POLYOMAVIRUS INFECTIONS OF RENAL ALLOGRAFTS

Volker Nickeleit ¹ and M.J. Mihatsch²

¹ Nephropathology Laboratory, Department of Pathology, The University of North Carolina, Chapel Hill, NC, USA
²Institute for Pathology, University of Basel, Switzerland

Volker_Nickeleit@med.unc.edu

Introduction:
Different viruses may infect kidney transplants and replicate in the allograft. Epstein-Barr Viruses causing lymphoproliferative diseases and polyomavirus infections with the BK-virus strain are clinically most significant. Infections with CMV or adenoviruses are rare. The following brief review will focus on nephropathies caused by BK-viruses.

Prior to the mid 1990s, polyomavirus infections of the kidney with the BK-Virus strain (i.e. BK-Virus nephropathy, BKN) did not gain any clinical attention and had been exceptionally rare in the 1970s and 80s ^{1, 2}. However, over the last few years the clinical scenario has fundamentally changed. Currently, the prevalence of BKN in different transplant centers varies between less than 1% and 5.5% ^3-8. At present, BKN is by far the most common viral disease affecting renal allografts (approximately 10 to 20 times more frequent than CMV). Thus, we are dealing with a new complication, and new risk profiles promoting BKN have to be considered.

In the immune competent human host polyomaviruses (BK- and JC-Virus strains) are without any clinical significance. Primary infections with polyomaviruses are common. They occur early during childhood with only minor clinical symptoms. Approximately 60 - 80 percent of asymptomatic adults in the western world have serological evidence of a previous infection with polyomaviruses. JC-Virus and BK-Virus are tropic for epithelial cells in the kidney and, in particular, for the transitional cell layer of the urogenital tract. Here, they commonly remain latent without causing any illness. In 0.5%-20% of immune competent hosts, polyomaviruses can be periodically re-activated and re-enter into a replicative cycle. Such re-activation is transient, asymptomatic and is morphologically characterized by the detection of intranuclear viral inclusion bearing cells in the urine (coined "decoy-cells"). Decoy-cells appear to originate from the transitional cell layer. Such asymptomatic re-activation of polyomaviruses can be seen during pregnancy, in patients suffering from diabetes mellitus or malignant tumors. Renal function remains unaltered. These general aspects of polyomavirus infections in immune competent individuals form the background against which pathophysiological aspects of disease, especially BKN, have to be studied.

Disease caused by polyomaviruses is only seen in immune compromised hosts. Depending on the viral strain (BK or JC) and the type of the underlying suppression of the immune system, different illnesses prevail. The BK-Virus strain typically causes BKN in renal allografts ^{2, 9, 10}. It is characterized by virally induced tubular injury.

In the setting of kidney transplantation, one should bear in mind that the prevalence of latent infections with polyomaviruses is high. Thus, latent polyomaviruses are likely often passed in the graft from the donor to the recipient (who also may have latent virus in the urogenital tract). Serological evidence of a BK-Virus infection is seen in up to 34% of patients post transplantation without any clinical symptoms ¹¹. Asymptomatic re-activation of polyomaviruses with shedding of decoy-cells in the urine is commonly also seen in healthy kidney transplant recipients (in up to 23% of patients; personal observation) .

Thus different "clinical phenotypes" of an infection should be considered: i) latent, asymptomatic polyomavirus infections lacking morphological and immunohistochemical signs of viral replication, ii) serological, asymptomatic evidence of an infection, iii) transient, asymptomatic re-activation of latent polyomavirus infections with morphological evidence of viral replication, i.e. with shedding of decoy cells in the urine, and iv) clinically symptomatic viral disease (i. e. BKN) with morphological evidence of virally induced renal injury. BKN is typically accompanied by signs of viral activation (decoy cells) - but not vice versa ^{4, 5, 12-15}. Latency and activation are common phenomena, disease is a rare event.

Clinical data:
BKN occurs many months after grafting, in our experience on average one year post transplantation (range: 40 days - 1377 days). Before the diagnosis of BKN many patients experience a complicated clinical course with rejection episodes and intensive immunosuppression often including tacrolimus (e.g. FK506) and/or mycophenolate mofetil (MMF, e.g. CellCept) ^{2, 4-6}. At time of the initial diagnosis of BKN, patients clinically present with varying degrees of allograft dysfunction, which is minimal at times ^{4, 5}. Occasionally, BKN is even seen in protocol biopsies of stable allografts ¹⁶. Decoy cells are typically detected in the urine and BK-Virus DNA can be found in the plasma by PCR ^{4, 13, 17, 18}. These latter findings constitute a clinical risk profile, which can help with patient management. Glomerular proteinuria, significant hematuria or signs of a generalized infection (i.e. fever) are typically absent. BKN is normally not associated with haemorrhagic cystitis or ureteral stenosis. In renal allograft recipients, BK-Virus generally does not spread to organs outside the graft 4, 5. BKN has a major detrimental impact on graft function and survival with a graft failure rate of up to 52% ^{4, 7, 8}.

