Blood, and Not Urine, BK Viral Load Predicts Renal Outcome in ...

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suppressive effects of leflunomide had not yet been evaluated in HSCT patients. There are reports of thrombotic microangiopathy in renal transplant pa-.
Blood, and Not Urine, BK Viral Load Predicts Renal Outcome in Children with Hemorrhagic Cystitis following Hematopoietic Stem Cell Transplantation Hilary L. Haines,1 Benjamin L. Laskin,2 Jens Goebel,2 Stella M. Davies,1 Hong J. Yin,3 Julia Lawrence,4 Parinda A. Mehta,1 Jack J. Bleesing,1 Alexandra H. Filipovich,1 Rebecca A. Marsh,1 Sonata Jodele1 BK virus is a significant cause of hemorrhagic cystitis after hematopoietic stem cell transplantation (HSCT). However, its role in nephropathy post-HSCT is less studied. We retrospectively evaluated clinical outcomes in pediatric HSCT patients with hemorrhagic cystitis. Although most of these patients had very high urine BK viral loads (viruria), patients with higher BK plasma loads (viremia) had significant renal dysfunction, a worse clinical course, and decreased survival. Patients with a peak plasma BK viral load of .10,000 copies/mL (high viremia) were more likely to need dialysis and aggressive treatment for hemorrhagic cystitis compared to patients with #10,000 copies/mL (low viremia). Conversely, most patients with low viremia had only transient elevations in creatinine, and less severe hemorrhagic cystitis that resolved with supportive therapy. Overall survival (OS) at 1 year post-HSCT was 89% in the low viremia group and 48% in the high viremia group. We conclude that the degree of BK viremia, and not viruria, may predict renal, urologic, and overall outcome in the post-HSCT population. Biol Blood Marrow Transplant 17: 1512-1519 (2011) Ó 2011 American Society for Blood and Marrow Transplantation

KEY WORDS: BK virus, BK nephropathy, Hemorrhagic cystitis, Stem cell transplantation, Thrombotic microangiopathy, Pediatrics

INTRODUCTION BK is a polyoma DNA virus that infects 90% of the population by adulthood and remains dormant in the uroepithelial cells of immune-competent hosts [1]. During periods of immunosuppression, such as following solid organ or hematopoietic stem cell transplantation (HSCT), BK reactivation may lead to organ dysfunction [1]. BK virus is a known cause of nephropathy following renal transplantation [2], but in HSCT recipients, it has generally been associated with isolated hemorrhagic cystitis [3]. However, more cases of BK virus–associated nephropathy have From the 1Division of Bone Marrow Transplantation and Immune Deficiency; 2Nephrology and Hypertension; 3Pathology; and 4 Department of Pharmacy, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio. Financial disclosure: See Acknowledgments on page 1518. Correspondence and reprint requests: Sonata Jodele, MD, Division of Bone Marrow Transplantation and Immune Deficiency, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, MLC 7015, Cincinnati, OH 45229 (e-mail: sonata. [email protected]). Received January 18, 2011; accepted February 16, 2011 Ó 2011 American Society for Blood and Marrow Transplantation 1083-8791/$36.00 doi:10.1016/j.bbmt.2011.02.012

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recently been described in the native kidneys of HSCT patients [4-7]. Urine and blood specimens can be monitored for BK virus using quantitative polymerase chain reaction (PCR) assays [8,9]. Using viruria (detection of virus in the urine) to assess for nephropathy is problematic, given the high rates of hemorrhagic cystitis and asymptomatic urinary shedding in the post-HSCT population [3,10,11]. Although only a small percentage of viruric renal transplant patients will develop viremia or overt nephropathy [12], the relationship between viruria and viremia has yet to be characterized in HSCT patients. Given the limitations of interpreting viruria, monitoring only BK viremia was recently shown to be costefficient after pediatric renal transplantation [13]. This practice has also been supported by adult kidney transplant guidelines [14]. Despite the growing literature in renal transplantation, there are few studies assessing BK viremia in the HSCT setting [15,16]. Finally, there are no current guidelines for the monitoring and treatment of BK virus infection after HSCT. We report a retrospective evaluation of the clinical impact of BK viruria, viremia, and the prevalence of associated nephropathy in a pediatric HSCT population. Our data emphasize the importance of plasma BK

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measurements in predicting renal, urologic, and overall clinical outcomes.

