Influenza is a respiratory disease that causes epidemics during the winter months. However, during these epidemics, respiratory viruses other than influenza are also circulating, such as human coronaviruses (hCoVs), rhinoviruses, and respiratory syncytial virus (RSV).1 This cocirculation makes the detection of coinfections of influenza and other respiratory viruses (ORVs) frequent.2,3

Currently, the microbiological diagnosis of respiratory viruses is mainly performed using molecular techniques. Among those methods, multiplex polymerase chain reaction (PCR) allows detection of several microorganisms in the same sample.4 In fact, a variety of molecular techniques for syndromic diagnosis of respiratory infections have proliferated in recent years. However, not all of laboratories have the same capacity, facilities, and economic and human resources to set up these multiplex techniques, and some of them have to use other, simpler methods that can only detect influenza or at most one more respiratory virus in the same reaction, frequently RSV.

The importance of detecting coinfections of influenza and ORVs has not been established clearly. In children, some studies have shown that coinfection is not associated with the severity of disease,5–7 although other studies have found an association with increased severity, especially if RSV or rhinovirus are involved.8–13 In adults, there seems to be greater consensus that coinfections are relevant, especially in patients who suffer from special clinical conditions, such as immunosuppression.14–17

The aim of our study was to describe the demographic and virological factors associated with the probability of detecting coinfection of influenza and ORVs though multiplex PCR methods, and to determine which patients would benefit most from this multiplex detection method.

We conducted a retrospective observational study that included 3623 respiratory samples (nasal wash in children aged less than 2 years and nasopharyngeal swab in older children) from patients with laboratory-confirmed influenza detected by multiplex RT-PCR obtained over 8 consecutive influenza seasons (2011–2012 to 2018–2019). The respiratory samples were collected from patients admitted to different hospitals of Castilla y León (Spain) and patients managed in sentinel outpatient sites of the Castilla y León Influenza Sentinel Network. Viral identification tests were performed at the National Influenza Centre-Microbiology Unit of the Hospital Clínico Universitario de Valladolid, Spain. For each patient, we collected demographic data (age, sex and type of patient [inpatient/sentinel]) and virological data (type/subtype of influenza [H1, H3 and B], ORV in coinfection) from the laboratory request forms. We defined coinfection as detection in a respiratory sample of 1 or more ORV in addition to influenza. The study adhered to the principles of the Declaration of Helsinki, and it was exempt from informed consent on account of the clinical and diagnostic use of the samples.

In many cases, clinicians do not consider the potential involvement of more than 1 virus in respiratory infections.19 However, the detection of various respiratory viruses in coinfection with influenza is frequent during epidemic seasons when the diagnosis is made by means of molecular multiplex techniques,19,20 so this possibility should be taken into account. Our study shows that coinfections of influenza and ORVs were present in approximately 2 out of 10 patients (17.8%) with a positive test for influenza, demonstrating that such coinfections are relatively frequent during influenza epidemics. Out of all coinfected patients, most (76.0%) had coinfection with influenza and only 1 ORV, and 24.0% had coinfection by influenza and 2 or more ORVs. Other studies have found similar proportions of coinfection (4%–33%).20–25

Our findings show that the respiratory viruses more frequently involved in coinfections of influenza were rhinovirus/enterovirus (24.0%), RSV (19.1%), bocavirus (19.1%) and human coronavirus (18.3%). These respiratory viruses are among the most prevalent throughout the year, especially in the winter months of the Northern Hemisphere,1 and also have been found in similar percentages in other studies.24 The PCR protocol used in our study cannot differentiate between rhinovirus and enterovirus, so the proportion of each viral genus in these respiratory infections cannot be established with certainty. However, a previous study conducted by our group found that rhinoviruses were involved in more than 90% of those samples, so that proportion was likely to be maintained in the study presented here.26

