University of Groningen Importance of molecular diagnostic of viral infections in renal transplant recipients Rurenga-Gard, Lilli

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Importance of molecular diagnostic of viral infections in renal transplant recipients

Rurenga-Gard, Lilli

DOI:

10.33612/diss.92267443

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Rurenga-Gard, L. (2019). Importance of molecular diagnostic of viral infections in renal transplant

recipients. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.92267443

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CHAPTER 7

Relevance of EBV load monitoring

in renal transplant recipients; a

retrospective cohort study

Lilli Gard

1

_{, Claudy Oliveira dos Santos}

1

_{, Willem van}

Doesum

2

_{, Hubert G.M. Niesters}

1

_{, Willem J. van Son}

2

_,

Arjan Diepstra

3

_{, Coen A. Stegeman}

2

_{, Henk Groen}

4

_{, Jan}

Stephan Sanders

2

_{, Annelies Riezebos-Brilman}

5

1_{Department of Medical Microbiology, Division of Clinical} Virology2_{, Department of Internal Medicine, Division of} Nephrology, 3_{Department of Pathology and Medical Biology,} 4_{Department of Epidemiology, University of Groningen,} University Medical Center Groningen, Hanzeplein 1, 9700 GZ Groningen, The Netherlands. 5_{Department of Medical} Microbiology, University Medical Center Utrecht, PO Box 85500, 3508 GA Utrecht, The Netherlands

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126 ABSTRACT

Background

EBV load monitoring after solid organ transplantation is used to predict post-transplant lymphoproliferative disorder (PTLD) development in high risk populations.

We studied in a retrospective single center cohort, the additive value of EBV monitoring in renal transplant recipients (RTR).

Methods

373 RTR were included who received a kidney transplant between 2010 and 2012. The incidence of EBV-viremia in whole blood, graft and patient survival, PTLD and biopsy proven acute rejection (BPAR) was studied.

Results

121 recipients were EBV-DNA negative in whole blood (32.4%), whereas EBV-DNA was detectable at least once in 252 recipients (67.5%). One patient developed histological proven PTLD (0.4%). Incidence of graft failure was not significantly different between the EBV-viremia group (n=25/247,10.1%) and the EBV-viremia negative group (n=7/121, 5.8%) (RR= 1.75, 95% CI 0.8-3.9, p=0.17). In recipients with EBV viral load > log 4cp/ml graft loss occurred significantly more often (p= 0.004). BPAR and mortality rate did not differ significantly between the groups with or without EBV-viremia.

Conclusion

EBV-viremia is common in RTR. With EBV viremia >log 4 cp/ml more graft loss was observed. However, no association with patient survival, PTLD and BPAR was found.

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127 INTRODUCTION

EBV reactivation is common in both immunocompetent and immunocompromised patients. In patients who received a solid organ transplant, it may cause post-transplant lymphoproliferative disorder (PTLD). The incidence of PTLD varies between different solid organ transplant recipients and is relatively low in renal transplant recipients (RTR) (1-5%) compared to heart and lung (2-10%) and intestinal as well as multivisceral transplant recipients (5-20%) 1_{. PTLD is} diagnosed by pathology, but clinical suspicion may develop by rising EBV load in whole blood/ EDTA-plasma together with relevant clinical symptoms and signs. These clinical features may vary from fever, weight loss and fatigue to extra nodal masses and dysfunction of involved organs. Currently, EBV load monitoring is used as a prognostic marker, at least, in the first year after transplantation, to predict PTLD development in high risk populations 2,3_.

Nevertheless, the implications of EBV-viremia after the first year of renal transplantation are not clear. Retrospective studies reporting on prevalence, patterns and long-term outcome of RTR demonstrate divergent associations of EBV-viremia involving graft loss, biopsy proven acute rejection (BPAR), renal function and other infections 4-7_.

The aim of this observational retrospective, single center, cohort study was to investigate the additive value of EBV monitoring in RTR, during the first-year post-transplant with up to six years follow-up, by investigating the effect of EBV-viremia on the outcome of graft and patient survival, and PTLD development.

MATERIAL AND METHODS

Patients

Patients who underwent a first, second or third renal transplantation between January 2010 and December 2012 at the University Medical Center Groningen (UMCG) were included. Exclusion criteria were primary non-function of the allograft or receiving no immunosuppressive therapy.

