Volume 64, Issue 7 pp. 520-531

ORIGINAL ARTICLE

Free Access

Effect of mTOR inhibitors during CMV disease in kidney transplant recipients: Results of a pilot retrospective study

Hannah Kaminski,

Hannah Kaminski

orcid.org/0000-0003-1604-3825

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

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Juliette Belanger,

Juliette Belanger

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

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Julien Mary,

Julien Mary

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

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Isabelle Garrigue,

Isabelle Garrigue

Laboratory of Virology, Pellegrin University Hospital, Bordeaux, France

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Mathieu Acquier,

Mathieu Acquier

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

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Julie Déchanet-Merville,

Julie Déchanet-Merville

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

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Pierre Merville,

Pierre Merville

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

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Lionel Couzi,

Corresponding Author

Lionel Couzi

[email protected]

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

Correspondence Lionel Couzi, Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Place Amélie Raba Léon, 33076 Bordeaux, France.

Email: [email protected]

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Hannah Kaminski,

Hannah Kaminski

orcid.org/0000-0003-1604-3825

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

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Juliette Belanger,

Juliette Belanger

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

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Julien Mary,

Julien Mary

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

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Isabelle Garrigue,

Isabelle Garrigue

Laboratory of Virology, Pellegrin University Hospital, Bordeaux, France

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Mathieu Acquier,

Mathieu Acquier

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

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Julie Déchanet-Merville,

Julie Déchanet-Merville

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

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Pierre Merville,

Pierre Merville

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

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Lionel Couzi,

Corresponding Author

Lionel Couzi

[email protected]

Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Bordeaux, France

CNRS-UMR 5164 ImmunoConcEpT, Bordeaux University, Bordeaux, France

Correspondence Lionel Couzi, Department of Nephrology, Transplantation, Dialysis and Apheresis, Pellegrin University Hospital, Place Amélie Raba Léon, 33076 Bordeaux, France.

Email: [email protected]

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First published: 06 April 2020

https://doi.org/10.1111/1348-0421.12794

Citations: 3

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Abstract

mTOR inhibitors exert a preventive effect on cytomegalovirus (CMV) disease in CMV seropositive (R+) kidney transplant recipients, but their impact during the curative treatment of CMV disease in high-risk kidney transplant recipients has not been investigated. We aimed to evaluate the efficacy and tolerance of mTOR inhibitors compared with mycophenolic acid in 63 consecutive kidney transplant recipients (80% of D+R−) suffering from CMV disease with a persistent or a recurrent CMV DNAemia. In this monocentric retrospective study, 16 had their treatment converted to mTOR inhibitors and 47 did not. The Kaplan–Meier curves did not show any significant differences in CMV DNAemia eradication (77% vs. 88% respectively; hazard ratio (HR), 1.648 [95% confidence interval (CI), 0.913–2.973]; log-rank test, P = .132), DNAemia recurrence (36% vs. 47%; HR, 1.517 [95% CI, 0.574–4.007]; log-rank test, P = .448) and CMV clinical recurrence (17% vs. 27%; HR, 1.375 [95% CI, 0.340–5.552]; log-rank test, P = .677) between patients who received mTOR inhibitors and those who did not. These results were confirmed in uni- and multivariate time-dependent Cox regressions. In summary, conversion from mycophenolic acid to mTOR inhibitors seems inadequate for improving CMV clearance or in better preventing CMV recurrences during severe or persistent CMV disease.

Abbreviations

ABMR: antibody-mediated rejection
CI: confidence interval
CMV: cytomegalovirus
CNI: calcineurin inhibitor
D+R−: donor with CMV status positive/recipient with CMV status negative
FCV: foscavir
GCV: ganciclovir
HR: hazard ratio
KTR: kidney transplant recipient
MPA: mycophenolic acid
mTORi: mTOR inhibitors
NA: not applicable
N: number
QNAT: quantitative nucleic acid testing
R+: recipient with CMV status positive
rATG: rabbit anti-thymocyte globulins
SD: standard deviation
TCMR: T cell-mediated rejection
VGCV: valganciclovir
Vδ2^neg T lymphocyte: V delta 2 negative T lymphocyte
vs: versus

1 INTRODUCTION

Despite the current prevention strategies, cytomegalovirus (CMV) is still a major issue in solid-organ transplant recipients. CMV disease is very frequent with an incidence reaching 15–20% in D+R−^1-3 and 5% in R+ kidney transplant recipients (KTR).⁴ Moreover, despite appropriate antiviral treatment during CMV disease, failure to clear CMV occurs in 30% of patients. Finally, DNAemia and clinical recurrences still occur in 30% and 15% of KTR, respectively.⁵ Therefore, there is an unmet need for new strategies to better treat CMV disease and to avoid recurrence.

Over the past years, a growing body of evidence, including randomized controlled trials, has shown that the use of mTOR inhibitors (mTORi) had anti-CMV properties. In vitro, mTORi could directly inhibit CMV replication, potentiate the generation of CMV-specific memory CD8+ T-cells, and deviate the CMV-mediated immune evasion by blocking mTORC1 activity in myeloid cells.^{6, 7} In randomized trials, a reduction of post-transplant CMV events was reported in kidney transplant recipients receiving de novo mTORi, either in association with mycophenolic acid (MPA),⁸ or with calcineurin inhibitors (CNI).^9-11 Based on these studies, CMV guidelines now propose the use of mTORi as a potential preventive approach to decrease CMV infection and disease in R+ kidney transplant recipients.¹²

While mTORi are able to exert a preventive effect on CMV disease in R+ kidney transplant recipients, their use during CMV disease to achieve faster CMV eradication has not been demonstrated. Two analyses have been made in 9 and 11 patients and it was reported that switching immunosuppression from CNI to a mTORi-based regimen could be proposed as a salvage therapy in ganciclovir-resistant CMV infection.^{13, 14} However, the use of mTORi in the clinical scenarios of CMV DNAemia persistence or recurrence has not been investigated, notably switching from MPA to mTORi with calcineurin inhibitors maintenance. Indeed, as mycophenolate mofetil (the precursor of MPA) has been shown to increase CMV invasive organ diseases in kidney transplant recipients,¹⁵ changing to mTORi could offer an option against the deleterious effect of MPA.

