Search

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.

West Nile Virus (WNV) is an arbovirus transmitted by mosquito bite involving birds as reservoirs, humans and equines as accidental hosts. Eight distinct lineages (WNV-1 to WNV-8) have been identified: WNV-1 and WNV-2 infect humans and animals, and WNV-3 to WNV-8 have been identified only in vectors. WNV has been implicated in neuroinvasives infections, especially meningitis and encephalitis. Tunisia experienced three epidemics in 1997, 2003 and 2012. Serological studies on humans, equines and birds as well as the detection of the virus in the vector favour a fairly frequent circulation in the country. A new epidemic has been observed in Tunisia between August and November 2018. The obtained sequences of the VWN from Tunisia 2018 grouped in a distinct monophyletic group within the Mediterranean subtype in Cluster 1, with a maximum of 2% nucleotide divergence. These sequences were clearly distinct from the Tunisia 1997, which grouped with sequences mainly from USA in Cluster 2. This work reports the genetic characterization of the Tunisia 2018 strain in comparison with the previously identified strains in Tunisia and worldwide. The epidemic virus Tunisia 2018 was genetically close to the Mediterranean basin and Eastern Europe sequences but distinct from the Tunisia 1997 closely related to the American sequences.

IntroductionSARS-CoV2 serology testing is multipurpose provided to choose an efficient test. We evaluated and compared 4 different commercial serology tests, three of them had the Food and Drug Administration (FDA) approval. Our goal was to provide new data to help to guide the interpretation and the choice of the serological tests.MethodsFour commercial tests were evaluated: Cobas®Roche®(total anti-N antibodies), VIDAS®Biomerieux®(IgM and IgG anti-RBD antibodies), Mindray®(IgM and IgG anti-N and anti-RBD antibodies) and Access®Beckman Coulter®(IgG anti-RBD antibodies). Were tested: a positive panel (n=72 sera) obtained from COVID-19 confirmed patients and a negative panel (n=119) of pre-pandemic sera. Were determined the analytical performances and was drawn the ROC curve to assess the manufacturer’s threshold.ResultsA large range of variability between the tests was found. Mindray®IgG and Cobas® tests showed the best overall sensitivity 79,2%CI95%[67,9-87,8]. Cobas® showed the best sensitivity after D14; 85,4%CI95%[72,2-93,9]. The best specificity was noted for Cobas®, VIDAS®IgG and Access® IgG(100%CI95%[96,9-100]). Access® had the lower sensitivity even after D14 (55,5% CI95%[43,4-67,3]). VIDAS®IgM and Mindray®IgM tests showed the lowest specificity and sensitivity rates. Overall, only 43 out of 72 sera gave concordant results (59,7%). Retained cut-offs for a significantly better sensitivity and accuracy, without altering significantly the specificity, were: 0,87 for Vidas®IgM(p=0,01), 0,55 for Vidas®IgG(p=0,05) and 0,14 for Access®(p−4).ConclusionAlthough FDA approved, each laboratory should realize its own evaluation for commercial tests. Tests variability may raise some concerns that seroprevalence studies may vary significantly based on the used serology test.

Introduction Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied RT-PCR in CSF, which often give negative results due to too short virorachia and late sampling.RT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples.Methods During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative RT-PCR was performed on both type of samples. Results WNV RNA was detected in 50,5% of urine samples (48/95) and in 2,8% of CSF samples (1/35). WNV RNA was detectable from 1 to 41 day from symptom onset, however, positive urine rate was 53,1% during the first 10 days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference ( p >0,005). Cycle threshold values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ct value was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases ( p Discussion/Conclusion Our study reports the use of RT-PCR on urine samples as a confirmatory diagnostic tool for WNV probable cases during an outbreak. Our findings underline the reliability, and rapidity of this confirmatory tool, even late and show its superiority on CSF investigation.