METHODS We retrospectively reviewed the charts of all pediatric patients undergoing HSCT at Cincinnati Children’s Hospital Medical Center (CCHMC) in the past 3.5 years, after institutional review board approval was obtained. The goal of the study was to evaluate the clinical impact of viremia and viruria in HSCT patients, and to assess the occurrence of BK-virus associated nephropathy. BK studies were performed in patients with hematuria or cystitis, and not as routine post-HSCT screening. Patient records were reviewed for evidence of hematuria or cystitis from January 2007 to June 2010, as BK PCR testing first became available for clinical use at our center in 2007. Hematuria was defined as .5 red blood cells per high-powered field (on microscopic analysis automatically triggered by a positive dipstick for blood) or documented gross hematuria with or without symptoms of cystitis (dysuria, urgency, bladder pain/spasms). Laboratory studies for plasma and urine viral PCRs were also reviewed in the asymptomatic HSCT population to ensure completeness of case identification. Pathogens related to the development of hemorrhagic cystitis were recorded. Patients having at least 1 urine or plasma BK PCR were included in the primary analysis. Quantitative BK virus PCR performed on plasma or urine samples had a detection threshold level of 1200 copies/mL. The maximum BK viral load in plasma and urine was documented. Patients with BK viremia were assigned to either the high viremia group (maximum BK viral load .10,000 copies/mL) or the low viremia group (maximum BK viral load #10,000 copies/mL). This cutoff level was based on the kidney transplant literature [12]. Transplant data, including complications and routine clinical and laboratory information, was collected for the identified cohort. Renal function was assessed by serum creatinine and pre-HSCT nuclear glomerular filtration rate (nucGFR), and compared to changes in serum creatinine after the diagnosis of BK virus infection. The maximum fold increase in serum creatinine from the pre-HSCT value was calculated by dividing the highest creatinine after BK virus detection by pre-HSCT creatinine value. Renal biopsy information, if available, was also reviewed. As there are no uniform guidelines for BK virus therapy following HSCT, we recorded treatments and medications prescribed at the discretion of the attending physician. Standard supportive HSCT care included pneumocystis pneumonia, fungal prophylaxis, and acyclovir prophylaxis if either recipient or donor was seropositive for cytomegalovirus (CMV) or herpes

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simplex virus (HSV). Intravenous immunoglobulin (IVIG) was provided to all patients to maintain a normal immunoglobulin level for age. Routine weekly viral monitoring included blood PCR testing for CMV, Epstein-Barr virus (EBV), and adenovirus. Graft-versus-host disease (GVHD) was graded according to the Glucksberg criteria [17]. Transplantassociated thrombotic microangiopathy (TA-TMA) was diagnosed based on currently accepted criteria including: de novo microangiopathic anemia (elevated lactate dehydrogenase [LDH], decline in hemoglobin and platelets, evidence of schistocytes on peripheral blood smear, decreased haptoglobin), and impaired renal or neurologic function [18,19].