We therefore conducted a retrospective study to evaluate the efficacy and tolerance of a conversion from MPA to mTORi in kidney transplant recipients suffering from CMV disease with a persistent or a recurrent CMV DNAemia.

2 PATIENTS AND METHODS

2.1 Study design

A monocentric retrospective study was conducted at the Bordeaux University Hospital, France, from April 2009 to April 2018. Among the consecutive 95 patients who underwent CMV disease during this period, first 20 patients were excluded with early CMV eradication without recurrence. Also excluded were the patients for whom mTORi had been already administered in other indications (n = 10), as well as multiple organ transplantations (n = 2). Consequently, 63 patients were included, either patients who had a persistent CMV DNAemia after 7 weeks of antiviral treatment (n = 22), based on the VICTOR study⁵ or those who had a CMV recurrence (n = 41; Figure 1). All patients were monitored for 1 year after meeting the inclusion criteria. This study was approved by the Institutional Review Board of the Bordeaux University Hospital.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Flow chart of study design. Repartition of patients are described. Between April 2009 and April 2018, 201 kidney transplant recipients (KTR) underwent CMV infection. Patients with asymptomatic CMV viremia were excluded (n = 106). Among 95 KTR with CMV disease, 20 were excluded because they had CMV eradication and no recurrence, 10 were excluded because were already treated with an mTOR inhibitor before inclusion for other reasons and 2 because of multiple transplantation. Twenty-two patients had CMV persistence of which 7 had mTORi conversion. Forty-one patients had CMV recurrence of which nine had mTORi conversion. Apr, April; CMV, cytomegalovirus; KTR, kidney transplant recipient; mTORi, mTOR inhibitor; n, number

2.2 Immunosuppression

Induction therapy was based on rabbit anti-thymocyte globulins (rATG) in sensitized patients, and basiliximab in non-sensitized patients. The immunosuppressive regimen was based on CNI with a tacrolimus target residual plasma concentration of 10–12 ng/mL for the first 3 months and then 5–10 ng/mL. The cyclosporine A target residual plasma concentration was 150–200 ng/mL for the first 3 months and then 75–125 ng/mL. MPA was used mainly as an antiproliferative drug until the clinical decision was made to switch to mTORi for the indication of CMV persistence or CMV recurrence during an outpatient visit. No standardized criteria were used to define patients eligible for mTORi conversion. Conversion from MPA to mTORi was decided clinically because mTORi has been demonstrated to prevent CMV infection better than MPA, to contribute to CMV-mediated evasion suppression,⁶ and consequently has been recently added in the latest CMV guidelines.¹²

We considered the beneficial effect of mTORi to outweigh its adverse events such aphtosis, edemas, and delay in healing. Those adverse events are described below. The mTORi target residual plasma concentration was then 4–7 ng/mL. All patients received everolimus as mTORi treatment, except one who received rapamycin.

The expanded criteria for donor and delayed graft function were defined as described previously.¹⁶ All acute rejections, which included both antibody-mediated and T cell-mediated acute rejections, were biopsy-proven. The glomerular filtration rate was expressed based on the MDRD formula.

2.3 CMV prevention

From April 2009 to June 2010, patients were preemptively followed and treated when the CMV quantitative nucleic acid testing (QNAT) was 2000 IU/mL as reported previously.¹⁷ From July 2010 to April 2018, D+R− and R+ patients received, respectively, 6 months and 3 months of universal prophylaxis using valganciclovir 900 mg once a day. The dose was carefully adjusted at each outpatient visit, following the manufacturer's recommendations, using the Cockcroft–Gault formula.

2.4 CMV monitoring

The CMV IgG serology (Enzygnost anti-CMV/IgM and IgG [Dade Behring, Marburg, Germany] and Acces CMV IgG and IgM [Beckman Coulter, Brea, CA]), were performed following the manufacturer's recommendations.

CMV QNAT was performed as described previously with a real-time polymerase chain reaction in whole blood,¹⁸ and from June 2012, using LightMix® kit human cytomegalovirus (hCMV; TIB MOLBIOL GmbH, Berlin, Germany). Before June 2012, QNAT was reported in copies/mL, however, the equivalence in IU/mL was retrospectively calculated by using the World Health Organization International Standard for hCMV, in order to homogenize all the results before performing the statistical analysis. The threshold of CMV DNAemia detectability was 250 IU/mL. The laboratory is following the program of Quality Control for Molecular Diagnostics (QCMD, Glasgow, Scotland) from 2004.

The baseline viral load was defined as the first positive DNAemia of the CMV event which led us to include the patient. During the viral monitoring of CMV disease, the assay was performed once a week during the first 7 weeks or until two consecutive negative CMV QNATs had occurred, then once a month until month 3. CMV QNAT was also systematically performed every year and when CMV disease was clinically suspected.

Antiviral drug resistance was suspected when persistent viral replication was observed after >2 weeks of appropriate antiviral therapy and was confirmed by full-length sequencing of the UL97 and UL54 genes,³ performed at the French National Cytomegalovirus Reference Center (Limoges, France).

2.5 Definitions of CMV events

CMV disease was defined as CMV syndrome or CMV tissue-invasive disease, consistent with the American Society of Transplantation and the CMV Drug Development Forum recommendations.¹⁹ Intravenous (i.v.) ganciclovir (5 mg/kg twice daily) or oral valganciclovir (900 mg twice daily) were given as the curative treatment. The dose was carefully adjusted at each outpatient visit, following the manufacturer's recommendations, using the Cockcroft–Gault formula.

“Post-prophylaxis” CMV disease was defined as the first episode of CMV disease occurring >3 months (100 days) after transplantation.

