J Am Soc Nephrol 10: 1080–1089, 1999 Polyomavirus Infection of Renal Allograft Recipients: From Latent Infection to Manifest Disease

VOLKER NICKELEIT,* HANS H. HIRSCH,† ISABELLE F. BINET,‡ FRED GUDAT,* OLIVIER PRINCE,* PETER DALQUEN,* GILBERT THIEL,‡ and MICHAEL J. MIHATSCH* *Institute for Pathology, †Institute for Medical Microbiology, and ‡Division of Nephrology, Kantonsspital, University of Basel, Switzerland.

Abstract. Polyomavirus (PV) exceptionally causes a morpho- crescents were found. In 54% of biopsies, infection was asso- logically manifest renal allograft infection. Five such cases ciated with pronounced inflammation, which had features of were encountered in this study, and were followed between 40 cellular rejection. All patients were excreting PV-infected cells and 330 d during persistent PV renal allograft infection. Trans- in the urine. PV infection was associated with 40% graft loss (2 plant (Tx) control groups without PV graft infection were of 5) and a serum creatinine of 484 Ϯ 326 ␮mol/L (mean Ϯ analyzed for comparison. Tissue and urine samples were eval- SD; 11 mo post-Tx). Tx control groups showed PV-infected uated by light microscopy, immunohistochemistry, electron cells in the urine in 5%. Control subjects had fewer rejection microscopy, and PCR. The initial diagnosis of PV infection episodes (P Ͻ 0.05) and stable graft function (P ϭ 0.01). It is with the BK strain was made in biopsies 9 Ϯ 2 mo (mean Ϯ concluded that a manifest renal allograft infection with PV (BK SD) post-Tx after prior rejection episodes and rescue therapy strain) can persist in heavily immunosuppressed patients with with tacrolimus. All subsequent biopsies showed persistent PV recurrent rejection episodes. PV mainly affects tubular cells infection. Intranuclear viral inclusion bodies in epithelial cells and causes necrosis, a major reason for functional deteriora- along the entire nephron and the transitional cell layer were tion. A biopsy is required for diagnosis. Urine cytology can histologic hallmarks of infection. Affected tubular cells were serve as an adjunct diagnostic tool. enlarged and often necrotic. In two patients, small glomerular