RESULTS Characteristics of the Study Population A total of 314 HSCTs were performed during the evaluation period (January 2007 to June 2010). Thirty-five HSCT patients (11%) had documented hemorrhagic cystitis. Urine was tested for BK virus in 33 of these 35 patients, and was positive in 30 (91%). One of the 2 patients had a positive plasma BK test without urine evaluation for BK virus, and the other patient had no documented infections and was not evaluated for BK virus. The mean urine BK viral load in all patients with viruria was 8 billion copies/mL with 73% (22/30) of patients having a maximum urine viral load .1 billion copies/mL. Sixty percent (21/35) of patients with hemorrhagic cystitis had plasma BK virus tested, and all of them had viremia. Of these 21 patients, 10 patients had high viremia (.10,000 copies/mL, ranging from 16 thousand to 30 billion copies/ mL) and 11 patients had low viremia (#10,000 copies/ mL, ranging from 1.25 to 9 thousand copies/mL). The transplant characteristics of the 21 patients with viremia are summarized in Table 1. All but 1 patient underwent allogeneic HSCT. The majority of allogeneic transplants were from unrelated donors (17/ 20, 85%). Bone marrow was the main source of stem cells (18/21, 86%). A total of 4 patients, 3 of whom were in the high viremia group, received reducedintensity conditioning regimens, and all others received myeloablative conditioning regimens. Transplant Comorbidities: GVHD, TA-TMA, and Other Viral Infections Most patients in both groups received cyclosporine and methylprednisolone (1-2 mg/kg/day) for GVHD prophylaxis. The prevalence of acute GVHD (aGVHD) was similar in the high viremia (7/9, 78%) and low viremia (8/11, 73%) groups, with 22% of patients in the high viremia group and 36% in the low viremia group having acute grade 3 GVHD.

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Table 1. Transplant Characteristics of 21 HSCT Patients with BK Viremia

Mean age at transplant (years) Sex Male Diagnosis Chronic granulomatous disease NEMO Fanconi anemia CML HLH Medulloblastoma XLP1 Myelodysplastic syndrome Congenital neutropenia CVID Metachromatic leukodystrophy Adrenoleukodystrophy X-linked Hyper IgM AML Transplant type Allogeneic Autologous Donor type Matched unrelated Mismatched unrelated Matched sibling Autologous Stem cell source Bone marrow PBSC Transplant chemotherapy Myeloablative Reduced intensity BMT regimens: Bu/Cy/ATG Campath/Flu/Mel Flu/ATG Cy/Bu/Flu/ATG Bu/Mel/Thio/A

High Viremia: >10,000 Copies/mL (n 5 10)

Low Viremia: #10,000 Copies/mL (n 5 11)

13

11.2

6 (60%)

10 (91%)

0 (0%)

3 (27%)

1 (10%) 2 (20%) 2 (20%) 0 (0%) 1 (10%) 1 (10%) 1 (10%) 1 (10%) 1 (10%) 0 (0%)

2 (18%) 0 (0%) 0 (0%) 2 (18%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (9%)

0 (0%) 0 (0%) 0 (0%)

1 (9%) 1 (9%) 1 (9%)

9 (90%) 1 (10%)

11 (100%) 0 (0%)

4 (40%) 3 (30%) 2 (20%) 1 (10%)

6 (55%) 4 (36%) 1 (9%) 0 (0%)

8 (80%) 2 (20%)

10 (91%) 1 (9%)

7 (70%) 3 (30%)

10 (91%) 1 (9%)

4 (40%) 3 (30%) 1 (10%) 1 (10%) 1 (10%)

10 (91%) 1 (9%) 0 (0%) 0 (0%) 0 (0%)

Bu indicates busulfan; Cy, cyclophosphamide; ATG, antithymocyte globulin; Flu, fludarabine; Mel, melphalan; Thio, thiotepa; A, amifostine (chemoprotectant); HSCT, hematopoietic stem cell transplant; AML, acute myelogenous leukemia; PBSC, peripheral blood stem cell; CML, chronic myeloid leukemia; CVID, common variable immunodeficiency; HLH, hemophagocytic lymphohistiocytosis.

GVHD preceded BK infection by 1 to 2 weeks in 5 of 7 (71%) patients in the high viremia group and in 5 of 8 (63%) patients in the low viremia group. Eightynine percent of patients in the high viremia group and 100% of patients in the low viremia group received methylprednisolone .2 mg/kg/day for GVHD therapy. Additional immunosuppressive therapy, mainly with monoclonal antibodies, was given to 6 of 7 (86%) patients with GVHD in the high viremia group and to 5 of 8 (63%) patients in the low viremia group. Of note, more patients in the high viremia group received infliximab (78% versus 36%) and basiliximab (33% versus 9%). Mycophenolate (MMF) was used in 4/10 (40%) of patients with high viremia and 2/11

