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Simple physico-chemical techniques can be used to evaluate the composition, structure, and characteristics of plant seeds to determine their viability, quality, and possible uses in agriculture. Advanced analytical techniques, including thermo gravimetric analysis (TGA), electron paramagnetic resonance (EPR), and high-pressure liquid chromatography (HPLC), provide completely new insights and more precise information. They can be integrated to build up seed quality profiles, with great advantage to assess water content, organic compounds, and inorganic metals without the need to carry out many extraction procedures, as requested by more conventional methods. In this study, seed lots from three different plant species such as Triticum turgidum L. subsp. durum (wheat), Trigonella foenum graecum L. (trigonella or fenugreek), and Atriplex halimus L. (saltbush or sea orach) have been used to test the potential of TGA, EPR, and HPLC to discriminate between seed-specific features. A key finding of this study is that HPLC is essential in Principal Component Analysis (PCA) because various seeds (from the same species or other species) may contain compounds with varying polarity groups. The reported data confirm the efficacy of this approach. These data, fully available for other users, are coherently constructed and provide a proof of concept for future seed quality control studies., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest Declarations of interest: none of the authors have any conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Members of the Bacillus cereus group are well-known opportunistic foodborne pathogens. In this study, the prevalence, hemolytic activity, antimicrobial resistance profile, virulence factor genes, genetic diversity by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) genotyping, and adhesion potential were investigated in isolates from a Tunisian dairy farm environment and raw milk. A total of 200 samples, including bedding, feces, feed, liquid manure, and raw bovine milk, were examined. Based on PCR test targeting sspE gene, 59 isolates were detected. The prevalence of B. cereus group isolates in bedding, feces, liquid manure, feed, and raw milk was 48%, 37.8%, 20%, 17.1%, and 12.5%, respectively. Out of the tested strains, 81.4% showed β-hemolytic on blood agar plates. An antimicrobial resistance test against 11 antibiotics showed that more than 50% of the isolates were resistant to ampicillin and novobiocin, while a high sensitivity to other antibiotics tested was observed in most isolates. The distribution of enterotoxigenic genes showed that 8.5% and 67.8% of isolates carried hblABCD and nheABC , respectively. In addition, the detection rate of cytotoxin K ( cytk ), enterotoxin T ( bceT ), and ces genes was 72.9%, 64.4%, and 5.1%, respectively. ERIC-PCR fingerprinting genotype analysis allowed discriminating 40 different profiles. The adhesion potential of B. cereus group on stainless steel showed that all isolates were able to adhere at various levels, from 1.5 ± 0.3 to 5.1 ± 0.1 log colony-forming unit (CFU)/cm 2 for vegetative cells and from 2.6 ± 0.4 to 5.7 ± 0.3 log CFU/cm 2 for spores. An important finding of the study is useful for updating the knowledge of the contamination status of B. cereus group in Tunisia, at the dairy farm level.

Background: Allogeneic hematopoietic stem cell transplantation (ASCT) induces acquired immunodeficiency, potentially altering vaccine response. Herein, we aimed to explore the clinical tolerance and the humoral and cellular immune responses following anti-SARS-CoV-2 vaccination in ASCT recipients., Methods: A prospective, non-randomized, controlled study that involved 43 ASCT subjects and 31 healthy controls. Humoral response was investigated using the Elecsys ® test anti-SARS-CoV-2. Cellular response was assessed using the QFN ® SARS-CoV-2 test. The lymphocyte cytokine profile was tested using the LEGENDplex™ HU Th Cytokine Panel Kit (12-plex)., Results: Adverse effects (AE) were observed in 69% of patients, encompassing pain at the injection site, fever, asthenia, or headaches. Controls presented more side effects like pain in the injection site and asthenia with no difference in the overall AE frequency. Both groups exhibited robust humoral and cellular responses. Only the vaccine transplant delay impacted the humoral response alongside a previous SARS-CoV-2 infection. Noteworthily, controls displayed a Th1 cytokine profile, while patients showed a mixed Th1/Th2 profile., Conclusions: Pfizer-BioNTech ® anti-SARS-CoV-2 vaccination is well tolerated in ASCT patients, inducing robust humoral and cellular responses. Further exploration is warranted to understand the impact of a mixed cytokine profile in ASCT patients.

