Immunosuppressive treatment predisposes to infectious complications in organ transplantation. One such infection seen after organ transplantation is BK polyoma virus infection (1–10). BK polyomavirus is a member of the papovavirus family. Asymptomatic infection can be seen in 70% of the normal population (11). Occasionally symptomatic infection can be seen in nontransplant immunosuppressed individuals (12,13). Polyoma virus (PV) has a potential to reside in the kidney or urinary tract and may reactivate with immunosuppression in transplant recipients (5,8). Because of its inherent ability to adhere to the urothelium, it can cause ureteral strictures leading to hydronephrosis (5,7). Occasionally, PV can cause interstitial inflammation in the allograft mimicking acute rejection (5–7). This may progress toward graft failure. It is thus important to identify features of PV infection that may differentiate it from acute kidney transplant rejection. We present our single center experience with PV infection. We estimated the outcome of the infected allografts. In addition, to differentiate PV infection from acute rejection we characterized the type of infiltrating lymphocytes in the allograft by immunophenotyping.
MATERIAL AND METHODS
We evaluated all kidney and kidney/pancreas transplants performed between January 1996 and July 1999 at the Medical College of Wisconsin. The diagnosis of PV infection was based on characteristic intranuclear inclusion in renal tubular epithelial cells identified in renal biopsies and the presence of viral cytopathic effect in the urine cytology specimen of each case. Cytomegalovirus (CMV) infection was excluded by direct fluorescence antibody test in peripheral lymphocytes. Immunostaining in renal tissues was performed to exclude CMV and herpes simplex virus (HSV) infections. In selected cases 4- to 5-μm sections of renal biopsies were deparaffinized and stained with CMV and HSV antibodies (Dako, Carpentina, CA) for immunoperoxidase staining to detect these viruses. Needle biopsy specimens from allograft kidneys were formalin-fixed and paraffin-embedded, sectioned at 4 μm, and stained with hematoxylin and eosin for light microscopic evaluation. Immunohistochemical staining for CD3, CD4, CD8 (T cells), CD20 (B cells), TIA-1 (natural killer [NK] and cytotoxic T cells), and CD56 (NK cells) was performed on paraffin-embedded tissue. Four-micrometer sections on poly l-lysine–coated glass slides were deparaffinized and treated with a mixture of methanol and hydrogen peroxide. Epitope (antigen) retrieval was achieved by microwaving in a buffer solution or by treating with proteinase K enzyme, and staining was performed using Dako Autostainer (Dako, Carpentina, CA). The percentage of lymphocytes staining for each antibody was estimated by two hematopathologists, independently and blindly. Biopsy specimens in six cases with PV infection were compared with six other recipients who had biopsy-proven acute rejection. Actuarial kidney survival was estimated by Kaplan-Meier method (14) for patients with PV infection. Mann-Whitney U test was used for statistical significance, and P <0.05 was considered significant.
There were 10 cases of histologically diagnosed PV infection between January 1996 and July 1999. The detailed demographics of the immunosuppressive regimen and the time to diagnosis and follow-up of these cases are shown in Table 1. The immunosuppressants consisted of mycophenolate mofetil (MMF) and tacrolimus (FK) in eight cases and MMF and cyclosporine (CsA) in two. The recipients who received CsA were switched over to FK before the diagnosis of PV infection. All recipients experienced renal dysfunction for which they underwent a renal biopsy. Hydronephrosis was not seen in these patients by ultrasound examination. Seven of the 10 cases had prior diagnosis of acute rejection for which they had received intravenous methylprednisolone. In addition, two of these seven received anti-T cell agents (OKT3/ATGAM) for the treatment of apparent acute rejection. All 10 recipients who had PV infection were also diagnosed to have concurrent acute rejection by light microscopic examination. Nine of these patients received intravenous methylprednisolone and none responded to treatment. One of these recipients also received ATGAM followed by OKT3 because of suboptimal response to intravenous methylprednisolone.
The histologic findings were characterized by typical homogenous basophilic intranuclear inclusion in renal tubular epithelial cells on light microscopic examination. There were also interstitial mononuclear infiltrates, tubular atrophy, and interstitial fibrosis. There was no evidence of CsA or FK toxicity. Urine cytology demonstrated typical decoy cells with the characteristic cytopathic effect of PV. Other infections such as CMV and HSV were excluded by immunohistochemistry of renal tissue, and peripheral lymphocytes were studied to exclude CMV by direct fluorescence antibody test.