(18%) of patients with low viremia. GVHD was the cause of death in 1 patient in each group. Seven of 10 (70%) patients in the high viremia group were diagnosed with transplant-associated thrombotic microangiopathy (TA-TMA) during BK viremia, whereas only 2 of 11 (18%) patients in the low viremia group had a concomitant diagnosis of TA-TMA. All patients with TA-TMA had significant hypertension requiring intensive medical therapy. Three patients in the high viremia group had renal biopsies diagnostic for BK virus–associated nephropathy. One of them also had histologic evidence of TA-TMA on the same biopsy specimen (Figure 1). In terms of coincident viral infections, 2 patients in the high viremia group had intermittent CMV and EBV viremia (treated with ganciclovir, foscarnet, cidofovir, rituximab) and 1 patient also developed adenoviremia. One patient in the low viremia group had CMV viremia and retinitis (treated with ganciclovir, foscarnet, cidofovir). Renal, Urologic, and Overall Outcome Hemorrhagic cystitis presented at a similar time post-HSCT in the high and low viremia groups (median 33 and 31 days post-HSCT). Eighty percent of patients in both viremia groups had very high urine viral loads of .1 billion copies/mL (Table 2). The lag time between presentation of hemorrhagic cystitis and the first documented positive urine BK PCR was \7 days in 80% of patients in both groups. The lag time between the first documented viruria and the first documented viremia was \7 days in 80% of patients with high viremia and 64% of patients with low viremia. Twenty of 21 (95%) patients were either receiving corticosteroids or had discontinued them within the previous 3 weeks prior to the onset of BK viruria. Clinical outcomes are summarized in Table 2. The majority of patients with BK infection presented with gross hematuria, with only 3 patients in the high viremia group and 2 patients in the low viremia group having microscopic hematuria. Five patients, all in the high viremia group, required aggressive surgical interventions for obstructive uropathy. One patient underwent bilateral nephrostomy tube placement and had multiple procedures to evacuate bladder clots. None of the patients in the low viremia group required surgical intervention for management of hemorrhagic cystitis. All 21 patients with viremia had baseline preHSCT creatinine that was normal for age, and all but 1 patient had baseline nucGFR .70 mL/min/1.73 m2. Patients with high viremia developed more significant impairment of renal function compared to those with low viremia. Specifically, patients in the high viremia group had a significantly higher serum creatinine elevation after BK virus detection (P \ 0.02) in comparison to their pre-HSCT baseline. Patients with

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Figure 1. Renal biopsy in patient with BK infection and TA-TMA: renal biopsy showed mild inflammatory infiltrate in the interstitium, mild interstitial fibrosis, and tubular atrophy. Most of the capillaries were patent, but with focal segmental mild thickening of glomerular capillary walls with a double contour appearance (A, trichrome stain, 60), consistent with expansion of the subendothelial zone. Electron microscopy showed irregular electron-lucent expansion of the subendothelial zone (double arrows) because of acute endothelial cell damage with neo-membrane formation. The capillary lumen was occluded by thrombi (B). With no immunoglobulin deposition, these changes are diagnostic for thrombotic microangiopathy. Many tubular epithelial cells had large, homogenous, intranuclear inclusions (arrows) with a ground-glass appearance (C, H&E, 40), representing aggregations of viral particles in the nucleus. BK virus in situ hybridization demonstrated positive staining in the nucleus (D, 40). Electron microscopic study also revealed 40- to 45-nm electron-dense virions in the nucleus (not shown).

low viremia also had creatinine elevations compared to their pre-HSCT baseline, but elevations were lower and transient, resolving with supportive care (Table 2). Seven of 10 patients (70%) in the high viremia group required renal replacement therapy (RRT). Six of these patients progressed to end-stage renal disease (ESRD), and 1 remains with stage 2 chronic renal disease (CKD). Four patients with ESRD died, and

