BK virus (BKV) belongs to the polyoma virus family and causes a subclinical infection in most of the general population within the first 10 years of life (1–3). The role of BKV infection in renal allograft dysfunction has been controversial, with studies reporting a range of clinical outcomes (4–7). However, recent studies, both from our center and other institutions, have shown that BKV infection can be associated with significant morbidity in renal transplant recipients (7–10). Central to the controversy surrounding the role of BKV in renal dysfunction is the difficulty in diagnosing and monitoring such infection (6–11). Serologic and traditional viral culture techniques are either not specific or not readily available and may not be practical in situations in which a rapid diagnosis is required to make patient management decisions.
However, even after the diagnosis of BKV-associated nephropathy (BKVAN) is made, clinical management remains a significant challenge. Various therapeutic strategies have been suggested and tried with variable and often dismal results (7,9). There are no clinically proven antipolyomavirus agents currently available for the management of BKVAN. In vitro studies have, however, demonstrated that several drugs, including cidofovir, can inhibit polyoma viral DNA replication (12,13). Given the challenges posed by the diagnosis and management of this infection, development of noninvasive, quantitative techniques to monitor viral load could have a significant impact on the clinical management of these cases (14). A recent report, analyzing retrospective samples, suggested the utility of monitoring plasma BKV load in patients with BKVAN (15). We describe the development of a quantitative polymerase chain reaction (PCR) assay for the detection of BKV in urine and blood and its potential clinical usefulness in the management of BKVAN. This assay was used to follow viral load prospectively in four renal transplant patients with BKVAN who were also treated with low-dose cidofovir therapy.
PATIENTS AND METHODS
We prospectively followed four patients diagnosed with BKVAN on renal allograft biopsies with quantitative viral load monitoring of their urine and plasma. These studies were approved by the University of Pittsburgh Medical Center Institutional Review Board and the Children’s Hospital of Pittsburgh Human Rights Committee. Samples of blood and urine were collected from the patients on the same day that they underwent renal allograft biopsies. Subsequent collections of urine and blood samples were performed during outpatient visits if the clinical situation of the patient changed or if any therapeutic changes were made. PCR assays for BKV in the blood and urine samples were performed as described next. The kidney biopsies were processed for polyoma viral studies as described previously (7,8). Serial renal function was monitored using clinically appropriate biochemical studies.
Blood and Urine Samples
The initial urine specimen was a collection of 50 to 100 cc fresh urine in a sterile container. Blood collection was 5 to 7 cc of venous blood in an ethylenediaminetetraacetic acid tube; 5 mL urine and 1 mL plasma were also stored at −50°C. Samples from outside of University of Pittsburgh were mailed at room temperature by overnight courier (FedEx) to Pittsburgh. DNA extraction was performed from 140 μL plasma or urine, using a commercially available kit, according to the manufacturer’s instructions (QiaAmp Viral RNA Minikit, Qiagen, Inc.). The DNA was extracted in a final volume of 140 μL water.
Renal Allograft Biopsy
The evaluation was performed by routine histology and immunohistochemical studies. Immunohistochemical demonstration of viral antigens was performed using an antibody directed against the SV40 simian polyoma virus, as described previously (7,8). In one patient, in situ hybridization for BKV was also performed using commercially available biotinylated DNA probes (Enzo Diagnostics, New York, NY). These methods of detecting viral antigens or DNA in biopsy tissue are not sensitive enough to detect latent viral infection and give positive reactions only in cases with histologically obvious intranuclear inclusions.
Quantitation of BK Virus DNA with TaqMan Real-Time Polymerase Chain Reaction
A BKV gene sequence from the VP-1 region was amplified by TaqMan real-time quantitative PCR (ABI Prism 7700 and 7000 Sequence Detection System) assay using the following primers and probe. The forward BKVN primer had the sequence 5′ TGATAGCCCAGAGAGAAAAATGC-3′ and a Tm of 59°C. The reverse BKVN primer had the sequence 5′-CCACAGGTTAGGTCCTCATTTAAA-3′ and a Tm of 59°C. The TaqMan probe had the sequence 5′-TTACAGCACAGCAAGAATTCCCCTCCC-3′ and a Tm of 68°C. The TaqMan probe was labeled at the 5′ end with a FAM dye and at the 3′ end with a TAMRA dye. The primers and probe were synthesized by Operon Technologies.
