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(Radiology. 2001;219:663-667.)
© RSNA, 2001


Vascular and Interventional Radiology

Renal Arterial Stenosis in Renal Allografts: Retrospective Study of Predisposing Factors and Outcome after Percutaneous Transluminal Angioplasty1

Nilesh H. Patel, MD, Rahul M. Jindal, MD, Terrence Wilkin, MD, Shane Rose, MD, Matthew S. Johnson, MD, Himanshu Shah, MD, Jan Namyslowski, MD, Kenneth P. Moresco, MD and Scott O. Trerotola, MD

1 From the Departments of Radiology (N.H.P., T.W., S.R., M.S.J., H.S., J.N., K.P.M., S.O.T.) and Surgery (R.M.J.), Indiana University Medical Center, 550 N University Blvd, Rm 0279, Indianapolis, IN 46202; and Department of Surgery, University of Glasgow, Scotland (R.M.J.). Received June 20, 2000; revision requested August 8; final revision received January 2, 2001; accepted January 22. Address correspondence to N.H.P. (e-mail: nhpatel@iupui.edu).


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine the predisposing factors to transplant renal arterial stenosis (TRAS) and assess the outcome of percutaneous transluminal angioplasty (PTA) as the primary treatment.

MATERIALS AND METHODS: Of 831 renal allograft recipients (584 cadaveric, 247 living related) between January 1991 and December 1998, 72 had hypertension and/or renal dysfunction. All 72 underwent arteriography, and their medical charts were retrospectively reviewed.

RESULTS: Prevalence of TRAS was 3.1% (26 of 831). Technical success rate of PTA was 94% (16 of 17), and clinical success rate was 82% (14 of 17). Those with renal dysfunction had a mean pre-PTA creatinine value of 2.6 mg/dL (230 µmol/L) ± 0.5 (SD) versus a 1-week post-PTA value of 1.7 mg/dL (150 µmol/L) ± 0.3 (P < .001). Of those with hypertension, all but one had substantial improvement in mean diastolic blood pressure. At 26.9 months mean follow-up in 16 patients with successful PTA, two stenoses reoccurred, and two grafts were lost to chronic rejection. TRAS was present in 14 of 45 end-to-side anastomoses and 12 of 27 end-to-end anastomoses (P = .31), and TRAS was more prevalent in cadaveric grafts (24 of 584) than in living related grafts (two of 247). In cadaveric grafts, the mean cold ischemia time was 29.0 hours ± 6.9 in those with TRAS (n = 24), as compared with 25.5 hours ± 8.1 in those with no TRAS (n = 39; P = .35). Seven of 17 patients with acute rejection and six of 35 with chronic rejection had TRAS.

CONCLUSION: Primary treatment of TRAS with PTA has good intermediate-term results. TRAS is more prevalent in cadaveric allografts with long cold ischemia time.

Index terms: Kidney, transplantation, 81.4557 • Renal arteries, stenosis or obstruction, 961.7212, 961.7213 • Renal arteries, transluminal angioplasty, 961.1282


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In renal allograft recipients, stenosis of the renal artery of a transplanted kidney is an important cause of hypertension and/or graft dysfunction. The prevalence of this disorder has varied in the literature, ranging from 1% to 23% (1). This discrepancy can be attributed to several factors. First, the definition of hemodynamically significant transplant renal arterial stenosis (TRAS) has not been standardized; investigators have used a range of narrowing of the arterial lumen from greater than 50% to greater than 80% (25). Second, the introduction of cyclosporine has been suggested as contributing to the increase in prevalence of TRAS, as this drug is known to cause vascular damage (6). Third, the ready availability of noninvasive screening modalities, such as color Doppler ultrasonography (US) and magnetic resonance (MR) angiography, may lead to an increase in frequency of their use in suspected cases of TRAS. This phenomenon may explain, in part, the recent apparent increase in the prevalence of TRAS. The aggressiveness with which investigations for TRAS are performed, varying surgical expertise, and cases of chronic rejection mistaken for TRAS may also play a role.

