Dynamic of SARS-CoV-2 variants circulation in Tunisian pediatric population, during successive waves, from March 2020 to September 2022.

The emergence of SARS-CoV-2 variants has led to several cases among children. However, limited information is available from North African countries. This study describes the SARS-CoV-2 strains circulating in Tunisian pediatric population during successive waves. A total of 447 complete sequences were obtained from individuals aged from 13 days to 18 years, between March 2020 and September 2022: 369 sequences generated during this study and 78 ones, available in GISAID, previously obtained from Tunisian pediatric patients. These sequences were compared with 354 and 274 ones obtained from Tunisian adults and a global dataset, respectively. The variant circulation dynamics of predominant variants were investigated during the study period using maximum-likelihood phylogenetic analysis. Among the studied population, adolescents were the predominant age group, comprising 55.26% of cases. Twenty-three lineages were identified; seven of which were not previously reported in Tunisia. Phylogenetic analysis showed a close relationship between the sequences from Tunisian adults and children. The connections of sequences from other countries were variable according to variants: close relationships were observed for Alpha, B1.160 and Omicron variants, while independent Tunisian clusters were observed for Delta and B.1.177 lineages. These findings highlight the pivotal role of children in virus transmission and underscore the impact of vaccination on virus spread. Vaccination of children, with booster doses, may be considered for better management of future emergences.

Development and evaluation of an easy to use real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus.

West Nile Virus (WNV) causes a serious public health concern in many countries around the world. Virus detection in pathological samples is a key component of WNV infection diagnostic, classically performed by real-time PCR. In outbreak situation, rapid detection of the virus, in peripheral laboratories or at point of care, is crucial to guide decision makers and for the establishment of adequate action plans to prevent virus dissemination. Here, we evaluate a Loop-mediated isothermal amplification (LAMP) tool for WNV detection. Amplifications were performed comparatively on extracted viral RNA and on crude samples using a classical thermal cycler and a portable device (pebble device). qRT-PCR was used as gold standard and two sets of urine samples (n = 62 and n = 74) were used to evaluate the retained amplification protocols and assess their sensitivity and specificity. RT-LAMP on RNA extracts and crude samples showed a sensitivity of 90 % and 87 %, respectively. The specificity was 100 % for extracts and 97 % for crude samples. Using the device, the RT-LAMP on extracted RNA was comparable to the gold standard results (100 % sensitivity and specificity) and it was a bit lower on crude samples (65 % sensitivity and 94 % specificity). These results show that RT-LAMP is an efficient technique to detect WNV. RT-LAMP provides a rapid, sensitive, high-throughput and portable tool for accurate WNV detection and has potentials to facilitate diagnostic and surveillance efforts both in the laboratory and in the field, especially in developing countries.

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-CoV-2 Lineage A.27: New Data from African Countries and Dynamics in the Context of the COVID-19 Pandemic.

SARS-CoV-2 is constantly evolving with lineages emerging and others eclipsing. Some lineages have an important epidemiological impact and are known as variants of interest (VOIs), variants under monitoring (VUMs) or variants of concern (VOCs). Lineage A.27 was first defined as a VUM since it holds mutations of concern. Here, we report additional lineage A.27 data and sequences from five African countries and describe the molecular characteristics, and the genetic history of this lineage worldwide. Based on the new sequences investigated, the most recent ancestor (tMRCA) of lineage A.27 was estimated to be from April 2020 from Niger. It then spread to Europe and other parts of the world with a peak observed between February and April 2021. The detection rate of A.27 then decreased with only a few cases reported during summer 2021. The phylogenetic analysis revealed many sub-lineages. Among them, one was defined by the substitution Q677H in the spike (S) gene, one was defined by the substitution D358N in the nucleoprotein (N) gene and one was defined by the substitution A2143V in the ORF1b gene. This work highlights the importance of molecular characterization and the timely submission of sequences to correctly describe the circulation of particular strains in order to be proactive in monitoring the pandemic.

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.

Molecular Epidemiology of SARS-CoV-2 in Tunisia (North Africa) through Several Successive Waves of COVID-19.

Documenting the circulation dynamics of SARS-CoV-2 variants in different regions of the world is crucial for monitoring virus transmission worldwide and contributing to global efforts towards combating the pandemic. Tunisia has experienced several waves of COVID-19 with a significant number of infections and deaths. The present study provides genetic information on the different lineages of SARS-CoV-2 that circulated in Tunisia over 17 months. Lineages were assigned for 1359 samples using whole-genome sequencing, partial S gene sequencing and variant-specific real-time RT-PCR tests. Forty-eight different lineages of SARS-CoV-2 were identified, including variants of concern (VOCs), variants of interest (VOIs) and variants under monitoring (VUMs), particularly Alpha, Beta, Delta, A.27, Zeta and Eta. The first wave, limited to imported and import-related cases, was characterized by a small number of positive samples and lineages. During the second wave, a large number of lineages were detected; the third wave was marked by the predominance of the Alpha VOC, and the fourth wave was characterized by the predominance of the Delta VOC. This study adds new genomic data to the global context of COVID-19, particularly from the North African region, and highlights the importance of the timely molecular characterization of circulating strains.