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., Competing Interests: Declaration of Competing Interest The author's declare no conflict of interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for serious respiratory infections in humans. Even in the absence of respiratory symptoms, gastrointestinal (GI) signs were commonly reported in adults and children. Thus, oral-fecal transmission was suspected as a possible route of infection. The objective of this study was to describe RNA shedding in nasopharyngeal and stool samples obtained from asymptomatic and symptomatic children and to investigate virus viability., Methods: This study included 179 stool and 191 nasopharyngeal samples obtained from 71 children, which included symptomatic ( n = 64) and asymptomatic ( n = 7) ones. They were collected every 7 days from the onset of the infection until negativation. Viral RNA was detected by real-time RT-PCR, targeting the N and ORF1 genes. Whole-genome sequencing was performed for positive cases. Viral isolation was assessed on Vero cells, followed by molecular detection confirmation., Results: All cases included in this study ( n = 71) were positive in their nasopharyngeal samples. SARS-CoV-2 RNA was detected in 36 stool samples obtained from 15 out of 71 (21.1%) children; 13 were symptomatic and two were asymptomatic. Excretion periods varied from 7 to 21 days and 7 to 14 days in nasopharyngeal and fecal samples, respectively. Four variants were detected: Alpha ( n = 3), B.1.160 ( n = 3), Delta ( n = 7), and Omicron ( n = 1). Inoculation of stool samples on cell culture showed no specific cytopathic effect. All cell culture supernatants were negative for RT-qPCR., Conclusion: Our study demonstrated nasopharyngeal and fecal shedding of SARS-CoV-2 RNA by children up to 21 and 14 days, respectively. Fecal shedding was recorded in symptomatic and asymptomatic children. Nevertheless, SARS-CoV-2 was not isolated from positive stool samples., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer SM declared a past co-authorship with the author SH-B to the handling editor., (Copyright © 2023 Khemiri, Gdoura, Ben Halima, Krichen, Cammà, Lorusso, Ancora, Di Pasquale, Cherni, Touzi, Sadraoui, Meddeb, Hogga, Ammi, Triki and Haddad-Boubaker.)

(1) Background: This study aimed to compare the immunogenicity of the mix-and-match CoronaVac/BNT162b2 vaccination to the homologous CoronaVac/CoronaVac regimen. (2) Methods: We conducted a simple-blinded randomized superiority trial to measure SARS-CoV-2 neutralization antibodies and anti-spike receptor binding domain (RBD) IgG concentrations in blood samples of participants who had received the first dose of CoronaVac vaccine followed by a dose of BNT162b2 or CoronaVac vaccine. The primary endpoint for immunogenicity was the serum-neutralizing antibody level with a percentage of inhibition at 90% at 21-35 days after the boost. A difference of 25% between groups was considered clinically relevant. (3) Results: Among the 240 eligible participants, the primary endpoint data were available for 100 participants randomly allocated to the mix-and-match group versus 99 participants randomly allocated to the homologous dose group. The mix-and-match regimen elicited significantly higher levels of neutralizing antibodies (median level of 96%, interquartile range (IQR) (95-97) versus median level of 94%, IQR (81-96) and anti-spike IgG antibodies (median level of 13,460, IQR (2557-29,930) versus median level of 1190, IQR (347-4964) compared to the homologous group. Accordingly, the percentage of subjects with a percentage of neutralizing antibodies > 90% was significantly higher in the mix-and-match group (90.0%) versus the homologous (60.6%). Interestingly, no severe events were reported within 30 days after the second dose of vaccination in both groups. (4) Conclusions: Our data showed the superiority of the mix-and-match CoronaVac/BNT162b2 vaccination compared to the CoronaVac/CoronaVac regimen in terms of immunogenicity, thus constituting a proof-of-concept study supporting the use of inactivated vaccines in a mix-and-match strategy while ensuring good immunogenicity and safety.