2 patients remain on RRT 717 and 536 days after HSCT. One patient in the low viremia group received RRT for 2 days prior to death from acute multiorgan failure secondary to GVHD and fungal infection. Overall survival (OS) at 100 days and 1 year postHSCT in the low viremia group was 100% and 89%, respectively, and in the high viremia group was 90% and 48%, respectively (Figure 2). Confidence intervals

Table 2. Clinical Outcomes in 21 HSCT Patients with BK Viremia

Urine BK >1 billion copies/mL Obstructive uropathy requiring surgical intervention Average max. fold increase in creatinine from pre-SCT TA-TMA Renal replacement therapy Biopsy proven polyoma virus–associated nephropathy End-stage renal disease Mortality attributed to BK virus

High Viremia: >10,000 Copies/mL (n 5 10)

Low Viremia: #10,000 Copies/mL (n 5 11)

P Value

8 (80%) 5 (50%) 6.4 7 (70%) 7 (70%) 3/3 samples 6 (50%) 3 (30%)

8 (80%) (n 5 10) 0 (0%) 2.1 2 (18%) 1 (9%)‡ none done 0 (0%) 0 (0%)

>0.99* 0.009* 0.02† 0.03* 0.006* n/a 0.003* 0.08*

TA-TMA indicates transplant-associated thrombotic microangiopathy; HSCT, hematopoietic stem cell transplantation; SCT, stem cell transplant. *Unconditional exact test, two-sided. †Wilcoxon two-sample test. ‡Not attributed to polyoma virus–associated nephropathy.

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were not calculated because of the small sample size and high censoring rate. Three of 6 deaths (50%) in the high viremia group were attributed to BK virus infection, including the only patient who died before 100 days post-bone marrow transplant (BMT) (day 198). Plasma viral load at the time of death for these 3 patients was 30 billion copies/mL, 950 million copies/mL, and 340 million copies/mL. The other 3 patients in the high viremia group died from multiorgan failure, pneumocystis pneumonia (both after resolution of BK viremia), and aGVHD (with a BK virus load of 104 million copies/mL at the time of death). One patient in the low viremia group died, secondary to aGVHD and fungal infection. None of these patients had primary disease relapse, or graft failure. BK Virus–Directed Therapy Sixty percent of patients in the high viremia group and 55% of patients in the low viremia group received cidofovir to treat BK virus infection. The decision to use cidofovir was at the discretion of the attending physician, and based on severity of clinical symptoms associated with BK virus infection, or for coinfections with CMV or adenovirus. Ninety-two percent of all cidofovir doses administered for BK infection were 1 mg/kg/day (with saline hydration and without probenecid) given 1 to 3 times per week. This dosing regimen was based on pediatric renal and adult HSCT literature described in the discussion. The average weekly dose was 1.1 mg/ kg in the high viremia group and 1.4 mg/kg in the low viremia group. The median number of cidofovir doses given over the course of therapy was 15 (range: 4-39) in the high viremia group and 3.5 (range: 2-57) in the

Figure 2. Overall survival (OS) after HSCT according to BK viremia level. Kaplan-Meier estimated OS was calculated starting from HSCT date (day 0, or stem cell infusion date) till the last date of contact or death for patients with low BK viremia (#10,000 copies/mL) and patients with high BK viremia (.10,000 copies/mL). Vertical hatched lines represent day 1100 and day 1365 following HSCT.

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low viremia group. The median cumulative cidofovir dose was 7.5 mg/kg (range: 0-75) in the high viremia group and 2 mg/kg (range: 0-55) in the low viremia group, with 2 patients in the high viremia group receiving 4 and 6 weekly doses of 5 mg/kg/dose and 1 patient in the low viremia group receiving 5 weekly doses of 5 mg/kg/dose for viral coinfections. Probenecid prophylaxis and saline hydration was used for 5 mg/kg/ dose cidofovir dosing. Two of 10 (20%) patients in the high viremia group and 4 of 11 (36%) patients in the low viremia group had documented BK viremia resolution by plasma PCR. All other patients had detectable viremia on the last available plasma PCR. One patient in the high viremia group received therapy with intravesical prostaglandins and intravesical cidofovir without any improvement. A second patient in the high viremia group was treated with intravenous estrogen for severe hemorrhagic cystitis with a partial response (50% reduction in PRBC transfusion requirements).

