Of 128 patients with identifiable respiratory viruses, univariate analysis revealed that patients with 1 of the following conditions were more likely to have non-influenza respiratory virus infections: immunocompromised state, chronic obstructive pulmonary disease (COPD), and chronic renal failure receiving dialysis (OR 5.4, 95% CI 1.2–25.5, P = 0.02). Multivariate analysis demonstrated that steroid use was an independent risk factor for rhinovirus infection (OR 15.3, 95% CI 1.5–154.7, P = 0.02), active malignancy was an independent risk factor for hMPV infection (OR 29.3, 95% CI 2.4–358.1, P = 0.008), and COPD was an independent risk factor for parainfluenza infection (OR 229.2, 95% CI 10.5–5020.8, P = 0.001).

While comparing the URTI and LRTI groups, factors found to be associated with LRTI by univariate analysis included old age (≥60 years), a high comorbidity index, congestive heart failure, COPD, malignancy, immunocompromised state, and detection of parainfluenza or EBV, whereas detection of influenza A was less frequently associated with LRTI. Codetection of respiratory virus was not associated with the development of LRTI. By multivariate analysis, only old age, immunocompromised state, and detection of parainfluenza remained 3 independent factors associated with LRTI (Table 3).

Cases that tested positive for any respiratory virus either by culture or by PCR/ESI-MS were analyzed. The positive detection rates declined with age: 55.3%, 41.7%, and 34.5% in the 18–39, 40–59, and ≥60-year-old groups, respectively (P = 0.02) (Figure 1A). A higher positivity rate was observed in patients with URTIs than that in patients with LRTIs (50.5% vs. 38.2%, P = 0.10) (Table 3 and Figure 1B). There were similar distributions of respiratory viruses in cases from the local clinical and the medical center (Table 2), and between patients from the 3 age groups (Figure 1A). Of 128 cases with identifiable respiratory viruses, non-influenza virus infection was more common in patients with LRTIs than those with URTIs (81.0% [17/21] vs. 48.6% [52/107], P = 0.007). Rhinovirus (12.7%), influenza A (10.9%), and parainfluenza (7.3%) were the 3 leading respiratory viruses involved in 55 cases of LRTIs, and parainfluenza was more frequently observed in the LRTI group than in the URTI group (Table 3 and Figure 1B). There was no seasonal variation in any individual respiratory virus over the 9-month period.

Of the 172 that tested positive for respiratory agents, there were 24 (14.0%) cases with mixed infections of two or three viruses. No patient was infected with more than three viruses. Twenty-one cases were infected with two viruses, with the most frequent mixture being adenovirus and influenza A virus (n = 5). Other frequent viral mixtures were adenovirus and rhinovirus A (n =3) and influenza A virus and coronavirus OC43 (n =3) (Table 3). Adenovirus was present in 62.5% (n = 15) of mixed infections which was almost a half (46.9%) of all detections for this virus. Respiratory syncytial virus B was the only aetiology that was not detected in a mixed infection.

This study identified viral aetiologies in 46.6% of all ILI cases at two health facilities in Kampala and Entebbe, Uganda, a prevalence level similar to that reported in other studies.22,23,24 The identified aetiologies include influenza A and B virus, adenovirus, rhinovirus A, coronavirus OC43 and 229E, parainfluenza virus 1, 2 and 3, respiratory syncytial virus A and B and human metapneumovirus. A number of these aetiologies have only recently been identified and their circulation in Uganda was unknown previously. These include human metapneumovirus, whose discovery as a causative agent for ILI has been recognised only in the last decade.25

Most of the detected viruses, including parainfluenza virus 1, 2 and 3, influenza B virus, adenovirus, human metapneumovirus and coronaviruses OC43 and 229E, were circulating at prevalence levels that were, in general, similar to those found elsewhere.12,14,26,27 Influenza A virus was detected in 19.2% of ILI cases, which was higher than the 12% prevalence level that was known to exist from previous observations in the same population.8 The higher prevalence observed for this virus could be attributed to the timing of this study which was conducted when rainfall is highest in Uganda. It is probable that an outbreak associated with this virus was ongoing during the study period as observed previously.4,5,6 Conversely, rhinovirus A was detected at 7.9% which is lower than the 10 % – 25% prevalence levels found in other ILI surveillance studies within sub-Saharan tropics.13,14,16,28 In the same studies, the prevalence of respiratory syncytial viruses A and B ranged between 5% – 21% which is higher than our 3.3% total prevalence for the same viruses. The low prevalence levels observed for these viruses could also be associated with their seasonality in the study population – a variable that could not be established with our current cross-sectional data. Also, our ILI case definition was focused more on influenza surveillance and could have been restrictive with regard to the signs and symptoms of other ILI aetiologies.29,30

Mixed infections amongst all cases that tested positive for respiratory agents accounted for 14.0% of the findings, with the majority being double infections. The prevalence of mixed infections ranging from 4.5% – 70% are reported from other studies, depending on the geographical location of the study area, the diagnostic method used or the general degree of illness in the study population.24,31,32,33 The high prevalence of mixed influenza A virus and adenovirus infections during a low circulation cycle of respiratory syncytial virus infection has previously been suggested.34 In our study, the number of mixed infections (n = 24) was not adequate to allow statistical analysis; a more comprehensive study is necessary in order to determine the associations and interactions between these viruses as well as the related clinical outcomes.

The mean age of the all infected patients was 57.1 years (range 19–84 years) with a male:female ratio of 1.9:1. The distribution of the underlying hematological disorders was similar to the overall patient population at the department of hematology. The majority of infected patients (n = 75/111) underwent stem cell transplantation (42 autologous, 48 allogeneic, 15 autologous and allogeneic); 13 patients were infected pre-engraftment, 62 post-engraftment. All but three patients were considered immunocompromised for one or more of the following reasons: uncontrolled hematological malignancy, chemotherapy, < 3 months after transplantation, prior steroid treatment, systemic immunosuppression, reduced CD4+ T-cell count, hypogammaglobulinemia. Details on clinical characteristics of infected patients are presented in Table 1.

Sixty four patients had URTI, 47 patients showed signs of LRTI, and 15 patients developed severe LRTI as defined by ICU treatment or fatal outcome. Thirteen infected patients with pneumonia died, despite ICU admission and supportive ventilation. Five of the fatal cases were infected with influenza virus, one with parainfluenza virus and seven with RSV. Oral tamiflu or ribavirin was given as treatment to 60 patients, immunoglobuline preparations to 13 patients, palivizumab was not administered. There were no significant differences in regard to demographic and clinical characteristics between influenza, parainfluenza and RSV infected patients. Furthermore, we could not identify any association between a specific virus group and a more severe course of illness. Co-infections were detected in 30 patients. A total of 44 different co-detected pathogens could be identified (bacterial 27, viral 11, fungal 6), e.g. Pseudomonas aeruginosa bacteremia, cytomegalovirus reactivation, and Aspergillus fumigatus pneumonia. Risk factor assessment amongst all infected patients showed by univariate analysis for the endpoint LRTI uncontrolled hematological disease (OR 4.39 [95% CI 1.70;11.84], p = 0.001), presence of co-infections (OR 5.82 [95% CI 2.07;17.82, p = 0.0002), and prior steroid therapy (OR 3.32 [95% CI 1.14;11.13], p = 0.02) as significant risk factors; a trend was seen for severe leukopenia (i.e. leukocytes <1/nl) >10 days (OR 3.22 [95% CI 0.88;13.34], p = 0.07). In regard of the endpoint severe LRTI only presence of co-infections (OR 4.89 [95% CI 1.31;19.39], p = 0.008) was a significant risk factor; a trend was seen for uncontrolled hematological disease (OR 3.42 [95% CI 0.93;13.28], p = 0.06). By multivariate analysis no significant influence factors could be identified.

