COVID-19 and Influenza Co-infection: A Systematic Review and Meta-Analysis

Background and Aim: Co-infection of COVID-19 with other respiratory pathogens which may complicate the diagnosis, treatment, and prognosis of COVID-19 emerge new concern. The overlap of COVID-19 and influenza, as two epidemics at the same time can occur in the cold months of the year. The aim of current study was to evaluate the rate of such co-infection as a systematic review and meta-analysis. Methods: A systematic literature search was performed on September 28, 2019 for original research articles published in Medline, Web of Science, and Embase databases from December 2019 to September 2020 using relevant keywords. Patients of all ages with simultaneous COVID-19 and influenza were included. Statistical analysis was performed using STATA 14 software. Results: Eleven prevalence studies with total of 3,070 patients with COVID-19, and 79 patients with concurrent COVID-19 and influenza were selected for final evaluation. The prevalence of influenza infection was 0.8% in patients with confirmed COVID-19. The frequency of influenza virus co-infection among patients with COVID-19 was 4.5% in Asia and 0.4% in the America. Four prevalence studies reported the sex of patients, which were 30 men and 31 women. Prevalence of co-infection with influenza in men and women with COVID-19 was 5.3 and 9.1%, respectively. Eight case reports and 7 case series with a total of 123 patients with COVID-19 were selected, 29 of them (16 men, 13 women) with mean age of 48 years had concurrent infection with influenza viruses A/B. Fever, cough, and shortness of breath were the most common clinical manifestations. Two of 29 patients died (6.9%), and 17 out of 29 patients recovered (58.6%). Oseltamivir and hydroxychloroquine were the most widely used drugs used for 41.4, and 31% of patients, respectively. Conclusion: Although a low proportion of COVID-19 patients have influenza co-infection, however, the importance of such co-infection, especially in high-risk individuals and the elderly, cannot be ignored. We were unable to report the exact rate of simultaneous influenza in COVID-19 patients worldwide due to a lack of data from several countries. Obviously, more studies are needed to evaluate the exact effect of the COVID-19 and influenza co-infection in clinical outcomes.


INTRODUCTION
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a rapid spreading viral pneumonia; known as coronavirus disease 2019 (COVID-19) originated from China in December 2019, and now become a worldwide public health emergency and finally becomes a global pandemic (1).
Recognition of COVID-19 is critical as it allows effective infection control measures and potentially beneficial antiviral therapy to be introduced, but the risk of COVID-19 co-infection should not be ignored.
COVID-19 co-infections with other respiratory pathogens which may complicate the diagnosis, treatment, prognosis of COVID-19 emerge new concern. These co-infections may even increase the disease symptom and mortality rate (2). The damage of respiratory ciliated cells due to some viral infections can also facilitate the conditions for an infection with SARS-CoV-2. The overlap of COVID-19 and influenza, as two epidemics at the same time can occur in the cold months of the year as seasonal influenza period begin. Both influenza virus A/B and SARS-CoV-2 are transmitted via close contact, respiratory droplets and contaminated surfaces, and cause a wide range of asymptomatic or mild to severe disease such as flu-like symptoms, pneumonia, loose of taste and smell, and even death (3)(4)(5).
There have been several studies published from different parts of world about COVID-19 co-infection with other pathogens especially influenza virus A/B. However, a systematic review that summarizes the results of existing studies has not yet been conducted.
Due to the importance of this issue, the aim of the present study was to systematically review the literature on COVID-19 co-infections with the influenza virus (type A and/or B) and potentially conduct a meta-analysis if possible.

METHODS
The present systematic review and meta-analysis was conducted according to the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) (6). In addition, the reference lists of covered studies were also checked to avoid missing suitable publications. Two different investigators independently checked this process (MG, AT). PICO algorithm was adopted to define inclusion and exclusion criteria for study selection. Accordingly, we evaluated the data on P (Patient, Population or Problem) = patients with COVID-19, I (Intervention or exposure) = influenza virus infection, C (Comparison) = not available, and O (Outcome) = co-infection COVID-19 and influenza A/B.

Search Strategy and Study Selection
All clinical studies investigating the presence of influenza virus infection in patients with COVID-19 were selected, except articles that reported only the prevalence of COVID-19 or the prevalence of influenza alone, review articles, abstracts presented in conferences, and duplicate studies. Relevant prevalence studies, case reports and case series were included. In the next step, titles and abstracts of all selected papers were screened by two investigators (SKN, PA).

Data Extraction
First author name; year of publication; type of study, country where the study was conducted; age and gender of patients, number of patients with confirmed COVID−19, number of patients with influenza co-infection and type of influenza virus were extracted from all eligible articles, and transfer to a data extraction form. To avoid any bias, two authors independently recorded the data (SKN, PA). The discrepancy was resolved by discussing between authors (BH, MD, and MG).

Quality Assessment
Quality assessment of included publications was performed using a checklist provided by the Joanna Briggs Institute (JBI) which assist in assessing the trustworthiness, relevance and results of published papers (7). Two investigators (HG, MV) checked the quality of included studies andthe publication bias independently.

