diagnosis with urine cytology, urine PCR and viremia In all 90 patients, one or more urine samples showed decoy cells within a week of the index biopsy. Decoy cells in urine preceded PVAN in 14 of the 90 patients and occurred 1 18 months before the diagnosis (mean 5.6 months). Quantitative urine PCR for BK virus was available concurrently with a biopsy showing PVAN in 43 patients and ranged from to BK viral copies per milliliter (mean ). Quantitative determinations of viremia at the time of the index biopsy were available in 25 patients and ranged from to copies of BK DNA/mL (mean ). The extent of involvement in the renal biopsy did not correlate with the level of viruria and viremia. Urine cytology and measurements of viremia after nephrectomy were consistently negative American Journal of Transplantation 2004; 4:

8 Histological Patterns of Polyomavirus Nephropathy Figure 4: (A) Index biopsy in a patient with pattern b of PVAN. The viral infection affects the right area of the tissue core that also shows prominent lymphocytic inflammation and tubular atrophy. At the time of the biopsy the viremia was BK viral copies/ml, viruria was and there were abundant decoy cells in urine cytology. (B) Follow-up biopsy 14 months after diagnosis of PVAN and decrease of immunosuppression. There is no evidence of PVAN. The biopsy shows patches of fibrosis (right of picture), consistent with sequela from healed PVAN. At the time of the biopsy, viremia was negative and viruria was absent (PCR and urine cytology). Discussion Infection with BK and JC polyomaviruses is endemic in the human population. Both viruses typically persist in the urogenital tract as the principal site. After the primary infection, BK and JC viral DNA is found throughout the kidney in randomly distributed foci (23 25). The polyomavirus infection remains latent and is of no clinical significance in the vast majority of individuals. Low-level viral replication, mostly limited to the urothelium and characterized by viruria, may sporadically occur in healthy individuals and is common in immunosupressed patients. In contrast, a status of high-level viral replication with associated tissue destruction occurs only in a minority of patients and it has been best characterized in renal allografts (PVAN). Pathological studies documenting the exact progression of polyomavirus reactivation in the kidney are lacking, although it has been speculated that viral reactivation starts in the urothelium (bladder, ureters) and renal medulla with later involvement of the rest of the kidney (18,19,27,28). Although in recent years the diagnosis of PVAN has been made with increasing frequency (26), the limited number of cases has so far precluded a methodical clinicopathological American Journal of Transplantation 2004; 4:

9 Drachenberg et al. Table 1: Histological findings in follow-up biopsies correlation with index biopsy, viremia and outcome Group 1 Group 2 Group 3 Group 4 Histological findings PVAN No PVAN PVAN, no tubulitis No PVAN, no tubulitis in follow-up biopsies Tubulitis (Banff t2, t3) Tubulitis (Banff t2, t3) # Patchy graft sclerosis Patchy graft sclerosis Number of patients 21 (33 biopsies) 8 (14 biopsies) 16(34 biopsies) 11 (19 biopsies) PVAN mean score, index 3.15 (2 4, 3.15) 1.57 (1 3, 1) 2.46 (1 3, 2) 2.18 (1 3, 2) biopsy (range/median) Graft loss 100% (n = 21) 12.5% (n = 1) 37.5% (n = 6) 18.8% (n = 2) Mean time of graft loss 15.7 months 11 months 11 months 10 months after diagnosis of PVAN (range 4 35) (range 3 33) (range 9 11) Graft function/mean Return to dialysis 2.48 mg/dl 3.1 mg/dl 2.65 mg/dl creatinine at end of (range ) (range ) (range ) (range ) follow-up Mean viremia at the time Negative of follow-up biopsy (range (range (4 of 11 patients) ) ) (range ) Decoy cells in urine at the Present Absent Present Present time of follow-up biopsy (4 of 11 patients) #In the absence of PVAN these features were consistent with acute rejection (type Ia, Ib) as defined by the Banff 97 scheme. Despite no histological evidence of PVAN, urine cytology and viremia indicated ongoing viral replication in 4 or 11 patients (2 of which lost graft function). Patients were followed for a mean of 34 months after transplantation. There was no difference in follow-up between the four groups. PVAN = Polyomavirus allograft nephropathy. Index biopsy: first renal biopsy revealing PVAN. At the time of the index biopsy all patients had decoy cells in urine and viremia performed on 25 patients was above BK viral copies/ml (range to , mean ). PVAN mean score, based on numerical values (1 5) assigned to histological patterns a, b1, b2, b3 and c, respectively. Graft sclerosis consisted of patches of interstitial fibrosis and tubular atrophy with sparse or absent chronic inflammation as seen in chronic allograft nephropathy. Graft loss defined by return to dialysis. The patients in group 1 (persistent PVAN and tubulitis) had worse outcome (graft loss) in comparison to the three other groups (p = ) and more advanced histological disease (p = ). In patients in all four groups, immunosuppression was reduced (see Materials and Methods). In addition, one of the patients in group 4 received cidofovir. He had persistent viremia and viruria and slow deterioration of graft function. assessment of this infection. The relatively large number of cases of PVAN in our center has provided for the unique opportunity, albeit in retrospective manner, to analyze the histopathological presentation, morphological patterns, clinical outcomes and potential surrogate markers of the disease. In contrast to the large proportion of graft loss reported initially, diagnosis of PVAN at earlier stages has resulted in improvement or stabilization of graft function in a significant proportion of patients (14 16). Prospective studies have depended on the performance of protocol biopsies or biopsies prompted by the presence of viruria or viremia (14 16). Although evaluation of an allograft biopsy has emerged as one of the most important tools in the successful management of patients with PVAN, our data show that the characteristic (multi-)focal nature of the disease may lead to tissue sampling errors. These findings have important implications for the muchneeded earlier diagnosis of PVAN, which is more likely to respond to intervention and allows for PVAN resolution without a high burden of residual parenchymal damage. We have previously proposed an algorithm for the early diagnosis of PVAN that recommends evaluation of a renal biopsy when there is persistence of decoy cells in urine or viremia, independently of the renal function (7,16). With this approach the diagnosis of PVAN can be potentially established earlier. On the other hand, a higher rate of false negative biopsies may be encountered in the early stages of the disease when the foci of parenchymal involvement are smaller. Progression of polyomavirus infection from a state of limited viral activation to PVAN is heralded by clinically silent but persistently large-scale viruria (viral DNA or RNA, decoy cells) (9,14). Whereas, most patients with sporadic viruria lack viremia and probably do not have significant renal parenchymal involvement, PVAN is associated with increasing viral load in plasma (4,7,14). Quantitative measurement of viruria and viremia as well as evaluation of urine cytology for decoy cells are increasingly used for screening, diagnosis and follow-up of PVAN in renal transplant patients (4,5,14,17). In the current study, we confirm the strong correlation between significant viruria and viremia with PVAN. Decoy cells were present in the urine 2090 American Journal of Transplantation 2004; 4:

10 Histological Patterns of Polyomavirus Nephropathy of all patients with PVAN and whenever determination of viremia was available, this was significant (> viral copies/ml) as previously proposed by Hirsch et al. (14). With the use of these surrogate PVAN markers, i.e. persistent viruria and increasing viremia, a diagnosis of presumptive PVAN can be established even if there is a negative renal biopsy (Hirsch et al., Multidisciplinary Consensus Statement, in preparation). We and others have proposed pre-emptive decrease of immunosuppression even in the absence of a histological diagnosis of PVAN, but this approach needs to be tested in larger controlled studies (7,17). Due to the extreme sensitivity and the wide range of values resulting from the molecular methods for the quantitation of viruria, we prefer to use urine cytology for the screening and follow-up of our renal transplant patients. Although no studies have specifically addressed this issue, urine cytology and quantitative measurements of viruria by PCR are likely comparable. The selection of one method over another will probably be dependent on the level of expertise and experience in each center. Patients with clearance of the PVAN characteristically show disappearance of the viral cytopathic changes in follow-up biopsies. In parallel with the histological lack of viral replication, the urine cytology and viremia also become negative. On the other hand, due to sampling errors, absence of viral cytopathic changes may be seen also in follow-up needle biopsies from patients that show persistent viruria and viremia (false-negative biopsy). The characteristic morphological findings of PVAN have been described previously, in particular by Nickeleit et al. (18,19,29). In the current study, we did not find any additional histological feature that can be used as a histological surrogate marker of PVAN in the absence of diagnostic polyomavirus cytopathic changes. On the other hand, we confirmed in our samples that features that have been attributed to acute rejection such as hemorrhage, edema and endotheliitis are not usually present in PVAN. The limited phenotypic studies of lymphocytes in our study showed larger numbers of B cells in PVAN, in comparison to biopsies with acute rejection. This has been previously described by Ahuja et al. (30). It is remarkable, however, that graft sclerosis unrelated to PVAN showed a similar increase in B cells, suggesting that B cell infiltrates may be an indication of chronic inflammatory processes without specific relationship to the underlying etiology. The puzzling role of tubulointerstitial inflammation and tubulitis in patients with PVAN (31,32) is highlighted by the current study. Active tubular inflammation (tubulitis) at the time of diagnosis of PVAN was associated with an increased incidence of graft loss. Active inflammatory infiltrates may suggest the possibility of PVAN arising in the context of ongoing acute allograft rejection. On the other hand, tubulitis in PVAN may represent anti-viral host immunity, or may be non-specific (i.e. secondary to tubular injury). Excessive tissue destruction may occur once the immune system recovers its capabilities (immune reconstitution response) (28,33 35). Irrespective of the underlying etiopathogenesis, florid inflammatory responses in PVAN are often associated with parenchymal fibrosis (scarring) (9,29). Therefore, as suggested previously it is possible that undiagnosed low-level PVAN may be the etiology of graft sclerosis in some patients (chronic allograft nephropathy) (23). In summary, the findings in this study indicate that due to the focal nature of PVAN, a negative biopsy cannot rule out the disease. However, evaluation of a renal biopsy in combination with the determination of viruria, viremia and assessment of renal function is necessary for the accurate determination of the activity of PVAN. Once the sampling limitations are understood, determination of the histological pattern of PVAN can optimize the diagnostic efficiency and prognostic value of a renal biopsy. Early diagnosis is important as more advanced PVAN in the initial biopsy correlates with significantly higher rates of failure to clear the infection and with increased rates of graft loss. Acknowledgments We wish to thank Dr. Jennifer Gordon (Temple University) and Dr. Volker Nickeleit (University of North Carolina) for helpful comments during the preparation of the manuscript. References 1. Purighalla R, Shapiro R, McCauley J, Randhawa P. BK virus infection in a kidney allograft diagnosed by needle biopsy. Am J Kidney Dis 1995; 26: Pappo O, Demetris AJ, Raikow RB, Randhawa PS. 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