Development and comparative evaluation of SARS-CoV-2 S-RBD and N based ELISA tests in various African endemic settings.

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.

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.

SARS-CoV-2 Serology: Utility and Limits of Different Antigen-Based Tests through the Evaluation and the Comparison of Four Commercial Tests.

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.

Development of an in-house quantitative ELISA for the evaluation of different Covid-19 vaccines in humans.

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.

Presumed Protective Role for Anti-Hepatitis B Virus Antibodies Against COVID-19 Severe Cases: A Clinical Study Confirming in silico Hypothesis

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.

Humoral and Cellular Immunogenicity of Six Different Vaccines against SARS-CoV-2 in Adults: A Comparative Study in Tunisia (North Africa).

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.

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.

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.