We retrospectively reviewed the records of renal allograft recipients who underwent arteriography for suspected TRAS to determine the presence of any predisposing factors to TRAS and to assess the outcome of percutaneous transluminal angioplasty (PTA) in the treatment of TRAS in terms of the effect on renal dysfunction and hypertension.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
From January 1, 1991, to December 31, 1998, 831 transplantations with renal allografts were performed at our institution, of which 584 were cadaveric and 247 were living related. In 72 of these cases, 26 patients had worsening renal function not explainable by other causes (rejection, drug toxicity, dehydration), 20 had acute onset or difficulty in controlling hypertension, and 26 had both renal dysfunction and hypertension. These 72 patients (41 male and 31 female patients; mean age, 38.2 years; age range 7–68 years) therefore underwent diagnostic renal arteriography for suspected TRAS, and their medical records were retrospectively reviewed (N.H.P., R.M.J., and T.W. or S.R.). The following data were recorded: cause of renal disease, date of renal transplantation, donor type, cold ischemia time, number of renal arteries and type of surgical anastomosis, presence of rejection at biopsy or result of cytomegalovirus (CMV) serology test performed within 1 month of arteriography, indication for and findings at diagnostic arteriography, treatment rendered, blood pressure and serum creatinine level before and 1 week after treatment, and duration of clinical follow-up.

In the 72 patients, diagnostic renal arteriography was performed by means of a femoral approach as previously described (7). In all cases, nonselective pelvic arteriography was performed to exclude inflow lesions. Then, the catheter tip was positioned just proximal to the transplant renal arterial anastomosis, and arteriography in multiple views was performed to profile the transplant renal artery and its anastomosis. A narrowing of greater than 50% of the luminal diameter was considered hemodynamically significant. If treatment with PTA was to be performed, a guide wire was advanced across the stenosis, heparin was intravenously administered (3,000–5,000 IU), dilation was performed with an appropriate-sized angioplasty balloon, and a postangioplasty arteriogram was obtained. Technical success of PTA was defined as a residual stenosis of less than 30% after angioplasty and no flow-limiting intimal flap. Clinical success was defined as (a) more than 15% reduction in serum creatinine level, (b) more than 15% reduction in mean diastolic blood pressure with the number of antihypertensive medications equal to that before PTA, or (c) more than 10% reduction in mean diastolic blood pressure with a reduction in the number of antihypertensive medications (1).

Also, we examined the association between (a) TRAS and the surgical technique used for the arterial anastomosis, (b) TRAS and CMV infection, and (c) TRAS and rejection episodes defined by means of allograft biopsy performed within 1 month prior to angiographic evaluation. The serum creatinine values before and after treatment of TRAS with PTA were compared by using a paired two-tailed Student t test, whereas the cold ischemia time in cadaveric allograft recipients with TRAS (n = 24) was compared with those without TRAS (n = 36) by using a two-sample two-tailed Student t test. A Fisher exact test was used to compare the frequency of TRAS by the type of anastomoses (end-to-end vs end-to-side). All values are expressed as the mean plus or minus SD. A P value of less than .05 was considered to indicate a statistically significant difference.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The causes of renal failure in the 72 patients were hypertension in 13, diabetes mellitus in 10, polycystic kidney disease in six, reflux nephropathy in two, reflux in two, congential dysplasia in two, lupus nephritis in two, and chronic and other glomerulonephritides in 35.

TRAS was seen in 26 (3.1%) of the 831 renal allografts: 24 (4.1%) of the 584 cadaveric recipients and two (0.8%) of the 247 living related recipients (Table). Two patients had positive serology test results for CMV, and arteriography showed numerous segmental stenoses suggestive of vasculitis. They were treated with anti-CMV medications, with improvement. Two patients with occlusion of the inflow artery and transplant renal artery, respectively, underwent transplant nephrectomy for severe renal dysfunction due to chronic rejection. In the remaining 22 patients, the initial treatment was PTA in 17 and surgical correction in five.


