© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Transplant Infectious Disease, ISSN 1398-2273
Two-year post-transplantation cytomegalovirus DNAemia in asymptomatic kidney transplant recipients: incidence, risk factors, and outcome B. Viot, I. Garrigue, B. Taton, T. Bachelet, J.-F. Moreau, J. DechanetMerville, P. Merville, L. Couzi. Two-year post-transplantation cytomegalovirus DNAemia in asymptomatic kidney transplant recipients: incidence, risk factors, and outcome. Transpl Infect Dis 2015: 17: 497–509. All rights reserved Abstract: Background. The incidence and the impact of asymptomatic cytomegalovirus (CMV) DNAemia occurring after the first year post transplantation is unknown. Methods. In this retrospective cross-sectional study, we analyzed the incidence, risk factors, and impact of 2-year post-transplantation asymptomatic CMV DNAemia (2YCD) on graft function. We included 892 consecutive asymptomatic kidney transplant recipients transplanted for at least 2 years and all were monitored using whole blood CMV quantitative nucleic acid amplification testing (CMV-QNAT). Results. Twenty-eight patients displayed 2YCD (3.1%). Using multivariate analysis in 578 patients, we found that female gender (odds ratio [OR] = 2.57, P = 0.02), a past history of CMV drugresistance mutation (OR = 8.73, P = 0.005), and corticosteroid use (OR = 2.37, P = 0.03) were independently associated with an increased risk of 2YCD. 2YCD was associated with an increased incidence of subsequent CMV disease over the year following its diagnosis (7% vs. 0.6%, P = 0.02). Patients with 2YCD also exhibited a declining estimated glomerular filtration rate more frequently (77%) than patients with a negative CMV-QNAT (56%, P = 0.02). Conclusion. 2YCD appears to be a rare entity, which appears to be associated with chronic graft dysfunction.
B. Viot1,2, I. Garrigue2,3,4, B. Taton1,2, T. Bachelet1,2,5, J.-F. Moreau2,5, J. Dechanet-Merville2,5, P. Merville1,2,5, L. Couzi1,2,5 Service de Nephrologie-Transplantation-Dialyse, Centre Hospitalier Universitaire de Bordeaux, H^opital Pellegrin, Bordeaux, France, 2Universite de Bordeaux, Bordeaux, France, 3Laboratoire de Virologie, Centre Hospitalier Universitaire de Bordeaux, H^opital Pellegrin, Bordeaux, France, 4Microbiologie Fondamentale et Pathogenicite, Unite Mixte de Recherche (UMR) 5234, Bordeaux, France, 5 Centre National de la Recherche Scientifique (CNRS)– Unite Mixte de Recherche (UMR) 5164, CIRID, Bordeaux, France 1
Key words: kidney transplantation; cytomegalovirus infection; CMV drug-resistance mutation; corticosteroid; chronic graft dysfunction Correspondence to: Dr Lionel Couzi, CHU Bordeaux – H^opital Pellegrin, Service de Nephrologie-Transplantation-Dialyse, Place Amelie Raba Leon, Bordeaux 33076, France Tel: 33 5 56 79 55 38 Fax: 33 5 56 79 61 07 E-mail: [email protected]
Received 26 December 2014, revised 6 April 2015, accepted for publication 14 May 2015 DOI: 10.1111/tid.12408 Transpl Infect Dis 2015: 17: 497–509
Cytomegalovirus (CMV) infection is one of the most common complications following kidney transplantation. It is associated with direct and indirect effects that can increase the risk of graft loss and death. Prevention strategies against CMV in kidney transplant recipients (KTR) remain a controversial issue, but the majority of centers favor the use of universal prophylaxis in the highest risk recipients (1). However, the well-recognized drawback of universal prophylaxis is the high incidence of late-onset CMV diseases that are associated with lower graft and patient survival (2–6), despite the proposal to extend the duration of prophylaxis to 6 months (7–9). The condition
defined by a positive CMV quantitative nucleic acid amplification test (CMV-QNAT) occurring after the discontinuation of prophylaxis (usually between 3 and 12 months post transplantation) in asymptomatic patients is called late-onset CMV DNAemia. Over the last few years, several reports have analyzed its impact on renal transplantation. In pediatric patients, subclinical late-onset CMV DNAemia was found to be associated with the development of moderate to severe interstitial fibrosis/tubular atrophy and declining glomerular filtration rate (GFR) (10). In adults, reduced graft survival was also observed in patients with late-
497
Viot et al: Two-year post-transplantation CMV DNAemia
onset DNAemia (11). However, as long-term CMV monitoring is not recommended after the first year post transplantation, no study has analyzed whether asymptomatic CMV DNAemia occurring 2 years after transplantation (or 2-year post-transplantation CMV DNAemia [2YCD]) is also associated with poor allograft outcome. The goals of this study were therefore to analyze in asymptomatic KTR the incidence, risk factors, and impact of 2YCD.
