|Year : 2017 | Volume
| Issue : 2 | Page : 42-48
Predictors of allograft survival and patient survival in living donor renal transplant recipients
Pinaki Mukhopadhyay1, KL Gupta1, Vivek Kumar1, Raja Ramachandran1, Manish Rathi1, Ashish Sharma2, Mukut Minz2, Harbir S Kohli1, Vivekanand Jha1, Vinay Sakhuja1
1 Department of Nephrology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Renal Transplant Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Web Publication||12-Sep-2017|
K L Gupta
Department of Nephrology, Postgraduate Institute of Medical Education and Research, Sector-12, Chandigarh - 160 012
Source of Support: None, Conflict of Interest: None
Background: Living donor renal transplantation is the dominant type of renal transplantation in developing countries such as India. We looked at factors affecting allograft and patient survival in such circumstances as these could be different owing to unique socioeconomic, demographic, and patient characteristics. Methods: We retrospectively analyzed data of living donor renal transplantation done at Postgraduate Institute of Medical Education and Research, Chandigarh, over 5 years (2002–2007) to ascertain the factors that affect allograft and patient survival. The relationship of pretransplant characteristics of patient and donor, comorbid conditions, posttransplant immunosuppressive drug regimens, and infectious and noninfectious complications to allograft survival and patient survival were assessed. Results: A total of 554 living donor renal transplantation surgeries were performed during this period. Rates of death-censored renal allograft survival at 1, 3, and 5 years after transplant were 94%, 90%, and 79%, respectively. Independent predictors of death-censored graft loss were BK virus nephropathy, episodes of rejection, and use of immunosuppressive drug protocols other than triple drug regimen of tacrolimus, mycophenolate mofetil, and prednisolone. The patient survival at 1, 3, and 5 years after transplant in our study was 92%, 87%, and 83%, respectively. Presence of cytomegalovirus disease, recipient age ≥50 years, unrelated transplant (spousal donor or donor beyond first-degree relative), and presence of any opportunistic infection were found to be significant independent predictors of patient survival. Conclusions: Although retrospective, our data have shown comparable rates for allograft and patient survival for living donor renal transplantation in India.
Keywords: Allograft survival, immunosuppression, kidney disease, mortality, renal transplantation
|How to cite this article:|
Mukhopadhyay P, Gupta K L, Kumar V, Ramachandran R, Rathi M, Sharma A, Minz M, Kohli HS, Jha V, Sakhuja V. Predictors of allograft survival and patient survival in living donor renal transplant recipients. Indian J Transplant 2017;11:42-8
|How to cite this URL:|
Mukhopadhyay P, Gupta K L, Kumar V, Ramachandran R, Rathi M, Sharma A, Minz M, Kohli HS, Jha V, Sakhuja V. Predictors of allograft survival and patient survival in living donor renal transplant recipients. Indian J Transplant [serial online] 2017 [cited 2018 Feb 21];11:42-8. Available from: http://www.ijtonline.in/text.asp?2017/11/2/42/214386
| Introduction|| |
Living donor renal transplantation is the most feasible and best option for end-stage renal disease (ESRD) in India as the deceased donor program is still in infancy and faces multiple barriers. Despite lack of adequate healthcare infrastructure, paucity of facilities for the treatment of ESRD, sociocultural barriers, and financial constraints, renal transplantation especially from live-related donors seems to be the only viable treatment option available to ESRD patients in this part of the world. The prevention of allograft rejection or allograft dysfunction with minimal side effects has been central to various forms of immunosuppressive drug protocols that have been used for organ transplant recipients. As death with a functioning renal allograft is a major cause of allograft loss in renal transplant recipients, there is need of improving not only allograft survival but also patient survival. In India, the lack of proper registries for chronic kidney disease patients or organ transplant recipients prevents an accurate assessment of the problems and outcomes of these patients. We present a retrospective analysis of the outcomes of living donor renal transplantation at a large tertiary care referral center in India and try to find factors that could predict graft and patient survival as these factors could become potential areas for intervention to increase graft and patient survival.
| Methods|| |
Patients who received living donor renal transplantation at Postgraduate Institute of Medical Education and Research, Chandigarh, India, over 5 years (2002–2007) were enrolled for this retrospective analysis. Nephrology Clinic and Transplant Clinic records were reviewed to determine renal transplant recipient's underlying primary renal disease, pretransplant comorbid illnesses, duration of dialysis, posttransplant immunosuppressive drug regimens and complications, and donor's characteristics.