Early-onset disease was defined as the first episode of CMV disease occurring <3 months (100 days) after transplantation.²⁰

CMV DNAemia eradication was defined as the occurrence of one negative CMV QNAT.

CMV DNAemia persistence was defined as no CMV eradication occurring after 7 weeks of antiviral treatment.

CMV DNAemia recurrence was assessed as a new positive CMV DNAemia (≥250 IU/mL) in patients with proven CMV DNAemia eradication. Clinical recurrence was defined as CMV DNAemia recurrence associated with viral syndrome or documentation of CMV in tissue from a relevant organ.¹²

2.6 Immunophenotyping

We have previously shown that Vδ2^neg T lymphocytes are involved in the control of CMV²¹ since their expansion is correlated with CMV infection resolution.²² Consequently, Vδ2^neg T lymphocytes kinetics were analyzed in patients with or without conversion to mTORi. The Vδ2^neg T lymphocyte count was obtained by immunophenotypic determination. Flow cytometry was carried out on 100 µL anticoagulated whole blood taking into account at least 5000 total lymphocytes stained with anti-CD45, anti-pan-δ (clone IMMU 510; Beckman Coulter, Krefeld, Germany), and anti-TCR Vδ2 (clone 15D; Thermo Fisher Scientific, Rockford, IL). The percentages of cell populations were obtained using CELLQUEST software (BD Bioscience), and absolute counts of lymphocytes were obtained using the Single–Platform Lyse/No–Wash Trucount (BD Bioscience). In our center, the surveillance of Vδ2^neg T lymphocytes was based on a measurement at day 0 of the graft; month 3, 6, and 12; and then every year. In the case of CMV persistence, the monitoring was performed at day 0 of CMV infection, day 49 and once a month in the 3 months following day 49. In the case of CMV recurrence, the surveillance of Vδ2^neg T lymphocytes was performed on that day and then once a month in the three following months.

2.7 Statistical analysis

The Mann-Whitney test and the χ² test were used when appropriate. Alternatively to the χ² test, the Fisher's test was used for a low number of patients. P < .05 was considered statistically significant. The survival curves were estimated with Kaplan–Meier method and compared with the log-rank test.

We performed a univariate time-dependent Cox regression analysis for CMV DNAemia eradication, CMV DNAemia recurrence and clinical recurrence. Then, covariates with a < 0.25 P-value and “mTORi conversion” were included in a multivariate time-dependent Cox regression analysis. The results were expressed as hazard ratios (HR) with 95% confidence intervals (95% CI). Finally, we separately analyzed patients with persistent and recurrent CMV disease.

Analyses were performed with conventional statistical methods using the GraphPad Prism (version 6.0; GraphPad Software, San Diego, CA) and the RStudio statistical software (Version 1.1.423 – © 2009-2018 RStudio, Inc).

3 RESULTS

3.1 Baseline characteristics of patients

Sixty-three KTR with CMV disease were included at the time of persistent CMV DNAemia diagnosis or at the time of CMV DNAemia recurrence. Baseline characteristics are described in Table 1. Seventy percent of the patients were males with an average age of 56 years. Forty-one percent received rATG as induction treatment, 79.4% received tacrolimus, while 17.5% received cyclosporine A. Seventy percent had corticosteroids and 92% had MPA. Acute antibody-mediated rejection occurred before inclusion in 1.6% of patients and acute T cell-mediated rejection in 11% of patients.

Table 1. Baseline characteristics of patients at inclusion

Characteristic	Value
Gender
Female/Male	19/63 (30%)/44/63 (70%)
Age (yr), mean ± SD	56 ± 13
Rank of transplantation
1	53/63 (84%)
>1	10/63 (15.8%)
Preemptive transplantation
No/Yes	7/63 (11%)/56/63 (89%)
Total ischemia time (hours), mean ± SD	15 ± 7.7
Delayed graft function
No/Yes	39/63 (62%)/24/63 (38%)
Expanded criteria donor
No/Yes	23/63 (37%)/34/63 (54%)
NA	6/63(9%)
Living donor
No/Yes	56/63 (89%)/7/63 (11%)
Nephropathy
Vascular	18/63 (29%)
Glomerular	17/63 (27%)
Unknown	8/63 (13%)
Tubulo interstitial/others	12/63 (19%)
Hereditary/others	8/63 (13%)
Immunosuppressive regimen
rATG induction
No/Yes	36/61 (59%)/25/61 (41%)
Cyclosporine A
No/Yes	52/63 (82.5%)/11/63 (17.5%)
Tacrolimus
No/Yes	13/63 (20.6%)/50/63 (79.4%)
MPA
No/Yes	5/63 (8%)/58/63 (92%)
Corticosteroids
No/Yes	19/63 (30%)/44/63 (70%)
Azathioprine
No/Yes	60/63 (95.2%)/3/63 (4.7%)
Previous ABMR
No/Yes	62/63 (98%)/1/63 (1.6%)
Previous TCMR
No/Yes	56/63 (89%)/7/63 (11%)

This table describes the baseline characteristics of patients at inclusion, regarding age, sex, rank of transplantation, graft characteristics, immunosuppressive regimen, and the history of acute rejection before inclusion.
Abbreviations: ABMR, antibody-mediated rejection; CMV, cytomegalovirus; rATG, rabbit anti-thymocyte globulins; TCMR, T cell-mediated rejection; SD, standard deviation.

Among the 63 included patients, 16 were converted to mTORi (Table 2). The conversion to mTORi occurred 31 days after inclusion on average (median 21; 1st–3rd quartile 8–49 days). Among the 16 patients converted to mTORi, 56% still received ganciclovir or valganciclovir at the time of the conversion, versus 45% in the patients without mTORi conversion (P = .675). In the other patients, antiviral therapy was stopped.

