Investigation of the Frequency of Respiratory Viral Co-Infection in Respiratory Samples of Suspected COVID-19 Patients

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogenic coronavirus that emerged in late 2019 and has caused a pandemic of acute respiratory disease, named ‘coronavirus disease 2019’ (COVID-19), which threatens human health and public safety. This virus has spread fast all over the world and declared a pandemic by the World Health Organization (WHO) (1). In early 2020 and recent literature has shown that co-infection of SARS-CoV-2 with other respiratory viruses might occur (2-4). The most common viral agents causing respiratory tract infection are influenza viruses (IFVs), adenoviruses (AdVs), parainfluenza viruses (PIVs), respiratory syncytial virus (RSV), and rhinoviruses (RVs). In addition, intensive investigations have led to the identification of new human respiratory viruses, including human metapneumovirus (hMPV) and human bocavirus (HBoV) (5). Therefore, identifying the epidemiological patterns of respiratory pathogens may be useful for stopping the epidemic spread of COVID-19, providing information for the development of optimal diagnosis (7). In this study, the presence of the respiratory syncytial virus, human metapneumovirus virus, adeno, boca, parainfluenza, influenza A and B, endemic coronaviruses including NL63, OC43, 229E, and HKU-1 among specimens with positive and negative SARS-CoV-2 tests was investigated.

This cross-sectional study was conducted at the beginning of the pandemic between December 2019 and March 2019. In this study, oropharyngeal/nasopharyngeal swab samples of patients suspected of COVID-19 were collected from the laboratory of Labafinejad Hospital in Tehran. A total of 197 samples (91 positive for SARS-CoV-2 and 106 negative) were included in the study. Total nucleic acids (DNA and RNA) were extracted from 200 µL oropharyngeal/nasopharyngeal swab specimens by GeneAll Ribospin vRD DNA/RNA Extraction Kit (Seoul, South Korea) according to the manufacturer’s instruction. The extracted specimens were tested by real-time reverse transcription PCR (rRT-PCR) with novel coronavirus (2019-nCoV) Nucleic Acid Diagnostic Kit (PCR-Fluorescence Probing) (Sansure Biotech Inc.) using the Rotor-Gene® Q instrument targeting the ORF1ab and N genes of SARSCoV-2 RNA. Internal control targeting the RNase P gene was used to monitor the sample collection and rRT-PCR process to avoid false-negative results. Afterward, the remaining total nucleic acids were stored at -80°C for analysis of viral respiratory pathogens. The residual total nucleic acids were subjected to the real-time PCR assay for the detection of respiratory viral pathogens using the real-time thermal cycler Mic qPCR instrument (BioMolecular Systems). In this study, Geneova HiTeq 17 Viro Respiratory Pathogen One-step RT-PCR Kit as a multiplex TaqMan one-step real-time PCR method was used. It can detect various respiratory viral pathogens including SARS-CoV-2, Flu/A, Flu/B, Flu H1N1, HCoV-NL63, HCoV-229E, HCoVHKU1, HCoV-229E, HCoV-OC43, PIV1/2/3, AdV, hRV, HBoV1/2/3, hMPV, and RSV. The reactions were named as an unknown sample, positive control, and negative control. Tube 1: Fam channel (SARS-CoV-2 virus), HEX channel (RNase P gene as internal control), Texas Red channel (Influenza A virus), and CY5 channel (Influenza B virus) were selected. Tube 2: channel FAM (influenza H1N1virus), channel HEX (HCoV-NL63 and HCov-229E viruses), channel Texas Red (metapneumovirus), and channel CY5 (respiratory syncytial virus) were selected. Tube 3: FAM channel (HKU-1 and OC-43 viruses), HEX channel (parainfluenza viruses 2, 1 and 3), Texas Red channel (bocavirus 2, 1 and 3), and CY5 channel (adenovirus) were selected.

In this study, the presence of several respiratory viruses on 197 nasopharyngeal/oropharyngeal swab samples collected from suspected COVID-19 patients was investigated. Among the 197 people who entered the study, 88 people (45%) were women and 109 people (55%) were men. The average age of the subjects was 58 ± 13 years. The most common clinical symptoms found in these patients were fever in 169 people (86%), cough in 133 people (68%), sore throat in 111 people (56%), shortness of breath in 51 people (26%), headache in 47 people (24%), diarrhea in 38 people (19%), and asthma in 18 people (9.1%). Among 197 patients, 64 (32%) had diabetes, 70 (36%) had high blood pressure, 49 (25%) had kidney disease, 24 (12%) had heart disease, and 9.1% had asthma. Therefore, high blood pressure was considered as the most common underlying disease. In general, there was no difference in basic characteristics including age, gender, underlying disease, and clinical manifestations between the two groups. Among 197 patients, 14 people (7.1 percent) were infected with human metapneumovirus, 4 people (2 percent) with RSV infection, 3 people (1.5 percent) with influenza A, B, 2 people (1 percent) with parainfluenza virus, 8 people (1 NL63,229E infection, 3 people (5.1%) had HKU1, OC43 infection, 2 people (1%) had adenovirus infection, and 1 person (0.5%) had bocavirus infection. In this study, a total of 37 viruses (18.78%) were detected in oropharyngeal/nasopharyngeal swab samples from 197 subjects suspected of COVID-19. The results showed that human metapneumovirus was the most common cause of the disease in both groups of SARS-CoV-2 positive people [7 (7.7%)] and SARS-CoV-2 negative people [7 (6.6%)]. In addition, the rate of viral co-infection in SARS-CoV-2 positive (18.68%) and SARS-CoV-2 negative (18.86%) patients was almost the same. In 91 people with positive SARS-CoV-2 test, the infection with other viral infections was in the following order: 7 people (7.7%) infection with metapneumovirus, 2 people (2.2%) with RSV, 1 person (1.1%) influenza A/B, 1 person (1.1%) parainfluenza, 3 people (3.3 NL-63,229E, 1 person (1.1%) HKU1, OC43, 1 person (1.1%) adenovirus, and 1 person (1.1%) also had co-infection with bocavirus.

The data presented in this study strengthened our understanding of the epidemiology of different types of viral respiratory pathogens in suspected patients with COVID-19 during the fall and winter of 2019 in Iran. Using the multiplex PCR method, we reported the rate of simultaneous viral respiratory infections of 18.78%, which was mainly human metapneumovirus. Therefore, simultaneous screening of other viral respiratory pathogens will be useful for clinicians and researchers interested in the treatment and control of viral respiratory tract infections. It seems that the reduction of the prevalence of the respiratory syncytial virus, influenza virus, and other respiratory viruses during the COVID-19 pandemic in Iran happened due to public health measures, maintaining social distancing, and wearing masks.