Abstract
Background
The COVID-19 pandemic changed the typical patterns of respiratory infections globally. While SARS-CoV-2 illness exhibited explosive growth since 2020, the activity of other respiratory viruses fell below historical seasonal norms. The objective of this study was to assess the prevalence of seasonal respiratory viruses during the COVID-19 pandemic in Tunisia.
Methods
This is a retrospective cross-sectional study including 284 nasopharyngeal samples tested negative for SARS-CoV-2 during the period October 2020–May 2021. All samples were screened for fifteen common respiratory viruses. Either a fast syndromic approach using Biofire FILM ARRAY respiratory 2.1 (RP2.1) Panel, or end-point multiplex RT-PCRs detecting RNA viruses and Real-Time PCR detecting Adenoviruses were used.
Results
Overall, 30.6% (87/284) of samples were positive for at least one virus. Mixed infections were detected in 3.4% of positive cases. Enterovirus/Rhinovirus (HEV/HRV) was the most detected virus throughout the study period, especially during December 2020 (33.3% of all HEV/HRV being detected). During the 2020–2021 winter season, neither Respiratory Syncytial Virus nor Influenza Viruses circulation was observed. Metapneumovirus and Parainfluenza Viruses infections were detected during the spring season. The highest rate of respiratory viruses detection was observed in children and adults aged [0–10] years (50%) and [31–40] years (40%). HEV/HRV was the most detected virus regardless of age group.
Conclusions
Public health measures used to prevent SARS-CoV-2 spread in Tunisia were also effective to reduce transmission of the other respiratory viruses, especially Influenza. The higher resistance of HEV/HRV in the environment could explain their predominance and continuous circulation during this period.
Keywords: Molecular assay, Prevalence, Respiratory viruses, SARS-CoV-2, Tunisia
1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in a cluster of pneumonia cases with unknown etiology in Wuhan city, China in late 2019.1 The World Health Organization (WHO) declared the Coronavirus Disease 2019 (COVID-19) outbreak as a pandemic on March 11, 2020.2 In response to the exponentially rising of confirmed cases and deaths throughout the world and to preserve public health systems, stringent non-pharmaceutical interventions (NPIs) were implemented in many countries. These NPIs included blocking the importation of the virus by border closure and quarantine for returning travelers, national lockdowns restrictions and schools closure, widespread rapid testing, isolation of confirmed cases and contact tracing.3 , 4 Individual control and prevention measures were also taken such as social distancing and banning large gatherings, hand cleaning, and mask wearing.5 , 6 In Tunisia, SARS-CoV-2 was first identified in a person who returned from Italy on March 2, 2020. Following the identification of the first 50 cases, NPIs were adopted in order to interrupt the transmission chain of the virus in the community. These strict movement restrictions combined with public compliance mitigated effectively the spread of COVID-19 and resulted in a downward trend of confirmed cases, achieving zero cases between 4 and 12 June 2020.7 It has been previously suggested that the implementation of NPIs was also associated with a significant decrease in respiratory tract infections caused by other respiratory viruses, which are characterized by a clinical presentation similar to COVID-19 infection.8 , 9
The aim of this study was to assess the prevalence of seasonal respiratory viruses among Tunisian patients with acute respiratory infections, during a period characterized by an intense circulation of SARS-CoV-2 in the country.
2. Methods
2.1. Study population
This is a retrospective cross-sectional study conducted in the Virology Unit of the laboratory of Microbiology-Habib Bourguiba University Hospital, Sfax, Tunisia. This study included patients who were hospitalized or presented at the different outpatient clinics of the two University Hospitals, Habib Bourguiba and Hédi Chaker, Sfax, Tunisia, between October 2020 and May 2021. All these patients developed acute respiratory symptoms compliant with COVID-19 illness within the last 7 days. These patients showed negative results when screened for SARS-CoV-2 acute infection by real-time RT-PCR. Epidemiological and clinical data of included patients were collected: gender, age, and date of sample collection. The enrolled patients were divided into 7 age groups (years): [0–10], [11–20], [21–30], [31–40], [41–50], [51–60], and more than 60 years of age.