Materials and methods Study design A total of 899 KTR at least 2 years post transplant and attending an annual visit at the outpatient clinic of the nephrology department of the Bordeaux University Hospital were consecutively included in this crosssectional retrospective study from January 1, 2010 to December 31, 2011 (Fig. 1). At the inclusion visit, all patients were monitored for CMV blood viral load using a whole blood CMV-QNAT, which was performed as previously described (12, 13). Asymptomatic patients with a positive CMV-QNAT were diagnosed as having 2YCD. All study procedures were approved by our local ethics committee.
Study end-points The incidence of 2YCD was determined at the inclusion visit in 899 patients. Our electronic database stores
clinical and biological data for all the KTR after the year 2000. For each patient, it is updated 2–4 times each year, either by a transplant physician in our center at each annual visit, or by a local nephrologist in hospitals and outpatient clinics. Data were available for 571 patients transplanted after 2000 and were considered missing for patients transplanted earlier. Additional details were retrieved from the medical record for 7 patients who presented a positive CMV-QNAT at the inclusion visit. The other end-points, including the risk factor analysis of 2YCD, were analyzed in 578 patients. The incidence of CMV disease was analyzed from the inclusion visit to the follow-up visit 12 months later. As previously reported (14), the evolution of renal function over the follow-up period was estimated as the change (D) in estimated GFR (eGFR) (by Modification of Diet in Renal Disease [MDRD] method) between the inclusion and follow-up visits: DeGFR = eGFR(followup) eGFR(inclusion). It was compared between patients with and without 2YCD (Fig. 1). Another CMV-QNAT was also performed in most patients at the follow-up visit 12 months later.
Pre- and post-transplant parameters included in the risk factor analysis of 2YCD The CMV prevention strategy (prophylaxis or preemptive strategy) has changed over time in our center. Before November 2006, CMV-positive donor for CMVnegative recipient (D+R ) and CMV-positive recipient (R+) patients receiving anti-lymphocyte globulins were given prophylaxis for 3 months post transplantation
Fig. 1. Study design. In total, 899 consecutive kidney recipients transplanted for at least 2 years were included at our outpatient clinic and were monitored for cytomegalovirus (CMV) using whole blood quantitative nucleic acid amplification testing (CMV-QNAT). They were followed up for 12 months. eGFR, estimate glomerular filtration rate.
498
Transplant Infectious Disease 2015: 17: 497–509
Viot et al: Two-year post-transplantation CMV DNAemia
with ganciclovir and then valganciclovir. The other R+ patients were treated preemptively. From December 2006 to December 2009, a preemptive strategy was used for both D+R and R+ patients. All patients transplanted after 2000 were monitored for CMV using a CMV-NAT until 2004, then with a CMV-QNAT after 2004, once a week for the first 3 months, then once a month between months 3 and 6, and finally every 2 months until 1 year post transplantation. The 7 patients transplanted before 2000 were monitored for CMV infection using pp65 antigenemia. Based on standardized criteria, post-transplant CMV manifestations were classified as CMV infection (CMV replication, i.e., positive blood CMV-NAT or pp65 antigenemia, without any symptoms) or CMV disease: namely CMV syndrome (fever, malaise, leukopenia, elevation of hepatic transaminases and/or thrombocytopenia, and CMV replication) or CMV tissue-invasive disease (proven CMV-related organ dysfunction or failure) (15). Late-onset CMV infection was defined as a CMV infection occurring between 3 and 12 months post transplantation, because routine CMV monitoring in asymptomatic patient stops after the first year post transplantation in our center. Late-onset CMV disease was defined as a CMV disease occurring 3 months post transplantation. Anti-CMV drug resistance was investigated when a significant increase in CMV viral load (>1 log10 copies/ mL) was observed despite antiviral therapy and was defined as the presence of resistance-associated mutations in UL97 and/or UL54 genes. Genotypic resistance testing was performed at the French National Cytomegalovirus Reference Center (Limoges, France) (16). All clinical rejection episodes were confirmed by a ‘for cause’ biopsy and classified according to the 2009 Banff classification (17). For each patient, the slope of the eGFR (by MDRD formula) from transplantation to the inclusion visit was calculated by least-square linear regression using all eGFR values available in the electronic database (eGFR (t) = a 9 t + b, a = slope, mL/min/1.73 m2/year) (Fig. 1).
