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HCV quasispecies variability represents the background for the selection of mutations and for the development of drug resistance. Natural aminoacid changes in NS3, associated with reduced protease inhibitor susceptibility, have been observed in treatment-naïve patients. Massively parallel sequencing has been used to analyze NS3 quasispecies in patients infected with HCV genotype 1, naive to anti-HCV treatment, with/without HIV-coinfection, to establish the genetic heterogeneity and the presence of amino acid substitutions at positions responsible for drug resistance. Genomes carrying substitutions represented either predominant or minority components of viral quasispecies, and were observed in 85.7% of patients. Multiple substitutions, frequently associated on the same haplotype, were observed in 46.4% of patients. High resistance combinations were not detected, neither on the same genome, nor in the whole quasispecies. Heterogeneity of HCV NS3 was lower in HIV-coinfected as compared to HCV-monoinfected patients, but factors underlying this difference remain to be established. Although the relevance of naturally occurring mutations with respect of resistance development and probability of success of direct acting antivirals is questioned, UDPS may be beneficial to help understanding viral dynamics, providing high resolution view of viral diversity.

Antiviral therapy has revolutionized treatment of chronic HBV infections. First generation compounds, lamivudine and adefovir, displayed a high rate of treatment failures, and have been replaced by more potent compounds with high genetic barrier to resistance. However, the evolution of the virus towards resistance due the use of first generation compounds may still provide useful information for a better management of current antivirals. A single center sequence database including 705 HBV reverse transcriptase sequences from patients failing antiviral treatments (2002-2012) has been statistically analyzed to highlight viral evolution in relationship to the use of antiviral compounds and to their associations/sequencing in those years. The influence of viral genotypes and polymorphisms on resistance-related mutational patterns was also investigated. This study documents how, after the first years of antiviral therapy, the use of adefovir as an add-on strategy allowed a consistent reduction treatment failures. It also documents the effects of the initial misuse of entecavir in lamivudine experienced patients. In the latest years, the correct use of entecavir and the introduction of tenofovir allowed further curbing of resistance-related treatment failures, which virtually disappeared. Furthermore, the study allows a better understanding of how viral genotype (A vs D) conditions specific mutational pathways to resistance against lamivudine and entecavir, and demonstrates that the use of adefovir in lamivudine experienced patients is associated to peculiar mutational patterns, in particular A181V + F/Y221L. Despite some concern may arise for patients previously treated with lamivudine/adefovir, in sequence or combination, where the virus may have developed a lower genetic barrier against resistance to tenofovir, the outlook of antiviral treatment of HBV infection should be quite optimistic.

Drug resistance is a crucial problem emerging frequently during treatment of hepatitis B, resulting in treatment failure and progression of liver damage. A direct sequencing method based on a nested PCR was established to detect mutations in samples with low viral load. Primers were designed to obtain an amplicon encompassing the A, B, C, D and E functional domains of HBV polymerase. Fifty-five samples were tested, containing HBV DNA ranging from 19 to 1700 IU/mL. Sixteen samples were also tested by the commercially available assay INNO-LiPA HBV DR v2. Sequencing was successful for all samples, and mutations were detected in 24/55 (43.6%). When used in parallel with DR v2, concordant results were found in 8/16 samples. In the eight discordant cases, four were resolved by sequencing and not by DR v2, and four had differences in the mutation patterns. Direct sequencing was able to show pol mutations not revealed by DR v2, such as rtV214A, rtQ215H/S, and rtM250V. Genotype and env variations were also established. This highly sensitive sequencing protocol, providing valuable sequencing data from samples with a low viral load, is suitable for detection of mutations at the very early signs of failure of treatment, thereby allowing to maximize the success of early treatment changes.

A retrospective review was performed comparing lamivudine-resistance mutation patterns between patients infected with hepatitis B virus (HBV) with or without human immunodeficiency virus (HIV) co-infection. Medical records that included a genotypic test of patients infected with HBV and treated with lamivudine as the only anti-HBV drug were reviewed. Pol gene mutations were assessed by direct sequencing of the reverse transcriptase fragment 125–213 aa. Eighty-nine patients infected with HBV (29 co-infected with HIV) with rtM204V or rtM204I mutations were included. Multiple mutations associated with the YMDD motif were observed in 33 (55%) of 60 patients infected with HBV only and in 28 (96.6%) of patients co-infected with HIV/HBV. In this latter group, the prevalence of the rtV173L + rtL180M + rtM204V triple mutation was 31% versus a prevalence of 3.4% observed among patients infected with HBV only. All patients with the triple mutational pattern showed sE164D + sI195M changes in the envelope gene. Multivariate analysis demonstrated that HIV co-infection (adjusted OR 11.2, 95% CI 2.0–61.0) and HBV genotype A (adjusted OR 7.2, 95% CI 1.5–34.8) were the only independent variables associated with the chance of harboring rtM204V. Patients with HBV genotype A or HIV co-infection were more likely to harbor the rtM204V mutation. Patients co-infected with HIV showed multiple mutations more frequently, including the triple mutation that may elicit a vaccine escape phenotype. Among patients co-infected with HIV/HBV, strict HBV DNA monitoring is essential to detect treatment failure and adapt therapy to avoid limitations of future therapeutic options or the emergence of a public health threat. J. Med. Virol. 81:1151–1156, 2009. © 2009 Wiley-Liss, Inc.

