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(Chest. 2005;128:1371-1378.)
© 2005 American College of Chest Physicians

Health-Related Quality of Life Following Single or Bilateral Lung Transplantation*

A 7-Year Comparison to Functional Outcome

Margaret W. Gerbase, MD, PhD; Anastase Spiliopoulos, MD; Thierry Rochat, MD; Marc Archinard, MD{dagger} and Laurent P. Nicod, MD

* From the Division of Pulmonary Medicine, Clinic of Thoracic Surgery and Unit of Liaison Psychiatry, University Hospitals of Geneva, Geneva, Switzerland. {dagger} In memoriam.

Correspondence to: Margaret W. Gerbase, MD, PhD, Division of Pulmonary Medicine, University Hospitals of Geneva, 24 rue Micheli-du-Crest, 1211 Geneva 14, Switzerland; e-mail: margaret.gerbase{at}hcuge.ch


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Objectives: To compare long-term health-related quality of life (HRQL) in single and bilateral lung transplant recipients independent of the underlying disease, and in a subset of patients with native pulmonary emphysema.

Methods: Forty-four lung transplant recipients (mean [± SD] age, 44.8 ± 11.6 years) were followed up for > 2 years after single lung transplantation (LTx) [14 recipients] or bilateral LTx (30 recipients). Data were prospectively collected, before undergoing LTx and annually after undergoing LTx, measuring FEV1, 6-min walk test (6MWT) results, and quality of life using the St. George respiratory questionnaire (SGRQ) and a visual analog scale (VAS). The SGRQ addresses three domains, namely, respiratory symptoms, accomplishment of routine activities, and disease impact on daily life.

Results: Statistically significant correlation coefficients were found comparing the SGRQ and the VAS (r = 0.812; p < 0.0001), the SGRQ and the 6MWT (r = 0.610; p < 0.0001), and the SGRQ and the FEV1 (r = 0.523; p < 0.0001) in all patients. Significant improvements on the FEV1, 6MWT, and SGRQ were observed after LTx in both single and bilateral LTx recipients. Increased risk for the development of bronchiolitis obliterans syndrome (BOS) [relative risk, 2.86; 95% confidence interval, 1.22 to 6.67; p = 0.03] and significantly lower FEV1 values were observed in patients following a single graft, compared to that in patients following a bilateral graft (p < 0.01). In contrast, the 6MWT and the SGRQ scores were not significantly different between recipients of single and double LTx. The same patterns of results were observed in comparisons between single and bilateral lung recipients with prior pulmonary emphysema.

Conclusions: Despite poorer FEV1 recovery and increased risk of BOS after LTx, single lung transplant recipients had comparable long-term exercise tolerance and quality-of-life scores as patients who received bilateral transplants. These results suggest the limited influence of functional performance on objective and subjective markers of HRQL recovery after LTx.

Key Words: exercise test • health-related quality of life • lung transplantation • spirometry


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Over the last two decades, lung transplantation (LTx) has become accepted as a realistic therapy for patients with end-stage pulmonary disease. During this period, considerable advances in organ preservation, surgical techniques, immunosuppression, and antibiotic therapy have contributed to improvements in both short-term and long-term postoperative survival for a significant number of patients undergoing LTx.12 Outcome measurements following LTx have been primarily based on survival and pulmonary function recovery. In the last several years, however, increasing attention has been given to assessments of the quality of survival, with particular importance given to the patients’ self-evaluations of recovery after the transplant procedure. In the absence of a specific validated tool, a variety of existing instruments have been used to evaluate the quality of life of these patients before and after LTx.

Health-related quality of life (HRQL) has been reported by several authors to improve significantly within the first year after LTx.34567 However, reports assessing HRQL beyond 2 years after LTx remain anecdotal, although this is the time frame in which major clinical complications such as bronchiolitis obliterans syndrome (BOS) may begin to develop and threaten the patients’ recovered autonomy.8 Previous studies910 have assessed the impact of BOS on HRQL after LTx, and the reported results have indicated significant impairment of physical mobility in lung transplant recipients. Other studies11 have reported that a transplant operation reduces the risk of BOS in double-lung transplant recipients. However, a comprehensive assessment of HRQL after LTx, to include pulmonary function, exercise capacity, and well-being status, has not yet been undertaken to compare these factors in single and bilateral lung transplant recipients.

