Introduction
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which belongs to the beta-coronavirus group of
Coronaviridae family was declared a pandemic by WHO on 11th March 2020 [
1]. Like any other respiratory viruses, this virus transmission occurs via contact with infected individuals and respiratory droplets. In the areas with poor ventilation and more than 30 min exposure with infected people, even the airborne transmission is possible [
2,
3].
During the pandemic era, the focus of the health centers was on SARS-CoV-2 detection, while the presence of co-infection or other common respiratory infections with or without SARS-CoV-2 has been neglected [
4].
The clinical manifestations of SARS-CoV-2 may range from asymptomatic infection (85–90% cases) to mild illness known as influenza like illness (ILI) and severe acute respiratory illness (SARI) requiring hospitalization [
5]. They include sore throat, cough, fever, sputum production, fatigue, muscle pain or joint pain, shortness of breath, chills, headache, and other atypical symptoms. These manifestations are not much different from common respiratory viral infections [
6]. These similar symptoms of COVID-19 and other respiratory infections can affect the diagnostic and therapeutic efficacy of the COVID-19 suspicious cases. Therefore, identification of the causative respiratory pathogens is of great importance, which contributes to the reduced duration of the patient’s isolation, especially for those who are infected with other common respiratory viruses except for SARS-CoV-2 [
7].
A systematic review of the causes of respiratory illness in 2015 confirmed that RSV was the most frequent pathogen detected among patients of different age groups [
8]. Later in 2018, a surveillance study on the epidemiology of respiratory illness among hospitalized young children in Chengdu from 2009 to 2014 reported ARI patients with human rhinovirus (HRV) (23.0%), respiratory syncytial virus (RSV) (22.7%), human parainfluenza virus (HPIV) (13.4%), and human bocavirus (HBoV) (8.4%) [
9].
Also, during COVID-19 pandemic several studies highlighted decrement in the rate of other respiratory viruses such as RSV [
10], influenza A and B, especially B yamagata [
11], influenza virus and RSV [
12], and influenza A and B RSV, parainfluenza, metapneumovirus, rhino/enterovirus, and seasonal coronaviruses [
13].
In this study we aimed to investigate the epidemiological, clinical and virologic characteristics of the suspected COVID-19 but SARS-CoV-2-negative patients, and clarify the infection rate of other respiratory pathogens in Iran during COVID-19 pandemic.
Materials and methods
Study design and patients
This cross-sectional study was conducted between January 2020 and January 2022. Totally, 1,002 patients with acute respiratory infections who were referred to the COVID-19 National Reference Laboratory at Pasteur Institute of Iran, the National Collaborating Laboratory of Influenza and COVID-19 National Reference Laboratory at Pasteur Institute of Iran and Valfajr Health Center at Iran University of Medical Sciences, were included. Nasopharyngeal and oropharyngeal swab samples were collected to detect 17 common respiratory viruses via TaqMan one-step real-time multiplex PCR. Demographic and clinical data of the participants were obtained from their electronic medical records.
The eligibility criteria for the patients enrolled in this study were as follows: (i) fever (≥ 37.0 °C) and/or respiratory symptoms (e.g., cough, sputum production, shortness of breath, wheezing, and chest pain, etc.) and (ii) with no other underlying pulmonary diseases. The detailed information, such as demographic and clinical characteristics were recorded in case report forms.
Viral RNA isolation and sample processing
Nasopharyngeal and oropharyngeal swab samples were collected from the patients. After sampling, the specimens were placed into collection tubes containing 2 mL virus transport media (VTM). Viral nucleic acid was extracted using High Pure Viral Nucleic Acid Kit (Roche, Switzerland) as instructed. Briefly, 200 µl of each sample was mixed thoroughly with the Binding Buffer in the microcentrifuge tube. Following 10 min incubation at room temperature and few steps ethanol precipitating and washing, the pure viral nucleic acid was isolated by adding elution buffer to the column tube and centrifugation. The viral nucleic acid was stored at -80 °C for further analysis.