CMV viral load quantitation is a powerful tool to assist clinicians in making accurate diagnoses, managing post-transplant CMV disease and monitoring antiviral therapy. The aim of this study was to evaluate the performance of Affigene ® CMV Trender for CMV viral load determination used in combination with a non-dedicated nucleic acid extraction system (BioRobot MDx) for high-throughput routine. Linearity, reproducibility and sensitivity were examined. Clinical samples were used to compare results obtained with the Affigene ® CMV Trender, with an “in house” nested PCR used for routine diagnosis and with pp65 antigenemia. The results indicated that the test is linear in the range of 1.81–5.18 Log copies/ml and that sensitivity is 77 copies/ml. The concordance of the Affigene ® CMV Trender with nested PCR was high, ( k = 0.91, IC 95% = 0.82–1.00), whereas a substantial concordance with pp65 antigenemia was observed ( k = 0.64, IC 95% = 0.54–0.73). In conclusion, combined use of a non-dedicated automated nucleic acid extraction method with the Affigene ® CMV Trender results in an accurate high throughput system, suitable for routine laboratory monitoring of CMV infection.

The role of hepatitis B virus (HBV) or hepatitis D virus (HDV) coinfections as determinants of hepatitis C virus (HCV) suppression in the setting of HIV-HCV coinfection are poorly understood. Our aim was to assess whether HCV viral replication may be affected by HBV or HDV coinfection in the setting of immunodeficiency driven by HIV.Among the 138 enrolled patients 28(20.3%) tested HCV RNA negative and 110 (79.7%) tested HCV RNA negative. The HCV RNA negative patients showed an higher rate of HBsAg positivity compared with those tested HCVRNA positive [12/28 (42.9%) and 5/110 (4.6%), respectively]. Patients with HCV-HBV-HDV coinfection had the highest chance of having an undetectable HCV RNA (adjusted odds ratio (AOR): 92.0, 95% confidence interval (CI) 5.7-1483.5, p

We compared two commercial assays for HBV DNA quantitation, Versant HBV 3.0, System 340 (bDNA; Bayer Diagnostics) and COBAS AmpliPrep-COBAS TaqMan HBV Test (TaqMan; Roche Diagnostics). Analytical sensitivity, calculated on WHO International Standard, predicted 95% detection rate at 11.4 and 520.2IU/ml for TaqMan and bDNA, respectively. Specificity, established on 50 blood donor samples, was 100% and 84% for TaqMan and bDNA, respectively. When using clinical samples, HBV DNA was detected by TaqMan in 21/55 samples negative to bDNA. Mean values of HBV DNA obtained with bDNA were higher than those obtained with TaqMan (4.09log(10)+/-1.90 versus 3.39log(10)+/-2.41, p0.001), and 24.4% of samples showed differences in viral load values0.5log(10), without association with HBV genotype. There was a good correlation for HBV DNA concentrations measured by the two assays (r=0.94; p0.001) within the overlapping range, and the distribution of results with respect to relevant clinical threshold recently confirmed (20,000 and 2000IU/ml) was similar. Approximately 50% of samples with low HBV DNA, appreciated by TaqMan but not by bDNA, were successfully sequenced in pol region, where drug resistance mutations are located.