Cytomegalovirus (CMV) is the virus most frequently identified cells in the urine (intranuclear inclusion cells, or so-called in kidney transplant recipients (1–3). An infection caused by “decoy cells”) (12,14). polyomavirus (PV) is exceptional (2,3). This rarity is also In immunocompromised patients, PV can cause a morpho- underscored by the observation that we had not detected mor- logically manifest renal infection with cytopathic signs and phologic evidence of a PV infection in renal allografts among functional impairment, a condition termed PV disease. In na- 616 kidney transplant recipients managed at our institution tive and transplanted kidneys, PV (BK virus strain) is found in between January 1985 and December 1995. areas of interstitial nephritis (15–19). In addition, renal allo- PV is a nonenveloped, double-stranded DNA virus. Two graft recipients were reported to suffer from PV (BK virus)- strains are associated with disease in humans: BK virus and JC associated ureteral stenosis, and bone marrow transplant recip- virus (4). In immunocompetent individuals, PV has no great ients from hemorrhagic cystitis (20–22). However, in the latter clinical significance, although 60 to 80% of adults are sero- group a causative pathobiologic association is disputed by some (12,23–25). A pathogenetic significance is well estab- logically positive (4–8). In healthy individuals, PV resides in lished for JC virus, which causes multifocal leukoencephalop- a latent state in the kidney (4,7,9–11) and can be activated athy, mainly in AIDS patients (4,7,26). without functional impairment or ill effects (12–14). Morpho- Although, PV disease is exceptionally rare, we encountered logic evidence of viral activation is the presence of PV-infected five cases in renal transplant recipients within 8 mo. Because detailed clinical and histologic information was available from all patients before and after manifestation of PV disease, we Received September 3, 1998. Accepted November 23, 1998. had a unique opportunity to characterize typical changes. This work was presented in part at the 87th annual meeting of the United States and Canadian Academy of Pathology (USCAP) in Boston, MA, February 28 to Materials and Methods March 6, 1998, and has been published in abstract form (Lab Invest 78: 148A, 1998). Histology and Urine Cytology Correspondence to Dr. Michael J. Mihatsch, Institute of Pathology, University Morphologic changes in tissue samples (25 biopsies and two graft of Basel, Schoenbeinstrasse 40, CH-4003, Basel, Switzerland. Phone: 41 61 nephrectomies) from five patients who contracted PV disease were stud- 265 2872; Fax: 41 61 2653194; E-mail: [email protected] ied. PV disease is defined as a morphologically manifest renal infection 1046-6673/1005-1080$03.00/0 with cytopathic signs accompanied by varying degrees of interstitial Journal of the American Society of Nephrology inflammatory cell infiltrates and functional impairment. Thirteen tissue Copyright © 1999 by the American Society of Nephrology samples (11 biopsies and two graft nephrectomies) were obtained during J Am Soc Nephrol 10: 1080–1089, 1999 Polyomavirus Infection of Renal Allografts 1081 the course of persistent PV disease (up to 330 d after the initial diagnosis). The outer primers 5Ј-AAG TCT TTA GGG TCT TCT AC-3Ј and Fourteen biopsies were taken before the initial manifestation, i.e., before 5Ј-GTG CCA ACC TAT GGA ACA GA-3Ј amplified a fragment of the first histologic diagnosis of PV infection (including negative workup 176 bp common to both viruses. The inner primer pair combined the by immunohistochemistry [IHC]; see below). common 5Ј-AAG TCT TTA GGG TCT TCT AC-3Ј with either the For comparison, 108 renal allograft biopsies from patients lacking any BK-specific primer 5Ј-GAG TCC TGG TGG AGT TCC-3Ј to obtain morphologic evidence of PV disease were evaluated (including negative a BK fragment of 149 bp, or the JC-specific primer 5Ј-GAA TCC workup by IHC; see below). All of the 133 biopsies were performed due TGG TGG AAT ACA-3Ј yielding a JC fragment of 146 bp. Both were to deterioration of graft function at similar time intervals posttransplan- confirmed by dideoxy sequencing (H.H.H., personal observation). tation. Thirteen distinct histologic criteria were analyzed and semiquan- DNA was prepared by protease K digestion and purification on silica titatively graded on a scale of 1 (minimal change) to 4 (marked change): spin columns (QIAmp, Qiagen, Basel, Switzerland) from the follow- extent of mononuclear cell infiltrate in the interstitium and tubulitis; ing sources: one nephrectomy from a patient with manifest PV disease vascular rejection (VR) with necrosis; transplant endarteritis; sclerosing and voided urine from five patients with PV disease. VR (“chronic vascular rejection”); transplant glomerulitis; transplant A total of 0.5 ␮g of DNA was used per reaction. Amplification was glomerulopathy; extent of interstitial fibrosis; ischemic tubular damage; performed after preincubation (37°C, 10 min) for uracyl-N-glycosy- cyclosporine-induced tubular damage; toxic arteriolopathy; arterioloscle- lase decontamination followed by 94°C at 30 s, 50°C at 45 s, and 72°C rosis; glomerulonephritis (immune complex-mediated); and pyelonephri- at 60 s (30 cycles, for both the outer and the