In the present study, clinical and laboratory characteristics of ADV infection in children were investigated, and the clinical impact of respiratory viral coinfection was determined. Respiratory viral coinfection caused more LRTI and a higher need for oxygen therapy than ADV single infection. However, coinfection did not lead to mortality, and the duration of fever and hospitalization was not significantly different between ADV and coinfection groups.

ADV infection in the present study occurred at a median age of 29 months, and in agreement with previous reports, occurred more frequently in males than in females. All children except one complained of fever on admission, with fever lasting for a median of 6 days, as is consistent with published reports. Respiratory symptoms were most frequent, followed by gastrointestinal symptoms, and this was comparable with previous data. In the present study, ADV caused more URTIs than LRTIs, even as the current literature provides contradictory evidences, with some reporting a predominance of URTIs, whilst others report a predominance of LRTIs.

Respiratory viral coinfections have been identified in 1.8% - 49.2% of respiratory tract infections in previous studies. The variability in the reported coinfection frequency could be due to the methods of viral detection, the number of detectable viruses, the characteristics of enrolled patients, the frequencies of underlying disorders, the seasons when the studies were performed, and the inclusion or exclusion of URTI. Most studies reported a higher frequency of respiratory viral coinfection in younger children than in older children and adults. This presumably was caused by immature immune systems, the lack of protective immunity arising from prior exposures to viruses, and prolonged excretion of respiratory viruses in younger children compared with older children and adults. Although respiratory viral coinfection increased the rate and duration of hospitalization, it eventually did not cause a poorer prognosis such as oxygen supplementation and ICU admission, compared with single viral infection. In a recent meta-analysis, the overall effect of respiratory viral coinfections on the disease severity and prognosis was not significant.

In the present study, respiratory viral coinfection was identified in 30.5% of the enrolled children with ADV infection. This coinfection frequency was within the reported range (9.4 - 69.6%). Rhinovirus was the most commonly coinfected virus in the present study with RSV being the second, and this was in concurrence with prior published reports.

Previous studies have reported significant association between younger age and coinfection in ADV infection, as is the case with other respiratory viral infections, and the present study also showed a significant increase in coinfection frequency in children younger than 24 months of age than that in older children. In addition, the increased LRTI cases we report in the coinfection group have been reported previously. In the present study, we observed a significant association between the presence of underlying medical conditions and coinfection, although this has not been a significant coinfection associated factor in some other previous studies. Eleven of 13 children with underlying medical conditions in the present study had a preterm birth history, and eight of them had a previous history of BPD. These medical conditions may affect the lower respiratory tract function during infancy and early childhood. Amongst younger age, LRTI, and underlying medical conditions, factors related to coinfection based on a univariate analysis, only the younger age was associated independently with coinfection following a logistic regression test.

Respiratory viral coinfection in ADV infection may cause prolonged hospitalization with or without increase in disease severity, represented by oxygen supplementation and ICU admission. Some other investigators have, on the other hand, reported no significant effect of coinfection on the duration of hospitalization and disease severity. In the present study, coinfection caused more LRTIs compared with ADV single infection, and therefore, manifested as more clinically severe and progressive disease than ADV single infection. In addition, oxygen therapy was administered more frequently in the coinfection group, especially in children coinfected with ADV and RSV, than in the ADV group. However, the oxygen supplementation duration in the coinfection group was less than 3 days, and there was no ICU admission and mortality in this group. Furthermore, the duration of hospitalization and fever was not significantly different between the coinfection and ADV groups. This suggested that even as respiratory viral coinfection may contribute to increased disease severity in children with ADV infection, appropriate treatment for such a condition can prevent further disease progression and poor prognosis.

The present study had several limitations, its retrospective nature being the first. We did not identify infecting ADV serotypes in our study, and the severity of ADV infection, as well as frequency of respiratory viral infection, varied according to the causative serotype. To evaluate the impact of specific ADV serotypes on clinical severity and frequency of coinfections, a multicenter study, involving several geographically-separated hospitals would need to be conducted for a sufficiently long period to capture multiple ADV outbreaks with different causative serotypes. Attending daycare centers may further increase the risk of exposure to a variety of respiratory viruses, and influence the frequency and severity of respiratory viral infections; however, this was not evaluated in the present study. We also could not determine the independent effect of each respiratory virus concurrently identified with ADV infection because of the small group size for each respiratory virus. Finally, the impact of coinfection on the long-term sequelae of ADV infection (e.g., bronchiectasis and bronchiolitis obliterans) could not be assessed.

In conclusion, respiratory viral coinfection in children with ADV infection occurred more frequently in children younger than 24 months of age compared with children aged 24 months or older, and respiratory viral coinfection increased the proportion of LRTI and oxygen therapy requirement. However, appropriate therapy prevented prolonged hospitalization and poor prognosis due to coinfection. Clinicians should thus be more mindful of younger children diagnosed with ADV infection, and be aware of possible coinfections and faster disease progression.

Thirty-two (30.5%) children were included in the coinfection group (Table 1). Of these, two viruses were simultaneously identified in 28 children (26.7%), and three viruses were simultaneously identified in 4 children (3.8%): ADV, rhinovirus and influenza B virus in one, ADV, rhinovirus and coronavirus in another, and ADV, rhinovirus and human bocavirus in the others. Rhinovirus (n = 15, 41.7%) and RSV (n = 7, 19.4%) were the most commonly coinfected viruses (Table 1).

The proportion of children younger than 24 months was significantly higher in the coinfection group compared with the ADV group (P <0.001, Table 2). The children in the coinfection group had a significantly higher tendency for underlying medical conditions (P = 0.020), LRTI (P = 0.011), and need for oxygen therapy (P = 0.029), compared with children in the ADV group (Table 2). However, the duration of fever and hospitalization was not significantly different between the two groups (Table 2). Among laboratory test results, only absolute lymphocyte count (ALC) showed a significant difference between the two study groups (Table 3). The ESR and CRP levels were lower in the coinfection group than in the ADV group; however, this difference was not statistically significant (Table 3). In a multivariate analysis, only the younger age was significantly associated with respiratory viral coinfection (P <0.001, Table 4).