Diagnosis and Morphology:
The diagnosis of BKN can only be established histologically in a renal biopsy ^{2, 4, 5, 9, 15, 19}. The morphological hallmarks of BKN are intranuclear viral inclusion bodies, seen exclusively in epithelial cells, and focal injury and necrosis of tubular cells. Due to the viral replicative cycle, intranuclear inclusion bodies can always be found, although, they are sometimes scarce. Inclusion bearing cells can be abundant in the medulla and distal tubules (possibly reflecting the ascending route of viral spread from the urothelium to the renal cortex). Viral replication ultimately results in the release of viral particles by lysis of the host cell. Thus, tubular epithelial cell injury and necrosis with denudation of basement membranes is a typical finding in BKN. Despite marked epithelial cell damage, tubular basement membranes remain intact. They can serve as a structural base for tubular regeneration ^{2, 4, 5, 9, 19, 20}. These morphological changes are not unique, but rather represent acute virally induced tubular necrosis (ATN). ATN is the morphological correlate for allograft dysfunction seen in BKN.

In the renal pelvis and ureters, viral inclusion bodies can be found in transitional cells ². If inclusion bearing transitional cells are shed into the urine, they can be detected in cytological preparations as decoy cells ^{4, 12, 13, 15, 17, 19}. Diagnostic confirmation of BK-viruses as causative agents can easily be achieved by immunohistochemistry or electron microscopy ^{2, 4}. Of note: In graft biopsies, immunohistochemistry and electron microscopy only help with confirming the light microscopical impression of a productive BK-virus infection. Their routine use to search for BKN is not recommended ^{2, 6}.

BKN, interstitial nephritis and rejection:
The correct diagnostic interpretation of "inflammatory changes" in the setting of BKN is difficult and has led to considerable controversy. Often, BKN is simply referred to as "BK-Virus interstitial nephritis" ⁹. Although, this descriptive term accurately describes the findings, it implies that the inflammatory changes observed are secondary to the viral replication. Using this terminology, one major differential diagnosis of "inflammation" in a renal allograft - rejection - remains without further consideration. In our opinion, it is crucial to establish a diagnosis of possible rejection in the setting of BKN since those concurrent rejection episodes can be treated with transient specific anti-rejection therapy that might include anti-lymphocytic preparations ^{4, 5, 17, 21-23}. We have gained good results with this strategy ^{4, 5, 17, 21}.

We use a stepwise approach to establish a diagnosis of acute rejection coinciding with BKN. 1) Light microscopy: We search for interstitial mononuclear inflammatory cell infiltrates and typical tubulitis in areas lacking cytopathic changes (morphological changes suggestive of Banff type I rejection). The detection of transplant endarteritis is diagnostic of acute rejection (Banff type II rejection). 2) Immunohistochemical "adjunct markers" of rejection: We perform additional studies to detect the tubular expression of MHC-class II (HLA-DR), and the accumulation of the complement degradation product C4d along peritubular capillaries. Both adjunct markers can be used in the setting of BKN to firmly establish a diagnosis of concurrent rejection ^{4, 5, 17, 21, 24, 25}.

Adjunct diagnostic tools:
Although the diagnosis of BKN has to be established in a graft biopsy, adjunct diagnostic tools are available to manage patients including urinalysis (in particular the screening for decoy-cells) and plasma PCR studies to search for BK-Virus DNA.

Decoy cells in urine cytology preparations:
The search for decoy-cells is a very useful and robust clinical tool that is currently underutilized ^{2, 4, 5, 12-15, 19}. For screening purposes the search for decoy cells is better suited than PCR analyses on urine samples ^{14, 26}. We set an arbitrary threshold level of more than 5 decoy cells per 10 high power fields for a urine specimen to qualify as "decoy cell positive". Using this approach, we established an overall sensitivity of decoy cells to predict BKN of 100%, a specificity of 95%, a positive predictive value of 27% and a negative predictive value of 100% ⁴. Thus, if patients are "decoy cell negative" they do not suffer from BKN (and other diagnoses should be considered). If, however, the urine is "decoy cell positive", BK-Virus has been activated and the patient is at potential risk for developing BKN. This patient should be closely followed (such as with plasma PCR studies). Of note: the detection of decoy cell casts is diagnostic for BKN. Decoy cells can also be detected in the urine sediment by phase contrast microscopy ¹².