inner amplification). tis. A diagnosis of acute renal allograft rejection was based on the criteria Ten-microliter aliquots of the inner PCR product were separated on of the Cooperative Clinical Trials in Transplantation classification NuSieve:Agarose (3%:1%) electrophoresis gels. The threshold of scheme (27,28). The histologic evaluation was performed according to detection was determined to be five genomes. Two parallel runs were standard protocols by light microscopy (hematoxylin and eosin, periodic analyzed for each result, and if equivocal, repeated. The results were acid-Schiff, trichrome, methenamine silver stains) accompanied by IHC interpreted as positive (2 of 2 runs positive); weakly positive (1 of 2 and electron microscopy. For routine IHC, a standard panel of antibodies runs positive, viral load close to detection threshold); negative (2 of 2 was used (directed against IgG, IgM, IgA, complement factors C3, C4, runs negative). C5b–9, C1q, and fibrin). Voided urine samples were analyzed at time of biopsies as a standard patient management procedure. Urine cytology reports were retrospec- Results tively evaluated from 483 renal transplant recipients (including five PV Disease patients with PV disease) managed in Basel between 1985 and 1997. The Patients. Five patients (mean age at transplantation, 46 Ϯ presence of PV-infected intranuclear inclusion cells, so-called “decoy 2 yr; range, 27 to 57; four men and one woman) showed a cells,” as a morphologic marker for viral replication was semiquantita- morphologically manifest renal allograft infection due to poly- tively recorded as absent, scant (1 to 4 per 10 high-power fields [HPF]), or abundant (Ͼ5 per 10 HPF). From all patients with abundant decoy cell omavirus (BK strain). PV infection became manifest and was excretion (Ͼ5 per 10 HPF in at least one urine sample), renal allograft histologically diagnosed several months after transplantation biopsies were (re-) examined (including IHC; see below) to search for (9 Ϯ 2 mo, mean Ϯ SD; range, 7 to 11; all between October cytopathic signs of a PV graft infection. 1996 and June 1997). All biopsies before the initial diagnosis of PV disease (n ϭ 14) lacked cytopathic signs of viral infec- Immunohistochemical Detection of Viruses tion by light microscopy or IHC. Three patients had received a PV was detected by indirect IHC on urine cytology, and tissue samples cadaver kidney, and two received organs from living unrelated (fresh frozen and formalin-fixed). Several mouse monoclonal antibodies donors. All of the five patients had a complicated posttrans- (mAb) were used to detect different PV strains: (1) mAb detecting both plantational course with recurrent rejection episodes. Before a BK and JC strains (Readysysteme, Bad Zurzach, Switzerland); (2) mAb manifest infection was diagnosed, immunosuppression had detecting JC strain (Readysysteme); (3) mAb detecting the SV40 large T been switched in all patients from cyclosporine to high-dose antigen (Calbiochem/Oncogene Research Products, Cambridge, MA). tacrolimus rescue therapy for 5 Ϯ 3 mo (mean Ϯ SD) (Figure The latter antibody reacts with primate (SV40) and human PV (BK and JC strains) and works after formalin fixation, using the microwave 1). Patients were kept on tacrolimus; three patients were Ϯ Ϯ technique for antigen retrieval (Tris-HCl buffer, pH 10.5, 15 min at switched back to cyclosporine 98 46 d (mean SD; range, 100°C, followed by overnight incubation with the primary antibody at 49 to 140 d) after the initial diagnosis of PV disease (Figure 1). 4°C, dilution 1:3200). All staining procedures were performed according Cytopathic Changes. PV disease was diagnosed histolog- to a previously described protocol with the avidin-biotin-peroxidase tech- ically. The morphologic characterization was based on light nique (29). DAB (diaminobenzidine tetrahydrochloride) and AEC (3- microscopy, immunohistochemistry and electron microscopy. amino,9-ethylcarbazole) were used as chromogens. All biopsies were The diagnostic hallmark was the detection of intranuclear viral also analyzed for the presence of CMV, Epstein–Barr virus (EBV), inclusions, which were exclusively found in epithelial cells herpes simplex virus (HSV) or adenovirus antigens (CMV [IEA]: mouse (with the exception of podocytes). All tissue samples (n ϭ 13) monoclonal antibody, Biosoft, Varilhes, France; CMV [EA] and EBV from patients with PV disease (n ϭ 5) showed viral inclusions. [LMP, ZEBRA, EBNA]: mouse monoclonal antibodies, Dako, Glostrup, Three types of intranuclear inclusion bodies were seen by light Denmark; HSV [types 1 and 2]: rabbit polyclonal antibody, Dako, Glostrup, Denmark; adenovirus: mouse monoclonal antibody, Chemicon, microscopy with similar frequencies along the entire nephron Temecula, CA). (Figure 2): (1) a homogeneous ground-glass appearance; (2)a central eosinophilic granular appearance with a (mostly) in- PCR Studies complete halo; (3) a more homogeneous, finely granular pat- The genome detection of polyomavirus JC strain and BK strain tern. Cells with viral changes were often (but not always) used a modified semi-nested format based on a previous report (30). enlarged with polymorphic nuclei. Frequently, tubular cells 1082 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1080–1089, 1999