As rhinovirus and RSV were the most common coinfected viruses, the ADV group was also compared with the ADV and rhinovirus coinfection group and the ADV and RSV coinfection group (Table 2). Children in the ADV and rhinovirus coinfection group tended to be younger and have more underlying medical conditions, and LRTIs compared with those in the ADV group; however, they did not receive more oxygen therapy. Similarly, children in the ADV and RSV coinfection group also tended to be younger, have more underlying medical conditions, and receive more oxygen therapy compared with those in the ADV group; however, the proportion of children with LRTIs was not significantly different between the two coinfection groups.

Two new polyomaviruses were identified in 2007 in respiratory tract samples following large scale molecular screening using high throughput DNA sequencing of random clones and have been named after the institutes where they were found: KI (Karolinska Institute) polyomavirus (KIPyV) and WU (Washington University) polyomavirus (WUPyV). Data on seroprevalence indicate that infection is widespread ranging from 54.1 and 67% for KI and from 66.4% and 89% for WU in North American and German blood donors. Since their first identification, KI and WU viral sequences have been confirmed worldwide in respiratory samples from children with respiratory tract disease ranging from 0.2% to 2.7% and from 1.1 to 7%, respectively. However WUPyV and KIPyV were found at similar frequencies in control groups without respiratory diseases so the link between these polyomaviruses and acute respiratory diseases remains speculative.

Careful analysis is complicated by high co-infection rates with other well-characterized viral respiratory pathogens. A co-detection rate of 74% has been observed for KIPyV and rates ranging from 68 to 79% for WUPyV. Therefore, in a recent study in Southern China, hospitalized children with WUPyV infection displayed predominantly cough, moderate fever, and wheezing, but were also diagnosed with pneumonia, bronchiolitis, upper respiratory tract infections and bronchitis. As in most of infected children a single WUPyV infection was detected, it was suggested that the newly described polyomavirus can cause acute respiratory tract infection with atypical symptoms, including severe complications. Nevertheless these data have to be confirmed in further studies.

The presence of WUPyV and KIPyV in samples from children but not from immunocompetent adults suffering from LRTIs suggests that these viruses primarily infect the young population. A correlation between immunosuppression and reactivation of the two novel polyomaviruses has been suggested in immunocompromised patients and in AIDS patients at the molecular level, but no evidence of a role of these viruses as opportunistic pathogens has been given.

Overall, these data support the hypothesis that, in analogy with BK and JC polyomaviruses, KIPyV and WUPyV can establish persistent infection, and that virus replication may increase in immunocompromised hosts. However, in a recent study on immunocompetent and immunocompromised adult patients, real-time PCR detected KIPyV and WUPyV in 2.6% and 4.6% of HIV-1–infected patients respectively and in 3.1% and 0.8% of blood donors respectively, while no association was found between CD4+ cell counts in HIV-1 positive patients and infection with KIPyV or WUPyV.

KIPyV and WUPyV are also incidentally detected in adults with community acquired pneumonia, in immunocompromised hosts, and in patients with lung cancer; they are more often found in patients suffering an underlying medical condition and coinfections with KIPyV and WUPyV with other respiratory viruses are common. A recent study evaluating the prevalence and viral load of WUPyV and KIPyV in respiratory samples from immunocompromised and immunocompetent children showed that the prevalence of WUPyV and KIPyV is similar in hematology/oncology patients compared with that of the general pediatric population. High co-detection rates with other respiratory viruses, mainly RSV and enterovirus or rhinovirus, were found for WUPyV and KIPyV in both groups, in analogy with previous reports. However, higher viral loads for KIPyV in the immunocompromised group were detected, suggesting that there may be an increased replication of this virus in this population.

A similar association was not observed for WUPyV. Furthermore, in the immunocompromised group, infection with either virus occurred in older children compared with controls, which may indicate viral-reactivation Table 1.

We studied whether the presence of specific viral agents was associated with clinical parameters such as respiratory rate (breaths/min), heart rate (beats/min), and peripheral capillary oxygen saturation (SPO2, %) or with blood test parameters such as white blood count, neutrophil count, or C-reactive protein (CRP). Elevated neutrophils were associated with adenovirus infection (OR = 1.4, p=0.006, Figure 3). There were no other noticeable associations.

CRP was slightly higher in the adenovirus and enterovirus/rhinovirus infection groups than in the negative group (p=0.29 and 0.32, respectively). Neutrophils were significantly higher in the adenovirus infection group (48.7%) than in the negative group (35.6%, p=0.0058).

The pathogenesis of hMPV infection is strongly affected by bacterial coinfections with pneumococcus. One study has shown that administration of a conjugate pneumococcal vaccine is sufficient to reduce the incidence of hMPV infection of the lower respiratory tract and the incidence of clinical pneumonia in both HIV positive and negative patients. These finding suggest that the incidence of hospitalizations in hMPV infections may be decreased by vaccination with a conjugate pneumococcal vaccine. Another case report of severe respiratory failure was found to be caused by coinfection with hMPV and Streptococcus pneumonia in a 64 year old patient. Both in vitro and in vivo studies have shown that infection with hMPV facilitates adhesion of pneumococcal bacteria, which may provide an explanation for the coinfection with pneumococcal strains and hMPV.

Viral coinfections between hMPV and RSV have been reported, but remain a contentious issue. The typical seasonal overlap of the two viruses has been suggested to promote viral coinfection. One study reported a 10-fold increase in risk of admission to an intensive care unit in pediatric patients coinfected with RSV and hMPV and associated the dual infection as capable of augmenting severe bronchiolitis. Other studies do not support this finding and further report a decreased correlation between hMPV-RSV coinfections and hospitalization and additionally lists dual infection, along with breastfeeding, as having protective effects.

The data was further analysed with regard to the age distribution of virus infection (see Table 2). In infants up to 3 months old, RSV was by far the most common pathogen (58.1%), followed by rhinovirus (20.3%) and PIV3 with 8.1% each. The incidence of RSV, however, decreases significantly with increasing age (p-value < 0.0001) dropping to 13% in children older than 3 years old, while the reverse relationship is observed for Influenza A and B and HAdV. Rhinoviruses, HBoV and enteroviruses are most frequently observed in children from 4 months to 3 years of age. The age dependency of the virus incidence is visualized in Fig 3 for the seven most frequently observed viruses. The positivity rate also showed a trend according to the age group dropping from 90.5% in the under 3-month old to 78.3% in the 4–12 years old (p-value = 0.020). This may point to an increasing role of pathogens not included in the assays, such as bacterial infections in older children.