The Ethics Committee of the UMCG decided that the study did not fall under the scope of the Medical Research Involving Human Subjects Act (METc 2015/448). Informed consent was not engaged with the study participants since this is not required in the Netherlands with retrospective studies.

Renal function and biopsy

Renal function was measured by using the estimated glomerular filtration rate (eGFR) (MDRD formula) at the following time points 3 (±3 weeks), 6 (±1.5 months), 12 (±3 months), 24 (±5

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months), 36 (±6 months), 48 (±6 months), and 60 (±6 months) months. At returning to dialysis or nephrectomy, recipients eGFR were imputated as 0 ml/min/1.73m2.

Renal biopsies were taken according to protocol at one year post transplantation and by indication. Renal biopsies were scored by a renal pathologist according to Banff classification 8_{. Biopsy proven acute T-cell mediated rejection (TCMR) was scored as Banff IA, IB, IIA, IIB or III.}

Clinical data

Retrospective virological, clinical and renal biopsy data were collected from the date of transplantation until 31 December 2016, using electronic patient files.

In the first year after transplantation, EBV was monitored by protocol, in the first month weekly, in the second and third month biweekly and thereafter monthly.

Virological Diagnostics

EBV-DNA was measured with an internally controlled quantitative in-house Real Time PCR (RT-PCR), targeting against the nonglycosylated membrane protein BNRF1 p143B and a detection limit of 100 cp/ml.

All samples were extracted according to the manufacturer’s instructions, using 190 µl whole blood samples with the addition of 10 µl seal herpes virus (PhHV) as an internal control 9_. Samples collected before January 2012 were extracted using the MagNaPure LC with the MagNa Pure LC Total Nucleic Acid Isolation kit (Roche Diagnostics, Germany). After January 2012, DNA was extracted with the MagNaPure 96 system using the MagnaPure 96 DNA and viral NA small volume kit (Roche Applied Bioscience, Mannheim, Germany).

The internal control PCR reaction was performed as described by Lollinga et al 10_{. EBV} PCR reactions were performed using a real time PCR as described by Niesters et al. 11 with minor modifications. Briefly, 20µl DNA and 30µl PCR mix, containing 2x Universal Mastermix (Thermofisher, USA), 5mg/ml bovine serum albumin (Roche Diagnostics, Germany), DNAse/RNAse free water (Sigma, The Netherlands), 900nM forward primer (5’-ggaacctggtcatcctttgc), 50nM reverse primer (5’-acgtgcatggaccggttaat) and 100nM probe (6FAM-cgcaggcactcgtactgctcgct-TAMRA). The ABI PRISM 7500 (Life Technologies, USA) was used for detection and amplification using the following thermal profile: 50°C for 2min, 95°C for 10min followed by 42 cycles of 95°C for 15sec, 60°C for 1min.

Viral loads were derived from the raw Ct-values using a previously established standard curve for copies/ml (based on electron microscopy counted EBV stock (Advanced Biotechnologies

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Incorporated, U.S.A.). Validity of these external standard curves was continuously monitored using a low and high range positive control calibrated to these standards.

EBV serology was routinely performed before transplantation. EBV VCA IgM, VCA IgG, and EBNA IgG antibodies determinations were performed on an automated immunoassay platform, the Liaison, using the EBV serology of the Liaison system. (Diasorin, Saluggia, Italy) In this two-step immunoassay these antibodies were being quantified using magnetic particles and a final chemiluminescence detection. The assay was performed in accordance with the manufacturers’ recommendations. The applied cutoffs for the Liaison assays were 40mU/liter for VCA IgM, 20 mU/liter for VCA IgG, and 20 mU/liter for EBNA IgG.

Patients EBV categorization

Patients were retrospectively categorised based on EBV-viremia in an EBV-PCR negative versus an PCR positive group. Viremia was defined if, at least once, in a whole blood sample EBV-DNA was measured above 100 cp/ml during the follow up period.

In the EBV-PCR positive group patients were also retrospectively categorised in high viral load, EBV-DNA measured above 10,000 cp/ml and low viral load, EBV-DNA measured between 100-10,000 cp/ml. The cut-off value of 100-10,000 cp/ml (log 4cp/ml) was selected according to a study of Bamoulid et al 4_.