Figure 1. Typical course of serum creatinine in a patient with polyomavirus (PV) disease. (Top) Arrows indicate the time of biopsy and the histologic diagnosis (PV: initial diagnosis of PV disease; VR: transplant endarteritis; ICR: interstitial cellular rejection). Light gray portionon the left: time before the diagnosis of PV disease; dark gray portion on the right: persistent morphologic evidence of PV allograft infection. (Bottom) Immunosuppressive treatment. were rounded-up, necrotic, and extruded from the epithelial seen (Figure 8). Crescent formation affected between 10 and cell layer into tubular lumens causing marked denudation of 20% of glomeruli. There was no evidence of an immune basement membranes (Figure 3). Although cytopathic signs complex-mediated glomerulonephritis. In the renal pelvis and were seen along the entire nephron, they were often abundant ureters (two nephrectomies), viral inclusion bodies were iden- in distal tubular segments and collecting ducts. Sporadically, tified in superficial transitional cells by light microscopy and infected cells were noted in the parietal epithelium lining additionally in the basal cell layer by IHC. Transitional cells Bowman’s capsule (Figure 4). By IHC, PV (BK strain) was almost exclusively showed intranuclear inclusions of the demonstrated (Figure 5; nuclear staining profile: AB detecting ground-glass type (in contrast to nephrons), closely resembling SV40 - positive; AB detecting BK/JC - positive; AB detecting decoy cells in urine cytology (Figure 9). JC - negative). Positive staining was also noted in tubular cells CMV, EBV, HSV, or adenovirus were not found. By stan- inconspicuous by light microscopy. IHC did not reveal PV in dard immunohistochemistry using a routine panel of antibodies nonepithelial cells and podocytes. By electron microscopy, directed against immunoglobulins and complement factors, a intranuclear viral particles were granular, sometimes arranged specific staining pattern was not present. as crystalloids (Figure 6). They measured between 35 and 40 Identification of BK Virus by PCR (Tissue, Urine). One nm in diameter, a size characteristic for PV (4,7,12,18). Viral nephrectomy specimen (Figure 10) and urine samples from particles were occasionally detected adherent to the external five patients were studied by PCR. All examined samples surface of cell membranes (Figure 7). In the cytoplasm, viral revealed abundant amplicons of 149 bp indicative of BK virus particles were only rarely noted by electron microscopy (Fig- genomes. JC virus was not detected. ure 7) or IHC. Ultrastructurally, PV was not seen in nonepi- PV Infection and Interstitial Inflammation. In seven thelial cells and podocytes. In three biopsies (two patients), biopsies (7 of 13; 54%), PV infection of tubular epithelial cells small glomerular crescents with inclusion-bearing cells were was associated with widespread inflammatory cell infiltrates in J Am Soc Nephrol 10: 1080–1089, 1999 Polyomavirus Infection of Renal Allografts 1083