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Arteriographic Findings and Treatment

 
In the PTA group (n = 17), three patients had renal dysfunction at presentation, three had new onset or uncontrolled hypertension, and 11 had both. Sixteen (94%) of the 17 hemodynamically significant stenoses were successfully treated with PTA. The one failure demonstrated no waist on inflation of the angioplasty balloon, and there was no change in appearance on the post-PTA arteriogram. At surgery, the artery was kinked in this location. In the patients with renal dysfunction, the mean serum creatinine level decreased from a pre-PTA value of 2.6 mg/dL ± 0.5 (SD) (230 µmol/L) to a 1-week post-PTA value of 1.7 mg/dL ± 0.3 (150 µmol/L) (P < .001). In the patients with new onset or uncontrolled hypertension, all but one had substantial improvement in mean diastolic blood pressure. The overall clinical success of PTA was 82% (14 of 17 patients).

At a mean follow-up of 26.9 months in the 16 patients with successful PTA, two patients (12%) had recurrent stenoses, which were successfully retreated with percutaneous endovascular techniques, and two patients lost function of their allografts owing to chronic rejection. One of the recurrent stenoses was initially a clinical failure after PTA. The patient presented again 2 months later with acute renal dysfunction superimposed on preexistent hypertension. Angiography showed a recurrent stenosis at the transplant arterial anastomosis. This was successfully treated with placement of a metallic stent (Figure). The patient’s hypertension improved, he required fewer antihypertensive medications, and his serum creatinine level decreased from 3.2 to 1.8 mg/dL (from 283 to 159 µmol/L). The postintervention course was complicated by chronic rejection of the renal allograft 9 months later. The second recurrent stenosis was at the transplant arterial anastomosis, which 6 months previously had been successfully treated with PTA. The stenosis was again treated with PTA, with improvement of the patient’s renal dysfunction and blood pressure. The one patient with PTA failure due to a kink in the renal transplant artery lost kidney function owing to acute rejection and underwent transplant nephrectomy 1 week later.



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Figure 1a. Cadaveric renal transplantation, with a single renal artery and end-to-side anastomosis to the right external iliac artery, in a 44-year-old man who had hypertension at presentation. (a) Anteroposterior digital subtraction arteriogram shows a severe stenosis (arrow) of the midportion of the transplant renal artery. (b) Anteroposterior digital subtraction arteriogram obtained after dilation with a 7-mm-diameter angioplasty balloon shows a patent transplant renal artery with no residual stenosis and a small non-flow-limiting intimal flap (arrow). The patient received systemic anticoagulation treatment overnight and was discharged the following day with antiplatelet medication. Two months later, he continued to have elevated blood pressure and also had renal dysfunction. (c) Repeat anteroposterior arteriogram shows recurrent stenosis (arrow). (d) Repeat anteroposterior arteriogram after treatment with a metallic stent (arrow) shows a widely patent artery. At clinical follow-up, hypertension was controlled with medications, and the patient’s serum creatinine level decreased from 3.2 to 1.8 mg/dL (from 283 to 159 µmol/L).

 


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Figure 1b. Cadaveric renal transplantation, with a single renal artery and end-to-side anastomosis to the right external iliac artery, in a 44-year-old man who had hypertension at presentation. (a) Anteroposterior digital subtraction arteriogram shows a severe stenosis (arrow) of the midportion of the transplant renal artery. (b) Anteroposterior digital subtraction arteriogram obtained after dilation with a 7-mm-diameter angioplasty balloon shows a patent transplant renal artery with no residual stenosis and a small non-flow-limiting intimal flap (arrow). The patient received systemic anticoagulation treatment overnight and was discharged the following day with antiplatelet medication. Two months later, he continued to have elevated blood pressure and also had renal dysfunction. (c) Repeat anteroposterior arteriogram shows recurrent stenosis (arrow). (d) Repeat anteroposterior arteriogram after treatment with a metallic stent (arrow) shows a widely patent artery. At clinical follow-up, hypertension was controlled with medications, and the patient’s serum creatinine level decreased from 3.2 to 1.8 mg/dL (from 283 to 159 µmol/L).

