A New Perspective; Co-Infection of Inuenza with COVID-19 During The COVID-19 Pandemic in Southern Iran

Background In the current COVID-19 pandemic, COVID-19 viral respiratory symptoms have been confused with other viral respiratory infections such as inuenza. Given that both viruses cause respiratory diseases, there are important differences between these two viruses in terms of how they are spread, controlled and treated. Due to these differences, a denitive diagnosis of each infection has important implications for the public health measures that can be implemented in response to the treatment of each virus. Method In this cross-sectional retrospective study from 4th September 2020 to 5th December 2020 (time period of inuenza outbreak in Iran, a total of 455 Severe Acute Respiratory Infections (SARI) patients were included. Two nasopharyngeal and one oropharyngeal throat swab samples were collected from all participants and evaluated for COVID-19 by real-time reverse transcriptase–polymerase-chain-reaction (RT-PCR) assay using the E-Gene specic primers/FAM probe and S Gene primers/ROX probe (Covitech, Iran) for SARS-CoV-2. Due to the concurrence of the study in autumn and the history of inuenza outbreak at this time in Iran. Nasopharyngeal samples were collected and tested for inuenza viruses A (H1N1, H3N2, seasonal u), and Inuenza B by one step qRT-PCR Master Mix (Invitrogen, United States) and AG synthesis probe and primers (Metabion, Germany) for Inuenza A (H1N1, H3N2, seasonal u) and B. Results In this study, 455 patients with SARI were hospitalized during September to December 2020. 203(44.61%) were infected with SARS-COV-2 and of these patients, one patient was positive for both COVID-19 and Inuenza. The mean age was estimated 54.93 ± 17.00 and 50.65 ± 17.71 in COVID-19 and non-COVID-19 groups, respectively which was signicantly different (P < 0.001). Sex distribution between two groups showed that most of COVID-19 patients were male, this is in contrast with the COVID-19 negative group, in which most of patients were female and these differences were statistically signicant. (P = 0.057). Clinical outcomes of patients with diagnosed SARI were measured. The main parameters were discharge from ICU and death during hospital admission. There was no signicant difference between the number of patients discharge from ICU who were COVID positive or COVID negative. In addition, there was no signicant difference between the number of patients who died who were COVID positive or COVID negative.

1 Introduction COVID-19 is a novel respiratory disease caused by a new coronavirus called SARS-CoV-2. The WHO rst became aware of this new virus on 31 December 2019, following the rst report of a cluster of infected cases of 'viral pneumonia' in Wuhan, People's Republic of China. [1,2] In the continuing COVID-19 pandemic, these viral symptoms have been confused with other viral respiratory infections such as in uenza. Given that both viruses cause respiratory diseases, there are important differences between these two viruses in terms of how they are spread, controlled and treated. Due to these differences, a de nitive diagnosis of each infection has important implications for the public health measures that can be implemented in response to the treatment of each virus. The World Health Organization's FluNet platform based on three Southern Hemisphere countries from Oceania (Australia), South America (Chile), and Southern Africa (South Africa) showed very low in uenza activity during the typical Southern Hemisphere in uenza season June-August 2020. Therefore, in the upcoming 2020-21 Northern Hemisphere in uenza season, countries where widespread personal protective and public health measures are maintained (e.g., face masks, social distancing, school closures, and teleworking) may have decreased in uenza circulation. According to the Infectious Diseases Society of America (IDSA) guidelines, nucleic acid ampli cation test (NAAT) is a recommended procedure for detection of infectious diseases (such as SARS-CoV-2) in symptomatic individuals and in the suspected infected population. [3] Currently, Quantitative real-time-PCR (RT-PCR) is widely used for NAAT due to its large dynamic range, high sensitivity, high sequence speci city, little to no post ampli cation processing, and sample throughput. [4][5][6] Co-infection may cause the disease to become more complicated and may increase the mortality rate. [2,7] Presently, diagnosis of the infectious agents is based on nucleic acid ampli cation is the gold standard technique and is very useful in the exact diagnosis of infectious diseases with similar presentations. Currently, a major concern is the co-concurrence of in uenza and COVID-19 in the current epidemic, the co-infection of COVID-19 with different infections is evaluated in several studies. Many studies have shown a pattern of decrease in in uenza incidence in 2020 due to public health measures. With respect to the importance of identifying the viral etiology in clinical practice, and for health policymakers in crisis management and supply of required protective equipment and medicines this study was designed to evaluate the co-infection of COVID-19 with in uenza virus type A (H1N1) in patients with the Severe Acute Respiratory Syndrome in Ali Asghar Hospital, Shiraz, Iran.