Management of the COVID-19 pandemic relies on molecular diagnostic methods supported by serological tools. Herein, we developed S-RBD- and N- based ELISA assays useful for infection rate surveillance as well as the follow-up of acquired protective immunity against SARS-CoV-2. ELISA assays were optimized using COVID-19 Tunisian patients' sera and prepandemic controls. Assays were further validated in 3 African countries with variable endemic settings. The receiver operating curve was used to evaluate the assay performances. The N- and S-RBD-based ELISA assays performances, in Tunisia, were very high (AUC: 0.966 and 0.98, respectively, p < 0.0001). Cross-validation analysis showed similar performances in different settings. Cross-reactivity, with malaria infection, against viral antigens, was noticed. In head-to-head comparisons with different commercial assays, the developed assays showed high agreement. This study demonstrates, the added value of the developed serological assays in low-income countries, particularly in ethnically diverse populations with variable exposure to local endemic infectious diseases., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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&#95;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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Haddad-Boubaker, Arbi, Souiai, Chouikha, Fares, Edington, Sims, Camma, Lorusso, Diagne, Diallo, Boubaker, Ferjani, Mastouri, Mhalla, Karray, Gargouri, Bahri, Trabelsi, Kallala, Hannachi, Chaabouni, Smaoui, Meftah, Bouhalila, Foughali, Zribi, Lamari, Touzi, Safer, Alaya, Kahla, Gdoura and Triki.)

Introduction: SARS-CoV-2 serology have several indications. Currently, as there are various types available, it is important to master their performance in order to choose the best test for the indication. We evaluated and compared four different commercial serology tests, three of them had the Food and Drug Administration Emergency Use Authorization (FDA-EUA). Our goal was to provide new data to help guide the interpretation and the choice of the serological tests. Methods: Four commercial tests were studied: Elecsys® Roche® on Cobas® (total anti-nucleocapsid (N) antibodies), VIDAS® Biomerieux® (IgM and IgG anti- receptor binding domain (RBD) antibodies), Mindray® (IgM and IgG anti-N and anti-RBD antibodies) and Access® Beckman Coulter® (IgG anti-RBD antibodies). Two panels were tested: a positive panel (n = 72 sera) obtained from COVID-19-confirmed patients with no vaccination history and a negative panel (n = 119) of pre-pandemic sera. The analytical performances were evaluated and the ROC curve was drawn to assess the manufacturer’s cut-off for each test. Results: A large range of variability between the tests was found. The Mindray®IgG and Cobas® tests showed the best overall sensitivity, which was equal to 79.2% CI 95% (67.9−87.8). The Cobas® test showed the best sensitivity after 14 days of COVID-19 molecular confirmation; which was equal to 85.4% CI 95% (72.2−93.9). The Access® test had a lower sensitivity, even after day 14 (55.5% CI 95% (43.4−67.3)). The best specificity was noted for the Cobas®, VIDAS®IgG and Access® IgG tests (100% CI 95% (96.9−100)). The IgM tests, VIDAS®IgM and Mindray®IgM, showed the lowest specificity and sensitivity rates. Overall, only 43 out of 72 sera (59.7%) showed concordant results by all tests. Retained cut-offs for a significantly better sensitivity and accuracy, without significant change in the specificity, were: 0.87 for Vidas®IgM (p = 0.01) and 0.14 for Access® (p < 10−4). The combination of Cobas® with Vidas® IgM and IgG offered the best accuracy in comparison with all other tests combinations. Conclusion: Although using an FDA-EUA approved serology test, each laboratory should carry out its own evaluation. Tests variability may raise some concerns that seroprevalence studies may vary significantly based on the used serology test.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Belaiba, Ayouni, Gdoura, Kammoun Rebai, Touzi, Sadraoui, Hammemi, Yacoubi, Abdelati, Hamzaoui, Msaddak Azzouz, Chouikha and Triki.)

The Bacillus cereus ( B. cereus ) group is a widespread foodborne pathogen with a persistent ability to form biofilm, and with inherent resistance to traditional treatment in the food industry. Bacteriophages are a promising biocontrol agent that could be applied to prevent or eliminate biofilms formation. We have described, in this study, the isolation from sewage samples and preliminary characterization of bacteriophages that are active against the B. cereus group. The effectiveness of phage treatment for reducing B. cereus attachment and biofilms on stainless steel surfaces has been also assessed using three incubation periods at different titrations of each phage. Out of 62 phages isolated, seven showed broad-spectrum lytic action against 174 B. cereus isolates. All selected phages appeared to be of the Siphoviridae family. SDS-PAGE proved that two phages have a similar profile, while the remainder are distinct. All isolated phages have the same restriction pattern, with an estimated genome size of around 37 kb. The isolated bacteriophages have been shown to be effective in preventing biofilm formation. Reductions of up to 1.5 log 10 UFC/cm 2 have been achieved, compared to the untreated biofilms. Curative treatment reduced the bacterial density by 0.5 log 10 UFC/cm 2 . These results support the prospect of using these phages as a potential alternative strategy for controlling biofilms in food systems.