Standards of care during study period: Pretransplant evaluation of patient and recipient
All ESRD patients and their donors were investigated according to the standard protocol before renal transplantation surgery, in close coordination with transplant surgery team. Patients who were diagnosed as suffering from tuberculosis in the pretransplant period were transplanted only after 2 months of rifampicin-based four-drug antitubercular therapy was completed. Patients with a diagnosis of pretransplant hepatitis B virus (HBV) infection or hepatitis C virus (HCV) infection were offered treatment for the same, but were not denied the right to renal transplantation if they could not afford it, and were counseled regarding the outcomes. Spousal donors or donors beyond first-degree relatives of recipients were considered as unrelated donors.
Standards of care during study period: Posttransplant care
Renal transplantation surgery was performed by the Department of Transplant Surgery, and posttransplant care was a coordinated effort of both nephrology and transplant surgery teams. The immunosuppressive drug protocol was individualized for each patient by taking into consideration type of donor, comorbid conditions, and financial implications; and the same was also explained to the patient. Induction therapy with basiliximab or daclizumab was given to few patients. The immunosuppressive drug regimen consisted of three drugs, namely calcineurin inhibitors, i.e., cyclosporine A (CAP) or tacrolimus, mycophenolic acid or azathioprine, and prednisolone. A minority of patients had received cyclosporine, sirolimus, and prednisolone. All patients received prophylaxis with co-trimoxazole for initial 6 months after transplant. Slow fall in serum creatinine was defined as serum creatinine of ≥3 mg/dl at postoperative day 5.
Acute rejection was suspected whenever renal dysfunction was present. After immediate exclusion of other possible causes, an allograft biopsy was performed. No protocol biopsies were done. All allograft biopsies, including those for indications other than suspected acute rejection, were interpreted according to the Banff 97 working classification. The need of change in patient's immunosuppressive drug therapy was based on clinical condition of the patient, allograft biopsy findings, and treating nephrologist's judgment.
All infectious and noninfectious complications occurring in posttransplant period were recorded. The diagnosis and treatment for them were based on available standards of care at that time. Graft loss was defined as need of chronic dialysis (hemodialysis or peritoneal dialysis) or repeat renal transplantation. The cause of graft loss was noted. In case the patient had died, the cause of death with underlying primary disease for the same was recorded.
The relationship of pretransplant characteristics of patient and donor, comorbid conditions, posttransplant immunosuppressive drug regimens, and infectious and noninfectious complications to allograft survival and patient survival were assessed.
Statistical software program SPSS (v16 for Windows, SPSS, Chicago, IL, USA) was used to analyze the collected data. Quantitative data were described as means and standard deviations with their 95% confidence intervals (CIs). Categorical data were shown as frequencies and proportions. Comparisons of different groups were carried out for various categorical variables using Chi-square test to find out any statistical association between categorical variables. The mean values of quantitative variables were compared between two groups using independent t-test. Pearson's correlation coefficients were also calculated between different quantitative variables. Univariate odds ratios and their 95% CIs were calculated to identify predictors for the outcomes of death and graft loss. Multivariate logistic regression analyses were further carried out to find significant independent predictors for the above-mentioned outcomes after adjusting for various confounding variables. Multivariate analysis was undertaken after checking the data for the assumptions of normality, homoscedasticity, and multicollinearity. Final models for predictors of each outcome were generated using backward stepwise logistic regression methods. For time to event data, survival analysis using life table actuarial approach and log-rank test was carried out. To account for the confounding effects of various covariates, multivariate hazards ratios (with their 95% CIs) were also calculated for time to outcomes of death and graft loss using Cox proportional hazard survival analysis. Assumption of proportionality of hazard over time was tested before undertaking Cox proportional hazard analysis. A P value (two-tailed) <0.05 was considered statistically significant.
| Results|| |
A total of 554 living donor renal transplantation surgeries were performed. Majority of the recipients were males with a ratio of 6:1 favoring males. The average age of the recipient was 33.6 ± 10.3 years (range: 14–58 years). The two most common underlying causes of ESRD were chronic glomerulonephritis and diabetes mellitus [Table 1]. Approximately 22% of the patients were on hemodialysis for >6 months. Majority (86%) of the patients had opted for hemodialysis as modality of renal replacement therapy in the pretransplant period. A diagnosis of HCV, HBV, or tuberculosis infection was made in 6.8%, 2.5%, and 4.6% of the patients, respectively, during pretransplant period.