Statistical analysis Comparisons between groups were performed using conventional statistics. The McNemar v2 or Fisher test for qualitative variables, and Student’s t-test or Wilcoxon rank-test were used when appropriate. The variables potentially associated with 2YCD or a declining eGFR after the inclusion visit were subjected to univariate
analysis using logistic regression. Associated risk factors with a P < 0.2 in univariate analysis were included in a multivariable model. Then, a backward selection procedure was used to select a final multivariate model including all significant variables with a P < 0.05. Analyses were performed with JMP.10 (2012; SAS Institute Inc., Cary, North Carolina, USA).
Results Incidence of 2YCD at the inclusion visit A total of 899 KTR, at least 2 years after transplantation, were included during an annual visit to the outpatient clinic and were monitored for CMV blood viral load (Fig. 1). Three of them (0.4%) were receiving curative anti-CMV therapy for an ongoing CMV disease (Fig. 2). Among the 896 patients without any anti-CMV therapy, 32 had a positive CMV-QNAT. A curative anti-CMV therapy was initiated in 4 of them (0.5%) who presented clinical symptoms and were therefore diagnosed with CMV disease. The characteristics of the 7 patients with CMV disease are presented in Table 1. Most of them had CMV colitis. None had received prophylaxis. Six of 7 had a past history of rejection. These 7 patients were excluded from the final analysis because they had a CMV disease. The 28 (3.1%) other patients, who remained asymptomatic while presenting a positive CMV-QNAT, were diagnosed with 2YCD. Their median viral load was 1355 (min–max: 499–14,900) copies/mL. None of them received curative anti-CMV therapy and immunosuppressive therapy was not reduced. The control group consisted of 864 asymptomatic patients with a negative CMV-QNAT (96%). Data were available for 550 control group patients transplanted after 2000. Final analyses were therefore carried out on 578 patients (Fig. 2). No association was found between 2YCD and time between transplantation and CMVQNAT (data not shown).
Risk factors for 2YCD at the inclusion visit The baseline characteristics, historical characteristics of past CMV events, history of rejection, immunosuppressive treatments, and renal function for patients with 2YCD (n = 28) and the control group (n = 550) are presented in Table 2. In univariate analysis, female gender (odds ratio [OR] = 2.81, P = 0.008), past history of CMV mutation (OR = 11.29, P = 0.001), past use of intravenous ganciclovir (OR = 3.51, P = 0.03), past use of foscavir
Transplant Infectious Disease 2015: 17: 497–509
499
Viot et al: Two-year post-transplantation CMV DNAemia
Patients with CMV disease who were excluded at the inclusion visit Patient no. 1
2
3
4
5
6
7
Age (years)
66
68
49
49
73
56
58
Gender (M/F)
M
M
F
M
M
F
F
CMV serostatus
D?R+
D R+
D+R
D+R
D+R+
D+R
D+R
Prophylaxis strategy for 3 months post transplantation
No
No
No
No
No
No
No
History of CMV mutation
No
No
UL97
No
No
No
No
Time between transplantation and the inclusion visit (years)
10
4
2
2
4
2
13
Duration of the ongoing CMV disease (days) (0 if diagnosed at the visit)
33
50
711
0
0
0
0
CMV-QNAT level at the inclusion visit (copies/mL)
0
1420
0
66,500
16,900
52,400
2930
Type of CMV disease
CMV Sd
Colitis
Retinitis
CMV Sd
Colitis
Colitis
Colitis
CMV disease under treatment at the inclusion visit
Yes
Yes
Yes
No
No
No
No
Intravenous ganciclovir
No
Yes
Yes
Valganciclovir
Yes
Yes
Yes
Foscavir
No
No
Yes
Drug received previously for the CMV disease
History of rejection
Yes
Yes
No
Yes
Yes
Yes
Yes
Time since last rejection episode (days)
3647
791
–
360
1317
356
2404
T cell-mediated
Yes
Yes
No
No
Yes
Yes
Yes
Antibody-mediated
No
No
No
Yes
Yes
No
No
M, male; F, female; CMV, cytomegalovirus; D, donor; R, recipient; CMV-QNAT, CMV quantitative nucleic acid amplification testing; Sd, syndrome.
