Tenofovir disoproxil fumarate (TDF) has demonstrated high antiviral efficacy in treatment-naive patients with chronic hepatitis B virus (HBV) infection but experience in nucleoside/nucleotide analogue (NA)-experienced patients is limited. In this retrospective multicenter study we therefore assessed the long-term efficacy of TDF monotherapy in patients with prior failure or resistance to different NA treatments. Criteria for inclusion were HBV DNA levels >4.0 log 10 copies/mL at the start and a minimum period of TDF therapy for at least 6 months. In all, 131 patients (mean age 42 ؎ 12 years, 95 male, 65% hepatitis B e antigen [HBeAg]-positive) were eligible. Pretreatment consisted of either monotherapy with lamivudine (LAM; n ‫؍‬ 18), adefovir (ADV; n ‫؍‬ 8), and sequential LAM-ADV therapy (n ‫؍‬ 73), or add-on combination therapy with both drugs (n ‫؍‬ 29). Three patients had failed entecavir therapy. Resistance analysis in 113 of the 131 patients revealed genotypic LAM and ADV resistance in 62% and 19% of patients, respectively. The mean HBV DNA level at TDF baseline was 7.6 ؎ 1.5 log 10 copies/mL. The overall cumulative proportion of patients achieving HBV DNA levels <400 copies/mL was 79% after a mean treatment duration of 23 months (range, 6-60). Although LAM resistance did not influence the antiviral efficacy of TDF, the presence of ADV resistance impaired TDF efficacy (100% versus 52% probability of HBV DNA <400 copies/mL, respectively). However, virologic breakthrough was not observed in any of the patients during the entire observation period. Loss of HBeAg occurred in 24% of patients and HBsAg loss occurred in 3%. No significant adverse events were noticed during TDF monotherapy. Conclusion: TDF monotherapy induced a potent and long-lasting antiviral response in NA-experienced patients with previous treatment failure. Our data may have implications for current add-on strategies. (HEPATOLOGY 2010;51: 73-80.)A major limitation of nucleoside and nucleotide analogues (NA) in hepatitis B virus (HBV) therapy is the selection of HBV resistance variants, which can lead to a rebound in HBV replication and exacerbation of HBV-related disease. HBV polymerase gene variants that mediate HBV resistance are known to confer cross-resistance to other NAs. Today, increasing numbers of patients have experienced NA treatment failure, mostly to lamivudine (LAM) or adefovir dipivoxil (ADV), which poses a growing problem for antiviral treatment. Add-on combination therapy with ADV plus LAM was shown to be effective in these patients but only when initiated during the early stages of resistance development. 1 Tenofovir disoproxil fumarate (TDF) was licensed in 2008 for the treatment of HBV infections in Europe and the Abbreviations: ADV, adefovir dipivoxil; ALT, alanine aminotransferase; HBeAg, hepatitis B e antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; human immunodeficiency virus; LAM, lamivudine; NA, nucleoside/nucleotide analogue; PCR, polymerase chain reaction; TDF, tenofovir disoproxil fumarate. ...

