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. 2024 Mar 20;344:199353. doi: 10.1016/j.virusres.2024.199353

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

Haifa Khemiri a,b,⁎, Iolanda Mangone c, Mariem Gdoura a,b, Khawla Mefteh d, Anissa Chouikha a,b, Wasfi Fares a,b, Alessio Lorusso c, Massimo Ancora c, Adriano Di Pasquale c, Cesare Cammà c, Samar Ben Halima a,b, Henda Krichen a,b, Hanen Smaoui d, Ilhem Boutiba Ben Boubaker e,f, Olfa Bahri g, Henda Touzi a,b, Amel Sadraoui a,b, Zina Meddeb a,b, Nahed Hogga a,b, Mouna Safer h, Nissaf Ben Alaya f,h, Henda Triki a,b,f, Sondes Haddad-Boubaker a,b,⁎
PMCID: PMC10966772  PMID: 38490581

Highlights

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    This study describes the SARS-CoV-2 strains circulating in Tunisian pediatric population during successive waves.

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    A total of 447 complete sequences were investigated in comparison to 628 sequences from Tunisian adult and worldwide.

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    Twenty-three lineages were identified; among them seven were not previously reported in Tunisia.

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    Phylogenetic analysis highlighted the pivotal role of children in virus transmission as well as the impact of vaccination on virus spread.

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    Continuous monitoring and vaccination should be considered to prevent child infection.

Keywords: Epidemiology, Pediatrics, Phylogenetic, Variants, SARS-CoV-2, Waves

Abstract

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.

1. Introduction

Coronavirus disease 2019 (COVID-19) appeared in 2019 in Wuhan, China, and caused a global health problem with more than 7031,216 deaths up to February 11, 2024 (WHO Coronavirus (COVID-19) Dashboard, 2023) https://covid19.who.int/. The causative agent is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a member of the family of Coronaviridae, subfamily Orthocoronavirinae, genus of Betacoronavirus and subgenus of Sarbecovirus, species SARS-related coronavirus (Wang et al., al., 2020). It is an enveloped, positive sense, single stranded RNA virus with a genome size of nearly 30 kb (Mercatelli et al., 2020); its genome contains structural and non-structural proteins. As other coronaviruses, its represented mainly by ORF1ab which codes for sixteen non-structural proteins as well as other genes encoding accessory proteins and four genes coding for four structural proteins (spike (S), envelope (E), membrane (M) and nucleocapsid (N)) (Yoshimoto, 2020).

Since its emergence, SARS-CoV-2 has rapidly evolved and undergone several mutations, resulting in the appearance of new variants. To distinguish and track global SARS-CoV-2 transmission lineage, a nomenclature system known as the Pango lineage nomenclature was developed by Rambaut (2020). This system complements two other SARS-CoV-2 nomenclatures: NextStrain and GISAID (Hadfield et al., 2018; Elbe and Buckland-Merrett, 2017). With the emergence of more virulent strains and to inform and prioritize the global response, SARS-CoV-2 variants were classified by the WHO into three groups: Variants of Concern (VOC), Variants of Interest (VOI), and Variants Under Monitoring (VUM) (WHO classification of SARS-COV-2 variants, 2022). As of the WHO update in March 2023, the Alpha, Beta, Gamma, Delta, and Omicron (B.1.1.529) variants were identified as previously circulating VOCs (Statement on the update of WHO's working definitions and tracking system for SARS-CoV-2 variants of concern and variants of interest, 2024) https://www.who.int/news/item/16-03-2023-statement-on-the-update-of-who-s-working-definitions-and-tracking-system-for-sars-cov-2-variants-of-concern-and-variants-of-interest.

At the beginning of the SARS-CoV-2 pandemic, COVID-19 in children was less common compared to adults. Various studies reported an underestimation of pediatric infections, which were mainly asymptomatic (Alteri et al., 2022; Zhu et al., 2021; Brinkmann et al., 2021). However, with the emergence of the Delta and Omicron variants, more symptomatic cases among children have been described, often leading to serious disease (Meyer et al., 2021; Yılmaz Çelebi et al., 2022; Clark et al., 2022; Lorthe et al., 2022). Despite this, limited information is available on the molecular epidemiology of the virus in infant populations (Alteri et al., 2022; Pandey et al., 2021), while a key role of children was suggested in the transmission of SARS-CoV-2 in middle and low-income countries (Laxminarayan et al., 2020; Kitano et al., 2021).