The primers were found to be specific for BKV by testing against JC and BKV stocks obtained from the American Type Culture Collection and by sequencing. The reaction mixture consisted of 12.5 μL TaqMan (2×) universal Master Mix (Applied Biosystems, Foster City, CA), 1 μL 10 mM forward primer, 1 μL 10 mM reverse primer, 1 μL 5 nM BKV probe, 5 μL sample, and water qs to 25 μL. The reaction mixture was cycled as follows: 50°C for 2 min; 95°C for 10 min; and 40 cycles of two-step PCR included with each cycle consisting of 95°C for 15 sec, followed by 60°C for 1 min. To generate a positive control, the BKV gene sequence from the VP-1 region amplified by the TaqMan forward and reverse primers was cloned in a vector using the pGEM-T PCR cloning system (Promega, Madison, WI). The inserted sequence was then quantitated using ultraviolet spectrophotometry to calculate the copy number for a master standard positive control. A 10-fold serial dilution of this positive control standard, with copies ranging from 107 to 10, was made and used to generate a standard curve with each quantitative assay. The samples were run in duplicate. The plasma and urinary viral load was expressed as BKV copies/μL of urine or plasma.
Figure 1 shows the results of a typical run with serially increasing copy numbers. The intra-assay coefficient of variation was 19%, and the interassay coefficient of variation was 30%, with a lower limit of detection of 10 viral copies. We found that the QiaAmp Viral RNA Minikit (Qiagen, Inc.) extracts both viral RNA and DNA and gave the maximum extraction of viral DNA among the various extraction procedures tried (data not shown).
The case summaries are presented below and the salient clinicopathologic characteristics of these patients are summarized in Table 1. All patients underwent kidney transplantation and were treated with intravenous (IV) cidofovir with subsequent clearance of BK viruria after 1 to 4 doses of this drug.
Case Report 1
This assay was initially developed to monitor a 6-year-old African American boy who was diagnosed with BKV infection in June 1999 at the Children’s Hospital of Pittsburgh. This child underwent cadaveric kidney transplantation with a single kidney from a 5-year-old donor in August 1997 for end-stage renal disease (ESRD) secondary to renal dysplasia and bilateral vesicoureteral reflux. The kidney functioned immediately. Immunosuppression was performed with tacrolimus and prednisone. The steroids were tapered over the first 6 months after the transplant and then discontinued; tacrolimus monotherapy was continued, with blood levels maintained in the 5 to 10 ng/mL range. The posttransplant course was relatively uncomplicated and without any episodes of rejection for almost 2 years until June 1999. During a routine clinic visit in June 1999, he was noted to have an elevated blood pressure (121/107 mm Hg) and an elevated serum creatinine (1.5 mg/dL vs. a baseline of 0.8–1.0 mg/dL), along with mild nonanion gap acidosis (HCO3=18 meq/L). He had experienced nasal congestion for 3 weeks and an episode of gastroenteritis 1 month before this visit. Other investigations at admission showed a tacrolimus serum level of 7.3 ng/mL; hematocrit, 28.9%; white blood cell, 9.0; 35% neutrophils; 51% lymphocytes; 6% monocytes; and 8% eosinophils. Cytomegalovirus immunoglobulin (Ig)G and IgM were 6 units and 0.2 units, respectively, and Epstein-Barr virus quantitative PCR was fewer than 8 copies per 100,000 white cells. An ultrasound of the transplanted kidney showed an indistinctly demarcated region of increased echogenicity involving the mid portion of the allograft (Fig. 2a). This region appeared hypoperfused on Doppler examination (Fig. 2b). A computed tomography scan showed the same region to have increased contrast enhancement. Two allograft biopsy specimens were taken, one from the echogenic mass and another from a normal-appearing area of the kidney. The histologic appearances of both specimens were similar, showing interstitial fibrosis, tubular atrophy, and focal areas of intense lymphocytic inflammatory infiltrate with mild tubulitis. There were many enlarged tubular epithelial cells with inclusions filling most of the nucleus, which stained positive for polyoma. The patient’s tacrolimus dose was reduced by 25% to achieve levels of approximately 5 ng/mL. A week later, the patient’s creatinine showed an increase from 1.5 mg/dL to 1.9 mg/dL, and he was readmitted. Repeat biopsy showed persistent BKV, and the immunosuppression was tapered further. He was then treated with a combination