Long-term Allograft Survival After Kidney Transplantation E. Gómez Gómez, J.P. Campos Hernández, F.J. Márquez López, J. Ruiz Garcia, V. Garcia Montemayor, F. Anglada Curado, M. Leva Vallejo, J.C. Regueiro López, M.D. Navarro Cabello, P. Aljama, and M.J. Requena Tapia ABSTRACT Background. Technical and medical advances over the past few years have produced an important increase in the functionality of renal allografts. The aim of this study was to identify the factors associated with allograft survival 15 years after transplantation in our series. Methods. A retrospective study of kidney transplantations was carried out at Reina Sofia Hospital in Cordoba from February 1979 to December 1997, with follow-up through June 2012. A subanalysis of the series was undertaken, and Kaplan-Meier analysis and Cox proportional hazards model regression used to achieve the main objective of the study. Results. A total of 487 renal allografts with a mean follow-up of 114 months were studied, of which 37% (n ¼ 180) survived for >15 years. Of the 180 patients, the main causes of graft failure were chronic allograft nephropathy in 29 (66%) and patient death in 13 (29.5%). Multivariate analysis identified the number of HLA mismatches (hazard ratio [HR] 1.25, 95% CI 1.01e1.56), panel reactive antibodies (HR 2.61, 95% CI 1.28e5.26), and delayed graft function (HR 11.25, 95% CI 1.33e95.28) as being significantly associated with graft loss after 15 years. Conclusions. The high immunologic risk of the patients was independently associated with graft loss. Delayed graft function was the most important factor in the speed of graft failure beyond 15 years.
K
IDNEY TRANSPLANTATION is nowadays the best treatment for patients who are in end-stage renal failure.1 Better knowledge about surgical techniques and immunosuppressive regimens have increased the allograft survival rate.2e4 Only a few researchers have studied and reported longterm graft survival in their series.4,5 The aim of the present study was to identify the factors associated with allograft survival in our series 15 years after transplantation.
grafts, excluding multiple organ transplantation. The patient demographic data were made available from our renal patient database. We considered graft failure to be the need to return to dialysis, the need to have a second transplantation, or patient death. Delayed graft function was the need for dialysis in the 1st week after transplantation. Before 1986, all patients were initially treated with azathioprine and prednisone. After 1986, cyclosporine was added to the immunosuppressive regimen.
PATIENTS AND METHODS We undertook a retrospective study of the kidney transplants carried out from February 1979 to December 1997 with follow-up through June 2012 at Reina Sofia Hospital in Cordoba. Because all transplantations were performed at a single center and there were a limited number of transplant surgeons, there was uniformity of care and treatment protocols. We included all of the first
From the Urology (E.G.G., J.P.C.H., F.J.M.L., J.R.G., F.A.C., M.L.V., J.C.R.L., M.J.R.T.) and Nefrology (V.G.M., M.D.N.C., P.A.) Units, Reina Sofía University Hospital, Córdoba, Spain. Address reprint requests to Enrique Gómez Gómez, Ramirez de Arellano N 1, Cordoba, Spain. E-mail: enriquegomezgomez@ yahoo.es
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.09.015
Transplantation Proceedings, 45, 3599e3602 (2013)
3599
3600
GÓMEZ, HERNÁNDEZ, LÓPEZ ET AL
Student t and chi-square analyses were used to compare demographic variables, both continuous and categoric, between the series. Kaplan-Meier and log rank were used to determine survival probabilities at various post-transplantation times in the group with a functioning kidney transplant after 15 years, based on different variables. Cox proportional hazards models were used in univariate and multivariate analyses to assess predictors of long-term survival. We used SPSS version 19.0 to analyze the data.
