Human polyoma virus in renal allograft biopsies: Morphological findings and correlation with urine cytology

Human Polyoma Virus in Renal Allograft Biopsies: Morphological Findings and Correlation With Urine Cytology CINTHIA B. DRACHENBERG, MD, CHRISTIAN O. BESKOW, MD, CHARLES B. CANGRO, MD, PHD, PATRICIA M. BOURQUIN, CT(ASCP), AYLIN SIMSIR, MD, JEFFREY FINK, MD, MATTHEW R. WEIR, MD, DAVID K. KLASSEN, MD, STEPHEN T. BARTLETT, MD, AND JOHN C. PAPADIMITRIOU, MD, PHD Human polyoma virus (PV) interstitial nephritis occurs in immunosuppressed patients after reactivation of latent virus in renal epithelium. Currently, there is neither general consensus about the incidence of clinically significant PV infection in renal transplants n o r conclusive evidence determining its significance in the long-term graft outcome. We evaluated 601 renal transplant biopsy specimens (from 365 patients) by routine light microscopy and immunoperoxidase stains with antibody against SV40 (which cross reacts with PV). We also examined urine samples from 200 patients (100 obtained concurrently with a renal biopsy in patients presenting with acute graft dysfunction and 100 from patients with stable graft function). Electron microscopic evaluation was performed in 50 renal biopsy specimens and in 23% of all urine samples. PVwas identified in 1.8% biopsy specimens (1.9% of patients). PV interstitial nephritis showed the typical viral cytopathic changes in tubular epithelial cells associated with marked tubular damage and a disproportionately mild degree of tubulitis. There was no difference in the incidence of PV in the urine of patients with acutely deteriorating versus stable renal function (18% and 19%, respectively); however, urines with large numbers of infected cells (> 10/cytospin) and inflammatory changes in the sediments corresponded invariably to patients with acute allograft dysfunction (8 of 8), and in most cases to biopsy specimens

showing PV interstitial nephritis (7 of 8). Based on these fmdings, urine samples seem to be the most sensitive and cost-effective screening method for PV infection; only urine samples with inflamed sediments and abundant infected cells correlate with clinically significant disease. In these cases, ex~minatinn of a renal biopsy is indicated. Immunohistochemical stains are useful to confirm the presence of PV but do not increase the sensitivity of diagnosis of PV if this is not already suspected on routine light microscopy. In our material, immunostains were helpful ruling out the presence of PV in a small number of biopsy specimens (2%) that showed markedly reactive tubular cells resembling PV infection. Most patients with PV interstitial nephritis responded to decreased immunosuppression; however, the decay in graft function (based on creafinine slopes) was significantly more rapid in these patients than in matched controls. Evidence of PV infection should be systematically sought in renal biopsy specimens and urine samples from renal aliograft recipients. HUM PATHOL 30:970-977. Copyright © 1999 by W.B. Saunders Company Key words: renal transplant, interstitial nephritis, SV40, viruria, nuclear inclusions. Abbreviation: PV, polyoma virus.

Human polyoma viruses (PV) are ubiquitous and accordingly positive serology is found in 65% to 100% of the population, TM depending on age-group and geographical location. The subgroup of polyoma consists of double-stranded DNA viruses, which measure 40 to 45 nm in diameter; included in this "family" are the JC and BK viruses that infect humans and the simian virus SV40.5 Although JC is primarily associated with progressive multifocal leukoencephalopathy,6 both JC and BK have been associated with pathological changes in the urinary tract. 7 Infection with BKvirus is believed to occur in childhood through the respiratory route ~,6,s

and in the latent stage the virus localizes in the renal epithelium. 2-9 Viral reactivation occurs when cellular immunity is lowered (through immunosuppression, pregnancy, uncontrolled diabetes, acquired immune deficiency syndrome, corticosteroid therapy, cytotoxic drugs).2,5,9,1°-12 Active infection and associated clinical disease have been demonstrated only in immunosuppressed patients) However, the incidence of positive serology for BK is the same in renal transplant recipients and in the general population. 12 Recently there has been renewed interest in the occurrence of PV-related interstitial nephritis and its differentiation from acute rejection 1~16; this preoccupation may be related to the potentially increased incidence of viral infections in renal transplant patients with the arrival of new and more potent immunosuppressive drugs. 13,14 The definite identification of PV infection in the urinary tract is done by conventional cell culture and viral isolation, indirect immunofluorescence, dot enzyme immunoassay, and DNA-DNA hybridization assay.~ In clinical practice, the cytological evaluation of urine samples is widely used for the diagnosis of PV infection. 17-19