of IVIg (0.5 gm/kg single dose), high-dose steroids (1,000 mg methyl prednisone IV, followed by 100 mg methyl prednisone IV the next day, and a taper over the next 6 days), and a single dose of cidofovir (1 mg/kg IV without probenecid). The urinary PCR for BKV showed a viral load of more than 107 copies/μL. The serum creatinine rose precipitously to 8 mg/dL. At this stage, a repeat biopsy showed no evidence of BKV and evidence of tubulitis. He was treated with steroids IV and an increase in tacrolimus. The serum creatinine fell and stabilized, although at a higher level than the previous baseline. Steroids were again eventually tapered and discontinued. Urine and blood samples were subsequently obtained 2 months later; both were not amplifiable for BKV. He continued to be viruria- and viremia-free 26 months after the diagnosis of BKVAN with a serum creatinine of 1.7 to 1.9 mg/dL. He developed viruria with a lower viral load (1,000–10,000 copies/μL) 30 months after the initial presentation but remained without viremia and showed no change in creatinine.
Case Report 2
This 54-year-old African American woman underwent cadaveric renal transplantation in October 1999 for ESRD secondary to diabetic nephropathy at the University of Pittsburgh Medical Center Hospital. Her immunosuppression regime consisted of tacrolimus, prednisone, and mycophenolate mofetil (MMF). She had received Thymoglobulin for steroid resistant rejection within the first month after transplantation, and her medical history included coronary artery disease, peripheral vascular disease, asthma, hypertension, and arthritis. She presented in July 2000 with a rise in creatinine to 2.8 mg/dL from a baseline of 2 to 2.2 mg/dL. There were no systemic symptoms; a kidney biopsy showed evidence of moderate rejection, which was treated with high-dose steroids (500 mg methyl prednisolone IV followed by a 5-day tapering course of oral prednisone) with only partial response. A follow-up biopsy 1 month later showed the presence of intranuclear inclusions with positive antibody staining and in situ hybridization for BKV (Figs. 4 and 5). The urinary PCR showed more than 107 copies/μL, and plasma PCR showed 3,700 copies/μL. The immunosuppression was reduced, but the serum creatinine continued to rise to 4.2 to 4.5 mg/dL. She was given an initial dose of cidofovir 0.25 mg/kg with a subsequent rise in the serum creatinine, which peaked at 5.1 mg/dL; however, the creatinine subsequently began to fall gradually. She was given 2 additional doses of cidofovir 0.5 mg/kg and 1 mg/kg (without probenecid) 2 and 10 weeks later as her serum creatinine continued to decrease. Her urinary PCR titers decreased over a 3-month period to levels with fewer than 100 copies/μL, and she became viremia free (Fig. 3b). Two years after transplantation and 15 months after the diagnosis of BKVAN, her serum creatinine is in the 3 to 3.2 mg/dL range.
Case Report 3
This 31-year-old white man underwent a second renal transplantation in March 1999 from a cadaveric donor for ESRD secondary to IgA nephropathy at Baylor University Medical Center in Dallas. His first transplant, which was lost to chronic rejection, was in 1986 from a living related donor. The patient was maintained on tacrolimus, MMF, and prednisone immunosuppression. He presented with rising creatinine in February 2000, 11 months after transplantation. He was empirically treated with high-dose steroids without response. He underwent an allograft biopsy after 3 weeks that showed the presence of intranuclear inclusions suggestive of BKV and stained positive for polyoma antibody. The immunosuppression was not changed, but his serum creatinine continued to increase over the next 8 weeks. He also developed systemic symptoms including low grade fever, diarrhea, and malaise. A repeat allograft biopsy, performed in April 2000, showed persistence of BKV. He initially received 3 doses of cidofovir (3 mg/kg per dose) with probenecid at 10- to 14-day intervals (Fig. 3c). A urinary BKV PCR checked at the end of the first 3 doses showed high levels of viruria (105 copies/μL), but the blood PCR was negative. However, his systemic symptoms resolved after starting cidofovir. A fourth dose of cidofovir was given (1 mg/kg per dose) with no probenecid. His urine PCR was negative 3 months after the last dose. A follow-up urine PCR obtained 3 months later showed a recurrence of viruria but at a lower viral load (1,000 copies/μL). However, as his serum creatinine remained stable (between 3.7 to 4.0 mg/dL), no further cidofovir was given.