Background: The mass vaccination campaign against SARS-CoV-2 was started in Tunisia on 13 March 2021 by using progressively seven different vaccines approved for emergency use. Herein, we aimed to evaluate the humoral and cellular immunity in subjects aged 40 years and over who received one of the following two-dose regimen vaccines against SARS-CoV-2, namely mRNA-1273 or Spikevax (Moderna), BNT162B2 or Comirnaty (Pfizer-BioNTech), Gam-COVID-Vac or Sputnik V (Gamaleya Research Institute), ChAdOx1-S or Vaxzevria (AstraZeneca), BIBP (Sinopharm), and Coronavac (Sinovac)., Material and Methods: For each type of vaccine, a sample of subjects aged 40 and over was randomly selected from the national platform for monitoring COVID-19 vaccination and contacted to participate to this study. All consenting participants were sampled for peripheral blood at 3-7 weeks after the second vaccine dose to perform anti-S and anti-N serology by the Elecsys ® (Lenexa, KS, USA) anti-SARS-CoV-2 assays (Roche ® Basel, Switzerland). The CD4 and CD8 T cell responses were evaluated by the QuantiFERON ® SARS-CoV-2 (Qiagen ® Basel, Switzerland) for a randomly selected sub-group., Results: A total of 501 people consented to the study and, of them, 133 were included for the cellular response investigations. Both humoral and cellular immune responses against SARS-CoV-2 antigens differed significantly between all tested groups. RNA vaccines induced the highest levels of humoral and cellular anti-S responses followed by adenovirus vaccines and then by inactivated vaccines. Vaccines from the same platform induced similar levels of specific anti-S immune responses except in the case of the Sputnik V and the AstraZeneca vaccine, which exhibited contrasting effects on humoral and cellular responses. When analyses were performed in subjects with negative anti-N antibodies, results were similar to those obtained within the total cohort, except for the Moderna vaccine, which gave a better cellular immune response than the Pfizer vaccine and RNA vaccines, which induced similar cellular immune responses to those of adenovirus vaccines., Conclusion: Collectively, our data confirmed the superiority of the RNA-based COVID-19 vaccines, in particular that of Moderna, for both humoral and cellular immunogenicity. Our results comparing between different vaccine platforms in a similar population are of great importance since they may help decision makers to adopt the best strategy for further national vaccination programs.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for COVID-19 disease which is known to have a broad clinical spectrum, from asymptomatic to critical presentation leading to death. Many researchers have investigated the factors impacting the course of the disease. Our previous in silico study suggested a possible protective effect of Hepatitis B, Tetanus and Measles vaccines against COVID-19. In continuity, we conducted a cross-sectional clinical study in order to confirm our in silico assumptions regarding the HBs-Ag antibodies., Methods: A representative sex- and age-matched sample of patients with confirmed COVID-19 was selected ( n = 340). All clinical presentations were equally represented. Using an ELISA test, each patient benefited of a serology for the detection and measurement of the anti-HBs specific IgG antibodies. The obtained results allowed determining the different correlations between these antibody titers and the disease severity. The R ® software and the MedCalc ® software served to calculate the Spearman's coefficient of rank correlation (rho) for the obtained titers per severity group as well as the different other calculations and figure representations., Results: A significant positive correlation was found with the anti-HBs titers (rho = 0.107; p = 0.04). High anti-HBs titers were significantly associated with the mild presentation of COVID-19. A significant difference was found between the obtained titers per severity class (chi-2 test, p = 0.03)., Discussion/conclusion: Our findings demonstrated that anti-HBs titers were significantly higher for patients having mild COVID-19 presentations. We presume that being immunized against the HB may play a protective role in the course of the disease. Our study provided more key elements in understanding the disparity of the clinical spectrum among regions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gdoura, Touati, Kalthoum, Ben Slama, Fatnassi, Mrad, Ammari, Brahmi, Ben Jazia, Hogga, Triki and Haddad-Boubaker.)