Table 2. Baseline characteristics relative to CMV disease

Characteristic	Conversion to mTORi, n = 16	No conversion to mTORi, n = 47	P-value
Recurrent/persistent	9/16 (56%)/7/16 (44%)	32/47 (68%)/15/47 (32%)	.391
CMV status D+R-/R+	13/16 (81%)/3/16 (19%)	36/47 (77%)/11/47 (23%)	.698
Preemptive versus universal prophylaxis	2/16 (12%)/14/16 (88%)	13/47 (28%)/34/47 (72%)	.315
Early-onset versus “post-prophylaxis” CMV disease	2/16 (12%)/14/16 (88%)	11/47 (23%)/36/47 (77%)	.486
No/VGV/GCV/FCV at inclusion			.675
	6/16 (37.5%)	26/47 (55%)
	7/16 (43.75%)	15/47 (32%)
	2/16 (12.5%)	6/47 (13%)
	1/16 (6.25%)	0
Baseline CMV DNAemia (IU/ml); mean ± SD	685720 ± 1447761	753024 ± 1924860	.869
Mutant strain	14/16 (88%)/2/16 (12%)	43/47 (91%)/4/47 (8.5%)
Lymphocyte count (/µL); mean ± SD	635 ± 647	785 ± 464	.058
rATG	7 (43.8)	18 (38.4)	.7

This table describes CMV baseline characteristics of the donor and recipient status, type of preventive strategy, inclusion for CMV persistence or recurrence, the antiviral treatment, virus characteristics, and lymphocyte count.
Abbreviations: CMV, cytomegalovirus; mTORi, mTOR inhibitors; rATG, rabbit anti-thymocyte globulins; SD, standard deviation.

The proportions of CMV diseases with persistent CMV DNAemia, D+/R− status, and of a mutant strain were not significantly different in the two groups (Table 2). The mean of the absolute lymphocytes counts was slightly higher in the group without mTORi conversion (635 vs. 785/µL, respectively), but it was not statistically significant (P = .058). The preventive strategy, the baseline viral load, the ratio of “post-prophylaxis” versus “early-onset disease” between the two groups were not significantly different either (Table 2).

3.2 mTOR inhibitors conversion is not associated with a faster CMV DNAemia eradication

Univariate analysis showed no significant association between mTORi conversion and CMV DNAemia eradication (HR, 0.835; [95% CI, 0.411–1.696]; P = .618; Table 3a). Analysis of the 1-year survival curve showed no significant difference of CMV DNAemia eradication between patients with or without mTORi conversion (77% vs. 88% respectively; HR, 1.648 [95% CI, 0.913–2.973]; log-rank test, p = .132; Figure 2a).

Table 3a. Uni- and multivariate analysis for CMV DNAemia eradication

Univariate analysis				Multivariate analysis
Covariate	HR	CI 95%	P-value	HR	CI 95%	P-value
Age	1.001	0.981–1.021	0.949
Sex	0.972	0.530–1.784	.928
rATG	0.861	0.479–1.549	.619
Corticosteroids	1.195	0.651–2.19	.566
mTORi conversion	0.835	0.411–1.696	.618
CMV status	1.194	0.609–2.338	.606
Preventive strategy	0.423	0.225–0.793	.007
Early-onset disease	0.429	0.255–0.95	.034
Mutant strain	0.540	0.194–1.504	.239
Baseline viral load	0.473	0.269–0.831	.009
Persistent versus recurrent	0.376	0.204–0.693	.001	0.438	0.221–0.718	.002
TCMR before CMV disease	1.526	0.644–3.609	.336
ABMR before CMV disease	3.011	0.401–21.58	.284
Lymphocyte count	1	1–1.001	.074	1.0005	1–1.0011	.068

Baseline viral load was converted in a binary variable, taking the median value of 58,000 IU/mL and analyzing the correlation between baseline viral load higher or lower than this cutoff and CMV DNAemia eradication. Seven variables were analyzed kept for the initial multivariate analysis and the model was convergent.
Abbreviations: ABMR, antibody-mediated rejection; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio; mTORi, mTOR inhibitors; rATG, rabbit anti-thymocyte globulins; TCMR, T cell-mediated rejection.

In the multivariate analysis, only the “persistent CMV DNAemia versus recurrent CMV infection” covariate was associated with a lower rate of CMV DNAemia eradication (HR, 0.438 [95% CI, 0.221–0.718]; P = .002; Table 3A).

3.3 mTOR inhibitors is not associated with CMV recurrence

We then excluded the patients for whom CMV DNAemia eradication was not obtained (n = 11, four with mTORi conversion and seven without mTORi conversion), in order to analyze the covariates associated with both CMV DNAemia and clinical recurrences.

mTORi conversion was not significantly associated with either CMV DNAemia recurrence (HR, 0.942; [95% CI, 0.314–2.831]; P = .916; Table 3b), or clinical recurrence (HR, 1.524; [95% CI, 0.408–5.693]; P = .531; Table 3c). The CMV DNAemia recurrence (36% vs. 47%; HR 1.517 [95% CI, 0.574–4.007]; log-rank test, P = .448; Figure 2b) and the clinical recurrence (17% vs. 27%; HR, 1.375 [95% CI, 0.340–5.552]; log-rank test, P = .677; Figure 2c) were not significantly different in patients with and without mTORi conversion in the 1-year survival curves.

Table 3b. Uni and multivariate analysis for CMV DNAemia recurrence

Univariate analysis				Multivariate analysis
Covariate	HR	CI 95%	P-value	HR	CI 95%	P-value
Age	1.006	0.974–1.038	.726
Sex	0.535	0.223–1.282	.161
rATG	3.956	1.625–9.63	.002	3.581	1.449–8.848	.005
Corticosteroids	1.402	0.546–3.595	.482
mTORi conversion	0.942	0.314–2.831	.916
CMV status	1.687	0.655–4.344	.278
Preventive strategy	0.682	0.277–1.675	.404
Early-onset disease	0.571	0.232–1.402	.221
Mutant strain	2.093	0.618–7.087	.235
Baseline viral load	1.197	0.518–2.762	.674
Persistent versus recurrent	0.569	0.209–1.547	.27
TCMR before CMV disease	0.295	0.039–2.196	.233
ABMR before CMV disease	1.086	0.988–1.122	.998
Lymphocyte count	0.680	0.294–1.572	0.368

Baseline viral load was converted in a binary variable, taking the median value of 58,000 IU/mL and analyzing the correlation between baseline viral load higher or lower than this cutoff and CMV DNAemia eradication. Five variables were kept for the initial multivariate analysis and the model was convergent.
Abbreviations: ABMR, antibody-mediated rejection; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio; mTORi, mTOR inhibitors; rATG, rabbit anti-thymocyte globulins; TCMR, T cell-mediated rejection.