The respiratory virus detected most frequently in coinfection with influenza varied by age group. Bocavirus was the most frequently detected in young children (0–4 years), rhinovirus/enterovirus in children (5–14 years) and young adults (15–39 years), hCoV in adults (40–64 years) and RSV in elderly patients aged 65 or more years. Some previous studies have found that rhinovirus was the virus detected most frequently in coinfections with influenza and with ORVs.12 Rhinoviruses play a relevant clinical role in both upper and lower respiratory tract diseases, and they are the leading cause of pneumonia in adults27 and the second leading cause in children.28 However, previous studies conducted in Spain have shown that RSV is the virus most frequently co-detected with influenza virus, especially in young individuals.24 Notwithstanding, in our study we found the highest proportion of coinfection with RSV in the elderly group, which suggests that this virus must be considered in adults, and not only in children.29

On the other hand, in 2020, the SARS-CoV-2 pandemic has highlighted the importance of hCoV in respiratory illness. Our results showed that hCoVs were the viruses most frequently detected in coinfection with influenza in adults (40-64 years), which is one of the age groups most affected by COVID-19 in the pandemic.30,31 The potential concurrence of influenza, ORV and SARS-CoV-2 must be taken into account in upcoming years, especially in the most vulnerable groups. Although our findings show that hCoVs are frequently co-detected with influenza in adults, we were not able to evaluate the potential involvement of SARS-CoV-2. However, some studies have been published by now that analysed coinfection by influenza and SARS-CoV-2 and suggest these coinfections may be common and be associated with increased severity in some cases.32,33

Our results showed a clear association between coinfections and age. The multivariate analysis showed that detection of coinfection by influenza and ORVs was 2.7 times more frequent in young children and 1.6 times more frequent in children. But also, coinfection seemed to be associated with the involved subtype of the influenza virus, as coinfections with ORVs were 1.4 times more frequent in patients infected by the A(H3N2) subtype.

There are several possible explanations for the higher probability of detection of coinfection in the youngest patients. One is that children have an immature immune system compared to adults, increasing the probability of infection by several respiratory viruses at the same time.34–36 Also, the greater interaction of children in schools also increases the opportunity of exposure to different viruses.13,37 The higher proportion of the A(H3N2) subtype in cases of coinfection could be explained by the greater virulence of this subtype compared to other influenza viruses.38,39 This greater virulence can cause changes in epithelial cells, weakening the immune response and facilitating infection by ORVs and bacteria.40 However, this issue requires further investigation.

Our results also showed that the prevalence of coinfection by 2 or more ORVs was two-fold in young children, two-fold in adults and 1.5-fold in female patients. Coinfections by 2 or more ORVs were particularly frequent adult women (40–64 years). One explanation could be the increased exposure of women to children, who are also affected also by multiple coinfections, due to their higher involvement in child care compared to men.41 In fact, the World Health Organization suggests that this factor is more important than differences in immunity related to sex.42 However, we did not have enough samples to test this issue thoroughly, so further research must be conducted in the future. On the other hand, the type of patient (hospitalized or sentinel) did not seem to be a factor associated with the probability of detection of coinfection by influenza and ORVs.

One of the main limitations of this study was its retrospective design, as we were unable to assess the outcomes of the included patients, such as duration of disease, admission to the ICU and mortality. Further studies are required to analyse outcomes and their association with the presence of coinfection by influenza and ORVs in detail. One of the main strengths of our study is that it covered a large number of influenza seasons, which compensated the variability intrinsic in each season and provided more accurate information on the factors that are truly involved in coinfection, such as age.

In summary, our study found that coinfections by influenza and ORVs were detected in nearly 2 out of 10 laboratory-confirmed influenza cases, and that such coinfections were more frequent in young children (0–4 years), children (5–14 years) and patients infected by the A(H3N2) influenza subtype. The virus detected most frequently in coinfection with influenza varied between age groups, with hCoV being the most frequently detected in adults (40–64 years). While sex was not associated with a higher incidence of coinfection, there was a higher frequency of coinfection by influenza and 2 or more ORVs in female patients, probably due to the greater involvement of mothers in caring for children, as children had the highest rates of coinfection, both by a single and by multiple ORVs. These results could be useful to guide the use of multiplex PCR for diagnosis of respiratory viruses during the influenza season in laboratories with low resources.