Case Report 4
An 11-year-old boy received a preemptive living donor renal transplant from his father in November 1999 for ESRD secondary to posterior urethral valves at the Royal Hospital for Sick Children, Yorkhill, Glasgow, United Kingdom. He experienced an initial period of delayed graft function, which was believed to be related to cyclosporine toxicity, and was switched to tacrolimus within the first week of transplantation. The immunosuppression subsequently was with tacrolimus (with blood levels maintained at approximately 10 ng/mL), MMF, and low-dose prednisone. He experienced an episode of biopsy-proven acute cellular rejection 4 months after transplantation, in March 2000, which responded well to methylprednisolone IV (600 mg/m2 per day for 3 days). Approximately 10 months after transplantation, he experienced a nonspecific illness characterized by low grade fever, myalgia, graft tenderness, and elevation in the serum creatinine from a baseline of 0.9 to 1 mg/dL to 1.3 to 1.5 mg/dL. A transplant biopsy was interpreted as acute tubulointerstitial rejection (Banff grade 1), and he responded partially to high-dose methylprednisolone IV (600 mg/m2 per day for 3 days). A repeat biopsy was performed 3 weeks later, which showed tubulointerstitial infiltrates and intranuclear inclusions, with negative Epstein-Barr virus, cytomegalovirus serologies, and PCR tests. He was again treated with methylprednisolone IV (600 mg/m2 per day for 3 days) with only incomplete response with serum creatinine remaining at approximately 1.5 to 1.7 mg/dL. At this stage, rapamycin was initiated and MMF was stopped. A repeat biopsy was performed 2 weeks later and showed findings similar to the previous biopsy. This biopsy stained strongly positive for polyoma viral antibody. The tacrolimus dose was reduced by 35% (from 5.5 mg/day to 3 mg/day), and rapamycin was discontinued. His renal function deteriorated with an increase in serum creatinine from 1.5 to 2 mg/dL at the onset of dysfunction to 2.5 to 3 mg/dL. The urinary PCR was strongly positive (>107 copies/μL), and plasma PCR showed 700 copies/μL. He was given a dose of cidofovir (0.5 mg/kg per day without probenecid) with no significant change in his creatinine. The same dose of cidofovir was repeated after 2 weeks. This was followed by a gradual decline in serum creatinine. A final dose of 1 mg/kg cidofovir was given after another 8 weeks as he showed persistent but declining viruria (Fig. 3d). He has remained free of viruria and viremia (<10 copies/μL) for 11 months after the last dose with his serum creatinine stabilizing in the 1.5 to 1.8 mg/dL range.
BKV was first isolated from the urine of a renal transplant recipient approximately 30 years ago. Since that time, evidence has accumulated to indicate that the seroprevalence rate in adults is between 60% to 80% (1,4,5,11). Several early studies did not report any biopsy findings or ascribe any renal allograft dysfunction to BKV, although more recent studies have shown that BKV can cause nephropathy in up to 5% of renal allografts (7,9). The diagnosis of BKV nephropathy is currently made by an allograft biopsy that shows viral inclusions and is often associated with a variable degree of tubulitis that is often mistaken for acute rejection (7,9,16). The diagnosis and management of BKVAN can be challenging, and the development of noninvasive quantitative techniques to monitor the viral load can have a significant impact on the clinical management of these cases (10). This study shows that viral load monitoring by quantitative PCR for BKV may serve as a tool in the diagnosis and subsequent management of this infection. It also reports the potentially beneficial effect of low-dose cidofovir therapy in the management of a subgroup of these patients.
