BK Polyomavirus in Pediatric Renal Transplantation. What We Know and What We Don’t.

1. Introduction

The Polyomaviridae is a family of small, non-enveloped viruses with circular dsDNA genomes of approximately 5 kbp. This family includes four genera whose members have restricted host range, infecting almost exclusively mammals and birds [1]. The first polyomavirus was identified in 1953 as filterable tumor-causing agent in mice, followed by Simian vacuolating virus (SV40) isolated from rhesus monkey kidney cells that had been used for poliovirus vaccine preparation in the sixties [2]. The first two human polyomavirus species, both named after the index case patients, were identified in 1971 independently in two different patients. BK polyomavirus (BKPyV) was isolated from a urine sample of a renal transplant patient [3] and JC virus was isolated from the brain tissue of a patient with progressive multifocal leukoencephalopathy [4]. BK and JC viruses have been the only well-known human polyomaviruses for decades. However, more than 50 years of research have provided crucial insights into the cellular and molecular biology of these viruses, and up to now at least 13 human polyomavirus species have been identified [1,5].

2. Pathogenesis

Primary BKPyV infection is mainly acquired early in life and is most likely transmitted horizontally by direct contact, by aerosol or fecal–oral routes [6]. Approximately 80% of the general population has detectable antibodies to BKPyV, which appear at a point in childhood and persist throughout life [7]. The mechanisms of persistence and reactivation of the virus remain poorly understood. The BKPyV is believed to firstly replicate in the respiratory tract and then to spread to other organs through the bloodstream, notably in renal tubular epithelial and urothelial cells [8]. BKPyV remains clinically latent in immunocompetent hosts, but after kidney transplantation, immunosuppressive treatment disrupts the equilibrium between virus replication and immune response resulting in potentially severe clinical complications.

3. Kidney Graft Involvement

Uncontrolled BKPyV replication leads to nephropathy (BKPyV nephropathy) which continues to be a real threat in the management of kidney transplantation. The first evidence of BKPyV reactivation in kidney transplant recipients is the detection of viral shedding in urine, which appears in 20% to 60% of patients [9]. BKPyV viremia may follow the replication in urine, and it is detected in 10-20% of kidney transplant recipients. BKPyV nephropathy occurs in 1-10% of renal allograft recipients, mainly within the first 2 years post-transplantation, causing graft loss in about half of those patients [10,11].

Virological and immunological determinants of the progression from self-limited BKPyV viruria or viremia to BKPyV nephropathy in kidney transplant recipients are poorly understood and the source of BKPyV could be either the urinary tract of the donor or the recipient, although both sources may not be mutually exclusive [12,13,14].

4. Immune Response

4.1. Humoral Immune Response

The role of BKPyV humoral immunity in the modulation of the clinical course of BKPyV infection is still debated. In 1983, Rosen et al. have reported a severe case of end-stage-renal-disease due to a BKPyV infection in a six-year-old immunocompromised boy with congenital dysgammaglobulinemia [15]. Renal biopsy at the time of his presentation revealed tubulo-interstitial lesions with mononuclear infiltrates and numerous BKPyV inclusions. A hypothesis raised that the humoral immune deficiency was responsible for the severity of the BKPyV infection in this child.

Pre-transplant serologies for cytomegalovirus and Epstein–Barr virus in donor and recipient are now performed routinely with the aim to estimate the risk of infection and to plan the prophylactic treatment. Similarly, could pre-transplant serology for BKPyV be used as a screening tool to evaluate the individual risk for BKPyV infection?

Serotypes I and IV represent more than 90% of strains found in kidney transplant patients. Studies have suggested that high BKPyV-specific antibody titers against the donor strain before transplantation might have a protective role. Wunderink et al. found that higher level of serotype I antibodies before transplantation was strongly correlated with a lower risk of BKPyV infection [16]. A study by Solis et al. in 2018 revealed the key role of the anti-BKPyV genotype–specific neutralizing antibody titers in protecting against BKPyV infection [17]. Recently, Dakroub et al. used a commercial antibody for enzyme-linked immunosorbent assay (ELISA) in order to assess the BKPyV serostatus of 329 adult kidney transplant recipients and 222 matched donors [18]. Their results showed a higher risk of developing post-transplantation BKPyV viremia in the R-/D+ group.

However, several authors estimate that humoral immune response does not play a major role in the control of the BKPyV virulence [9,19,20,21,22,23].

4.2. Cellular Immune Response

Already 40 years ago, Drummond and al showed that BKPyV-specific cell-mediated immune response was the main mechanism in controlling viral replication [24]. This finding was further supported by a study on the cellular immune response to JC virus in patients with progressive multifocal leuko encephalopathy. In those patients, JC virus-specific cytotoxic T lymphocytes were found to be a key factor in containing the disease [25].