In a review of the literature, only one study (to our knowledge)12 was found that compared quality of life in patients following a single or a bilateral LTx, and its results were less favorable in single LTx recipients. To date, no cohort study is available to longitudinally address HRQL in lung transplant recipients following a single or a bilateral lung graft. We performed a longitudinal follow-up of 44 patients who underwent single or bilateral sequential LTx at our institution, obtaining prospective data from spirometry, 6-min walk tests (6MWT), and HRQL questionnaires. To reduce the potential bias from comparing different underlying diseases, the outcomes were also compared in a subset of patients with prior pulmonary emphysema who had undergone single or bilateral LTx.


   Materials and Methods
TOP
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Between June 1993 and June 2004, 90 LTxs, including 5 retransplantations, were performed at our institution. Adult patients who were able to self-complete the HRQL instruments and who had undergone > 2 years of follow-up after LTx were considered to be eligible for the analysis. Forty-four consecutive adult lung transplant recipients (mean [± SD] age, 44.8 ± 11.6 years) with > 2 years of follow-up after LTx (median follow-up time, 63 months; range, 24 to 84 months) qualified for the study. The following patients were excluded from the study: 18 patients who died during the first 2 years following LTx; 3 children; 1 patient who underwent LTx at our institution, but was followed up elsewhere after the procedure; 1 Vietnamese patient who required the presence of a translator and was, therefore, unable to self-complete the quality-of-life instruments; and 18 patients who had not completed 2 years of follow-up after LTx by the time the analyses began. Nineteen of the 44 patients (43%) were women. Of 44 lung transplant recipients, 14 (32%) had received a single lung transplant and 30 (68%) had received a bilateral sequential transplant. Indications for single LTx were pulmonary emphysema (10 patients), idiopathic pulmonary fibrosis (3 patients), and pulmonary fibrosis following paraquat intoxication (1 patient). Indications for bilateral LTx were pulmonary emphysema (14 patients), cystic fibrosis (9 patients), idiopathic pulmonary fibrosis (2 patients), primary pulmonary hypertension (2 patients), thromboembolic disease (1 patient), bronchiolitis obliterans (1 patient), and lymphangiomyomatosis (1 patient). Patients with pulmonary emphysema included the diagnoses of emphysema and {alpha}1-antitrypsin deficiency. The indication for single or bilateral LTx was based on the underlying disease, organ availability, and length of time spent on the waiting list. The protocol for the study was approved by the ethical committee of the institution. Informed consent was obtained before LTx from all patients.

Immunosuppression Protocol and Clinical Follow-up After LTx
Standard immunosuppression therapy including steroids, cyclosporine A, and azathioprine was used between 1993 and 1999. Details on the initial immunosuppression regimen were presented in another report.13 Over time, the immunosuppression protocol has been changed at our institution to include new drugs that became available. Consequently, cyclosporine A and azathioprine have been gradually replaced by tacrolimus and mycophenolate mofetil in the majority of our patients since 1998. Doses of tacrolimus were adjusted to maintain serum levels between 7 and 10 µg/L as determined by immunoassay (Abbot Laboratories; Abbot Park, IL). Doses of mycophenolate mofetil (25 to 35 mg/kg/d, with a maximum of 2 g/d) were adjusted to maintain trough levels of mycophenolic acid of > 1.0 mg/L, and were monitored by assay (Emit Syva Dade Assay; Behring Diagnostics, Inc; San Jose, CA).

Before LTx, patients were regularly seen at the outpatient clinic, with the exception of one patient who had undergone LTx as a salvage therapy after massive paraquat poisoning. Following hospital discharge after LTx, patients were scheduled for outpatient visits twice a month for clinical follow-up and therapeutic adjustments. Comprehensive clinical evaluations were performed at 3, 6, 9, and 12 months during the first year after LTx, and every 6 months thereafter, or whenever needed for the investigation of potential clinical complications, such as respiratory tract infections or acute rejection. These clinical evaluations included, among others, spirometry, 6MWT, and fiberoptic bronchoscopy with transbronchial biopsies. The diagnosis of BOS followed the criteria recommended by the International Society for Heart and Lung Transplantation.1415