Primary EBV infection was diagnosed in sero-negative recipients without EBV IgG antibodies before transplantation EBV-DNA was measured after transplantation.

Immunosuppression

The immunosuppressive regimens consisted, for the majority of patients, of quadruple immunosuppression with basiliximab induction, a calcineurin-inhibitor, i.e. Tacrolimus (Tac) or Cyclosporine A (CsA), MPA, i.e. mycophenolate mofetil (MMF) or mycophenlate sodium (MPS) and prednisolone. By protocol, RTR received CsA before January 2012 and Tac from January 2012 onwards. Based on clinical indication, some recipients received Tac before January 2012 or steroid-free immunosuppression. MPA was started at 2000 mg/day and tapered to1500 mg/ day when combined with tacrolimus, and subsequently 1000 mg/day or 500 mg/day based on clinical indications, especially if side-effects to MPA were present. By protocol, co-trimoxazole was given as PCP prophylaxis after transplantation for the duration of six months.

PTLD treatment consisted of lowering immunosuppression, Rituximab treatment, or a combination of both. Anti-rejection therapy consisted of methylprednisolone intravenously

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on three consecutive days, if a vascular rejection occurred or in case of steroid resistance rescue therapy with Anti-Thymocyte Globulin (ATG) was started.

Tac trough drug levels (µg/l) and CsA trough drug levels (µg/l) were collected at time points 2 weeks (±2 days), 6 weeks (±2 weeks), 3 months (±3 weeks) and 12 months (±3 months).

Statistics

Statistical analyses were performed, and figures were created using SPSS IBM Statistics 22 (IBM, USA) and Graphpad Prism 5.01 (GraphPad Software, Inc, USA). P-values <0.05 were considered significant.

Baseline characteristics between the two EBV groups were compared using Chi-square test for categorical variables and the independent sample t-test for continuous variables. Occurrence of EBV-viremia was compared by Chi-square test at time point 3 months and 12 months between Tac and CsA trough levels above and below the median.

Kaplan-Meier analysis were used for graft loss according to EBV-viremia. For this analysis only EBV-viremia before graft loss was included. The following variables were used for the cox regression analysis: recipient sex and age, donor sex and age, cold ischemia time, type of donor (living, deceased), and BPAR.

Longitudinal analyses were performed using generalized estimating equations (GEE) with an exchangeable correlation matrix and the resulting estimated marginal means (EMM) with 95% confidence intervals were plotted in graphs. The effect of EBV-viremia on the renal function was analyzed using GEE over a time period from t=3 to t=60 months. Adjusted for recipient sex and age, donor sex and age, cold ischemia time, type of donor (living, deceased), BPAR, graft loss, HLA mismatch AB/DR, primary disease, reduction/stop MPS/MMF .

RESULTS

Baseline characteristics

From 384 RTR transplanted in the UMCG between 2010 and 2012, 373 were included in the analysis. Six recipients had no allograft function and 5 recipients received no immunosuppressive therapy and were therefore excluded. Five recipients of the EBV-viremia group, became EBV positive after graft loss. They were excluded from the analysis for graft survival, BPAR and renal function.

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Baseline characteristics did not differ significantly between the EBV-PCR negative versus the EBV-PCR positive group (Table 1), with an exception for the type of donor (living versus postmortem) and the age of recipient.

Table 1. Baseline characteristics

EBV neg (N = 121) EBV pos (N = 252) P-value Male, n (%) 71 (58.7) 136 (54.0) 0.39 Age, years ± SD 47.54 ± 13.8 52.0 ± 13.2 0.003 Caucasian n (%) 109 (92.4) 225 (91.8) 0.86

HLA mismatch AB, mean ± SD 1.79 ± 1.1 1.81 ± 1.16 0.85

HLA mismatch DR, mean ± SD 0.83 ± 0.62 0.82 ± 0.67 0.85

Induction, n (%) 0.89

ATG 1 (0.8) 3 (1.2)

Basiliximab 118 (97.5) 246 (97.6)

Other 2 (1.7) 3 (1.2)

Primary disease, n (%) 0.63

Primary glomerular disease/

glomerulonephritis 40 (33.1) 69 (27.4)

