Abstract
The prevalence of coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza virus among referred patients in Hamadan province, Iran, from November 2, 2021, to January 30, 2022, was evaluated. Samples were obtained from 14,116 individuals with COVID-19 symptoms and screened for SARS-CoV-2 and influenza viruses using a multiplex real-time PCR panel assay. Of these patients, 14.19%, 17.11%, and 1.35% were infected with influenza virus, SARS-CoV-2, and both viruses, respectively. The majority of the coinfected patients were female outpatients aged 19–60 years.
Keywords: SARS-CoV-2, Influenza, Coinfection, COVID-19
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has spread rapidly around the world [1]. Simultaneously with the outbreak of the COVID-19 pandemic, the prevalence of influenza showed a sharp decline in different parts of the world due to people’s compliance with health principles [2]. The seasonal influenza A and B viruses continue to cause significant complications and deaths worldwide even though annual influenza vaccines have been introduced in recent decades. Influenza is a worldwide public health concern and has a high prevalence of associated conditions including cardiovascular conditions, chronic respiratory syndrome, and malnutrition. Moreover, it creates economic hardships and puts a financial burden on individuals [3, 4]. Influenza and COVID-19 are both contagious respiratory illnesses. Damage to respiratory ciliated cells due to influenza virus in the cold months of the year can also increase the severity of COVID-19 infection [4]. Coinfection with SARS-CoV-2 and other respiratory pathogens can make diagnosis, treatment, and prognosis difficult. Influenza virus infection in COVID-19 patients may increase hospitalization, disease symptoms, and the mortality rate [4, 5]. Influenza virus and SARS-CoV‐2 are transmitted via contact, aerosolized or respiratory droplets, and contaminated surfaces. COVID-19 and influenza have a wide range of symptoms from mild to severe [6, 7]. Although several studies from various parts of the world have investigated coinfection with SARS-CoV-2 and influenza virus, there are no definitive clinical symptoms that enable physicians to distinguish between COVID-19 and influenza [7], making it challenging to select the best medical treatment option. As a result, paying attention to coinfection with SARS-CoV-2 and other respiratory viruses is critical for early diagnosis and treatment. For this reason, in the present study, we evaluated the coinfection rate of influenza virus and SARS-CoV-2 in patients with COVID-19-like symptoms in Hamadan province, Iran, from November 2, 2021 to January 30, 2022.
The study samples were obtained retrospectively from a population (n = 14,116) in Hamadan province, Iran. All of the study subjects were inpatients or outpatients with COVID-19-like symptoms. Nasopharyngeal (NP) and oropharyngeal (OP) swabs were obtained using personal protective equipment during sampling. The patients were enrolled during the COVID-19 pandemic from November 2, 2021, to January 30, 2022, in Hamadan province. The collected swabs were stored in viral transport medium (VTM) at 4°C. RNA was extracted from 200 µL of pooled nasopharyngeal (NP) or oropharyngeal (OP) swab samples using a BehGen Virus RNA Extraction Kit (Tehran, Iran). A multiplex real-time qPCR assay for detecting the influenza virus and SARS-CoV-2 was performed by QIAquant96 Plex, using the RNA Hana Gene Kit (Tehran, Iran) according to the manufacturer’s protocol with RdRp- and N-gene-specific primers and probes for SARS-CoV-2 and M2- and NP-gene-specific primers and probes for influenza virus.) Briefly, 8 µL of the extracted RNA was added to 11 µL of PCR mix. Then, 1 µL of enzyme mix was added to a final volume of 20 µL. The thermal cycling conditions were 50°C for 10 min, 95°C for 3 min, and a standard two-step protocol of 42 cycles of 95°C for 10 s, 60°C for 30 s, and 37°C for 30 s. The categorical variables were reported as frequency and percentage, whereas the continuous variables were reported as mean and standard deviation (SD). The proportions of infection with influenza virus, infection with SARS-CoV-2, and coinfection with SARS-CoV-2 and the influenza virus were reported with a 95% confidence interval. The chi-square test was used to assess the associations between the categorical variables of the infection rates. In addition, a logistic regression model was used to evaluate the association between the data of the studied viruses. STATA (version 16.0; Stata Corporation, College Station, TX, USA) software was used for statistical analysis. P ≤ 0.05 was considered statistically significant.