From the Departments of Pathology, Medicine, and Surgery, University of Maryland School of Medicine, Baltimore, MD 21201. Accepted for publication April 20, 1999. Supported by a grant from the National Kidney Foundation of Maryland. Address correspondence and reprint requests to John C. Papadimitriou, MD, PhD, Director, Surgical Pathology, University of Maryland Hospital, 22 South Greene St, NBW43, Baltimore, MD 21201. Copyright © 1999 by W.B. Saunders Company 0046-8177/99/3008-0014510.00/0

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HUMANPOLYOMAVIRUS(Drachenberg et al) In the evaluation of urine samples for the purpose of identifying PV infection, it has b e e n emphasized that healthy individuals may shed virally infected cells sporadically. This is particularly true in 2% to 3% of p r e g n a n t women.17, 20 Several studies have surveyed the incidence, diagnosis, and clinical significance of PV infection in renal transplant recipients. 3,7,12-16,19,21-~ These reports highlight the i m p o r t a n c e of PV as a p a t h o g e n in renal transplants, but by describing the variability of the clinical, serological, and symptomatological correlations, they underscore the difficulty in the evaluation of these patients. T h e c u r r e n t study h a d four objectives: (1) To p e r f o r m a systematic a n d prospective evaluation of the incidence of PV in renal transplant biopsy specimens by light microscopic, immunohistochemical, and ultrastructural studies; (2) T h r o u g h the evaluation of a large n u m b e r of consecutive biopsy specimens to evaluate the potential limitations of the morphological identification of PV in routinely stained renal biopsy specimens, a n d to explore the additional value of i m m u n o p e r o x i d a s e stains and electron microscopy to identified virally infected cells; (3) To evaluate the incidence of PV-infected cells in urine samples and correlate these findings with renal biopsy specimens; a n d (4) To correlate the morphological findings with the clinical presentation.

the hospital with acute graft dysfunction (acute rise in serum creatinine). Also studied were urine samples from 100 patients with stable graft function that were evaluated in the outpatient clinic; in 14 of these patients concurrent biopsies performed as part of various protocols were available. For cytological evaluation, the urine samples were concentrated by centrifugation at 900g for 10 minutes. After decanting the supernatant, the sediment was mixed with an equal volume of fixative (Shandon cytospin collection, Shandon lnc, Pittsburgh, PA). From each specimen, two cytospin slides (Shandon Inc, Pittsburgh, PA) prepared with 8 drops of sediment each were prepared at 25gfor 5 minutes and stained with Papanicolaou stain. Electron microscopic evaluation was performed in all urine samples showing light microscopic evidence of viral infection (n = 37), and randomly in 10 negative samples. For the ultrastructural evaluation of urine samples, half of the material available per case was centrifuged at 900g for 15 minutes, and the pellet was embedded as previously described. 25 Statistical comparison of the least means square slopes of 1/creatinine versus time (in months) was done between patients with histologically and cytologically documented PV infection and a matched control group with negative urines and biopsies, using the Kruskal-Wallis test.

RESULTS Incidence of Polyoma Virus (PV) in Renal Biopsy Specimens

MATERIALS AND METHODS Six hundred one (601) consecutive, adequate renal transplant biopsies performed at the University of Maryland Hospital between August 1, 1997 and November 30, 1998 were prospectively evaluated with hematoxylin and eosin (12 sections), periodic acid-Schiff, periodic acid methenamin silver, and Masson's trichrome stains and with the peroxidaseantiperoxidase technique with SV40 antibody (Lee Biomolecular Research Laboratories, San Diego, CA, dilution 1:1,500, performed on 3 sections). Because of significant nucleotide sequence homology (69% to 75%), this antibody cross reacts with both BK andJC viruses. 24 The biopsies corresponded to 365 patients (127 females, 238 males), with a mean age of 46 years (range, 9 to 77). The racial distribution was 199 white, 163 black, and 3 Asians. The biopsies were done at 1 week to 15.5 years posttransplantation (mean, 22.4 months). Each patient had 1 to 4 biopsies during the study period (mean, 1.8). The biopsies were performed for acute (73%) or chronic (23%) deterioration of the renal function (increase in serum creatinine above baseline), or in anticipation of scheduled changes in the immunosuppression regimen (4%). Electron microscopy was performed in all cases with evidence of polyoma virus infection on light microscopy (n = 11), in 27 additional cases randomly chosen independently of the initial light microscopic findings, and in 12 samples showing tubular nuclear atypia suspicious for but not diagnostic of PV. For ultrastructural studies, the specimens were submitted using the procedure previously described2~; 36 samples were fixed directly in 4% formaldehyde and 1% glutaraldehyde, and 14 samples were obtained from the paraffin-embedded tissue. In each case, a minimum of 20 tubular cross sections was evaluated. Urine samples (20 to 100 mL each) were obtained concurrently with a renal biopsy in 100 patients admitted to