CLART® Pneumovir array is a qualitative, highly sensitive and high-throughput technology which allows rapid detection of a broad spectrum of human respiratory viruses at a time, particularly to detect viruses that which are not culturable in the laboratory, and also those that are not detectable by the standard multiplex RT-PCR (29, 30). In the present study, CLART® Pneumovir array was used to detect 17 common respiratory viruses on 400 throat or nasopharyngeal swab specimens of Iranian military trainees which were collected during January to March 2017. Our findings have shown that 31% of all patients with respiratory infection were associated with different viral agents. According to our results, rhinovirus, respiratory syncytial virus A, and influenza B virus have been responsible for ~66% of viral respiratory infections among military trainees.

Previous studies have shown that adenoviruses, influenza A and B viruses, human coronaviruses, rhinoviruses, and at lower frequencies, respiratory syncytial viruses and human parainfluenza viruses are the most common viruses leading to respiratory infection in the military environment worldwide (1, 31). Similar to our results, respiratory syncytial virus was the main causative agent of acute febrile illness in the Republic of Korea Air Force boot camp from May to July 2011 (32). O'Shea et al. showed that among 54 British military recruits receiving basic training with respiratory symptoms, adenovirus, influenza viruses and respiratory syncytial virus were found in 35%, 19% and 14%, respectively (33). It is notable that among influenza viruses, influenza B was more associated with symptomatic infections, which was consistent with our findings. In a large study in the Singapore military population (13), viral etiology of respiratory illnesses was investigated among 7733 cases of febrile respiratory illness (FRI) using the multiplex PCR. Their results have indicated that 49% of cases were positive for viral infection and the most commonly detected viruses among FRI cases were influenza A H1N1(13%), influenza B (13%), and coxsackievirus (9%).

The low prevalence of viral infections in our study in comparison with the other parts of the world can be attributed to the existence of natural immunity to respiratory viral infections. The Iranian Muslims have several organized mass gatherings such as Hajj pilgrimage and Jumu'ah (Friday prayers), likely leading to transmission of respiratory viruses between people and consequently, developing of natural immunity to them. This hypothesis can be evaluated in seroepidemiological studies.

The overall prevalence of respiratory syncytial virus (types A and B) (8.7%) in our study was lower than that found in the previous works (34, 35). We believe that the rate of respiratory syncytial virus in our study could be even higher since infection with respiratory syncytial virus in a significant proportion of patients is asymptomatic. In accordance with our findings, some studies also found that although both subtypes are infectious and circulate annually, but subtype A is the main cause of respiratory syncytial virus infection which can cause more severe complications (36). Cough, rhinorrhea, nasal congestion, sore throat, and fever were most frequently symptoms of respiratory syncytial virus infection in our investigated population. It was interesting that the majority of dyspnea cases (n=5; 62.5%) was seen in respiratory syncytial virus infection in compared with other viral infections.

Our study revealed that influenza viruses (particularly influenza B) also have an important role in causing respiratory viral infection along with respiratory syncytial virus with prevalence rate of 9.7% among military trainees. Interestingly, despite the lack of adenovirus vaccination program in Iranian military health system, we reported a low rate of adenovirus (n=6; 1.5%) in military trainees. It may be explained by the fact that influenza infection tends to occur in the winter, while adenovirus infection is more likely to occur in the spring, summer, and fall seasons. In fact, seasons can be used to help the in differential diagnosis of FRI among military trainees. It should be noted that our investigation was only performed during the winter months.

There is no report on the prevalence of respiratory viruses in the military population in Iran and other countries in the Middle East. However, several studies have been performed among the general population. For example, in a cross-sectional study in pediatric patients with respiratory symptoms in Shiraz (37), adenovirus was detected in 22% of nasopharyngeal swabs samples. In another study by Barati et al., adenovirus was detected in 6.3% of nasopharyngeal secretions samples from 160 children with upper respiratory tract infection, and cervical adenopathy was the most common symptom in adenovirus infection (38). The higher prevalence of adenovirus infection in children can be explained by the fact that adenovirus shedding is more prolonged in children than the adults.

This study is the first extensive laboratory surveillance of the etiology of respiratory viruses in ILI patients through teaching hospital-based influenza surveillance in South Korea. The influenza A virus was the predominant agent and the majority of influenza A virus was A(H3N2) during the 2011–2012 season. In addition, 23.1% of the respiratory viruses detected in respiratory specimens from adult patients with ILI were viruses other than influenza viruses.

The respiratory virus detection rates in respiratory specimens from ILI patients ranged from 15.6% to 78.7%, depending on the study period, the characteristics of the study population including age, type of specimens, circulating viruses, and analysis method–[11]. At least one virus was detected in 52.1% of samples in this study. Influenza virus was the predominant agent, and this finding is consistent with other reports–[9]. Since ILI is a clinical definition designed to detect potential influenza cases, the use of ILI as a case-definition makes influenza viruses the viruses most likely to be identified. This case-definition is suitable for the purpose of HIMM surveillance, which is focused on the detection of influenza. Yu et al. investigated respiratory viral etiology in adults with acute respiratory tract infections visiting an ED in China, and the parainfluenza virus was found to be the dominant agent. In their study, enrollment criteria included respiratory symptoms, a body temperature above 37.5°C, and a normal or low leukocyte count, but not radiographic abnormalities on chest. There is a difference in the respiratory viral etiology between children and adults. RSV was the most prevalent virus and was associated with substantial morbidity in children with respiratory virus infection–[15]. In this research, the study population included only adult patients, and the viral etiology of pediatric patients with ILI was not evaluated.

The co-incidence rates of respiratory viruses in ILI patients vary according to study (0.7–15.3%)–[11]. In this study, multiple respiratory viruses were detected in 5.6% of positive specimens and 2.9% of total ILI patient samples. The clinical significance of the co-incidence of respiratory viruses has not been clearly determined. In infants with bronchiolitis, dual viral infection was a risk factor for ICU admission and co-infection with human metapneumovirus and RSV was strongly associated with disease severity,. Additionally, viral co-infections were related to an increased probability for hospitalization in children with respiratory infection. However, the impact of detection of multiple respiratory viral infections on clinical outcome has rarely been investigated in adults. In this study, the disease severity or outcome were not different significantly between patients with single virus and patients with multiple respiratory viruses. In addition, the detection of viral nucleic acid in respiratory specimens does not always suggest that it is the causative agent of the apparent infection. Among respiratory viruses, rhinovirus was the most common virus detected in influenza patients. In one patient, six respiratory viruses were detected simultaneously. This 20 years old female patient was previously healthy, and the outcome was good without hospitalization or any complication or sequelae. Among 58 co-incidence cases, two patients were hospitalized (influenza A virus and rhinovirus, influenza A virus and RSV A). Further research on viral interference, especially between influenza virus and rhinovirus, is required in both animal models and humans.

This study has some limitations. First, the surveillance population was limited to adults who visited an ED with ILI. Second, the evaluation of the causative agents of ILI covered only 15 respiratory viruses, and other bacterial or viral pathogens which can cause acute febrile respiratory illnesses were not investigated. Third, there is a possibility that a low viral titer could not be detected by RT-PCR. Fourth, patients with acute febrile illness but without respiratory illness could be enrolled in this study because the recruitment depended on whether cases met the established ILI criteria or not. Finally, this study described only the result of laboratory data. However, this study is valuable for understanding the respiratory viral etiology of ILI patients during the 2011–2012 season. This study is noteworthy in that it is the first extensive laboratory surveillance of adult ILI patients through a teaching hospital-based influenza surveillance scheme in South Korea.

The rate of concurrent serious bacterial infections with viral illness is appreciable. Similar emphasis must be given to the prevention and treatment of viral illnesses, especially in young children. Furthermore, health care providers should emphasize to parents on the importance of clinical follow-up of infants and young children diagnosed with VRTI. Moreover, the introduction of MxA in the diagnosis of viral illnesses in children is promising.

Most children (62%) missed two or fewer days of school due to their medically attended ARI. The 2012‐2013 season and peak influenza season were associated with prolonged absence of >2 days (Table 2). Longer duration of illness, being fatigued, and having a fever were also associated with prolonged absence. There were no differences in prolonged absence by age group, sex, reported health status prior to illness, influenza vaccination status, or receipt of antivirals.