Impairment of cell-mediated immune response allows the BKPyV reactivation, which begins with the active viral replication in the graft, followed by viral shedding in the urine, and eventually viremia, nephropathy and, in the worst-case scenario, graft loss [21,26]. In kidney transplant recipients low levels of BKPyV-specific interferon gamma-producing T cells correlate with progression to BKPyV nephropathy [27].

Currently the plasma BKPyV-DNA load is used after kidney transplantation to identify patients at risk of BKPyV nephropathy. However, some authors advocate that the BKPyV viremia alone is not an accurate predictor of the clinical outcome and that more reliable parameters are needed to distinguish patients with self-limiting viremia from those with higher risk of BKPyV nephropathy [28]. Therefore, monitoring pre- and post-transplant BKPyV-specific T cells was suggested as a potential accurate marker to identify those kidney transplant recipients at increased risk.

Data on children are very scarce. As for the adult population, in a recent pediatric study high levels of BKPyV-specific CD4 and/or CD8 T-cells were found in cases of self-limiting viremia, whereas lack or low levels of BKPyV-specific T cells were associated with long-term viremia and florid BKPyV nephropathy [29].

5. Diagnosis

5.1. Urine Cytology

Decoy cells are characterized by a ground-glass appearance with intra-nuclear inclusions. Their presence in urine has been used for decades as a marker for BKPyV infection, although their predictive value is limited by the high intraobserver variability [10]. Moreover, urine decoy cells are not specific for BKPyV infection, having also been described in adenovirus and cytomegalovirus infections [30]. However, many institutions continue to screen renal transplant recipients through the detection of decoy cells and they measure BKPyV-DNA in the plasma only if decoy cells persist. The main reason is that in many countries the public health system and/or insurance policy's do not cover the cost of PCR testing [31].

5.2. BKPyV Viruria

Urine BKPyV-PCR has been proposed as a more reliable screening tool in renal transplant recipients, as this method could detect the infection at an early stage. Thresholds used for the risk of viremia and BKPyV nephropathy are commonly > 10⁷ copies/mL [32]. Adult data shown that a BKPyV viruria was associated with BKPyV nephropathy in only one third of cases, while no patients with a viruria under this cut-off developed the disease [33]. Therefore, this test is only useful when it is negative, as it makes the diagnosis of BKPyV nephropathy improbable.

Few data concern the pediatric population. In the CERTAIN Registry study, about one quarter of children developed a high level BKPyV viruria within the first post-transplantation year, while only 3% ended with a BKPyV nephropathy [34]. In our ongoing Belgian study on 55 transplanted children between 2010 and 2022, 18% developed a high level BKPyV viruria already within the first 6 months post-tranplantation, while during the same early period, 6% progressed towards a high-level viremia and none to BKPyV nephropathy (Data presented at the 37^th congress of the French Pediatric Nephrology Society, Nice 15-17 November 2023).

5.3. BKPyV Viremia

Regular screening for BKPyV replication by plasma viral load is now universally recommended in kidney transplant recipients [35,36]. A recent Belgian study with systematic biopsy and BKPyV-DNA load follow-up has shown a very high negative predictive value (0.989) when the plasma viral load was below 10⁴ copies/mL. Performing a biopsy for confirmation of BKPyV nephropathy is therefore not necessary in cases of low viral load [37].

Conversely, persistent high-level BKPyV viremia, (> 10⁴ copies/mL) is associated with an increased risk of BKPyV nephropathy, and it currently even defines a presumptive BKPyV nephropathy [9,37,38]. Therefore, in this scenario, guidelines universally recommend a reduction of maintenance immunosuppression [38].

As regards as the children population, in the CERTAIN Registry study 14% of patients developed a high-level viremia (presumptive BKPyV nephropathy) within the first year and needed a therapeutic intervention [34]. However, many reports have observed self-limiting BKPyV viremia in kidney recipients without any immunosuppression reduction [38,39,40]. Therefore, in these cases, pre-emptive reduction and/or modification of immunosuppressive drugs could not only be unnecessary but potentially at increased risk of rejection.

5.4. BKPyV-Specific Cell Immune Monitoring

BKPyV-specific cell-mediated immune response has been a recent area of research as BKPyV-specific cellular immunity seems to play an important role in controlling viral replication. Adult and pediatric studies observed that an increase in BKPyV-specific T cells was correlated with the viral clearance in kidney transplant recipients [27,29]. BKPyV-specific cell immune monitoring has been therefore suggested as a prognostic tool to identify patients who are at risk of BKPyV nephropathy [40].