 


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Figure 1c. Cadaveric renal transplantation, with a single renal artery and end-to-side anastomosis to the right external iliac artery, in a 44-year-old man who had hypertension at presentation. (a) Anteroposterior digital subtraction arteriogram shows a severe stenosis (arrow) of the midportion of the transplant renal artery. (b) Anteroposterior digital subtraction arteriogram obtained after dilation with a 7-mm-diameter angioplasty balloon shows a patent transplant renal artery with no residual stenosis and a small non-flow-limiting intimal flap (arrow). The patient received systemic anticoagulation treatment overnight and was discharged the following day with antiplatelet medication. Two months later, he continued to have elevated blood pressure and also had renal dysfunction. (c) Repeat anteroposterior arteriogram shows recurrent stenosis (arrow). (d) Repeat anteroposterior arteriogram after treatment with a metallic stent (arrow) shows a widely patent artery. At clinical follow-up, hypertension was controlled with medications, and the patient’s serum creatinine level decreased from 3.2 to 1.8 mg/dL (from 283 to 159 µmol/L).

 


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Figure 1d. Cadaveric renal transplantation, with a single renal artery and end-to-side anastomosis to the right external iliac artery, in a 44-year-old man who had hypertension at presentation. (a) Anteroposterior digital subtraction arteriogram shows a severe stenosis (arrow) of the midportion of the transplant renal artery. (b) Anteroposterior digital subtraction arteriogram obtained after dilation with a 7-mm-diameter angioplasty balloon shows a patent transplant renal artery with no residual stenosis and a small non-flow-limiting intimal flap (arrow). The patient received systemic anticoagulation treatment overnight and was discharged the following day with antiplatelet medication. Two months later, he continued to have elevated blood pressure and also had renal dysfunction. (c) Repeat anteroposterior arteriogram shows recurrent stenosis (arrow). (d) Repeat anteroposterior arteriogram after treatment with a metallic stent (arrow) shows a widely patent artery. At clinical follow-up, hypertension was controlled with medications, and the patient’s serum creatinine level decreased from 3.2 to 1.8 mg/dL (from 283 to 159 µmol/L).

 
Surgery was performed as the initial treatment for hilar stenosis (n = 2), kink in the transplant artery (n = 1), endarterectomy of internal iliac arterial stenosis (n = 1), and transplant arterial anastomotic stenosis in the immediate perioperative period (n = 1). One patient with a hilar stenosis was treated with a distal saphenous venous bypass graft. Three months later, she again had renal dysfunction and hypertension. Arteriography showed a hemodynamically significant stenosis of the distal bypass graft anastomosis. This was treated with PTA, with clinical improvement of the patient’s hypertension and reduction in her serum creatinine level from 2.3 to 1.9 mg/dL (from 203 to 168 µmol/L).

In the 72 patients, TRAS was present in 14 (31%) of 45 end-to-side arterial anastomoses and in 12 (44%) of 27 end-to-end arterial anastomoses (P = .31). Sixty-three of the 72 patients had cadaveric transplants; the mean cold ischemia time was 29.0 hours ± 6.9 in those with TRAS (n = 24), as compared with 25.5 hours ± 8.1 in those with no TRAS (n = 39; P = .35). Sixty-one of the 72 patients underwent biopsy of the allograft within 1 month of arteriography. There were 17 cases of acute rejection and 35 cases of chronic rejection among the biopsy specimens. Of the cases of TRAS, seven were associated with acute rejection and six with chronic rejection.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Renal arterial stenosis is not uncommon after renal transplantation, and it should be considered in patients who have severe hypertension and/or renal dysfunction. Hypertension may be due to chronic rejection, cyclosporine toxicity, steroid use, recurrence of glomerulonephritis, diseased native kidney, or TRAS (8). Both patient and allograft survival rates are lower in patients with TRAS than in those without TRAS (6). The cause of TRAS is multifactorial and includes surgical technique, type of allograft, immunologic factors, and CMV infection. The aggressiveness with which investigations are performed, particularly with the recent use of noninvasive screening modalities such as MR angiography, Doppler US, and computed tomographic angiography, has led to an apparent increase in cases of TRAS being detected and treated (914). In a study reported by Wong et al (6), the prevalence of TRAS was 2.4% before and 12.4% after the introduction of "screening" color Doppler US. However, there is also evidence that the clinical effect of TRAS might be overestimated in patients who have renal dysfunction and chronic rejection of the allograft (9).