Table 3c. Uni- and multivariate analysis for clinical recurrence

Univariate analysis				Multivariate analysis
Covariate	HR	CI 95%	P-value	HR	CI 95%	P-value
Age	1.013	0.968–1.06	.578
Sex	0.290	0.092–0.911	.0341
rATG	6.386	1.718–23.73	.005	7.723	2.0422–29.2	.002
Tacrolimus	1.233	0.333–4.558	.754
Corticosteroids	2.559	0.559–11.71	.226
mTORi conversion	1.524	0.408–5.692	.531
CMV status	2.256	0.677–7.516	.185
Preventive strategy	5.07	0.652–39.37	.121
Early-onset disease	4.317	0.555–33.54	.162	5.806	0.7313–46.1	.096
Mutant strain	3.014	0.656–13.84	.156
Baseline viral load	1.294	0.417–4.012	.656
Persistent versus recurrent	0.894	0.241–3.313	.867
TCMR before CMV disease	1.184e-08	0-Inf	.998
ABMR before CMV disease	2.959e-07	0-Inf	.998
Lymphocyte count	0.323	0.097–1.077	.066

Baseline viral load was converted in a binary variable, taking the median value of 58,000 IU/mL and analyzing the correlation between baseline viral load higher or lower than this cutoff and CMV DNAemia eradication. We put the seven most significantly associated variables to obtain a convergent model.
Abbreviations: ABMR, antibody-mediated rejection; CI, confidence interval; CMV, cytomegalovirus; HR, hazard ratio; mTORi, mTOR inhibitors; rATG, rabbit anti-thymocyte globulins; TCMR, T cell-mediated rejection.

In the multivariate analyses, only rATG was significantly associated with an increased risk of both DNAemia (HR, 3.581; [95% CI, 1.449–8.848]; P = .005; Table 3b) and clinical recurrence (HR, 7.723; [95% CI, 2.042–29.2]; P = .002; Table 3c). The percentage of patients receiving rATG was not different in the patients with and without mTORi conversion (43.8% vs. 38.4% respectively, P = .7).

3.4 Analysis of the anti-CMV immune response using the Vδ2^neg T lymphocyte expansion

Baseline characteristics were similar in patients with or without mTORi conversion in the two subgroups: “CMV persistence” and “CMV recurrence”, with the exception of lymphocyte counts which were lower in patients converted to mTORi in the CMV persistent subgroup (326 vs. 430/µL, respectively, P = .01; Table 4a, b).

Table 4a. Baseline characteristics in “CMV persistent” group

Characteristics	Conversion to mTORi, n = 7	No conversion to mTORi, n = 15	P-value
CMV status D+R-/R+	1 (14.3)/6(85.7)	12 [80]/3 (20)	.746
Preemptive versus universal prophylaxis	1 (14.3)/6(85.7)	0/15	.318
Early-onset versus “post-prophylaxis” CMV disease	1 (14.3)/6(85.7)	0/15	.318
No/VGV/GCV/FCV at inclusion	3 (42.8)/2 (28.2)/2 (28.2)/1 (14.3)	2 (13.3)/10(66.7)/3(20)/0	.166
Baseline CMV DNAemia (IU/mL); mean ± SD	1.44 10⁶ ± 2.39 10⁵	1.62 10⁶ ± 2.56 10⁵	.679
Mutant strain	1 (14.3)/6(85.7)	2 (13.3)/13 (86.7)
One year CMV DNAemia eradication	5/7 (71.4)	10/15 (66.7)	.823
One year CMV DNAemia recurrence	2/5 [40]	3/10 (30)	.387
Lymphocyte count (/µL), mean ± SD	326 ± 198	430 ± 327	.01

Abbreviations: CMV, cytomegalovirus; HR, hazard ratio; mTORi, mTOR inhibitors; SD, standard deviation.

Table 4b. Baseline characteristics in “CMV recurrent” group

Characteristic	Conversion to mTORi, n = 9	No conversion to mTORi, n = 32	P-value
CMV status	7 (77.8)/2(22.8)	23 (71.8)/9(28.2)	.724
Preemptive versus universal prophylaxis	1 (11.1)/8(89.9)	9(28.2)/23(71.8)	.293
Early-onset versus “post-prophylaxis” CMV disease	1 (11.1)/8(89.9)	8(25)/24[75]	.999
Baseline CMV DNAemia (IU/mL); mean ± SD	2.20 10⁵ ± 3.24 10⁵	5.3 10⁶ ± 1.91 10⁵	.504
Mutant strain	1 (11.1)/8(89.9)	0/32	.219
One year CMV DNAemia eradication	6/9 (66.7)	30/32 (93.7)	.061
One year CMV DNAemia recurrence	2/6 (33.3)	15/30 [50]	.661
Lymphocyte count (/µL); mean ± SD	1 024 ± 848	762 ± 453	.731

This table describes the baseline CMV characteristics among patients with CMV recurrence regarding donor and recipient status, type of preventive strategy, inclusion for CMV persistence or recurrence, the antiviral treatment, virus characteristics, and lymphocyte count.
Abbreviations: CMV, cytomegalovirus; HR, hazard ratio; mTORi, mTOR inhibitors; SD, standard deviation.

One-year CMV DNAemia eradication and CMV DNAemia recurrence were also similar in patients with or without mTORi conversion in these two subgroups (subgroup CMV persistence; P = .823 and .387 respectively. Subgroup CMV recurrence; P = .061 and .661 respectively, Table 4a, b).