The Delta variant wave in Tunisia: Genetic diversity, spatio-temporal distribution and evidence of the spread of a divergent AY.122 sub-lineage.

Introduction: The Delta variant posed an increased risk to global public health and rapidly replaced the pre-existent variants worldwide. In this study, the genetic diversity and the spatio-temporal dynamics of 662 SARS-CoV2 genomes obtained during the Delta wave across Tunisia were investigated. Methods: Viral whole genome and partial S-segment sequencing was performed using Illumina and Sanger platforms, respectively and lineage assignemnt was assessed using Pangolin version 1.2.4 and scorpio version 3.4.X. Phylogenetic and phylogeographic analyses were achieved using IQ-Tree and Beast programs. Results: The age distribution of the infected cases showed a large peak between 25 to 50 years. Twelve Delta sub-lineages were detected nation-wide with AY.122 being the predominant variant representing 94.6% of sequences. AY.122 sequences were highly related and shared the amino-acid change ORF1a:A498V, the synonymous mutations 2746T>C, 3037C>T, 8986C>T, 11332A>G in ORF1a and 23683C>T in the S gene with respect to the Wuhan reference genome (NC_045512.2). Spatio-temporal analysis indicates that the larger cities of Nabeul, Tunis and Kairouan constituted epicenters for the AY.122 sub-lineage and subsequent dispersion to the rest of the country. Discussion: This study adds more knowledge about the Delta variant and sub-variants distribution worldwide by documenting genomic and epidemiological data from Tunisia, a North African region. Such results may be helpful to the understanding of future COVID-19 waves and variants.

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.

Whole genome sequencing and phylogenetic analysis of six SARS-CoV-2 strains isolated during COVID-19 pandemic in Tunisia, North Africa.

BACKGROUND: In Tunisia a first SARS-CoV-2 confirmed case was reported in March 03, 2020. Since then, an increase of cases number was observed from either imported or local cases. The aim of this preliminary study was to better understand the molecular epidemiology and genetic variability of SARS-CoV-2 viruses circulating in Tunisia and worldwide. METHODS: Whole genome sequencing was performed using NGS approach on six SARS. CoV-2 highly positive samples detected during the early phase of the outbreak. RESULTS: Full genomes sequences of six Tunisian SARS-CoV-2 strains were obtained from imported and locally transmission cases during the COVID-19 outbreak. Reported sequences were non-identical with 0.1% nucleotide divergence rate and clustered into 6 different clades with worldwide sequences. SNPs results favor the distribution of the reported Tunisian sequences into 3 major genotypes. These SNP mutations are critical for diagnosis and vaccine development. CONCLUSIONS: These results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level.

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.

Circulation and Molecular Epidemiology of Enteroviruses in Paralyzed, Immunodeficient and Healthy Individuals in Tunisia, a Country with a Polio-Free Status for Decades.

This report is an overview of enterovirus (EV) detection in Tunisian polio-suspected paralytic cases (acute flaccid paralysis (AFP) cases), healthy contacts and patients with primary immunodeficiencies (PID) during an 11-year period. A total of 2735 clinical samples were analyzed for EV isolation and type identification, according to the recommended protocols of the World Health Organization. Three poliovirus (PV) serotypes and 28 different nonpolio enteroviruses (NPEVs) were detected. The NPEV detection rate was 4.3%, 2.8% and 12.4% in AFP cases, healthy contacts and PID patients, respectively. The predominant species was EV-B, and the circulation of viruses from species EV-A was noted since 2011. All PVs detected were of Sabin origin. The PV detection rate was higher in PID patients compared to AFP cases and contacts (6.8%, 1.5% and 1.3% respectively). PV2 was not detected since 2015. Using nucleotide sequencing of the entire VP1 region, 61 strains were characterized as Sabin-like. Among them, six strains of types 1 and 3 PV were identified as pre-vaccine-derived polioviruses (VDPVs). Five type 2 PV, four strains belonging to type 1 PV and two strains belonging to type 3 PV, were classified as iVDPVs. The data presented provide a comprehensive picture of EVs circulating in Tunisia over an 11-year period, reveal changes in their epidemiology as compared to previous studies and highlight the need to set up a warning system to avoid unnoticed PVs.

Identification of two novel hepatitis C virus subtype 2 from Tunisia (2v and 2w).