Figure 2. Types of intranuclear viral inclusion bodies. (a) Homogeneous ground-glass configuration (long arrow) and eosinophilic granular appearance with an incomplete halo (thick arrow) encountered in the same tubular cross section (hematoxylin and eosin [H&E] stain, ϫ400); (b) Homogenous finely granular pattern (arrow; periodic acid-Schiff [PAS] stain, ϫ400). Figure 3. Tubules with infected cells. (a) An infected cell is rounded up and extruded into the lumen (arrow). The interstitium shows only minimal inflammation (PAS stain, ϫ251). (b) Necrotic cells with cytopathic signs sloughed off into the lumen. The tubular basement membrane is denuded (arrow, H&E stain, ϫ160) . Figure 4. Intranuclear inclusion bodies in parietal epithelial cells lining Bowman’s capsule (arrow, H&E stain, ϫ160). Figure 5. Polyomavirus detected by immunohistochemistry. Individual tubular epithelial cells show a positive staining reaction (formalin-fixed and paraffin-embedded tissue, antibody detecting the SV40 large T antigen, ϫ160). 1084 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1080–1089, 1999

Figure 6. Electron micrographs showing intranuclear viral inclusion bodies. (A) Dense granular viral particles in a transitional cell (ϫ3800). (B) A tubular cell with loosely arranged granular viral particles (ϫ5600). (Inset) A crystalloid arrangement of polyomavirus with particles measuring between 35 and 40 nm (ϫ97,000).

the interstitium and tubulitis. Inflammatory infiltrates were initial diagnosis of PV disease). The percentage of crescents predominately composed of lymphocytes and histiocytes in remained unchanged over time (Ͻ20%); however, they be- combination with some polymorphonuclear leukocytes and came increasingly fibrotic with pronounced layering of the plasma cells. Focally, plasma cells were abundant. In four basement membrane of Bowman’s capsule associated by an biopsies (4 of 13; 31%), the infiltrate was sparse with minute inflammatory cell infiltrate (Figure 12). tubulitis (Յ1 nonatrophic tubular cross section involved). The The gross appearance of two graft nephrectomies revealed infiltrating cells were randomly distributed and not primarily kidneys slightly reduced in size with a smooth, focally finely associated with virally affected tubules (Figure 11a). Mononu- granular outer surface. The parenchyma was homogenously clear cell infiltrates and tubulitis were often noted in areas ochre brown; the corticomedullary junction was ill-defined. lacking viral inclusion bodies. Some infected tubules, espe- Decoy Cells in the Urine. Urine samples from all patients cially in the medulla, did not show an adjacent inflammatory with persistent PV disease revealed decoy cells with ground- response (Figure 11b). In two biopsies (2 of 13; 15%), there glass type intranuclear inclusions (Figure 9) positive for PV by was negligible interstitial inflammation. IHC and electron microscopy. Decoy cells were typically ex- Morphology of Persistent PV Disease. After the initial creted in large numbers. Small numbers (Ͻ5 per 10 HPF) were histologic diagnosis of PV disease had been made, eight sub- only sporadically noted during the course of disease. The sequent tissue samples were examined during the course of excretion of decoy cells vanished in one patient after transplant persistent graft infection (range, 40 to 330 d postdiagnosis; six nephrectomy (starting 2 d postoperatively; follow-up: 4 mo). In biopsies and two graft nephrectomies; five patients). All tissue two patients tested, urine samples revealed large numbers of samples showed the three types of viral inclusion bodies, decoy cells 10 and 5 mo before manifestation of PV disease severe focal tubular injury, and varying degrees of inflamma- when the corresponding biopsies lacked cytopathic signs (see tory cell infiltrates (see above). Chronic changes increased: (1) above). marked interstitial fibrosis (three patients; six samples); (2) marked arteriolosclerosis (one patient, one sample); (3) marked Differences between PV Disease Group and “cyclosporine-type” arteriolopathy (two patients, two sam- Control Subjects ples); (4) intimal fibrosis (“chronic vascular rejection;” two Decoy cell excretion was a typical finding in patients with patients, two samples). PV disease. To evaluate the specificity of decoy cell excretion, In one patient, PV-associated crescent formation was ob- urine cytologies from 483 renal allograft recipients (including served in two subsequent biopsies (50 d and 330 d after the five patients with PV disease) were analyzed. Abundant decoy J Am Soc Nephrol 10: 1080–1089, 1999 Polyomavirus Infection of Renal Allografts 1085