Majority (76.3%) of the donors were first-degree relatives of the corresponding recipients. Spouses as donors were present in 17.2% of the cases whereas distant relatives or unrelated persons constituted the rest. The average age of the donor was 42.36 ± 11.27 years (range: 18–68 years) and approximately two-third of the donors were females.
Induction therapy with basiliximab or daclizumab was given in 8.1% and 1.1% of the patients, respectively. All the patients received three drugs as part of immunosuppressive drug protocol. Cyclosporine A along with azathioprine and prednisolone was the most commonly used drug during this period [Table 2]. Mean duration of follow-up was 13.98 ± 20.99 months (range: 0–84 months; 95% CI: 29.2–32.7 months).
|Table 2: Initial immunosuppressive drug regimens in renal transplant recipients|
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During follow-up, 162 patients were diagnosed as having infectious complications at some point during their posttransplant course. Tuberculosis, HCV, and HBV infections were diagnosed in 6.1%, 6.7%, and 0.5% of the patients, respectively. Cytomegalovirus (CMV) disease was seen in 20 (3.6%) patients. Herpes zoster infection was noted in 17 (3.1%) patients whereas BK virus (BKV) infections involving the allograft were diagnosed in 12 (2.2%) patients. One case each of dengue virus, Epstein–Barr virus, and varicella infection was also seen.
The records revealed that there were thirty cases of fungal infections in the study population till last follow-up visit, with oral candidiasis constituting majority of them. Other fungal infections diagnosed in the patients were cryptococcosis (five cases), pneumocystis carinii (five cases), mucormycosis (two cases) aspergillosis (three cases), and histoplasmosis (one case). Nocardiosis was diagnosed in three patients whereas tularemia was seen in one patient.
About 4% of the patients had one or more episodes of urinary tract infection (UTI). The most common etiological organisms causing UTI were Escherichia More Details coli, followed by Klebsiella species. The average time after transplant at which the first infection was diagnosed was 15.2 ± 19.7 months.
New-onset diabetes after transplantation
About 8.5% of the renal transplant recipients were diagnosed as having new-onset diabetes after transplantation (NODAT). Majority of them received only oral hypoglycemic agents for control of hyperglycemia. Only nine patients were prescribed insulin injections to control elevated blood sugar levels.
A total number of 142 episodes of acute rejection were documented. Up to 90% cases of acute rejection responded favorably to intravenous methylprednisolone therapy. Antithymocyte globulin was given to only five patients and all of them responded to it. About 70% of the rejection episodes occurred within 1 month of renal transplantation surgery. The mean time to first episode of rejection was 14.1 ± 5.36 weeks.
A total of 46 living patients had suffered graft loss. The two most common causes of graft loss were chronic allograft nephropathy and drug noncompliance [Table 3]. Overall rates of death-censored graft survival at 1, 3, and 5 years after renal transplant were 94%, 90%, and 79%, respectively. Average time to graft loss was 33 ± 11.05 months (range: 17–56 months).
There were six predictors (use of immunosuppressive drug protocol other than combination of tacrolimus, mycophenolate mofetil, and prednisolone [TMP]; use of tacrolimus, azathioprine, and prednisolone over TMP; use of CAP over TMP, episodes of rejection, and BKV nephropathy; use of azathioprine over mycophenolate mofetil; presence of opportunistic infections) that were statistically significant in univariate analysis [Table 4]. Multivariate logistic regression analysis was done to find out independent predictors [Table 5]. A backward logistic regression method was used to arrive at a final model. The final model retained only three variables as having strongest and independent predicting ability for the outcome of graft loss. They were BKV nephropathy, episodes of rejection, and use of immunosuppressive protocol other than combination of TMP. Overall diagnostic accuracy of this three predictor model was 92%, and it explained 15% of total variability in the outcome (Nagelkerke's R2 = 0.153).
