Whole genome analysis of hepatitis B virus before and during long-term therapy in chronic infected patients: Molecular characterization, impact on treatment and liver disease progression.

Hepatitis B virus (HBV) infection remains a serious public health concern worldwide despite the availability of an efficient vaccine and the major improvements in antiviral treatments. The aim of the present study is to analyze the mutational profile of the HBV whole genome in ETV non-responder chronic HBV patients, in order to investigate antiviral drug resistance, immune escape, and liver disease progression to Liver Cirrhosis (LC) or Hepatocellular Carcinoma (HCC). Blood samples were collected from five chronic hepatitis B patients. For each patient, two plasma samples were collected, before and during the treatment. Whole genome sequencing was performed using Sanger technology. Phylogenetic analysis comparing the studied sequences with reference ones was used for genotyping. The mutational profile was analyzed by comparison with the reference sequence M32138. Genotyping showed that the studied strains belong to subgenotypes D1, D7, and D8. The mutational analysis showed high genetic variability. In the RT region of the polymerase gene, 28 amino acid (aa) mutations were detected. The most significant mutations were the pattern rtL180M + rtS202G + rtM204V, which confer treatment resistance. In the S gene, 35 mutations were detected namely sP120T, sT126S, sG130R, sY134F, sS193L, sI195M, and sL216stop were previously described to lead to vaccine, immunotherapy, and/or diagnosis escape. In the C gene, 34 mutations were found. In particular, cG1764A, cC1766G/T, cT1768A, and cC1773T in the BCP; cG1896A and cG1899A in the precore region and cT12S, cE64D, cA80T, and cP130Q in the core region were associated with disease progression to LC and/or HCC. Other mutations were associated with viral replication increase including cT1753V, cG1764A/T, cC1766G/T, cT1768A, and cC1788G in the BCP as well as cG1896A and cG1899A in the precore region. In the X gene, 30 aa substitutions were detected, of which substitutions xT36D, xP46S, xA47T, xI88F, xA102V, xI127T, xK130M, xV131I, and xF132Y were previously described to lead to LC and/or HCC disease progression. In conclusion, our results show high genetic variability in the long-term treatment of chronic HBV patients causing several effects. This could contribute to guiding national efforts to optimize relevant HBV treatment management in order to achieve the global hepatitis elimination goal by 2030.

Overview of the epidemic history of Hepatitis C uncommon subtypes 2i and 4d in Tunisia and in the world.

The impressive improvements in qua therapy efficacy alone are not sufficient to substantially reduce the Hepatitis C Virus burden because of the usually very long asymptomatic phase of the infection. In turn, this renders prevention of infection of great importance. The value of learning how the virus has spread in the past is that this can provide clues as to what routes the virus likely spreads through today, which can feedback into prevention policy. In Tunisia, HCV subtypes 2i and 4d are minor circulating subtypes. Here, we applied a Bayesian Markov Chain Monte Carlo method for visualization of spatial and temporal spread of HCV-2i and 4d in Tunisia and some other countries in the world. Our analysis included sequences retrieved from Genbank and isolated from several countries in the world; 21 HCV-NS5B subtype 2i genome sequences obtained during the period 2002-2020 and 206 HCV-NS5B-4d sequences detected between 2000 and 2019. Phylogenetic analysis revealed that two geographical clusters could be identified in HCV-2i tree with two clearly distinguished clusters in HCV-4d Tree. The estimated time for the most recent common ancestor suggested that current HCV-2i strains emerged in 1963 [1930, 1995] and current HCV-4d strains emerged in 1992 [1988, 1996] in Tunisia and other countries from the world investigated in the present study.

SARS-CoV2 RT-PCR assays: In vitro comparison of 4 WHO approved protocols on clinical specimens and its implications for real laboratory practice through variant emergence.