Table 1
(OR = 11.29, P = 0.001), corticosteroid use (OR = 2.65, P = 0.01), and eGFR slope from transplantation to the inclusion visit (OR = 0.94, P = 0.01) were associated with an increased risk of 2YCD. In contrast, CMV serostatus, preemptive strategy, past history of CMV infection or disease, past history of late-onset CMV infection or disease, past history of rejection, induction therapy, calcineurin inhibitors, and mycophenolate mofetil, or eGFR at the inclusion visit were not associated with an increased risk of 2YCD (Table 3). The time of occurrence of CMV infection or disease post transplantation, the time elapsed between past CMV infection or disease and positive CMV-QNAT, the period of time during which the patients were given anti-CMV therapy, and the time between last rejection and positive CMV-QNAT were not included in the univariate analysis because these data were limited to patients with past CMV events (n = 93) or rejection (n = 129). Instead, they were compared with conventional tests, and we did not observe any difference between patients with 2YCD and the control group (P = 0.4, 0.07, 0.4, and 0.07, respectively).
500
As foscavir was given to patients with a history of CMV mutation, it was not included in the multivariate model. Using multivariate analysis, we found that the parameters independently associated with 2YCD were female gender (OR = 2.57, P = 0.02), past history of CMV drug-resistance mutation (OR = 8.73, P = 0.005), and steroid treatment at the inclusion visit (OR = 2.37, P = 0.03). eGFR slope from transplantation to the inclusion visit was no longer associated with 2YCD in the multivariate analysis.
2YCD and subsequent very late-onset CMV disease We next analyzed the incidence of CMV disease over the year following the inclusion visit, in an attempt to determine whether 2YCD could predict subsequent CMV disease (Fig. 1). Among the 578 patients, 11 were lost to follow-up. We observed that the incidence of very late-onset CMV disease over the year following the visit was very low (0.9%, 5/567). However, it was higher in patients with 2YCD (7.4%, 2/27) than in the control group
Transplant Infectious Disease 2015: 17: 497–509
Viot et al: Two-year post-transplantation CMV DNAemia
Fig. 2. Flowchart of the study design at the inclusion visit. A total of 899 consecutive kidney recipients transplanted for at least 2 years were included at our outpatient clinic. They were monitored for cytomegalovirus (CMV) using whole blood quantitative nucleic acid amplification testing (CMV-QNAT), and were classified according to the presence of curative anti-CMV therapy and CMV-associated clinical symptoms.
(0.6%, 3/540) (P = 0.02). The 2 patients with 2YCD who developed CMV disease had very low CMV DNAemias at the inclusion visit (1060 and 1240 copies/mL).
2YCD and eGFR evolution at the follow-up visit 1 year later After the inclusion visit, 1-year patient survival was 96.3% (26/27) and 99.6% (538/540) in the 2YCD and control groups, respectively (P = 0.1). One-year graft survival was 96.3% (26/27) and 98.3% (531/540) in the 2YCD and control groups, respectively (P = 0.4). After the inclusion visit, eGFR evolution was measured using the DeGFR (change in eGFR from the inclusion visit to the follow-up visit). A poor correlation was observed between the eGFR slope before the inclusion visit and the DeGFR over the following year (r = 0.08), demonstrating that trends in kidney function vary throughout the post transplantation period.