SARS-CoV-2 real-time quantitative PCR
SARS-CoV-2 real-time Reverse-transcription PCR (real-time RT-PCR) was performed by 2019-nCoV Nucleic Acid Diagnostic kit (Sansure biotech, Changsha, China) targeting two genomic regions of the virus: open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) according to the manufacturer’s guide. Real-time RT-PCR was done as per the manufacturer’s protocol [
14]. Briefly, 30 µL PCR master mix was added to 20 µL RNA template and primers. Thermal cycling was performed at 50 °C for 30 min (for reverse transcription), followed by 95 °C for 1 min (for cDNA pre-denaturation) and 45 cycles of 95 °C for 15 s and 60 °C for 30 s using the Rotor-Gene Q instrument (Qiagen, Germany).
The extracted RNAs of all SARS-CoV-2-negative samples were tested by three-reaction HiTeq 17 Viro Respiratory Pathogens Multiplex Detection Kit (GeneovA, Iran) for simultaneous identification of 17 respiratory viruses including SARS -CoV − 2, Influenza A, Influenza H1N1, Influenza B, HCoV -HKU1, HCoV -OC43, HCoV -NL63, HCoV − 229E, Metapneumovirus, Respiratory Syncytial Virus, Human Bocavirus 1–3, Human Parainfluenza 1–3, and Adenovirus. The analytical sensitivity and specificity of the tests are 95 and 100%, respectively. Thermal cycling was performed at 50 °C for 15 min (for cDNA synthesis), followed by 94 °C for 3 min (for holding) and 46 cycles of 94 °C for15 s and 60 °C for 30 s on the Rotor-Gene Q instrument (Qiagen, Germany).
Statistical analysis
Data was analyzed in 3 time periods, before scale-up of the lockdowns (January - March 2020), during lockdowns (March 2020 - March 2021), and after relaxing lockdown measures (March 2021 - Jan 2022) in Iran [
15]. Descriptive analyses were performed to characterize the specimens concerning age, gender, underlying disease, and clinical manifestations. Continuous variables were expressed as the mean (± SD) and categorical ones as the number and percentage (%). Logistic regression analysis was used to compare the proportion of respiratory viruses identified in different time periods over the course of the pandemic, as well as in different age groups (toddlers, children and adolescents, adults and elderlies), and diseases outcomes (outpatients, inpatients, and deaths). A P-value of ≤ 0.05 was considered significant. The Stata version 14 software was used for all data analyses.
Discussion
The most common causes of various infections in the lungs and respiratory system are respiratory viruses; moreover, their prevalence through the year varies in different seasons, which is conducted by the retrospective epidemiologic studies. Since the beginning of the COVID-19 pandemic in the early 2020, the non-SARS-CoV-2 respiratory infections decreased dramatically, which resulted in a significant drop in the diagnosis of such viruses mentioned in several publications like RSV [
10], influenza B, that its extinction was suggested [
11], RSV and influenza virus together [
12], and also common respiratory viruses such as influenza A and B viruses, RSV, parainfluenza, metapneumovirus, rhino/enterovirus, and seasonal coronaviruses [
13]. The global dominance of SARS-CoV-2 infection, and restrictions applied against COVID-19 such as quarantines, social distancing, virtual gatherings, and hygiene instructions, repetitive hand washings, high-level surface disinfections, and wearing masks led to the alterations in the pattern of other respiratory viruses distribution all over the world [
16].
In the second year of pandemic, the governments started to reduce the restriction guidelines gradually; therefore, it resulted in the growth of other viral respiratory infection cases by the following year [
17].
In this study, the oropharyngeal/nasopharyngeal swab samples of the patients with respiratory symptoms but negative COVID-19 PCR test results, were assessed for other 17 respiratory viruses. Most of the results of this study belong to the year 2020 and 2022, which corresponds to the time of legal restrictions reduction all over the world, including Iran. The gender composition and the percentage of inpatient-outpatient cases were chosen relatively equal to reduce the analytical error. The positivity of 28 samples (21.8%) for at least one viral infection other than SARS-CoV-2 in the period of our sampling confirms the fact that even in a viral pandemic, the rest of the viruses are still able to circulate and cause disease.
