A 30-year-old male patient initiated a hemodialysis treatment in August 1995. At that time, he was negative for hepatitis B surface antigen (HBsAg) as well as for anti-HBc and anti-HIV antibodies. The patient received three blood transfusions during the first trimester of 1996 and developed a posttransfusional hepatitis. He became HBsAg and anti-HIV positive and remained positive for both markers in all routine tests performed between 1996 and 2002. Data of other tests, such as serum transaminases, CD4 levels, and HIV load, performed in external laboratories between 1995 and 2002, were available. Resistance to different HIV therapies was evaluated by the medical staff based on HIV load and CD4 levels. HIV treatment started in April 1996, with a combination of zidovudine (AZT) and didanosine (ddI). In November 1996, this treatment was modified to a combination of AZT and lamivudine which was maintained until March 1997, when treatment was once again changed to triple therapy with AZT, lamivudine and invirase. In May 1999, high HIV load (3.1 x 105 copies/ml) was observed, indicating HIV resistance. At that time the patient started an anti-tuberculosis treatment with rifampicin. However, due to interaction of antiretroviral drugs with rifampicin, the administration of antiretroviral drugs was discontinued. This resulted in a decrease of CD4 counts to 156 cells/ml, while elevated levels of HIV persisted at the end of 1999. Antiretroviral treatment resumed in January 2000, composed of four drugs, namely ddI, stavudine, nevirapine (NVP) and nelfinavir (NFV). As a consequence, CD4 cell counts increased noticeably, and HIV RNA became undetectable. Unfortunately, resistance to therapy was observed during the second semester of 2001, when HIV load reached 1.2 x 105 copies/ml and CD4 counts decreased to 120 cells/ml. The treatment was then replaced by another including lamivudine associated to AZT, NVP and NFV. No HIV resistance was observed until the end of the follow-up (November 2002). The patient died of renal failure at the end of 2003.
Three serum samples collected in March 1999, August 2000 and November 2002, were available for HBV DNA analysis. Sera were submitted to DNA extraction by the phenol-chloroform method after treatment with proteinase K, as described previously. HBV pre-S/S genomic region was amplified by PCR using sense primer PS1 (5'-CCATATTCTTGGGAACAAGA-3', nt 2826-2845) and a mix of antisense primers S2 (5'-GGGTTTAAATGTATACCCAAAGA-3', nt 841-819) and S22 (5'-GTATTTAAATGGATACCCACAGA-3', nt 841-819), able to amplify all HBV genotypes. PCR assays were performed under the following conditions: 94 C, 30 s; 52 C, 1 min; 72 C, 2 min; 35 cycles, followed by a final elongation of 7 min at 72 C. Amplification products (10 muL) were loaded on a 2% agarose gel, electrophoresed, stained with ethidium bromide, and visualized under UV light.
Quantification of HBV DNA was performed by endpoint dilution. DNA samples were diluted to a tenfold series up to 10-8 dilution. Each dilution was submitted to PCR with oligonucleotide pairs PS1-PS2, designed in the pre-S region and C1-C2 (core region), as described previously. Serial dilutions and PCR assays for HBV DNA quantification were performed in triplicate. The sensitivity of the method has been estimated to about 100 copies per PCR reaction, equivalent to 104 HBV genome copies per milliliter of serum. Quantitative results were estimated by dividing 104 copies/ml by the last positive dilution.
Pre-S/S PCR products were cloned into pCRII plasmid vector using TA cloning kit (Invitrogen, San Diego, CA). For nucleotide sequencing, recombinant plasmid DNAs were purified by a commercially available kit (Plasmid midi kit, Qiagen, Hilden, Germany). Nucleotide sequences were determined using the Cy5 auto read sequencing kit (Amersham Biosciences, Little Chalfont, UK) with M13 universal and reverse primers, as well as internal, HBV specific primers. For direct sequencing (S region), PCR products were extracted from low melting agarose gels (Qiaquick gel extraction kit, Qiagen). Sequencing reactions with HBV specific primers were done using the thermo sequenase Cy5 dye terminator sequencing kit and analyzed on an ALFexpress automated sequencer (Amersham Biosciences). Independent plus and minus strand sequencing was completed.
Nucleotide sequences were aligned using PILEUP (Wisconsin Sequences Analysis Package GCG, Madison, WI). A phylogenetic tree was generated by neighbour-joining analysis of genetic distances, using the TREECON software package for Windows. HBV sequences available from the GenBank database (accession numbers AY090458, M57663, U55220-U55222, J02201 and X51970) were used for the construction of a phylogenetic tree.