Material And Methods
In this cross-sectional retrospective study from 4th September 2020 to 5th December 2020 (time period of in uenza outbreak in Iran, a total of 455 Severe Acute Respiratory Infections (SARI) patients were included. All patients were hospitalized in Ali Asghar Hospital, Shiraz, Iran which is a liated with Shiraz University of Medical Sciences, Shiraz, Iran. Two nasopharyngeal and one oropharyngeal throat swab samples were collected from all participants and evaluated for COVID-19 by real-time reverse transcriptase-polymerase-chain-reaction (RT-PCR) assay using the E-Gene speci c primers/FAM probe and S Gene primers/ROX probe (Covitech, Iran) for SARS-CoV-2. Nasopharyngeal samples were collected and analyzed for in uenza. Due to the concurrence of the study in autumn and the history of in uenza outbreak at this time in Iran. All nasopharyngeal samples were tested for in uenza viruses A (H1N1, H3N2, seasonal u), and In uenza B by one step qRT-PCR Master Mix (Invitrogen, United States) and AG synthesis probe and primers (Metabion, Germany) for In uenza A (H1N1, H3N2, seasonal u) and B. All tests were performed under standard procedures in the referral laboratories of Fars Province, Iran.. Demographic data and clinical speci cations of the patients include age, sex, comorbidities, presenting symptoms, travel history, and contact history were collected in standard questionnaires and analyzed.
Informed consent was obtained from all participants of this study, in case of dead participants informed consent was obtained from legally authorized representatives. This study was approved by Shiraz University of Medical Sciences Ethical Committee, with ethics code IR.SUMS.REC.1398.1173. In addition, all methods were carried out in accordance with the relevant hospital clinical and ethical guidelines and regulations 3 Results In the current study, 455 patients with SARI were hospitalized during September to December 2020. 203(44.61%) were infected with SARS-COV-2 and of these patients, one patient was positive for both COVID-19 and In uenza. The mean age was estimated 54.93 ± 17.00 and 50.65 ± 17.71 in COVID-19 and non-COVID-19 groups, Table 4 − 1, respectively which was signi cantly different (P < 0.001). Sex distribution between two groups showed that most of COVID-19 patients were male, Fig. 3 − 1, this is in contrast with the COVID-19 negative group, which most of patients were female, Fig. 3 − 2, and these differences were statistically signi cant. (P = 0.057).

Discussion
The current study evaluated the co-infection of SARS-CoV-2 and In uenza virus in u epidemic season during the rst year of COVID-19 pandemic in the Southern Iran, in 455 patients with a SARI. In this study total of 203 patients were positive for COVID-19, only one patient was infected with In uenza H1N1 simultaneously (co-infected with COVID19). It was expected that other respiratory illnesses, such as the in uenza, would be signi cantly reduced due to preventive policies related to the current COVID-19 crisis.
Our study is consistent with several studies in other parts of the world. positive individuals in the study was concomitantly positive for In uenza A virus. [10,11] In the rst large case series study in the US, a total of 5700 patients hospitalized with COVID-19 in the New York City Area were considered where it was shown that the rate of respiratory virus co-infection is only 2.1 percent. [12] In a study in Japan by Sakamoto et al. it was shown that seasonal in uenza activity was lower in 2020 than in previous years in that region. [13] On the other hand, Yue and his research team have found that co-infection of SARS-CoV-2 and in uenza viruses were common in Wuhan, China, between 12th January and 21st February 2020 during the COVID-19 outbreak. They have found that co-infection of SARS-CoV-2 and in uenza viruses is highly prevalent during the early time of the COVID-19 outbreak in Wuhan (12th January-21st February 2020). [14] But it should be noted that this difference might be caused by other factors such as underlying diseases in infected patients or the different geographical circulation of respiratory viruses which may change during the COVID-19 epidemic continues as we exit out of the classical in uenza season. [15] Another important point in reducing respiratory diseases, including in uenza, is to follow the guidelines related to global COVID-19 risk control, which includes mask-wearing, hand washing, self-isolation as well as social distancing between symptomatic individuals. [14,16] Because of these results, it is not yet clear whether initial testing should include both COVID-19 and in uenza viruses especially at the time of in uenza outbreak or whether the in uenza testing can be added after the SARS-CoV-2 negative results return. Concerning COVID-19 risk factors, in agreement with our ndings and previous studies, it can be suggested that COVID-19 has more effect on males than females, and increasing age is associated with increased odds of a positive SARS-Cov2 test result. [17][18][19][20] Also in systematic reviews, it has been shown that chronic comorbidities such as hypertension and diabetes mellitus especially uncontrolled forms are more at risk for severe COVID-19 disease [21,22] Based on this information and the aggregation of results obtained so far, it seems that the current policies on respiratory diseases, especially in the eld of diagnosis and vaccination, should be revised.

Conclusion
Page 6/14 The decline in In uenza incidence and coinfection with COVID-19 appears to be signi cant due to its concurrence with the COVID-19 pandemic and general population awareness on observing the instructions for personal respiratory protection e.g mask-wearing, hand washing, self-isolation and public health measures. Therefore, routine testing and empirical treatment for suspected in uenza coinfection in COVID-19 patients is not recommended. Sex distribution between two groups showed that most of COVID-19 patients were male, gure 3-1 Figure 2 this is in contrast with the COVID-19 negative group, which most of patients were female, gure 3-2, and these differences were statistically signi cant. (P=0.057).