We tested 14,116 samples for the presence of SARS-CoV-2 and the influenza virus from November 2, 2021, to January 30, 2022. Tables 1 and 2 present demographic and clinical information about the patients, 2409 (17.11%) and 2004 (14.19%) of which were positive for SARS-CoV-2 and influenza virus, respectively. Only 191 (1.35%) patients were infected with both viruses, most of whom were outpatients. The majority of the coinfected patients were women aged 19–60 years. Clinical symptoms were more likely to be present in patients with SARS-CoV-2 infection than in those without SARS-CoV-2: fever (n = 1614 [36%] vs. n = 4200 [36%], p < 0.05), cough (n = 4120 [35%] vs. n = 988 [41%], p < 0.05), headache (n = 4433 [38%] vs. n = 1132 [47%], p < 0.05), difficulty in breathing (n = 9101 [78%] vs. n = 2071 [86%], p < 0.05). Difficulty in breathing was less common among influenza-virus-infected patients than those without influenza virus infection (n = 9087 [75%] vs. n = 1282 [64%], p < 0.05). Patients coinfected with SARS-CoV-2 and influenza virus were more likely to have a fever, cough, runny nose, headache, and difficulty in breathing than patients without coinfection. Runny nose was more likely to be present in influenza-virus-infected patients than in those without influenza virus infection (n = 1262 [63%] vs. n = 5331 [44%], p < 0.05). However, it was less likely in SARS-CoV-2-infected patients than in those without SARS-CoV-2 infection (n = 97 [51%] vs. n = 6547 [47%], p < 0.05). We found a higher prevalence of coinfection with SARS-CoV-2 and influenza virus in the city of Hamadan than in the other cities in the province (Table 2). However, it should be noted that the total number of cases was higher in Hamadan than in the other cities. The adjusted odds of coinfection in people aged 19–60 and over 60 years were 2.68 and 3.86 times higher, respectively, than those in people younger than 18 years (Table 3). The odds of coinfection in men were 13.0% lower than those in women, adjusted for age and whether they were inpatients or outpatients (Table 3). The odds of coinfection were 13.0% lower in outpatients than in inpatients, adjusted for age and gender (Table 3).
Table 1.
The demographic and clinical characteristics of the patients with SARS-CoV-2 and influenza virus infection
| Characteristic | SARS-CoV-2 infection | Influenza virus infection | Coinfection with influenza virus and SARS-CoV-2 | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Negative n (%) |
Positive n (%) |
p-value | Negative n (%) |
Positive n (%) |
p-value | Negative n (%) |
Positive n (%) |
p-value | |
| Frequency | 11668 (82.89) | 2409 (17.11) | - | 12117 (85.81) | 2004 (14.19) | - | 13930 (98.65) | 191 (1.35) | - |
| Sex | 0.0001 | 0.0001 | |||||||
| Female | 5325 (45.66) | 1214 (50.39) | 5750 (47.47) | 806 (40.22) | 6460 (46.39) | 96 (50.26) | 0.287 | ||
| Male | 6338 (54.34) | 1195 (49.61) | 6362 (52.53) | 1198 (59.78) | 7465 (53.61) | 95 (49.74) | |||
| Age group | 0.0001 | 0.0001 | 0.0001 | ||||||
| ≤ 18 | 1890 (16.20) | 169 (7.02) | 1717 (14.17) | 349 (17.42) | 2055 (14.75) | 11 (5.76) | |||
| 19–60 | 8472 (72.61) | 1746 (72.48) | 8753 (72.24) | 1497 (74.7) | 10108 (72.56) | 142 (74.35) | |||
| ≥ 60 | 1306 (11.19) | 494 (20.51) | 1647 (13.59) | 158 (7.88) | 1767 (12.68) | 38 (19.90) | |||
| Type of referral | 0.0001 | 0.0001 | 0.314 | ||||||
| Inpatient | 1646 (14.11) | 560 (23.27) | 2010 (16.59) | 206 (10.28) | 2181 (15.66) | 35 (18.32) | |||
| Outpatient | 10022 (85.89) | 1847 (76.73) | 10105 (83.41) | 1798 (89.72) | 11747 (84.34) | 156 (81.68) | |||
| Symptoms | 0.0001 | 0.0001 | 0.0001 | ||||||
| Fever | 4200 (36) | 1614 (67) | 4726 (39) | 47 (47) | 5711 (41) | 108 (56) | |||
| Cough | 4120 (35) | 988 (41) | 3756 (31) | 521 (26) | 3900 (28) | 59 (31) | |||
| Runny nose | 5017 (43) | 602 (25) | 5331 (44) | 1262 (63) | 6547 (47) | 97 (51) | |||
| Headache | 4433 (38) | 1132 (47) | 3392 (28) | 601 (30) | 504 (36) | 78 (41) | |||
| Difficulty in breathing | 9101 (78) | 2071 (86) | 9087 (75) | 1282 (64) | 10865 (78) | 156 (82) | |||
Table 2.