|Table 4: Univariate analysis for predictors of graft loss and death as outcomes|
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|Table 5: Multivariate logistic regression analysis for outcome of graft loss|
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In Cox regression proportional hazard analysis for the outcome of time to graft loss, recipient's age ≥50 years, episodes of rejection, and presence of BKV nephropathy were found to be statistically significant [Table 6].
|Table 6: Cox regression proportional hazard analysis for the outcome of time to graft loss|
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There were 54 deaths among 554 patients in the study group [Table 7]. Presumed or proven septicemic illness was the most common cause and accounted for 36 deaths. Acute myocardial infarction and hepatic encephalopathy accounted for four patients' death in each category. The mean time to death was 18 ± 19.05 months (range: 0–76 months). Overall patient survival rates at 1, 3, and 5 years after renal transplant were 92%, 87%, and 83%, respectively.
There were 12 predictors (recipient's age ≥50 years, unrelated transplant, presence of pretransplant diabetes, presence of pretransplant tuberculosis, presence of pretransplant comorbid illness, use of induction therapy, chronic allograft nephropathy, presence of opportunistic infection, posttransplant tuberculosis, BKV nephropathy, CMV disease, and presence of NODAT) that came out to be significant in univariate analysis [Table 4]. Subsequent multivariate logistic regression analysis with the use of backward logistic regression method for building a model to predict the outcome of death retained only five variables as having independent predicting ability [Table 8]. These were CMV infection, unrelated transplant, presence of NODAT, recipient's age ≥50 years, and presence of opportunistic infections. Overall diagnostic accuracy of this five predictor model was 92% and it explained approximately 45% of variability in the outcome of death (Nagelkerke's R2 = 0.448).
In Cox regression proportional hazard analysis for the outcome of time to death, CMV disease, recipient's age ≥50 years, presence of opportunistic infections, and unrelated transplant were found to be statistically significant [Table 9].
|Table 9: Cox regression proportional hazard analysis for the outcome of time to death|
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| Discussion|| |
In our study, rates of death-censored renal allograft survival at 1, 3, and 5 years after transplant were 94%, 90%, and 79%, respectively. The patient survival at 1, 3, and 5 years after transplant in our study was 92%, 87%, and 83%, respectively.
ESRD has become a major health concern all over the world. In ESRD patients, renal transplantation not only offers a survival advantage over dialysis, but it is also economical with a much-improved quality of life., Improving allograft survival and thus decreasing the need of retransplantation can help in tackling the global donor organ shortage. The disturbing downward trend for living donor renal transplantation in developing countries could be due to aging recipient population, changing economic factors, improvements in deceased donor program, and lack of emphasis on living donation. This is despite the fact that 1, 5, and 10 years patient and graft survival statistics are best for living donor transplantations followed by nonexpanded criteria donor deceased donor renal transplant recipients.
In India, the social and family structure revolves around males as they are earning members, and important family decisions are also usually taken by them. In this study, majority of the recipients (86.6%) were young adult males and majority of the donors (67.5%) were females. This is a reflection of the prevailing sociocultural environment and may not be necessarily a result of willful gender discrimination.
The 2009 Annual Report of the Organ Procurement and Transplantation Network (OPTN) and Scientific Registry of Transplant Recipients (SRTR) has shown unadjusted graft survival rates at 1, 5, and 10 years after living donor kidney transplantation to be 96%, 81%, and 59%, respectively. The 33rd Annual Report of Australia and New Zealand Dialysis and Transplant Registry (ANZDATA) has reported graft survival of 95.9% and 87.7% at 1 and 5 years after transplant for living donor primary renal transplants done from 2000 to 2004. Data from a large tertiary care military hospital in North India have revealed estimated graft survival rates at 1, 5, and 10 years after transplant to be 95.4%, 80.5%, and 51.3%, respectively. Only 1.4% of the renal transplants were cadaveric donations in this group. The rates for death-censored graft survival at 1, 5, and 10 years after transplant were 97%, 89%, and 78%, respectively, in another living donor renal transplant program at one of the other big centers in India. Therefore, the graft survival at our center is comparable to what has been reported from other centers. Out of six predictors of death-censored graft loss in univariate analysis, only three were found to be independent predictors after multivariate logistic regression analysis. These were BKV infection, episodes of rejection, and use of immunosuppressive drug protocol other than combination of TMP.