The percentage of lymphocytes positive by immunostaining for CD3, CD4, CD8 (T cells), CD20 (B cells), CD56 (NK cells), and TIA-1 (NK and cytotoxic T cells) for patients with acute rejection and PV infection (n=6 each) is shown in Figure 1. In acute rejection, the average values for CD3, CD4, and CD8 were 91 (range, 81–95), 66 (range, 26–70), and 33 (range, 25–40), respectively, and the corresponding values for PV interstitial nephritis were 78 (range, 60–95), 56 (range, 40–65), and 22 (range, 15–30). The CD3 (P =0.1017), CD4 (P =0.7619), and CD8 (P =0.2316) values were not significantly different between the two groups (Fig. 1). However, the percentages of CD20-positive cells in renal biopsies of patients with acute rejection and PV interstitial nephritis were 6% (range, 0–10%) and 21% (range, 5–40%), respectively, higher in the latter group (P =0.039;Fig. 1). Immunostaining for TIA-1 (NK and cytotoxic T cells) and CD56 (NK cells) was used to quantify NK cells and cytotoxic T lymphocytes. Because CD56-positive cells were negligible, all cells positive for TIA-1 were considered to be cytotoxic T cells. As shown in Figure 1, there were significantly lower cytotoxic T lymphocytes in PV-infected allografts (7%; range, 2–15%) compared with those with acute rejection (24%; range, 15–30%;P =0.0159).
Figure 2 depicts the interstitial inflammation in typical examples of PV infection and acute rejection. Under light microscopy, both show prominent infiltration of the interstitium by lymphocytes and variable edema. Figure 3 shows the pattern of immunostaining for CD20 and CD3 in representative cases of PV infection and acute rejection. In acute rejection, the B-cell component (CD20-postive cells) is markedly decreased compared with PV infection. Table 2 shows the clinical features and outcome of six patients each with acute rejection and PV interstitial nephritis. Among six patients with acute rejection, there were two irreversible graft failures (one chronic rejection and one died with a functioning graft). The remaining four patients have stabilized renal functions. However, four of six recipients with PV infection lost their allograft, and two have a functioning kidney.
Table 1 also shows the time from transplantation to diagnosis of PV in these recipients, renal function (plasma creatinine) at the time of diagnosis, follow-up, and current graft status for all 10 recipients. The median time from renal transplantation to diagnosis of PV infection was 9.5 months (range, 6–32 months). Immunosuppression was uniformly reduced in all 10 patients. Both MMF and FK doses were halved, and OKT3/thymoglobin were not administered after the diagnosis of PV infection. The median time from PV infection to graft failure was 4 months (range, 2–33 months). The median time from the time of transplantation to graft failure was 14 months (range, 9–40 months). Progression toward kidney failure was rapid in six of seven cases. The 1-, 2-, and 3-year posttransplant kidney allograft survival for those PV-infected allografts was 78.9%, 54.7%, and 36.4%, respectively. Three grafts are still functioning 1, 4, and 10 months after diagnosis. These patients were switched from FK to CsA after the diagnosis of PV infection. The remaining seven are alive on maintenance dialysis therapy. None of our patients have yet been retransplanted. Transplant nephrectomy has been performed to prevent reinfection after second transplantation.
PV can be seen in urine in normal as well as immunosuppressed individuals (11). However, PV infection causing renal damage is seen in immunosuppressed transplant recipients (4–10). Reactivation of latent virus causing interstitial nephritis is thought to be a mechanism involved in transplant recipients (5,8). Histologically, it mimics acute rejection and progresses toward renal scarring (5). PV infection has been recognized only in the last 10 years. Immunosuppressants such as FK have been implicated in the pathogenesis of PV infection (5,6,9,10). Stabilization of renal function has been achieved with CsA treatment (10).
Diagnosis of this infection in our patients is based on renal histology and urine cytology. Typical homogenous basophilic viral inclusions involving the renal tubular epithelial cells was seen and is characteristic of this infection. These characteristic findings have been illustrated in other studies (5–7). In our series we have also ruled out CMV and HSV as a cause of interstitial inflammation. In addition, in all cases urine cytology documented the presence of the classic PV cytopathic effect.
The lymphocyte infiltrates of PV infection have not been characterized previously. In comparison to patients with acute rejection, there were more B cells (CD20) and fewer cytotoxic T cells. This is an important diagnostic tool that differentiates this infection from acute rejection. Proper diagnosis of PV infection avoids unnecessary antirejection treatment. The characterization of interstitial lymphocytic infiltrates for patients with renal dysfunction should be considered for renal transplant recipients who are not responding to antirejection treatment. Pathogenesis of interstitial nephritis secondary to PV has not been studied. Viral infection in the presence of immunosuppressive state may be stimulating B-cell response resulting in interstitial nephritis. Poor response to methylprednisolone and or anti-T cell agents is not surprising owing to enhanced B-cell activity.
From this current series, 7 of these 10 patients (70%) developed irreversible graft failure. A similar degree of graft failure (67%) was seen by Radhawa et al. (5). Most of our grafts were lost within 6 months after diagnosis. Switching patients to CsA may be beneficial as three of our recipients have stabilized renal function with CsA.
In conclusion, poor response to antirejection treatment should raise the suspicion for PV infection in renal allografts. Reduced allograft survival is seen with PV infection. More B cells (CD20) and fewer cytotoxic T cells in allografts seems to be characteristic of PV infection. Diagnosis with immunostaining for T and B cells and cytotoxic T cells can be used as a guide to differentiate PV infection from acute rejection.
The authors thank Linda Eisert for manuscript preparation.
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