RESULTS
We studied 487 kidney transplantations performed in our hospital from February 1979 to December 1997 with follow-up through June 2012, with a mean follow-up of 114 months. The mean recipient age was 38 years (range, 6e64) and the majority received deceased-donor kidney transplants (n ¼ 476; 97.7%). The demographic characteristics are presented in Table 1. A total of 180 patients (36.9%) maintained graft function after 15 years (GS). At the time of the study, 136 patients (27%) still maintained graft function. The principal cause of graft failure in the main series (patients with graft function after 15 years) was chronic graft nephropathy (CGN) in 29 patients (66%), followed by the death of 13 patients with functioning grafts (29.5%; Table 2). Regarding the immunosuppressive regimen, the introduction of cyclosporine was associated with improved results in graft survival in all patients, with a median of 210.3 months (95% CI, 181.5e239.1) and a difference of 79 months compared with the group not treated with cyclosporine (P ¼ .009). However, this difference was not observed in the group with graft function after 15 years, as presented in Table 3. In Cox proportional hazards ratio analysis, immunologic factors and delayed graft function (DGF) were the variables associated with better graft survival 15 years after transplantation. DGF was the most important factor, with a hazard ratio (HR) of 11.25 (95% CI, 1.33e95.28), followed by panel reactive antibody (PRA; HR 2.61; 95% CI, 1.28e5.26), and finally the number of HLA mismatches (HR 1.25; 95% CI, 1.01e1.55; Table 3). Table 1. Clinical Characteristics of Patients
Variable
NoneGraft Survivors (n ¼ 307)
Donor age (y) Recipient age (y) Male Time on dialysis (mo) Cold ischemia (h) HLA mismatches PRA 15 y P (n ¼ 180) Value
32.7 14.9 35.5 12.8 112 (62.2%) 41.1 37.4 22.2 7.1 2.4 1.4 122 (67.8%) 0.2 0.4 8 (4.5%) 2 (1.1%)
.08 .00 .72 .46 .74 .03 .02 .03 .02 .00
All Patients (n ¼ 487)
34.4 45.6 38.1 13.1 308 (63.2%) 40.1 15.9 22.1 1.2 2.6 6.3 295 (60.6%) 0.3 3.5 11 (2.3%) 40 (8.2%)
Abbreviations: PRA, panel reactive antibody; DGF, delayed graft function.
Table 2. Causes of Graft Failure Cause of graft failure
Chronic allograft nephropathy Patient death Other
Graft survivors >15 y; n ¼ 44 (24% of 487)
29 (66%) 13 (29.5%) 2 (4.5%)
NoneGraft Survivors; n ¼ 263 (59% of 487)
124 (47.2%) 89 (33.8%) 50 (19%)
In GS the probability of maintaining graft function was 97.2% (95% CI, 95e99.6%) and 76.9% (95% CI, 69e83%) at 1 and 5 years, respectively (16 and 20 years after transplantation; Fig 1). Probabilities changed to 100% and 91.7% in the group with 1 mismatches compared with 96.3% and 70% in the other group (2 mismatches; P ¼ .03). These differences in the survival rate for the same period were also evident in log rank analysis based on DGF, with probabilities of 97.2% and 76.5% in the group with no DGF compared with 100% and 50% in the group with DGF (P ¼ .002), and on PRA, with probabilities for the group with 50% PRA of 93.9% and 54.1% after a period of 1 and 5 years, respectively (P ¼ .04).
DISCUSSION
There is a large young population with end-stage renal failure who require a kidney transplant early in their lives, so identifying the factors associated with long-term graft survival is highly beneficial. Various series show long-term graft survival, such as that of Traynor et al with 23.5% (n ¼ 1081) functional grafts up to 20 years after the transplantation.4 In our series, with 487 patients, 37% had a functional graft for up to 15 years and almost 28% for up to 20 years. In the literature, recipient age, donor age, and recipient sex are factors associated with graft survival.4,6e8 Nevertheless, we did not find any association in the speed of the loss of the graft function in GS. Another factor associated with long-term graft survival is a living-donor renal allograft.4,9 Despite having only a few living-donor renal allografts in our study period, we found a higher percentage of this kind of graft in GS. However, this factor did not influence the graft loss rate in this group. In agreement with our study, and as reported in other publications, immunologic factors are important in graft failure.10e12 We found an independent association between the number of HLA mismatches and a large PRA percentage with the speed of graft loss after 15 years. DGF is a controversial factor in graft survival.13,14 Lauri et al found a significant influence in graft survival, relating it to different factors.6,14 Our data show an association between DGF and long-term graft survival, specifically 15 years after the transplantation.