Recently, Ahlenstiel-Grunow and colleagues performed a detailed analysis of the BKPyV-specific cellular immune response in a German cohort of transplanted children at the onset of BKPyV viremia [29]. They found that lack or low levels of BKPyV-specific CD4 and CD8 T cells were associated with an increased risk of persistent viremia, florid BKPyV nephropathy, and the urgent need of a reduction of immunosuppression. Whereas patients with adequate BKPyV-specific CD4 T cells (≥ 0.5 cells/µL) and/or CD8 T cells (≥ 0.1 cells/µL) showed transient, self-limiting viremia without BKPyV-associated complications.

Although the analysis of BKPyV-specific CD4 and CD8 T cells might offer an accurate prognostic tool in order to tailor the level of immunosuppression in kidney transplant recipients, up to now, the price, availability and technical hitches make these tests impractical for use in routine care.

5.5. Allograft Biopsy

Standardized graft biopsy using immunohistochemistry (SV40 T antigen staining or in situ hybridization) remains the gold standard to confirm a BKPyV nephropathy [35,36]. The Banff working group on BKPyV nephropathy classified the disease into 3 grades based on histology to allow comparative analyses and improvement in predicting clinical presentation and outcome [41]. Typical findings are focal interstitial mononuclear inflammatory cell infiltrates, necrotic tubular epithelium, and presence of homogenous intra- nuclear inclusions. Tubular atrophy and interstitial fibrosis remain the most important predictors of poor outcome [42].

In the CERTAIN Registry study, biopsy-proven BKPyV nephropathy was diagnosed in 4.5% of children during a 5-year post-transplantation period [34]. Unlike in adults, more than 20% of these patients developed a BKPyV nephropathy beyond the second year post-transplant [34]. Although current guidelines recommend to screen for BKPyV replication only once per year after the second year post-transplant, these pediatric observations suggest a more vigilant approach.

Negative biopsy results do not necessarily rule out BKPyV nephropathy, as the disease can be focal at start, and sampling errors are always possible [43]. Two cores containing medulla are required for an adequate biopsy sample [36]. Therefore, in patients with a negative kidney biopsy and a BKPyV viremia (> 10⁴ copies/mL) for more than 4 weeks, the diagnosis of “presumptive BKPyV nephropathy” should be retained [35].

6. Risk Factors of BKPyV Nephropathy in the Immunosuppressed Patient

It is widely agreed that the introduction of more potent immunosuppression, both for the induction and the maintenance phase, has increased the risk of BKPyV nephropathy in transplanted kidneys [29,34,44].

6.1. Immunosuppression

Thymoglobuline (ATG): As for the induction therapy, ATG has been clearly incriminated when compared to no induction or induction with IL-2 receptor blockers [9,44].

Tacrolimus (TAC): Results from the large Organ Procurement and Transplantation Network (OPTN) Registry found an association between the TAC-based maintenance regimens and a higher incidence of treatment for BKPyV nephropathy within the first 2 years post-transplantation; and this when compared to cyclosporine (CsA)-based regimens [44]. TAC has also been incriminated in more recent prospective studies on the adult population [45,46].

The pediatric cohort analysis of the United Network for Organ Sharing (UNOS) database on children followed for 5 years post-transplantation, found that TAC therapy at the time of discharge was significantly associated with graft failure due to BKPyV nephropathy as compared to graft failure due to other causes [47]. The CERTAIN Registry study also found that TAC-based immunosuppression was independently associated with presumptive BKPyV nephropathy in transplanted children [34].

Mycophenolate mofetil (MMF): The OPTN registry study found an association between the MMF-based regimen and a higher incidence of treatment for BKPyV nephropathy within the first 2 years post-transplantation when compared to azathioprine-based regimen [44].

Not surprisingly, the combination TAC-MMF has been associated with a particularly higher risk of BKPyV infection [9,44], even in children [34].

Inhibitors of mammalian target of rapamycin (mTORi): Studies have found that mTORi have an antiviral effect without losing their immunosuppressive action against graft rejection [48]. In vitro experiments using BKPyV-infected renal epithelial cell lines, found that sirolimus reduces BKPyV large T-antigen expression and its genome replication by blocking the protein-kinase Akt/mTOR pathway [49,50]. In clinics, an important role of mTORi in decreasing BKPyV infection has been observed [44,51], especially when combined with low-dose calcineurin inhibitors CNI [51,52,53,54,55]. Up to now, no reliable data exist in transplanted children.