In Tunisia, reported data focused on the general population and described various SARS-CoV-2 lineages circulating from March 2020 to December 2021 (Fares et al., 2021a; Chouikha et al., 2022; Fares et al., 2021b; Haddad-Boubaker et al., 2023). The first and second waves occurred from March to June 2020 and from July 2020 to January 2021, respectively, characterized by the circulation of different imported lineages. The third and fourth waves, from February to May 2021 and May to December 2021, were caused by the Alpha and delta variants, respectively (Chouikha et al., 2022; Haddad-Boubaker et al., 2023). Since December 2021, the Omicron variant has been detected, causing the fifth wave, which has been poorly documented.

The present work provides complementary information on the different lineages of SARS-CoV-2 circulating in Tunisia from March 2020 to September 2022, including the under-reported Omicron wave. The genetic diversity of SARS-CoV-2 strains in the pediatric population was investigated by phylogenetic analysis of each SARS-CoV-2 variant and subvariant.

2. Materials and methods

2.1. Ethical statement

This study was approved by the local Medical Ethics Committee of Bechir Hamza Children's Hospital of Tunis, Tunisia (12/2021) and the Bio-Medical Ethics Committee of Pasteur Institute of Tunis, Tunisia (2020/14/I/LR16IPT/V1). It was performed under ethical standards according to the 1964 Declaration of Helsinki and its later amendments. Informed and written consent was obtained from the parents or legal tutors of children. All samples were investigated after de-identification with respect of patient anonymity.

2.2. Viral sequences

A total of 369 sequences were obtained from nasopharyngeal and fecal samples of pediatric cases (≤ 18 years old), in accordance with the World Health Organization's definition (World Health Organization (WHO), 2024) https://iris.who.int/bitstream/handle/10665/346552/WHO-2019-nCoV-Sci-Brief-Children-and-adolescents-2021.1-fre.pdf?sequence=2, from April 2020 to February 2022. The investigated specimens included samples from children in contact with positive adults (n = 121), collected at the Pasteur Institute of Tunis; samples from home-quarantined children (n = 118), collected by the Tunisian Ministry of Health staff; and others acquired from children hospitalized at Bechir Hamza Children's Hospital of Tunis (n = 130). All samples underwent immediate RNA extraction using the QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany), according to the manufacturer's instruction. Genome detection was performed using SARS-CoV-2-specific real-time Reverse Transcription PCRs (RT-qPCR) according to WHO-approved protocols, either by Hong Kong University (HKU) protocol, or China protocol (singleplex nucleoprotein (N) and singleplex Open reading frame Orf1b) (Chu et al., 2020) or Berlin protocol (singleplex envelop (E) and singleplex RNA-dependent RNA polymerase (RdRp)) (Corman et al., 2020).

2.3. Whole genome sequencing

Whole genome sequencing was carried out at two international collaborating institutes: the Istituto Zooprofilattico Sperimentale dell' Abruzzo e del Molise (Teramo, Italy) (IZS-Te) (n = 281) and the Quadram Institute (Norwich, United Kingdom) on behalf of the UK Health Security Agency (UKHSA) New Variant Assessment Platform (NVAP) (n = 88). The 369 sequenced samples presented a cycle threshold (CT) cutoff value of less than 33. Both sequencing sites used nearly equivalent sequencing protocols to those previously described (Chouikha et al., 2022; Fares et al., 2021b; Haddad-Boubaker et al., 2023; Molini et al., 2022).

Whole genome sequencing was assessed using the Illumina COVIDSeq assay (Curini et al., 2023). The extracted RNAs were reverse-transcribed into single-stranded cDNA, which was then amplified using CPP1 and CPP2 primers. Subsequently, the library underwent preparation and purification steps using Illumina Tune Beads (Illumina Technology, USA). The quantification of amplification products was measured using the Qubit 2.0 fluorometer (ThermoFisher Scientific, USA) and qualified on an Agilent Technologies 2100 Bioanalyzer using a high-sensitivity DNA chip, following the manufacturer's instructions. The generated libraries were pooled, denatured, diluted to 1.4pM and sequenced using NextSeq 500/550 and 1000 (Illumina, Inc, USA), providing 150 bp paired-end reads.