Mean DeGFR after the inclusion visit was higher in patients with 2YCD ( 6.5 21.2 mL/min/1.73 m2) than in control group patients ( 1.2 12.3 mL/min/ 1.73 m2), but the difference was not significant (P = 0.07). Patients were then classified into a stable/ improving eGFR group (DeGFR ≥0), and a declining eGFR group (DeGFR
Two-year post-transplantation cytomegalovirus DNAemia in asymptomatic kidney transplant recipients: incidence, risk factors, and outcome B. Viot, I. Garrigue, B. Taton, T. Bachelet, J.-F. Moreau, J. DechanetMerville, P. Merville, L. Couzi. Two-year post-transplantation cytomegalovirus DNAemia in asymptomatic kidney transplant recipients: incidence, risk factors, and outcome. Transpl Infect Dis 2015: 17: 497–509. All rights reserved Abstract: Background. The incidence and the impact of asymptomatic cytomegalovirus (CMV) DNAemia occurring after the first year post transplantation is unknown. Methods. In this retrospective cross-sectional study, we analyzed the incidence, risk factors, and impact of 2-year post-transplantation asymptomatic CMV DNAemia (2YCD) on graft function. We included 892 consecutive asymptomatic kidney transplant recipients transplanted for at least 2 years and all were monitored using whole blood CMV quantitative nucleic acid amplification testing (CMV-QNAT). Results. Twenty-eight patients displayed 2YCD (3.1%). Using multivariate analysis in 578 patients, we found that female gender (odds ratio [OR] = 2.57, P = 0.02), a past history of CMV drugresistance mutation (OR = 8.73, P = 0.005), and corticosteroid use (OR = 2.37, P = 0.03) were independently associated with an increased risk of 2YCD. 2YCD was associated with an increased incidence of subsequent CMV disease over the year following its diagnosis (7% vs. 0.6%, P = 0.02). Patients with 2YCD also exhibited a declining estimated glomerular filtration rate more frequently (77%) than patients with a negative CMV-QNAT (56%, P = 0.02). Conclusion. 2YCD appears to be a rare entity, which appears to be associated with chronic graft dysfunction.
B. Viot1,2, I. Garrigue2,3,4, B. Taton1,2, T. Bachelet1,2,5, J.-F. Moreau2,5, J. Dechanet-Merville2,5, P. Merville1,2,5, L. Couzi1,2,5 Service de Nephrologie-Transplantation-Dialyse, Centre Hospitalier Universitaire de Bordeaux, H^opital Pellegrin, Bordeaux, France, 2Universite de Bordeaux, Bordeaux, France, 3Laboratoire de Virologie, Centre Hospitalier Universitaire de Bordeaux, H^opital Pellegrin, Bordeaux, France, 4Microbiologie Fondamentale et Pathogenicite, Unite Mixte de Recherche (UMR) 5234, Bordeaux, France, 5 Centre National de la Recherche Scientifique (CNRS)– Unite Mixte de Recherche (UMR) 5164, CIRID, Bordeaux, France 1
Key words: kidney transplantation; cytomegalovirus infection; CMV drug-resistance mutation; corticosteroid; chronic graft dysfunction Correspondence to: Dr Lionel Couzi, CHU Bordeaux – H^opital Pellegrin, Service de Nephrologie-Transplantation-Dialyse, Place Amelie Raba Leon, Bordeaux 33076, France Tel: 33 5 56 79 55 38 Fax: 33 5 56 79 61 07 E-mail: [email protected]
Received 26 December 2014, revised 6 April 2015, accepted for publication 14 May 2015 DOI: 10.1111/tid.12408 Transpl Infect Dis 2015: 17: 497–509
Cytomegalovirus (CMV) infection is one of the most common complications following kidney transplantation. It is associated with direct and indirect effects that can increase the risk of graft loss and death. Prevention strategies against CMV in kidney transplant recipients (KTR) remain a controversial issue, but the majority of centers favor the use of universal prophylaxis in the highest risk recipients (1). However, the well-recognized drawback of universal prophylaxis is the high incidence of late-onset CMV diseases that are associated with lower graft and patient survival (2–6), despite the proposal to extend the duration of prophylaxis to 6 months (7–9). The condition
defined by a positive CMV quantitative nucleic acid amplification test (CMV-QNAT) occurring after the discontinuation of prophylaxis (usually between 3 and 12 months post transplantation) in asymptomatic patients is called late-onset CMV DNAemia. Over the last few years, several reports have analyzed its impact on renal transplantation. In pediatric patients, subclinical late-onset CMV DNAemia was found to be associated with the development of moderate to severe interstitial fibrosis/tubular atrophy and declining glomerular filtration rate (GFR) (10). In adults, reduced graft survival was also observed in patients with late-
497
Viot et al: Two-year post-transplantation CMV DNAemia
onset DNAemia (11). However, as long-term CMV monitoring is not recommended after the first year post transplantation, no study has analyzed whether asymptomatic CMV DNAemia occurring 2 years after transplantation (or 2-year post-transplantation CMV DNAemia [2YCD]) is also associated with poor allograft outcome. The goals of this study were therefore to analyze in asymptomatic KTR the incidence, risk factors, and impact of 2YCD.