Figure 7. Electron micrographs demonstrating PV in different cellular compartments. (A and B) Tubular cells with intranuclear and cytoplasmic (arrow) viral particles. One cell is sloughed into the lumen (asterisk; ϫ5800 in A; ϫ25,000 in B). (C) Viral particles adherent to the external cell membrane of a tubular cell (ϫ47,000), and (D) PV in Bowman’s space sticking to podocytes. Viral particles were not seen within visceral epithelial cells (ϫ47,000). cells were found in 28 recipients (6%) and scant decoy cells in tial rejection and transplant glomerulitis were particularly 72 (15%). PV disease was associated with abundant decoy cell frequent in the disease group before PV infection became excretion in five of 28 (18%) recipients; twenty-three of twen- manifest. ty-eight (82%) were free of disease (no cytopathic changes in graft biopsies by light microscopy or IHC). Outcome Tissue samples from patients with and without PV disease Renal function in patients with PV disease deteriorated quite (n ϭ 27 versus n ϭ 108) differed in only two histologic rapidly. Two patients (2 of 5; 40%) lost their grafts within 40 parameters from 13 analyzed (P Ͻ 0.05, Fisher exact test) and 113 d postdiagnosis of infection. The serum creatinine (Table 1): Interstitial cellular rejection and transplant glomer- level of the remaining three patients was 484 Ϯ 326 ␮mol/L ulitis were more frequently found in patients with PV disease (mean Ϯ SD; 11 mo posttransplantation). In comparison, the (60 and 29%) than without (35 and 4%, respectively). Intersti- 23 transplant recipients with abundant decoy cells in urine 1086 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1080–1089, 1999

Morphologic evidence of viral renal allograft disease is excep- tional, in particular a manifest PV infection (2,18). Although we did not see a single patient with PV disease among 616 renal allograft recipients between 1985 and 1995, we suddenly encountered a cluster of five patients. PV renal allograft disease can only be diagnosed histologically. Severe tubular injury with cellular enlargement, frank epithelial necrosis, denudation of tubular basement membranes, and distinct intranuclear inclusion bodies in epithelial cells including transi- tional cells suggest an infection with PV. Infected parietal epithe- lial cells occasionally form glomerular crescents, a highly excep- tional finding in the absence of an underlying glomerular tuft pathology (glomerulonephritis). The morphology of a PV infec- tion is typical, however, not pathognomonic, since adenovirus and HSV may cause a similar pattern. Diagnostic confirmation is easily achieved by immunohistochemistry and electron micros- copy, which shows viral particles of approximately 40 to 50 nm typical for PV. The detection of urinary decoy cells is helpful in identifying PV activation and replication. However, decoy cells alone do not necessarily indicate PV disease (31,32). The false Figure 8. Glomerular crescent. Intranuclear viral inclusion (arrow) in positive rate in our series was high (82%; sensitivity: 100%), a small cellular crescent (asterisk: glomerular tuft; PAS stain, ϫ400). underscoring the necessity of a renal biopsy for a definitive diagnostic workup. cytology but without PV disease had a mean serum creatinine PV renal allograft disease was associated with deteriorating level of 207 Ϯ 177 ␮mol/L (mean Ϯ SD; 57 mo posttrans- graft function. Two factors contributed to functional impair- plantation; correlation with disease group P ϭ 0.01, t test). ment: tubular necrosis induced by PV infection and sclerosing Two of the latter 23 patients (9%) had lost their grafts (corre- allograft nephropathy. PV infection caused frank tubular ne- lation with disease group P Ͻ 0.01, ␹2 test). crosis and, thus, altered renal function significantly. Sloughed necrotic epithelial cells formed intraluminal casts leaving be- Discussion hind denuded tubular basement membranes. Denuded areas of Renal allograft recipients require permanent immunosup- basement membranes permit leaking of fluid into the intersti- pression and, therefore, are at an increased risk for infections. tial compartment, which is associated with functional impair-