Regarding multiple infections, children less than 3 month of age and those older than 4 years had a significantly smaller risk to present with multiple infections as compared to the other two age groups (p-value = 0.014).

A reason for this could be that very young children have limited contact to others reducing thereby the chance for a co-infection, whereas children older than 3 years already established immunity to an increasing number of viruses encountered previously.

HRVs are currently classified in the Picornaviridae family, genus Enterovirus, that includes 3 species: HRV-A, HRV-B, and HRV-C. Within each species there are multiple HRVs designated as “serotypes”, “types”, or “strains”. Several recent epidemiological studies suggest that HRV-A and HRV-C are the predominant species associated with acute respiratory illnesses in hospitalized children and adults, compared to HRV-B which are rarely detected.

The new HRV lineage designated HRV-C has been identified using molecular methods and associated with severe clinical presentations in infants and immunocompromised adults. Symptoms of patients infected with this new strain were mainly bronchiolitis, wheezing, and asthmatic exacerbation in cases from Australia and Hong Kong, which peaked in fall and winter whereas in New York the new rhinovirus genotype was detected in cases of influenza like illness (ILI) that were clustered within an 8-week period from October to December. A recent study describes a clinical case of severe respiratory and pericardial disease in an infant infected by HRV-C suggesting tha viral tropism is not strictly restricted to the respiratory tract. A study focusing on the global distribution of novel rhinovirus indicates its association with community outbreaks and pediatric respiratory disease also in Africa and in symptomatic subjects living in remote locations having limited contacts with other human populations. Moreover evidence for a role of HRV-C in lower respiratory tract disease and febrile wheeze in infants and asthma exacerbations in older children was reported. Recent studies making comparisons between HRVs species, found the HRV Cs more so than As or Bs as the major contributors to febrile wheeze and asthma exacerbation in infants and children, respectively . However, the severity of clinical manifestations for HRV-C is comparable to that for HRV-A in children with community-acquired pneumonia. In HRV C studies so far, no clear clinical difference has been noted between patients with single or mixed HRV-C infection. In a study, monoinfection was observed in more than half of cases and was more common than RSV monoinfection in patients with upper RTD, however the duration of hospitalizations was not significantly different between the HRV-C monoinfection group, HRV-A or HRV-B monoinfection group and RSV group suggesting that HRV-C is an important etiological factor in children with RTI. Most HRV-C co-detections are with RSV, however in a large study HRVs were statistically the least likely virus of 17 examined to be associated with co-infections Table 1.

In this study, we calculated the frequency of different respiratory viruses present in children with lower respiratory tract infection at the Vietnam National Children's Hospital. The high proportion of viruses detected in pediatric ARI patients agreed with previous studies done in Vietnam. Tran et al. found that in the South of Vietnam, human rhinovirus (HRV) and human bocavirus were associated with the severity of children with respiratory infections, while those viruses were detected rarely in the North of Vietnam due to the difference of the location and climate between the South and North of Vietnam.. RSV and EV/Rhi were the most frequent viruses, which was similar to the findings of a study done in Southern Vietnam.

RSV, rhinoviruses, influenza viruses, parainfluenza viruses, enteroviruses, coronaviruses, and certain strains of adenovirus are the leading causes of viral respiratory infections in children. The nasal or respiratory secretions from children with viral respiratory tract infections contain more viruses than those from infected adults. The increased output of viruses, together with an overall reduced attention to hygiene, makes children more likely to spread their infection to others.

When viruses invade the cells of the respiratory tract, they trigger inflammation and mucus production, which causes nasal congestion, runny nose, scratchy throat, and cough. The small airways of young children can be significantly narrowed by inflammation and mucus, making breathing difficult. Airway problems are most common in infections caused by parainfluenza viruses, RSV, and human metapneumovirus [1, 8, 17]. Bronchiolitis occurs predominantly in the first year of life and with decreasing frequency in the second and third years. It is characterized by inflammatory obstruction of the small airways and hyperinflation of the lungs and typically presents along with breathing problems and wheezing. RSV is the primary causative agent of bronchiolitis worldwide, causing between 70 or 80 percent of ARIs during the high season [18–20]. RSV was the only virus associated with bronchiolitis in this study.

Neutrophils are immune cells that are present in many lung diseases associated with acute respiratory distress syndrome (ARDS) and may contribute to acute lung injury. Neutrophils are poorly studied with respect to viral infection. We observed an association between elevated neutrophil count and adenovirus infection, which might indicate an association between neutrophil count and damage to the alveolar epithelium.

Finding biomarkers to diagnose specific viral infections is essential to improve patient care. CRP is an acute phase protein synthesized by the liver in response to IL-6 increase, which is used as a biomarker of inflammation and to distinguish between bacterial and viral infections. It is not well known if CRP levels differ between different viral respiratory infections. In this study, we found no significant association between CRP and a specific virus.

HBoV1 is a respiratory pathogen affecting all regions of the globe and is associated with approximately 2%–19% of all upper and lower respiratory tract conditions. HBoV1 productively infects human airway epithelium cell cultures and leads to damage of airway epithelial cells, which supports clinical observations that infection does result in respiratory disease. In contrast, hBoV2–4, are found in the gastrointestinal tract and hBoV2, and possibly hBoV3, are associated with gastroenteritis. Interestingly, HBoV2 is the only enteric bocavirus to be isolated from nasopharygeal aspirates and may, therefore, also be associated with respiratory disease. HBoV1 is detected in all age groups, but predominantly in young children between the ages of 6–24 months and is rarely detected in adults. Transmission and infection occurs throughout the year, but predominantly during winter and spring months. Seroprevalence studies suggest that maternal antibodies, which provide protection, are present in infants younger than 2 months of age, after which seropositivity decreases with low levels of detection until 6–12 months. Virtually 100% of children aged 6 are seroconverted for hBoV1 and as reinfection occurs throughout life this remains into adulthood. The presence of the three enteric bocaviruses does however complicate the findings of seroconversion as cross-reactivity does exist.

