SDC, MATERIALS AND METHODS
Description of the CERTAIN Registry
The CERTAIN Registry24,25 is a voluntary scientific registry used for clinical research in the field of pediatric renal transplantation. Its dataset supplies essential information on generic kidney transplantation-related topics and also captures specific pediatric parameters such as growth, physical and psychosocial development and adherence. To avoid redundant data input, CERTAIN has set up data exchange interfaces with Eurotransplant, Collaborative Transplant Study (CTS) and the registry of the European Society of Pediatric Nephrology (ESPN) and European Renal Association - European Dialysis and Transplant Association (ERA-EDTA) (ESPN/ERA-EDTA registry).
Data entry follows a specified chronology, starting with the initial visit at renal transplantation surgery, ensued by 5 follow-ups in the first posttransplantation year and 2 annual follow-ups thereafter. The CERTAIN dataset also facilitates a detailed and flexible documentation of the posttransplant follow-up by introducing continuous entries which enable any number of relevant values (eg, laboratory values and medication history) to be documented. A dataset is divided into 2 sections: minimal and extended. The minimal dataset is mandatory for all participating centers, only data fulfilling these minimum requirements being incorporated into the research database. The minimal data set must be complete; missing data are not allowed. The extended dataset provides deeper insight into patients’ treatment and supports documentation of additional items partly predefined and partly definable by the participating center itself.
For research purposes, an analysis of the multinational dataset requires approval of the predefined study protocol by the CERTAIN Registry Steering Committee. The dataset is automatically validated during data entry. Additionally, the system has an integrated manual quality assurance process. First, a documented follow-up visit requires local approval on site;
second, a data quality manager at the registry headquarters checks data plausibility and accepts them for research purposes. Only data passing quality assurance are incorporated into the research database. For the present study, additional source data verification was carried out by one of the investigators (L.S.). The CERTAIN web application (accessible via http://www.certain-
registry.eu/RegApp) therefore supports not only data input, presentation, visualization, and export, but also automatic and manual data validation. These functions are active at any time and location, requiring only a common web browser and internet access.
The range of functionalities available to the logged-on user varies in dependence upon his/her function in the system; predefined roles encompass study nurse, clinician, supervising clinician, data quality manager and registry administrator. Patient data, including the patients’
identity, are accessible only to staff members of the center involved in the care of the respective patient. For quality assurance and research purposes, only medical data cleared of any identifying elements are presented and used. CERTAIN meets all regulatory and ethical requirements of the European Union and Germany, in particular those regarding patients’ data privacy and security.
BKPyV replication and BKPyVAN
Quantitative nucleic acid testing (QNAT) in urine and/or plasma was performed locally according to the manufacturers’ instructions including RealStar® BKV PCR Kit (Altona Diagnostics) in 8 contributing centers, BK Virus R-gene® (Bio-Mérieux) in 3, TIB LightMix® BK kit (TIB Molbiol) in 2, GeneProof BK/JC Virus (BK/JC) PCR Kit (Medac Diagnostika) in 2 centers, and BKV ELITe MGB® Kit (EliTech Group) in 1 contributing center.
