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Supplemental Digital Content 1 Detailed description of Patients and Methods
of the study.
Patients and Methods
The case records of all the patients who underwent kidney transplantation from
January 1985 to December 2008 and followed up at the Nephrology unit of Queen
Mary Hospital were reviewed. Among these kidney transplant recipients, those
tested positive for hepatitis B surface antigen (HBsAg+ve) were included. Patients
who tested positive for anti-HCV or HCV RNA, or had significant alcohol consumption,
were excluded from analysis. In our centre, patients with histological evidence of
advanced liver cirrhosis were excluded from kidney transplantation. The control
subjects were HBsAg-ve age- and sex-matched kidney transplant recipients chosen in
a 1:1 ratio to the HBsAg+ counterparts. In addition, these control subjects received
their kidney allograft in the same year as their HBsAg+ counterparts (+/- 1 year if no
matched control transplanted in the same year was found).
Prophylactic immunosuppression consisted of prednisolone with cyclosporine or
tacrolimus, with or without mycophenolate mofetil. None received thymoglobulin or
anti-CD3 antibody induction. Maintainence immunosuppression consisted of
low-dose prednisolone and cyclosporine or tacrolimus, with or without
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mycophenolate mofetil. 4% of lamivudine-treated subjects had steroid withdrawal.
All of the lamivudine-treated patients were on maintenance corticosteroid.
Patients were seen every 2-4 weeks for 6 months, then every 4-6 weeks for 6 months,
then every 6-8 weeks in the second year, then every 8-12 weeks. At each follow-up
visits clinical events were recorded, and the levels of alanine aminotransferase (ALT),
HBV DNA, and serum creatinine were measured. Chronic hepatitis was defined as
abnormal ALT levels (ALT > 58 iu/L for men and 45 iu/L for women) lasting more than
6 months. Circulating HBV DNA level was measured using quantitative LightCycler
real-time PCR, with a sensitivity limit of 100 copies/mL [10]. All HBsAg+ve subjects
underwent liver stiffness measurement using transient elastography (Fibroscan R).
Cirrhosis based on FibroScanR result was defined as a stiffness score higher than
11kPa, which correlated with a METAVIR stage 4 fibrosis [26].
Lamivudine was available in our unit since January 1996, adefovir since December
2004, entecavir since September 2005 and telbivudine since November 2007.
Lamivudine was given as first-line treatment for hepatitis B before the availability of
entecavir and telbivudine, and treatment was started when HBV DNA level was at or
above 105 copies/mL, or in the range of 102 to 105 copies/mL if there was
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concomitant aminotransferase abnormalities (ALT >58 iu/L for men and >48 iu/L for
women). Lamivudine was started at 100mg daily and dosage was adjusted if there is
allograft dysfunction. Salvage anti-viral treatment with adefovir or entecavir was
given to patients who developed lamivudine resistance. Resistance to anti-viral
treatment was defined as resurgence of HBV DNA to 105 copies/mL or higher in at
least two consecutive blood samples following a period of suppression to <1.00 x10 2
copies/mL for at least one month during treatment. YMDD mutations in
lamivudine-resistant HBV variants were characterized by the INNO-LiPA HBV DR line
probe assay (Innogenetics N.V., Ghent, Belgium) according to the manufacturer’s
instructions. Salvage therapy with either adefovir or entecavir was initiated when
reappearance of serum HBV DNA levels exceeds 105 copies/mL after successive viral
suppression irrespective of the ALT levels. Rescue anti-virals were also commenced if
the reappearance of serum HBV DNA levels were between 102 to 105 copies/mL with
concomitant abnormal aminotransferase levels (ALT >58 iu/L for men and >45 iu/L for
women). Adefovir was administered at 10mg daily as salvage therapy. Entecavir was
started at 0.5mg daily for treatment-naïve individuals and 1 mg daily for
lamivudine-resistant subjects. Dosages of adefovir or entecavir were again adjusted
with respect to the patient’s allograft function. We avoided adefovir in patients with
serum creatinine >150 μmol/L or creatinine clearance <40 mL/min. Patients who
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developed lamivudine resistance before the availability of adefovir or entecavir were
maintained on lamivudine. These subjects were switched to salvage therapy only if
the aforementioned criteria for rescue therapy were met after the availability of
adefovir and entecavir.
Statistical analysis
Continuous variables are expressed as mean±SD, unless otherwise specified.
Longitudinal data between groups was compared using Wilcoxon rank-sum test.
Intra-group comparison of paired variables was performed by Wilcoxon signed ranks
test. HBV DNA values were log-transformed before analysis. Patient survival was
estimated by multivariate Cox regression using sex, age, donor type, year of
transplantation, CMV status, dialysis vintage, diabetes mellitus, primary cause of
renal failure and episodes of acute rejection as covariates. The Cox regression
model was further adjusted for the baseline serum HBV DNA and ALT values when
comparing patient survival between subjects who had or had not developed
lamivudine resistance. FibroscanR scores were compared by analysis of covariance
with HBV DNA and ALT values included as covariates. Data analyses were generated
by SPSS for windows version 17 and Prism version 5 and P values were two-sided. In
some analyses, patients were segregated according to transplantation before or after
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1996, i.e. before and after the availability of anti-viral treatments.