Materials and methods Study design A total of 899 KTR at least 2 years post transplant and attending an annual visit at the outpatient clinic of the nephrology department of the Bordeaux University Hospital were consecutively included in this crosssectional retrospective study from January 1, 2010 to December 31, 2011 (Fig. 1). At the inclusion visit, all patients were monitored for CMV blood viral load using a whole blood CMV-QNAT, which was performed as previously described (12, 13). Asymptomatic patients with a positive CMV-QNAT were diagnosed as having 2YCD. All study procedures were approved by our local ethics committee.
Study end-points The incidence of 2YCD was determined at the inclusion visit in 899 patients. Our electronic database stores
clinical and biological data for all the KTR after the year 2000. For each patient, it is updated 2–4 times each year, either by a transplant physician in our center at each annual visit, or by a local nephrologist in hospitals and outpatient clinics. Data were available for 571 patients transplanted after 2000 and were considered missing for patients transplanted earlier. Additional details were retrieved from the medical record for 7 patients who presented a positive CMV-QNAT at the inclusion visit. The other end-points, including the risk factor analysis of 2YCD, were analyzed in 578 patients. The incidence of CMV disease was analyzed from the inclusion visit to the follow-up visit 12 months later. As previously reported (14), the evolution of renal function over the follow-up period was estimated as the change (D) in estimated GFR (eGFR) (by Modification of Diet in Renal Disease [MDRD] method) between the inclusion and follow-up visits: DeGFR = eGFR(followup) eGFR(inclusion). It was compared between patients with and without 2YCD (Fig. 1). Another CMV-QNAT was also performed in most patients at the follow-up visit 12 months later.
Pre- and post-transplant parameters included in the risk factor analysis of 2YCD The CMV prevention strategy (prophylaxis or preemptive strategy) has changed over time in our center. Before November 2006, CMV-positive donor for CMVnegative recipient (D+R ) and CMV-positive recipient (R+) patients receiving anti-lymphocyte globulins were given prophylaxis for 3 months post transplantation
Fig. 1. Study design. In total, 899 consecutive kidney recipients transplanted for at least 2 years were included at our outpatient clinic and were monitored for cytomegalovirus (CMV) using whole blood quantitative nucleic acid amplification testing (CMV-QNAT). They were followed up for 12 months. eGFR, estimate glomerular filtration rate.
498
Transplant Infectious Disease 2015: 17: 497–509
Viot et al: Two-year post-transplantation CMV DNAemia
with ganciclovir and then valganciclovir. The other R+ patients were treated preemptively. From December 2006 to December 2009, a preemptive strategy was used for both D+R and R+ patients. All patients transplanted after 2000 were monitored for CMV using a CMV-NAT until 2004, then with a CMV-QNAT after 2004, once a week for the first 3 months, then once a month between months 3 and 6, and finally every 2 months until 1 year post transplantation. The 7 patients transplanted before 2000 were monitored for CMV infection using pp65 antigenemia. Based on standardized criteria, post-transplant CMV manifestations were classified as CMV infection (CMV replication, i.e., positive blood CMV-NAT or pp65 antigenemia, without any symptoms) or CMV disease: namely CMV syndrome (fever, malaise, leukopenia, elevation of hepatic transaminases and/or thrombocytopenia, and CMV replication) or CMV tissue-invasive disease (proven CMV-related organ dysfunction or failure) (15). Late-onset CMV infection was defined as a CMV infection occurring between 3 and 12 months post transplantation, because routine CMV monitoring in asymptomatic patient stops after the first year post transplantation in our center. Late-onset CMV disease was defined as a CMV disease occurring 3 months post transplantation. Anti-CMV drug resistance was investigated when a significant increase in CMV viral load (>1 log10 copies/ mL) was observed despite antiviral therapy and was defined as the presence of resistance-associated mutations in UL97 and/or UL54 genes. Genotypic resistance testing was performed at the French National Cytomegalovirus Reference Center (Limoges, France) (16). All clinical rejection episodes were confirmed by a ‘for cause’ biopsy and classified according to the 2009 Banff classification (17). For each patient, the slope of the eGFR (by MDRD formula) from transplantation to the inclusion visit was calculated by least-square linear regression using all eGFR values available in the electronic database (eGFR (t) = a 9 t + b, a = slope, mL/min/1.73 m2/year) (Fig. 1).