Plasma PCR analyses to detect BK-Virus DNA:
BKN is characterized by viral replication in tubular cells, epithelial cell injury, cell lysis and ATN. BK-Virus likely gains access to the blood stream through injured tubular walls and via peritubular capillaries. Indeed, PCR analyses to detect BK-Virus in plasma are useful clinical tools since positive PCR results mirror the time course of viral nephropathy. The patients we studied converted from plasma negative to plasma positive by PCR after transplantation, remained plasma positive during the course of histologically proven BKN and became plasma negative after BKN had been overcome ¹³. In our experience and that of others, all patients with BKN had detectable BK-Virus in the plasma ^{13, 17, 18, 21}. However, the detection of BK-Virus in plasma can also yield false positive results. Plasma PCR studies to predict BKN have a sensitivity of 100%, a specificity of 88%, a positive predictive value of 50% and a negative predictive value of 100% ¹⁷. Currently, considerable effort is being put into the development of quantitative plasma PCR tests in order to establish diagnostically relevant cut-off points for viral loads. One report suggested a threshold of 7700 BK-copies per ml serum as diagnostic significant ¹⁷. However, at present, those cut-off points have not been reliably established and thus, general clinical guidelines cannot be rendered.

Risk factors:
Likely, BKN is promoted by a currently poorly defined multifactorial process. Over the last decade, new potent immunosuppressive drugs - foremost tacrolimus and mycophenolate-mofetil - have been introduced into the immunosuppressive protocols. Their worldwide use has been linked to the outbreak of BKN ^{2, 4, 5, 12, 27}. High dose tacrolimus (trough levels over 8 ng/ml) and/or mycophenolate-mofetil immunosuppression increases the relative odds ratio of developing BKN by 13 times 6. The observed clinical differences in the prevalence of BKN seem to be linked to the preferential (center dependent) administration of these newer immunosuppressive drug regimens. However, the clinical use of tacrolimus and mycophenolate mofetil does not fully explain the entire "risk profile" since only a minority of renal allograft recipients treated longterm with these drugs ultimately develop viral nephropathy. Other, currently poorly defined risk factors have to be defined in future studies. Although conceptually promising, no solid evidence has been found so far to link specific gene mutations of the BK-viruses to increased pathogenicity and the development of BKN ^{10, 28}.

Clinical management:
Virally induced ATN is the fundamental histological concept behind the pathophysiology of BKN. The degree of allograft dysfunction (if rejection is absent) is proportionate to the number of injured tubules (i.e. the degree of ATN). Rejection, of course can have an additional impact on allograft function ²¹. Under optimal conditions, virally induced ATN (BKN) may heal and grafts can function very well in the long run ^{4, 5, 16, 21}. The duration of ATN determines the extent of irreversible secondary damage, i.e. interstitial fibrosis and tubular atrophy, which have a major impact on long term graft function and survival ¹⁵.

The management of patients suffering from BKN is a very difficult clinical challenge, and despite all efforts, outcome is often ominous. At present, specific anti-viral drugs to treat BKN are not available. Anecdotal, although encouraging therapeutic attempts have been reported with cidofovir (administered at 0.25 mg/kg up to 1 mg/kg every 2 to 3 weeks without probenecid) and possibly also leflunomide ^29-33.

Currently, the most frequently used clinical approach to overcome viral nephropathy is to lower baseline immunosuppression with the hope that the host immune system will subsequently "clear the virus" ^{5, 8, 16, 21, 34}. However, as of yet specific recommendations how to approach this task are not available, i.e. to which degree should immunosuppression be lowered, for how long should this be done and are conventionally used drugs like cyclosporine or azathioporine better suited for this task. Pre-requisite for lowering immunosuppression is of course the absence of rejection.

In general the clinical management of renal allograft recipients who are at risk for developing BKN has two major goals: 1) The diagnosis of BKN should be established early (when histological changes/ATN are potentially fully reversible and before interstitial fibrosis and tubular atrophy have occurred). This represents the patient screening period. 2) BKN should be "treated" long enough (presumably for many weeks) to achieve complete cessation of viral replication, i.e. "full viral clearance". This represents the patient monitoring period. Both objectives can be met with the help of surrogate markers.

High risk patients (e.g. those coming out of rejection episodes and being treated with high dose tacrolimus and/or mycophenolate mofetil) should be regularly screened. The screening period should include the search for decoy cells in urine cytology preparations, and PCR analyses on plasma samples. If both tests are positive on repeat analysis, a graft biopsy should be performed to establish a definitive diagnosis of BKN (in particular in the absence of allograft dysfunction). If BKN is diagnosed histologically the patient should be treated. In cases with concurrently diagnosed rejection, we choose to transiently treat the rejection episode first, before therapeutic attempts are made to treat BKN (the Basel "two step approach").

While patients are being treated for BKN, plasma PCR testing as well as the screening for decoy cells are especially useful to monitor for viral clearance. If both tests turn from positive to negative and remain negative on repeat testing, it can be assumed that BKN has resolved (i.e. viral replication has ceased). Viral resolution may be further confirmed in a repeat graft biopsy. Using this approach for patient management, the duration of low-dose immunosuppression, which might trigger rejection episodes, can be optimized. We have been trying this "clinical algorithm" over the last years with very good success ^{4, 5, 13, 17, 21}. Once BKN has been overcome, allografts can have a good longterm prognosis ^{4, 5, 21}. If grafts are lost due to BKN, re-transplantation is an option even without graft nephrectomy ^{35, 36}.

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