Statistical Analysis
Statistical analyses were performed with STATA (version 14, IC; Stata Corporation, College Station, TX, USA). Meta-analysis of the continent-, country-, age-, sex-, and influenza virus type-specific prevalence rates of co-infection observed in the considered studies were conducted. We estimated the pooled proportion of co-infected patients. The pooled frequency with 95% confidence intervals (CI) was assessed using the fixed-effects (FEM), and the random effects models (REM). We assessed statistical heterogeneity using the I 2 statistical method. Cochran's Q and the I2 statistic were used to determine between-study heterogeneity. Begg's and Egger's tests were used to measure publication bias statistically (p < 0.05 was considered statistically relevant publication bias).

RESULTS
Our initial search retrieved 2,412 articles, after removing duplicates, 1,530 articles remain for the secondary screening. Following title and abstract screening 1,250 of the chosen articles were excluded. Guidelines, review articles, duplicate articles, systematic reviews, unrelated articles, and articles in languages other than English were among the excluded articles. After reviewing the full text of 280 studies, eventually, 26 studies met the inclusion criteria and were included for the final analysis (Figure 1). Final selected articles encompass 11 prevalence studies, 15 case report/case series. Tables 1, 2 summarized the characteristics of the included articles.

Prevalence Studies
Eleven prevalence studies were evaluated in the present review [4 reported from China (36.3%), 3 from USA (27.3%), and Brazil, Switzerland, Italy, and Iran each reported 1 study (9.1%)]. These studies had 3,070 participants with COVID-19 of which 79 patients had influenza co-infection. Sixty seven and 9 patients had an influenza co-infection A or B, respectively. The pooled prevalence of Influenza co-infection among patients with COVID-19 was 0.8 % (95% CI: 0.4-1.3).   An examination of publication bias was carried out by visual observation of the funnel plot (Supplementary Figure 1). Given the small number of studies, the funnel plots revealed some asymmetry. Then, to provide statistical evidence of funnel plot asymmetry, Egger's and Begg's tests were used. The results showed no signs of publication bias The meta-analysis of prevalence studies revealed that the frequency of influenza virus co-infection among patients with COVID-19 was 4.5% (95% CI 0.1-7.9) in Asia (5 studies, 70 patients), and 0.4 % (95% CI 0.0-0.7) from the American continent (4 studies, 6 patients). There were no reports of coinfection with influenza virus A/B in patients with COVID-19 from Africa or Oceania at the time of this study. The prevalence of co-infection with influenza in men (30 patients in 4 studies) and women (31 patients in 3 studies) with COVID-19 was 5.3 and 9.1%, respectively. The rate of co-infection in the age groups of <50 years, and more than 50 years was 1.7 and 4.6%, respectively. Table 3 shows more details of subgroup analysis of the studies.

Case Reports/Case Series Studies
Eight case reports (i.e., total 13 patients) and seven case reports (i.e., total 110 patients) highlighted an influenza co-infection in    As the details of these studies are listed in Table 4, it can be seen that hypertension (58.6%) and diabetes mellitus (48.3%) were among the most prevalent co-morbidities of co-infected patients. Haemodialysis with the prevalence of 17.2% was the third most common co-morbidity in these patients.
The clinical symptoms were also checked in patients with COVID-19 and influenza virus co-infection. Fever, cough, and shortness of breath were the most common clinical manifestations reported in this group of patients with a frequency of 89.4, 79.3, and 24.1 percent, respectively ( Table 5). Based on the results of included studies, nasopharyngeal swabs were the most common method of sampling for molecular evaluations (52%). Throat swab and sputum were only used for two and one patient, respectively.
After reviewing the laboratory findings recorded in the evaluated articles, it was found that the use of nasopharyngeal swabs was the most common method of sampling to diagnose the infection in patients. It was also found that elevated Creactive protein (CRP) (55.2%), erythrocyte sedimentation rate (ESR) (34.5%), and increased D-dimer (24.1%) were the most commonly reported findings, respectively. Leukopenia (3.4%) was the least reported abnormality.
The outcomes of COVID-19-Influenza co-infection were reported in 11 studies. According to them, 2 of 29 patients died (6.9%), and 17 out of 29 patients recovered (58.6%). Table 6 provides more information in this regard.
In the case reports/case series studies, the drugs used for treatment of patients with COVID-19 co-infected with influenza virus divided into sections such as antiviral drugs, antibacterial drugs, and a combination of drugs ( Table 5). Oseltamivir was the most widely used antiviral drugs reported in 8 studies. Hydroxychloroquine was also one of the most widely used drugs, as reported by 4 studies, 31% of evaluated patients received this agent. Among the antibacterial drugs azithromycin and ceftriaxone were more common antibiotics each was used in 4, and 3 studies, respectively. Details of the treatment regimens used in the reviewed studies are presented in Table 7.