2.2. Sample collection and detection of respiratory viruses
Nasopharyngeal swabs from enrolled patients were collected by trained medical personnel using biosafety measures. Samples were put in a viral transport medium and transported within 24 h in a cold chain to the Laboratory of Microbiology. Viral genome extraction was performed using Chemagic Viral DNA/RNA kit special H96 (PerkinElmer, Inc.) on the Chemagic™ 360 instrument. Detection of SARS-CoV-2 was carried out using real-time RT-PCR commercialized kits targeting the nucleocapsid (N) and the Open Reading Frame 1 ab (ORF1ab) genes. The amplification reaction was performed on the Applied Biosystems QuantStudio 5 instrument (Applied Biosystems™, ThermoFisher Scientific). SARS-CoV-2 negative samples were then screened for fifteen common respiratory viruses including Influenza Viruses (IFVA, IFVB and IFVC), Human Respiratory Syncytial Virus (RSV), Human Metapneumovirus (HMPV), Human Parainfluenza Viruses 1, 2, 3 and 4 (PIVs), Human Enterovirus/Rhinovirus (HEV/HRV), Adenoviruses (ADV) and Human Coronaviruses (HCoV-NL63, HCoV-OC43, HCoV-E229, HCoV-HKU1). Simultaneous detection of these pathogens was performed using either end-point RT-PCR assay and Real-Time PCR (for Adenoviruses) as previously described,10, 11, 12, 13, 14 or a fast syndromic approach using BIOFIRE® FILMARRAY® Respiratory 2.1 (RP2.1) panel (BioFire Diagnostics). This latter allowed the detection of 4 additional respiratory pathogens (Bordetella parapertussis, Bordetella pertussis, Chlamydia pneumoniae, and Mycoplasma pneumoniae) (Table 1 ).
Table 1.
Molecular assays used for respiratory pathogens detection.
| Respiratory Pathogens | End-point Multiplex RT-PCR | Real Time PCR | Biofire FILM ARRAY Panel RP.2.1 |
|---|---|---|---|
| Multiplex 1 | |||
| Influenza Virus A (IFVA) | ✓ | ✓ | |
| Influenza Virus B (IFVB) | ✓ | ✓ | |
| Human Respiratory Syncytial Virus (RSV) | ✓ | ✓ | |
| Human Metapneumovirus (HMPV) | ✓ | ✓ | |
| Multiplex 2 | |||
| Human Parainfluenza Virus 1 (PIV1) | ✓ | ✓ | |
| Human Parainfluenza Virus 2 (PIV2) | ✓ | ✓ | |
| Human Parainfluenza Virus 3 (PIV3) | ✓ | ✓ | |
| Human Parainfluenza Virus 4 (PIV4) | ✓ | ✓ | |
| Multiplex 3 | |||
| Human Enterovirus/Rhinovirus (HEV/HRV) | ✓ | ✓ | |
| Influenza Virus C (IFVC) | ✓ | ||
| Multiplex 4 | |||
| Human Coronavirus (HCoV-NL63) | ✓ | ✓ | |
| Human Coronavirus (HCoV-E229) | ✓ | ✓ | |
| Human Coronavirus (HCoV-OC43) | ✓ | ✓ | |
| Human Coronavirus (HCoV-HKU1) | ✓ | ✓ | |
| Adenovirus (ADV) | ✓ | ✓ | |
| Bordetella parapertussis (IS1001) | ✓ | ||
| Bordetella pertussis (ptxP) | ✓ | ||
| Chlamydia pneumoniae | ✓ | ||
| Mycoplasma pneumoniae | ✓ | ||
3. Results
3.1. Positivity rate of respiratory viruses
Overall, 284 patients were tested during the period October 2020–May 2021. The sex-ratio was 0.97 and the median age was 45 years (range, 1–95). The positivity rate for at least one pathogen was 30.6% (87/284). The HEV/HRV was found to have the highest detection rate (63%), followed by HCoV-NL63 (11%) and HMPV (9%). Viral co-infections were detected in three cases (3.4%): (HEV/HRV, ADV, and HCoV-NL63); (HEV/HRV and PIV1); (IFVA and HCoV-NL63). Moreover, neither IFVC nor the four additional bacteria (when screened) were detected throughout the study. The distribution of detected respiratory viruses is shown in Fig. 1 .
Fig. 1.
Positivity rate of respiratory viruses from October 2020 to May 2021.