DISCUSSION BK virus is an increasingly recognized cause of significant complications following HSCT [16,20-22]. BK virus reactivation during periods of immunosuppression can be associated with significant organ dysfunction, most commonly in the lower urinary tract and kidneys [2]. BK-associated hemorrhagic cystitis is rare after renal transplantation, but this complication is common after HSCT [10]. The reason for this difference is not known, but may be related to cyclophosphamide exposure or GVHD (which leads to profound immune suppression) in the HSCT population [21,23,24]. Earlier renal transplant literature associated MMF with a higher rate of BK nephropathy [25]; however, more recent reviews suggest that the depth of overall immune suppression is more important than the particular immunosuppressant agent [1,26]. This correlation with MMF has not been described in HSCT patients, and less than one-third of patients in our cohort received MMF, limiting any conclusions. Similarly, even though more patients in the high viremia group received basiliximab and infliximab, the small numbers of patients receiving these monoclonal antibodies preclude any correlation between these medications and severity of BK virus infection. BK virus infection after HSCT can be diagnosed with urine PCR testing. Several studies have correlated quantitative urinary viral loads with the severity of hemorrhagic cystitis. Not surprisingly, patients with higher, sustained levels of viruria were more likely to be diagnosed with hemorrhagic cystitis and have a worse outcome [16,27]. The relationship between viremia, nephropathy, and overall outcome has been well documented after

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renal transplant [1,12,28] but is less understood in HSCT patients [6]. Erard et al. [15] found that HSCT patients with a BK plasma load .10,000 copies/mL had a higher rate of hemorrhagic cystitis. In 2009, O’Donnell et al. [16] prospectively found that prolonged BK viremia was an independent risk factor for the development of post-HSCT renal impairment, but these authors did not report any correlation between plasma viral load and outcome. In the present study, we found that urine BK levels did not correlate with clinical prognosis, as almost all our patients with hemorrhagic cystitis had very high levels of viruria, but did not necessarily have a poor outcome. However, in agreement with O’Donnell et al. [16], we found that patients with viremia had impaired renal function after diagnosis. Expanding on their findings, we demonstrated that a BK plasma level of 10,000 copies/mL could differentiate patients with a poor renal, urologic, and overall outcome from those with a self-limited, milder course. Specifically, although all patients with viruria who were evaluated with BK plasma PCR had viremia, patients in the high viremia group had worse renal function, a greater need for dialysis, more aggressive surgical interventions for hemorrhagic cystitis, and a higher mortality compared to patients in the low viremia group. This 10,000 copy/mL cutoff level has also been shown to predict the development of nephropathy after kidney transplant [12]. It is important to note that renal dysfunction following HSCT is multifactorial. Hemorrhagic cystitis can lead to renal insufficiency via urinary tract obstruction. Nephrotoxic medication use is common following HSCT and includes chemotherapy, calcineurin inhibitors, antibiotics, and even antiviral therapy with cidofovir for BK virus infection. Furthermore, complications such as aGVHD and TA-TMA are also associated with significant short- and long-term renal impairment [29]. Interestingly, 70% of our patients in the high viremia group were also diagnosed with TA-TMA during periods of BK viremia. Viral infections associated with the development of TA-TMA include CMV, adenovirus, HHV-6, and parvovirus B19 [18,30-33]. To the best of our knowledge, there are only 3 reports of TA-TMA associated with BK virus infection [7,34,35]. Even though BK virus traditionally damages renal tubular cells and TA-TMA affects the glomerular endothelium, we found both of these processes occurring concomitantly (Figure 1). Although this association could merely be coincidental, it is possible that BK virus is a novel trigger for TA-TMA, contributing to the severe renal injury observed in our high viremia group patients. The optimal treatment for BK associated hemorrhagic cystitis or nephropathy in HSCT patients is currently not known. Treatment for hemorrhagic cystitis is generally supportive and includes pain control,