More than 40% of children with influenza reported prolonged absence (B/Yamagata 60%, B/Victoria 59%, A/H3N2 47%, A/H1N1 41%) compared to other viruses (range: from 21% for parainfluenza to 34% for human metapneumovirus). The odds of experiencing prolonged absence was 3.7 (95% CI: 2.3, 6.1), 3.5 (95% CI: 2.1, 5.9), and 2.2 (95% CI: 1.5, 3.2) for influenza B/Yamagata, B/Victoria, and A/H3N2, respectively, compared to children with no viral infection (Figure 2A). Children with B/Yamagata infection were more likely to report prolonged absence than children with A/H1N1 (OR=2.1, 95% CI: 1.0, 4.5, P=.05) or A/H3N2 (OR=1.7, 95% CI: 1.0, 2.9, P=.04) infection. Vaccination status was not associated with prolonged absence among children with influenza (Figure 2B). When we excluded children vaccinated with LAIV, the odds of prolonged absence due to A/H1N1 among vaccinated children was attenuated, but confidence intervals overlapped the odds for all vaccinated. Results for A/H3N2 did not change when the 2014‐2015 season was excluded.

Results for influenza did not change in sensitivity analysis excluding the 20 known coinfections with influenza.

Corollary and secondary bacterial infections in patients with viral diseases are known to coexist. A study conducted by O’Brien et al56 showed that the influenza virus (H1N1) was the culprit of the severe pneumococcal pneumonia outbreak among children that occurred in Iowa in the mid-1990s. Other studies have shown that almost one-third of children with community acquired pneumonia (CAP) had mixed (viral and bacterial) infection.57,58 Moreover, a study in France showed that influenza virus infection was the direct cause of CAP in 12% of children.59 Syrjanen et al60 found in their study that the isolation of S. pneumonia from the nasopharyngeal area was higher during respiratory infection without concomitant AOM. Viral respiratory infection due to RSV, influenza virus (type A or B), and adenovirus increase the incidence of otitis media (OM) and recurrent OM in children.61,62 Ruuskanen et al63 found that there is a concrete association between AOM and 57% of children with RSV, 33% with parainfluenza type 3 virus, 30% with adenovirus, 35% with influenza A virus, 28% with parainfluenza type 1 virus, 18% with influenza B virus, and 10% with parainfluenza type 2 virus infections; the most common bacteria isolated from tympano-centesis were H. influenzae, S. pneumoniae, Branhamella catarrhalis, and Mycoplasma pneumonia. Another study showed that the rates of bacteremia and OM were 18% and 44%, respectively, in children with viral-induced bronchiolitis.11 The highest incidence of AOM is usually 2–5 days after an upper respiratory infection.64,65 Isolation of viruses alone from sinus aspirates or in concomitance with bacteria proposes the role of viruses in the induction of bacterial sinusitis,62 with rhinovirus and parainfluenza viruses being the culprits.66 The rate of bacteremia in children with acute bronchiolitis ranges from 0.2% to 1.4%.67–75 In addition, the rate of bacterial urinary tract infection (UTI) in children with bronchiolitis can be as high as 11.4%.67 In a recent study, Hendaus et al76 assessed the prevalence of UTI in infants and children with bronchiolitis. The study included 835 hospitalized children with acute bron-chiolitis. The results disclosed that UTI was found in 13.4% with bronchiolitis triggered by a respiratory viruses such as rhinovirus (31%), adenovirus (14%), parainfluenza virus type 4 (14%), bocavirus (10%), human metapneumovirus (10%), coronavirus (7%), parainfluenza virus type 3 (3.4%), parainfluenza virus type 2 (3.4%), parainfluenza virus type 2 (3.4%), and H1N1 (3.4%). Rittichier et al77 have researched the effect of respiratory viruses on the risk of acquiring serious bacterial infection, including UTI. The study concluded that febrile infants with enterovirus had a coexisting rate of serious bacterial infection of 6.6%.

We wanted to determine whether bacterial or viral agents were responsible for causing the majority of CAP cases in the enrolled patients. Table 2 shows the broad category of etiological agents identified in pediatric CAP patients from Beijing Children's Hospital. Infection by a single bacteria was observed in 6.5% (n = 24) of the patients, infection by multiple bacteria was observed in 2.2% of patients (n = 8), infection by both bacterial and viral agents was observed in 17.8% of patients (n = 66), infection by a single viral agent was observed in 35.3% of patients (n = 131), and finally, infection by multiple viruses was observed in 11.6% of patients (n = 43). In approximately one quarter (26.7%, n = 99) of the patients the category of the etiological agent was not identifiable.

We next want to identify the prevalence of specific viral and/or bacterial pathogens that were contributing to CAP. Table 3 shows the bacterial and/or viral pathogens likely to be the causative agent for disease identified in the CAP patients. Bacterial infections accounted for 30.5% (n = 113) of the total CAP cases. The most prevalent were S. pneumoniae (20.8%; n = 77) and H. influenzae (9.7%; n = 36). Of the cases associated with S. pneumoniae, 77.9% (n = 60) were co-infected with viruses and/or bacteria. The majority (n = 45) were viral coinfection. In the CAP cases associated with H. influenzae, 80.1% (n = 29) were coinfections with viruses and/or bacteria. Viral coinfection occurred in 14 cases (48.3%).

In fact, the majority of CAP cases were associated with viral infection. Viral agents were detected in 330 of the patients (88.9%). As shown in Table 3, the predominant infection was with respiratory syncytial virus which accounted for 43.9% (n = 163) of the viral infections. Of the remaining viruses rhinovirus was associated with 14.8% (n = 55) of the CAP cases, parainfluenza virus with 9.4% (n = 35), adenovirus with 8.6% (n = 32), human bocavirus with 3.8% (n = 14), human coronaviruses (NL63, OC43, 229E and HKU1) with 3.8% (n = 14), influenza virus B with 1.9% (n = 7), and human metapneumovirus with 1.3% (n = 5). Influenza infections were detected in only five of the CAP patients. The seasonal influenza (H3N2) strain was detected in three patients (0.8%) and swine influenza (H1N1) was detected in two patients (0.5%). In the majority of influenza virus B (57.1%), respiratory syncytial virus (51.5%), and human coronavirus (42.9%) infections only a single agent was detected which was higher than for other viral infections. In contrast, the four major viral pathogens (respiratory syncytial virus, rhinovirus, parainfluenza virus, and adenovirus) were detected more frequently in coinfections with other bacterial or viral agents. In patients infected with rhinovirus, parainfluenza virus, and adenovirus a greater percentage of coinfections were with viruses than bacteria.

Influenza and Bocavirus were identified throughout the year, though cases of Influenza were commonly found in May, August and September, and cases of Bocavirus were commonly found in June and November. Influenza B was identified only in the second semester, while the two cases of Influenza A/H2N3 were seen in May. Adenovirus was seen only during the rainy season (April to May and August to November); most cases of Metapneumovirus, Parainfluenza and RSV were seen only in the first semester, specifically in April and May; the two cases of Coronavirus were identified in May (Fig 2).