Finally, CMV-specific Vδ2^neg T lymphocyte expansion were also similar in patients with or without mTORi conversion in these two subgroups (Figure 3a, b)

3.5 Rejection and graft survival

Since tacrolimus was mainly used, we compared the tacrolimus trough levels between the two groups at 1-, 3-, 6-, and 12-months post-inclusion, and observed that the tacrolimus trough level at month 3 was significantly lower in patients with mTORi conversion (Figure S1). Patients converted to mTORi exhibited few adverse events, as described in Table S1. Finally, acute rejection, eGFR, graft, and patient survival were compared 1-year post-inclusion and no significant difference was noted between the two groups (Table S2).

4 DISCUSSION

It is now widely accepted that the use of mTORi is a potential approach to decrease the incidence of CMV disease in R+ kidney transplant recipients. On the contrary, this retrospective study shows that the conversion from MPA to mTORi seems inadequate in improving CMV clearance or in better preventing CMV recurrences of persistent and recurrent CMV disease. Hypotheses can be proposed to explain this lack of efficacy in those scenarios.

First of all, this finding was observed in a very carefully selected population, displaying the most difficult CMV diseases to treat. For patients with persistent CMV DNAemia after 7 weeks of antiviral treatment, the management based on international recommendations remains controversial. Treating patients until obtaining a CMV QNAT below the lower limit of quantification on one highly sensitive assay, or two consecutive negative results if the assay is not highly sensitive is recommended.¹² However, a cumulative (val)ganciclovir exposure exceeding 6 weeks is a strong risk factor for CMV antiviral drug resistance due to the emergence of UL97/UL54 mutations.^{3, 12} There is therefore an urgent need for new strategies in this scenario.

CMV recurrence is also a major issue. It is very frequent in patients with a lack of prior CMV immunity (D+/R−), a high baseline viral load (100,000 copies/mL), a failure to eradicate DNAemia by day 21 post-treatment, a weak viral load decrease during CMV therapy, or lymphopenia.^{23, 24} Our cohort of 63 kidney transplant recipients exhibited all of these characteristics. About 10% of our cohort exhibited CMV diseases with mutant strains which are also associated with a higher risk of recurrences.²⁵ Testing a new strategy to improve CMV DNAemia eradication and to prevent recurrence in these patients was therefore very relevant but also very challenging.

Secondly, the lack of mTORi conversion efficacy could be related to the absence of tacrolimus minimization. In Transform and Athena studies, the lower incidence of CMV events could be related to everolimus or to the reduced dose of tacrolimus.^{9, 26, 27} In our study, most of the patients converted to everolimus had a tacrolimus blood concentration over 5 ng/mL because physicians are reluctant to decrease the tacrolimus trough level. Indeed, it has been associated with de novo donor-specific antibodies emergence and a lower graft survival.²⁸ It probably resulted in a state of over-immunosuppression which could have contributed to this absence of effect on CMV.

Thirdly, the analysis of the CMV-specific Vδ2^neg T lymphocyte expansion following the CMV disease or recurrence showed that conversion to mTORi was not associated either with a stronger or with a faster anti-CMV immune response.²⁰ The protective effect of mTORi against CMV could depend on the immunological status of the recipient, and mainly benefit patients with preformed CMV-specific immunity. Two randomized studies have shown that mTORi attenuated the incidence of CMV disease mainly in R+ patients.^{10, 11} At the time of transplant, most R+ patients had memory CMV-specific T cells that could control the virus.²⁹ mTORi may influence this immune-mediated response in R+ recipients, since it has been reported that sirolimus exerted dose-dependent immunostimulatory effects on CD8+ memory T cells in both mice and macaques exposed to viral pathogens.^{7, 30} More recently, an in vitro study demonstrated that mTORi significantly improved preformed CMV-specific effector memory T-cell function.³¹ Here, we focused mainly on naive D+R− KTR, for whom mTORi have controversial effects.^{30, 32, 33} From day 0 post transplantation to the end of universal prophylaxis, most of the D+R− KTR did not develop any CMV-specific T cells.³⁴ Moreover, persistent CMV DNAemia or recurrence is also observed in D+R− patients with a lack of CMV immunity.²² Interestingly, mTORi did not influence naive T cells in vitro, nor their overall activation marker expression after CD3/CD28 coactivation.³¹ Our clinical data also suggest an inability of mTORi to control CMV infection in naive patients.

Finally, our study has some limits. It is a retrospective and monocentric study with a low number of patients. Very few R+ recipients develop CMV persistence or recurrence. CMV diseases in these patients are usually easily manageable. It is therefore impossible to include more R+ patients displaying the chosen inclusion criteria. Even in D+R− patients, these difficult CMV scenarios (CMV persistence or CMV recurrence) are not frequent enough to include more patients.

Moreover, the absence of standardized criteria to convert patients to mTORi could introduce potential hidden bias. One may expect a trend to consider patients with more difficult CMV disease to treat as eligible for conversion to mTORi but no difference in clinical CMV parameters was observed between the two groups. Prophylactic strategies changed over time but were equally balanced between the two groups with or without mTORi conversion. The assay to monitor DNAemia also changed over time but the threshold of CMV DNAemia detectability of 250 IU/mL did not change, which allowed us to analyze equally the eradication and recurrences all over the period. We may also point out a delay in the conversion to mTORi after inclusion. However, despite these drawbacks, our study is the first clinical experiment of the use of mTORi, compared with maintaining the standard immunosuppressive regimen: mTORi were quite well tolerated with no impact on graft function, rejection, and graft survival but showed no effect in such challenging scenarios.

In conclusion, the use of mTOR inhibitors did not lead to any improvement in the outcomes of CMV disease with persistent DNAemia or recurrence in that high-risk population.

Testing the conversion of mTORi in less severe CMV diseases, in R+ patients with preformed CMV immunity, or at an earlier stage of CMV disease (as soon as the first positive CMV QNAT is detected) should be tested in the future.