LONG-TERM ALLOGRAFT SURVIVAL
3601
Table 3. Results of univariate and multivariate Cox regression Univariate
Multivariate
Variable
HR
P Value
95% CI (HR)
HR
P Value
95% CI (HR)
Recipient age Donor age Male Immunosuppression* PRA (50%) DGF HLA mismatches Rejections Time on dialysis Living donors Cold ischemia
1.01 1.01 0.72 0.79 2.74 12.93 1.28 1.19 1.01 0.68 1.03
.85 .28 .31 .46 .01 .02 .02 .58 .49 .59 .18
0.98e1.03 0.99e1.03 0.38e1.36 0.42e1.48 1.36e5.51 1.61e104.21 1.05e1.56 0.64e2.24 0.99e1.01 0.16e2.80 0.99e1.07
2.61 11.24 1.25
.01 .03 .04
1.28e5.26 1.33e95.27 1.0e1.55
Likelihood ratio test ¼ 15.068; P ¼ .005; GL ¼ 4. *Cyclosporine-based immunosuppression versus not cyclosporine-based.
The most common causes of graft loss are usually patient death and chronic graft nephropathy (CGN), the first being more relevant in short- and long-term follow-up series.4,15 In our overall series, the data agree with both causes, but CGN becomes more important in GS (66% vs 47%) according to Nankivell et al,16 probably because of latent subclinical damage. The introduction of cyclosporine into immunosuppressive regimens significantly improved the graft survival rate, which was a relevant contribution.2 A secondary analysis of our global series produced results in agreement with this fact, although we did not find any association with the graft survival rate in GS, probably because some patients
Fig 1. Kaplan-Meier survival after 15 years. The mean survival was estimated to be 210.26 (95% CI, 182.46e238.08) months.
subsequently started cyclosporine (data not controlled for that in this study). Despite the need to interpret this information cautiously owing to the possibility of bias because of the retrospective character of the study, we are able to emphasize the importance which the initial transplant management, the immunologic characteristics, and DGF have on long-term graft function. We should also bear in mind that nowadays the transplant population has changed, with older donors, more expanded-criteria donors, and more livingdonor renal allografts. REFERENCES 1. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med. 1999;341:1725e1730. 2. Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med. 2000;342:605e612. 3. Matas A, Gillingham K, Humar A, et al. 2,202 kidney transplant recipients with 10 years of graft function: what happens next? Am J Transplant. 2008;8:2410e2419. 4. Traynor C, Jenkinsonb A, Williamsa Y, et al. Twenty-year survivors of kidney transplantation. Am J Transplant. 2012;12: 3289e3295. 5. Bererhi L, Pallet N, Zuber J, et al. Clinical and immunological features of very long-term survivors with a single renal transplant. Transpl Int. 2012;25:545e554. 6. Kyllonen LEJ, Salmela KT, Eklund BH, et al. Long-term results of 1047 cadaveric kidney transplantations with special emphasis on initial graft function and rejection. Transplant Int. 2000;13:122e128. 7. Serur D, Saal S, Wang J, et al. Deceased donor kidney transplantation: improvement in long-term survival. Nephrol Dial Transplant. 2011;26:317e324. 8. Meier-Kriesche HU, Ojo AO, Leavey SF, et al. Gender differences in the risk for chronic renal allograft failure. Transplantation. 2001;71(3):429e432. 9. El-Agroudy AE, Bakr MA, Hassan NA, et al. Characteristics of long-term live-donor renal allograft survivors. Am J Nephrol. 2003;23:165e171. 10. Loupy A, Hill GS, Jordan SC. The impact of donor-specific anti-HLA antibodies on late kidney allograft failure. Nat Rev Nephrol. 2012 Apr 17;8(6):348e357.
3602 11. Varghese Z. Immunologic and nonimmunologic correlates of chronic renal allograft dysfunction. Transplant Proc. 1999;31: 3356e3358. 12. Opelz G, Döhler B. Effect of human leukocyte antigen compatibility on kidney graft survival: comparative analysis of two decades. Transplantation. 2007;84:137e143. 13. Marcen R, Orofino L, Pascual J, et al. Delayed graft function does not reduce the survival of renal transplant allografts. Transplantation. 1998;66:461e466.
