Treatment of polyomavirus infection with cidofovir in a renal-transplant recipient

Ola Bjorang1,, Hilde Tveitan1, Karsten Midtvedt2, Lars U. Broch2, Helge Scott1 and Per A. Andresen1

1 Department of Pathology and 2 Department of Nephrology, National University Hospital, Oslo, Norway

Keywords: biopsy; BKV; cidofovir; immunosuppression; JCV; polyomavirus



   Introduction
 Top
 Introduction
 Materials and methods
 Discussion
 References
 
Asymptomatic infection with human polyomaviruses BK (BKV) and JC (JCV) is common and usually acquired during the first decade of life (~60–70% of healthy adults are serologically positive) [1]. Recently, there has been an increased awareness of BK-virus nephropathy (BKN) as a new complication affecting renal allografts and causing graft dysfunction. Manifestations of the disease in renal-transplant recipients include interstitial nephritis, ureteric stenosis, renal dysfunction, and in extreme cases graft loss [24]. Distinction of polyomavirus infection from acute allograft rejection is of particular importance and may represent a diagnostic challenge. Patients at risk of BKN are diagnosed by the presence of intranuclear viral inclusion bodies in cells shed in urine and in renal epithelial cells (‘decoy cells’). Molecular diagnosis based on real-time quantitative polymerase chain reaction (Q-PCR) on urine/blood strengthens the suspicion. Final diagnosis is made by specific findings in the allograft biopsy, in particular positive immunohistochemical staining for viral proteins.

There is a possible association between the use of tacrolimus (Prograf®) and/or mycophenolate mofetil (MMF) (CellCept®) after transplantation and the increasing frequency of BKN [3]. If an ongoing polyomavirus infection is detected, a reduction of the current immunosuppressive medication is recommended. The goal of decreased immunosuppressive therapy is to restrain viral activity without triggering rejection. Reduction of immunosuppression may stabilize renal function when instituted early; however, BKN may progress to graft loss. Specific antiviral therapy is needed. Cidofovir, a nucleotide analogue of cytosine that is active against various DNA viruses, is an agent with activity against polyomaviruses [57]. We hereby report our experience with intravenous (i.v.) cidofovir in a renal-transplant recipient with an ongoing polyomavirus infection.



   Materials and methods
 Top
 Introduction
 Materials and methods
 Discussion
 References
 
DNA was extracted from urine sediment and blood plasma by the use of a QIAamp DNA Blood Mini Kit (Qiagen). Q-PCR was performed by an established real-time PCR using an ABI Prism 7900HT Sequence Detector (Applied Biosystems). Primers and probe specific for BKV large T antigen (GenBank Accession No. V01109) were used; PYV.for 5'-TAG GTG CCA ACC TAT GGA ACA GA-3', PYV.rev 5'-GAA AGT CTT TAG GGT CTT CTA CC-3' primers [8], TaqMan-probe 5'-FAM-CAT TAA AGG AAC TCC ACC AGG ACT CCC ACT C-TAMRA-3'. A standard curve was constructed by plotting the CTs against the logarithmic values of a serial dilutions (0.05–5000 fg) of the plasmid standard pBKV(34–2) (ATCC No. 45025). All patient samples were tested in triplicate, their respective CTs were determined, and the copies of BKV per millilitre of urine or blood plasma were calculated from the standard curve [9].

Case
We present a 21-year-old Caucasian female with chronic pyelonephritis who had received a renal transplant from her father in 1998 (HLA 2–1 mismatch, CMV neg->pos). Initial immunosuppression consisted of cyclosporin (Neoral®), prednisolone, and azathioprine. She experienced four biopsy-proven acute rejection episodes during the first 2 months after transplantation. The three first rejection episodes were successfully treated by i.v. methylprednisolone. The fourth was steroid resistant but successfully treated by i.v. OKT3 and a switch from cyclosporin to tacrolimus. Serum-creatinine (s-creatinine) stabilized at 110–130 µmol/l. Twenty-two months after the transplantation there was an acute increase in s-creatinine to 170 µmol/l. A graft biopsy showed an interstitial nephritis and tubulitis very similar to an acute cellular rejection, but with a number of epithelial cells with large nuclei, raising the suspicion of a polyomavirus infection. Our knowledge of polyomavirus infection in renal transplant recipients at that time was rather limited. It had been stated that immunosuppression should be reduced, so oral prednisolone was reduced from 10 to 7.5 mg/day, whereas tacrolimus (5 mgx2/day, with trough values around 7 ng/ml) and azathioprine (50 mg/day) were unaltered. S-creatinine stabilized at 150–160 µmol/l. Eight months later there was another increase in s-creatinine to 300 µmol/l. A new graft biopsy showed an interstitial nephritis plus development of chronic allograft nephropathy with fibrosis. Unfortunately, the interstitial nephritis was not thought to be related to polyomavirus (no staining was performed at that time) but was rather a sub-acute rejection. The patient received methylprednisolone i.v. and was changed from azathioprine to MMF. S-creatinine declined to around 200 µmol/l for a short period but 4 months later (January 2001) she was re-submitted with an increase in s-creatinine, now to 310 µmol/l. Once again a renal graft biopsy was performed. At that time in situ hybridization, immunohistochemistry and PCR-based analysis for polyomavirus had been established at our pathology department. Massive BK virus infiltration was detected in the renal biopsy (Figure 1Go), BK virus was detected in plasma and high titres of BK virus were confirmed and monitored in urine by real time PCR (Figure 2Go).