BACKGROUND: Hepatitis C virus (HCV) has a high genetic diversity. Eight genotypes and 90 subtypes are currently described. Genotypes are clinically significant for therapeutic management and their determination is necessary for epidemiological studies. METHODS: Tunisian patients plasma samples (n = 6) with unassigned HCV-2 subtype using partial sequencing in the NS5B and Core/E1 regions were analyzed by realizing whole-genome sequencing analysis. Phylogenetic analyses were performed to assign subtypes. RESULTS: Phylogenetic analysis of the full genome sequences of Tunisian strains shows two subtypes within HCV-2. These later were genetically distinct from all previously established HCV-2 subtypes with nucleotide divergence greater than 15% (20% -31%). These two subtypes are proposed as new subtypes 2v and 2w. CONCLUSIONS: The discovery of two new HCV-2 subtypes circulating in the Tunisian population confirms the great diversity of HCV-2 viruses and increases the total number of HCV-2 subtypes from 21 to 23.

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 viruses take advantage of traditional practices to increase the burden of hepatocellular carcinoma in Tunisia.

Hepatocellular carcinoma (HCC) is a major public health issue in Africa. In Tunisia, hepatitis B virus (HBV) is known to be an important risk factor for HCC in the south of the country, but the role played by hepatitis C virus (HCV) still remains unclear. The aim of the current case-control study was to identify risk factors for HCC development in the northern part of the country. Clinical and biological data including viral hepatitis status (serological and molecular) and non-infectious risk factors from 73 patients with HCC and 70 control subjects without hepatic diseases were collected. The mean age of the patients was 63 ± 10 years, and the ratio of males to females was 1.1. HCC occurred in cirrhotic liver in 72.0% of the cases. HCV infection was the dominant risk factor (64.3% of cases); the presence of HBV was observed in 53.4% of the cases. Occult hepatitis B and C were implicated, respectively, in 30.1% and 9.6% of the cases. HCV genotype 1b was predominant. Patients originating from western Tunisia formed a homogeneous group, characterized by significantly higher rates of tattoos or scarifications (83%) and HCV infection (80%) than those from other parts of the country. Chronic HCV infection is currently the primary risk factor for HCC in Tunisia; HBV infection remains frequent in its overt or occult infection forms. Traditional esthetic practices apparently contribute to increasing the burden of terminal liver diseases in western 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.

Phylogenetic Analysis and Epidemic History of Hepatitis C Virus Genotype 2 in Tunisia, North Africa.

HCV genotype 2 (HCV-2) has a worldwide distribution with prevalence rates that vary from country to country. High genetic diversity and long-term endemicity were suggested in West African countries. A global dispersal of HCV-2 would have occurred during the 20th century, especially in European countries. In Tunisia, genotype 2 was the second prevalent genotype after genotype 1 and most isolates belong to subtypes 2c and 2k. In this study, phylogenetic analyses based on the NS5B genomic sequences of 113 Tunisian HCV isolates from subtypes 2c and 2k were carried out. A Bayesian coalescent-based framework was used to estimate the origin and the spread of these subtypes circulating in Tunisia. Phylogenetic analyses of HCV-2c sequences suggest the absence of country-specific or time-specific variants. In contrast, the phylogenetic grouping of HCV-2k sequences shows the existence of two major genetic clusters that may represent two distinct circulating variants. Coalescent analysis indicated a most recent common ancestor (tMRCA) of Tunisian HCV-2c around 1886 (1869-1902) before the introduction of HCV-2k in 1901 (1867-1931). Our findings suggest that the introduction of HCV-2c in Tunisia is possibly a result of population movements between Tunisia and European population following the French colonization.

Clinical epidemiological and molecular aspects of rubella outbreak with high number of neurological cases, Tunisia 2011-2012.

BACKGROUND: A large and unusually prolonged rubella outbreak occurred in Tunisia from April 2011 to July 2012 and was characterized by a high number of neurological cases. OBJECTIVES: To describe the outbreak and to perform virus genotyping of isolated virus strains. STUDY DESIGN: From January 2011 to December 2012, 5000 sera for serological diagnosis of acute rubella and 31 cerebrospinal fluid from patients with neurological symptoms were tested for the presence of rubella immunoglobulins G and M. Real-time PCR was performed on 49 throat swabs, 21 cerebrospinal fluid and 27 serum samples. Positive samples were assessed for virus genotyping by sequencing and the obtained sequences were compared to those previously isolated in the country. RESULTS: Acute rubella was confirmed in 280 patients including 15 neonates, 217 children and adults with mild rash and 48 patients with severe rubella (mainly encephalitis, n = 39). Most of acquired rubella cases (60.7%) were aged over 12 years with a male predominance observed in the age group 12-25 years (79%). Females belonged essentially to the unvaccinated age groups under 12 and over 25 years. Among the 23 samples tested positive by real-time PCR, six could be genotyped and clustered with either the 1E genotype, previously detected in Tunisia, or the 2B genotype which has never been isolated in Tunisia before. CONCLUSIONS: Gender and age distributions of the patients reflect the impact of the selective rubella vaccination program adopted in Tunisia since 2005. Genotype 1E continues to circulate and genotype 2B was probably recently introduced in Tunisia.