Statistical analysis Comparisons between groups were performed using conventional statistics. The McNemar v2 or Fisher test for qualitative variables, and Student’s t-test or Wilcoxon rank-test were used when appropriate. The variables potentially associated with 2YCD or a declining eGFR after the inclusion visit were subjected to univariate
analysis using logistic regression. Associated risk factors with a P < 0.2 in univariate analysis were included in a multivariable model. Then, a backward selection procedure was used to select a final multivariate model including all significant variables with a P < 0.05. Analyses were performed with JMP.10 (2012; SAS Institute Inc., Cary, North Carolina, USA).
Results Incidence of 2YCD at the inclusion visit A total of 899 KTR, at least 2 years after transplantation, were included during an annual visit to the outpatient clinic and were monitored for CMV blood viral load (Fig. 1). Three of them (0.4%) were receiving curative anti-CMV therapy for an ongoing CMV disease (Fig. 2). Among the 896 patients without any anti-CMV therapy, 32 had a positive CMV-QNAT. A curative anti-CMV therapy was initiated in 4 of them (0.5%) who presented clinical symptoms and were therefore diagnosed with CMV disease. The characteristics of the 7 patients with CMV disease are presented in Table 1. Most of them had CMV colitis. None had received prophylaxis. Six of 7 had a past history of rejection. These 7 patients were excluded from the final analysis because they had a CMV disease. The 28 (3.1%) other patients, who remained asymptomatic while presenting a positive CMV-QNAT, were diagnosed with 2YCD. Their median viral load was 1355 (min–max: 499–14,900) copies/mL. None of them received curative anti-CMV therapy and immunosuppressive therapy was not reduced. The control group consisted of 864 asymptomatic patients with a negative CMV-QNAT (96%). Data were available for 550 control group patients transplanted after 2000. Final analyses were therefore carried out on 578 patients (Fig. 2). No association was found between 2YCD and time between transplantation and CMVQNAT (data not shown).
Risk factors for 2YCD at the inclusion visit The baseline characteristics, historical characteristics of past CMV events, history of rejection, immunosuppressive treatments, and renal function for patients with 2YCD (n = 28) and the control group (n = 550) are presented in Table 2. In univariate analysis, female gender (odds ratio [OR] = 2.81, P = 0.008), past history of CMV mutation (OR = 11.29, P = 0.001), past use of intravenous ganciclovir (OR = 3.51, P = 0.03), past use of foscavir
Transplant Infectious Disease 2015: 17: 497–509
499
Viot et al: Two-year post-transplantation CMV DNAemia
Patients with CMV disease who were excluded at the inclusion visit Patient no. 1
2
3
4
5
6
7
Age (years)
66
68
49
49
73
56
58
Gender (M/F)
M
M
F
M
M
F
F
CMV serostatus
D?R+
D R+
D+R
D+R
D+R+
D+R
D+R
Prophylaxis strategy for 3 months post transplantation
No
No
No
No
No
No
No
History of CMV mutation
No
No
UL97
No
No
No
No
Time between transplantation and the inclusion visit (years)
10
4
2
2
4
2
13
Duration of the ongoing CMV disease (days) (0 if diagnosed at the visit)
33
50
711
0
0
0
0
CMV-QNAT level at the inclusion visit (copies/mL)
0
1420
0
66,500
16,900
52,400
2930
Type of CMV disease
CMV Sd
Colitis
Retinitis
CMV Sd
Colitis
Colitis
Colitis
CMV disease under treatment at the inclusion visit
Yes
Yes
Yes
No
No
No
No
Intravenous ganciclovir
No
Yes
Yes
Valganciclovir
Yes
Yes
Yes
Foscavir
No
No
Yes
Drug received previously for the CMV disease
History of rejection
Yes
Yes
No
Yes
Yes
Yes
Yes
Time since last rejection episode (days)
3647
791
–
360
1317
356
2404
T cell-mediated
Yes
Yes
No
No
Yes
Yes
Yes
Antibody-mediated
No
No
No
Yes
Yes
No
No
M, male; F, female; CMV, cytomegalovirus; D, donor; R, recipient; CMV-QNAT, CMV quantitative nucleic acid amplification testing; Sd, syndrome.