Figure 9. Transitional cells infected by PV compared with “decoy cells” in urine cytology. (A) Low power view of the urothelium showing cytopathic signs in superficial transitional cell layers (arrow, H&E stain, ϫ62) pictured in Panel B with high magnification (arrow, H&E stain, ϫ400). (C) Immunohistochemistry reveals a positive staining pattern not only in superficial transitional cells with cytopathic signs, but also in occasional basal cells that were unremarkable by light microscopy. The lamina propria is without inflammation (formalin-fixed and paraffin-embedded tissue, antibody detecting the SV40 large T antigen, ϫ160). (D) Urine cytology with a PV-infected cell, so-called “decoy cell,” showing a ground-glass type intranuclear inclusion body (arrow, Papanicolaou stain, ϫ400). J Am Soc Nephrol 10: 1080–1089, 1999 Polyomavirus Infection of Renal Allografts 1087

Figure 10. PCR detection of BK virus genomes in a nephrectomy specimen from a patient with PV disease. Tissue samples from the renal cortex and medulla were macroscopically separated followed by DNA extraction. Agarose gel showing a 149-bp amplicon of BK virus genomes (arrow) in the renal cortex (lanes 12, 13, 17, and 18) and medulla (lanes 7 and 8). Lanes 1 and 22, size standards; lane 2, human DNA control; lanes 3 and 4, external positive BK DNA controls; lanes 5, 6, 9, 11, 14, 16, 19, and 21, negative H2O controls; lanes 7 and 8, renal medulla; lane 10, renal medulla with external positive BK DNA control; lanes 12, 13, 17, and 18, renal cortex; lanes 15 and 20, renal cortex with external positive BK DNA control. With excess target, the semi-nested PCR technique gives rise to a double band. The human control DNA preparation from peripheral blood mononuclear cells (lane 2) consistently yields an unspecific band of 130 bp. ment (33). In addition, tubular casts may cause an obstructive endarteritis led to interstitial and intimal fibrosis (i.e., “chronic component. Such pathways are not unique to PV disease but allograft rejection”). However, the prevalence of chronic are well described in other forms of acute tubular necrosis (33). changes did not differ significantly from control subjects, Because PV never cleared from the kidneys, tubular injury did emphasizing that tubular necrosis is a main factor for func- not heal, and therefore functional impairment persisted. In tional deterioration. addition, over time recurrent rejection episodes with transplant There is controversy about whether the interstitial inflamma-

Figure 11. Inflammation and viral inclusion body. (a) There is diffuse interstitial inflammation associated with tubulitis, features suggestive of cellular rejection. Only one intranuclear viral inclusion is present (arrow, H&E stain, ϫ80); (b) Cytopathic signs in the medulla. Low power view of the renal medulla showing scattered inclusion bodies in tubular cells (arrowhead). There is no interstitial inflammatory response (H&E stain, ϫ25). Figure 12. Glomerular crescent in a follow-up biopsy 330 d after the initial diagnosis of PV disease. The basement membrane of Bowman’s capsule is seen twice as an inner and outer circular line (arrow) associated with cellular aggregates forming a crescent. The remaining glomerular tuft is seen in the center (asterisk). Same patient as pictured in Figure 8. (PAS stain, ϫ125). 1088 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 1080–1089, 1999

Table 1. Differences of histologic diagnoses in patients with and without polyomavirus (PV) disease

PV Disease Group (total n ϭ 27 tissue samples) Control Diagnosis Before Diagnosis of Persistent Biopsies PV Disease PV Disease (total n ϭ 108) (n ϭ 14) (n ϭ 13)