Data analysis was carried out using the Genpat platform (Di Pasquale et al., 2021) and Nextflow/ viralrecon pipeline (version 1.0.0) (Ewels et al., 2020). Quality control of sequences was conducted with FastQC (version 0.11.9) and filtered using Trimmomatic (version 0.39) and fastp (version 0.23.4) with a Phred quality score of 30 as the threshold. Consensus sequences were generated using Bowtie (version 2.5.3) and Ivar (version 1.4.2) tools by mapping to the SARS-CoV-2 reference genome (NC_045512). The obtained sequences were submitted to the GISAID database (https://www.gisaid.org) (Elbe and Buckland-Merrett., 2017) (Supplementary Table S1).

2.4. Variant identification

Sequences obtained in FASTA format were used for lineage and sub-lineage assignment using Pangolin (version 3.1.17) (https://cov-lineages.org/pangolin.html) (Rambaut et al., 2020).

2.5. Phylogenetic analysis

A total of 706 sequences available on GenBank (NCBI) (https://www.ncbi.nlm.nih.gov/labs/virus) and GISAID (https://www.gisaid.org) were considered to study the relatedness of Tunisian pediatric sequences with sequences from Tunisian adults and other regions of the world. These included previously reported Tunisian sequences (78 from children and 354 from adults) and 274 sequences from different geographic regions: African countries (n = 31), Asian countries (n = 71), European countries (n = 116), North American countries (n = 34), Oceanic countries (n = 4) and South American countries (n = 18) as indicated in Supplementary Table S2. Only sequences with less than 10% ambiguous nucleotides were included (Supplementary Table S3).

The phylogenetic analysis focused on Variants of Concern (VOCs) and the most frequently circulating lineages during the investigated period. For each variant, the sequences were aligned using Mafft online platform (Version7) (https://mafft.cbrc.jp/alignment/software/) (Katoh et al., 2019) with default parameters. The generated file was used to construct a Maximum-likelihood phylogenetic tree using IQ-TREE multicore program (v1.6.12) with 1000 bootstrap replicates (http://iqtree.cibiv.univie.ac.at/) (Minh et al., 2020), and the tree was visualized using Figtree software (Version 1.4.4) (Rambaut et al., 2010).

2.6. Statistical analysis

The statistical analysis was carried out using SPSS (version 20) via the ANOVA test corresponding to the analysis of variances (Allen and Bennett, 2012). This test determines whether there is a difference between the means of three or more groups. If a significant difference is found, the post hoc (Tukey) test is used to distinguish and specify the groups (Kim, 2017).

2.7. Data availability

The complete genome SARS-CoV-2 sequences generated and used in this study, which contained less than 10% ambiguous nucleotides (Ns), were submitted to the GISAID database (https://www.gisaid.org). Accession numbers are available in the Supplementary Table S1.

3. Results

3.1. Epidemiological features of collected samples

The epidemiological features of Tunisian SARS-CoV-2 sequences obtained from the pediatric population (n = 447) were investigated. This included 369 sequences obtained as part of the present study and 78 previously published in GISAID. The Tunisian pediatric cases were collected from March 2020 to September 2022. They comprised symptomatic patients presenting with mild COVID-19 clinical forms (n = 273), moderate forms (n = 9) and severe forms (n = 154), as well as asymptomatic individuals (n = 11) sampled in contact with confirmed adult cases. The study population included 234 males and 213 females (sex ratio equal to 1.09). They were aged from 13 days to 18 years old with an average and median age of 9.28 and 11 years, respectively. Case distribution by age, according to the World Health Organization (World Health Organization (WHO), 2024) https://iris.who.int/bitstream/handle/10665/346552/WHO-2019-nCoV-Sci-Brief-Children-and-adolescents-2021.1-fre.pdf?sequence=2, shows that the majority of cases belonged to adolescents [10–18 years] (55.26%) followed by children [1 year- 9 years] (23.71%), and newborns and infants groups [0– 11 months] (21.03%) (Fig. 1). A significant difference was detected among the three age groups (p-value < 0.05) (Supplementary Table S4).

Fig. 1.

Fig 1

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Distribution of SARS-CoV-2 sequences according to age in the total pediatric population.