Reliable serological assays are needed to understand the real impact of COVID-19. In order to compare the efficiency of different COVID-19 vaccines used in the National Vaccination Program in Tunisia, we have developed a quantitative in-house ELISA. The ELISA is based on the ectodomain of the SARS-CoV-2 Spike Baculovirus recombinant protein. We used a panel of 145 COVID-19 RT-PCR positive serum samples and 116 pre-pandemic serum samples as a negative panel. The validation was carried out by comparison to four commercial techniques (Vidas SARS-CoV-2 IgG anti-RBD Biomérieux, Elecsys Anti-Nucleocapsid of SARS-CoV-2 Roche, cPass GenScript and the quantitative Elecsys Anti-RBD of SARS-CoV-2, Roche). For the evaluation of the National Vaccination campaign, we have included 115 recipients who received one of the approved vaccines. The qualitative performances of the developed ELISA gave 96% sensitivity, 97.5% specificity and 0.968 accuracy. For the evaluation of the different brand of vaccines in recipients not previously infected with SARS-CoV-2, it seems that mRNA vaccine of Pfizer/BioNTech has shown a higher efficacy compared to inactivated virus vaccines. COVID-19 convalescent individuals have generated poor antibody responses. Nevertheless, when they are vaccinated with any brand of the COVID-19 vaccines, many of them mounted an exponential increase of the induced immune responses, qualified as a "hybrid vigor immunity". Our developed in-house ELISA seems to be very efficient in evaluating the effectiveness of anti-COVID-19 vaccination. Platforms based on mRNA vaccine are better performing than those based on inactivated virus., (© 2022. The Author(s).)

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 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., 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 rRT-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 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)., Discussion/conclusion: 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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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., (© 2022. The Author(s).)

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.

Urinary tract infections (UTIs) are the most frequent human infections in community and hospitals. This study aimed to determine the distribution of bacterial uropathogens among urinary tract infections diagnosed within the regional hospital Houcine Bouzaiene (Gafsa, South West Tunisia) during a survey of 54 days from the 8th of November to the 31st of December 2017. Enterobacterales strains were tested for antimicrobial resistance by disk diffusion method and extended-spectrum β-lactamase (ESBL) production was tested by double-disc synergy test. Strains were further subjected to a molecular assessment of ESBL and AmpC β-lactamase production by PCR. Overall, 173 bacterial isolates were studied, out of which 91.3% were Enterobacterales. Escherichia coli was the dominant pathogen, followed by Klebsiella pneumoniae. High to moderate resistance rates were observed, ranging from 66% to 90.7% for penicillins, from 6.7% to 18.6% for cephalosporins and from 16.2% to 25.4% for fluoroquinolones. Enterobacterales with decreased susceptibility to third-generation cephalosporins (3rd GC) carried several resistance genes: blaCTX-M group 1 and group 9, and ACC and FOX AmpC β-lactamase genes. Overall, ESBLs and AmpC β-lactamases were detected in 57% and 14% of the 3rd GC-resistant isolates, respectively. This study proved the high potential of K. pneumaniae species to develop resistance against commonly used antibiotics. Thus, rigorous monitoring of the antibiotic resistance of clinical pathogens have to be implemented in Tunisia. Our results are very relevant to evaluate efficiency of the Tunisian therapeutic strategies against UTIs and adapt them to the emerging problem of antimicrobial resistance.

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.

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., Competing Interests: The authors declare that Prochidia Laboratory provided genotyping kits for free to the Laboratory of Clinical Virology to conduct the present work., (Copyright © 2021 Chouikha, Ghrabi, Ghodbane, Hammemi, Khedhiri, Sadraoui, Touzi, Hassine, Maatoug, Bensaoud, Abdelhak, Bouarrouj, Gdoura, Chaouachi and Triki.)

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., (© 2021. The Author(s).)

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., (© 2020 Wiley-VCH GmbH.)

Background: Despite the importance of the B. cereus group as major foodborne pathogens that may cause diarrheal and/or emetic syndrome(s), no study in Tunisia has been conducted in order to characterize the pathogenic potential of the B. cereus group. The aim of this study was to assess the sanitary potential risks of 174 B. cereus group strains isolated from different foodstuffs by detecting and profiling virulence genes (hblA, hblB, hblC, hblD, nheA, nheB, nheC, cytK, bceT and ces), testing the isolates cytotoxic activity on Caco-2 cells and antimicrobial susceptibility towards 11 antibiotics., Results: The entertoxin genes detected among B. cereus isolates were, in decreasing order, nheA (98.9%), nheC (97.7%) and nheB (86.8%) versus hblC (54.6%), hblD (54.6%), hblA (29.9%) and hblB (14.9%), respectively encoding for Non-hemolytic enterotoxin (NHE) and Hemolysin BL (HBL). The isolates are multi-toxigenic, harbouring at least one gene of each NHE and HBL complexes associated or not to bceT, cytK-2 and ces genes. Based on the incidence of virulence genes, the strains were separated into 12 toxigenic groups. Isolates positive for cytK (37,9%) harbored the cytK-2 variant. The detection rates of bceT and ces genes were 50.6 and 4%, respectively. When bacteria were incubated in BHI-YE at 30 °C for 18 h and for 5 d, 70.7 and 35% of the strains were shown to be cytotoxic to Caco-2 cells, respectively. The cytotoxicity of B. cereus strains depended on the food source of isolation. The presence of virulence factors is not always consistent with cytotoxicity. However, different combinations of enterotoxin genetic determinants are significantly associated to the cytotoxic potential of the bacteria. All strains were fully sensitive to rifampicin, chloramphenicol, ciprofloxacin, and gentamycin. The majority of the isolates were susceptible to streptomycin, kanamycin, erythromycin, vancomycin and tetracycline but showed resistance to ampicillin and novobiocin., Conclusion: Our results contribute data that are primary to facilitate risk assessments in order to prevent food poisoning due to B. cereus group.