TABLE S1. BKPyV surveillance strategies
ParameterPatients (n, %) 1st year posttransplant
Patients (n, %) 5-year observation period
Centers (n, %)
DNA in plasma 290/313 (92.7) 311/313 (99.4) 15/16 (93.8)
DNA in urine 150/313 (47.9) 152/313 (48.6) 6/16 (37.5)
DNA in plasma and urine 130/313 (41.5) 152/313 (48.6) 6/16 (37.5)
Decoy cells in urine 3/313 (1.0) 3/313 (1.0) 1/16 (6.3)
BKPyV, BK polyomavirus; DNA, desoxyribonucleic acid
TABLE S2. BKPyV surveillance frequencies
Time posttransplant DNA in plasma DNA in urine
1st year 9.01 ± 4.17 9.03 ± 4.09
2nd year 3.22 ± 2.17 3.99 ± 2.48
3rd year 2.82 ± 2.33 3.31 ± 2.23
4th year 2.94 ± 2.51 3.58 ± 2.95
5th year 2.71 ± 2.01 3.83 ± 3.50
BKPyV, BK polyomavirus; DNA, desoxyribonucleic acid
TABLE S3. Definition of the pediatric Vasudev score
Immunosuppressant“Vasudev score”1 dose per unit (mg/d)
Pediatric Vasudev score2 dose per unit (mg/m²·d)
Immunosuppressive unit
TAC 2 1.2 1
CSA 100 58 1
EVR 2 1.2 1
SRL 2 1.2 1
MMF 500 290 1
AZA 100 58 1
Prednisone equivalent 5 2.9 1
TAC, tacrolimus; CSA, ciclosporin microemulsion; EVR, everolimus; SRL, sirolimus; MMF, mycophenolate mofetil;
AZA, azathioprine.
1See reference 31,32
2The pediatric score, modified according to Vasudev et al, for assessment of the overall immunosuppressive load, was calculated by adjustment of the adult score to a body surface area (BSA) of 1.0 m², assuming an adult BSA of 1.73 m². Induction therapy with an interleukin-2 receptor antagonist and antirejection treatment with steroid pulse therapy were included in this score and assigned to 2 immunosuppressive units each. See also reference 31,32.
TABLE S4. Characteristics of patients with biopsy-proven BKPyVAN.
Patients are listed according to functional outcome: recovery, stabilization, deterioration Age
at RTx
Onset of BKPyV viruria
Onset of BKPyV viremia
Histopathological findings1
eGFR loss
(%)2 Therapy
Outcome (% of baseline eGFR)
3.0 yrs n.d. Viremia
- 4 months p.t.
High-level viremia - 6 months p.t.
BKPyVAN grade A - 13 months p.t.
Banff IB rejection3 - 26 months p.t.
12% Ciprofloxacin Recovery
(100%)
Decrease of viremia (from 106 to 103 copies/mL) 2.8 yrs Viruria
- 37 months p.t.
High-level viruria - 37 month p.t.
Viremia
- 39 months p.t.
High-level viremia - 39 months p.t.
BKPyVAN grade A Banff IA rejection4 - 44 months p.t.
BKPyVAN grade A - 46 months p.t.
47% 1. TAC dose reduction 2. MMF withdrawal 3. IVIG
4. Levofloxacin
Recovery (97%)
Clearance of viremia (from 107 copies/mL) Decrease of viruria (from 109 to 104 copies/mL) 5.0 yrs Viruria
- 18 months p.t.
High-level viruria - 20 month p.t.
Viremia
- 21 months p.t.
High-level viremia - 26 months p.t.
BKPyVAN grade A - 43 months p.t.
37% 1. TAC dose reduction 2. MMF withdrawal
Recovery (93%)
Clearance of viremia (from 105 copies/mL) Clearance of viruria (from 109 copies/mL)
17.0 yrs n.d. Viremia
- 11 months p.t.
High-level viremia - 11 months p.t.
BKPyVAN grade A - 12 months p.t.
11% 1. TAC dose reduction 2. MMF dose reduction
Recovery (89%)
Decrease of viremia (from 106 to 103 copies/mL)
17.0 yrs Viruria
- 23 months p.t.
High-level viruria - 34 month p.t.
Viremia
- 34 months p.t.
High-level viremia - 34 months p.t.
BKPyVAN grade C Acute (II) and chronic ab-mediated rejection5
- 35 months p.t.
42% 1. TAC dose reduction
2. Conversion from TAC/MMF to TAC/EVR
3. Cidofovir
Stabilization (77%)
Decrease of viremia (from 105 to 103 copies/mL)
Decrease of viruria (from 1010 to 106 copies/mL)
15.0 yrs n.d. Viremia
- 3 months p.t.