GÓMEZ, HERNÁNDEZ, LÓPEZ ET AL 14. Shoskes DA, Cecka JM. Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection. Transplantation. 1998;66(12):1697e1701. 15. El-Zoghby ZM, Stegall MD, Lager DJ, et al. Identifying specific causes of kidney allograft loss. Am J Transplant. 2009;9: 527e535. 16. Nankivell BJ, Borrows RJ, Fung CL, et al. The natural history of chronic allograft nephropathy. N Engl J Med. 2003; 349(24):2326e2333.
K
IDNEY TRANSPLANTATION is nowadays the best treatment for patients who are in end-stage renal failure.1 Better knowledge about surgical techniques and immunosuppressive regimens have increased the allograft survival rate.2e4 Only a few researchers have studied and reported longterm graft survival in their series.4,5 The aim of the present study was to identify the factors associated with allograft survival in our series 15 years after transplantation.
grafts, excluding multiple organ transplantation. The patient demographic data were made available from our renal patient database. We considered graft failure to be the need to return to dialysis, the need to have a second transplantation, or patient death. Delayed graft function was the need for dialysis in the 1st week after transplantation. Before 1986, all patients were initially treated with azathioprine and prednisone. After 1986, cyclosporine was added to the immunosuppressive regimen.
PATIENTS AND METHODS We undertook a retrospective study of the kidney transplants carried out from February 1979 to December 1997 with follow-up through June 2012 at Reina Sofia Hospital in Cordoba. Because all transplantations were performed at a single center and there were a limited number of transplant surgeons, there was uniformity of care and treatment protocols. We included all of the first
From the Urology (E.G.G., J.P.C.H., F.J.M.L., J.R.G., F.A.C., M.L.V., J.C.R.L., M.J.R.T.) and Nefrology (V.G.M., M.D.N.C., P.A.) Units, Reina Sofía University Hospital, Córdoba, Spain. Address reprint requests to Enrique Gómez Gómez, Ramirez de Arellano N 1, Cordoba, Spain. E-mail: enriquegomezgomez@ yahoo.es
ª 2013 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/13/$esee front matter http://dx.doi.org/10.1016/j.transproceed.2013.09.015
Transplantation Proceedings, 45, 3599e3602 (2013)
3599
3600
GÓMEZ, HERNÁNDEZ, LÓPEZ ET AL
Student t and chi-square analyses were used to compare demographic variables, both continuous and categoric, between the series. Kaplan-Meier and log rank were used to determine survival probabilities at various post-transplantation times in the group with a functioning kidney transplant after 15 years, based on different variables. Cox proportional hazards models were used in univariate and multivariate analyses to assess predictors of long-term survival. We used SPSS version 19.0 to analyze the data.
RESULTS
We studied 487 kidney transplantations performed in our hospital from February 1979 to December 1997 with follow-up through June 2012, with a mean follow-up of 114 months. The mean recipient age was 38 years (range, 6e64) and the majority received deceased-donor kidney transplants (n ¼ 476; 97.7%). The demographic characteristics are presented in Table 1. A total of 180 patients (36.9%) maintained graft function after 15 years (GS). At the time of the study, 136 patients (27%) still maintained graft function. The principal cause of graft failure in the main series (patients with graft function after 15 years) was chronic graft nephropathy (CGN) in 29 patients (66%), followed by the death of 13 patients with functioning grafts (29.5%; Table 2). Regarding the immunosuppressive regimen, the introduction of cyclosporine was associated with improved results in graft survival in all patients, with a median of 210.3 months (95% CI, 181.5e239.1) and a difference of 79 months compared with the group not treated with cyclosporine (P ¼ .009). However, this difference was not observed in the group with graft function after 15 years, as presented in Table 3. In Cox proportional hazards ratio analysis, immunologic factors and delayed graft function (DGF) were the variables associated with better graft survival 15 years after transplantation. DGF was the most important factor, with a hazard ratio (HR) of 11.25 (95% CI, 1.33e95.28), followed by panel reactive antibody (PRA; HR 2.61; 95% CI, 1.28e5.26), and finally the number of HLA mismatches (HR 1.25; 95% CI, 1.01e1.55; Table 3). Table 1. Clinical Characteristics of Patients
Variable
NoneGraft Survivors (n ¼ 307)
Donor age (y) Recipient age (y) Male Time on dialysis (mo) Cold ischemia (h) HLA mismatches PRA 15 y P (n ¼ 180) Value
32.7 14.9 35.5 12.8 112 (62.2%) 41.1 37.4 22.2 7.1 2.4 1.4 122 (67.8%) 0.2 0.4 8 (4.5%) 2 (1.1%)
.08 .00 .72 .46 .74 .03 .02 .03 .02 .00
All Patients (n ¼ 487)
34.4 45.6 38.1 13.1 308 (63.2%) 40.1 15.9 22.1 1.2 2.6 6.3 295 (60.6%) 0.3 3.5 11 (2.3%) 40 (8.2%)
Abbreviations: PRA, panel reactive antibody; DGF, delayed graft function.
