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.

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.

Metagenomic analysis identifies human adenovirus 31 in children with acute flaccid paralysis in Tunisia.

A variety of viruses can cause acute flaccid paralysis (AFP). However, the causative agent, sometimes, remains undetermined. Metagenomics helps in identifying viruses not diagnosed by conventional methods. Stool samples from AFP (n = 104) and non-AFP (n = 114) cases that tested enterovirus-negative by WHO standard methods were investigated. A metagenomics approach, first used on five pools of four samples each, revealed the presence of adenovirus sequences. Amplification in A549 cells and full-genome sequencing were used for complete virus identification and for designing a PCR assay to screen individual related samples. Metagenomic analysis showed that adenovirus sequences that were closely to the A31 and A61 genotypes were the most abundant. Two out of the corresponding 20 individual samples were found positive by PCR, and isolates were obtained in cell culture. Phylogenetic analysis based on complete genome sequences showed that the viruses belong to HAdV-A31 genotype (98-100% nucleotide sequence identity). PCR analysis of stool samples from all AFP and non-AFP cases revealed that a larger proportion of the positive samples were from AFP cases (17.3%) than from non-AFP cases (2.4%). These results open the way to studies aiming to test a possible role of HAdV-A31 in the pathogenesis of AFP.

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.

Iterative Excretion of Human Cosaviruses from Different Genotypes Associated with Combined Immunodeficiency Disorder.

BACKGROUND: Human cosaviruses (HCoSVs) are newly discovered enteric viruses in the Picornaviridae family. They have been described in non-polio acute flaccid paralysis, diarrheal patients, and healthy individuals. They remain rarely documented in immunodeficient patients. OBJECTIVES: This study reports iterative excretion of HCoSVs in a patient with major histocompatibility complex (MHC) class II combined immunodeficiency, a relatively common primary immunodeficiency in consanguineous settings. METHODS: A total of 35 samples were collected from a patient followed for oral polio vaccine strains detection in stool samples during a 57-month period. Detection of HCoSVs in stools was performed by nested RT-PCR in the 5' noncoding region. The genotype identification and screening for recombinant strains was performed by sequencing in the VP1 and 3D genomic regions followed by phylogenetic analysis. RESULTS: The patient was infected with HCoSVs twice at a 3-year interval. The excreted viruses belonged to 2 different genotypes with 2 probable recombinant viruses. During HCoSV infections, the patient was also excreting Sabin-related polioviruses. CONCLUSIONS: This study describes excretion kinetics and genetic characteristics of HCoSVs in a patient with combined immunodeficiency due to MHC class II expression defect. The patient did not have concomitant symptoms related to the HCoSV infection.

Prevalence of human cosaviruses in Tunisia, North Africa.

A new picornavirus, named human Cosavirus (HCoSV) was isolated recently from stools of children with acute flaccid paralysis (AFP) and healthy children in Pakistan and Afghanistan. Since then, it was also isolated from patients from other countries. Five species are presently identified forming a new genus in the Picornaviridae family. This study reports the detection of HCoSV in stool specimens collected as part of the National Poliovirus surveillance Program in Tunisia, between 2011 and 2012, from patients with AFP and healthy individuals among their contacts. One hundred and ninety four stool samples were investigated by RT-PCR in the 5' non-coding region of the genome. A total of 64 specimens (33%) tested positive for HCoSV. HCoSV positive specimens were found in 36 cases with neurological syndromes and 28 of their healthy contacts. The highest rate of HCoSV infection (62.5%) occurred in children younger than 6 years of age. The sampling date of stool specimens suggested that HCoSV infection occurred regularly over time. Also, the sampling origin of stool specimen showed that HCoSV infection was detected in almost all the governorates of Tunisia from the North to the South of the country. This study is the first report of HCoSV prevalence in the North African region. It contributes to a better knowledge on the geographic distribution and the epidemiology of these viruses.

Molecular epidemiology of coxsackievirus type B1.