As with many respiratory viruses, clinical differentiation with hBoV1 infection is not possible by symptomatic presentation. Common features of infection of the upper respiratory tract include common cold-like symptoms with cough, rhinorrhoea and acute otitis media. Infection of the lower respiratory tract in children is associated with pneumonia, acute wheezing, asthmatic exacerbations and bronchiolitis, but life-threatening complications are rare with hBoV1 infection. Although hBoV1 has been isolated from stool samples, there is no statistical evidence to associate hBoV1 with gastrointestinal disease. HBoV1 has not only been found in the upper and lower respiratory tract and gastrointestinal specimens, but also in urine samples, serum, saliva, and tonsils. Rather than having a role in disease pathogenesis, this viraemia and systemic spread may be a feature common to all Parvoviruses as they require proliferating host cells for replication.

Interestingly, hBoV appears to be more than just a respiratory or gastrointestinal virus. In a recent study, hBoV was identified in 18.3% of lung (n = 11/60) and 20.5% of colorectal (n = 9/44) tumors screened. Unfortunately, the study did not investigate whether the hBoV genomes were in fact incorporated into the host genome as reported for other known Parvoviruses. Therefore, based on their observations as well as previous studies on other parvoviruses, the authors speculate that hBoV could contribute to the development of some lung and colorectal tumors. However, they do also acknowledge that these tumors could simply be providing an optimal environment for hBoV replication and more conclusive studies are required to resolve this issue.

In this study, 24.6% of patients with serious lower respiratory tract illness were detected having HBoV in their respiratory tract aspirates. We confirmed that HBoV was frequently detected in children with severe LRTIs. Wheezing was one of the most common symptoms presented by HBoV-positive patient. HBoV at a high load could be an etiologic agent for LRTIs, which led to more severe lower respiratory tract symptom, longer hospitalization, and longer duration of wheezing.

HBoVs have been detected worldwide, low HBoV viral load was usually more frequently detected than high HBoV viral load in children,,, its role as a pathogen in respiratory tract was often questioned. However, HBoV at a high load were clearly suggested for being an etiologic agent for respiratory tract disease. In our study, fifteen out of thirty-one HBoV-positive patients who had high HBoV viral loads, presented acute primary infection. Symptoms, duration of hospitalization were compared among patients who had LRTI with or without HBoV infection. We found that in patients of LRTIs with HBoV infection needed longer hospital stay. These findings suggested that HBoV was one of the common pathogens for children with serious LRTIs.

Although, increasing number of studies suggested the pathogenic potential of HBoV in young children with respiratory tract infections, however HBoVs were frequently detected in asymptomatic children's respiratory tract secretions, which may raise some justifiable concerns for etiology,–[29]. The variation of HBoV detection in these studies was likely attributable to multiple factors, such as patient's age, season, geographic location, primer sensitivity, laboratory technique, true variation in incidence of HBoV, and methods used in sample collection. Some studies suggested that persistent virus shedding was therefore being a reasonable explanation for the high occurrence of HBoV in healthy subjects. Moreover failure for follow-up on HBoV-positive asymptomatic children also led to high occurrence of HBoV detection in asymptomatic children. Recently, an in vitro study of HBoV infection in pseudostratified epithelial cells was established to confirm the virus uptake, transcription, and replication. This study supports the concept that HBoV is a respiratory pathogen.

In agreement with previous studies, co-infection with other pathogens was common in HBoV-positive patients in our study, but no significant differences were found in term of frequencies of specific respiratory symptoms, duration of hospitalization, wheezing and the cytokine production in patients with HBoV infection only and patients with co-infection with other viruses or bacteria. Clearly, our results indicated potential co-infections will not influence clinical outcome of HBoV infection in respiratory tract. This finding strongly suggested the pathogenic potential of HBoV in young children with LRTI.

Because virus co-infection would not increase illness duration or severity in virus-induced respiratory disease in our study or the others,–[34]. Therefore the more severe lower respiratory tract symptom presented in high HBoV viral load patients may solely depend on HBoV viral load. In our study, HBoV viral load did not show significant influence on the presentations of upper respiratory tract infection symptoms, such as cough, rhinorrhea, however high HBoV viral load led to more severe lower respiratory tract symptoms and longer hospitalization. It was also indicated in vitro studies that viral infection resulted in the high IL-8 level and other pro-inflammatory molecules; these cytokines were likely to bring in additional neutrophils and also to cause hyper-responsiveness in bronchus, which could contribute to the severity of both upper and lower respiratory symptoms during the viral infection. In our study, the IL-8 level was significantly higher in patients with high HBoV viral loads that those who with low viral load. Overall, our findings indicated that the high HBoV viral loads played an important role in the severity of LRTIs, the symptoms and the duration of hospital stay.

No previous studies showed the distinctive clinical signs that help to differentiate HBoV-positive infections from other viral infections,. In our study, main clinical symptoms in HBoV-positive patients included cough (100%), wheezing (90.32%), and fever (41.94%). Although the existence of HBoV was not directly associated with the illness, some studies showed wheezing was the main manifestation of HBoV infection,,–[39]. Our data was consistent with the findings in these studies and support the fact that wheezing was one of the most common symptoms presented in HBoV- positive patients. We also investigated the relationship between HBoV viral load and duration of wheezing, we found that the days of wheezing correlated with viral load. Emily et al documented that persistent HBoV shedding, may increase the duration of respiratory symptoms. This report may explain the link between HBoV load and persistent wheezing.

Because our study is a comprehensive clinical observation on HBoV- positive patients with severe LRTIs, failure of follow-up was one of the limitations to our study, which may led to the loss of some important data, such as the impact of persistent viral shedding on the infection prognosis during the acute viral infection. It is also important to assess the asthma development in these cases if possible.

High HBoV viral load was detected in 15 patients. The comparisons of demographics and clinical data among patients who were shedding high HBoV (>104 copies/mL of respiratory tract aspirates) and those who were shedding low HBoV were shown in table 5. In HBoV-positive patients, the distributions of age and gender were not different between the two groups. Almost all of HBoV-positive patients presented cough and wheezing. Non-specific lower respiratory tract symptoms, such as fever and rhinorrhea, had no significant correlation with HBoV viral load. However, the symptoms observed in patients with serious LRTIs, like tachypnea, dyspnea and cyanosis, were presented more frequently in children with high HBoV viral loads, and the mean duration of hospitalization in these children was 14.67±1.93 days, which was significantly longer than in children with low HBoV viral load.