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Eleven of 601 renal transplant biopsy specimens (1.8%) f r o m 7 patients (1.9%) showed cytopathic changes in tubular cells consistent with PV infection. Randomly distributed cells (0 to 6 p e r high-power field) with the characteristic " s m o o t h " a n d gelatinous basophilic i n t r a n u c l e a r inclusions with n o p r o m i n e n t s u r r o u n d i n g halo were present; the morphological diagnosis of PV infection was c o n f i r m e d with i m m u n o histochemical stains and electron microscopy in all cases (see below). In addition to the viral inclusions, the nuclei of the tubular cells frequently showed m a r k e d variation in size (anisonucleosis), m a r k e d h y p e r c h r o m a sia, and smudging or clumping of c h r o m a t i n (Figs 1 and 2). Rare tubular cells had very large nuclei measuring u p to 40 p m (karyomegaly). In addition, 10% to 80% of the tubules showed evidence of tubular injury, consisting of tubular cell apoptosis, cell dropout, desquamation, and flattened epithelial lining. Scattered tubules showed flat, dense, and pyknotic d e g e n e r a t e d / d e s q u a m a t e d cells that a p p e a r e d to b e c o m e i n c o r p o r a t e d into thick intraluminal casts (Fig 1B). T h e tubular cells sometimes displayed a " h o b n a i l " shape. Multinucleation was not frequently seen in the cases with PV infection. This was in contrast to cases with reactive atypia but not PV infection (Fig 3). T h e a m o u n t of cytoplasm of the affected tubular cells varied significantly, but often cells with very large nuclei had proportionally a b u n d a n t a m p h o p h i l i c or basophilic cytoplasm. T h e distribution of the viral inclusions was r a n d o m , and all affected biopsy specimens showed, to some degree, " s k i p p e d " uninvolved areas. Patchy, m i x e d tubulointerstitial infiltrates were present in areas of tubular damage. Five p e r c e n t to 20% of

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FIGURE 3. Left, reactive changes in acute tubular necrosis. An intranuclear cytoplasmic inclusion (arrow) resembles polyoma virus nuclear inclusion. Right, multinucleation and nuclear vesicular changes (clearing) in chronic rejection. (H&E stains, original magnification × 150.)

FIGURE I. (A) Marked tubular anisocytosis, polyoma virus intranuclear inclusion (arrow), and apoptotic changes (arrowhead). Minimal inflammation and tubulitis are present. (H&E stain, original magnification ×150.) (B) Tubular necrosis and cast formation. Note anisocytosis, hyperchromasia, and karyomegaly (arrow), polyoma virus intranuclear inclusion (arrowhead). (H&~=stain, original magnification ×150.)

abnormal tubules showed permeation of their basement membranes by lymphocytes (tubulitis). Many tubules lined by virally infected cells did not appear to elicit any significant inflammatory response. Neutrophils were present in interstitium and tubules in areas with marked tubular damage. Arterial endotheliitis was not identified in any of the 11 biopsy specimens with PV inclusions. Biopsy specimens showing PV infection were invariably accompanied by urine samples showing moderately