Hepatitis C virus (HCV) variability is mainly attributed to the ability of the virus to respond to host immune pressure, acting as a driving force for the evolution of quasispecies. This study was aimed at studying the changes in HVR-1 heterogeneity and the evolution of HCV quasispecies in HIV/HCV-coinfected patients according to the pattern of response to highly active antiretroviral therapy (HAART). Sixteen HIV/HCV-coinfected patients harbouring HCV genotype 1 and who had been on HAART for at least 1 year, 8 showing increasing CD4+T-cell counts (immunological responders) and 8 showing a stable or decreasing CD4+ T-cell counts (immunological non-responders), were selected from a prospective cohort study. After 1 year of HAART, 11 patients showed HIV viral load 10 cp/ml (virological responders), and 5 showed HIV viral load above this value (virological non-responders). Plasma samples, collected before starting therapy and after 1 year of HAART, underwent clonal sequence analysis for HVR-1 region of HCV. Non-synonymous/synonymous substitutions ratio (Ka/Ks), aminoacidic complexity (normalized Shannon entropy) and diversity (p-distance), were considered as parameters of quasispecies heterogeneity. After 1 year of HAART, heterogeneity of HVR-1 quasispecies significantly decreased in virological non-responders, whereas the heterogeneity tended to increase in virological responders. The differences in the evolution were less stringent, when considering immunological response. On the other hand, profound qualitative modifications of HVR-1 quasispecies were observed only in patients with both immunological and virological HAART response. On the whole, these findings suggest that, in patients undergoing HAART, the extent of HCV variability and the evolution of HVR-1 quasispecies is influenced by the pattern of response to antiretroviral therapy.

IntroductionIn western countries the transmission of hepatitis B virus (HBV) transmission through multi-patients lancing devices has been inferred since early '90s, however no study has ever provided biological evidence which directly link these device with HBV cross-infection. Here we present results of an outbreak investigation which could associate, by molecular techniques, the use of lancing device on multiple patients with HBV transmission in an Italian oncohematology unit.MethodsThe outbreak investigation was designed as a retrospective cohort study to identify all potential cases. All cases identified were eventually confirmed through molecular epidemiology techniques. Audit of personnel including extensive review of infection control measures and reviewing personnel's tests for HBV was done identify transmission route.ResultsBetween 4 May 2006 and 21 February 2007, six incident cases of HBV infection were reported among 162 patients admitted in the oncohematology. The subsequent molecular instigation proved that 3 out 6 incident cases and one prevalent cases (already infected with HBV at the admission) represented a monophyletic cluster of infection. The eventual environmental investigation found that an identical HBV viral strain was present on a multi-patients lancing device in use in the unit and the inferential analysis showed a statistically significant association between undergoing lancing procedures and the infection.DiscussionThis investigation provide molecular evidence to link a HBV infection cluster to multi-patients lancing device and highlights that patients undergoing capillary blood sampling by non-disposable lancing device may face an unacceptable increased risk of HBV infection. Therefore we believe that multi-patients lancing devices should be banned from healthcare settings and replace with disposable safety lancets that permanently retract to prevent the use of the same device on multiple patients. The use of non-disposable lancing devices should be restricted to individual use at patients' home.

We describe a simple, sensitive, and reproducible method for using whole blood collected onto filter paper (dried blood spots) for detection and genotyping of hepatitis C virus RNA that can be useful in large field studies, particularly in settings where collection, preparation, storage, and shipment of samples at controlled temperature can be difficult.

Direct population sequencing and reverse hybridization (line probe assay [LiPA])-based methods are the most common methods for detecting hepatitis B virus (HBV) drug resistance mutations, although only mutations present in viral quasispecies with a prevalence of ≥20% can be detected by sequencing, and only known mutations are detected by LiPA. Massively parallel ultradeep pyrosequencing (UDPS; GS FLX platform) was used to analyze HBV quasispecies in reverse transcriptase (RT) and hepatitis B S antigen (HBsAg) from five drug-naive patients and eight drug-resistant patients. Eight primer pairs were used to obtain partially overlapping amplicons, covering the RT gene from codons 1 to 288 and the complete overlapping HBsAg sequence. A 1% mutation frequency was selected as the cutoff based on an error rate estimated on plasmid DNA. This technology enabled simultaneous analysis of between 2,852 and 18,016 clonally amplified fragments from each patient. The results indicate that UDPS has a relative sensitivity much higher than both direct sequencing and LiPA. In addition, the UDPS results are quantitative, allowing establishment of the relative frequency of both known mutations and novel substitutions. Some of the detected RT substitutions led to changes also in HBsAg. On the whole, genotype D presented a higher heterogeneity than genotype A. Considering the high quantity of information that can be provided by a single test from one patient, the short turnaround time, the information on substitution frequency, and the detection of rare variants, there are strong advantages conferred by UDPS, and the new method could play a relevant role in the clinical management of HBV infection and therapy.