Regarding seasonality, different patterns of circulations could be observed for RSV, rhinoviruses and influenzaviruses (A and B combined) (Fig 2), with RSV and influenza exhibiting a clear seasonality with marked peaks in January/February, while rhinovirus infections did not exhibit a pronounced seasonality being detected almost throughout the year. However, as more than 100 different rhinovirus strains have been identified to be circulating worldwide in parallel and successively, a potential seasonality of individual rhinovirus serotypes may be masked by overlapping patterns.

Following the discovery of WUPyV in Australia, the virus was detected in specimens from patients with respiratory tract disease on all continents suggesting a worldwide distribution [10,29–31]. So far, WUPyV-DNA was reported to be found in respiratory tract specimens (e.g. nasopharyngeal washes, tracheal secretion, BAL), serum, and faeces. The virus could not be detected in urine or from UV light-associated primary malignant lymphomas. Specimens from other malignant diseases have not been investigated. The use of tracheal secretion for diagnostics has been shown to lead to an underestimation of the rate of positive specimens compared to other respiratory materials for HBoV. This may be true for WUPyV, too.

The data available are mainly based on retrospective studies exclusively including symptomatic patients. The detection rate in respiratory samples from children with respiratory disease varies from 0.4% to 11.5%. The age of WUPyV infected patients ranged from a few weeks to 53 years, children <3 years of age were dominating. Infections were predominantly detected in late winter, spring, and early summer. High infection rates were reported for study populations preselected for lack of immunocompetence. HIV positive patients had detection rates of up to 35.7% in respiratory tract specimens and 8.3% in blood. The rates of co-infection with established respiratory viruses lay between 30.8% and 91.7%, commonly exceeding 50%. WUPyV was detectable in blood, possibly indicating its potential for systemic infections.

Le and co-workers presented evidence for viral persistence, Wattier et al. for nosocomial infections with WUPyV.

The real time protocols available allow the quantification of WUPyV. Quantification of viral loads in respiratory tract specimens revealed viral titers up to 1010 copies/ml, but low and medium viral loads were dominating. No correlation between viral load and the rate of co-infection or clinical diagnoses was observed.

Few studies included asymptomatic control groups [34,36,42–44]. The results were not concordant and reached from higher detection rates in the control group to higher detection rates among the group of patients with respiratory tract diseases.

Prospective studies have been published only recently. Van der Zalm and co-workers reported the detection of WUPyV in a cohort of 18 children. Their parents were contacted twice a week over a 6-months period (November to April) and asked for symptoms of respiratory tract disease in their children. Every two weeks respiratory tract specimens were collected, regardless of respiratory symptoms. 11.5% of the specimens of children with symptoms were WUPyV positive, but only 3.1% of specimens of healthy children, indicating at least an association of WUPyV with disease.

Respiratory tract infections are a leading cause of morbidity in children.

Studies conducted in industrialized countries report a prevalence of respiratory tract infections ranging from 3.4 to 32.1% in the first year of life (1–4). Respiratory tract infections are also a major reason for hospitalization in children younger than 5 years (5–7).

Different studies have explored and confirmed the role of clinical and socioeconomic risk factors for respiratory tract infections, including birth weight, gestational age, socioeconomic status, ethnicity, number of siblings, day care attendance, and parental smoking (4, 8, 9). Breastfeeding is included among the protective factors for respiratory infections in infants. The protective role of breastfeeding against respiratory infections has been repeatedly demonstrated for children living in developing countries (10–12). Although breastfeeding is described as protective also in industrialized countries, different study designs, definitions (e.g., for infant feeding and kind of infection), timing of evaluation of exposure (feeding), and outcome (infection), have been used in studies performed this setting.

Most studies confirm a protective role of breastfeeding against respiratory infections in the long term, as the outcomes are often measured after 6 months of age, or even at 1, 2, or 6 years (13), showing a persistent protective effect even after breastfeeding has been stopped.

In fact, protection seems to be time dependent: in a large cohort of infants in the UK, those who were breastfed for <4 months had a higher risk of hospitalization for infectious diseases in the first year of life compared with those who were breastfed for more than 4 months (14). In addition, infants who were breastfed for 4–6 months showed a higher risk of both pneumonia and recurrent otitis media compared to those who were breastfed for 6 months or longer (15–17).

Fewer are the studies analyzing the protective role of breastfeeding in the first 3 months of life. While Duijts et al. report a protective effect of breastfeeding in children exclusively breastfed until 4 months of age compared to never breastfed infants (1) other studies report a weaker protection for children younger than 3 months (18) or no protection at all (19).

We report here the results of a case-control study exploring the association of breastfeeding with viral respiratory infections in a metropolitan area, in children younger than 12 months of age.

We found a very high rate of bacterial colonisation in HCWs, especially Streptococcus pneumonia, with fluctuation in infections over a period of weeks. Almost 88% of all HCWs had bacteria detected in the nasopharynx at baseline, the end of the study period or both. This is a much higher rate of colonisation compared to other studies of adults. For example, other studies of adults show rates of 5–20% [27, 28]. We have previously shown only 0.3% of elderly subjects carry pneumococcus in the nasopharynx. The finding of such a high rate in this HCW population may reflect greater exposure to respiratory infections in the hospital setting and confirms the continual, ongoing risk to HCWs in the hospital setting.

Respiratory infections in hospital HCWs are of particular concern due to the risk of transmission to patients who are ill and/or immunocompromised. Respiratory tract infections generally present with symptoms such as fever, tachypnea, shortness of breath and cough. However the relationship of bacterial colonization to symptomatic illness has not been studied extensively. We found a very high and dynamic rate of bacterial colonisation in hospital HCWs, with changes from baseline to the end of the follow up period in the individuals with infection as well as the types of infection.