ACKNOWLEDGMENTS

We thank Catherine Rio, Bordeaux's center nurse coordinator. We also acknowledge the technicians of the Bordeaux University Hospital Laboratories of Virology for their significant contribution to this study, and the medical doctors involved in patient care.

CONFLICT OF INTEREST

The authors declare that there are no conflict of interests.

Supporting Information

REFERENCES

1Khoury JA, Storch GA, Bohl DL, et al. Prophylactic versus preemptive oral valganciclovir for the management of cytomegalovirus infection in adult renal transplant recipients. Am J Transplant. 2006; 6(9): 2134-43.
10.1111/j.1600-6143.2006.01413.x
CAS PubMed Web of Science® Google Scholar
2Reischig T, Jindra P, Hes O, Svecova M, Klaboch J, Treska V. Valacyclovir prophylaxis versus preemptive valganciclovir therapy to prevent cytomegalovirus disease after renal transplantation. Am J Transplant. 2008; 8(1): 69-77.
10.1111/j.1600-6143.2007.02031.x
CAS PubMed Web of Science® Google Scholar
3Couzi L, Helou S, Bachelet T, et al. High incidence of anticytomegalovirus drug resistance among D+R- kidney transplant recipients receiving preemptive therapy. Am J Transplant. 2012; 12(1): 202-9.
10.1111/j.1600-6143.2011.03766.x
CAS PubMed Web of Science® Google Scholar
4Witzke O, Hauser IA, Bartels M, Wolf G, Wolters H, Nitschke M. Valganciclovir prophylaxis versus preemptive therapy in cytomegalovirus-positive renal allograft recipients: 1-year results of a randomized clinical trial. Transplantation. 2012; 93(1): 61-8.
10.1097/TP.0b013e318238dab3
CAS PubMed Web of Science® Google Scholar
5Asberg A, Humar A, Jardine AG, et al. Long-term outcomes of CMV disease treatment with valganciclovir versus IV ganciclovir in solid organ transplant recipients. Am J Transplant. 2009; 9(5): 1205-13.
10.1111/j.1600-6143.2009.02617.x
CAS PubMed Web of Science® Google Scholar
6Poglitsch M, Weichhart T, Hecking M, et al. CMV late phase-induced mTOR activation is essential for efficient virus replication in polarized human macrophages. Am J Transplant. 2012; 12(6): 1458-68.
10.1111/j.1600-6143.2012.04002.x
CAS PubMed Web of Science® Google Scholar
7Araki K, Turner AP, Shaffer VO, et al. mTOR regulates memory CD8 T-cell differentiation. Nature. 2009; 460(7251): 108-12.
10.1038/nature08155
CAS PubMed Web of Science® Google Scholar
8Mallat SG, Tanios BY, Itani HS, et al. CMV and BKPyV infections in renal transplant recipients receiving an mTOR inhibitor-based regimen versus a CNI-based regimen: a systematic review and meta-analysis of randomized, controlled trials. Clin J Am Soc Nephrol. 2017; 12(8): 1321-36.
10.2215/CJN.13221216
CAS PubMed Web of Science® Google Scholar
9Pascual J, Berger SP, Witzke O, et al. Everolimus with reduced calcineurin inhibitor exposure in renal transplantation. J Am Soc Nephrol. 2018; 29(7): 1979-91.
10.1681/ASN.2018010009
CAS PubMed Web of Science® Google Scholar
10Tedesco-Silva H, Felipe C, Ferreira A, et al. Reduced incidence of cytomegalovirus infection in kidney transplant recipients receiving everolimus and reduced tacrolimus doses. Am J Transplant. 2015; 15(10): 2655-64.
10.1111/ajt.13327
CAS PubMed Web of Science® Google Scholar
11de Sandes-Freitas TV, Pinheiro PMA, Sales M, Girao CM, Campos EF, Esmeraldo RM. The impact of everolimus in reducing cytomegalovirus events in kidney transplant recipients on steroid-avoidance strategy: 3-year follow-up of a randomized clinical trial. Transplant Int. 2018; 31(12): 1345-56.
10.1111/tri.13313
PubMed Web of Science® Google Scholar
12Kotton CN, Kumar D, Caliendo AM, et al. The Third International Consensus Guidelines on the management of cytomegalovirus in solid-organ transplantation. Transplantation. 2018; 102(6): 900-31.
10.1097/TP.0000000000002191
PubMed Web of Science® Google Scholar
13Sabe N, Gonzalez-Costello J, Rama I, et al. Successful outcome of ganciclovir-resistant cytomegalovirus infection in organ transplant recipients after conversion to mTOR inhibitors. Transplant Int. 2012; 25(7): e78-82.
10.1111/j.1432-2277.2012.01489.x
CAS PubMed Web of Science® Google Scholar
14Ozaki KS, Camara NO, Nogueira E, et al. The use of sirolimus in ganciclovir-resistant cytomegalovirus infections in renal transplant recipients. Clin Transplant. 2007; 21(5): 675-80.
10.1111/j.1399-0012.2007.00699.x
CAS PubMed Web of Science® Google Scholar
15Sarmiento JM, Dockrell DH, Schwab TR, Munn SR, Paya CV. Mycophenolate mofetil increases cytomegalovirus invasive organ disease in renal transplant patients. Clin Transplant. 2000; 14(2): 136-8.
10.1034/j.1399-0012.2000.140206.x
CAS PubMed Web of Science® Google Scholar
16Metzger RA, Delmonico FL, Feng S, Port FK, Wynn JJ, Merion RM. Expanded criteria donors for kidney transplantation. Am J Transplant. 2003; 3(Suppl 4): 114-25.
10.1034/j.1600-6143.3.s4.11.x
PubMed Web of Science® Google Scholar
17Reischig T, Hribova P, Jindra P, et al. Long-term outcomes of pre-emptive valganciclovir compared with valacyclovir prophylaxis for prevention of cytomegalovirus in renal transplantation. J Am Soc Nephrol. 2012; 23(9): 1588-97.
10.1681/ASN.2012010100
CAS PubMed Web of Science® Google Scholar