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Fig. 1.  BKV T antigen (brown staining) in nuclei of tubular cells. Indirect immunoperoxidase technique, monoclonal antibody, SV40 T Ag (PAb416, OncogeneTM) cross-reacts with BKV T antigen, as primary reagent. (Original magnification 600x)

 


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Fig. 2.  Copies of BK virus per millilitre urine/plasma from the patient. Urine samples were analysed between January 2001 and May 2002. Plasma samples were analysed between June 2001 and May 2002. The period with cidofovir treatment are indicated by arrows.

 
We decided to withdraw MMF therapy and to reduce tacrolimus, aiming at trough values of 3–5 ng/ml and in addition to try i.v. cidofovir therapy. Cidofovir is known to be highly nephrotoxic. Treatment involved 2 h of hydration (1000 ml 0.9% normal saline i.v.) preceding i.v. infusion of 0, 25 mg/kg (15 mg) cidofovir in 1000 ml 0.9% normal saline. The patient tolerated the infusions well, with no noticeable side-effects, specifically no proteinuria. Cidofovir was later increased to i.v. 0.42 mg/kg (25 mg) every 2 weeks (total dose 295 mg over a period of 7 months). A subsequent fall in urine viral load during treatment was observed (Figure 2Go). The virus was also cleared from the blood (PCR negative) during this time. The patient has now been followed up for 4 months after the last cidofovir infusion. S-creatinine is stable at 300 µmol/l and there is still no detectable BKV in plasma; however, there are persistently low levels of BKV in the urine.



   Discussion
 Top
 Introduction
 Materials and methods
 Discussion
 References
 
The tremendous rise in BKN over the last few years could be attributed to an increased use of biopsies and improved methods for identifying the cause of allograft dysfunction. It is, however, more likely to be a consequence of the introduction of new and more potent immunosuppressive drugs with an increase in overall immunosuppression as a result [24].

However, a reduction of the dose of immunosuppressive drugs decreases the viral load, but increases the risk of rejection. A close follow-up of the patients is necessary. It is likely that the interstitial nephritis associated with polyomavirus infection may lead to progressive fibrosis and chronic allograft nephropathy as in our patient, since the long-term outcome in BKN patients is worse than in patients without active polyomavirus infection [24]. It is therefore important to diagnose these patients early, before tubulointerstitial damage with progression to fibrosis occurs. Although reduction in immunosuppression often leads to stabilization of renal function, no actual improvement in renal function in BKN has been reported. Cidofovir has proved to be effective against polyomavirus in vitro and in vivo and several groups have reported on cidofovir treatment of BKV-associated haemorrhagic cystitis [57]. However, this is the first report on cidofovir treatment of a renal transplant recipient with ongoing polyomavirus infection. As shown in Figure 2Go there was a fall in urine viral load during therapy. We cannot, however, relate this fall solely to the use of cidofovir, since immunosuppression was reduced simultaneously. The urine viral load also remained low after cidofovir therapy was terminated. In contrast to a recent report on renal failure induced by cidofovir treatment [10], cidofovir was not found to be nephrotoxic in our study. Still, graft function was not improved during cidofovir treatment.

In conclusion, cidofovir used as described in this report can reduce the viral load in renal transplant recipients with polyomavirus infection and possibly improve the long-term prognosis. Molecular diagnostic testing is a useful method to monitor affected patients during treatment. Further studies with a larger number of patients are needed to monitor and confirm the effect of cidofovir treatment.



   Acknowledgments
 
The authors thank Dr Christine H. Rinaldo, University of Tromsoe, Norway, for the plasmid standard pBKV(34–2).



   Notes
 
Correspondence and offprint requests to: Ola Bjorang, MSc, Department of Pathology, National University Hospital, N-0027 Oslo, Norway. Email: ola.bjorang{at}rikshospitalet.no Back



   References
 Top
 Introduction
 Materials and methods
 Discussion
 References
 

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Received for publication: 29. 4.02
Accepted in revised form: 4. 7.02