numerous to abundant virally infected cells in their sediment (Fig 4). Immunoperoxidase stains for SV40 (Fig 5) were positive in tubular cell nuclei and in intraluminal contents of the tubules, showing the changes described. The staining pattern was diffuse nuclear or confined to well-circumscribed intranuclear inclusions; occasional cells displayed a "nucleolar-like" staining pattern (small viral inclusions). The cytoplasm of tubular cells was positive, with a granular pattern only in a minority of cells. Definite endothelial or glomerular inclusions were not identified in any case. Electron microscopy examination showed focal severe changes in tubular cells changes consisting of (1) viral inclusions predominantly in nuclei and less often in the cytoplasm of tubular cells. The viral particles measured 39 to 42 nm in diameter (Figs 6 and 7); (2) reactive/degenerative (injury-related) changes, including isolated tubular cell necrosis or apoptosis, cytoplasmic vacuolization, blebbing, loss of brush border, prominent lysosomal inclusions, luminal protein, and cellular casts. In addition, cytoskeletal reorganization, binucleafion, prominent nucleoli and reduplication of the tubular basal lamina were noted (Fig 6). The presence of viral inclusions in urine was confirmed with electron microscopy in all samples (Fig 7). R e a c t i v e Tubular Cells in A c u t e a n d C h r o n i c A I I o g r a f t R e j e c t i o n Simulating PV I n f e c t i o n

Tubular reactive and degenerative changes that had to be differentiated from PV infection were seen in 12 of 601 biopsy specimens (2%) (Fig 3). These biopsy specimens corresponded to chronic rejection in 3 cases, acute rejection in 7 cases (types IB and IIa, Banff scheme 1997),26 acute tubular necrosis in I case, and an otherwise unremarkable histology in 1 case. These cases showed focal reactive and degenerative nuclear and cytoplasmic changes in epithelial ceils in 0.5% to 40% of the tubules. The tubular cells displayed moderate to marked anisocytosis and hyperchromasia; the nuclei often contained nuclear cytoplasmic inclusions as well as vesicular changes in the chromatin (Fig 3). Smudg-

FIGURE 2. Rare infected cells in biopsy specimens from patients 2 to 4 months after reduction of immunosuppression. (H&E stain, original magnification × 150.)

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HUMAN POLYOMA VIRUS (Drachenberg et al)

Immunoperoxidase stains for SV40 performed in 2 to 3 sections of each of these cases and electron microscopic studies failed to show viral particles. Ultrastructurally, the tubular epithelial cell nuclei showed reactive features (prominent nucleoli and vesicular chromatin or evenly dispersed euchromatin). The urine samples obtained concurrently with the 12 biopsy specimens with atypical cells but no immunohistochemical or ultrastructural evidence of PV infection showed no cytological evidence of vitally infected ceils. Electron microscopy p e r f o r m e d from urine samples from 6 of these patients also did not show viral particles. PV Inclusions in Urine; C o r r e l a t i o n With Biopsy Findings

Urine samples from 100 patients with acute ailograft dysfunction and 100 patients with adequate graft function and stable creatinines for at least 3 months contained PV-infected cells in 18 and 19 cases (18% and 19%), respectively. Concurrent biopsies in the 18 patients with acute graft dysfunction showed PV infection in 7 patients (39%); the remaining biopsy specimens showed acute rejection (n = 7), thrombotic microangiopathy (n = 1), pyelonephfitis (n = 1), and acute tubular necrosis (n = 2). Of the 19 patients with stable graft function that had P¥ in urine, 14 had biopsies, and none showed PV in renal tissue (0%). In the urine, the infected cells displayed rounded nuclei that varied in size but were generally larger than the normal transitional and tubular cells. PV cytopathic changes appeared as the typical "gelatinous," dense, or granular basophilic inclusion often occupying most of the nucleus and with no definite surrounding halo (Fig 4A and B). Often a rim of granular chromatin was seen

FIGURE 4, (A and B) Urine sediment: Typical gelatinous intranuclear inclusions (arrow). There is no "halo." The cytoplasm in most cells shows degenerative changes. Note large, atypical cell with clumped chromatin. (Papanicolaou stain, original magnification x400.) (C) Urine sediment: Cellular cast with nuclei showing features of polyoma virus infection. (Papanicolaou stain, original magnification x250,)

ing and smooth distribution of the chromatin resembling PV inclusions were rarely seen (<2% of atypical cells). Tubular cell multinucleation was common, particularly in the cases with chronic rejection. Thick intraluminal casts were occasionally seen. Tubulointersfifial infiltrates and marked tubulifis were present in association with the reactive/degenerative tubular epithelial changes in the cases of acute rejection. Neutrophils were present in the intersfifium and tubules in areas with marked tubular damage. Plasma cells were seen in larger numbers than in cases with PV infection. Sparse interstitial inflammation (lymphocytes and plasma cells) was seen in cases of chronic rejection.