3.2. Variant assignment

Using Pangolin (https://cov-lineages.org/pangolin.html, 433 out of 447 sequences were assigned to 23 variants, including 17 sub-variants as described in Supplementary Table S5. These consist of three VOCs: Alpha (n = 62), Delta (n = 178), and Omicron (n = 108), three VOIs: A.27 (n = 3), P2 (n = 1) and B.1.525 (n = 1) and other lineages: B (n = 1), B.1 (n = 14), B.1.1 (n = 8), B.1.1.1 (n = 1), B.1.22 (n = 2), B.1.1.189 (n = 3), B.1.1.198 (n = 2), B.1.160 (n = 28), B.1.177 (n = 12), B.1.362.2 (n = 1), B.1.395 (n = 1), B.1.428.2 (n = 2), C.36.3 (n = 1), B.1.1.50 (n = 1), B.1.597 (n = 1), B.1.551 (n = 1) and B.1.620 (n = 1). Within the Delta and Omicron variants, our sequences were divided in four sub-variants: AY.122 (n = 169), AY.4 (n = 5), AY.34 (n = 1) and B.1.617.2 (n = 3) for Delta and BA.1 (n = 64), BA.2 (n = 24), BA.4 (n = 4), and BA.5 (n = 16) for Omicron (Fig. 2).

Fig. 2.

Fig 2

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A maximum likelihood phylogenetic tree based on 433 SARS-CoV-2 sequences collected from March 2020 to September 2022 and a reference sequence (Wuhan, NC 045512) as an out-group. The most detected variant in the Tunisian pediatric population was represented with triangles. Variants of Concern (VOCs) were represented with red triangles: Alpha, Delta and Omicron. Other common variants were represented with blue triangles: B.1.160 and B.1.177. Other lineages present in small number, which circulated in Tunisia during the study period, are shown in black.

Variants detected in the pediatric population were predominantly Delta (39.8%), followed by Omicron (24.2%) and Alpha (13.9%). Two other non-VOC variants were recorded: B.1.160 (6.3%) and B.1.177 (2.7%). All the remaining variants were present in small proportions (Supplementary Fig. S1).

3.4. Timeline distribution of the SARS-CoV-2 lineage during the COVID-19 waves

From March to June 2020, during the first COVID-19 wave in Tunisia, the B.1.597 and B.1.1 lineages were detected in pediatric population. During the second wave, from July 2020 to January 2021, different lineages, with a limited number of strains, were detected: B.1, B.1.1, B.1.1.1, B.1.22, B.1.1.189, B.1.1.198, B.1.160, B.1.177, B.1.362.2, B.1.428.2 and B.1.395. The third wave, from February to May 2021, was characterized by the emergence of the Alpha (B.1.1.7) variant and the restricted circulation of others: B.1.620, P.2, A.27, B.1.525, B, and C.36.3. The fourth wave, from May to December 2021, showed the emergence of the Delta variant. Two Delta peaks appeared, including AY.122, AY.4, AY.34 and B.1.617.2 sub-variants. At the same time, other minor lineages circulated: B.1.1.50 and B.1.551. The fifth wave, from January 2022 to September 2022, was characterized by the emergence of the Omicron variant. Two Omicron peaks involved BA.1 and BA.2 sub-variants in the first and BA.4 and BA.5 sub-variants in the second. B.1.160 and B.1.177 circulated discreetly during all the waves (Supplementary Table S5) (Fig. 3).

Fig. 3.

Fig 3

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SARS-COV-2 variants circulating in the Tunisian pediatric population between March 2020 and September 2022. In the background, is the number of positive cases detected in Tunisia according to the statistics published in the World Health Organization (WHO) website (https://covid19.who.int/region/emro/country/tn).

3.5. Phylogenetic tree

The phylogenetic analysis included Delta, Omicron, and Alpha VOCs, as well as the B.1.160 and B.1.177 variants, which were most representative of our pediatric population.

3.5.1. Delta variant

Phylogenetic analysis included sequences from the Tunisian pediatric population (n = 163), Tunisian adults (n = 163), and other countries (n = 117) (Supplementary Table S3). The tree displays three major groups corresponding to the three sub-variants: AY.122, AY.4 and B.1.617.2 (Fig. 4-A). For the AY.122 subvariant, Tunisian sequences were strongly related to each other and genetically independent from other sequences of the world. They formed three major phylogenetic clusters (Fig. 4-B). The AY.4 and B.1.617.2 groups involved a limited number of Tunisian pediatric sequences related to other European sequences.

Fig. 4.