Cystic kidney disease 22 (18.2) 37 (14.7)

Renovascular disease/ hypertension 11 (9.1) 29 (11.5)

Diabetic nephropathy 10 (8.3) 21 (8.3) Tubulo-interstitial nephritis 3 (2.5) 3 (1.2) Urological complications 4 (3.3) 10 (4.0) Other 31 (25.6) 83 (32.9) First transplantation, n (%) 101 (84.2) 209 (83.3) 0.83 BKV viremia, n (%) 21 (17.4) 60 (23.8) 0.16 Primary immuunsuppression, n (%) 0.14 CyA + MMF 71 (58.7) 120 (47.6) Tac + MMF 46 (38.0) 121 (48.0) Other 4 (3.3) 11 (4.4) CMV serogroup at transplantation, n (%) 0.07 D-/R- 37 (30.6) 52 (20.6) D-/R+ 24 (19.8) 58 (23.0) D+/R- 16 (13.2) 56 (22.2) D+/R+ 42 (34.7) 77 (30.6) unknown 2 (1.7) 9 (3.6)

EBV IgG pos, n (%) 109 (95.6) 222 (92.9) 0.32

Donor characteristics

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Table 1. Continued EBV neg (N = 121) EBV pos (N = 252) P-value Male, n (%) 59 (48.8) 126 (50.2) 0.80 Age, years ± SD 49.3 ± 13.4 51.6 ± 12.3 0.12 Type of transplantation, n (%) 0.027 Living 70 (57.9) 110 (43.7)

Donation after brain death 29 (24.0) 90 (35.9)

Donation after cardiac death 22 (18.2) 52 (20.7)

Cold ischemia time, deceased donors only,

min ± S.D. 379.1 ± 488.1 477.3 ± 475.98 0.067 Delayed Graft function,n (%) 30 (24.8) 57 (22.6) 0.64

Chi-squared test was used for the donor and recipient baseline characteristics.

EBV-viremia

With regular screening in the first year 4858 of the 6341 (76.6%) EBV load measurements were performed.

121 recipients were EBV-DNA negative (32.4%), whereas EBV-DNA was detectable in 252 recipients (67.5%) with a median EBV load of log 3.38 cp/ml (range: log 2.18 - log 5.9 cp/ml). First EBV-viremia occurred mostly within 30 days post-transplantation (<1 month, 50.8%, 1-3 month, 17.8%, 3-6 month, 9.9%, >6 month, 21.4%), as depicted in Fig 1A. Fig 1B. shows that in recipients with EBV-viremia, 54% achieved the highest load after a period of 6 months.

Fig 1. Course of EBV viral load.

A) Percentage of first EBV load measure in whole blood in time (<1 month, 50.8%, 1-3 month, 17.8%, 3-6 month, 9.9%, >6 month, 21.4%), every recipient is included once. B) Highest EBV load (cp/ml) in time. Every recipient is included once. The bar depicts the mean viral load with SD.

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22 recipients had no detectable EBV IgG antibodies (another 20 were unknown) before transplantation. Seventeen of these recipients developed a primary EBV infection within 3 months post-transplantation (<1 month, 29.4%; 1-3 months, 11.8%; 3-6 months, 41.2%; >6 months, 17.7%).

Primary outcome: graft and patient survival

From the 373 recipients, 55 (14.7%) deceased during the study period. Fifteen (12.4%) in the EBV negative group and 40 (15.9%) in the group with EBV-viremia (p=0.38).

Graft loss occurred in 25 (10.1%) recipients with EBV-viremia and in 7 (5.8%) recipients without EBV-viremia (p=0.17). Using Kaplan Meier analysis, no difference was observed in the occurrence of graft loss over time in recipients with or without EBV-viremia (death censored p=0.108; uncensored p=0.180) (Fig 2A. and Fig 2B.).

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Fig 2. Kaplan-Meier occurrence of graft loss over 72 months.