High-level viremia - 3 months p.t.
BKPyVAN grade A - 3 months p.t.
57% 1. TAC dose reduction 2. MMF dose reduction
Stabilization (49%)
Clearance of viremia (from 104 copies/mL) 3.4 yrs Viruria
- 3 months p.t.
High-level viruria - 3 months p.t.
Viremia
- 3 months p.t.
High-level viremia - 3 months p.t.
BKPyVAN grade A - 8 months p.t.
BKPyVAN grade A - 10 months p.t.
70% 1. Conversion from TAC/MMF/MPR to CSA/EVR/MPR
2. CSA withdrawal 3. Levofloxacin
Stabilization (82%)
Decrease of viremia (from 105 to 103 copies/mL)
Decrease of viruria (from 1010 to 106 copies/mL) 2.3 yrs Viruria
- 1 month p.t.
High-level viruria - 3 months p.t.
Viremia
- 4 months p.t.
High-level viremia - 4 months p.t.
BKPyVAN grade A Banff IB rejection6 - 9 months p.t.
BKPyVAN grade A - 13 months p.t.
BKPyVAN grade C - 18 months p.t.
87% 1. TAC withdrawal 2. MMF withdrawal 3. Conversion to EVR/MPR 4. IVIG
5. Cidofovir
Stabilization (27 %)
Clearance of viremia (from 107 copies/mL) Decrease of viruria (from 1011 to 103 copies/mL)
14.5 yrs Viruria
- 5 months p.t.
High-level viruria - 5 months p.t.
Viremia
- 5 months p.t.
High-level viremia - 5 months p.t.
BKPyVAN grade B - 6 months p.t.
30% 1. Conversion from TAC/MMF/MPR to CSA/EVR/MPR
Stabilization (71%)
Ongoing Viremia (106 - 105 copies/mL) Ongoing viruria (1010 - 109 copies/mL) 1.1 yrs Viruria
- 1 month p.t.
High-level viruria - 1 month p.t.
Viremia
- 3 months p.t.
High-level viremia - 6 months p.t.
BKPyVAN grade B - 12 months p.t.
67% 1. MMF dose reduction 2. TAC dose reduction 3. Cidofovir
4. Conversion from TAC/MMF to TAC/EVR
5. IVIG
6. Ciprofloxacin
Stabilization (44%)
Clearance of viremia (from 105 copies/mL) Decrease of viruria (from 1011 to 107 copies/mL)
4.0 yrs n.d. Viremia
- 3 months p.t.
High-level viremia - 10 months p.t.
BKPyVAN grade A - 13 months p.t.
BKPyVAN grade A - 20 months p.t.
41% Conversion from TAC/MMF to CSA/EVR Stabilization (59%)
Decrease of viremia (from 105 to 103 copies/mL) 8.3 yrs Viruria
- 9 months p.t.
High-level viruria - 9 month p.t.
Viremia
- 9 months p.t.
High-level viremia - 10 months p.t.
BKPyVAN grade A - 20 months p.t.
41% 1. IVIG
2. TAC dose reduction 3. MMF dose reduction
4. Conversion from TAC/MMF to TAC/EVR
Stabilization (57%)
Clearance of viremia (from 105 copies/mL) Decrease of viruria (from 109 to 105 copies/mL)
8.0 yrs n.d. Viremia
- 18 months p.t.
High-level viremia - 18 months p.t.
BKPyVAN grade A - 18 months p.t.
Banff IA rejection7 31 months p.t.
19% 1. IVIG
2. TAC dose reduction
3. Conversion from TAC/MMF to TAC/EVR
Deterioration (56%)
Decrease of viremia (from 105 to 103 copies/mL)
14.4 yrs Viruria
- 5 months p.t.
High-level viruria - 6 month p.t.
Viremia
- 6 months p.t.