Southern Italy shows the highest rates of liver cancer for Europe, mainly related to infection with hepatitis viruses. We thus described incidence rates of liver cancer and investigated prevalence and determinants of HCV and HBV infections in 4496 individuals randomly selected from the general population of the province of Naples. 7.5% was infected with HCV and 27.6% with HBV (2.2% was HBsAg-positive). Prevalence of both infections increased with age, 23.2% of those aged 65 years or older was HCV-positive and 47.9% were HBV-positive. Intravenous drug use (odds ratio (OR) = 16.4 for anti-HCV and 4.7 for anti-HBc), history of blood transfusions (OR = 2.8 and 1.5, respectively) and surgery, and household contacts with infected people (OR = 2.1 and 1.6, respectively) increased risks for both infections. Sexual intercourse with HCV-positive individuals conveyed a 3-fold higher risk of HCV infection. This study quantified the spread of HCV and HBV in the population of southern Italy heavily affected by liver cancer.

Intra-hepatic levels of mRNA for IFN-a, IFN-y, IFN type 1 receptor (IFNAR-1) and PKR were determined in hepatitis C virus (HCV)/HIV co-infected and HCV mono-infected patients. In co-infected patients, IFN-a mRNA was upregulated and correlated with HIV-1 viraemia. IFN-y, IF-NAR-1 and PKR mRNA were detected in mono-infected, but not in co-infected patients. These findings suggest that in HCV/HIV co-infected individuals the ability to respond to IFN-a is impaired, probably because of the absence of its receptor.

Background/Aims HVR-1 quasispecies composition and evolution were investigated in patients chronically infected with genotype 1b HCV, treated with PEG-IFN α2b or STD-IFN α2b plus RBV. Methods HVR-1 heterogeneity was assessed by calculating nucleotidic complexity, diversity, synonymous (S) and non-synonymous (NS) substitutions at baseline, after 4 weeks of therapy ( T 1) and at follow-up ( T 18). Evolution of viral quasispecies was analysed by constructing phylogenetic trees. Results No correlation of baseline viremia with heterogeneity was observed. Nucleotidic complexity was lower in patients showing early virological response, and tended to be inversely correlated to viral load decline at 4 weeks of treatment. In the majority of SR, profound changes of quasispecies composition occurred during 4 weeks of treatment, while in NR virtually no major changes of pre-therapy variants were observed. Relapse showed both patterns of quasispecies evolution. Virus quasispecies after follow-up was similar to that found at T 1 in both Relapsers and NR patients. Conclusions Baseline parameters of HVR-1 heterogeneity seem to be involved in the early response to treatment, and early response is associated with profound variations in the HVR-1 quasispecies. Viral quasispecies surviving early therapeutic pressure are most likely able to give rise to either virus rebound or persistence at T 18.

Background: Hepatitis virus infections continue to be a major concern in the dialysis setting. We studied levels of hepatitis B surface antigen (HBsAg) and hepatitis C virus (HCV) RNA contamination in dialysis units to better define the role of the dialysis environment and machines in the nosocomial transmission of hepatitis viruses. Methods: Possible contamination by hepatitis B virus (HBV) and HCV was studied by collecting environmental samples in 3 dialysis units located in Rome, Italy. Samples and controls were tested for HBsAg by a microparticle enzyme immunoassay, and for HCV RNA, by qualitative transcription-mediated amplification assay. Results: HCV RNA and HBsAg were detected in 1 of 64 (1.6%) and 1 of 64 samples (1.6%), respectively. The only HCV RNA-positive sample was found in 1 dialysis unit on the external surface of the dialysate (inlet-outlet) connector of a dialysis machine used for HCV-negative patients. The only HBsAg-positive sample was found in another dialysis unit on the internal surface of the blood pressure monitor cuff of a dialysis bed dedicated for HBsAg-positive patients. Conclusion: A segregation policy for HBsAg-positive patients is a necessary measure despite its high cost-effectiveness; we found HBsAg contamination in the segregated HBV-infected room. Conversely, the finding of HCV RNA contamination on a dialysis machine not dedicated to HCV-positive patients suggests that isolation of HCV-infected dialysis patients and use of dedicated machines are unjustified. Major attention should be given to strict adherence to infection control measures in the dialysis setting.