Acute bronchiolitis was diagnosed in 316 infants younger than 24 months of age. Overall, at least one respiratory virus was detected in 75% (237/316) of the cases at Ege University Children’s Hospital. The results of the incidence of respiratory viruses in infants hospitalized with AB during the study period are shown in Table 3. RSV was identified in 127 (40.1%) of the 316 infants hospitalized for AB, and RSV was the most common agent. Of the 127 RSV-positive infants, 76 (59.8%) were sole pathogen for RSV and 51 infants had RSV-coinfection, which was responsible for respiratory tract infection. The second prevalent viral agent was rhinovirus, identified in 78 (24.6%) of the subjects, and as a single pathogen in 35 (44.8%). Furthermore, in this study, influenza virus types A and B (n = 28/316, 8.8%), human metapneumovirus (n = 27/316, 8.5%), adenovirus (n = 23/316, 7.2%), human bocavirus (n = 20/316, 6.3%), parainfluenza virus (n = 18/316, 5.6%), and human coronavirus (n = 10/316, 3.1%) were determined (Table 3). A single virus in 156/316 (49.3%), dual infections in 67/316 (21.2%), triple infections in 13/316 (4.1%), and quadruple infections in 1/316 (0.3%) were detected. Of the 81 infants with multiple agents, RSV (51 cases) was the most common virus followed by rhinovirus (n = 43, 53%), influenza virus (n = 18, 22.2%), human metapneumovirus (n = 16, 19.7%), PIV (n = 10, 12.3%), adenovirus (n = 16, 19.7%), human bocavirus (n = 13, 16%), parainfluenza virus (n = 10, 12.3%), and human coronavirus (n = 8, 9.8%). Among dual co-infections, the combination of RSV and rhinovirus had the the highest number (n = 15, 22.3%). The age distribution of infants is shown in Figure 1 for RSV, rhinovirus, adenovirus, and dual infections. In particular, most viral agents were identified in children of all months, whereas RSV was detected in infants aged under 6 months (n = 93/127, 73.2%) and in 15.7% (n = 20/127) of the 127 RSV-positive infants aged under 1 year of age, which was the case in most of our patients. The monthly incidence of bronchiolitis in infants due to common respiratory pathogens is shown in Figure 2. The majority of the cases were admitted to hospital in the winter time with a peak occurring in January, February, and March; detection rates were also high during these months (26.2%, 25.3%, and 17%, respectively). Other respiratory virus epidemics were seen also during the winter season (Figure 3). Fifteen cases infected with parainfluenza virus, adenovirus, and human bocavirus were seen in the summer time.

From October 2008 to March 2017 a total of 9,472 participants were enrolled in the

study from five hospital-based surveillance sentinel sites. Majority of participants

were children under 5 years n= 8,169 (86.2%). 615 (6.5%) samples tested positive for

influenza A, while 385 (4.1%) tested positive for influenza B viruses and 10 (0.1%)

were co-infections between influenza A and B. Of the 2,062 influenza negative

samples, results indicated positivity for the following organisms; adenoviruses

(9.4%), respiratory syncytial B (7.3%), parainfluenza-3 (4.5%), parainfluenza-1

(4.3%), respiratory syncytial A (3.5%), human bocavirus (1.7%), human

metapneumovirus (1.7%), human coronavirus (1.5%), parainfluenza-4 (1.4%) and

parainfluenza-2 (0.9%) by PCR.

There were 58 cases (5.6%) in which two or more respiratory viruses were detected from respiratory specimens. In 52 cases, two respiratory viruses were detected concurrently (Table 3). Five patients with influenza A also were found to have two other viruses: rhinovirus and RSV, rhinovirus and adenovirus, rhinovirus and parainfluenza virus, influenza B virus and rhinovirus, and influenza B virus and parainfluenza virus. In one case, six respiratory viruses were found simultaneously: influenza A virus, influenza B virus, rhinovirus, RSV A, coronavirus OC43, and human metapneumovirus. Rhinovirus (19/58, 32.8%) was the most frequently detected concurrent virus in influenza patients. There were 11 cases of co-incidence of influenza A and B.

In comparison of demographic characteristics between patients with single virus and those with multiple viruses in respiratory specimens, patients with hematologic malignancy (1.1% vs. 16.7%, P = 0.02) and organ transplantation recipients (0% vs. 8.3%, P = 0.04) had higher rate of co-incidence (Table 4). However, there was no significant difference in clinical outcomes including hospitalization rate and complications.

In total, 2295 days of school were missed by our study population over three influenza seasons; 175 (17%) children did not miss any days. The 2012‐2013 season, reported fair/poor health status prior to illness, enrollment during the peak influenza season, and receipt of antivirals were associated with greater mean days absent from school (Table 2). Longer duration of illness, being fatigued, having a fever, being short of breath, and having a sore throat were also associated with greater mean days absent. There were no differences in mean days absent by age group, sex, race/ethnicity, or influenza vaccination status.

Influenza infection accounted for 39% of ARI visits and 47% of all days absent (A/H3N2, 20%; B/Yamagata, 12%; B/Victoria, 11%; A/H1N1, 4%). Those with no infection detected accounted for 24%, and other viruses accounted for between <1% (adenovirus) and 7% (RSV) of all days absent. Mean days absent was highest for influenza and was not significantly different by subtype or lineage (Figure 2A, range: 0.96‐1.19). Mean days absent was higher among children with influenza A/H3N2, B/Yamagata, and B/Victoria compared to those with RSV (P≤.04), coronavirus (P≤.0004), parainfluenza (P≤.04), and rhinovirus (P≤.01). Among children with influenza, there was no difference in mean days absent between vaccinated and unvaccinated children with medically attended influenza (Figure 2B). When we excluded children vaccinated with LAIV, the mean days absent due to A/H1N1 among vaccinated children was lower, but confidence intervals overlapped the mean days absent for all vaccinated children. For H3N2, exclusion of the 2014‐2015 season resulted in lower mean days absent for both vaccinated and unvaccinated children.

During the study period, 381 mPCR tests were performed among 344 children. According to our definition of repeated samples, children cannot be included a second time in the 30-day period following the first sample. The median interval between two inclusion for a same child (n = 37) was 76 days (Inter-Quartile Range [IQR] = 52–168). The children median age at inclusion was 3.2 months [IQR = 0.8–23.7], 48.8% were female. Global positivity rate was 51.4% (196 samples), 9.2% (35 samples) corresponded to viral co-infections. Frequencies of positive and viral co-infection results across age groups are depicted in Fig 1. The identified viruses were picornavirus (n = 125, 52.7%), RSV (26, 11.0%), adenovirus (26, 11.0%), parainfluenza (17, 7.2%), metapneumovirus (14, 5.9%), coronavirus (13, 5.5%), influenza (8, 3.4%) and bocavirus (8, 5.2% of all identified viruses with Respifinder® 22 only able to detect this virus group). In comparison, 3142 adults’ samples were included during the same period among 2103 patients. The median delay between two inclusions for a same patient (n = 1039 samples) was 106 days [IQR = 57–243]. No patient was included a second time if there is less than 30 days since the previous sample. The adult population showed a lower positivity rate (51.4 vs 33.1%, p = 0.009) and a lower viral co-infection rate (9.2 vs 1.9%, p<0.001). Viral distribution was statistically different between adults and children (p<0.001) (Fig 2). Three virus groups were more represented in children: adenovirus (11 vs 3.9%, p<0.001), picornavirus (52.7 vs 34.4%; p<0.001) and bocavirus (3.4 vs 0.6%; p = 0.002). On the contrary, two viruses’ groups were less represented among children: influenza (3.4 vs 21.5%; p<0.001) and coronavirus (5.5 vs 12.6%; p = 0.002). RSV, metapneumovirus and parainfluenza groups presented similar prevalence in both populations.

Several differences appear for viruses’ distribution among children in paediatric units and adults. For example, RSV had a tendency to be more frequent in children < 36 months than in all patients > 36 months old (16.8 vs 10.6%, p<0.10), as classically described. More interestingly, adenoviruses were more frequent in children between 6 and 36 months old than in all other patients (23.3 vs 4.3%, p<0.0001). Influenza was also very low for all children strata but highly frequent among adults (3.4 vs 21.5%, p<0.0001). Some temporal variations between children and adults can also be observed during the three years of this study (Fig 3). Thus, from May to October 2011, the adenovirus group was largely present in children but absent in adults. Inversely, parainfluenza viruses were absent in children from September 2013 to April 2014, despite an active circulation among > 15 years old patients.

Neonatology and paediatric unit showed slightly similar positivity and co-infection rates as well as virus distribution. Thus, among the 151 children <6 months old in neonatology unit, global positivity and viral co-infection were 24.5 and 1.3%, respectively. In comparison, among the 51 children of the same age group in paediatric unit, global positivity and co-infection were statistically higher 56.9 and 7.8%, respectively (p<0.001). The distribution of viral groups between these units presented some differences that did not reach a significant level (p = 0.3). The most frequent viral group was picornavirus in both populations but was more represented in neonatology than in paediatric units (78.0 vs 54.1%) (Fig 1 and Table 1). This higher representation of picornavirus group may be explained by the asymptomatic carriage of such viruses among adults in neonatology units.