The demographic characteristics of the patients with SARS-CoV-2 and influenza virus infection in different cities of Hamadan province
| City | SARS-CoV-2 infection | Influenza virus infection | Coinfection with influenza virus and SARS-CoV-2 | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Positive n (%) |
Negative n (%) |
p-value | Positive n (%) |
Negative n (%) |
p-value | Positive n (%) |
Negative n (%) |
p-value | |
| Asadabad | 88 (3.65) | 304 (2.61) | 0.0001 | 52 (2.59) | 343 (2.83) | 0.0001 | 10 (5.24) | 385 (2.76) | 0.0001 |
| Bahar | 56 (2.32) | 257 (2.20) | 58 (2.89) | 263 (2.17) | 4 (2.09) | 317 (2.28) | |||
| Dargazin | 38 (1.58) | 163 (1.40) | 39 (1.95) | 162 (1.34) | 6 (3.14) | 195 (1.40) | |||
| Famenin | 336 (2.88) | 26 (1.08) | 122 (6.09) | 240 (1.98) | 7 (3.66) | 355 (2.55) | |||
| Hamadan | 1,261 (52.35) | 6,525 (55.92) | 1141 (56.94) | 6649 (54.87) | 115 (60.21) | 7675 (55.1) | |||
| Kabudrahang | 109 (4.52) | 767 (6.57) | 199 (9.93) | 677 (5.59) | 7 (3.66) | 869 (6.24) | |||
| Malayer | 402 (16.69) | 1,635 (14.01) | 125 (6.24) | 1913 (15.79) | 9 (4.71) | 2029 (14.57) | |||
| Nahavand | 226 (9.38) | 714 (6.12) | 106 (5.29) | 836 (6.9) | 6 (3.14) | 936 (6.72) | |||
| Razan | 48 (2.97 ) | 347 (1.99) | 95 (4.74) | 301 (2.48) | 6 (3.14) | 390 (2.8) | |||
| Tuyserkan | 155 (6.43 ) | 620 (5.31) | 67 (3.34) | 733 (6.05) | 21 (10.99) | 779 (5.59) | |||
Table 3.
The relationship of the studied factors with influenza virus infection, SARS-CoV-2, and coinfection
| Variable | Odds ratio | 95% confidence interval | P>|z| | |
|---|---|---|---|---|
| Lower | Upper | |||
| Influenza virus infection | ||||
| Age (years) | ||||
| <19 | 1.00 | |||
| 19–60 | 0.77 | 0.68 | 0.88 | < 0.001 |
| ≥60 | 0.52 | 0.42 | 0.63 | < 0.001 |
| Gender | ||||
| Female | 1.00 | |||
| Male | 1.32 | 1.19 | 1.45 | < 0.001 |
| Type | ||||
| Inpatient | 1.00 | 1.37 | ||
| Outpatient | 1.61 | 1.89 | < 0.001 | |
| Influenza virus and SARS-CoV-2 coinfection | ||||
| Age (years) | ||||
| <19 | 1.00 | |||
| 19–60 | 2.68 | 1.44 | 4.98 | 0.002 |
| ≥60 | 3.86 | 1.96 | 7.62 | < 0.001 |
| Gender | ||||
| Female | 1.00 | |||
| Male | 0.87 | 0.65 | 1.16 | 0.36 |
| Type | ||||
| Inpatient | 1.00 | |||
| Outpatient | 0.87 | 0.58 | 1.31 | 0.52 |
| SARS-CoV-2 infection | ||||
| Age (year) | ||||
| <19 | 1.00 | |||
| 19–60 | 2.64 | 2.23 | 3.13 | < 0.001 |
| ≥60 | 3.80 | 3.14 | 4.60 | < 0.001 |
| Gender | ||||
| Female | 1.00 | |||
| Male | 0.85 | 0.77 | 0.93 | < 0.001 |
| Type | ||||
| Inpatient | 1.00 | |||
| Outpatient | 0.55 | 0.49 | 0.62 | < 0.001 |
COVID-19 is a highly contagious viral disease that can be severe and sometimes fatal, and many studies have been conducted to identify factors associated with disease severity and mortality [6, 8]. The coincidence of other diseases with COVID-19, especially viral infections, can make the diagnosis of the COVID-19 more difficult [9]. We evaluated the prevalence of coinfection with SARS-CoV-2 and influenza virus in people with COVID-19 symptoms from November 2, 2021, to January 30, 2022. In our study, the coinfection rate was 1.35%. Studies in China and the USA showed a similarly low coinfection rate (0.4% and 0.9%, respectively) among the patients. The low coinfection rate may be due to competition or interference between influenza virus and SARS-CoV-2 [10, 11]. The results of a meta-analysis of 30 studies showed that the rates of viral and bacterial coinfections with COVID-19 