Incomplete virological response to adefovir dipivoxil (ADV) has been observed in patients with lamivudine-resistant hepatitis B virus (HBV) infection and may be associated with developing resistance and disease progression. We therefore investigated whether the efficacy of viral suppression could be improved by replacing ADV with tenofovir disoproxil fumarate (TDF). Twenty patients with chronic HBV infection (18 HBeAg؉), viral breakthrough during lamivudine therapy, and persistent viral replication (>10 4 copies/mL) after 15 months of ADV monotherapy (range 4-28 months) were treated with TDF 300 mg daily and were retrospectively analyzed. A screening for nucleoside/nucleotide analogue resistance mutations within the HBV polymerase gene was performed in all patients by direct sequencing. Within a median of 3.5 months, application of TDF led to undetectable HBV DNA in 19 of 20 patients, as demonstrated by suppression of HBV DNA below the detection limit of 400 copies/mL. Initially elevated ALT levels had normalized in 10 of 14 patients by the end of follow-up (median 12 months, range 3-24 months). Four patients lost HBeAg, after 3, 4, 5, and 16 months, and one patient seroconverted to anti-HBs after 16 months of TDF therapy. Lamivudine-associated mutations (rtV173L, rtL180M, rtM204V/I) could be detected in 6 patients at baseline of TDF, but this obviously did not influence the response. ADV-resistant mutations were not detected. No side effects were reported. In conclusion, these preliminary observations strongly suggest that TDF might be a highly effective rescue drug for HBV-infected patients with altered responsiveness to treatment with lamivudine and ADV. (HEPATOLOGY 2006;44:318-325.) See Editorial on Page 309 C omplete and sustained suppression of viral replication remains the most important goal in the treatment of patients with chronic hepatitis B virus (HBV) infection. 1,2 In this respect, the introduction of the nucleoside analogue lamivudine and the nucleotide analogue adefovir dipivoxil (ADV) largely improved the outcome in these patients, also by preventing hepatitic decompensation or the development of hepatocellular carcinoma. [3][4][5] These compounds also have a place when interferon-alpha based therapy is contraindicated, as in patients with acute liver failure, decompensated cirrhosis, or extrahepatic manifestations.However, a major disadvantage of nucleoside/nucleotide analogues is that resistant hepatitis B virus mutants develop during long-term treatment, limiting the efficacy of these analogues in suppressing viral replication. As demonstrated in recent studies, approximately 70% of patients receiving lamivudine therapy for more than 4 years suffer the emergence of lamivudine-resistant HBV Abbreviations: ADV, adefovir dipivoxil; HBV, hepatitis B virus; TDF, tenofovir disoproxil fumarate; HBeAg, hepatitis B virus e antigen. From the

The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis of Staphylococcus epidermidis biomaterial-related infections. Using biofilm-producing S. epidermidis 1457 and transposon Tn917 carried on plasmid pTV1ts, we isolated nine isogenic biofilm-negative transposon mutants. Transduction by S. epidermidis phage 71 was used to prove the genetic linkage of transposon insertions and altered phenotypes. Mapping of the different transposon insertions by Southern hybridization and pulsed-field gel electrophoresis indicated that these were inserted in four unlinked genetic loci. According to their phenotypes, including quantitative differences in biofilm production in different growth media, in the amount of the polysaccharide intercellular adhesin (PIA) produced, in the hemagglutination titers, and in the altered colony morphology, the mutants could be separated into four phenotypic classes corresponding with the genetic classes. Synthesis of PIA was not detectable with class I and II mutants, whereas the amount of PIA produced reflected the residual degree of biofilm production of class III and IV mutants in different growth media. Chromosomal DNA flanking the transposon insertions of five class I mutants was cloned and sequenced, and the insertions were mapped to different locations of icaADBC, representing the synthetic genes for PIA. Expression of icaADBC from a xylose-dependent promoter in the different isogenic mutant classes reconstituted biofilm production in all mutants. In a Northern blot analysis no icaADBC-specific transcripts were observed in RNA isolated from mutants of classes II, III, and IV. Apparently, in addition to icaADBC, three other gene loci have a direct or indirect regulatory influence on expression of the synthetic genes for PIA on the level of transcription.