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FIGURE S. Immunoperoxidase stain for SV40, Strong staining of nuclei and intraluminar contents. Cytoplasm stains weakly, (H&E stain, original magnification x400.)

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FIGURE 6. Electron micrograph showing marked tubular damage. Cell apoptosis and dropout (asterisks), loss of brush border (arrows), intranuclear viral particles (V). Reduplication of basal lamina (arrowheads). (Original magnification x7,000.)

around the inclusion. Other cells had vesicular nuclei, with clumped chromatin. One or multiple moderately large, red nucleoli were seen in cells with slightly clumped and "washed-out" chromatin. The cytoplasm of many epithelial cells in urine appeared vascuolated, foamy, and often had globular clear or eosinophilic inclusions. In the urines with abundant virally infected cells, the background also contained transitional cells, tubular cells isolated or forming part of casts (Fig 4C), clumps of amorphous basophilic material, and inflammatory cells. Extremely large nuclei that simulated a malignant neoplastic process were present in two cases

FIGURE 7. Electron micrographs of infected cell in unne. (Original magnification x 17,000; Insert, x72,000.) 974

(Fig 4A); these cells had degenerative changes in their cytoplasm. Electron microscopy studies of urines showing no viral inclusions on light microscopy were consistently negative; the opposite was true for urine samples containing viral inclusions on light microscopy. In all of these cases, viral particles with the typical features of PV infection were seen ultrastructurally. Correlation B e t w e e n Q u a n t i t a t i v e Findings in Urine C y t o l o g y a n d Clinical Presentation

When the urine samples were divided according to the n u m b e r of infected cells in urine sediment (rare, up to 4 and more than 10/cytospin) and the quality of the background (inflammatory versus noninflammatory), it was found that patients with scanty viral inclusions in urines and sediments with no inflammation were less likely to have biopsy specimens with PV infection than those with abundant infected cells in urine and inflammatory sediments (0 of 24 v 7 of 8; P < .001, Fisher's exact test). Decay in renal function based on the creatinine slopes showed that patients with few (up to 4/cytospin) virally infected cells and noninflamed sediments were no different from negative controls after 10 months to 4 years of follow-up. In contrast, patients with abundant (>10/cytospin) PV-infected cells in urine, inflammatory sediments, and biopsy-proven PV interstitial nephritis had significantly greater decay in renal function than patients with no evidence of PV infection in urines and biopsy specimens (P = .013).

HUMANPOLYOMAVIRUS(Drachenberg et al) Correlation Between Morphological Evidence of Polyoma Virus Infection and Clinical Presentation The 11 positive biopsy specimens corresponded to 7 patients (1.9%, 6 men and 1 woman), with a mean age of 52.4 (range, 35 to 70). The biopsies were performed 6 to 42 months posttransplantation (mean, 21.2). Six of the patients had acute rise in creatinine from a mean baseline of 2.2 m g / d L to 3.4 m g / d L . The remaining patient had a slow rise in creatinine (1.9 to 2.5) over 2 months. One patient who presented with abundant virally infected cells in urine but a negative biopsy had an acute rise in creatinine from 2.3 to 2.9. Generalized infectious symptoms, hemorrhagic cystitis, and ureteral stenosis were not identified in any of the patients. Five of six patients presenting with acute allograft dysfunction were initially treated with high-dose intravenous corticosteroids before the diagnosis of PV nephritis was made; immunosuppression was then decreased (50% to 75% of standard doses). In 5 of these patients, creatinine values returned to baseline within 3 weeks (mean, 2.1; 80%). In the other patient, a component of concurrent acute allograft rejection was suspected, and aggressive antirejection treatment with antilymphocytic therapy was instituted for 8 to 10 days; this treatment was accompanied by a persistent rise of creatinine. Later reduction in immunosuppression in this patient resulted in stabilization of the renal function with a serum creatinine of 5.4 m g / d L . Follow-up biopsies in 4 of 7 patients (1 to 6 months after diagnosis of PV infection) showed marked decrease in the n u m b e r of PV-infected cells and associated tubular damage in 3 patients (Fig 2) and persistent unchanged PV interstitial nephritis in 1. Once more, a trial of antirejection treatment failed to improve renal function in this patient, and immunosuppression was again reduced with stabilization of creatinine at 4.9. Three to 18 months after diagnosis of PV infection (mean, 11 months), these 7 patients have mean serum creatinine of 3.2 m g / d L .