The current series of cases illustrates that BKVAN can present in many different ways. Case 1 presented with an echogenic lesion on ultrasound after the onset of a brief, nonspecific prodromal illness. We speculate that this child may have experienced a community-acquired primary infection with BKV. Although serologic evidence to support our contention is not available, several aspects of this case support this hypothesis. The fact that both the donor and recipient were young increases the likelihood that both were seronegative at the time of transplant. Further, the delay in onset of symptoms of BKV nephropathy until 2 years posttransplant (compared with the other three cases that presented in the first year after transplantation) could also be explained by acquisition of primary BKV infection in the community long after transplant. Finally, the presence of a nonspecific illness suggestive of a viral infection several weeks before the onset of allograft dysfunction may also be consistent with a primary BKV infection in this child. In addition, prospective observations documenting the serologic status against BKV in pediatric renal transplant recipients and their donors at the time of transplant need to be performed to determine the frequency and spectrum of disease associated with primary BKV infection in this population.
It is of interest that presentation of BKVAN in association with an echogenic mass with the allograft, as seen in our first patient, has not been described. Kidney biopsies from the echogenic mass and a normal appearing allograft showed similar histologic findings consistent with BKV. Serial ultrasound examinations after treatment with cidofovir and improved renal function showed disappearance of the mass. Inasmuch as the two children reported in this series represent the first reports of BKVAN in pediatric renal transplant recipients, the range of clinical disease associated with primary infection needs to be investigated further. Thus, presentations with an echogenic mass may become recognized as a typical manifestation of primary BKV infection and may join posttransplant lymphoproliferative disease in the differential diagnosis of such lesions.
Presently, infection with BKV has not been associated with a well-defined clinical illness. However, nonspecific “viral” syndromes before or near the time of diagnosis of BKVAN were noted in three of our four patients. In the first patient, a prodromal illness consisting of several weeks of nasal congestion and transient gastroenteritis were noted. The third patient initially presented with an asymptomatic rise in his serum creatinine; however, he subsequently developed systemic symptoms consisting of low-grade fever, diarrhea, and malaise after several months of illness progression. The fourth patient presented with a low-grade fever, myalgia, and graft tenderness when an elevation in his serum creatinine as a result of BKV was recognized. Unfortunately, although these groups of symptoms may in fact be attributable to BKV, the lack of specificity of these symptom complexes are unlikely to differentiate BKV disease from other entities, including rejection. The lack of specificity of these symptoms (alone or in the presence of allograft dysfunction) indicates that relying on clinical symptoms alone is unlikely to result in being able to accurately predict the presence of BKVAN. Three of the four patients had been diagnosed with and treated for acute rejection in the months preceding the diagnosis of BKVAN. Thus, it is the lack of complete response to antirejection therapy that would most likely raise the possibility of BKV infection. In previous reports and series, BKV infection has presented with features that were variably diagnosed, including acute rejection (7,8), interstitial nephritis (7,9,16), and ureteric stenosis (17). In addition, BKVAN has been diagnosed in asymptomatic patients (5,6,11,18). Thus, a high index of suspicion is needed for diagnosis of BKVAN. In episodes of acute rejection that are refractory to steroid therapy, BKV infection should be considered a possibility, especially in patients presenting with late acute rejection (>6 months–1 year posttransplantation).
The quantitative PCR assays that we have developed were designed to be specific for BKV and were sensitive enough to detect as few as 10 copies of viral genome. BKV viruria was found in all the patients even when tested 2 months or more after the biopsy diagnosis (case 3). However, the plasma PCR was initially negative in case 3, and clearance of viremia preceded clearance of viruria in three of the four patients. In case 1, plasma PCR was not tested early in the course of the disease, because the assay was still in developmental stages, and plasma samples were not available for retrospective analysis. However, blood PCR may turn out to be a helpful test in distinguishing patients with more severe or later stages of the disease. These findings generally agree with those reported by Limaye et al. and Nickeleit et al. (15,19). However, those two studies did not report any BKVAN patients with a negative plasma PCR. Differences in viral load, stages of the disease (and possibly differences in viral strains or PCR amplification conditions), or primers used in the studies may explain some of these discrepancies. Further studies are needed to establish cutoff criteria for viral load in both urine and blood and to establish the range of values found in asymptomatic and healthy posttransplant controls.