Regarding mPCR results across children age strata in paediatric units, positivity rates were statistically different with the highest rates observed in the 6–36 months group: 81.0% and 25.0% for positivity and coinfection rates, respectively. Main children characteristics are depicted in Table 1. Despite some variations, the viral distribution was not statistically different across age groups. Yet, RSV presented a slightly decreasing prevalence among older children with prevalence at 16.2% of all identified viruses in the 0–6 months old strata to 13.7 and 7.7% in the 6–36 months and 3–15 years strata, respectively. The adenovirus group had a slight tendency to be more frequently identified in the 6–36 months group (18.9%) compared to the 0–6 months and 3–15 years groups (5.4 and 9.6%, respectively, p = 0.09).

In order to estimate how many diagnoses would have been missed by the use of a specific RSV or influenza detection during their epidemic periods, we analysed separately all the results obtained during the active circulation of each of these virus according to the data obtained through the French RENAL hospital surveillance network for influenza and RSV. During their respective epidemic periods, RSV and influenza always encountered for 10 to 40% and 10 to 50% of all viruses identified in our laboratory, respectively. During RSV epidemic periods 63 children were sampled in paediatric units. 18/63 (28.6%) were positive for RSV, including 3 viral coinfections (2 with picornavirus and 1 with metapneumovirus), and 30/60 (50.0%) were positive for other respiratory viruses. Among the latter, 38 viruses have been identified: 21 picornaviruses, 5 coronaviruses, 4 adenoviruses, 3 influenza viruses, 3 metapneumoviruses, 1 parainfluenza and 1 bocavirus. During the influenza epidemic periods, 58 children were sampled in paediatric units. 4/58 (6.9%) were positive for influenza, including one coinfection with a picornavirus, and 34/58 (58.6%) were positive for other respiratory viruses. Among the latter, 46 viruses have been identified: 17 picornaviruses, 8 metapneumoviruses, 7 coronaviruses, 7 adenoviruses, 3 bocaviruses, 2 RSV and 2 parainfluenza.

The proportion of each virus group identified in viral co-infections was statistically different between virus groups (p = 0.02, Table 2), ranging from 19.2% for RSV to 65.4% for adenovirus. Two virus groups presented a statistically lower level of viral co-infections compared to all other viral groups: RSV (19.2%, p = 0.04) and picornaviruses (20.8%, p<0.0001). On the contrary, only adenovirus presented a statistically higher proportion of viruses identified among such coinfections than in other viral groups (p = 0.02). No specific viral association emerged and all respiratory viruses seem able to cohabit with any other virus.

Respiratory pathogens were detected in 741 of 1222 patients (60·6%): viruses in 716 (58·6%), atypical bacteria in 39 (3·2%) and both in 14 (1·2%; Figure1). No clear seasonality of the number of patients enrolled per month was seen during the year that the samples were collected.

The viruses detected were rhinoviruses in 229 of 1222 patients (18·7%), bocaviruses in 200 (16·4%), RSV in 144 (11·8%), parainfluenza viruses in 140 (11·5%; PIV1: 32; PIV2: 12; PIV3: 71; PIV4: 25), adenovirus in 102 (8·4%), influenza viruses in 93 (7·6%; influenza A: 77; influenza B: 16), human metapneumoviruses in 22 (1·8%), coronaviruses in 23 (1·8%; OC43: 9; E229: 14), enteroviruses in 53 (4·3%) and parechoviruses in 5 (0·4%).

Detected bacterial pathogens were M. pneumoniae (n = 33, 2·7%), C. psittaci (n = 2), C. pneumoniae (n = 1), B. pertussis (n = 1) and L. pneumophila (n = 2). Fluctuations in frequencies were found in particular months for several viruses, with RSV occurring from July to November 2008 and rhinovirus peaking in March 2009 (Figure2).

Due to differences in age group representation, the number of viral agents detected per patient was lower in Indonesia (60/225, 0·27) than in Vietnam (800/826, 0·97) and Thailand (151/171, 0·88). Likewise, the proportion of identified viral agents detected that were RSV was higher in Thailand and Vietnam than in Indonesia (24/151, 15·9%; 117/800, 14·6%, respectively, compared with 3/60, 5%) whilst coronavirus was a greater proportion of identified viral agents in Indonesia (7/60, 11·7%) than Thailand (3/151, 2·0%) and Vietnam (11/800, 1·4%). See Table S1 for pathogens detected by country.

There were 234 of 1222 patients (19·2%) who had at least two pathogens detected (bacterial and/or viral). Of these, 170 patients were found to have two pathogens, 54 patients had three pathogens, nine patients had four pathogens, and one patient had five pathogens. The frequency of each pathogen is shown in Table S2. The pathogens most commonly detected in patients with two or more pathogens were bocavirus (n = 132) and rhinovirus (n = 123). The proportion of patients who had either a virus or a bacterium detected appeared to decrease with age, with the highest proportion of pathogens detected in the 0–4 age group, but increased again in those older than 65. Most viral pathogens were more common in children, with parechovirus and parainfluenza virus 4 only found in children (Table2). M. pneumoniae was found only in patients younger than 45 years, whilst both cases of C. psittacii occurred in patients 45 years and above.

The ICU admissions for each country are shown in Table1. The number of patients who were mechanically ventilated was 42 of 826 (5·1%) in Vietnam and 21 of 171 (12·3%) in Thailand (data not available for Indonesia). For the Vietnamese data set, where data were most complete, ICU admission was related to age; 15 of 616 children (2·4%) aged under 5 years, three of 36 (8·3%) aged 5–14 years, 69 of 106 (65·1%) aged 15–44 years, 33 of 45 (73·3%) aged 45–64 years and 20 of 23 (87%) aged over 65 years were admitted to ICU (χ2 for trend P < 0·0005). Logistic regression was performed on this data set including the covariates: pathogen type (virus, bacterial, both and neither), age and sex. The analysis showed that only higher age was associated with ICU admission (adjusted OR for ICU admission in those aged >64 years compared to those aged <5 years: 243·79, P < 0·001, Table3). No specific pathogen was associated with ICU admission.

There were only sufficient data available to analyse the duration of hospitalisation for patients in Vietnam. The median duration of hospitalisation was 5 days (IQR: 3–7 days). Multivariate Cox regression analysis for hospitalisation days by pathogen type (virus, bacterial, both and neither), sex and age was performed, and both pathogen type (P = 0·0004) and age category (P = 0·015) had a significant effect. It was found that patients in the >65-year group were hospitalised longer, but did not differ by sex. Influenza A virus-positive patients were admitted for a median of 4 days (IQR: 3–8 days) versus 5 days (IQR: 3–8 days) for patients where influenza A was not detected. A similar pattern was seen for coronavirus e229: median of 3 days (IQR: 2–4 days) for positive patients versus 5 days (IQR: 3–8 days) in the negative group. Patients who were M. pneumoniae-positive were admitted longer: a median of 9 days (IQR: 7–12 days) versus a median of 5 days (IQR: 3–8 days). There were 29 deaths and a viral pathogen was detected in 12 (41%) of these: influenza virus A (n = 3), rhinovirus (n = 3), bocavirus (n = 1), coronavirus (n = 2), parainfluenza virus (n = 1), RSV (n = 1) and one combination of influenza A with adenovirus and rhinovirus. Data concerning the cause of death were not collected in this study; consequently, conclusions about causation cannot be drawn.

