Mariacarmela Solmone

Use of Massively Parallel Ultradeep Pyrosequencing To Characterize the Genetic Diversity of Hepatitis B Virus in Drug-Resistant and Drug-Naive Patients and To Detect Minor Variants in Reverse Transcriptase and Hepatitis B S Antigen

ABSTRACT 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.

Contamination by hepatitis B and C viruses in the dialysis setting.

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.

Role of hepatitis C virus (HCV) viremia and HCV genotype in the immune recovery from highly active antiretroviral therapy in a cohort of antiretroviral-naive HIV-infected individuals.

BACKGROUND The roles of hepatitis C virus (HCV) viremia and HCV genotype in the immune response to highly active antiretroviral therapy (HAART) are poorly understood. Our aim was to assess the CD4+ cell count recovery after HAART in human immunodeficiency virus (HIV)-infected patients with HCV viremia and HIV-infected patients who tested negative for HCV antibody (HCV-Ab). We also aimed to assess whether the response to HAART in these patients varied according to HCV genotype. METHODS The analysis focused on 1219 HCV-Ab-negative patients and 284 HCV-viremic patients from a cohort of HIV-infected subjects that includes persons who were antiretroviral naive before initiating HAART after cohort enrollment. HCV RNA load and HCV genotype were determined in plasma specimens obtained and stored during the 6-month period preceding the initiation of HAART. RESULTS The chance of achieving a CD4+ cell count increase of > or = 100 cells/microL from the pre-HAART level tended to be poorer in HCV-viremic patients than in patients who tested negative for HCV-Ab (adjusted relative hazard [RH], 0.82; 95% confidence interval [CI], 0.66-1.01; P = .06). In contrast, a comparison of patients who had a HCV RNA load >1 x 10(6) IU/mL with patients who had a HCV RNA load of 5-1 x 10(6) IU/mL revealed no significant association between HCV RNA load and achievement of an increased CD4+ cell count (adjusted RH, 0.97; 95% CI, 0.75-1.27; P = .83). There was no clear association between HCV genotype and the probability of achieving a CD4+ cell count increase. CONCLUSIONS An association between the presence of HCV-Ab and immune reconstitution after HAART has been shown elsewhere. Results of our large, prospective study support a direct role of HCV viremia in the CD4+ cell count response to HAART. Moreover, our results underline the fact that, in individuals coinfected with HIV and HCV, the goal of treating HCV infection is to eradicate HCV, to both slow the rate of HCV progression and limit potential interference with the response to HAART.

Epidemiology of viral hepatitis infections in an area of southern Italy with high incidence rates of liver cancer

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.

Simple and Reliable Method for Detection and Genotyping of Hepatitis C Virus RNA in Dried Blood Spots Stored at Room Temperature

ABSTRACT 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.

Extent of HCV NS3 protease variability and resistance-associated mutations assessed by next generation sequencing in HCV monoinfected and HIV/HCV coinfected patients

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.

Trends in HIV prevalence among pregnant women in Italy, 1994 to 2002.

Objective: The aim of the study was to analyze the trends in HIV prevalence among childbearing women through unlinked anonymous testing of newborns. Methods: Dried-blood specimens, residual to routine metabolic screening of newborns, were collected in Italy during the last quarter of each year, from 1994 to 2002. These samples were tested anonymously for HIV antibodies. Results: Of 844,311 specimens tested, 703 (0.83/1000; 95% confidence interval, 0.76-0.88) resulted as HIV antibody-reactive. Prevalence was 1.05 per 1000 in 1994 and decreased significantly over the following 3 years, reaching 0.60 per 1000 in 1997. Thereafter, a seroprevalence increase was recorded with a figure of 1.01 per 1000 in 1999. A decrease was again observed in 2001 and in 2002, when the seroprevalence rate was significantly lower than that recorded in 1994. During the entire study period, higher prevalence rates were observed in northern Italian regions, where the highest AIDS incidence rates are recorded, and in metropolitan compared with nonmetropolitan areas. The estimated number of children born each year to an HIV-positive mother ranged from 313 to 546. Conclusion: The number of children born to an HIV-positive mother calls for continued efforts to prevent vertical transmission of the infection.

A sensitive direct sequencing assay based on nested PCR for the detection of HBV polymerase and surface glycoprotein mutations.

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.

Effect of HIV co-infection on mutation patterns of HBV in patients with lamivudine-resistant chronic hepatitis B.

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.

Role of hepatitis B virus, hepatitis D virus and other determinants on suppression of hepatitis C viraemia in HIV infected patients with chronic HCV infection: A longitudinal evaluation

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<0.0001). Furthermore, HBV coinfection per se was also found to be independently associated with negative HCV viraemia (AOR: 18.5, 95% CI 2.4–143.5, p<0.0001). HBsAg-positive patients with negative HCV viraemia maintained undetectable levels over time. Our results support a direct role of HBV and HDV coinfections in suppressing HCV viraemia in HIV infected patients. This effect is durable over time, and is not influenced by HAART including anti-HBV drugs.