TRAS may be related to surgical techniques used during organ removal and transplantation. Intimal tears to the transplant renal artery may arise from excessive traction of the artery during removal or from cannulation for organ perfusion. Trauma to the endothelial lining of the allograft artery may occur from a vascular clamp. Faulty technique for suturing of the anastomosis may cause stenosis at the suture line. An immediate stenosis can occur from kinking of the artery, a problem that is more apt to occur with a right renal allograft in which the artery is longer than the vein. In our series, we had two such cases. Surgical correction is the preferred method for treatment of kinking of the proximal transplant renal artery (9).

It has been suggested that an end-to-side anastomosis is prone to the development of TRAS owing to turbulent blood flow as a result of a hyperacute angle between the donor renal artery and the recipient iliac artery (15). However, results of several subsequent studies (1519) have shown no difference in the prevalence of TRAS between the end-to-side and end-to-end techniques.

Other researchers have proposed a correlation between TRAS and cadaveric transplants as opposed to living related transplants (9). Greater cold ischemia time, which is inevitable in cadaveric transplants, compounded by the use of pulsatile perfusion systems, may contribute to endothelial damage in the renal artery. The reported prevalence rates of TRAS in cadaveric transplants from two recent studies were 4.5% (8) and 6.5% (20). In contrast, in a retrospective study of 1,000 living related transplants, Mammen et al (21) found a prevalence of TRAS of 1.7%. Roberts et al (22) found a 1.5% prevalence of TRAS in 2,002 renal allograft recipients, of which 67% were in those who received a cadaveric transplant. In a retrospective analysis of 1,262 renal allograft recipients, Sankari et al (23) found that the prevalence of TRAS was 2% in cadaveric transplants and 0.3% in living related transplants. However, Greenstein et al (16), in a review of 547 renal transplant recipients, and Fauchald et al (24), in a review of 1,141 renal transplant recipients, found that the prevalence of TRAS (7.1% and 2.2%, respectively) was equally distributed between the cadaveric and living related transplants. Our overall prevalence of TRAS was 3.1% and was higher in cadaveric transplants (4.1%) than in living related transplants (0.8%).

Experimental and clinical data have suggested an immunologic cause for TRAS (4,17,25,26). Macia et al (27) found a significant association between TRAS and acute rejection through a retrospective review of 110 consecutive renal transplantations in which 8.2% of their patients had TRAS associated with rejection episodes. Wong et al (6), in a report of 77 cases of TRAS among 917 renal transplant recipients, found a significantly higher prevalence of TRAS in the acute cellular rejection group, as compared with that in the control group matched for age, year of transplantation, sex, number of previous allografts, and class I human leukocyte antigen matching. These findings suggested that renal vessels are subjected to rejection-induced inflammatory processes that can eventually lead to stenosis.

However, the similar prevalence of TRAS in human leukocyte antigen–identical living related donor allografts and the fact that rejection affects small vessels, sparing major vessels such as the renal artery, argue against immunologic factors as a major causative determinant (16,20,28). In the setting of chronic rejection and TRAS, frequently it may be difficult to ascribe the renal dysfunction to one specific cause over the other. It may be argued that in these patients TRAS is an incidental finding, thus resulting in its "overdiagnosis" as the cause for renal dysfunction.

An association of TRAS with CMV infection was reported by Pouria et al (29) in a study of 917 renal transplantations. They proposed that CMV-induced large vessel damage might occur through local infection and mitogenic action of viral gene products, similar to cardiac allograft vasculopathy and restenosis of native coronary arteries after angioplasty (30). These findings were confirmed by the detection of CMV DNA products in the vessel wall of restenotic coronary arterial lesions in humans (31).

Investigators at the University of Minnesota (32) reviewed the cases of 2,013 adult kidney transplantations performed between 1984 and 1998 and discovered an overall prevalence of TRAS of 2.3%. The pretransplantation CMV status was not shown to affect the occurrence of TRAS; however, recipients who were CMV negative and then became CMV positive after transplantation had a significantly higher prevalence of TRAS versus those who remained seronegative (4.6% vs 1.9%; P = .02). Notably, other risk factors were identified in their retrospective analysis, including multiple arterial anastomoses, use of hypogastric artery versus external iliac artery, and cadaveric versus living transplants. The routine use of anti-CMV prophylaxis for positive donors to negative recipients may account for the low prevalence of CMV in the organ transplant population in our study.