Fig 4

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Phylogenetic tree generated with Delta variant sequences and a reference sequence (Wuhan, NC 045512) as an out-group. The blue branches correspond to the Tunisian pediatric population. The red branches represent Tunisian adults. Black branches correspond to sequences from the world. A: corresponds to the three Delta sub-variants: AY.122 is highlighted in gray, the AY.4 sub-variant is highlighted in yellow and B.1.617.2 sub-variant is highlighted in green. B: corresponds to the AY.122 subvariants. Tunisian sequences formed 3 clusters highlighted in pink, yellow and green, respectively.

3.5.2. Omicron variant

The phylogenic analysis included sequences from the Tunisian pediatric population (n = 95), Tunisian adults (n = 95), and other countries (n = 94) (Supplementary Table S3). The tree displayed four clusters involving BA.1, BA.2, BA.4, and BA.5 sub-variants, respectively (Fig. 5). In the four sub-variant groups, sequences from Tunisian pediatrics and adults were closely related to worldwide sequences.

Fig. 5.

Fig 5

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Phylogenetic tree generated with Omicron variant sequences and a reference sequence (Wuhan, NC 045512) as an out-group. The blue branches correspond to the Tunisian pediatric population. The red branches represent sequences from Tunisian adults. The black branches correspond to sequences from different regions of the world. BA.1is highlighted in gray; BA.2 is highlighted in yellow; BA.4 is highlighted in green and BA.5 is highlighted in pink.

3.5.3. Alpha variant

The phylogenetic analysis included sequences from Tunisian pediatric population (n = 58), Tunisian adults (n = 58), and other countries (n = 37) (Supplementary Table S3). The tree displays five major clusters. One cluster included only Tunisian pediatric and adult sequences and 4 clusters involved Tunisian sequences with others from different regions (Fig. 6).

Fig. 6.

Fig 6

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Phylogenetic tree generated with Alpha variant sequences and a reference sequence (Wuhan, NC 045512) as an out-group. The blue branch corresponds to the Tunisian pediatric population. The red branch presents sequences from Tunisian adults. The black branch corresponds to sequences from different regions of the world. Cluster 1 represents the Tunisian sequences only (pediatrics and adults), highlighted in gray. Clusters 2, 3, 4 and 5 correspond to both Tunisian sequences and sequences from other countries, highlighted in yellow, pink, purple and green, respectively.

3.5.4. B.1.160 variant

The phylogenetic analysis included sequences from the Tunisian pediatric population (n = 26), Tunisian adults (n = 26), and other countries (n = 16) (Supplementary Table S3). The tree displays three major clusters: two clusters correspond to the pediatric and adult Tunisian sequences genetically close to each other and one cluster includes Tunisian sequences and others from different regions of the world (Fig. 7).

Fig. 7.

Fig 7

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Phylogenetic tree generated with B.1.160 sequences and a reference sequence (Wuhan, NC 045512) as an out-group. The blue branch corresponds to the Tunisian pediatric population. The red branch represents Tunisian adults. The black branch corresponds to sequences from other countries. Cluster 1 and 3 included Tunisian pediatric and adult sequences, respectively, highlighted in gray and yellow. Cluster 2 included both Tunisian sequences and sequences from different countries, highlighted in green (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

3.5.5. B.1.177 variant

The phylogenetic analysis included sequences from the Tunisian pediatric population (n = 12), Tunisian adults (n = 12), and other countries (n = 10) (Supplementary Table S3). The tree displays two major clusters. One cluster included the Tunisian pediatric and adult sequences and the second included sequences from other regions (Fig. 8).

Fig. 8.

Fig 8

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Phylogenetic tree generated with sequences of B.1.177 variants and a reference sequence (Wuhan, NC 045512) as an out-group. The blue color corresponds to the Tunisian pediatric population. The red color presents sequences from Tunisian adults. The black color corresponds to sequences from different regions of the world. Cluster 1 represents the Tunisian sequences only (pediatrics and adults), highlighted in gray. Cluster 2 represents sequences from other regions of the world, highlighted in yellow.

4. Discussion

In the beginning of the COVID-19 pandemic, the molecular epidemiology of SARS-CoV-2 was mainly based on sequences obtained from the general population, especially from adult cases, as children very rarely developed symptomatic clinical forms with the first SARS-CoV2 variants (Boshier et al., 2021; Oude Munnink et al., 2021). Thus, the investigation of sequences from pediatric populations may provide valuable complementary information, helping to draw more comprehensive representation of circulating variants and lineages. Furthermore, it can help further the understanding of the potential role of children in the dynamics of virus transmission (Zhu et al., 2021; Meyer et al., 2021; Yılmaz Çelebi et al., 2022; Clark et al., 2022; Lorthe et al., 2022; Pandey et al., 2021).