Table 2. Causes of Graft Failure Cause of graft failure
Chronic allograft nephropathy Patient death Other
Graft survivors >15 y; n ¼ 44 (24% of 487)
29 (66%) 13 (29.5%) 2 (4.5%)
NoneGraft Survivors; n ¼ 263 (59% of 487)
124 (47.2%) 89 (33.8%) 50 (19%)
In GS the probability of maintaining graft function was 97.2% (95% CI, 95e99.6%) and 76.9% (95% CI, 69e83%) at 1 and 5 years, respectively (16 and 20 years after transplantation; Fig 1). Probabilities changed to 100% and 91.7% in the group with 1 mismatches compared with 96.3% and 70% in the other group (2 mismatches; P ¼ .03). These differences in the survival rate for the same period were also evident in log rank analysis based on DGF, with probabilities of 97.2% and 76.5% in the group with no DGF compared with 100% and 50% in the group with DGF (P ¼ .002), and on PRA, with probabilities for the group with 50% PRA of 93.9% and 54.1% after a period of 1 and 5 years, respectively (P ¼ .04).
DISCUSSION
There is a large young population with end-stage renal failure who require a kidney transplant early in their lives, so identifying the factors associated with long-term graft survival is highly beneficial. Various series show long-term graft survival, such as that of Traynor et al with 23.5% (n ¼ 1081) functional grafts up to 20 years after the transplantation.4 In our series, with 487 patients, 37% had a functional graft for up to 15 years and almost 28% for up to 20 years. In the literature, recipient age, donor age, and recipient sex are factors associated with graft survival.4,6e8 Nevertheless, we did not find any association in the speed of the loss of the graft function in GS. Another factor associated with long-term graft survival is a living-donor renal allograft.4,9 Despite having only a few living-donor renal allografts in our study period, we found a higher percentage of this kind of graft in GS. However, this factor did not influence the graft loss rate in this group. In agreement with our study, and as reported in other publications, immunologic factors are important in graft failure.10e12 We found an independent association between the number of HLA mismatches and a large PRA percentage with the speed of graft loss after 15 years. DGF is a controversial factor in graft survival.13,14 Lauri et al found a significant influence in graft survival, relating it to different factors.6,14 Our data show an association between DGF and long-term graft survival, specifically 15 years after the transplantation.
LONG-TERM ALLOGRAFT SURVIVAL
3601
Table 3. Results of univariate and multivariate Cox regression Univariate
Multivariate
Variable
HR
P Value
95% CI (HR)
HR
P Value
95% CI (HR)
Recipient age Donor age Male Immunosuppression* PRA (50%) DGF HLA mismatches Rejections Time on dialysis Living donors Cold ischemia
1.01 1.01 0.72 0.79 2.74 12.93 1.28 1.19 1.01 0.68 1.03
.85 .28 .31 .46 .01 .02 .02 .58 .49 .59 .18
0.98e1.03 0.99e1.03 0.38e1.36 0.42e1.48 1.36e5.51 1.61e104.21 1.05e1.56 0.64e2.24 0.99e1.01 0.16e2.80 0.99e1.07
2.61 11.24 1.25
.01 .03 .04
1.28e5.26 1.33e95.27 1.0e1.55
Likelihood ratio test ¼ 15.068; P ¼ .005; GL ¼ 4. *Cyclosporine-based immunosuppression versus not cyclosporine-based.