Table 1
(OR = 11.29, P = 0.001), corticosteroid use (OR = 2.65, P = 0.01), and eGFR slope from transplantation to the inclusion visit (OR = 0.94, P = 0.01) were associated with an increased risk of 2YCD. In contrast, CMV serostatus, preemptive strategy, past history of CMV infection or disease, past history of late-onset CMV infection or disease, past history of rejection, induction therapy, calcineurin inhibitors, and mycophenolate mofetil, or eGFR at the inclusion visit were not associated with an increased risk of 2YCD (Table 3). The time of occurrence of CMV infection or disease post transplantation, the time elapsed between past CMV infection or disease and positive CMV-QNAT, the period of time during which the patients were given anti-CMV therapy, and the time between last rejection and positive CMV-QNAT were not included in the univariate analysis because these data were limited to patients with past CMV events (n = 93) or rejection (n = 129). Instead, they were compared with conventional tests, and we did not observe any difference between patients with 2YCD and the control group (P = 0.4, 0.07, 0.4, and 0.07, respectively).
500
As foscavir was given to patients with a history of CMV mutation, it was not included in the multivariate model. Using multivariate analysis, we found that the parameters independently associated with 2YCD were female gender (OR = 2.57, P = 0.02), past history of CMV drug-resistance mutation (OR = 8.73, P = 0.005), and steroid treatment at the inclusion visit (OR = 2.37, P = 0.03). eGFR slope from transplantation to the inclusion visit was no longer associated with 2YCD in the multivariate analysis.
2YCD and subsequent very late-onset CMV disease We next analyzed the incidence of CMV disease over the year following the inclusion visit, in an attempt to determine whether 2YCD could predict subsequent CMV disease (Fig. 1). Among the 578 patients, 11 were lost to follow-up. We observed that the incidence of very late-onset CMV disease over the year following the visit was very low (0.9%, 5/567). However, it was higher in patients with 2YCD (7.4%, 2/27) than in the control group
Transplant Infectious Disease 2015: 17: 497–509
Viot et al: Two-year post-transplantation CMV DNAemia
Fig. 2. Flowchart of the study design at the inclusion visit. A total of 899 consecutive kidney recipients transplanted for at least 2 years were included at our outpatient clinic. They were monitored for cytomegalovirus (CMV) using whole blood quantitative nucleic acid amplification testing (CMV-QNAT), and were classified according to the presence of curative anti-CMV therapy and CMV-associated clinical symptoms.
(0.6%, 3/540) (P = 0.02). The 2 patients with 2YCD who developed CMV disease had very low CMV DNAemias at the inclusion visit (1060 and 1240 copies/mL).
2YCD and eGFR evolution at the follow-up visit 1 year later After the inclusion visit, 1-year patient survival was 96.3% (26/27) and 99.6% (538/540) in the 2YCD and control groups, respectively (P = 0.1). One-year graft survival was 96.3% (26/27) and 98.3% (531/540) in the 2YCD and control groups, respectively (P = 0.4). After the inclusion visit, eGFR evolution was measured using the DeGFR (change in eGFR from the inclusion visit to the follow-up visit). A poor correlation was observed between the eGFR slope before the inclusion visit and the DeGFR over the following year (r = 0.08), demonstrating that trends in kidney function vary throughout the post transplantation period.
Mean DeGFR after the inclusion visit was higher in patients with 2YCD ( 6.5 21.2 mL/min/1.73 m2) than in control group patients ( 1.2 12.3 mL/min/ 1.73 m2), but the difference was not significant (P = 0.07). Patients were then classified into a stable/ improving eGFR group (DeGFR ≥0), and a declining eGFR group (DeGFR