INTRODUCTION: RT-PCR testing on nasopharyngeal swabs is a key component in the COVID-19 fighting, provided to use sensitive and specific SARS-CoV2 genome targets. In this study, we aimed to evaluate and to compare 4 widely used WHO approved RT-PCR protocols on real clinical specimens, to decrypt the reasons of the diverging results and to propose recommendations for the choice of the genome targets. METHODS: 260 nasopharyngeal samples were randomly selected among the samples tested between Week-16, 2020 and week-16 2021, in the Institut Pasteur de Tunis, Tunisia, one of the referent laboratories of COVID-19 in Tunisia. All samples were tested by Charité, Berlin protocol (singleplex envelop (E) and singleplex RNA-dependent RNA polymerase (RdRp)), Hong Kong Universiy, China protocol (singleplex nucleoprotein (N) and singleplex Open reading frame Orf1b), commercial test DAAN Gene® (using the CDC China protocol), (triplex N, Orf1ab with internal control) and Institut Pasteur Paris protocol (IPP) (triplex IP2(nsp9) and IP4 (nsp12) with internal control). For IPP, a selection from samples positive by IP2 but negative with IP4 was re-tested by exactly the same protocol but this time in singleplex. New results were described and analyzed. RESULTS: In vitro analysis showed discordant results in 29.2% of cases (76 out of 260). The most discordant protocol is DAAN Gene® due to the false positive late signals with N target. Discordant results between the two protocol's targets are more frequent when viral load are low (high Ct values). Our results demonstrated that the multiplexing has worsened the sensitivity of the IP4 target. CONCLUSION: We provide concise recommendations for the choice of the genome targets, the interpretation of the results and the alarm signals which makes suspect a gene mutation.

Sequencing Using a Two-Step Strategy Reveals High Genetic Diversity in the S Gene of SARS-CoV-2 after a High-Transmission Period in Tunis, Tunisia.

Recent efforts have reported numerous variants that influence severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral characteristics, including pathogenicity, transmission rate, and detectability by molecular tests. Whole-genome sequencing based on next-generation sequencing technologies is the method of choice to identify all viral variants; however, the resources needed to use these techniques for a representative number of specimens remain limited in many low- and middle-income countries. To decrease sequencing costs, we developed a primer set allowing partial sequences to be generated in the viral S gene, enabling rapid detection of numerous variants of concern (VOCs) and variants of interest (VOIs); whole-genome sequencing is then performed on a selection of viruses based on partial sequencing results. Two hundred one nasopharyngeal specimens collected during the decreasing phase of a high-transmission COVID-19 wave in Tunisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allow the detection of first introductions in the country of already-known VOCs and VOIs, as well as other variants that have interesting genomic mutations and need to be kept under surveillance. IMPORTANCE The method of choice for SARS-CoV-2 variant detection is whole-genome sequencing using next-generation sequencing (NGS) technologies. Resources for this technology remain limited in many low- and middle-income countries, where it is not possible to perform whole-genome sequencing for representative numbers of SARS-CoV-2-positive cases. In the present work, we developed a novel strategy based on a first partial Sanger screening in the S gene, which includes key mutations of the already known VOCs and VOIs, for rapid identification of these VOCs and VOIs and to help better select specimens that need to be sequenced by NGS technologies. The second step consists of whole-genome sequencing to allow a holistic view of all variants within the selected viral strains and confirm the initial classification of the strains based on partial S gene sequencing.

Distribution of HCV Genotypes Among People Who Inject Drugs in Tunisia: New Evidence for Scaling Up Prevention and Treatment Toward National Elimination Goal.

Little is known about the distribution of hepatitis C virus (HCV) genotypes among people who inject drugs (PWID) in North African countries, including Tunisia. This study aims to describe HCV genotypes circulating among Tunisian PWID. A cross-sectional study was conducted, and 128 HCV-positive PWID were recruited between 2018 and 2019 from community-based harm reduction centers. After informed consent, sociodemographic characteristics and risk behavior data were obtained using an interviewer-administrated questionnaire. Blood samples were collected for further serological and molecular testing. Overall, five women and 123 men were included. The median age was 39.5 years. The majority of PWID (56.3%) had less than a secondary level of education, were single (57%), were unemployed (65.6%), were incarcerated at least once (93.0%), and had a history of residency in at least one foreign country (50.8%). During the previous 12 months, 82.0% reported having reused syringes at least once, 43.8% shared syringes at least once, while 56.2% had at least one unprotected sexual relation, and 28.1% had more than two different sexual partners. Tattooing was reported among 60.2%. All positive results for HCV-infection by rapid testing were confirmed by enzyme-linked immunosorbent assay (ELISA). HCV-RNA was detectable in 79.7%. Genotyping showed a predominance of genotype 1 (52%) followed by genotype 3 (34%) and genotype 4 (10%). Four patients (4%) had an intergenotype mixed infection. Subtyping showed the presence of six different HCV subtypes as follows: 1a (53.2%), 1b (6.4%), 3a (33.0%), 4a (3.2%), and 4d (4.3%). This is the first study describing circulating HCV genotypes among PWID in Tunisia. The distribution of HCV genotypes is distinct from the general population with a predominance of subtypes 1a and 3a. These findings can be used to guide national efforts aiming to optimize the access of PWID to relevant HCV prevention and treatment measures including pangenotypic regimens for patients infected with HCV genotype 3.

