Hepatitis B, D, and C, and HIV serology were assessed by commercial immunoassays. Serum HBV-DNA was measured by a hybridization method (Hybrid Capture® System HBV DNA assay; Digene Corporation, Gaithesburg, MD, USA) with a sensitivity of 5 pg/ml. Representative stored serum samples were later studied by a quantitative real-time polymerase chain reaction (PCR) assay, using TaqMan probes (HBV PCR kit, Abbott Laboratories, Hamburg, Germany) in an ABI PRISM 7000 Sequence Detection Systems. This method has a detection range of 4 UI/ml to 3 × 107 UI/ml. Nucleic acid extraction was performed with the QIAamp DNA Blood Mini Kit, (Qiagen, Hilden, Germany).
Resistant mutants to lamivudine were studied by the Inno-LiPA HBV DR assay (Innogenetics N.V., Ghent, Belgium).
A man with no known allergies, ex-smoker and a moderate consumer of alcohol, was diagnosed in 1975 at the age of 26 with asymptomatic chronic hepatitis B infection. In 1996 he required hospitalization for abdominal pain and diarrhea. Abdominal CT showed superior mesenteric vein thrombosis that resolved spontaneously 1 year later. The study of thrombotic risk was negative. Laboratory analyses were normal except for mild liver enzyme alterations. Viral hepatitis serology was as follows: HBsAg positive, anti-HBs negative, anti-HBcIgM negative, anti-HBcIgG positive, HBeAg positive, antiHBe negative, HBV-DNA negative (by hybridization), anti-HCV negative, and anti-Delta negative. The patient was monitored on an outpatient basis with periodic abdominal ultrasound studies and laboratory analyses.
In 1998 analytical monitoring detected elevated transaminases [ALT 1.1 ukat/l (normal <0.73 ukat/l) and AST 1.34 ukat/l (normal <0.5 ukat/l)], thrombocytopenia at 43 000 cells/mm3 and viral DNA at 144 ng/ml. The diagnosis of hepatic cirrhosis was established on radiological and analytical criteria. Abdominal ultrasound showed a small liver, morphologically consistent with chronic disease and no space occupying lesions. Portal vein diameter was increased and the patient presented splenomegaly without ascites. Interferon treatment was not contemplated because of the patient's thrombocytopenia and decompensated liver disease. In December 1998 the patient was admitted to the hospital for hepatic encephalopathy.
In March 1999 he started lamivudine at 100 mg daily within the program of compassionate use, with a favorable initial clinical and serological response. Viral DNA tested negative at the fourth month of treatment. The response persisted up to April 2000, at which time YMDD lamivudine-resistant mutants emerged. The patient experienced upper gastric tract hemorrhage secondary to grade II esophageal varices; Child B score was 7 points.
In June 2002 the patient was started on adefovir therapy at 10 mg/day combined with lamivudine, within the compassionate treatment program. The antiviral response was progressive but slow. HBV-DNA was still positive by hybridization after 8 months, and according to our hospital policy he could not be accepted as a liver transplant recipient. The patient presented ascites and a first episode of spontaneous bacterial peritonitis (SBP) related to Streptococcus bovis. He suffered hepatic encephalopathy and progressive worsening of Child-Pugh status.
In January 2003 he was classified as Child Pugh C. Additionally, the patient presented kidney failure and refractory ascites, which was treated with draining paracentesis and albumin. He could not be discharged home and remained in the hospital on the liver transplant waiting list. However, he was not eligible for transplantation because of detectable HBV-DNA. Laboratory tests on February 3, 2003 were as follows: creatinine 99 umol/l, urea 5.3 mmol/l, Na+ 135 mmol/l, K+ 5.2 mmol/l, albumin 27 g/l, glucose 4.9 mmol/l, AST 1.05 ukat/l, ALT 0.5 ukat/l, total bilirubin 171 umol/l, GGT 0.27 ukat/l, alkaline phosphatase 1.7 ukat/l, leukocytes 2280 cells/mm3 (N 54.5%, L 26.8%, M 13.6%), erythrocytes 2980 × 106/l, Hb 10.3 g/l, hematocrit 29.9%, Platelets 37 200 cells/mm3, INR 2, fibrinogen 1.08 g/l.
The patient was started on tenofovir at 245 mg/day within the program of compassionate use on February 27, 2003. HBV-DNA measured 12 pg/ml and lamivudine-resistant mutations (L180M and M204V/I) were detected in serum. Administration of tenofovir while continuing with oral adefovir 10 mg daily and oral lamivudine 100 mg daily resulted in a fast decline of hepatitis B viremia out of the limit of detection by hybridation test in 4 weeks with negative results on March 24, 2003. At that time the patient was accepted as a liver transplant candidate. Table 1 shows the results of serology, ALT and DNA-HBV determinations.
Table 1. Analytical and therapeutical evolution before liver transplantation.
|DNA-VBH ng/ml†||144||40||−||1039||577||674||591|| ||66||13|| ||12||−|| |
|DNA-VBH UI/ml‡|| || || || || || || || || || ||20 241||15 914||771||315|
|YMDD Lam-R mutants|| || ||+||+|| || || || ||+|| || ||+|| || |
|Lamivudine 100 mg/day|| ||Start||Yes||Yes||Yes||Yes||Yes||Yes||Yes||Yes||Yes||Yes||Yes||Yes|
|Adefovir 10 mg/day|| || || || || || || ||Start||Yes||Yes||Yes||Yes||Yes||Yes|
|Tenofovir 235 mg/day|| || || || || || || || || || || ||Start||Yes||Yes|
The patient underwent transplantation on April 19, 2003 without complications. The donor was 42 years old and HBV markers were negative. The initial immunosuppressive treatment used was cyclosporine and basiliximab. The anti-HBV prophylactic regimen consisted of intramuscular HBIg and lamivudine plus adefovir.
Outcome after LT has been excellent. At 21 months the patient has an active, normal life and he has not needed additional hospitalizations. He is now receiving immunosuppressive treatment with cyclosporine and mycophenolate mofetil and anti-HBV prophylaxis. Liver enzymes are normal, HBsAg remains negative and anti-HBs levels are 200 IU/l. Analytical results in the most recent post-LT follow-up (December 2004) were as follows are: creatinine 127 umol/l, urea 6.3 mmol/l, albumin 46 g/l, glucose 5.7 mmol/l, AST 0.63 ukat/l, ALT 0.72 ukat/l, total bilirubin 28 umol/l, GGT 0.63 ukat/l, alkaline phosphatase 1.1 ukat/l.