HRQL and Pulmonary Function Assessments
HRQL was evaluated before and longitudinally after LTx using two different assays, the French-validated version of the St. George respiratory questionnaire (SGRQ) and a visual analog scale (VAS). These two assays were simultaneously performed throughout the follow-up period. The SGRQ contains questions addressing the following three main domains: respiratory symptoms; daily activities limitation; and disease impact on social and psychological functioning.1617 Partial scores corresponding to each domain of the questionnaire were calculated. A derived global score of well-being (ie, SGRQ global score), ranging from 0 to 100%, is calculated from the difference between the highest possible score and the sum of the scores obtained in the three domains contained in the instrument. For intraindividual comparisons, a 4-point (4%) change between scores obtained during follow-up is indicative of clinical relevance.16 For the purpose of the present study, a global score of 100% was defined as an optimal state of well-being. The scores were prospectively compiled for each patient each time the questionnaire was completed.

In addition to the SGRQ, patients were invited to self-assess their global state of well-being using a VAS. For this purpose, the EuroQoL VAS18 was calibrated from 0 to 100 mm, and was labeled respectively as the worst and best possible state of health imaginable. The results obtained by the SGRQ scores were compared to the rates of the VAS.

The spirometry (FEV1) and 6MWT findings were used as clinical indicators of outcome. The FEV1, 6MWT, SGRQ, and VAS were performed simultaneously within the year preceding LTx, then at 6 and at 12 months post-LTx, and then once a year thereafter for the analyses.

Statistical Analysis
The data obtained in this study presented a normal distribution. The results are shown as absolute values, percent predicted values (FEV1), the mean ± SEM, and median (range). Unpaired t tests were used to compare demographic data between single and bilateral lung transplant recipients. Repeated-measures analysis of variance was used to compare the FEV1 values, the 6MWT results, and the SGRQ global and domain-specific scores between patients following a single or a bilateral LTx. Post hoc Bonferroni analyses were applied to compare outcomes between the groups studied at each time point. Stratified analyses of variance with repeated measures were performed to compare (1) outcomes at 3 years after LTx in the overall group of patients between those who had undergone LTx before 1998 and patients transplanted after 1998, when changes in the immunosuppression protocol had been made and (2) between single and bilateral lung transplant recipients in the same time periods. The Pearson correlation test was used to compare the scores obtained from the simultaneous completion of the SGRQ with the measurement of FEV1, 6MWT results, and VAS score throughout the follow-up period. The data are presented as the overall correlation coefficients, irrespective of time after LTx. The relative risk and the 95% confidence interval were calculated to estimate the impact of BOS between single and double lung transplant recipients. The Fisher Exact Test was employed to compare the occurrence of infections and acute rejections following a single or a bilateral LTx. Adjustments were made to p values for the multiple comparisons. Data analyses were performed using a statistical software package (GraphPad PRISM, version 3.00 for Windows; GraphPad Software; San Diego, CA). A p value of ≤ 0.05 was considered to be statistically significant.


   Results
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After LTx, patients were followed up for a mean duration of 4.9 ± 1.2 years (median duration, 5.3 years; range, 2 to 7 years). The demographic characteristics and longitudinal results obtained for the FEV1, 6MWT, and SGRQ scores in the overall group of lung recipients and in patients with prior pulmonary emphysema following single or bilateral LTx are summarized in Table 1 . As shown, age at transplantation was not significantly different between groups, but the length of follow-up was significantly longer in recipients of a single lung graft. No statistically significant differences were observed in FEV1, 6MWT distance, and SGRQ scores before LTx comparing single and bilateral lung recipients, both in the overall group and in patients with prior pulmonary emphysema. Statistically significant improvements at 1 year post-LTx were observed on the FEV1, 6MWT, and SGRQ in all groups. Using the same outcome parameters, the long-term results were not statistically different when compared to the values at the 1-year follow-up after LTx in all groups. The long-term comparison of outcomes between single and bilateral lung transplant recipients is displayed in Figure 1 . It shows significantly lower FEV1 values in single lung transplant recipients, compared to those receiving a bilateral graft, most notably during the first 4 years after LTx. In contrast, the 6MWT distance and SGRQ scores were not significantly different at any time point throughout the follow-up period between the two groups of lung recipients. However, the absolute differences of the mean SGRQ values between bilateral and single graft patients exceeded the minimal clinically relevant difference of 4%, most notably beyond 4 years of follow-up. These findings were even more pronounced in the subset of patients with prior pulmonary emphysema.