Coxsackievirus type B1 (CVB1) has emerged globally as the predominant enterovirus serotype and is associated with epidemics of meningitis and chronic diseases. In this report, the phylogeny of CVB1 was studied based on the VP1 sequences of 11 North African isolates and 81 published sequences. All CVB1 isolates segregated into four distinct genogroups and 10 genotypes. Most of the identified genotypes of circulating CVB1 strains appear to have a strict geographical specificity. The North African strains were of a single genotype and probably evolved distinctly. Using a relaxed molecular clock model and three different population models (constant population, exponential growth and Bayesian skyline demographic models) in coalescent analysis using the BEAST program, the substitution rate in CVB1 varied between 6.95 × 10(-3) and 7.37 × 10(-3) substitutions/site/year in the VP1 region. This study permits better identification of circulating CVB1, which has become one of the most predominant enterovirus serotypes in humans.

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.

SARS-CoV-2 excretion kinetics in nasopharyngeal and stool samples from the pediatric population.

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.

[Genetic variability of type 3 echoviruses]. / Variabilité génétique des échovirus de type 3.

The aim of the present work was to assess the genetic and antigenic variability in the VP1 region of type 3 echviruses (E-3), an enterovirus serotype associated to meningitis, neuro-muscular diseases and type 1 diabetes in human. Forty-six VP1 sequences of E-3, among which 9 were isolated in tunisian infants, were included. Phylogenetic analyses and nucleotidic divergence rates were studied in the complete VP1 region and in a 290-nucleotides fragment in the 5' part of the P1. Aminoacid sequences were deduced in the aim to identify genotype-specific antigenetic determinants. E-3 sequences divided into two genogroups, I and II; the genetic variability within the E-3 serotype reached 29.1%. Genogroup I included sequences with a relatively high genetic diversity among each other, some of them grouped in one genotype with at most 15.1% divergence rate. The sequences included in Genogroup II have a maximum of 13.8% divergence corresponding probably to only one genotype. The two genogroups have a concomitant circulation and a wide geographical and temporal distribution. Aminoacid substitutions that may be specific to genogroups, genotypes and special variant were noted. This work provides a first tentative of classification of E-3 into genogroups and genotypes and reports new E-3 sequences from North Africa. It contributes to a better understanding of the molecular epidemiology of human enteroviruses, and of Echoviruses type 3 in particular, a serotype that remains insufficiently studied in the international literature.

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.

Typing of Human Cosaviruses by sequencing of full VP1: Update on global genetic diversity and identification of possible new genotypes circulating in Tunisia, North Africa.

Human Cosaviruses (HCoSVs) are relatively newly characterized picornaviruses; they have been described in non-polio acute flaccid paralysis, diarrheal patients, and healthy individuals. Previous studies showed HCoSV circulation in Tunisia and only six genotypes circulating in the country were reported. In the present study, we sequenced 27 complete VP1 genomic region from HCoSV isolates in human feces from healthy individuals and patients with acute flaccid paralysis in Tunisia. Most of the Tunisian sequences belong to species A (78%, 21 out of 27). Three sequences belong to species B, two to species E and one sequence to species D. The Tunisian sequences belonged to genotype A6, A7, A8, A10, A1, A17 and E2. Based on genetic distance criteria for assigning genotypes corresponding to neutralization serotypes in enteroviruses we also identified 4 new HCoSV genotypes named A25, B2, B3 and D6. Our study updates the genetic classification of HCoSVs, proposes new genotypes within species A, B and D and contributes to a better knowledge of the HCoSV circulation throughout the world.

Cytopathic effect of Human cosavirus (HCoSV) on primary cell cultures of human embryonic lung MRC5.

Human cosaviruses (HCoSVs) are newly discovered viruses in Picornaviridae family. Until now, most published studies reported HCoSV detection using molecular techniques and genetic characterization of the virus. Nevertheless, no laboratory has yet reported the replication of these viruses in cultured cell lines. In the present work, the propagation of HCoSV strains isolated from human fecal specimens in MRC5 cell line and their induced cytopathic effects (CPE) was studied. The first sign of virus growth was observed 24-48h after inoculation. The cells rounded up and clumped together rapidly; empty areas became visible and, on the third day of CPE, a remarkable decrease in live cells was observed. This represents the first report on in vitro model of HCoSV replication which opens up opportunities for future investigations of these new viruses.