Viral differences among lamivudine resistant hepatitis B (HBV) genotypes have not been yet investigated. Therefore, we analyzed the characteristics of these viral strains in vivo. Fortyone patients carrying lamivudine resistant HBV were enrolled. Twenty-six patients (63%) carried resistant HBV genotype A (group A) and 15 patients (37%) carried resistant HBV genotype D (group D). The rate of reverse transcriptase 204I mutants was significantly higher in group D (67%) compared with group A (19%), whereas rt204V mutants (81% in group A vs 33% in group D; P ‫؍‬ .006) and rt180M mutants (81% in group A vs 40% in group D, P ‫؍‬ .015) prevailed in group A. The median time of shift from rt204I to rt204V mutants was significantly shorter in group A (4 months in group A, >12 months in group D, P < .001). Additional resistance associated mutations were detected exclusively in group D (P ‫؍‬ .004). In a multivariate analysis, HBV genotype (P ‫؍‬ .039) and pretreatment serum HBV DNA (P ‫؍‬ .001) were independently associated with emerging rt204I or rt204V mutants, respectively. Serum HBV copy numbers after emergence of resistance were higher in group A (mean log 10 6.99 copies/ml; range 3-9) compared with group D (mean log 10 6.1 copies/ml; range 3.3-8; P ‫؍‬ .04). There was no difference between both groups regarding core promoter/precore mutations, viral turnover, and number of flares or disease progression during follow-up. T he emergence of drug resistant hepatitis B virus (HBV) during lamivudine treatment for chronic hepatitis B is a major problem with an incidence of 14 -36% after 1 year of treatment. [1][2][3][4] This frequency increases to 38%, 49%, and 66% after 2, 3, and 4 years of treatment, respectively. 5-7 Lamivudine resistant HBV is characterized by amino acid variations in the reverse transcriptase domain of the HBV polymerase. In particular, an exchange of the methionine within the YMDD motif by an isoleucine or a valin (rtM204I/V mutants) is associated with lamivudine resistance. Breakthrough of these drug-resistant HBV mutants leads to a viral rebound to baseline levels, 8,9 to a decrease in the rate of loss of hepatitis B e antigen (HBeAg), 10 a high rate of relapses of serum alanine transaminase (ALT) levels, 11,12 and worsening liver histology. 13 Therefore, the emergence of viral resistance is one of the critical issues in the longterm outcome of patients treated for chronic hepatitis B. On the other hand, lamivudine resistant HBV is considered to have reduced viral fitness due to less replication efficiency in vitro 14 and lower ALT levels in vivo as compared with baseline levels. 4,15,16 This led to the recommendation to continue lamivudine treatment despite the emergence of resistant variants as long as benefit to the patient is maintained. 17 Taken together, it would be useful to identify factors which are associated with a better Abbreviations: HBV, hepatitis B virus; HBeAg, hepatitis B e antigen; ALT, serum alanine transaminase; PCR, polymerase chain reaction; CP, core promoter. From the

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Inspection of the amino acid sequence of the non-structural region of the hepatitis C virus (HCV) gene product reveals a sequence of 14 amino acids, Argl487-Arg-Gly-Arg-Thr-Gly-Arg-Gly-Arg-Ai-gGly-Ile-Tyr-Arg1500, located in the non-structural protein, NS3. This sequence is highly similar to the inhibitory site of the heat-stable inhibitor of CAMP-dependent protein kinase (PKA) and to the autophosphorylation site in the hinge region of the PKA type I1 regulatory domain. A synthetic peptide that corresponds to the HCV sequence above and a set of shorter analogues act as competitive inhibitors of PKA. A 43.5-kDa fragment of NS3 that consists of residues 1189-1525 of the HCV polyprotein inhibits PKA in a similar range to the investigated synthetic peptides. In contrast to the short peptides, which show competitive inhibition, HCV-polyprotein-( 11 89-1525) influences PKA in a mixed-inhibition-type manner. A possible mechanism explaining these differences is the formation of complexes that consist of the protein substrate, the enzyme and the HCV-polyprotein-(l189-1525). Binding studies with PKA and Keywords: non-structural protein 3 ; protein phosphorylation ; CAMP-dependent protein kinase; chaperone.Hepatitis C virus (HCV) was identified as the major cause of non-A, non-B liver infections [ l ] that result in more than 50% of the investigated cases of chronic liver disease that lead to cirrhosis and hepatocellular carcinoma [2]. The mechanism by which HCV gene products exert their pathogenic effects is not understood. However, there are some indications that the interaction of viral antigens with intracellular proteins, after infection of the cell, could result in viral diseases of chronic nature. Since protein phosphorylation is an important step in the regulation of cell metabolism, differentiation and tumor promotion, the disturbance of this post-translational modification of proteins is often considered to be a possible mechanism for pathogenic pathways that are not understood. Examples in the literature include a number of viral antigens that interfere with intraCorrespondence to P.

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