In November and December 2012, a few sporadic cases of patients with respiratory virus infection were detected by PCR. Escalated screening of all patients to be admitted was performed starting in week 8 to week 16 in 2013, when a significant increase of infected patients was observed. The total number of patients admitted for any reason to one of the four hematologic wards was 421 (148 female, 273 male) from November 2012 to January 2013, and 341 (123 female, 218 male) from February to April 2013. Between November 2012 and April 2013, a total of 672 patients with hematological disorders were tested for presence of influenza virus, parainfluenza virus or RSV, 111 patients were found to be infected, 40 patients were with influenza virus, 13 patients with parainfluenza virus and 64 patients with RSV, six patients had co-infections (influenza virus/RSV: n = 5; parainfluenza virus/RSV: n = 1). According to the case definition, 30 of 40 (75%) influenza infections, 11 of 13 (84%) parainfluenza infections and 36 of 64 (56%) RSV infections were community-acquired. The epidemiological curve of respiratory infections is shown in Fig 1.

Respiratory viruses are the most common cause of infection among the general population. Every year, they cause millions of illnesses, thousands of hospitalizations and deaths,4 and impose a significant economic burden on health care systems.5 Respiratory viral infections are highly contagious, and the lack of a vaccine (with the exception of influenza) and absence of long‐lasting immunity favors the continued recurrence of outbreaks of these pathogens. Despite this ubiquitous circulation, very little is known about the prevalence of these infections among the global population, as the available estimates are based on syndromic surveillance (eg, in the United States, the CDC‐based National Respiratory and Enteric Virus Surveillance System (NREVSS) and FLUVIEW1, 2) and prospective studies are usually restricted to groups at risk.

Most of the existing literature focuses on young children because acute respiratory infections are a leading cause of childhood hospitalization and mortality worldwide.6, 7 Early life respiratory viral infections, principally due to rhinovirus and RSV, have been shown to be associated with the development of recurrent wheezing and asthma in infants and children.8, 9, 10 Conversely, the impact of these infectious agents on healthy adults and the role of asymptomatic infections on transmission dynamics have not been properly investigated. A recent study found high levels of asymptomatic respiratory infection among an ambulatory adult population in New York City.11

The work presented here is part of larger project attempting to document the prevalence of respiratory viral infections among different strata of the population, with a specific focus on the environmental, demographic, and genetic factors affecting susceptibility, symptomology and transmission. Here, we have shown that respiratory virus infections are present among all age groups, with almost all participants presenting with at least one viral infection per year and an overall rate of 17.5% of positivity among all collected samples. Infection appeared to be strongly connected with age, with young children presenting more than double the number of infections of other age groups. Adults with daily contact with children (parents and pediatric doctors) also had a higher number of infections than their counterparts without daily contact with children. Moreover, the distribution of respiratory virus infections for parents and pediatric doctors was very similar to the distribution observed in the children. These observations suggest children are a principal source of respiratory infection and confirm earlier studies that found day cares to be optimal environments for transmission.12, 13 Self‐identification with American Indian or Alaskan native race was also a factor influencing the number of respiratory viral infections. This association was likely due to the non‐mixed nature of our population, as nearly all of the participants self‐identifying as Alaskan native or American Indian were children or parents from one of the day care settings. Children were also associated with a higher risk for co‐infection than adults and teenagers, as has also been shown in earlier studies.14

A larger variety of viruses was found in children and their close contacts; however, rhinovirus and coronaviruses were the most frequently identified viral respiratory pathogens in all age groups. Together, these two viruses accounted for more than 70% of positive results. The presence of multiple subsequent infections with the same virus in many individuals suggests short‐lasting immunity or potential low cross‐immunity among multiple co‐circulating serotypes of the same pathogen. Previous studies on HRV report up to 20 different rhinovirus types (among more than one hundred known) circulating in a community during one season. Further, the prevailing strains can differ widely between locations, across seasons, and switch almost completely from year to year.15

Our estimates of incidence rates differ markedly from those built on syndromic surveillance data.16, 17 Among patients seeking care, some viruses like influenza are overrepresented and others, like coronaviruses, profoundly underrepresented. This asymmetry is likely due to the different pathogenicity of the viruses causing respiratory infections and underscores the importance of using of non‐syndromic surveillance data to capture the true overall prevalence of respiratory virus infection within the general population.

A limitation of this study is the low frequency of sampling in late spring/summer months, due to decreased participation of the enrolled individuals. Despite the lower number of samples collected during these months, seasonal and non‐seasonal patterns are clearly identifiable. Some viruses (influenza, RSV, coronavirus and HMPV) had a distinct peak during winter months, whereas others circulated year round. Such assessment of seasonality for different pathogens is important for planning vaccination and control strategies and to understand the dynamics of transmission.

Future work should involve analyses of differences in pathogenicity among respiratory viruses, as well as the impact of genetic, demographic, and environmental features on pathogenicity. Moreover, longitudinal sampling coupled with information on symptomology should be used to analyze the impact of asymptomatic infections and the role of asymptomatic carriers on transmission dynamics.

In this study we found: a) a high frequency of AECOPD due to viral infections in elderly and non-elderly patients without differences between the two groups, b) more frequent infections due to human Parainfluenza virus (hPIV) and influenza in elderly patients compared to non-elderly longer and c) lengthier hospital stays for the elderly patients.

In a recently published study where we evaluated the epidemiology of viral infections in patients with AECOPD a high rate of viral infections (53.8%) was detected which was in accordance with other previously published reports. In this study when comparing the frequency of viral infections among elderly and non-elderly patients no differences were detected. A possible explanation for this could be the fact that COPD is a chronic systemic inflammatory syndrome affecting the immune response independently of the age and making these patients more prone to such infections. The elderly is a large and even growing population, proportional to the age of the general hospitalized population. This group of patients is characterized by a decline of the immunological response to infection, principally due to functional insufficiency of monocytes and macrophages that results to inadequate phagocytosis, by the lack of antigen presenting cells, such as dendritic cells (so are naive T-cells due to thymus gland involution), by the loss of memory capacity of mature T-cells exhibiting a poor and/or altered cytokine production and by the decrease of the number of circulating B-cells resulting in a weaker response to antigenic challenges through immunoglobulin production. Elderly usually have higher rates of vaccination against influenza from their primary care physicians than non-elderly (although both are at risk). However, no difference was detected either in influenza virus or other viruses’ detection rates. This could be explained by the fact that immune responses to vaccination decline substantially with age thus the elderly have impaired humoral and cell mediated immune responses to influenza vaccines compared with younger adults. It also points to the need for better prevention measures against respiratory viruses for this population.

A strong association between comorbidities (number and type) and older age is well known.

COPD patients usually have increased number of comorbitities (cardiovascular diseases, respiratory tract diseases, metabolic diseases, haematological diseases / coagulopathies,

musculoskeletal diseases, gastro-intestinal diseases, renal diseases, psychiatric diseases, neoplasias) known as “COPD comorbidome” which are considered as COPD- related (e.g. respiratory failure, pulmonary heart disease cachexia) or COPD-non related (eg obesity, diabetes mellitus, arterial hypertension). In our patients cardiac/ renal failure and diabetes mellitus were the most frequent detected comorbitities. This increased number could be explained by the orientation of our center as is a specialized site on cardio-respiratory diseases in Greece.

The infections due a bacterial pathogen were more common in elderly subjects and the hospital stay was lengthier. These patients need more time to recover because of the complexity of COPD ( either a systemic oxidative stress syndrome or an inflammatory process or a combination of those), the weaker immune response as result of the inflammatory process and the frequent colonization by bacteria that lead to bacterial infections or co-infections (viral+bacterial). This is a main statement in a recently published study where a longer hospital stay in AECOPD patients with co-existed candidiasis, anemia, psychological disorder, atrial fibrillation and congestive heart disease, asthma, respiratory failure and cachexia was detected. We didn’t find any difference in the mortality in elderly AECOPD patients with a viral infection compared to the non-elderly although mortality is higher in COPD patients with lung cancer, pulmonary heart disease, heart failure, atrial fibrillation, obstructive sleep apnea, obesity, osteoporosis and asthma.