There was no significant difference in the incidence of graft loss over 72 months between the EBV positive and EBV negative group. A) Graft loss death censored. (EBV-positive group: 0 months, n= 212; 12 months, n=204; 24 months, n= 198;

36 months, n=194; 48 months, n=191; 60 months, n=189; 72 months, n=186). (EBV negative group: 0 months, n=

106; 12 months, n=105; 24 months, n= 102; 36 months, n=101; 48 to 72months, n=101). B) graft loss not censored for death. (EBV-positive group: 0 months, n= 252; 12 months, n=242; 24 months, n= 235; 36 months, n=230; 48 months,

n=227; 60 months, n=225; 72 months, n=222). (EBV negative group: 0 months, n= 121; 12 months, n=118; 24

months, n= 115; 36 months, n=114; 48 to 72months, n=114)

Graft loss occurred significantly more often in recipients with a viral load > log 4cp/ml. Respectively, in 13 (7%) recipients with a viral load < log 4cp/ml and in 12 (19.7%) recipients with a viral load > log 4cp/ml (p= 0.004).

Outcome in terms of occurrence of graft loss was not significantly different between recipients with an EBV primary infection (13.6%, n=3) and recipients with an EBV reactivation (9.4%, n=31).

PTLD

In total, 189 (75%) recipients had, at least one episode of detectable EBV load <log 4cp/ml, whereby 63 (25%) recipients had, at least once, EBV load > log 4cp/ml. In the latter group, eleven recipients were suspected of having PTLD by rising EBV load combined with clinical symptoms and signs. One recipient developed a histological proven B cell PTLD (0.4%). Histology showed

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a polymorphic PTLD with EBER and CD20 positive B-cells. This patient was treated by lowering immunosuppression and Rituximab. In two recipients, Rituximab was started pre-emptively and in eight recipients, immunosuppression was lowered based upon rising EBV viral loads. Subsequently, additional diagnostic tests (PET) were performed, but none of these recipients developed PTLD.

Transplant function and BPAR

The univariate longitudinal course of eGFR rates was not different in the group with EBV-viremia and the EBV PCR negative group (Fig 3.).

Fig 3. eGFR between 3 and 60 months in the EBV group.

There was no significant difference in the eGFR between 3 and 60 months between the EBV-viremia and EBV negative group. GEE with an exchangeable correlation structure were used for calculating the P-value. (3 months, n=364; 6 months, n= 356; 12 months, n= 357; 24 months, n= 305; 36 months, n= 304; 48 months, n= 275; 60 months, n= 176)

In 59 recipients BPAR occurred, 3 recipients with antibody mediated rejection and 56 with TCMR. Recipients with antibody mediated rejection had an EBV-viremia.

In 35 recipients with an EBV-viremia, TCMR occurred (14.4%), which was treated. In 17 of these 35 recipients (48.6 %), EBV-DNA was detected first and in 18 (51.4%) TCMR was diagnosed first. Comparable TCMR rates were seen in 21 (17.4%) recipients of the group without EBV-viremia. Of these 56 recipients with treated TCMR, 29 patients (51.8%) were treated with methylprednisolone; 19 recipients (54.3%) in EBV-viremia and 10 recipients (47.6%) in the EBV negative group, respectively; 18 recipients with TCMR (32.1%) received ATG, in general first recipients received methylprednisolone and by no response escalated to ATG. Nine recipients received another treatment.

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Outcome in terms of occurrence of TCMR was not significantly different between recipients with an EBV primary infection and recipients with an EBV reactivation (18.2%, n=4 vs.15.6%, n=51).

Occurrence of TCMR in recipients with viral load < log 4 cp/ml (n= 23, 12,5%) or > log 4 cp/ml (n= 12, 20.3%) was comparable (p= 0.14)

EBV and immunosuppression

Differences in immunosuppressive treatment initiated directly after transplantation, had no effect on the occurrence of EBV-viremia. EBV load was at least once detectable in 120 (62.8%) RTR treated with CsAM compared to 121 (72.5%) RTR treated with TacM (p=0.053). Tac and CsA trough levels were analyzed during the first 12 months after transplantation. The median Tac and CsA trough level were comparable at t=3 months and t=12 months for RTR with and without EBV-viremia (Supplement Table 1). Also, no difference could be observed in EBV loads above log 4cp/ml in RTR with Tac and CsA trough levels above the median at t=3 and 12 months. MPA/MPS was, significantly, more often stopped or reduced in the group with EBV-viremia (p=0.002). In most of these RTR (n=230, 91.3%) MPS/MMF was either stopped or reduced in response to EBV-viremia detection.