The most common causes of graft loss are usually patient death and chronic graft nephropathy (CGN), the first being more relevant in short- and long-term follow-up series.4,15 In our overall series, the data agree with both causes, but CGN becomes more important in GS (66% vs 47%) according to Nankivell et al,16 probably because of latent subclinical damage. The introduction of cyclosporine into immunosuppressive regimens significantly improved the graft survival rate, which was a relevant contribution.2 A secondary analysis of our global series produced results in agreement with this fact, although we did not find any association with the graft survival rate in GS, probably because some patients
Fig 1. Kaplan-Meier survival after 15 years. The mean survival was estimated to be 210.26 (95% CI, 182.46e238.08) months.
subsequently started cyclosporine (data not controlled for that in this study). Despite the need to interpret this information cautiously owing to the possibility of bias because of the retrospective character of the study, we are able to emphasize the importance which the initial transplant management, the immunologic characteristics, and DGF have on long-term graft function. We should also bear in mind that nowadays the transplant population has changed, with older donors, more expanded-criteria donors, and more livingdonor renal allografts. REFERENCES 1. Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all patients on dialysis, patients on dialysis awaiting transplantation, and recipients of a first cadaveric transplant. N Engl J Med. 1999;341:1725e1730. 2. Hariharan S, Johnson CP, Bresnahan BA, et al. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med. 2000;342:605e612. 3. Matas A, Gillingham K, Humar A, et al. 2,202 kidney transplant recipients with 10 years of graft function: what happens next? Am J Transplant. 2008;8:2410e2419. 4. Traynor C, Jenkinsonb A, Williamsa Y, et al. Twenty-year survivors of kidney transplantation. Am J Transplant. 2012;12: 3289e3295. 5. Bererhi L, Pallet N, Zuber J, et al. Clinical and immunological features of very long-term survivors with a single renal transplant. Transpl Int. 2012;25:545e554. 6. Kyllonen LEJ, Salmela KT, Eklund BH, et al. Long-term results of 1047 cadaveric kidney transplantations with special emphasis on initial graft function and rejection. Transplant Int. 2000;13:122e128. 7. Serur D, Saal S, Wang J, et al. Deceased donor kidney transplantation: improvement in long-term survival. Nephrol Dial Transplant. 2011;26:317e324. 8. Meier-Kriesche HU, Ojo AO, Leavey SF, et al. Gender differences in the risk for chronic renal allograft failure. Transplantation. 2001;71(3):429e432. 9. El-Agroudy AE, Bakr MA, Hassan NA, et al. Characteristics of long-term live-donor renal allograft survivors. Am J Nephrol. 2003;23:165e171. 10. Loupy A, Hill GS, Jordan SC. The impact of donor-specific anti-HLA antibodies on late kidney allograft failure. Nat Rev Nephrol. 2012 Apr 17;8(6):348e357.
3602 11. Varghese Z. Immunologic and nonimmunologic correlates of chronic renal allograft dysfunction. Transplant Proc. 1999;31: 3356e3358. 12. Opelz G, Döhler B. Effect of human leukocyte antigen compatibility on kidney graft survival: comparative analysis of two decades. Transplantation. 2007;84:137e143. 13. Marcen R, Orofino L, Pascual J, et al. Delayed graft function does not reduce the survival of renal transplant allografts. Transplantation. 1998;66:461e466.
GÓMEZ, HERNÁNDEZ, LÓPEZ ET AL 14. Shoskes DA, Cecka JM. Deleterious effects of delayed graft function in cadaveric renal transplant recipients independent of acute rejection. Transplantation. 1998;66(12):1697e1701. 15. El-Zoghby ZM, Stegall MD, Lager DJ, et al. Identifying specific causes of kidney allograft loss. Am J Transplant. 2009;9: 527e535. 16. Nankivell BJ, Borrows RJ, Fung CL, et al. The natural history of chronic allograft nephropathy. N Engl J Med. 2003; 349(24):2326e2333.