The isolated type of viruses in two groups didn’t present any difference (Table 3) except hPIV3 detection. Human parainfluenza viruses (hPIVs) are considered to be one of the most common causes of lower respiratory tract infections in children but it is difficult to understand their biologic significance in this cohort. One possible explanation could have been the occurrence of a clonal hPIV outbreak during the study period. When analyzing the seasonal pattern of viral infections no statistically differences were noted although is well known, that COPD patients present more commonly exacerbations during the cold seasons (winter-autumn) and viral infections have a higher prevalence during this period associated with a longer recovery period, longer in house stay and increased likelihood of hospital admission. Also, despite the important role of viral infections, in this study it was difficult to examine their true pathogenic role since for some of these viruses there is an increasing evidence that they could colonize the respiratory tract; such colonizers may act as modulators of the local immune response in a subsequent bacterial upper tract infection in an already susceptible patient but the exact interaction of bacterial with viral colonizers is an issue for further debate [36, 37].

This study has some limitations. First, no quantitative PCR techniques were performed in order to test the modifications of the viral load of a specific virus which could be indicatives of the (re)activation of the virus in a previously colonized patient before the exacerbation. Second, patients with AECOPD frequently receive home care for moderate- mild episodes and for this reason we could have missed a fraction of similar episodes occurring in the community. Third, because of the low yield of sputum cultures regarding the confirmation of bacterial infections we could have missed some bacterial pathogens inhabiting or infecting the respiratory tract. Forth, there is no evident a convinced explanation for the high detection of RSV.

In conclusion, in a significant percentage of elderly people with AECOPD a viral pathogen was detected in their upper respiratory tract. Human parainfluenza viruses and mixed viral infections were more common in elderly subjects but the exact role of the different viral species is a matter for further research.

In this study, we found that PCR had higher detection rates compared with traditional antigen tests and viral cultures (75.3% vs. 48.3%). RSV, RV, and PIV3 were the leading pathogens detected in pediatric RTI patients. However, FluA, ADV, and EV were more prevalent in children older than 5 years. Knowledge of epidemiology contributes to the awareness of pathogen, accurate diagnosis, and prompt management. We also found that approximately one-quarter of specimens were coinfected with two or more viruses. However, no obvious differences in clinical manifestations and laboratory tests were found in individual virus infection or between single infection and coinfection; the clinical significance of coinfection was not fully elucidated.

A rapid and accurate diagnosis of respiratory viruses is increasingly important in clinical settings. The availability of rapid diagnostic assays is essential for optimizing the efforts of infection control teams to reduce the transmission of virulent or resistant pathogens in hospitals. Nucleic acid amplification tests are the new gold standard for the diagnosis of respiratory viruses. Our study has shown high detectability of PCR for respiratory viruses, suggesting that PCR-based diagnostic tools may be practical for detecting a wide range of respiratory viruses. Viral infection can be fatal, especially in premature infants and infants with congenital heart disease. In a previous study, symptomatic and asymptomatic premature infants were prospectively screened in a neonatal ICU using multiplex PCR twice weekly; respiratory viruses were identified in 52% of prematurely born infants during their birth hospitalization. Their length of hospital stay was significantly longer (70 days vs. 35 days), and bronchopulmonary diseases were more frequent in infected infants. In adult and pediatric patients, the major impact of respiratory viral infections with hematologic malignancies, hematopoietic stem cell transplantation, and solid organ transplantation has been recognized over the past decade. In the most immunocompromised populations, respiratory viruses have a high rate of progression to pneumonia (20%–40%). The mortality among those patients ranged from 30% to 50%. The application of multiplex PCR for respiratory virus detection in high-risk groups has been proved to be valuable. Our study showed a high detectability of PCR for respiratory viruses, suggesting that PCR-based diagnostic tools may be helpful for detecting a wider range of respiratory viruses. We also showed a high consistency of PCR with virus cultures, except for FluB, suggesting the accuracy of the PCR method. Virus culture is time-consuming and not feasible for clinical practice. It was even impossible to detect some viruses by virus cultures, e.g., coronaviruses (229E, OC43, NL63, and HKU-1), PIV4, RV, and Boca. Approximately just over half of the viruses could be detected after the wide application of PCR. These results re-enforce the importance of PCR-based diagnosis.

Viral infections are ubiquitous and may present with fever and respiratory symptoms. It is sometimes difficult to differentiate between bacterial infections and viral infections, and thus the use of unnecessary antibiotics is common. Antimicrobial resistance (AMR) has been increasing worldwide, resulting in poor treatment responses and deplorable clinical outcomes. The problem of AMR is an urgent and critical health threat and is directly associated with the overuse of antibiotics. Antibiotic treatment does not improve the clinical outcomes of viral infections. Decreasing the use of unnecessary antibiotics is the key to combating AMR, and accurate and rapid diagnosis is crucial to decrease antibiotic prescriptions with a minimized risk. The present study demonstrates that PCR has higher detectability for respiratory viruses compared to traditional antigen tests and viral cultures. PCR-based viral detection may help physicians to make appropriate decisions and decrease unnecessary antibiotic use. Furthermore, the precise diagnosis of certain viruses may contribute to timely antiviral agent treatment, e.g., oseltamivir against influenza infections. We discovered that influenza is common among pediatric patients (11.6% of respiratory specimens) and is the most commonly detected pathogen in older children (27% in children aged 5–9 years and 16.7% in children older than 10 years). Rapid diagnosis of influenza viruses and early treatment with oseltamivir or peramivir is crucial. In addition, prompt diagnosis of respiratory viruses also contributes to appropriate infection control measures and isolation care. In recent years, the cost of PCR testing has decreased, and the availability and feasibility has been largely improved. Some commercialized PCR machines are increasingly available and may serve in point-of-care testing. Hence, the widespread use of PCR-based detection of respiratory viruses is increasing and may become more practical.

The incidence of etiologic pathogens differs between adults and children. It has been reported by Jain et al. that, among the hospitalized adults with CAP, pathogens were detected in 38% of patients, and the leading pathogens were RV (9%) and influenza viruses (6%). By contrast, pathogens were detected in 81% of the hospitalized CAP children, and the leading pathogens were RSV (28%), RV (27%), and MPV (13%). Generally speaking, viral infections are more prevalent in children than in adults. The leading pathogens may also differ according to geographical region, climate, season, and year. The leading pathogens detected in our study were RSV (23.8%), RV (15.2%), and PIV3 (11.2%). A previous study conducted in Taiwan found that RSV was the most common pathogen (41.7%), followed by MPV (27.1%), Boca (6.3%), and EV (6.3%). Some important studies investigating the epidemiology of respiratory tract infection are summarized in Table 4. RSV is always the most common pathogen in young children worldwide, but the accompanying pathogens are not always the same. Virus detection was more common in summer and autumn in our study. Taiwan is located in a subtropical zone, where there are no swift changes in temperature amplitudes. Although RSV infections occur biennially, with peaks reported in the spring and autumn in Taiwan, variations in RSV infections are not particularly large. Detection of respiratory viruses could enable estimation of the local epidemiology of respiratory viral infection and help pediatricians to improve their clinical judgments.

One-quarter of the positive specimens were coinfected with other respiratory viruses in our study. A similar prevalence was found in previous studies. The rates of coinfection were between 18.8% to 36.2% in previous studies (Table 4). With the advances in diagnostic testing, the number of detectable viruses will increase. However, the clinical significance of coinfection remains unclear. Some studies reported increased severity of coinfection, but the impact of coinfection was not particularly obvious in other studies. Diversities in the study design, population, and detection methods may be the reason for this inconclusiveness. When we compared the clinical manifestations and laboratory tests for patients with negative detection, single infection, and coinfections, we found no statistically significant differences in age, body weight, hospitalization duration, ICU stay, CRP level, and complete blood cell counts; although higher platelet counts were observed in patients with coinfection. Further studies are required to clarify the clinical significance of our findings.