High-level viremia - 6 months p.t.
BKPyVAN grade A Borderline rejection8
- 19 months p.t.
59% 1. IVIG
2. Conversion from TAC/EVR/MPR to CSA/EVR/MPR
3. CSA withdrawal
Graft loss
Ongoing viremia (107 copies/mL) Ongoing viruria (109 copies/mL) BKPyVAN, BK polyomavirus-associated nephropathy; RTx, renal transplantation; BKPyV, BK polyomavirus; eGFR, estimated glomerular filtration rate; yrs, years; n.d., not done;
p.t., posttransplant; TAC, tacrolimus; MMF, mycophenolate mofetil; MPR, methylprednisolone; CSA, ciclosporin microemulsion; EVR, everolimus; IVIG, intravenous immunoglobulin; ab, antibody
1According to the Banff classification (ref.29,30,63)
2Prior to treatment of BKPyVAN
3Rejection therapy consisted of a conversion from MMF to EVR which did not lead to any significant change of BKPyV plasma viral loads.
4Rejection was treated by a steroid pulse therapy which resulted in an intermittent rise of urinary and plasma BKPyV viral loads from 107 to 108 and from 104 to 105 copies/mL, respectively.
5Rejection was treated by rituximab and had no significant impact on urinary or plasma BKPyV viral loads.
6Rejection therapy consisted of steroid pulses which led to an intermittent increase of urinary (from 108 to 1010 copies/mL) and plasma (from 104 to 106 copies/mL) BKPyV viral loads.
7Rejection was treated by steroid pulse therapy without any significant changes of plasma BKPyV viral loads.
8Rejection was treated by thymoglobulin administration which led to a rise of urinary BKPyV viral loads from 108 to 109 copies/mL and an increase of plasma BKPyV viral loads from 106 to 107 copies/mL.
TABLE S5. Risk factors for developing biopsy-proven BKPyVAN (including ureteral stent placement at transplantation)
Variable
Unadjusted OR
(95% CI) p value
Adjusted OR
(95% CI) p value
Younger age at RTx (years) 1.08 (0.98 – 1.20) 0.122
Male gender 3.70 (0.81 – 16.8) 0.091
Obstructive uropathy 9.00 (2.88 – 28.2) <0.001 13.5 (3.09 – 58.5) 0.001 Ureteral stent at RTx1 2.00 (0.42 – 9.48) 0.383
Transplant pyelonephritis 1.53 (1.11 – 2.11) 0.009 Acute rejection 4.06 (1.24 – 13.2) 0.020
Pediatric Vasudev Score2 1.26 (1.10 – 1.45) <0.001 1.29 (1.09 – 1.52) 0.003 BKPyVAN, BK polyomavirus-associated nephropathy, RTx, renal transplantation
1Duration of ureteral stent (> 1week vs. ≤ 1 week posttransplant) was also evaluated but did not prove to be an independent risk factor.