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137 DISCUSSION

Several studies have reported on EBV-viremia and the development of PTLD in RTR recipients as the standard procedure of monitoring and acting on EBV results, especially with the relative low incidence of PTLD in this group. However, this remains a subject of debate.

Therefore, in this retrospective cohort study, we investigated the effect of EBV-viremia in 373 RTR on the outcome of graft, patient survival and PTLD development. EBV-viremia was found in 67.5% of the RTR, with only one proven PTLD case (0.4%). EBV-viremia was associated with increased graft loss in recipients with EBV viral load > log 4 cp/ml, but not associated with increased BPAR, although in this group MPS/MMF was stopped more often.

The incidence of EBV-viremia, 50.8% in the first month, is consistent with other studies of RTR 4,12_{. The occurrence of PTLD (0.4% histologically proven) in this study, was low, but in line with} others 13,14_{. We observed no difference in incidence of EBV-viremia between treatment with} CsAM or TacM, initiated directly after transplantation.

These findings are, beside the effect on renal function and graft survival, in line with a study of Morton et al. In their study, including 499 RTR, no differences were observed for patient, rate of kidney function decline and patient reported symptoms between the group with and without EBV-viremia 15_{. In contrast, Bamoulid et al. showed a significant association of} EBV-viremia in the first 6 months after transplantation and the occurrence of graft loss 4_{. Here we} found that graft loss was significant higher in recipients with an EBV viral load >log 4 cp/ml. Smith and colleagues found an association of EBV-viremia and decrease in renal function in pediatric renal transplant recipients 7_{. In our study renal function also decreased in the first 3} months post-transplantation in RTR with EBV-viremia. After the first 3 months there was no further decline in renal function, this was not significantly different between the EBV-viremia and EBV negative group.

The influence of the newer immunosuppressive drugs and the occurrence of EBV-viremia remains unclear. Bamoulid and colleagues state that EBV reactivation reflects over-immunosuppression, as their study demonstrated that EBV-viremia is more frequent in ATG treated RTR and that persistent EBV-viremia is associated with the occurrence of other opportunistic infections 4_. These findings strengthen the earlier study of San-Juan et al., showing that EBV-viremia could be an early surrogate marker of the net state of immunosuppression 16_{. Our data did not confirm} this, as, at t= 3 and 12 months, since no significant differences in EBV loads were seen between recipients with Tac/CsA drug trough levels above versus under the median.

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This study has two limitations. First, the retrospective design of the study, which could have influenced the outcome by multiple sequential events that are not attributed directly to the virus, but rather to clinical decisions that cascade from the detection of the virus. This retrospective design limits the power of the study, effects the results from unmeasured confounders, lacks a standardized approach and data of the EBV serostatus of the donor. Nevertheless, our study shows results derived from real time clinical practice, making them relevant for transplant care.

Second, the significant difference between the donor types. The EBV-viremia group had less living and more post-mortem donations. The difference in quality of donor kidneys could have played a role in the rate of EBV reactivation and difference in renal function. Yet, to exclude this effect the analysis were adjusted for the type of transplantation.

To date a reliable prognostic marker for PTLD detection is lacking. For this, although it is not the optimal prognostic marker, EBV viral load is currently being used as monitoring tool for PTLD development. When EBV loads are rising or remain persistent detectable, pre-emptive treatment with Rituximab is being considered to prevent PTLD 17,18_{. Additionally, EBV viral loads} in peripheral blood can be negative in RTR patients who develop PTLD in the central nervous system 19_.

In the future, more specific markers may become available to differentiate between patients at high risk and low risk for developing PTLD. Possibly, EBV miRNA’s could fulfill this role 20_. However, future studies should further focus on the biological mechanisms and interactions of the viruses with the immune system to gain more insight in the management and usage of immunosuppression.

This study demonstrated that EBV-viremia is common in RTR, with an association with graft loss in recipients with EBV viral load >log 4 cp/ml and no association with BPAR. Routine EBV screening should be performed, at least in the first year, in EBV seronegative recipients as they are prone for a primary infection, hence at greater risk for developing PTLD. For all other RTR, though, this study shows that EBV load monitoring has limited to no additive value besides screening for symptoms and clinical examination.

ACKNOWLEDGMENTS

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