Colonisation is important as this may progress to invasive disease. Bacterial colonisation may be an important source of horizontal spread of infection within the community. Among 170 HCWs with positive bacterial result at baseline, 68 (40%) became negative at the end of the study. Natural clearance of bacteria in asymptomatic and symptomatic subjects has not yet been studied. The rates of bacterial colonisation in symptomatic HCWs were higher than in asymptomatic HCWs, but this was not significant. Bacterial colonisation in the majority of the HCWs resolved without any treatment or development of symptoms. We found 12 cases of CRI developed over 4 weeks, 11 of which had bacterial colonisation at baseline. If bacterial shedding occurs asymptomatically, then a large amount of undetected transmission may be occurring in hospitals. This may be important for bacteria such as pneumococcus, where the transition from carriage to invasive disease is thought to occur soon after acquisition of infection.

Of interest, we identified 5 cases of asymptomatic viral infection - four rhinovirus/enterovirus and one influenza A(H3N2). Few studies have been conducted on the incidence of asymptomatic viral infection, and of these, the results are often inconsistent. One study examined the rate of asymptomatic infection resulting from inoculation and found that 1/3 of participants did not develop any symptoms whereas a more recent study found the rate of respiratory illness attributable to influenza infection to be 27 respiratory illnesses per 100 persons. Our findings indicated a high rate of asymptomatic infection at baseline, being cleared without the development of symptoms. The clinical significance of such findings is still unknown with limited information on viral shedding and transmission in asymptomatic subjects. It is well known that influenza virus is shed from the respiratory tract in the incubation period in asymptomatic subjects, and asymptomatic infection has also been observed with parainfluenza virus infection. It has also been found that viral shedding of influenza occurs on average for 5 days after infection, indicating that some positive tests could have been in HCWs recovering from influenza. Asymptomatic viral infections pose a significant risk of nosocomial transmission to both patients and HCWs.

We found many co-infections in this study. Previous studies have demonstrated that a viral infection may facilitate bacterial colonisation or co-infection with S. pneumoniae. This may be a significant concern as such co-infection has been associated with significantly higher morbidity and mortality. A growing body of evidence suggests that the risk of bacterial respiratory infections is increased by co-infection with viruses and vice-versa, however bacterial respiratory tract infections are generally not considered a major occupational hazard. Despite documented outbreaks of Bordetella pertussis, Chlamydia pneumoniae and Mycoplasma pneumoniae [32–36], there are few prospective studies of bacterial respiratory infections or colonization, nor consideration of the clinical implications for HCWs. The risk of co-infection has been reported in schools and daycare centres with subsequent community transmission, but not in HCWs. It has also been suggested that viral infection may facilitate bacterial colonisation of the respiratory tract particularly with S. pneumoniae. Studies in mice have found that influenza virus infection increases the transmission and burden of pneumococcal disease. Similar findings have been reported in other studies demonstrating significantly higher morbidity and mortality of cases with influenza virus co-infection with S. pneumoniae. This is suggestive that the role and significance of viral infection in the nasopharynx may be complex, highlighting the need for further research into this topic.

Being a healthcare provider has been identified as a major risk factor for respiratory infections [38, 39], however even within HCWs, the risk varies significantly. Hand hygiene, use of personal protective equipment (PPE) and working on intensive care units (ICUs) have been associated with risk of influenza. Interestingly, factors such as vaccination status, performing high-risk procedures, working on respiratory and paediatric wards and smoking were not found to be significant in predicting bacterial colonisation in this study. Smoking, influenza vaccination status and ward type in hospitals have been previously identified as risk factors for respiratory infection in various groups [40, 41] however our findings suggest that such risk factors may not be absolute and may vary in different situations. The effect of vaccination also needs to be studied. Some studies show that pneumococcal vaccination may reduce colonisation with vaccine-serotype pneumococcal infection, though replacement by other strains reduces the overall effect. Previous studies showed that medical masks and respirators reduce the risk of bacterial respiratory infections, which further supports the occurrence of nosocomial transmission of bacteria.

The limitations of this study include that we did not test for bacterial or viral infection at the time of reported symptoms. This would confirm that an infection was the cause of symptom development and also ensure that no other infections were missed within the 4 weeks. Our sample size may have also been too small to detect differences in colonisation between symptomatic and asymptomatic subjects, or for analysis of risk factors such as smoking and underlying disease as there were very few participants in these categories. We were unable to recruit the initially planned sample size so a larger scale study is warranted. The selected follow up period of 4 weeks was the maximal period of follow up possible within the available resources for the study, but longer follow up would be valuable. Finally, these results may not be generalised due to varying geographical distribution of pathogens and vaccine uptake by country.

The clinical diagnosis in order of frequency were rhinopharyngitis in 67%, pharyngitis in 18%, influenza-like illness, pharyngotonsillitis, and laringotracheitis in 3%, followed by bronchiolitis and rhinitis in 2%, rhinosinusitis in 0.6%, and pertussis-like syndrome in 0.2% (Table 3). All children were managed in an ambulatory fashion, and none required hospitalization. The most frequent signs and symptoms were cough in 85%, rhinorrea in 81%, nasal congestion in 70%, nasal discharge in 68%, fever in 68% and odynophagia in 61%. Other less frequent signs and symptoms were headache in 36%, myalgia in 25%, nausea in 21%, vomiting in 22%, dysphonia in 17%, respiratory distress in 13%, diarrhea in 11%, dyspnea in 11% and conjunctivitis in 8% of the children (Table 3).

We found differences in the pathogens detected among single viral infections according to the clinical diagnosis. In the children with rhinopharyngitis the most frequent pathogen was RSV A and B in 20%, followed by RV in 13.5%, FluA in 5.4%, AdV in 3.7%, and HMPV in 3.4%. In contrast, in children with pharyngitis the most frequent pathogens were RV in 10.5%, EV in 8.4%, FluA in 7.4%, HuCoV 229E/NL63 in 6.3%, and HMPV and PIV-3 in 4.2%. In children with rhinitis the pathogens detected were RSV-A in 27.3%, RV in 9.1%. The pathogens that affected the larynx, trachea and bronchiols were RSV, RV, and FluA (Table 4).

In summary, we have found very high rates (almost 88%) of bacterial colonisation, viral infection and co-infections in hospital HCWs, far higher than rates previously described in adults. Most studies show that adults have rates much lower than children, yet the rates we demonstrated exceeded even colonisation rates in children, possibly reflecting hospitals being a high exposure setting. Respiratory tract infections were also dynamic and changing over time, with different HCWs infected at baseline and the end of the study, with different pathogens. We were unable to determine the relationship of symptoms to colonisation because of a small sample size, but suggest larger studies are warranted. Our results suggest there is a continual ongoing risk of respiratory infection in hospitals HCWs.