Routine laboratory screening is based on the detection of WNV specific IgM and IgG in blood and cerebrospinal fluid. Confirmation is then classically applied by real time RT-PCR (rRT-PCR) in Cerebrospinal fluid (CSF), which often gives negative results due to too short virorachia and late sampling. rRT-PCR was applied-for the first time for routine diagnosis purpose-on urine samples.During 2018 outbreak in Tunisia, 107 patients presented WNV neurologic symptoms and were positive for WNV serology. Of them, 95 patients were sampled for urine and 35 were sampled for CSF. Qualitative rRT-PCR was performed on both type of samples.WNV RNA was detected in 50.5% of urine samples (48/95) and in 2.8% of CSF samples (1/35). WNV RNA was detectable from day 1 to day 41 from symptom onset, however, positive urine rate was 53.1% during the first 10 days from symptom onset. The proportions of urine-positive and urine-negative samples, based on day of collection, showed no statistical difference (p 0.005). Cycle threshold (Ct) values ranged from 12 to 39, with no correlation with the day of collection. The lowest Ct value was detected for urine sampled on day 5 after symptom onset. A statistically significant difference was found between age groups of confirmed and non confirmed cases (p 0.001).Our study reported the use of rRT-PCR on urine samples as a confirmatory diagnostic tool for WNV "probable cases" during an outbreak. Our findings underlined the reliability and the rapidity of this confirmatory tool, even late, and showed its superiority on CSF investigation.

Recent efforts have reported numerous variants that influence SARS-CoV-2 viral characteristics including pathogenicity, transmission rate and ability of detection by molecular tests. Whole genome sequencing based on NGS 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 cost, we developed a couple of primers allowing to generate partial sequences in the viral S gene allowing 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 and one nasopharyngeal specimens collected during the decreasing phase of a high transmission COVID-19 wave in T unisia were analyzed. The results reveal high genetic variability within the sequenced fragment and allowed the detection of first introduction in the country of already known VOCs and VOIs as well as others variants that have interesting genomic mutations and need to be kept under surveillance.ImportanceThe method of choice for SARS-CoV-2 variants detection is whole genome sequencing using 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 number 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 including key mutations of the already known VOCs and VOIs for rapid identification of these VOCs and VOIs and helps to better select specimens that need to be sequenced by NGS technologies. The second step consisting in whole genome sequencing allowed to have a holistic view of all variants within the selected viral strains and confirmed the initial classification of the strains based on partial S gene sequencing.

International audience; 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 results indicate multiple introductions of the virus in Tunisia and add new genomic data on SARS-CoV-2 at the international level.

Objective: A recently discovered non-A-E hepatitis virus has been designated as human Pegivirus (HPgV). HPgV is prevalent in high-risk groups such as patients with hepatitis C virus (HCV), and it is of interest for patients who are at risk for transmitted infections. The aim of this study was to evaluate the prevalence of HPgV as well as the genotype distribution among patients in the Tunisian population who are infected with HCV and also in multitransfused patients. Methods: A total of 144 patients were screened using RTPCR/nested PCR of the 5′-untranslated region (UTR); 14 cases were sequenced and phylogenetically analyzed. Results: Seven (14.9%) subjects from the multitransfused group and 7 (7.2%) patients infected with HCV, respectively, were found positive for HPgV RNA. Sequencing and phylogenetic analysis of the 14 cases revealed that genotype 2a was the main genotype circulating in Tunisian patients. Genotype 2b was found in the amplified samples of 2 HCV-infected patients. Conclusion: This study enriches the limited data on HPgV prevalence in Tunisia, and shows, for the first time, the molecular epidemiology of the circulating strains in this country.

Resume Objectif Cette etude avait pour but d’etudier les profils serologiques et moleculaires de l’infection delta chez des patients tunisiens. Patients et methodes Notre travail a porte sur 215 sujets antigene HBs (AgHBs) positifs, incluant 176 porteurs asymptomatiques issus de regions d’endemicite variables pour le virus de l’hepatite B (VHB) et 39 malades atteints d’hepatite B chronique et a serologie delta positive. Une recherche de l’antigene delta, des anticorps delta et de l’antigene HBe a ete effectuee chez tous les patients ; une recherche et un genotypage de l’ARN du virus de l’hepatite delta (VHD) ainsi qu’une recherche de l’ARN du VHB ont ete conduites chez les patients du second groupe. Resultats Douze sujets (6,8 %) sur 176 des porteurs asymptomatiques testes ont une serologie VHD positive. La prevalence a ete relativement plus elevee dans les localites de forte endemicite pour le VHB que dans celles d’endemicite moyenne ou faible. La moyenne d’âge des sujets delta positifs a ete de cinq ans superieure a celle de la population globale (30 ans versus 24,4 ans). Pour les malades a hepatite B chronique et a serologie delta positive, l’ARN du VHD a ete detecte chez 53,8 % des cas, une replication simultanee des deux virus VHB–VHD a ete observee chez 38,4 % des cas. Le genotype 1 a ete retrouve pour un des echantillons amplifies. Conclusions Les resultats de notre etude enrichissent les donnees encore tres limitees de la litterature sur la prevalence de l’infection par le VHD en Tunisie et sur l’epidemiologie moleculaire de souches circulantes.