The strength of our study lies in the comprehensive detection of respiratory viruses and further comparison of the clinical manifestations and laboratory tests in single and coinfection. Our study is subject to some limitations that warrant discussion. Firstly, although our findings were consistent with those of previous studies, respiratory specimens were not collected in all patients with respiratory symptoms. The prevalence of EV was underestimated because the clinical diagnosis of EV infection relies mainly on the presence of oral vesicles. Further virus culture might not be performed when vesicles over oropharynx were found. Secondly, we did not include bacteria in our detection spectra. Some bacteria such as Mycoplasma pneumoniae and Streptococcus pneumoniae also play an important role in respiratory infections and commonly cause coinfections with other pathogens. Furthermore, some respiratory viruses were not included in our testing, such as the Middle East respiratory syndrome coronavirus and human polyomaviruses KI and WU.

Throughout the surveillance period, a total of 548 episodes of ILI were reported among 476 (9.7%) individuals from 330 (33.2%) households. The estimated incidence of ILI for the metropolitan Vientiane population, adjusted for demographic structure within the study area, was 10.7 (95%CI: 9.4–11.9) episodes per 100 person-years. On average, each participant reported 0.11 (range: 0–3) episodes of ILI, with a mean of 0.55 (range: 0–8) episodes per household. Fever and cough were the two commonest symptoms, reported by 92.0% and 89.6% of cases respectively. Sore throat, headache and myalgia were reported by over 60% of cases.

The results of the multivariate models for each of ILI outcomes are presented in Table 2. Variables were considered for inclusion if they showed associations with P <0.1 in the bivariate models (S2 Table). Males and young adults (25–44 years) were significantly less likely to report ILI. The number of bedrooms was inversely associated with ILI (adjusted odds ratio, AOR 0.89 per room increase; 95% CI: 0.79–1.00) and bacteria-positive ILI (AOR: 0.82; 95% CI:0.67–0.99). Having a pre-existing chronic condition was significantly associated with ILI (AOR: 1.43; 95% CI: 1.01–2.09) and virus-positive ILI (AOR:1.78; 95%CI: 1.15–2.77) but not with bacteria-positive ILI. Meanwhile, low SES and recent history of influenza vaccination were significant predictors for bacteria-positive ILI, but not ILI or virus-positive ILI.

During the follow-up period, 3 cases (0.5%) were hospitalized for their respiratory illness. Two had tested positive for Coronavirus 63, from the disease investigation (i.e. prior to their hospitalization), while in the third patient no pathogens were detected. All 548 cases were alive at the point of follow-up.

In this study on the coast of Kenya, we describe the frequency of occurrence, and the spatial and temporal distribution of 15 viruses associated with respiratory illness in the outpatient setting. We also compare the distribution of viruses associated with respiratory infection among children under 5 years of age in the outpatient and inpatient settings.

To provide baseline data for investigating the transmission patterns and pathways of spread for common respiratory viruses, we used molecular techniques to detect viruses circulating throughout a well-defined population of nearly 300,000 inhabitants in an area of ~900 km

2 in rural coastal Kenya. We observed a prevalence of 42% virus infection among the selected patients presenting with symptoms of ARI in outpatient departments of nine health facilities. These findings indicate that viruses are prominently associated with respiratory tract infections of sufficient severity for individuals to seek medical attention within this community.

Consistent with observations elsewhere

2,

5,

24, most of the ARI outpatients in this study were young children under 5 years; 50.3% of the participants in this age group had a virus positive NPS sample. This demonstrates that the under 5’s are a highly vulnerable age group for medically important ARI. The results are comparable to those from the outpatient health facilities in refugee camps and at a national referral hospital, in Kenya, where the proportions of virus-positive NPS samples in those under 5 years old with cases of ARI was 49.8%

25 and 54%

26, respectively. This age group appears to experience the highest burden of acute illnesses linked to the studied respiratory viruses and should therefore be a focus for future intervention strategies. However, additional data should be collected on the social and economic burden of these viruses, such as days of school missed, medical costs and time off work for caregivers.

Of all virus-positive patients presenting to the outpatient facilities, only a small proportion (30%) were in education (mostly kindergarten or primary.) This is of interest as the school age groups might be expected to be important agents in the community spread of infectious disease, certainly given their high rates of contact through which transmission is presumed to be effected

27,

28. There is need for empirical evidence to define the relationship between contact rates and respiratory virus transmission.

The most frequently detected virus in this study was rhinovirus. This is in accordance with previous community-based studies, for example the Tecumseh project in Michigan

29. Serological and molecular epidemiological studies show rhinoviruses to exist as many types (currently over 160)

10,

21, with little cross-type protection (amongst those classified immunologically), which may explain the high prevalence and absence of seasonality in respiratory illness caused by rhinovirus infection in the community. Persistence of rhinovirus might be ascribed to the frequent introduction of new virus strains into the community unconstrained by prior circulation of other types.

In this study, we did not see any major difference in distribution of viruses across the health facilities. This could have been attributed to the relatively small size of the demographic surveillance system area (891 km

2). This allows for the possibility of population mixing and frequent interactions, especially during social events, leading to the rapid spread of viruses across the KHDSS area. However, definitive understanding of the temporal and spatial patterns of spread requires the addition of sequence data to infer relatedness of circulating viruses.

Influenza, RSV and coronavirus exhibit a clear seasonality pattern in occurrence, whereas rhinovirus and adenovirus are detected throughout the year. Of note is that during the first quarter of the year, other than rhinovirus, RSV is predominant amongst the detected viruses. Currently little is known about the mechanisms underlying virus dominance, interaction, co-existence and competition. Studies are warranted to investigate occurrence and interactions of multiple respiratory viruses in the nasopharynx of the individual over time (i.e. across the seasons), and to explore the possible effect of eliminating a virus such as RSV through vaccination. Such information will be useful to guide policy on priority respiratory viruses to focus for intervention.

We also find the wide use of antibiotics to treat majority of patients presenting with symptoms of ARI most likely caused by viruses. This raises concern over antimicrobial stewardship, with increased risk of antimicrobial resistance to first-line antibiotic agents and unnecessary use of expensive second-line antibiotics in treating mild acute respiratory disease.

In contrast to the outpatient setting, where rhinovirus is the most common virus associated with ARI among children under 5 years; in the inpatient setting, RSV and adenovirus are the leading cause of severe respiratory illness. From the long-term in-patient surveillance at KCH the observation for RSV is not unusual, but 2016 had an unusually high occurrence of adenovirus cases (data not shown for other years). The pattern in distribution of virus load (equated to Ct values) suggests that the cut off of 35.0 for the MPX real time PCR diagnostic method is generally suitable for all viruses, both in outpatient and inpatients (i.e. irrespective of disease severity and possible viral load), excepting for HMPV, PIV-1 and CoV 22E in outpatients and PIV-2 in inpatients and outpatients, where sensitivity may be an issue and a contributor to low prevalence in this study.

The major limitation of this study is that data are for 1 year only and caution should be applied in inferring seasonal patterns. This is exacerbated by the low numbers of participants recruited in December due to countrywide industrial action by nurses. In addition, there is competition for viruses due to the design of sampling we used, of selected 15 samples per facility per week. An epidemic for one target virus might influence the proportions of other viruses observed but this might not necessarily imply seasonality. The detection of multiple viruses in one individual makes it difficult to determine the viral pathogen responsible for the respiratory illness at the time of recruitment. Only a sub-sample of all ARI presentations were recruited and the underlying denominator not recorded, which prevents an estimation of community incidence of presentations that would be useful for comparative purposes.

To determine whether the etiological agent for CAP varied with age, we compared the prevalence of viral and bacterial pathogens in each age group. Table 4 shows the distribution of each pathogenic agent according to CAP patient age. In the pre-kindergarten group, the most common pathogens were respiratory syncytial virus (n = 108, 44.6%), rhinovirus (n = 31, 12.8%) and S. pneumoniae (n = 24, 9.9%). In the kindergarten group, S. pneumoniae (n = 14, 38.9%), respiratory syncytial virus (n = 11, 30.6%), H. influenzae (n = 7, 19.4%), and adenovirus (n = 7, 19.4%) were the most common infections. Finally, in the school-age group, respiratory syncytial virus (n = 44, 47.3%), S. pneumoniae (n = 39, 41.9%), and rhinovirus (n = 20, 21.5%) accounted for the greatest number of infections.