Pseudo-TRAS refers to a stenosis of the native iliac artery proximal to the transplant renal arterial anastomosis secondary to peripheral arterial disease. In a retrospective analysis of 819 renal allograft recipients by Becker et al (5), 92 patients suspected of having TRAS underwent MR angiography or conventional arteriography. They found TRAS in 24 patients, pseudo-TRAS in 15 patients, and both in five patients. A multivariate analysis revealed that insulin-dependent diabetes mellitus, panel reactive antibody, increasing weight at transplantation, and donor age were significantly associated with an increased risk of pseudo-TRAS. In our series, three patients had hemodynamically significant peripheral arterial disease involving the native iliac artery proximal to the transplant renal artery. Two patients lost function of their allografts, requiring hemodialysis. One patient with a severe internal iliac arterial stenosis and well-developed collateral vessels did well after surgical endarterectomy.

The treatment of TRAS with both surgical and endovascular techniques has been evaluated in multiple studies. Surgical correction of TRAS has a 66%–90% initial success rate, and one series reported a 12% recurrence rate (9). In a review of 1,200 renal allograft recipients by Benoit et al (25), of the 88 TRAS cases, 39 underwent surgical repair and 49 underwent PTA. The immediate and long-term success rates were 92.1% and 81.5%, respectively, for surgical repair, and 69% and 40.8%, respectively, for PTA. Nonetheless, these authors and others (9,20) still favor PTA as the first-line treatment of TRAS because of the technical demand of the vascular surgery and risk of allograft loss, ureteral injury, and surgical mortality. Others have reported more favorable initial success rates of 60%–90% for PTA (9,16). Authors of all but one study found a low restenosis rate, approximately 10% (6,16,17). Our technical success rate was 94%, and our clinical success rate was 82%. At a mean follow-up of 26.9 months, there were two (12%) recurrent stenoses, which were successfully retreated by using percutaneous endovascular techniques. The role of PTA in "incidental" TRAS that is occasionally seen in chronic rejection of renal allografts is not clear. We believe that retransplantation may be the best alternative in this subset of patients.

Arteriography is considered the diagnostic standard for TRAS. Unfortunately, it exposes patients suspected of having TRAS to the potential risk of nephrotoxicity from iodinated contrast material (33). To profile the transplant renal arterial anastomosis, multiple views and complex angulations may be required, thus increasing the volume of iodinated contrast material used. Since 1997, we have used intraarterial carbon dioxide and gadolinium chelates to exclude TRAS (7). In cases in which endovascular intervention was performed, the volume of iodinated contrast material was minimized (7).

In conclusion, the intermediate-term success of PTA for treatment of TRAS is good and is comparable with the results reported in the literature for surgical correction. The role of stent placement for the treatment of TRAS and its effect on long-term patency have yet to be investigated. TRAS is more prevalent in cadaveric allografts with long cold ischemia time.


    FOOTNOTES
 
Abbreviations: CMV = cytomegalovirus, PTA = percutaneous transluminal angioplasty, TRAS = transplant renal arterial stenosis

Author contributions: Guarantors of integrity of entire study, N.H.P., R.M.J.; study concepts and design, N.H.P., R.M.J.; literature research, N.H.P., R.M.J.; clinical studies, N.H.P., R.M.J.; data acquisition, N.H.P., R.M.J., T.W., S.R.; data analysis/interpretation, N.H.P., R.M.J.; statistical analysis, N.H.P., R.M.J.; manuscript preparation, N.H.P., R.M.J., T.W.; manuscript definition of intellectual content, N.H.P., R.M.J.; manuscript editing, S.R., M.S.J., H.S., J.N., K.P.M., S.O.T.; manuscript revision/review, N.H.P., R.M.J.; manuscript final version approval, N.H.P., T.W.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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