18Garrigue I, Doussau A, Asselineau J, et al. Prediction of cytomegalovirus (CMV) plasma load from evaluation of CMV whole-blood load in samples from renal transplant recipients. J Clin Microbiol. 2008; 46(2): 493-8.
10.1128/JCM.01499-07
PubMed Web of Science® Google Scholar
19Ljungman P, Hakki M, Boeckh M. Cytomegalovirus in hematopoietic stem cell transplant recipients. Hematol Oncol Clin North Am. 2011; 25(1): 151-69.
10.1016/j.hoc.2010.11.011
PubMed Web of Science® Google Scholar
20Razonable RR, Blumberg EA. It's not too late: a proposal to standardize the terminology of “late-onset” cytomegalovirus infection and disease in solid organ transplant recipients. Transplant Infect Dis. 2015; 17(6): 779-84.
10.1111/tid.12447
CAS PubMed Web of Science® Google Scholar
21Couzi L, Pitard V, Moreau JF, Merville P, Dechanet-Merville J. Direct and indirect effects of cytomegalovirus-induced gammadelta T cells after kidney transplantation. Front Immunol. 2015; 6: 3.
10.3389/fimmu.2015.00003
PubMed Web of Science® Google Scholar
22Kaminski H, Garrigue I, Couzi L, et al. Surveillance of gammadelta T cells predicts cytomegalovirus infection resolution in kidney transplants. J Am Soc Nephrol. 2016; 27(2): 637-45.
10.1681/ASN.2014100985
CAS PubMed Web of Science® Google Scholar
23Eid AJ, Arthurs SK, Deziel PJ, Wilhelm MP, Razonable RR. Clinical predictors of relapse after treatment of primary gastrointestinal cytomegalovirus disease in solid organ transplant recipients. Am J Transplant. 2010; 10(1): 157-61.
10.1111/j.1600-6143.2009.02861.x
CAS PubMed Web of Science® Google Scholar
24Martin-Gandul C, Perez-Romero P, Blanco-Lobo P, et al. Viral load, CMV-specific T-cell immune response and cytomegalovirus disease in solid organ transplant recipients at higher risk for cytomegalovirus infection during preemptive therapy. Transplant Int. 2014; 27(10): 1060-8.
10.1111/tri.12378
CAS PubMed Web of Science® Google Scholar
25Fisher CE, Knudsen JL, Lease ED, et al. Risk factors and outcomes of ganciclovir-resistant cytomegalovirus infection in solid organ transplant recipients. Clin Infect Dis. 2017; 65(1): 57-63.
10.1093/cid/cix259
CAS PubMed Web of Science® Google Scholar
26Sommerer C, Suwelack B, Dragun D, et al. An open-label, randomized trial indicates that everolimus with tacrolimus or cyclosporine is comparable to standard immunosuppression in de novo kidney transplant patients. Kidney Int. 2019; 96(1): 231-44.
10.1016/j.kint.2019.01.041
CAS PubMed Web of Science® Google Scholar
27Asberg A, Jardine AG, Bignamini AA, et al. Effects of the intensity of immunosuppressive therapy on outcome of treatment for CMV disease in organ transplant recipients. Am J Transplant. 2010; 10(8): 1881-8.
10.1111/j.1600-6143.2010.03114.x
CAS PubMed Web of Science® Google Scholar
28Wiebe C, Rush DN, Nevins TE, et al. Class II Eplet mismatch modulates tacrolimus trough levels required to prevent donor-specific antibody development. J Am Soc Nephrol. 2017; 28(11): 3353-62.
10.1681/ASN.2017030287
CAS PubMed Web of Science® Google Scholar
29Bestard O, Lucia M, Crespo E, et al. Pretransplant immediately early-1-specific T cell responses provide protection for CMV infection after kidney transplantation. Am J Transplant. 2013; 13(7): 1793-805.
10.1111/ajt.12256
CAS PubMed Web of Science® Google Scholar
30Ferrer IR, Araki K, Ford ML. Paradoxical aspects of rapamycin immunobiology in transplantation. Am J Transplant. 2011; 11(4): 654-9.
10.1111/j.1600-6143.2011.03473.x
CAS PubMed Web of Science® Google Scholar
31Bak S, Tischer S, Dragon A, et al. Selective effects of mTOR inhibitor sirolimus on naive and CMV-specific T cells extending its applicable range beyond immunosuppression. Front Immunol. 2018; 9: 2953.
10.3389/fimmu.2018.02953
CAS PubMed Web of Science® Google Scholar
32Cristelli MP, Esmeraldo RM, Pinto CM, et al. The influence of mTOR inhibitors on the incidence of CMV infection in high-risk donor positive-recipient negative (D+/R−) kidney transplant recipients. Transplant Infect Dis. 2018; 20(4):e12907.
10.1111/tid.12907
PubMed Web of Science® Google Scholar
33Tedesco-Silva H, Pascual J, Viklicky O, et al. Safety of everolimus with reduced calcineurin inhibitor exposure in de novo kidney transplants: an analysis from the randomized TRANSFORM study. Transplantation. 2019; 103: 1953-63.
10.1097/TP.0000000000002626
CAS PubMed Web of Science® Google Scholar
34Lucia M, Crespo E, Melilli E, et al. Preformed frequencies of cytomegalovirus (CMV)-specific memory T and B cells identify protected CMV-sensitized individuals among seronegative kidney transplant recipients. Clin Infect Dis. 2014; 59(11): 1537-45.
10.1093/cid/ciu589
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume64, Issue7

July 2020

Pages 520-531

Filename	Description
mim12794-sup-0001-200207_supporting_information.docx28.9 KB	Supplementary information
mim12794-sup-0002-supplFig1.tif979.3 KB	Supplementary information
mim12794-sup-0003-SupMat.docx25.3 KB	Supplementary information

Effect of mTOR inhibitors during CMV disease in kidney transplant recipients: Results of a pilot retrospective study