Bacillus cereus group is widespread in nature and foods. Several members of this group are recognized as causing food spoilage and/or health issues. This study was designed to determine the prevalence and genetic diversity of the B. cereus group strains isolated in Tunisia from different foods (cereals, spices, cooked food, fresh-cut vegetables, raw and cooked poultry meats, seafood, canned, pastry, and dairy products). In total, 687 different samples were collected and searched for the presence of the B. cereus group after selective plating on MYP agar and enumeration of each sample. The typical pink-orange uniform colonies surrounded by a zone of precipitate were assumed to belong to the B. cereus group. One typical colony from each sample was subcultured and preserved as cryoculture. Overall, 191 (27.8%) food samples were found positive, giving rise to a collection of 191 B. cereus -like isolates. The concentration of B. cereus -like bacteria were below 10 3 cfu/g or ml in 77.5% of the tested samples. Higher counts (>10 4 cfu/g or ml) were found in 6.8% of samples including fresh-cut vegetables, cooked foods, cereals, and pastry products. To verify whether B. cereus -like isolates belonged to the B. cereus group, a PCR test targeting the sspE gene sequence specific of the group was carried out. Therefore, 174 isolates were found to be positive. Food samples were contaminated as follows: cereals (67.6%), pastry products (46.2%), cooked food (40.8%), cooked poultry meat (32.7%), seafood products (32.3%), spices (28.8%), canned products (16.7%), raw poultry meat (9.4%), fresh-cut vegetables (5.0%), and dairy products (4.8%). The 174 B. cereus isolates were characterized by partial sequencing of the panC gene, using a Sym'Previous software tool to assign them to different phylogenetic groups. Strains were distributed as follows: 61.3, 29.5, 7.5, and 1.7% in the group III, IV, II, and V, respectively. The genetic diversity was further assessed by ERIC-PCR and PFGE typing methods. PFGE and ERIC-PCR patterns analysis allowed discriminating 143 and 99 different profiles, respectivey. These findings, associated to a relatively higher prevalence of B. cereus group in different foods, could be a significant etiological agent of food in Tunisia.

The application of natural zeolite for water and wastewater treatment has been carried out and is still a promising technique in environmental cleaning processes. Natural zeolite can be used to improve the purification process of clams (Ruditapes decussatus). Thus, our study aimed at improving the clam purification system in order to reduce Escherichia coli and eliminate Salmonella in samples artificially contaminated with this bacterium using a natural zeolite to replace the biological filter. The results showed that zeolite used in a depuration system improved the clam purification process. Moreover, natural zeolite exhibited high performance in the adsorption of bacteria and allowed to reduce the Escherichia coli abundance in 24 h, thus ensuring purified clams conformity with the ISO 16649-3 standard. These results indicate the beneficial effects of using zeolite in the adsorption of bacteria and the reduction in the abundance of Escherichia coli and set the Salmonella from marine organisms.