Interstitial rejection 9 7 38 P Ͻ 0.05 Transplant glomerulitis 6 2 5 P Ͻ 0.05

tory cell infiltrate in PV disease represents virally induced inter- disease before the infection became manifest than in the con- stitial nephritis or cellular rejection. We do not think that the virus trol group. Tubular injury has been demonstrated as a promot- stimulates a marked inflammatory response, because a manifest ing factor for a morphologically manifest PV renal infection in infection with intranuclear inclusion bodies is often associated mice (35). with an inconsistent, randomly distributed, and occasionally even A major factor involved in the manifestation of PV disease is scant inflammatory reaction. Particularly in the medulla and high-dose immunosuppression. Therapeutic rescue attempts with urothelium, inflammation might be very minimal. If a mixed tacrolimus, a new immunosuppressive drug, seem particularly inflammatory cell infiltrate containing scattered polymorphonu- important since all of our patients were switched from cyclospor- clear leukocytes is present in areas with tubular injury, it is likely ine to tacrolimus months before manifestation of infection (36). due to urine back leak, thus representing “nonspecific” severe Tacrolimus as a possible risk factor has been suggested in the tubular damage (33). Cellular rejection should be considered if past—all eight previously reported patients with PV allograft abundant cortical mononuclear cell infiltrates and typical tubulitis disease were on a tacrolimus-based immunosuppressive regimen are found, randomly affecting tubules with and without cytopathic (17–19). However, tacrolimus cannot be the only factor, because signs. This interpretation is supported by the observation that we recently diagnosed PV disease in a patient on cyclosporine and tubulitis was seen in areas lacking cytopathic changes, thus mak- mycophenolate mofetil, not treated with tacrolimus (consultation ing a diagnosis of virally induced nephritis less likely. We diag- practice M.J.M.). One previous report (19) described (histologic) nosed interstitial cellular rejection in 54% of tissue samples during clearance of PV from the graft and stabilization of renal function the course of PV disease. Virally induced nephritis may coexist, after stopping tacrolimus and reintroducing cyclosporine. We but seems to be of minor significance. could not confirm this finding. In three of our patients who were What are the steps involved in PV activation and manifes- switched back to cyclosporine, the virus persisted and renal func- tation of disease? It seems surprising that PV does not affect tion deteriorated (36). renal transplant recipients more often, since approximately 60 In summary, we propose the following evolution and profile to 80% of healthy adults are serologically positive for PV of PV disease of renal allografts: (1) reactivation of latent BK (4–8). Under normal immunosurveillance, PV is latent in the virus with replication (decoy cells in urine); (2) long-lasting kidneys (4,7,9–11) and, therefore, renal allografts carry the tubular injury, such as recurrent “therapy-resistant” rejection risk for viral transmission (8,34) and manifestation of PV episodes; and (3) therapeutic rescue attempts with high-dose disease. Despite the high prevalence of latent virus, PV acti- immunosuppression, frequently tacrolimus-based. vation is uncommon, even in immunosuppressed renal allo- A definitive diagnosis of PV disease requires a renal biopsy, graft recipients. We found abundant decoy cells as a morpho- ideally triggered by the detection of large numbers of decoy cells logic marker of viral activation and replication (12,14,32) in in the urine. PV disease is a rare but serious cause for transplant only 6% of 483 kidney transplant recipients, and manifest renal failure, mainly due to virally induced severe tubular injury. allograft disease was only seen in 1%. Decoy cells were found in all of our patients with persistent PV disease; they were present before the disease became manifest and disappeared Acknowledgments after transplant nephrectomy. Activation of latent virus and The authors thank U. Du¨rmu¨ller, R. Epper, and C. Lautenschlager replication with excretion of decoy cells likely represents an for superb technical assistance and Dr. S. Regauer (University of initial, still balanced, and reversible step along the way to Graz, Austria) for critical review of the manuscript. manifest PV disease. Manifest renal allograft infection can develop in an ascending manner via cell-to-cell spread, which References is suggested by the predominance of viral inclusions in distal 1. Rubin RH, Tolkoff-Rubin NE: Viral infections in the renal tubular segments and viral particles adherent to outer cell transplant patient. Proc Eur Dial Tranplant Assoc 19: 513–526, membranes. Recurrent rejection with long-lasting tubular in- 1982 jury could render tubular cells susceptible to viral replication. 2. Colvin RB, Fang LST: Interstitial nephritis. In: Renal Pathology Cellular rejection was more common in our patients with PV with Clinical and Functional Correlations, 2nd Ed., edited by J Am Soc Nephrol 10: 1080–1089, 1999 Polyomavirus Infection of Renal Allografts 1089

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