BKV nephropathy has been reported to be seen in 1%–10% of the kidney transplant recipients., It has been recognized as an important cause of graft loss and the association of different histological patterns with graft loss varies from 13% in Class A to as high as 100% in Class C. In our study also, 10 out of 12 patients who were diagnosed as having BKV nephropathy either suffered graft loss or died. The odds ratio for death-censored graft loss in patients with BKV nephropathy was very high at 9.8 and the time to graft loss was almost 6 times early as compared to patients without BKV nephropathy.
Over the last two decades, introduction of new drugs and immunosuppressive drug protocols has led to decrease in the incidence of acute rejection, but there has not been any impressive change in long-term graft survival. Acute rejection within 1st year after renal transplant has been associated with shorter time to death-censored graft loss whereas the presence of NODAT has a strong association with outcome of death with functioning graft. In our study, the odds ratio for death-censored graft loss in patients with episodes of acute rejection was 2.9 and these patients were likely to develop graft loss 2.4 times earlier than patients without rejection. Our analysis also showed NODAT to be a predictor of death with an odds ratio of 2.2.
In a large observational series of protocol biopsies performed in renal transplant recipients, combination of tacrolimus and mycophenolate mofetil was shown to have significantly lower incidence of subclinical rejection when compared to cyclosporine and azathioprine. In our study, use of initial immunosuppressive drug protocols other than triple drug regimen of TMP came out as a strong predictor for graft loss with odds ratio of 5.5. At present, TMP regimen is the most commonly used regimen at our center. The use of induction agents has been low owing to higher financial costs involved. The 2009 Annual Report of the OPTN and SRTR has shown a trend toward more use of induction agents (81% living donor transplants in 2008) and less use of steroids in the United States. In 2008, the most common dual therapy used in the United States was a combination of tacrolimus and mycophenolate mofetil.
The 2009 Annual Report of the OPTN and SRTR has reported patient survival rates of 99%, 91%, and 77% at 1, 5, and 10 years, respectively, after living donor renal transplant. The 33rd Annual Report of ANZDATA has reported patient survival of 98.5% and 94.3% at 1 and 5 years after transplant for living donor primary transplants done from 2000 to 2004. Estimated patient survival of 93.2% at 1 year after renal transplant has been reported from North India. Presence of CMV disease, recipient age ≥50 years, unrelated transplant, and presence of any opportunistic infection were found to be significant independent predictors of patient survival in multivariate analysis of 12 factors that came out to be significant in univariate analysis.
The association of both donor and recipient age with outcome has been studied in the past. Donor age has been found to be the most important factor affecting graft survival at 5 years. This analysis included both living donor and deceased donor recipients with functioning graft at 1 year. In our study, donor age did not come out to be a significant predictor either for the outcome of graft loss or death. Transplantation in the elderly population is characterized by low incidence of acute and chronic rejection and higher death-censored graft survival as compared to younger patients. The most common cause of graft loss in this population is death of the patient. Therefore, older recipients are more likely to die with a functioning allograft than younger recipients. Multivariate logistic regression analysis of our data revealed that recipient age ≥50 years was a predictor for outcome of death with an odds ratio of 2.8 but was not so for death-censored graft loss.
Unrelated transplants came out to be an independent predictor of recipient's death in our study population. However, one of the transplant centers from North India has reported no significant differences between related and unrelated renal transplants with respect to incidence of infection, rejection, graft dysfunction, graft loss, or death.
The management of infectious complications arising due to therapeutic immunosuppressed state is an important aspect of posttransplant care and is constantly evolving. Unusual or complicated presentations, need of prompt definitive diagnosis, and risk of drug interactions make their management complex and difficult. Infections (either proven or presumed infectious illness) were the most common cause of death in our transplant recipient population. In multivariate logistic regression analysis, the odds ratio for death in recipients with infection was 2.1 and they were two times more likely to die earlier than recipients without infections. Furthermore, CMV disease came out to be an independent predictor of outcome of death. The recipient and donors were never screened for the presence of CMV infection in the pretransplant period, and therefore, only recipients with symptomatic CMV disease were detected and treated. CMV disease has been known to have a variety of indirect effects in the transplant recipient, which predispose to other infections and altered immune responses.