3.2. Seasonal distribution
The highest detection rate of respiratory viruses was found in December 2020 (51.1%), followed by April and May 2021 (45.4% and 38.7% respectively) (Table 2 ). The lowest positivity rate was observed in January, February and March 2021. HEV/HRV infections were detected throughout the study period, with a sharp increase in December 2020 (33.3% of all HEV/HRV being detected). HMPV and PIVs infections were predominantly detected during the spring season. Remarkably, the circulation of IFVs and RSV during 2020–2021 winter season was almost inexistent (Fig. 2 ).
Table 2.
Number of detected respiratory viruses per month during October 2020–May 2021.
| Month | Negative | Positive | Total | Positivity (%) |
|---|---|---|---|---|
| October | 24 | 5 | 29 | 17.2 |
| November | 21 | 7 | 28 | 25 |
| December | 21 | 22 | 43 | 51.1 |
| January | 19 | 7 | 26 | 26.9 |
| February | 25 | 6 | 31 | 19.3 |
| March | 50 | 13 | 63 | 20.6 |
| April | 18 | 15 | 33 | 45.4 |
| May | 19 | 12 | 31 | 38.7 |
| Total | 197 | 87 | 284 | 30.6 |
Fig. 2.
Monthly distribution of detected viruses.
3.3. Age group distribution
The highest positivity rate of respiratory viruses was observed in the age groups [0–10] and [31–40] years (50% and 40%, respectively), whereas the lowest positivity rate was found in the age group >60 years (14.2%) (Table 3 ). HEV/HRV prevalence was higher than the other respiratory viruses regardless of the age group (Fig. 3 ).
Table 3.
Number of detected respiratory viruses according to the age group.
| Age groups | Negative | Positive | Total | Positivity (%) |
|---|---|---|---|---|
| 0y-10y | 18 | 18 | 36 | 50 |
| 11y-20y | 21 | 11 | 32 | 34.3 |
| 21y-30y | 26 | 11 | 37 | 29.7 |
| 31y-40y | 27 | 18 | 45 | 40 |
| 41y −50y | 27 | 14 | 41 | 34.1 |
| 51y-60y | 36 | 8 | 44 | 18.1 |
| >60y | 42 | 7 | 49 | 14.2 |
Fig. 3.
Distribution of respiratory viruses according to the age group.
4. Discussion
The massive rollout of NPIs during the COVID-19 pandemic was shown to provide a large preventive effect not only on the SARS-CoV-2 spread worldwide, but also on the circulation of other respiratory viruses.15 The NPIs implemented changed over time and differed according to the epidemic evolution, which is specific to the concerned region.5
Indeed, in Tunisia, the first wave ranged from March to June 2020 and was characterized by a small number of circulating lineages, followed by the second wave ranging from July 2020 to January 2021, which was characterized by a higher genetic diversity with the circulation of at least 20 different lineages. The first emergence of variants of concern (VOCs) and variants of interest and/or under monitoring was reported during the third wave (especially the Alpha VOC), ranging from February to May 2021. During the beginning of the fourth wave (June–July 2021), the Delta B.1.617.2 VOC rapidly became the dominant variant in the country, resulting in an upsurge of COVID-19 cases.7 Therefore, several public health measures were implemented in response to these epidemics (Table 4 ). In the present study, we retrospectively assessed the prevalence of seasonal respiratory viruses among adults and children with acute respiratory illness, during a period that was characterized by an intensive circulation of SARS-CoV-2 (second and third waves) and a generalized application of restriction measures in the country.
Table 4.
Timeline of COVID-19 related public health measures implemented in Tunisia.