It also indicates that the majority of the studied population (about 80%) in our study got sick with other infectious agents including the rest of the viruses other than SARS-CoV-2 and bacteria. The number of viral infection cases is generally predominant compared to the bacterial ones with symptoms such as sepsis, sore throat, and yellowish sputum [
18,
19], but the incidence of bacterial secondary infection as super-infection is also very common. Therefore, detection of causative agents of the remaining 80% of the cases is very important, which might include high percentages of other infections that were not detected in our study such as common rhinoviruses that was not included in our detection kit panel.
Based on our study, among the samples with at least one viral infection, the highest rate was allocated to influenza A virus, which is in accordance with other studies [
18,
19]. It was followed by the seasonal coronaviruses [
20,
21], and the least infection was developed by adenoviruses which was in contrast to the results before the COVID-19 outbreak over the world [
19,
22‐
24].
Generally, most of the cases were young infants age < 2y (approx. 55%), which was predictable based on their immune system evolution [
25]. Although, it was expected to have the same frequency with the elderly adults age > 65y according to their vulnerability, underlying diseases, and insufficient immune responses; the least number of the patients were identified in this category: however, it must be noticed that most of the consequential preventive guidelines were applied to elderly people during the pandemic period; in addition, an effective healthcare services were applied to them even when the signs were not much life-threatening.
The significant increase in the hospitalized patients compared to the outpatients is also of importance. This is debatable that either these types of viral infections caused patients’ hospitalization, or hospitalization made the patients expose with various viral agents. However, the latter might be supported by the viral diversity (9 types) among the hospitalized participants.
Our study implicitly confirmed the proper and reliable function of the virological laboratories. It was previously undervalued by the public or some physicians that claimed the lack of precision and profession in the laboratories in addition to COVID-19 PCR test inefficiency caused the negative results of the patients with severe symptoms in the outbreak. Although the functionality and reliability of some laboratories and tests could be criticized in general but the study revealed that even in the middle of a pandemic other non-related infectious agents may have the ability to circulate and cause infection or disease as well.
The results of this study underline the importance of using molecular techniques with the ability to simultaneously detect multiple infectious agents (such as multiplex qRT-PCR) for syndromic surveillance of respiratory viral infections. The method used in this study, in addition to rapid detection of various viral agents, has also provided the possibility of detecting simultaneous infections (co-infection) of two or more viruses [
21,
26].
It is necessary to mention the constraints of the study. Small sample size in some periods of time due to COVID-19 preventive measures, and the lack of proper access to the respiratory samples are the most limitations of our study. The results would have been more reliable and meaningful if samples could be collected weekly over different years, but due to access limit and economic constraints, this was not feasible to conduct. Also, one of the other important limitations of this study was the inability of the used diagnostic kit to detect rhinoviruses, while rhinoviruses are the most common cause of common cold through the population annually.
Conclusion
In this study, the prevalence of respiratory viruses other than SARS-CoV-2 was investigated during the COVID-19 pandemic. This is one of the first studies investigated simultaneous detection during SARS-CoV-2 infection in Iran. In this period of time, influenza A virus and adenovirus showed the highest and the lowest prevalence, respectively. By increasing the age, the prevalence of respiratory viruses decreased mostly due to the preventive measures applied to the elder population more strictly. During the peak of SARS-CoV-2 prevalence (March 2020-March 2021), due to applying the quarantine, travel restriction, face mask, etc., the only common viral pathogen detected in our study was parainfluenza virus. Simultaneous screening of common viral respiratory pathogens will help to expand our understanding about the epidemiology and dynamics of common viral types during pandemics and also help the clinicians to diagnose the exact viral disease to assist patients both during pandemic periods and in non-pandemic contexts.
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