2According to reference31,32
TABLE S6. Comparison between patients with late (> 2 years posttransplant) BKPyV viremia and those without BKPyV viremia
Parameter
Patients with late BKPyV viremia
(n = 11 )
Patients without viremia
(n = 198) p value
Age at RTx, years 9.7 ± 5.1 11.1 ± 5.4 0.395
Donor age, years 37.1 ± 17.4 32.2 ± 16.6 0.340
Cold ischemia time, hours 9.2 ± 8.4 8.9 ± 6.9 0.918
HLA mismatches, n 2.6 ± 1.7 2.6 ± 1.2 0.989
Male gender, n (%) 7 (63.6) 119 (60.1) 0.816
Living-related RTx, n (%) 5 (45.5) 75 (37.9) 0.615
Obstructive uropathy, n (%) 1 (9.1) 14 (7.1) 0.801
Transplant pyelonephritis, n (%) 2 (18.2) 32 (16.2) 0.860
eGFR (ml/min·1.73 m²)1 78.5 ± 29.1 78.7 ± 30.7 0.985
BPAR, n (%)2 5 (45.5) 79 (39.9) 0.715
TAC, n (%)3 10 (90.9) 133 (67.2) 0.107
CSA, n (%)3 1 (9.1) 57 (28.8) 0.151
MMF, n (%)3 10 (90.9) 153 (77.3) 0.288
AZA, n (%)3 0 (0.0) 4 (2.0) 0.634
EVR, n (%)3 1 (9.1) 37 (18.7) 0.422
Steroids, n (%)3 9 (81.8) 163 (82.3) 0.966
Pediatric Vasudev Score 7.6 ± 2.8 6.5 ± 2.7 0.221
BKPyV, BK polyomavirus; RTx, renal transplantation; HLA, human leukocyte antigen; eGFR, estimated glomerular filtration rate;
BPAR, biopsy-proven acute rejection; IL-2, interleukin 2; TAC, tacrolimus; CSA, ciclosporin microemulsion; MMF, mycophenolate mofetil; AZA, azathioprine; EVR, everolimus
1Defined as eGFR62 at 30 days posttransplant. eGFR was calculated according to the revised Schwartz formula62: eGFR = (mL/min·1.73 m²) = 0.413 x [height (cm)/serum creatinine (mg/dL)].
2Within the first 2 years post-transplant; including borderline changes (Banff 97 ’09 update63,64)
3Comparison of the immunosuppressive regimen between patients with BKPyV viremia (within 6 months prior to BKPyV viremia) and those without BKPyV viremia (at 2 years posttransplant)
TABLE S7. Therapeutic strategies and impact on BKPyV replication
Therapeutic strategies BKPyV viruria BKPyV viremia
CNI (TAC) dose reduction only (n = 14) Clearance (n = 9)1 Clearance (n = 14)5 Decrease (n = 2)2
Ongoing (n = 2)3,4
MMF dose reduction only (n = 7) Clearance (n = 2)1 Clearance (n = 4)5 Ongoing (n = 1)3,6 Ongoing (n = 2)4,7,8 CNI (TAC) and MMF dose reduction (n = 7) Clearance (n = 4)1,9 Clearance (n = 6)5
Ongoing (n = 1)10
MMF withdrawal only (n = 3) n.a.11 Clearance (n = 2)5
Ongoing (n = 1)7 Other combinations (n = 33)12 Clearance (n = 6)1 Clearance (n = 20)16
Decrease (n = 12)13 Decrease (n = 5)17 Ongoing (n = 5)14,15 Ongoing (n = 6)18,19
BKPyV, BK polyomavirus; CNI, calcineurin inhibitor; TAC, tacrolimus; CSA, ciclosporin microemulsion; MMF, mycophenolate mofetil; EVR, everolimus
1maximal urine BKPyV viral load 1010 copies/mL
2from 109 to 103 copies/mL
3urine BKPyV viral load 107 – 109 copies/mL
4no measurement of urinary BKPyV viral load in 1 patient
5maximal plasma BKPyV viral load 105 copies/mL
6no measurement of urine BKPyV viral load in 4 patients
7plasma BKPyV viral load 103 – 104 copies/mL
8no BKPyV viremia in 1 patient
9no measurement of urinary BKPyV viral load in 3 patients
10plasma BKPyV viral load 105 – 106 copies/mL
11no measurement of urine BKPyV viral load
12including dose reduction, withdrawal, switch of TAC to CSA and/or MMF to EVR as well as adjunct therapy
13from 1011 to 103 copies/mL
14urine BKPyV viral load 107 – 1010 copies/mL
15no measurement of urine BKPyV viral load in 10 patients
16maximal plasma BKPyV viral load 107 copies/mL
17from 106 to 103 copies/mL
18plasma BKPyV viral load 104 – 107 copies/mL
19no BKPyV viremia in 2 patients