Finally, even after BKVAN has been properly diagnosed, clinical management remains a significant challenge. To date, the efficacy of antiviral agents against BKVAN has not been systematically evaluated, although in vitro studies have demonstrated that several drugs, including retinoic acid derivatives, DNA gyrase inhibitors, cytosine arabinoside, and cidofovir, inhibit polyoma viral DNA replication (12,13,20–25). Although various therapeutic strategies have been suggested and tried, the results are often variable and dismal. Therapy for BKVAN is usually based on renal allograft biopsy findings. The difficulty in clinical management is compounded by the fact that even when biopsies show tubulitis, indicating the possibility of underlying rejection, there is little or no response to corticosteroids in most cases. Another possibility for clinical management is to reduce immunosuppression (7). Although reducing immunosuppression decreases the viral load, it can increase the risk of rejection. Hence, an area of critical need is the development and evaluation of drugs with potential efficacy against polyomavirus. The response of our four patients shows that low-dose cidofovir may be efficacious in the treatment of BKVAN. Cidofovir [HPMPC, Vistide, (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine] is an acyclic nucleoside phosphonate with broad-spectrum activity against a variety of DNA viruses (12). It has previously been reported to be anecdotally effective in patients diagnosed with BKV-associated cystitis and progressive multifocal leukoencephalopathy caused by JC virus (13,21). A meta-analysis of 5 years of data indicates that cidofovir may be the most reasonable treatment option for progressive multifocal leukoencephalopathy in human immunodeficiency virus-infected individuals (21). It is usually administered in a dosage of 5 mg/kg of body weight with the antidiuretic probenecid to reduce renal excretion and increase tissue levels of the drug. Approximately 75% to 80% of the cidofovir dose excreted in the urine unchanged within 24 hr of administration. Cidofovir is nephrotoxic and, therefore, is contraindicated in patients with impaired renal function. Nevertheless, cidofovir was used, albeit at low doses, in our four kidney transplant recipients diagnosed with BKVAN. It was administered every 2 to 3 weeks at a dose of 0.25 to 1 mg/kg, which is 5% to 20% of the usual recommended dose. Because most of the cidofovir dose is excreted unchanged in the urine, the infection was hypothesized to respond to the reduced dosage because of its localization in the kidney. We decided not to give the patients any probenecid, because we wanted as much of the drug to be excreted by the kidney as possible. One patient (case 3), however, had initially received 3 doses of cidofovir (3 mg/kg per dose) with probenecid before receiving a lower dose without probenecid. The higher doses were prescribed by one of the authors before we became aware of the utility of low doses given without probenecid. All four patients showed clearance of BK viruria after 1 to 4 doses of this drug, although two of the four patients were detected to have recurrence of viruria. However, all the patients currently have a lower serum creatinine than at the initiation of the therapy or at the peak of the disease. No persistent nephrotoxicity was seen with the doses used in this study. This apparent efficacy of cidofovir treatment is a promising finding, which, if confirmed in additional patients, may improve the prognosis in these difficult cases. In contrast with our experience with these four patients, the untreated patients experienced prolonged persistence of BK viruria for periods of more than a year (2,26). Quantitative PCR studies can thus help monitor the response to cidofovir and the course of the infection. Because cidofovir is nephrotoxic, quantitative PCR testing could help ensure that exposure to this drug is no more than necessary to control the infection. A multicenter, randomized case-control study may be needed to study this drug or other candidates.
In summary, this study shows that quantitative PCR monitoring for BKV can be a useful tool in the diagnosis and management of BKVAN. Quantitation of the viral load may allow a physician to monitor a patient’s response to specific antiviral therapy or allow a reduction of immunosuppression sufficient to permit stimulation of antiviral immunity without reducing it to a point that would precipitate acute rejection. Also, cidofovir therapy may be useful in the treatment of a subgroup of these patients. Its role needs to be further investigated in a larger cohort of patients, perhaps in a multicenter study. The ability of viral load to predict clinical BKVAN during pretransplant evaluation and in posttransplant follow-up of asymptomatic patients also needs further investigation.
The authors thank Susan Frank, RN, for her help, all the patients for their participation, and Ms. Bobbi Wiercioch for her secretarial assistance.
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