were 3% and 7%, respectively [9]. Influenza virus and SARS-CoV-2 are both airborne pathogens. Alveolar type II cells (AT2 pneumocytes) are the primary location for the proliferation of influenza virus and are also preferentially targeted by SARS-CoV-2 [12, 13], and respiratory symptoms are common in both COVID-19 and influenza. The mechanism of transmission, clinical manifestations, and seasonal emergence of influenza virus are often similar to those of SARS-CoV-2. Therefore, simultaneous infection with SARS-CoV-2 and influenza virus can be effectively prevented by treating influenza. Coinfection can increase the severity of the disease and the risk of death among high-risk COVID-19 patients [6, 9]. The COVID-19 pandemic and seasonal influenza could put a large number of individuals at risk of contracting both viruses at the same time [14]. In this study, people aged 19–60 years were more likely to get infected with SARS-CoV-2 and the influenza virus simultaneously. Based on the results of a study in Bangladesh, age > 60 years was considered a significant risk factor for COVID-19 [15]. In our study, males were more likely to be infected with influenza virus, whereas females were more likely to be coinfected with SARS-CoV-2 and influenza virus. Our results conflict with findings in Italy [16] and Bangladesh [17], in which more males were infected with SARS-CoV-2 than females. We found that more outpatients were coinfected with SARS-CoV-2 and influenza virus than inpatients. Receiving supportive medications and intensive care in the hospital may reduce the number of coinfections among hospitalized patients. Furthermore, considering Hamadan’s large population, the probability of detecting coinfection with SARS-CoV-2 and influenza virus is higher in Hamdan than in the other cities in the province. A limitation of our study is that there was no information about the history of any underlying disease such as hypertension, diabetes, or the use of the seasonal influenza vaccine. In conclusion, our data show that coinfection with influenza virus and SARS-CoV-2 was not very common in Hamadan province. This is likely due to the fact that seasonal influenza virus circulation was less intense during the COVID-19 pandemic. Health care during the COVID-19 pandemic may have contributed to the reduced seasonal influenza virus transmission.
Acknowledgement
The authors are grateful to the Health Department, Hamadan University of Medical Sciences, Hamadan, Iran, for their cooperation.
Authors’ contributions
Design and implementation of the research: DR, FAJ, and NA. Contribution to experimental work: DR and SB. Analysis of the results: SB and DR. Draft manuscript preparation: DR and NA. Funding acquisition: FTA. All authors reviewed the results and approved the final version of the manuscript.
Funding
This work was supported by the Deputy of Health of Hamadan University of Medical Sciences.
Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Declarations
Conflict of interest
The authors have no relevant financial or non-financial interests to disclose.
Ethical approval
This study was approved by the Research and Ethics Committee of Hamadan University of Medical Sciences (Hamadan, Iran).
Footnotes
Handling editor: William G Dundon
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
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Data Availability Statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.