Vibrio spp. have emerged as a serious threat to human health worldwide. V. parahaemolyticus , V. cholerae , and V. vulnificus pose a considerable public health risk in Tunisia because they cause sporadic and epidemic foodborne infections associated with the consumption of raw or undercooked contaminated seafood. More recently, toxR-positive V. alginolyticus was also reported to be a potential source of contaminated seafood. A total of 247 samples, including 113 fishes ( Labrus viridis , Penaeus kerathurus , Diplodus annularis , Diplodus sparaillon , Scorparna porcus , Sarpa salpa , Dentex dentex , Scorparna scrofa , Sardinella aurita , Trachurus trachurus , Synodus saurus , Pagellus erythrinus , and Metapenaeus monoceros ), 83 clams ( Ruditapes decussatus species), 30 seawater samples, and 21 sediment samples were analyzed using traditional culture methods (ISO/TS 21872-1; International Organization for Standardization 2007) and a conventional PCR method for Vibrio spp., Identification: A rapid, sensitive, and highly reproducible real-time PCR assay was developed to detect the three major Vibrio spp. pathogenic for humans in Tunisian seafood products and sediments. A conventional culture method found 102 (41.3%) of 247 analyzed samples positive for Vibrio spp.; a conventional PCR method found 126 (51%) of the 247 samples positive. Real-time PCR assay found 126 (51.1%) samples positive; V. alginolyticus toxR was the most common, found in 99 (78.57%) of samples, followed by V. parahaemolyticus in 26 (20.63%) and V. cholerae in 1 (0.7%). All culture-positive samples were PCR positive. However, 24 samples that were positive by conventional PCR and real-time PCR were culture negative. Our findings indicate that retail seafood is commonly contaminated with Vibrio spp. and presents a potential risk to human health in Tunisia. These data also indicate that real-time PCR can provide sensitive species-specific detection of Vibrio spp. in seafood without prior isolation and characterization of the bacteria by traditional microbiological methods.

Subclavian vein stenosis is a classical complication of longterm venous catheterization in hemodialysis. We report the case of a 74 years-old woman, operated for multiple arteriovenous fistulae, admitted to hospital with upper-limb oedema. Venous angiography demonstrated subclavian stenosis. Surgical treatment was performed by transposition of the external jugular vein onto the subclavian vein with a good result.

We report 4 cases of subclavian vein stenosis after hemodialysis occurring in 4 women with a mean age of 36 years. The duration of subclavian catheterization was 1 to 11 months. The diagnosis was established by angiography or phlebography. The treatment was always surgical: axillo-internal jugular bypass: 1 case; deviation of the internal jugular vein: 1 case; basilo-internal jugular bypass: 1 case; axillo-axillary bypass: 1 case.

We report a new case of persistent sciatic artery. An 18-year-old girl was hospitalized with ischemia of the left foot. An arteriogram demonstrated a sciatic artery completely persistent. The superficial femoral artery was hypoplasic. A left femoro-popliteal by pass with saphenous vein graft was practised. After the operation, the patient did well and had no complications. We present this observation with a literature review about clinical arteriographic and surgical aspects of this vascular structure and its complications.

We report a case of successful management of a false aneurysm of the superior mesenteric artery. The only helpful clinical manifestations were episodes of previous abdominal pain and a history of bacterial endocarditis. The surgical management involved endo-aneurysmorrhaphy. The patient's post-operative course was unremarkable.

37 cases of diabetic arteritis of lower limbs in diabetic patients were retrospectively studied, from 1988 to 1991. Mean age was 66 years. Masculine predominance was evident with a sex ratio of 1/10. The majority of our patients have consulted at a late stage with trophic disturbance of inferior limbs, indeed 18 patients were seen at the 4th stage. Mortification was observed in 55% cases. Arteriography has allowed to show an elective localization of lesions at the femoralis arteria, profunda femoralis arteria and distal axes. Operative indication was made in 22 patients. Conservative surgery was performed for 50% of the operated patients. Conservative surgery results had been relatively less satisfactory than for others arteritis, particular because of late consultation and the condition of diabetic patients.

A study of 11 cases of Takayasu's disease, collected from the cardio-vascular surgical service of Sfax, has been realized during the 4 year-period from 1988 to 1991. Womanly predominance was neat. The average age was 40 years, with extremes of 24 and 56 years. Dominant revealing symptoms was upper limb ischaemia found in 9 cases, a reno-vascular hypertension rebelled to medical treatment was noted in 2 cases. Arteriography have showed a preferential localization of lesions in the subclavian artery essentially at the post-vertebral segment. Lesions types were dominated by stenosis and obliteration, then aneurysms were rare. Operative indication was posed in 9 cases. 11 revascularisations procedures were performed of which 10 arterial by passes and one resection graft. Two bypasses were obstructed, the 9 remaining bypasses have a good previous checked clinically and by angiography (or echo-doppler) with a mean follow-up of 14 months.