Noncompliance with drugs was the second most common cause of graft loss in our study population. The importance of noncompliance is especially more relevant in the current era of potent immunosuppressive drugs as it is an important potentially modifiable risk factor for development of rejection and graft loss., The main reason for noncompliance in our study population was lack of financial resources to support the cost of immunosuppressive drugs. Nevertheless, there remains scope for better patient education with respect to financial implications of immunosuppressive drug costs, need of life-long therapy, and risks of noncompliance.
Solid organ transplant registries in developed countries are the major source of data and trends regarding renal transplant recipients. The renal transplant programs in the developing countries are different from developed world. Poor rates of deceased organ donation, lack of awareness, scarcity of health care resources, and financial constraints are important concerns in the developing countries. The need to individualize therapy is even more important in view of these concerns. Our study suffers from all the drawbacks associated with a retrospective study. Despite deficiencies in data collection, small sample size, and short follow-up duration, our study highlights the relatively good success of live renal donation in developing countries and identifies areas for potential interventions to improve allograft and patient survival.
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Conflicts of interest
There are no conflicts of interest.
| References|| |
Jha V. Current status of end-stage renal disease care in South Asia. Ethn Dis 2009;19 1 Suppl 1:S1-27-32.
Wolfe RA, Ashby VB, Milford EL, Ojo AO, Ettenger RE, Agodoa LY, 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;341:1725-30.
Laupacis A, Keown P, Pus N, Krueger H, Ferguson B, Wong C, et al.
Astudy of the quality of life and cost-utility of renal transplantation. Kidney Int 1996;50:235-42.
Campistol JM. Conclusions from the symposium. Transplant Proc 2010;42:S47-8.
Varma PP, Hooda AK, Sinha T, Chopra GS, Karan SC, Sethi GS, et al.
Renal transplantation – An experience of 500 patients. Med J Armed Forces India 2007;63:107-11.
Hirsch HH, Steiger J. Polyomavirus BK. Lancet Infect Dis 2003;3:611-23.
Pham PT, Schaenman J, Pham PC. BK virus infection following kidney transplantation: An overview of risk factors, screening strategies, and therapeutic interventions. Curr Opin Organ Transplant 2014;19:401-12.
Drachenberg CB, Papadimitriou JC, Hirsch HH, Wali R, Crowder C, Nogueira J, et al.
Histological patterns of polyomavirus nephropathy: Correlation with graft outcome and viral load. Am J Transplant 2004;4:2082-92.
Meier-Kriesche HU, Li S, Gruessner RW, Fung JJ, Bustami RT, Barr ML, et al.
Immunosuppression: Evolution in practice and trends, 1994-2004. Am J Transplant 2006;6:1111-31.
Cole EH, Johnston O, Rose CL, Gill JS. Impact of acute rejection and new-onset diabetes on long-term transplant graft and patient survival. Clin J Am Soc Nephrol 2008;3:814-21.
Nankivell BJ, Borrows RJ, Fung CL, O'Connell PJ, Allen RD, Chapman JR, et al.
The natural history of chronic allograft nephropathy. N
Engl J Med 2003;349:2326-33.
Jassal SV, Opelz G, Cole E. Transplantation in the elderly: A review. Geriatr Nephrol Urol 1997;7:157-65.
Gulati S, Gupta S, Kher V, Gupta A, Ahlawat R, Arora P. Outcome of live related and live unrelated renal transplants. Nephrology 1997;3:563-7.
Martin-Gandul C, Mueller NJ, Pascual M, Manuel O. The impact of infection on chronic allograft dysfunction and allograft survival after solid organ transplantation. Am J Transplant 2015;15:3024-40.
Nevins TE, Matas AJ. Medication noncompliance: Another iceberg's tip. Transplantation 2004;77:776-8.
Williams AF, Manias E, Gaskin CJ, Crawford K. Medicine non-adherence in kidney transplantation. J Ren Care 2014;40:107-16.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9]