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  		Table 1.. Demographic, Clinical, and Quality of Life Data From Single and Double Lung Transplant Recipients Irrespective of the Underlying Disease (All Patients) and in Patients With Prior Pulmonary Emphysema*

 


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  		Figure 1.. Time-dependent changes in FEV1 percent predicted (top, A), distance walked during the 6MWT [middle, B], and SGRQ scores (bottom, C) measured before LTx (Pre) and longitudinally after LTx in single lung transplant recipients (S) and double lung transplant recipients (D). The results at different time points are shown as the mean ± SEM. The number of patients at each time point, as indicated (numbers in bold correspond to single lung lung transplant recipients). * = p < 0.05 for FEV1, comparing S vs D; {ddagger} = p < 0.01 for FEV1, comparing S vs D.

 
Table 2 shows the correlation coefficients of the comparison between the SGRQ scores and the FEV1, 6MWT distance, and VAS scores in single and bilateral lung transplant recipients. Strong and significant correlations were found between the SGRQ and the VAS scores in both single and bilateral lung transplant recipients. Furthermore, significant correlations were obtained comparing the SGRQ to the FEV1 and to the 6MWT in double lung transplant recipients; however, the comparisons between the SGRQ and the FEV1, and between the SGRQ and the 6MWT, were not statistically significantly in single lung transplant recipients.


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  		Table 2.. Correlation Coefficients Between SGRQ Scores and VAS Scores, Percent Predicted FEV1, and 6MWT Distance From Single and Double Lung Transplant Recipients*

 
Figure 2 presents the scores of the three SGRQ domains, as well as the global scores. Graph bars represent the mean scores obtained during the follow-up of patients before and after undergoing single or bilateral LTx. The results are presented in the overall group (top, A) and in patients with prior pulmonary emphysema (bottom, B). Low SGRQ global scores are seen in the year preceding LTx; this result relates to the increased presence of respiratory symptoms and the impact of the disease in the performance of routine daily activities. After LTx, significant improvements on the three domains of the SGRQ were observed in patients following a double lung graft. However, patients receiving a single lung graft reported a lack of significant improvement in respiratory symptoms after LTx.



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  		Figure 2.. Time-dependent changes in SGRQ scores measured before LTx (Pre) and sequentially after single or double LTx. Each column represents the proportion corresponding to the three domains of the SGRQ and the global score. Results are shown for the overall group (top, A) and for patients with prior pulmonary emphysema (bottom, B). Comparing single lung transplant recipients to double lung transplant recipients at each time point throughout follow-up in graphs top, A, and bottom, B, the p value was not significant; p < 0.001 for the three SGRQ domains, comparing pre-LTx and any time point after LTx in double lung transplant recipients; p < 0.01 for two SGRQ domains (activities limitation and disease impact), comparing pre-LTx and any time point after LTx in single lung transplant recipients; the p value was not significant for one SGRQ domain (respiratory symptoms), comparing pre-LTx and any time point after LTx in single lung transplant recipients. See the legend of Figure 1 for abbreviations not used in the text.

 
Fourteen patients (32%) developed BOS during follow-up (median, 24.5 months post-LTx; range, 18 to 66 months post-LTx), 8 of whom were (57%) single lung transplant recipients and 6 of whom (20%) were bilateral lung recipients (relative risk, 2.86; 95% confidence interval, 1.22 to 6.67; p = 0.03). In approximately half of the patients developing BOS, evidence of BO was also found in transbronchial biopsy specimens. Six patients died during follow-up. Among them, three single lung transplant recipients developed BOS and died from progressive pulmonary dysfunction at 3, 7, and 8 years after undergoing LTx. Three bilateral lung recipients died at 3 years (two patients) and 6 years (one patient) after undergoing LTx as a result of acute cardiovascular disorders.