This work describes the SARS-CoV-2 variants circulating in the Tunisian pediatric population from March 2020 to September 2022. Twenty-three different variants were detected including three VOCs (Alpha, Delta, Omicron), VOIs (P.2, A.27, B.1.525) and other minor variants (n = 17). Previous published data from Tunisia during the period of March 2020 to December 2021, reported the circulation of the same VOCs and VOIs (Chouikha et al., 2022; Haddad-Boubaker et al., 2023). However, different SARS-CoV-2 variants were identified as part of the present study. During the first wave, different variants were detected among which were B.1.597 and B.1.1. In the previous study, such variants were later detected, during the second wave. Our findings also highlight the detection of additional variants during the second, the Alpha and the Delta waves: B.1, B.1.362.2, B.1.395 during the second wave; B.1.620, B, C.36.3 during the Alpha wave and B.1.1.50, B.1.551 during the Delta wave (Fares et al., 2021a; Chouikha et al., 2022; Haddad-Boubaker et al., 2023). Furthermore, during the delta wave, only four sub-lineages (B.1.617.2, AY.4, AY.34 and AY.122) were detected among children with two peaks in contrast with previous published data describing more sub-lineages and only one Delta peak among the global population (Haddad-Boubaker et al., 2023). The first peak corresponds to the emergence of the Delta variant in May 2021 followed by a decrease in pediatric cases since June 2021 and a second increase of the case number since September 2021. The fluctuation of pediatric cases may coincide with the scholar activities in Tunisia, with the beginning of the summer holidays in June and class entry in September. Other studies have also reported an association between case frequency among the pediatric population and school activities and a notable rise in Delta variant cases within primary and secondary schools (Khemiri et al., 2022; Mensah et al., 2021; Torjesen, 2021).

In contrast to the first COVID-19 waves, the Omicron wave was relatively poorly documented in Tunisia and our study provides valuable data, starting from January 2022 to September 2022. Among the pediatric population, two distinct peaks were observed: the first, in February 2022, including the BA.1 and BA.2 sub-variants, and the second, in July 2022, with the BA.4 and BA.5 sub-variants. The majority of European and North American countries reported the circulation of the same sub-variants during the same periods (Konyak et al., 2022; Setiabudi et al., 2022; Chatterjee et al., 2023; Maldonado-Cabrera et al., 2023; Dhawan et al., 2022). Notably the BA.3 sub-variant, which was first detected in South Africa and then circulated in multiple countries, was not detected in our series (Konyak et al., 2022; Setiabudi et al., 2022; Chatterjee et al., 2023; Maldonado-Cabrera et al., 2023; Dhawan et al., 2022) Moreover, as of January 17, 2024, there is no available data on BA.3 sequences from Tunisia published on GISAID.

For better comprehension of circulation dynamics of SARS-CoV-2 variants among children and adults and in the context of a global dataset, deep phylogenic analyzes were achieved for the five most common variants (Alpha, Delta, Omicron, B.1.160 and B.1.177). A close relationship was observed between Tunisian pediatric and adult's sequences among these four variants. These findings highlight the pivotal role of children in the transmission of SARS-CoV-2 either in school and household environments as well as within community. Previous studies pointed to the key role of children in the SARS-CoV-2 transmission in middle-income countries, facilitated by special cultural, economic and sociological conditions (Laxminarayan et al., 2020; Kelly, 2020). Children can potentially transmit the virus through close contacts with other children and adults, especially in crowded areas such as scholar structures and districts with poor social economic conditions (Laxminarayan et al., 2020; Kelly, 2020). Therefore, adequate measures, such as vaccination, should be adapted to prevent infection among children and also to limit their role in the virus spread (Maldonado-Cabrera et al., 2023; Bard et al., 2021).