Background Our aim is to detect the infection by Toscana virus (TOSV) and other Phleboviruses in the sera and cerebro-spinal fluid (CSF) of patients with meningitis in Tunisia. We examined various species of phlebotomus present in Tunisia to determine whether or not a direct relationship exists between cases of meningitis and the viruses circulating in the insect vectors. Methods Patients with the meningeal syndrome were tested for anti-TOSV IgM and IgG using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) and for the presence of TOSV and other Phleboviruses using a RT-PCR test. An entomological study was carried out using CDC light traps to trap sandflies in different bioclimatic zones of Tunisia. Collected sandflies were tested by RT-PCR for the presence of TOSV and other Phleboviruses and subsequently by viral isolation on Vero cells. Results Of 263 patients were tested using ELISA of which 12.16% (n = 32/263) were IgM positive for anti TOSV. Of these 32 patients, 78% (n = 25/32) were IgG positive. 12.86% (n = 18/140) of the CSF samples tested by RT-PCR were positive for the Toscana virus. One CSF sample tested by RT-PCR revealed the presence of Sandfly Fever Sicilian Virus (SFSV). The Punique virus was identified in one sandfly pool. Conclusions This study confirms, for the first time, that TOSV is involved in a neurological disorder in North Africa. The incidence of this involvement in Tunisia conforms with observations made in other Mediterranean countries. Moreover, for the first time, a molecular approach was used to detect SFSV in a Tunisian patient displaying neurological symptoms. Electronic supplementary material The online version of this article (doi:10.1186/s12879-014-0598-9) contains supplementary material, which is available to authorized users.

This report is an overview of enterovirus epidemiology in Tunisia during a 12-year period from 1992 to 2003. A total of 4700 clinical samples were collected as part of the national poliovirus surveillance programme and the routine diagnostic programme for aseptic meningitis. Enterovirus detection was performed by isolation on cell culture according to World Health Organization recommended protocols. Serotype identification was performed by seroneutralization of the cytopathic effect using pools of specific antisera and sequencing in the VP1 region of the genome. Poliovirus isolates were assessed for their wild or vaccine-related origin by standard World Health Organization recommended methods (PCR, probe hybridization and ELISA). The results confirm the interruption of wild poliovirus circulation since 1995. A total of 236 non-polio enterovirus (NPEV) strains were isolated; seroneutralization allowed typing of 93 % (219 out of 236) of them. The antisera used allowed the identification of the most common enterovirus serotypes. The remaining 17 isolates were sequenced; 16 of them belonged to enterovirus serotypes that were not targeted by the antisera pools used. A total of 29 different serotypes of NPEV were detected in the country during the study period. Echoviruses of serotypes 6, 11 and 30 were the most frequently isolated, almost every year; other serotypes had a cyclic occurrence and others were detected during a limited period with very few isolates. The NPEV isolation rate varied from year to year but was steadily under 10 %, suggesting a relatively low prevalence of these viruses in comparison to that in other developing countries. A seasonal variation was also noted; the high transmission period starts in March and peaks in September-November. This study is the first report of the epidemiology of NPEV in Tunisia. These viruses are associated with various diseases and epidemiological data may help to clarify their impact on human health.

In an attempt to explain temporal and geographical rabies incidence fluctuations in Tunisia, a molecular epidemiological study of rabies virus (RV) was carried out. A panel of RV isolates from dogs, collected between 1992 and 2003, from different regions in Tunisia have been analysed by direct sequencing of PCR-amplified products coding for the nucleoprotein gene. New sequences have been compared to prototype sequences of Lyssavirus species and nine lineages of species 1. All Tunisian isolates belonged to species rabies virus and segregated into two rabies lineages geographically distinct: NCS lineage characterizing Northeast, Central and Northern areas of the country and NW lineage more restricted to the North-Western regions. Phylogenetic analyses showed that Tunisian RV clustered most closely to Africa 1a lineage: NCS lineage showed nucleic affiliation with isolates from Algeria and Morocco, whereas, NW lineage shared a strong relationship with Ethiopian and Sudanese strains.

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.