DISCUSSION The true incidence of h u m a n polyoma virus excretion in the urine of healthy individuals is difficult to determine; a study of over 7,000 urine samples from nontransplant patients 2,1a found that 0.3% to 0.85% of urine samples contained PV-infected cells. Only a minor proportion of these patients were immunosuppressed, but they had urinary tract symptoms (mostly hematuria) that prompted the evaluation of urine samples and therefore can hardly be considered a healthy population. Conversely, several studies have consistently shown that urinary excretion of PV in renal transplant patients is high, with occurrences reported between 8% and 26%.12,17,19,21,27 Patients with negative serology for PV do not have evidence of viruria9; however, only some patients with serological studies indicating active PV infection (5% to 68%) excrete viral particles in urine. 12,21 It has been

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speculated that patients with serological evidence of active infection and no viruria could have involvement of organs other than the urinary tract. 21 In bone marrow transplant patients, hemorrhagic cystitis and transient hepatic dysfunction have been attributed to PV infection. 5 The definite association between hemorrhagic cystitis and PV, 9'28"3° however, has been questioned and considered rather coincidental. Favoring the latter idea is the fact that hemorrhagic cystitis has not been identified as part of the clinical picture in renal transplant patients with PV infection. The deleterious effects of PV in the kidney have been well documented in transplant recipients. 7,12-16,23 One of the best studied cases exemplifying renal involvement by PV concerned a child with congenital immunodeficiency that developed extensive tubulointerstitial and urothelial damage with progressive renal failure. 29 The manifestations of PV infection in renal transplant recipients consist of ureteral stenosis and interstitial nephritis. 9,1° In one study, ureteral stenosis and renal failure were associated statistically with urinary excretion of PV, whereas antibody rise to BK was associated with rising creatinine and need for transplant biopsy. 21 Another study, however, failed to show clinically significant findings in 24 patients who excreted virus in urine, a9 The marked variability in the degree of renal involvement could be explained by the possibility of occurrence of mutated strains of PV with the potential to alter viral DNA replication and enhance pathogenicity. 1° Ureteric obstruction was described in the first reported patients with BK infection but was seen only in a small proportion of the cases in subsequent reports~,7&18,19,22,31; none of our patients with viruria had evidence of ureteral stenosis. It has been speculated that ischemic or inflammatory damage makes ureteral epithelium more susceptible to PV infection. 3 In the state of latency, polyoma virus is distributed throughout the kidney in random foci. 13,2° The locality of PV infection can explain the one case in our study with acute increase in creatinine that had abundant virally infected cells in urine but a negative renal biopsy. It may be speculated also that the infected ceils derived from the patient's native kidney. Cytological evaluation of urine samples is routinely used in clinical practice for the diagnosis of PV infection. The infected exfoliated cells have cytological features consistent with tubular epithelial originag; a significant proportion of the cells present in our samples appeared to derive from tubules and were in some cases associated with well-formed casts, also indicating an origin in the nephron. This is consistent with the high degree of correlation between renal biopsy specimens showing PV infection and positive urine samples. Conversely, almost one fifth of the patients with stable renal function and negative biopsies had rare PV-infected ceils in urine. We can conclude based on this study that the routine cytological evaluation of urine sediment (with or without additional electron microscopic evaluation) appears to be for practical purposes the most sensitive m e t h o d to determine the presence of PV in the