In comparison with worldwide sequences, our findings highlighted three patterns of circulation, likely related to the local epidemiological context. First, for B.1.160 and Alpha variants, heterogenous groups including Tunisian and worldwide strains as well as homogenous groups including mainly Tunisian ones were observed. This finding suggests the occurrence of multiple importation events at the same time with the establishment of autochthonous circulation. Indeed, after a national drastic lockdown period, from March to July 2020, the reduced international travel restrictions contributed to new variants’ introductions and the relaxation of preventive measures within the general population allowed the establishment of autochthonous circulation. The occurrence of virus importation events is heightened by Tunisia's geographical position at the core of the Mediterranean Sea and its active international trade with Europe, Africa and also Asia (Fares et al., 2021a, 2021b; Chouikha et al., 2022; Haddad-Boubaker et al., 2023).

The autochthonous circulation was mainly observed with the B.1.177 variant and the AY.122 Delta subvariant introduced in September 2020 and May 2021, respectively. Indeed, our phylogenetic analysis of the latter variants found the presence of genetic groups exclusive to Tunisian sequences. Such findings were previously observed in the global Tunisian population for the Delta variant and for other underlying variants in Italy and Dominican Republic (Alteri et al., 2022; Haddad-Boubaker et al., 2023; Paulino-Ramírez et al., 2023). Overall, the autochthonous circulation of B.1.177, AY122 Delta sub-lineage and some sub-lineages of the Alpha and B.1.160 variants may be the consequence of the decline in prevention measures especially among children, as part of their scholar and social activities. The absence of SARS-CoV-2 vaccination for pediatric population and the low coverage for adults’ vaccination during this period may have contributed to the establishment of autochthonous circulation (Haddad-Boubaker et al., 2023). Indeed, SARS-CoV-2 vaccination in Tunisia only started in August 2021 for age group 15–17 years and, in November 2021, for those aged 12–14 years (Strategie vaccinale contre la COVID-19 en tunisie, 2022) http://www.santetunisie.rns.tn/images/strategie-vaccination-covid-19mars2022.pdf.

In contrast, regarding the Omicron variant, close genetic connections were observed with sequences from different regions of the world, with no clustering of Tunisian sequences in independent groups. Such a finding suggests the occurrence of multiple importation events with limited autochthonous spread among children. The use of SARS-CoV-2 vaccination targeting children aged over 12 as well as adult groups likely limited virus circulation among children (Haddad-Boubaker et al., 2023). According to the World Health Organization (WHO) and the Centers for Disease Control and Prevention (CDC), opting for a COVID-19 vaccine proves to be a safer and more reliable approach for fortifying immunity and preventing disease (Safety of COVID-19 Vaccines, 2024; CDC Centers for Disease Control and Prevention, 2023) https://www.who.int/news-room/feature-stories/detail/safety-of-covid-19-vaccines.

Nevertheless, phylogenetic analysis of some variants relies on a relatively limited number of sequences, due to data availability constraints. A larger dataset would amplify the statistical robustness of findings and provide a more understanding of the evolutionary relationships and dynamics associated with these variants.

5. Conclusion

In conclusion, this study complements previous findings on the molecular epidemiology of SARS-CoV-2 variants in Tunisia and underscores the circulation of the Omicron variant and its sub-variants within the pediatric groups. It offers valuable information on the circulation of SARS-CoV-2 strains within the pediatric population and their key role in the virus transmission. It is worthy to note that the pediatric population requires continuous monitoring and vaccination in the aim to prevent child infection and to achieve better management of the pandemic.

Funding statement

This study was co-funded by the Tunisian Ministry of Health, the Tunisian Ministry of Higher Education and Research (LR20IPT02), World Health organisation Tunisia office and the Italian Ministry of Health (IZS AM 03/22 RC dal titolo "OneCoV: coronavirus animali emergenti e impatto nella Salute Pubblica) the project MEDNET.

This research was also partially supported by EU funding within the Next Generation EU-MUR PNRR Extended Partnership initiative on Emerging Infectious Diseases (Project no. PE00000007, INF-ACT).

Institutional review board statement

This study was approved by the local Medical Ethics Committee of Bechir Hamza Children's Hospital of Tunis, Tunisia (12/2021) and the Bio-Medical Ethics Committee of Pasteur Institute of Tunis, Tunisia (2020/14/I/LR16IPT/V1).

Informed consent statement

The parents or legal tutor of children provided informed and written consent to collect samples and data specifically for this study.

Author statement

During the preparation of this work, the authors did not use the generative artificial intelligence (AI) and AI-assisted technologies in the writing process.

The views expressed in this article are those of the author(s) and are not necessarily those of UK Health Security Agency or the Department of Health and Social Care.