Evidence of Circulation of Several HAV Genetic Variants and Emergence of Potential Antigenic Variants in an Endemo-Epidemic Country before Vaccine Introduction.

Similar to several other countries in the world, the epidemiology of hepatitis A virus changed from high to intermediate endemicity level in Tunisia, which led to the occurrence of outbreaks. This study aimed to determine the genetic and antigenic variability of HAV strains circulating in Tunisia during the last few years. Genotyping using complete VP1 gene and VP1-2A junction confirmed the predominance of genotype IA, with co-circulation of several genetic and antigenic variants. Phylogenetic analysis including Tunisian and strains from other regions of the world showed the presence of at least two IA-variants within IA subgenotype. Amino-acid analysis showed several mutations in or close to epitope regions in the VP1-region. This study provides a baseline on the genetic and antigenic variability of HAV circulating strains before the introduction of vaccination into the national immunization schedule.

Focus on hepatitis C virus genotype distribution in Tunisia prior to elimination: a 16-year retrospective study.

With the introduction of direct-acting antiviral treatment (DAA), Tunisia has committed to achieving the international goal of eliminating viral hepatitis. Because the specific DAA prescribed depends on viral genotype, viral genotyping remains of great importance. The aim of the present study was to outline the trends in the distribution of HCV genotypes from 2002 to 2017 in the Tunisian general population in order to guide authorities towards the most appropriate therapeutic strategies for preventing HCV infection. A total of 2532 blood samples were collected over a 16-year period and from all regions of Tunisia. Genotyping showed that genotype 1 (subtype 1b) was the most prevalent genotype in the country (n = 2012; 79.5%), followed by genotype 2 (n = 339; 13.3%). Genotypes 3, 4 and 5 were detected in 4.8%, 2.2% and 0.1% of the country's population, respectively. Mixed infections with different HCV genotypes were detected in 0.1% of the population (one case each of genotypes 1b + 4, 1b + 2 and 2 + 4). Interestingly, a significant increase in genotypes 2, 3 and 4 was observed over time (p = 0.03). Sixteen different subtypes were detected over the study period, most of which were subtypes of genotype 2, and some of these subtypes appeared to be new. Patients infected with genotypes 1a, 3 and 4 were significantly younger than those infected with genotypes 1b and 2 (p < 0.01). Furthermore, genotypes 1b and 2 were detected more often in women than men, while genotypes 1a and 3 were detected mostly in men (P < 0.01). Our study confirms a large predominance of genotype1/subtype1b in Tunisia and shows a significant increase in the prevalence of other genotypes over time. These findings reinforce the need for an additional HCV genotype survey to improve the design of treatment strategies in Tunisia.

Hepatitis C virus epidemiology in Central-West Tunisia: a population-based cross-sectional study.

This study aimed to assess the seroprevalence, viraemia and genotype distribution of hepatitis C virus (HCV) in a region in Central-West Tunisia. A door-to-door cross-sectional study was conducted on a randomly selected sample. A total of 3178 individuals aged 5 to 74 years and members of 935 families were investigated. Seroprevalence of HCV was assessed using ELISA tests. The viral load was determined by real-time RT-PCR, and HCV genotyping was conducted by amplification and sequencing in the NS5b genomic region. The global prevalence of HCV antibodies was 3.32% (95% confidence interval [CI]: 2.72-4.00). It was significantly higher in women: 4.47% vs. 2.16% in men, p = 0.001. Seroprevalence increased with age, and the highest rates were found in the 50- to 59-year-old age group (12.90%, 95% CI: 9.45-16.86), suggesting a cohort effect with very low contribution of intrafamilial transmission. Genotyping showed a predominance of subtype 1b (84.6%), with cocirculation of subtypes 2c (9.6%), 1a (1.9%), 1d (1.9%) and 2k (1.9%), similar to the previously reported genotype distribution in Tunisia and with no genetic clusters specific to the study region. These results indicate a higher endemicity of HCV infection when compared to the previously reported nationwide surveillance data. This study provides valuable data that can contribute to current strategies to eliminate hepatitis C.