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transfusion support, intravenous fluids, and bladder irrigations to prevent urinary tract obstruction [23]. Cidofovir, or (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl) cytosine, is a broad-spectrum anti-DNA virus agent active against CMV, adenovirus, and BK virus, and exhibits dose-related nephrotoxicity, limiting its widespread use. High-dose cidofovir therapy (3-5 mg/kg/dose) prescribed for CMV and adenovirus infections requires probenecid administration for renal function protection via reduction of intracellular accumulation of cidofovirphosphocoline in the proximal tubule. Intermediate-dose cidofovir (1 mg/kg/dose) every 1 to 3 weeks with saline hydration and without probenecid prophylaxis had been successfully used in pediatric renal transplant patients with BK-associated nephropathy [36,37]. All these patients had impaired renal function when cidofovir was initiated. These studies demonstrated that cidofovir at this dose was effective in reducing BK viremia without further deterioration or even with improvement in renal function. Ganguly et al. [38] and Savona et al. [39] each reported a series of adult HSCT patients with hemorrhagic cystitis and BK viremia treated with intermediate-dose cidofovir regimens weekly. In these patients, there was no creatinine increase, and approximately 70% of patients had a decrease in viruria. As there is no wellestablished cidofovir dosing regimen for BK infection in pediatric HSCT patients, our patients were treated with intermediate-dose cidofovir based on the above outlined data. Even though cidofovir-related nephrotoxicity cannot be excluded, renal biopsies obtained in our patients with high BK viremia demonstrate BK virus inclusions in tubules and/or glomerular injury from TA-TMA, supporting pathogenesis of renal injury other than medication induced. Well-designed trials of other potential antiviral therapies, including leflunomide, ciprofloxacin, and intravenous immune globulin have not been performed. A reduction in immunosuppression, first-line therapy for BK allograft nephropathy after renal transplant, is effective to control BK virus infection in that clinical setting [40], but is often not feasible postHSCT because of the risk of GVHD. Illustrating this point, in our cohort, BK virus infection often followed a diagnosis of GVHD, where immunosuppression is increased. Leflunomide, an antimetabolite with antiviral and immunosuppressive properties, might be an attractive option for patients with BK viremia and GVHD [41]. Leflunomide is also effective against CMV and might be indicated in CMV- and BK virus–infected patients [42]. Significance of myelosuppressive effects of leflunomide had not yet been evaluated in HSCT patients. There are reports of thrombotic microangiopathy in renal transplant patients receiving leflunomide, so HSCT patients receiving leflunomide should be monitored for TA-TMA

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[43]. However, it is plausible that TA-TMA may be triggered not by leflunomide, but by BK virus itself, a possibility supported by the fact that none of our patients with TA-TMA had received leflunomide. Our study is limited by its retrospective design and BK diagnostic testing in only symptomatic patients. We found a very high prevalence (∼90%) of BK virus infection in those with hemorrhagic cystitis. Although most patients with hemorrhagic cystitis had high urine viral loads, those with high levels of viremia (.10,000 copies/mL) had worse outcomes. Based on our results, we advocate a single urine BK PCR test to confirm the viral etiology in patients with clinical concern for hemorrhagic cystitis. Following a positive urine test, plasma BK viral monitoring may assist with prognosis and guide the use of antiviral therapies. Furthermore, BK plasma testing should be considered for HSCT patients with unexplained renal dysfunction, even in the absence of obvious hemorrhagic cystitis. Prospective clinical trials are urgently needed to confirm our findings, determine the potential role of TA-TMA and GVHD in HSCT-associated BK infection and nephropathy, and establish optimal monitoring and treatment protocols.

ACKNOWLEDGMENTS We acknowledge the physicians, nurses, hemodialysis staff, pharmacists, care managers, data managers, and Hoxworth Stem Cell Therapy staff involved in our patient care. We thank Dr. Mi-Ok Kim and Ms. Chunyan Liu for helping with statistical analysis. Financial disclosure: None of the authors have conflicts of interest to disclose.

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