HCV and interferon: viral strategies for evading innate defence mechanisms in the virus-host battle

May HCV VL or HCV genotype affect the immune response after starting HAART in HCV/HIV co-infected subjects? G Antonueci I A Cozzi-Lepri 2, E Girardi 1, MR Capobianchi ], M Solmone 1, F Carletti 1, M. Puoti 3, A d ' ~ i o Monforte 4, G Ippollto, and the ICoNA Study Group INMI S p a l l ~ , Rome, I; Royal Free and Univ. College, London, LIK2; University of Brescia3; University o f Milan 4, and the ICoNA Study Group Baekgreund and aim: Little is known about whether HCV viremia or HCV genotype alter the progression o f HIV infection in HAA T-treated co-infected patients. The identification of differeat categories o f coinfected subjects with different cell recoveries after HAART could be very useful to define the optimum time to start anti-HCV as well as antiHIV treatments. We assessed the CD4 response to HAART among coinfected subjects enrolled in the I.Co.N.A. cohort according to either HCV viremia or genotype. Methods: Prospective, observational study o f 319 HIV/HCV co-infected subjects with HCV RNA, and HCV genotype determined from plasma stored within 6 months prior to HAART initiation. Time to CD4 count response was defined as the time to achieve a rise o f 200 ceUs/ml. Results: Of the 319 patients included, 61.4% were IDUs and 77.0% males. Regarding HCV infection, subjects were grouped as follows: 40 (12.5%) had HCV RNA _200 cell/ml [adjusted RH=0.78 per lOgl0 higher HCV RNA, 95% CI: 0.63-0.97;/3=0.02]. Moreover, the CD4+ cell count response after HAART tended to he poorer in HCV genotype 3 compared to subjects with genotype 1 (adjusted RH of CD4+ cell count response=0.71, 95% CI:0.48-1.06, p=0.09). Conelasion: Our data carried strong evidence that different levels of plasma HCV RNA are predictive o f different response o f CD4+ following initiation o f HAART in subjects who responded virologically to HAART within the first 24 weeks. This finding is compatible with the hypothesis e r a modification o f CD4+ cells kinetics driven by HCV.

The prognostic role of GB virus type C (GBV-C) viraemia in HIV-infected subjects treated with highly active antiretroviral therapy (HAART) is still undefined, The aim of this analysis is to assess the relationship between GBV-C infection and response to antiretroviral therapy among HIV-infected subjects initiating HAART when antiretroviral-naive. A prospective, observational study of 400 HIV-infected patients with measurements of GBV-C RNA, hepatitis C virus (HCV) antibodies and HCV RNA determined from plasma stored prior to HAART initiation, Time to virological (achieving HIV RNA ≤500 copies/ml) and immunological success (a CD4+ count increase of ≥200cells/μl), and the time to virological relapse (confirmed HIV RNA >500 copies/ml) were assessed by Kaplan-Meier methods and Cox proportional hazard regression model. Of the subjects, 117 (29.3%) were GBV-C positive and, overall, 351 (87.8%) patients achieved virological success, After controlling for a number of confounders including HCV RNA, GBV-C viraemic patients experienced a significantly lower risk of HIV rebound than those who were GBV-C negative [relative hazard (RH)=0.56, 95% CI: 0.34–0.93, P=0.03], Conversely, the probability of achieving initial virological success or CD4+ count response after HAART did not differ between GBV-C-negative and -positive subjects, These results suggest that GBV-C coinfection may play a role in determining the rate of HIV rebound possibly by competing with HIV replication after HIV load has been successfully suppressed by HAART.

The aim of the study was to characterize the interferon sensitivity determining region (ISDR) mutation pattern and its changes at 4 weeks of treatment in a population of patients infected with hepatitis C virus (HCV) genotype 1b receiving standard or PEG-IFN plus ribavirin (RBV), to find possible early correlates of therapy outcome.Forty-five patients with chronic hepatitis due to HCV 1b were treated by PEG-IFN-α2b (n=23) or IFN-α2b (n=22) plus RBV 1000–1200 mg/day. They were classified 24 weeks after stopping therapy as sustained responders (SR), relapsers (REL) or non-responders (NR). Sixteen patients were SR, 12 REL and 17 NR. ISDR mutations were evaluated by direct sequencing at baseline in all and after 4 weeks in patients with detectable viraemia (n=30).The frequency of the three ISDR types was 26.7% wild-type, 64.4% intermediate-type and 8.9% mutant-type, without significant difference in their frequency in SR, REL and NR, independent of IFN formulation. Average numbers of mutations in SR, REL and NR were 1.88 ±0.54, 1.33 ±0.33 and 0.94 ±0.25, respectively, P>0.05. The baseline number of ISDR mutations was not related to the extent of viral load decline in the first month of therapy. Sequence analysis of ISDR region performed 4 weeks after starting therapy revealed qualitative or quantitative changes of ISDR sequence in only seven patients, without correlation with response.Thus, in our patients the baseline pattern of ISDR was unrelated to treatment outcome. Selection towards a dominant IFN-resistant strain did not occur under standard or PEG-IFN plus RBV.