Respiratory tract infections and acute rejections, which could have a potential impact on pulmonary function decline and the development of BOS, were recorded throughout the follow-up period. Thirty-five severe, mainly pulmonary, viral and bacterial infections requiring hospital admission were diagnosed during the follow-up period. Twelve infections (0.86 per patient) occurred in single lung transplant recipients, and 23 infections (0.77 per patient) occurred in bilateral lung transplant recipients (difference was not significant). A total of 65 acute rejections (International Society for Heart and Lung Transplantation score, A2 or greater) were diagnosed during the follow-up period. There were 1.6 acute rejections per patient among single lung transplant recipients and 1.4 acute rejections per patient among bilateral lung transplant recipients (difference was not significant).


   Discussion
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study has focused on the long-term comparison of outcomes in single and bilateral lung transplant recipients, as assessed by FEV1, 6-min walking capacity, and self-estimated HRQL measures. As anticipated, lower FEV1 values were found in patients after a single lung graft. In contrast, the 6MWT results and SGRQ scores were not statistically different between single and bilateral lung transplant recipients. The correlation coefficients for the comparisons between the SGRQ and the FEV1, and between the SGRQ and the 6MWT, were not statistically relevant in unilateral lung transplant recipients, as opposed to bilateral lung recipients. This observation indicates that the patient’s perception of quality of life and ability to exercise after LTx transcend the actual functional capacity measured by spirometry, particularly in recipients of a single lung graft.

A number of studies4567 have shown that HRQL is recovered rapidly after LTx, a finding that is corroborated by our results. However, the improvement of HRQL may be limited in recipients of a single lung due to functional restrictions. In a literature review, we were able to identify only one previous study12 comparing quality of life between single and bilateral lung transplant recipients in a cross-sectional fashion. Using a generic tool, the authors of that study reported superior quality-of-life scores in double lung transplant recipients. However, the assignment of older lung transplant candidates with poorer quality of life to the single lung transplant group biased the results against these patients compared to double lung recipients. In contrast, our data show significant improvements in FEV1, 6MWT distance, and SGRQ scores in both single and bilateral lung transplant recipients after undergoing LTx, compared to values obtained before LTx. Moreover, the long-term results remained fairly unchanged throughout the follow-up period in the two groups of patients when compared to values at 1 year after LTx, and despite the lower FEV1 values resulting from the single lung graft.

No statistical differences were observed in HRQL, as opposed to functional status, over time between patients who had undergone single and bilateral lung grafts. The potential explanations for this observation could be the equivalent load of medication and medical visits required for the routine follow-up that are independent of the type of transplant procedure. Moreover, the rates of infections and acute rejection requiring additional hospitalizations, which may affect HRQL, were similar between groups. In addition, as shown in Figure 2, limitations in the performance of daily routine activities were similar between single and double lung transplant recipients, both in the overall group and in patients with native emphysema. On the other hand, substantially lower SGRQ scores were seen in single lung graft patients, particularly beyond 4 years of follow-up. These results largely surpass the minimal clinically relevant difference established for the SGRQ tool, thus indicating a reduced HRQL in these patients compared to bilateral recipients.

Improvements in HRQL after surgery may be limited by the onset of clinical complications. Progressive deterioration of HRQL after the development of BOS was reported in lung transplant recipients, most notably in the physical mobility domain.9 The risk of BOS has been reported to be higher in single lung transplant recipients,11 a finding that is consistent with our results. It is commonly agreed that a double lung graft confers better mechanical function and may also prevent immunologic mechanisms predisposing the patient to the development of BOS.11 In the absence of a functional native lung, the single allograft contributes to a maximum of 60% of the predicted FEV1. As such, recipients of a single lung transplant remain limited by a marginal functional reserve, unlike their counterparts who benefit from a bilateral graft. As a consequence, the impact of even a mild functional loss is greater in single lung graft recipients who may, therefore, reach the scoring threshold established for the diagnosis of BOS earlier than bilateral lung transplant recipients. As observed by Nathan and coworkers,19 criteria for the early diagnosis of BOS might induce an overestimation of incidence in single lung transplant recipients. Regardless, our data show that, although the recovery of respiratory symptoms and of FEV1 were limited in single lung transplant recipients, these limitations did not significantly affect their quality-of-life scores or walking capacity.