The following supporting information can be downloaded at: Supplementary Table S1: Accession numbers and names of complete genome SARS-CoV-2 sequences generated in this study and submitted in GISAID; Supplementary Table S2: Geographical distribution of used sequences; Supplementary Table S3: Description of sequences used for phylogenetic analysis; Supplementary Table S4: Statistical analysis between the 3 age groups using ANOVA test; Supplementary Table S5: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage distribution in Tunisia from March 2020 to September 2022 according to month of sample collection. Variants of concern (VOCs) and variants of interest (VOIs) are shown in red and in blue, respectively; Supplementary Fig. S1: Frequency of SARS-CoV-2 variants detected in the Tunisian pediatric population.

CRediT authorship contribution statement

Haifa Khemiri: Writing – original draft, Visualization, Resources, Investigation, Data curation. Iolanda Mangone: Supervision, Resources, Investigation. Mariem Gdoura: Resources, Investigation, Data curation. Khawla Mefteh: Resources, Investigation. Anissa Chouikha: Resources, Investigation. Wasfi Fares: Resources, Investigation. Alessio Lorusso: Writing – review & editing, Validation, Funding acquisition. Massimo Ancora: Resources, Investigation. Adriano Di Pasquale: Writing – review & editing, Validation, Supervision, Resources, Investigation, Funding acquisition. Cesare Cammà: Writing – review & editing, Resources, Investigation, Funding acquisition. Samar Ben Halima: Resources, Investigation. Henda Krichen: Resources, Investigation. Hanen Smaoui: Resources, Investigation. Ilhem Boutiba Ben Boubaker: Resources, Investigation. Olfa Bahri: Resources, Investigation. Henda Touzi: Resources, Investigation. Amel Sadraoui: Resources, Investigation. Zina Meddeb: Resources, Investigation. Nahed Hogga: Resources, Investigation. Mouna Safer: Resources, Investigation. Nissaf Ben Alaya: Resources, Investigation. Henda Triki: Writing – review & editing, Validation, Supervision, Funding acquisition, Conceptualization. Sondes Haddad-Boubaker: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Funding acquisition, Data curation, Conceptualization.

Declaration of competing interest

Declarations of interest: none of the authors have any conflict of interest.

Acknowledgments

The authors gratefully acknowledge the Instituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise G. Caporale (IZSAM), Teramo, Italy, and the UK Health Security Agency (UKHSA) New Variant Assessment Program (NVAP) on behalf of the International COVID-19 response working to strengthen genomic surveillance of SARS-CoV-2 and data sharing, for their contribution in the whole-genome sequencing (WGS) as well as the WHO office in Tunisia for covering sample shipment fees to IZSAM and NVAP, respectively. The authors also acknowledge the dedication of the technical staff of the service of external consultants at Pasteur Institute of Tunis for their efforts in samples and information collection, and the National Observatory of New and Emerging Diseases (ONMNE) for coordinating sample and data collection.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.virusres.2024.199353.

Contributor Information

Haifa Khemiri, Email: haifa.khemiri@pasteur.utm.tn.

Sondes Haddad-Boubaker, Email: sondes.haddadboubaker@pasteur.tn.

Appendix. Supplementary materials

mmc1.docx (58.3KB, docx)
mmc2.xlsx (16.9KB, xlsx)
mmc3.docx (37.5KB, docx)
mmc4.docx (21.8KB, docx)
mmc5.xlsx (23.2KB, xlsx)
mmc6.docx (38.3KB, docx)

Data availability

  • I have shared the file containing the accession numbers of sequences generated in the study (Supplementary Table S1. Further data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (58.3KB, docx)
mmc2.xlsx (16.9KB, xlsx)
mmc3.docx (37.5KB, docx)
mmc4.docx (21.8KB, docx)
mmc5.xlsx (23.2KB, xlsx)
mmc6.docx (38.3KB, docx)

Data Availability Statement

The complete genome SARS-CoV-2 sequences generated and used in this study, which contained less than 10% ambiguous nucleotides (Ns), were submitted to the GISAID database (https://www.gisaid.org). Accession numbers are available in the Supplementary Table S1.

  • I have shared the file containing the accession numbers of sequences generated in the study (Supplementary Table S1. Further data will be made available on request.

Articles from Virus Research are provided here courtesy of Elsevier

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