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urinary tractZ,S; the clinical significance of this finding, however, depends on the viral load and the quality of the sediment. Patients with large numbers of infected cells and evidence of inflammatory damage to renal epithelium presented with acute allograft dysfunction and in most cases had correlative biopsy findings. It has been reported previously that the appearance of large numbers of cells with PV carries a gloomy prognosis. 3 In a study by Coleman, 22 although 29% of patients excreted virus in urine, only 6.7% of these patients had large numbers of inclusions. 22 In our study, we found evidence of more rapid deterioration of graft function in patients with large numbers of inclusions in urine and PV interstitial nephritis (in spite of being treated with reduced immunosuppression) when compared with controls. From the morphological point of view, cells infected by PV need to be differentiated from cytomegalovirus cytopathic changes and from malignancy. 32,3sThe findings in our cases are consistent with previous studies indicating that malignant cells have more hyperchromatic nuclei with coarser chromatin that contrasts with the structureless nucleus in cells infected with PVi; also, the severe cytoplasmic degeneration characteristic of PV infection is helpful in the diagnosis. Typical cytomegalovirus inclusions in the nucleus are usually smaller and orangeophilic, surrounded by a clear halo, and concurrent cytoplasmic inclusions are common. 2,19 PV excretion in urines from almost 20% of renal transplant recipients with stable graft function may suggest that PV infection is common and harmless to the graft. The findings of our study, however, indicate that PV infection can cause important structural damage to the kidney and can be associated with acute allograft dysfunction that does not respond to antirejection treatment but rather to decrease in the immunosuppression regimen. Similar conclusions were reached in a recent study of a larger n u m b e r of infected patients by Randhawa et al. 13 The association between tacrolimus and increased incidence of PV infection in renal transplant patients has been recently emphasized) TM The differentiation between PV infection and tubular epithelial atypia secondary to other causes can be achieved with systematic evaluation of the morphological features and with the aid of immunohistochemical stains for SV40 or in situ hybridization for BK andJC. 1~ These ancillary studies are not indicated as screening tools but rather should serve as a confirmation of the diagnosis of PV infection when suspicious nuclear features are seen on routine stains. A definite distinction between PV interstitial nephritis and acute allograft rejection may occasionally be impossible because tubulitis is a feature c o m m o n to both processes. In our experience, the presence of numerous virally infected cells with associated marked tubular damage, disproportionally mild tubulitis, and lack of vascular rejection warrants decrease of the immunosuppression regimen. The presence of large amounts ofvirally infected cells in urine and an inflammatory sediment are also a sign that an active PV nephritis is taking place.

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We propose that because renal biopsies are now accepted as the gold standard for the diagnosis of acute allograft rejection, 26 in patients with acute allograft dysfunction, concurrent semiquantitative evaluation of a cytological urine sample also should be performed to evaluate for PV infection. This procedure is inexpensive and easy. Although light microscopic findings are definitive in most cases, part of the urine samples can be routinely saved in electron microscopic fixative (4F1G) that can be stored indefinitely. If on the urine sample there are numerous PVinfected cells, a renal biopsy is indicated. Renal biopsy specimens from patients with significantly atypical tubular cells and biopsy specimens from patients with positive urine samples should be examined with immunoperoxidase stain for SV40 or in situ hybridization for BK orJC to further evaluate for PV infection.

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HUMAN POLYOMA VIRUS (Drachenberg et al) 18. Minassian H, Schinella R, Reilly JC: Polyomavirus in the urine: Follow-up study. Diagn Cytopathol 10:209-211, 1994 19. Traystman MD, Gupta PK, Shah KV, et al: Identification of viruses in the urine of renal transplant recipients by cytomorphology, Acta Cyto124:501-510, 1980 20. Chesters PM, Heritage J, McCance DJ: Persistence of DNA sequences of BKandJC virus in normal human tissues and in diseased tissues.J Infect Dis 147:676-684, 1983 21. Hogan TF, Borden EC, McBain JA, et al: Human polyomavirus infections with JC virus and BK in renal transplant patients. Ann Intern Med 92:373-378, 1980 22. Coleman DV, Mackenzie EFD, Gardner SD, et al: Human polyomavirus (BK) infection and ureteric stenosis in renal allograft recipients.J Clin Patho131:338-347, 1978 23. Purighalla R, Shapiro R, McCauleyJ, et al: BKvirus infection in a kidney allograft diagnosed by needle biopsy. Am J Kidney Dis 26:671-673, 1995 24. Shinohara T, Matsuda M, Cheng SH, et al: BKvirus infection of the human urinary tract. J Med Viro141:301-305, 1993 25. Papadimitriou JC, Drachenberg CB: Giant mitochondria with paracrystalline inclusions in paraganglioma of the bladder: Correlation with mitochondrial abnormalities in paragangliomas of other sites. Ultrastruct Patho118:563-568, 1994 26. Solez K, Benediktsson T, Cavallo T, et al: Report of The Third Banff Conference on Allograft Pathology (July 20-24, 1995) on

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