Survival after LTx has been reported20 to not be significantly superior in lung transplant recipients with prior pulmonary emphysema in comparison with other transplant indications. Conversely, better survival despite lesser functional recovery has been shown in patients with native pulmonary emphysema when compared to patients with prior idiopathic pulmonary fibrosis. In this latter comparison, the volume of the native emphysematous lung, albeit small, was thought to be contributive to the overall functional performance after a single lung graft.21

Greater functional improvement after LTx has been reported in bilateral lung transplant recipients with COPD when compared to recipients of a single LTx,22 although the available data are limited to the first 2 years following LTx. However, an assessment of HRQL in these patients has not been reported to date, though HRQL has been suggested for use as a complementary measure in the evaluation of lung transplant recipients who are affected by this condition.23 Our results show a significant improvement in HRQL after LTx, as assessed by the SGRQ scores. Moreover, positive changes were seen in the specific domains assessed by the SGRQ in both single and bilateral lung transplant recipients who were previously affected by emphysema. Indeed, these patients reported improvements in the performance of routine activities and a lesser impact of the disease after LTx. However, the recovery of respiratory symptoms was less marked in patients who had received a single lung graft, which was possibly determined by the native lung and by the partial FEV1 gain after LTx.

One interesting observation within our results is the comparison between the two methods of HRQL assessment. The SGRQ has been validated over the last several years in different language environments, and has been proven to be useful for the evaluation of patients who are severely affected by chronic respiratory disorders, notably COPD. The questions addressed by the questionnaire are, therefore, potentially suitable for lung transplant candidates. However, to our knowledge, the SGRQ has never been used before to assess quality of life after a transplant procedure or to evaluate the long-term follow-up of lung transplant recipients. Our cohort study allowed patients to serve as their own control subjects, thus decreasing the potential bias related to a tool that was not specifically constructed to assess quality of life successively after an intervention. The comparison between the SGRQ and the VAS scores showed a strong and statistically significant correlation; this result suggests a good association between the two instruments that is reassuring to those searching for appropriate assessment tools in these populations. When applied to the clinical setting, the two tests may prove to be complementary. While the VAS is a simple tool that rapidly yields information about the patient’s overall state of well-being, the SGRQ allows a deeper insight into three different components of the HRQL.

No significant differences between single and double lung transplant recipients were found for age, gender, FEV1, 6MWT distance, and quality of life status before LTx in our study. However, other confounding factors may have occurred. Despite a prospective design, the number of patients who were followed up in a single center remained small. Furthermore, changes to the protocol were gradually introduced during the patients’ follow-up to integrate newly acquired experience and therapeutic options. In fact, the majority of our single lung transplant recipients had undergone LTx during the first few years that transplantation became available at our institution. Nevertheless, the immunosuppression scheme used during the first 2 years after LTx was standardized for all patients included in this study, independent of the type of operation carried out. In addition, no statistical differences were found in stratified analyses of outcomes according to the "era" of transplantation which addressed the changes in the immunosuppression protocol, as previously described (data not shown). Clinical complications, such as infections or acute rejections that could have had an impact on outcome, were also not significantly different between groups.

In conclusion, this study shows that, despite poorer FEV1 recovery and increased risk of BOS, single lung transplant recipients can maintain long-term exercise capacity and quality-of-life scores approaching the performance of patients benefiting from a bilateral graft. Furthermore, the assessment of quality of life before transplantation may add to the decision-making process in choosing between single or double LTx by identifying patients who, despite severe functional impairment, surpass in other domains equally contributive to posttransplant recovery, such as muscular and psychosocial endurance. If confirmed by larger studies, our data obtained from lung transplant candidates with emphysema suggest that single lung transplant recipients can have long-term survival with a satisfactory HRQL, provided that assignment to the single lung graft is not reserved for patients with presumably the worst prognosis.


   Footnotes
 
Abbreviations: BOS = bronchiolitis obliterans syndrome; HRQL = health-related quality of life; LTx = lung transplantation; 6MWT = 6-min walk test; SGRQ = St. George respiratory questionnaire; VAS = visual analog scale

This study was partially supported by grants from the Swiss Wilsdorf Foundation and the Geneva Pulmonary League.

Received for publication October 26, 2004. Accepted for publication January 24, 2005.


   References
TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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