6.2. Other Risk Factors

In addition to immunosuppression, other independent risk factors for BKPyV infection have been suggested, mainly recipient characteristics such as male gender [45], young [44] and advanced age [45], obstructive uropathy [34], and prior graft loss due to BKPyV nephropathy [28]. Also specific graft characteristics have been suspected such as organs from BKPyV seropositive donors to seronegative recipients, number of HLA mismatches, ischemic injury and ureteral stent placement at transplantation [19,34].

Transplanted children have an increased risk of developing a BKPyV serious infection. The possible explanation is that those patients have not been previously exposed to the virus, and when immunosuppressed, they acquire a primary and more severe infection [34,44]. On the contrary, the increased rate of BKPyV nephropathy in the elderly is plausible because of the reduction with increasing age in global humoral and cellular immunity to viruses [45]. It is therefore strongly recommended a more frequent surveillance in these two extreme age populations.

7. How to Manage BKPyV in Pediatric Kidney Transplantation

BKPyV screening: Our own experience on 55 transplanted children between 2010 and 2022, showed that 12% of patients progressed towards a high-level viremia (presumptive BKPyV nephropathy) within the first 2 post-transplant years, with 50% of them already affected within the first 6 months and nearly 85% within the first year [Data presented at the 37^th congress of the French Pediatric Nephrology Society, Nice 15-17 November 2023].

Therefore, we suggest that kidney transplanted children should be screened for plasma BKPyV-DNA monthly the first year, then every 3 months the second year, and afterward every 6 months.

If BKPyV viremia load is > 10³ copies/mL in two measurements at less than 3 weeks of interval, BKPyV load should be monitored for potential infection with frequent plasma sampling, at least every two weeks. If the viremia load is > 10⁴ copies/mL in two measurements, a presumptive BKPyV nephropathy should be considered even in the absence of renal biopsy and the first step of management should be taken [37] (Figure 1).

Immunosuppression management: The first step in the management of presumptive or proven BKPyV nephropathy is reducing maintenance immunosuppression as CNI and/or MMF. TAC trough levels are commonly targeted to <6 ng/ml, cyclosporine to <150 ng/ml, and MMF equal or less than half the daily maintenance dose. During reduction of immunosuppressive therapy, plasma BKPyV-DNA should be tested every 2-3 weeks, as well as the levels of BKPyV-specific T cells when possible.

If necessary, a following step could be switching to mTORi plus low-dose CNI after cessation of MMF [56]. Unfortunately, this aggressive reduction in immunosuppression may inevitably lead to acute rejection and/or graft loss [57].

Adjunctive therapies: In patients with persistent BKPyV nephropathy, despite adequately reduced immunosuppression, the use of adjunctive therapies may be considered [28,35,36]. However, no randomized controlled trials could provide evidence that their use alone or in combination, is superior to the reduction of immunosuppression alone [28].

Intravenous immunoglobulin (IVIG): IVIG could play a broad anti-inflammatory, immunomodulator role or directly neutralize viral activity. Clinical studies showing clearance of plasma BKPyV-DNA following IVIG administration do exist [58], though their real efficacy is difficult to evaluate as other antiviral interventions are always performed concomitantly [28]. IVIG have been administered in doses ranging from 0.1 to 2 g/Kg in conjunction with reduced immunosuppression.

Cidofovir: Cidofovir is a nucleoside analog of cytosine which has an antiviral activity inhibiting the DNA polymerase. Although randomized clinical trials assessing its efficacy are pending, few adult studies did report a stabilization of renal function and/or clearance of viremia [59]. In addition, several side effects have been reported, including nephrotoxicity, myelosuppression, nausea, diarrhea, anterior uveitis [28]. At our best knowledge, no robust pediatric studies have been published up to now [60].

Leflunomide: Leflunomide has unique antiviral and immunosuppressive properties, inhibiting the pyrimidin synthesis and protein kinase activity. It has been orally administered as a replacement for discontinued MMF, with a loading dose of 100 mg for 3 to 5 days, followed by a maintenance dose de 20 to 60 mg daily [61]. Significant toxic effects have been reported, including hepatitis, hemolysis, thrombotic microangiopathy, bone marrow suppression [9,28]. In children, very scarce case reports reporting the efficacy of Leflunomide have been published [62,63].

In order to avoid rejection, return to routine maintenance immunosuppression after successful clearance of BKPyV viremia should be considered under careful monitoring plasma viral loads. In case rejection appears, it should be treated according to standard protocol.

8. Conclusion

Due to limited number of pediatric studies, diagnosis and treatment of BKPyV-infections vary considerably between centers and countries [64]. The vast majority of children are managed according to adult guidelines. We have nevertheless attempted in the following flow-chart to summarize a sensible clinical attitude in order to screen and manage BKPyV infection in kidney transplant children (Figure 1).