In summary, we compared the spectrum, seasonality, age distribution, and coinfection of respiratory virus infections in children with URTIs and LRTIs. Our data show that the viral epidemiology differed between URTIs and LRTIs. It is necessary to monitor respiratory viruses over long periods of time to determine their epidemiology.

According to the multivariable analysis, having at least one sibling was associated to a higher risk of viral respiratory infection (OR 3.6; 95% CI 2.14–5.92) as well as having a smoking mother (OR 2.6; 95% CI 1.33–4.89). Being exclusively breastfed at symptom onset was associated with a higher risk of viral respiratory infection (3.7; 95% CI 1.64–8.41) but protection increased with breastfeeding duration (OR 0.98; 95% CI 0.97–0.99) (Table 3).

In our study it was possible to identify common respiratory viruses in samples of the lower respiratory tract of adults under invasive mechanical ventilation, regardless of the presence or absence of acute lower respiratory infection, through real-time PCR techniques. In the field of pulmonary virology investigation, several factors make this study innovative: the age and type of patients, the inclusion of two groups of patients, with (WRI group) and without acute lower respiratory infection (WORI group), and also the type of samples (protected samples from the distal lower respiratory tract). According to Luyt et al, BAL is the respiratory sample of choice to evaluate lung infection.

Nucleic acid extraction was performed with the same commercial kit used in the study of Wang et al, who analized the respiratory virome of healthy and severe acute respiratory infection children through metagenomic analysis. We used “in-house” real-time PCR and RT-PCR techniques that were tested and validated long before.

Thirty percent of the patients belonging to WORI group had common respiratory viruses in the distal lower respiratory tract. According to data from the pediatric population, lung virome of asymptomatic individuals is less diverse than that of infected individuals, and consists mostly, of members of the Anelloviridae family, with a lower percentage of common epidemic respiratory viruses. In the study of Wang et al, samples from infected patients had six to seven-fold more viral pathogens than the WRI group. These authors suggested that the viral infection may be asymptomatic and, occasionally prolonged, making controversial the interpretation of a positive PCR for some viruses.

. In a study conducted by Choi et al, the main respiratory viruses associated with severe pneumonia in adults admitted in ICU were HRV (23.6%), HPIV (20.8%), HMPV (18.1%), influenza (16.7%) and RSV (13.9%), according to data obtained from real-time PCR analysis in bronchoalveolar lavage samples. In the study by Xu et al, 368 samples collected with nasopharyngeal swab from infected children were analyzed with real-time PCR, using a panel of 18 respiratory viruses. The percentage of positive samples was 58.97%.

In our study, half of the WRI group patients had positive samples. Comparing the two groups, influenza AH3 was the most prevalent virus, coinciding with the peak of influenza in Portugal. RSV, HMPV and HRV were common to both groups. Differences were found with HPIV 1/3, which was more prevalent in uninfected patients, and HEV and HBoV that were, only found in WRI group. The analysis of the Ct values showed similar viral loads in both groups, although with a slightly tendency for lower Ct values in WORI group. The small number of samples did not allow us to draw conclusions about possible differences in viral loads between infected and uninfected individuals.

It may be hypothesized that some respiratory viruses, such as influenza, RSV, HMPV and HRV, may transiently colonize the mucosa of the tracheobronchial tract at times of increased viral activity, whereas others, such as HPIV 1/3, might be prolonged colonizers. However, only a longitudinal study could determine the extent of the colonization period and thus solve this important issue. In addition, we need to know the meaning of its presence, if they are only bystanders, even during an acute respiratory infection, or if they are responsible for symptomatic infections of the lower respiratory tract.

Our study has some limitations, including a small sample size and a limited number of centers involved, which does not allow us to make inferences about mortality between the groups and subgroups. In addition, all the patients in both groups were severely ill. Therefore, the observations made in this study may not be generalizable to other groups, in particular to healthy populations.

Although signs and symptoms of respiratory infection were excluded at admission in the WORI group, viral respiratory infections in the last weeks preceding hospitalization cannot be excluded, and therefore viral detection in some of the cases could be the result of a recent infection and not an extended stay in the lower respiratory tract.

In this study a protected mini-BAL (with a double catheter, Combicath® kit) was used in order to reduce the upper level contamination. However, it is possible that contamination from upper respiratory secretions, mainly due to the ventilation process, may have had an important contribution to the detection rate observed in this study.

Another limitation relates with the methodology used: although real-time PCR is currently the gold-standard for the diagnosis of viral infections, this methodology is limited to specific target sequences. In fact, only the most common respiratory viruses were investigated. According to Willner et al, PCR-based studies confer an incomplete airway virome picture and little opportunity for the discovery of new agents, as compared to metagenomics, a technique independent of genomic sequences. In addition, viruses like herpesvirus simplex, cytomegalovirus or torque teno virus can be present in the distal airway mucosa [13, 20–22], and therefore may have been missed with our PCR strategy. However, despite this limitation, the main respiratory viruses were properly searched, and this is the group of major concern, when dealing with respiratory infections. Another important advantage of this study was the focus on a population undergoing invasive mechanical ventilation, allowing the collection of samples from the distal airways, although blindly, which is usually a limitation in this type of studies.

Seasonality is well described for several viral respiratory pathogens [5, 6]. In our study there was a seasonal predominance for RSV-A from September to December, FluA was more frequent from December to February, FluB from December to April, and HMPV predominated from February to May. In contrast, RV, EV, AdV, and PIV-3 were present during all the study period (Figure 2).

Our understanding of EBOV transmission in humans mainly relies on epidemiological observations. Contact with bodily fluids from EVD patients remains the most likely route of transmission. Notably, the number of past outbreaks and associated epidemiological studies hat carefully examine transmission patterns is small. Therefore, conclusions about transmission are based on relatively limited data sets. Interestingly, 18 (6.6%) of the 2774 cases in the 1976 SUDV outbreak in Nzara, Sudan, and 55 (17.4%) of the 316 cases during the 1995 EBOV outbreak in Kikwit, DRC, had no direct or physical contact with an infected person or known infected dead body, thus pointing to other possible routes of transmission, e.g., human to human respiratory tract infection through droplet and aerosols. During the 2013–2016 Western Africa epidemic, more than 890 health care workers (HCW) were infected, with a case fatality rate of 57%, whereas during the current 2018–2019 outbreak in DRC, as of 22 July 2019, 140 HCW have been already affected (5.4% of total cases).