DISCUSSION
COVID-19 as a highly transmissible viral infection which led to thousands of deaths worldwide has challenged the world's healthcare systems (34). Therefore, several studies have been performed to identify how the infection occurs, its symptoms and complications, as well as the factors involved in increasing its severity and mortality, and it is still the subject of studies. Cooccurrence of infections can be one of the causes of exacerbation of this disease. Lansbury et al. in their recent meta-analysis of 30 studies reports the pooled proportions of bacterial and viral co-infections in patients with COVID-19 were 7 and 3%, respectively (35).
In some viral infections, simultaneous infections with other bacterial and viral agents can exacerbate complications and increase disease mortality. The same can be said for SARS-CoV2 infection; however, there is not enough evidence to suggest that such concurrent infections increase disease morbidity or mortality.
COVID-19 often presents with nonspecific flu like respiratory symptoms. Influenza virus infection is thought to be similar to COVID-19 in clinical presentation, transmission mechanism, and seasonal coincidence. Simultaneous infection with influenza and SARS-CoV2 can interfere with the diagnosis and treatment of patients. In addition, this co-infection, especially in highrisk groups of patients, may aggravate the symptoms and complications of the disease (35).
SARS-CoV-2 and influenza virus are both airborne pathogens that affect the respiratory tract. Furthermore, SARS-CoV-2 appears to preferentially infect alveolar type II cells (AT2 pneumocytes), which are also the primary site of influenza virus replication. This can exacerbate the side effects of COVID-19 if there is a concomitant flu infection (36,37). As a result, the COVID-19 pandemic and seasonal influenza could put a large population at risk of contracting both viruses at the same time. To confirm the role of seasonal flu in the severity of the COVID-19 pandemic, Bai et al. in their research provide the first experimental evidence and reported that the preinfection with influenza virus strongly promotes SARS-CoV-2 virus entry and infectivity in cells and animals. They demonstrated that among the viruses tested; only IAV enhanced SARS-CoV-2 infection (38). This underscores the importance of the risk of influenza infection in patients with COVID-19. Especially in people with underlying factors, the occurrence of such a concomitant infection can aggravate the complications caused by  Due to the onset of influenza infection in the cold seasons of the year, scattered reports have been published about the influenza co-infection in patients with COVID-19 from the countries of the Northern Hemisphere that were in the cold seasons at the time of performing this study. Since there are no accurate statistics on the rate of such concurrent infections, herein, we evaluated the results of prevalence studies as well as case reports and case series in this field in the form of metaanalysis and systematic review.
According to a study recently published from China, concurrent infection of SARS-CoV-2 and influenza virus was common during the initial COVID-19 outbreak in Wuhan, and patients who had co-infection encounter a higher risk of poor health outcomes (39).
Our meta-analysis indicated that overall 1.2% of COVID-19 patients had influenza co-infection. Reports of the frequency of co-infection between COVID-19 and influenza vary from different parts of the world. Lansbury et al. estimated that 3% of patients hospitalized with COVID-19 were also co-infected with another respiratory virus. Based on their analysis, respiratory syncytial virus and influenza-A being the most common viral pathogens in co-infected patients (35).
It should be noted that all studies reviewed in the present study used the molecular method to confirm the presence of SARS-CoV-2 as well as influenza viruses, except for one study in which the presence of infection was diagnosed serologically through detection of IgM. Since serological test is not highly specific and may result in overestimation of infections the overall rate of co-infection reported in that study was higher than others (12).
Overall, the rate of co-infection with COVID-19 and influenza has been reported between 0.24 and 44% in the reviewed studied. This dispersion and difference can be due to the study population, the underlying conditions of the patients, the method of confirming the infection as well as the time and place of the investigation in terms of the prevalence of influenza.
Influenza co-infection among patients with COVID-19 was more reported from Asia and China country. Given the high population of Asia, as well as China, it is reasonable to be likely that patients with COVID-19, as well as influenza in the region be more prevalent, other studies have reported similar statistics too (40,41).
Based on the results of prevalence studies that reported the influenza virus type involved in the development of infection in patients with COVID-19, it was found that the prevalence of type A virus was higher than type B. This was to be expected due to the higher overall prevalence of influenza virus type A than type B (42).
Finally we should mention the limitations of our study. Since there is not enough information from many countries, we were not able to fully demonstrate the prevalence of influenza infection in COVID-19 patients worldwide. Many COVID-19 patients with influenza may not have been hospitalized and most of them could have been treated at home. Also, some articles lacked the necessary information to be added to the present study, and we had to exclude them. In addition, only studies published in English were included, which may have caused important studies to be missed. Finally, the heterogeneity exists among the included publications. Despite the random effects model allows for the presence of heterogeneity, there may still be some controversy about combining study estimates in its presence.

CONCLUSION
Although transmission of other respiratory viruses, including influenza, has decreased with the implementation of preventive measures such as social distancing and mask wearing, however, due to the importance of the issue and the possibility of severe complications in a group of high-risk or hospitalized patients influenza vaccination, especially in the elderly, is strongly recommended.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
MD and BH designed the study. BH and MG conducted the search strategy. SK, PA, AT, and MV performed the data extraction. HG and BH wrote and edited the manuscript. MD carried out the statistical analysis. All authors contributed to the article and approved the submitted version.