| Date | Public health COVID-19 restriction measures |
|---|---|
| 13 March 2020 | Wave1, Level 2 alert, Partial border closure, cancellation of event, school closure, banning gathering, official mask wearing, curfew from 8pm to 6am |
| 22 March 2020 | National lockdown for two weeks |
| 4 May 2020 | Lightened Curfew from 11pm to 5am, school opening |
| 14 June 2020 | Restrictions end for movement and business work |
| 27 June 2020 | Sea, land and Air border opening |
| 25 August 2020 | Curfew from 10pm to 5am, 10 days quarantine for not fully vaccinated travelers |
| 10 October 2020 | Wave 2, Curfew for high risk regions from 8pm to 5am |
| 28 October 2020 | Public school closure, Curfew for all governorate from 9pm to 5am |
| 7 December 2020 | Curfew prolongation to 15 January from 8pm to 5am |
| 15 January 2021 | Total Curfew for 4 days and then Curfew from 8pm to 5am from 23 January to 14 February, schools closure until 24 January |
| 18 January 2021 | Closure of weekly markets, Consumption ban on the premises in cafes, Suspension of gatherings until 24 January |
| 25 January 2021 | Regional travel ban, Extended suspension of gatherings until 14 February, Reopening of schools |
| 20 February 2021 | Wave3, first cases of Alpha variants in Tunisia |
| 8 March 2021 | Lightened Curfew from 10pm to 5am, inter-governorate movement for 3 weeks |
| 13 March 2021 | National company of vaccination beginning, intensive circulation of Alpha variant |
| 09 April 2021 | Ban Gathering and closure of weekly markets, Curfew from 10pm to 5am with traffic restriction starting at 7pm |
| 18 April 2021 | School closure, curfew maintained, restriction for circulation of private vehicles from 7pm |
| 17 May 2021 | Reopening of schools, Curfew maintained, restriction end for circulation of private vehicles |
The detection rates of seasonal respiratory viruses in the present study were relatively low throughout the study period (except for December, April, and May), ranging from 17.2% to 26.9%, suggesting that the adopted NPIs, since the beginning of the second wave, might have affected, at least in part, the circulation of other respiratory viruses. Interestingly, our results showed that the circulation of IFVs and RSV was the most affected, with a prevalence estimated only at 2% and 4%, respectively.
Actually, in Tunisia, limited data are available regarding the epidemiology of seasonal respiratory viruses before the emergence of SARS-CoV-2, especially for non-influenza viruses, and these data are in the most of cases related to specific populations.11 , 16 , 17 In a previous study conducted in 368 children hospitalized for community-acquired lower respiratory tract infection between January 2009 and March 2010 in Sfax, Tunisia, the positivity rate of at least one pathogen was high, estimated to 86.7%, with RSV being the predominant virus (42.7%), followed by Rhinoviruses (32.9%).11 In addition, according to a study evaluating the Influenza-like illness (ILI) surveillance system in our country during 2012–2015, IFVs alone were found to account for 27.6% of cases,17 whereas in the present study, the overall positivity rate for all respiratory viruses do not exceed 30.6%. Altogether, these findings highlight the potential impact of COVID-19 pandemic especially on IFVs and RSV circulation in our region, as it was previously reported worldwide by other studies, mainly due to the application of restriction measures.3 , 9 , 18
While a very weak activity of IFVs was noticed during the study period, HEV/HRV, which was the most prevalent virus (63%), showed sustained and strong circulation in both children and adults. Indeed, HEV/HRV is a non-enveloped virus resulting in a strong resistance to environmental factors. The virus resistance, in addition to prolonged shedding, could explain its persistence despite the application of NPIs, with less susceptibility to viral elimination by hand cleaning with water and soap.19 , 20 Although the NPIs generalized use worldwide is thought to play a major role in reducing significantly the circulation of other seasonal respiratory viruses, the impact of the introduction of the new pandemic virus SARS-CoV-2 should be considered to improve understanding of the epidemiological situation. Indeed, the interaction between the pandemic virus and the different circulating respiratory viruses may affect their usual epidemiological trend. In this context, it was previously suggested that a rhinovirus epidemic may have interfered with the spread of pandemic Influenza after the end of the summer in 2009, leading to a decreased circulation of Influenza during this period.21 , 22 The limited available data supporting the possibility of such interaction between respiratory viruses stresses the need to carry out further investigations to better understand the inter-viral complexities that govern community circulation, notably according to the different phases of the natural evolution of SARS-CoV-2.
5. Conclusion
This study highlights the low prevalence of respiratory viruses during the season 2020–2021 in Tunisia, a period that corresponds to the massive circulation of SARS-CoV-2 in the country. Our findings showed that the activity of IFVs was deeply affected; however, the higher resistance of HEV/HRV in the environment could explain their predominance and continuous circulation during this period.
Funding
This study was funded by the Habib Bourguiba University-Hospital of Sfax.
Informed consent statement
Patient consent was waived. According to the approval of the institutional review board and as samples were fully anonymized, the bioethics committee waived the requirement for informed consent to be able to manage the pandemic.
Declaration of competing interest
There are no conflicts of interest.
Acknowledgments
The authors acknowledge the technical staff of the Laboratory of Microbiology of the Habib Bourguiba University Hospital of Sfax for their efforts in COVID-19 and respiratory viruses diagnosis.
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