Currently, full body protection is recommend by WHO and CDC. All HCW involved in the care of EVD patients must receive training and demonstrate competency in performing all Ebola related infection control practices and procedures, specifically in proper donning and doffing PPE even if using an N95 mask or a powered air-purifying respirator (PAPR). The risk of infection via inhalation of contaminated aerosols from exposed individuals has not been documented. However, droplets containing EBOV that have become aerosolized (e.g., from coughing sneezing, vomiting, invasive medical or surgical procedures, or surfaces) may have the potential to come into contact with a person’s mucous membrane in their nose or mouth or non-intact skin. Therefore, respiratory protection may be helpful in providing a barrier to help prevent infectious materials from contacting a wearer’s mucous membranes.

Finally, the epidemiologic and viral evidence of EBOV detection and replication in the respiratory tract raise concerns on the need of strict application of cough etiquette for patients and of droplet and/or respiratory precautions for all HCW involved in the clinical management of EVD suspected and confirmed cases.

Acute respiratory tract infections (ARTIs) remain a leading cause of mortality, morbidity, and economic loss, and viruses are one of the main causes of such disease. WHO estimates that ARTIs cause nearly four million deaths per year, a rate of more than 60 deaths/100,000 people. The microbial etiology of ARIs is varied, with viruses being the most common cause in humans, leading to a high level of awareness and the necessity to develop countermeasures to control them (Table 3).

Filoviruses are not commonly considered to be viruses responsible for ARIs, even if respiratory symptoms may be present as a consequence of diffuse systemic alterations. Interestingly, evidence collected in animal studies, in the epidemiological analysis of transmission chains, and in the most recent Ebola outbreaks suggests that EBOV may be able to cause primary pulmonary infection. This evidence highlights the ability of the virus to be shed in the lung, suggesting a role in lung pathogenesis. Specifically, the relevant proportion of EVD patients without any epidemiologic link to the exposure to contaminated biological samples or fomites, or to any contact with EVD patients; the evidence of respiratory signs and symptoms commonly reported all over the clinical course; the abundance of viral antigens in the lungs in animal necropsies; the prolonged persistence of EBOV detection and replication within the respiratory tract days after undetectable EBOV viral load in plasma; and similar clinical patterns in several other viral respiratory tract infections are all different parameters with consistent evidence of a major role in the pathogenesis of EVD in respiratory tissues.

On the other hand, there is no evidence of aerosol transmission in EVD. However, different studies addressing this issue have been performed, and aerosol transmission was considered a possibility as a consequence of epidemiological observations in past outbreaks, where people showed signs of EVD even in the absence of a direct or physical contact with an infected person or known infected dead body. This hypothesis was corroborated by other studies, in which the presence of free viral particles in alveoli and within intra-alveolar macrophages demonstrated a pulmonary involvement.

From a clinical point of view, the 2013–2016 EBOV outbreak underlined the lung involvement in EVD pathogenesis. In fact, only a few patients treated in Europe and USA had a cough and difficulty breathing at admission. Nevertheless, during the clinical progression, half of the patients experienced hypoxemia while breathing room air, one third had respiratory failure, and one fourth received invasive or non-invasive mechanical ventilation. In the Italian experience at the National Institute for Infectious Diseases “L. Spallanzani” (INMI), respiratory symptoms were present in both patients, in the absence of other common respiratory pathogens. One case required mechanical ventilation and the other presented EBOV replication markers in the lungs even after clearance of the virus from the blood. The INMI experience suggests a direct role of the virus in lung pathogenesis.

Although lung pathogenesis in EVD may be secondary to systemic alterations (correlating with general pathogenic mechanisms) the direct presence of the virus is undisputable in the lung, and its interaction with the immune system, whose hyper-activation may be the most likely explanation of the lung damage, is also indisputable. Further research will be needed to better understand the potential role of pulmonary involvement in EVD and whether it may be a factor in the transmission of the virus from one human to another.

In all 323 patients with LRTIs, a total of 93 and 48 specimens were positive for pathogenic bacteria and copathogenic bacteria, respectively. Streptococcus mitis, Streptococcus viridans, and Klebsiella pneumonia were the prevalent bacteria with positive rates of 31.91%, 21.99%, and 19.15%, respectively (Table 5). Group D streptococci were the most likely pathogens to be coinfecting, with the positive rate of S. mitis of 12.06%. The percentage of viral-bacterial confections was 27.24% and most often viral copathogen was RV, PIV, and RSV. RV was not found to coinfect with group streptococci. PIV was also absent from infections with S. mitis and group D streptococci. Contrary to PIV, HBoV was the most frequent viral pathogen to coinfect with S. mitis and group D streptococci.

An HBoV1 low load was found significantly more frequently than was HBoV1 with a high viral load in children. Of the HBoV1 single detection occurrences, a high viral load was more prevalent among children with dyspnea and wheezing than was a low viral load (42.9% vs. 23.7%, P = 0.036; 60.7% vs. 31.6%, P = 0.018). In clinical severity, a significant difference was recorded (25.0% vs. 5.3%, P = 0.003) between the high viral load and low viral load group (Table 3). We analyzed 18 HBoV1-positive patients who suffered from SRTI to study the association between HBoV1 and SRTI. Fourteen children had a diagnosis of severe pneumonia, 3 children had severe bronchiolitis, and 1 child had plastic bronchitis. Ten of 18 (55.6%) patients had a high HBoV1 load and only 3 of 10 children had other co-detected viruses, whereas 7 in 10 children with HBoV1 single detected were diagnosed with SRTI. The other 8 children who were diagnosed with SRTI had a low HBoV1 load. Among these 8 children, only 2 children were with HBoV1 single detected, whereas the other 6 children were co-detected with ADV, RSV or RV. HBoV1 single infection was more prevalent in the children with a high viral load than in those with a low viral load (70% vs. 25%) in the severe cases.