Acute infections of the respiratory tract are a major cause of morbidity and mortality in humans worldwide and approximately 80% are caused by viruses [1, 2]. The viruses most frequently associated with respiratory tract infections include respiratory syncytial virus (RSV), parainfluenza viruses (PIV), influenza viruses (Flu), adenoviruses (AdV), human rhinoviruses (hRV), and enteroviruses, and less commonly, human metapneumovirus (hMPV), human bocavirus (HBoV), and human coronaviruses (HCoV). Until recently, it was believed that HCoVs were responsible for the common cold syndrome and were the cause of only mild upper respiratory tract infections (URTIs) [3]. After the SARS epidemic in 2003, it was established that these viruses can also be associated with severe, life-threatening, or even fatal respiratory infections [4]. Stewart at al. have also suggested a possible relationship between HCoV infections and the development of extrarespiratory symptoms, including some involving the central nervous system [5]. The role of HCoV in human gastrointestinal infections also awaits more detailed exploration [6, 7].

The aim of this study was to estimate the prevalence of HCoV in a series of hospitalized infants and young children with symptoms of respiratory tract disease at the University Children’s Hospital in Ljubljana from June 2007 to May 2008. The main focus of the present study was to detect all four human coronaviruses in hospitalized children. A proportion of the patients included in this study have been partially described in a separate publication [8].

From June 2007 to May 2008, 664 specimens were collected from 592 children under six years of age with ARTI. The median age was 17.7 months (interquartile range: 9–28 months). The female: male ratio was 1:1.3 (256/592; 43% females). The 664 samples comprised 542 (81.5%) nasopharyngeal swabs, 102 (15.4%) throat swabs, and 20 (3%) other respiratory samples (i.e., tracheal aspirates).

Virus detection

Overall, in 70.6% of samples, one or more viruses were confirmed with the combined results of RT–PCR and DFA. The prevalence of RSV, FluA, FluB, PIV1, PIV3, and AdV according to DFA was 12%, 0.1%, 0.1%, 1.2%, 0.6%, and 1%, respectively. PIV2 was not detected during the study. Human coronavirus RNA was detected in 40 (6%; 95% CI: 4.3%–8.1%) of the 664 samples, 33/40 (82.5%) in nasopharyngeal swab, 6/40 (15%) in throat swabs and 1/40 (2.5%) in bronchoalveolar lavage. Of the 40 specimens positive for coronavirus, 21/40 (52.5%) were HKU1, 7/40 (17.5%) were OC43, 6/40 (15%) were 229E, and 6/40 (15%) were NL63. All specimens were also tested with real-time RT–PCR for hRV, HBoV, and hMPV. The most common virus detected was hRV (37.6%), followed by HBoV (20.9%) and hMPV (11.3%).

Most HCoV infections were detected in winter (22/40, 55%) and spring (10/40, 25%), whereas 6/40 (15%) positive samples were collected in autumn and only 2/40 (5%) in summer. The association between HCoV positivity and seasonality was statistically significant (P < 0.001). February had the highest number of HCoV-positive specimens (12/40, 30%), whereas HCoV was not detected in June or July (Figure 1).

Of the 40 HCoV-positive samples, only 12 (30%) were shown to involve only one virus, including 3/6 229E, 2/7 OC43, 5/21 HKU1, and 1/6 NL63. Species 229E was most frequently detected as a monoinfection (50%), but one or more other viruses were identified in the majority (28/40, 70%) of coronavirus-positive samples: 21/40 (52.5%) were dual infections, 6/40 (15%) were triple infections, and four viruses were detected simultaneously in one infection (2.5%). The viruses most frequently detected with HCoV were hRV (42.8%), HBoV (32.1%), RSV (28.6%), hMPV (21.4%), and AdV (3.6%).

On average, children with HCoV infections were older than those negative for HCoV (median ages in months: 23 vs 18 months, respectively), but infection with HCoV and age were not significantly associated (regardless of whether age was treated as a continuous or a categorical variable; data not shown). However, age was associated with the estimated quantity of HCoV viral nucleic acids present (measured as Ct). The association was nonlinear (P < 0.001) and the highest estimated viral load (lowest Ct) was detected in children around 10 months old. The estimated viral load increased with age for children younger than 10 months, whereas it decreased between 10 and 24 months. The estimated shape of the association is shown in Figure 2.

In our study, human coronavirus RNA was detected in 6% of respiratory samples from hospitalized children with ARTI. This prevalence was lower than we have found previously [8] and similar to that in other studies (from 2.6% to 8.7%) [17–20]. The majority of positive samples contained the HKU1 (52.5%) and the other coronavirus species were detected less frequently. A similar prevalence has been reported by Kuypers, and the prevalences of the HCoV species were reported in the same order [11]. There may be regional and annual variations in the circulation of different coronavirus species, as have been described by others [19]. HCoV was most often detected in winter and spring (80%), with the maximum number of cases presenting in February, whereas only 20% of samples were positive for HCoV in autumn and summer. Coronavirus infections have also been strongly associated with seasonality in previously published studies [20–23].

The children included in our study were under six years of age (median, 17.7 months). The probability of infection with HCoV was not significantly associated with age. However, the HCoV-infected patients in our study were older than those negative for HCoV. Some studies have included populations from all age groups [21, 24, 25], and coronaviruses have been detected in adults [26–29], young children [30–34], and neonates [35].

The association between age and the estimated viral load (from the Ct value) was interesting. The highest viral load was estimated for children about 10 months old (lower viral loads were detected in HCoV-infected children younger or older than 10 months). One reason for the highest viral load at this age may be the presence of maternal antibodies directed to HCoVs in younger infants [36].

The small number of cases and high number of HCoV coinfections with other respiratory viruses limited our ability to determine whether specific clinical signs or symptoms were associated with coronavirus infections. We compared only group of children infected with HCoV and those without HCoV infections. In our analysis children negative to all tested viruses were not included, because there is still uncertainty about possible infection with some of the newly described viruses (which were not included in our testing array). Children infected with HCoV required significantly less oxygen support, and had less cough, dyspnea, wheezing, LRTI, and bronchiolitis than the HCoV-negative children. URTIs were more frequent among HCoV-positive patients with monoinfections than among HCoV-negative children. As in other similar studies, we had only a relatively small number of coronavirus-positive samples. So far, only a few studies have included sufficient patients to permit a statistical comparison of the clinical outcomes of infections with different HCoV species [19, 21, 37]. It has recently been reported that coronavirus-infected children have LRTI less frequently than those infected with other viruses [38]. Children with coinfections had less severe disease than those with single virus infections. Children under 5 months of age had higher prevalence of single virus infections than older children [39]. In other recently published study the patients with coronavirus and influenza A virus coinfections had more severe disease than those with influenza virus infection only [40].

In the majority of our HCoV-positive specimens (70%), at least one other viral nucleic acid was present. To distinguish possible differences in the clinical presentations of children with a single infection from those of children with coinfections, and between the clinical presentations of infections with specific HCoV species, a study with more patients is required.

All HCoV-positive children included in our study were hospitalized with ARTI: 70.3% had LRTIs and 29.7% had URTIs. Pneumonia was diagnosed in 13.5% of children and 56.7% of children had bronchiolitis; 29.7% of HCoV-positive children had an underlying medical condition. As reported by others, single infections with HCoV were more often detected in children with an underlying medical condition than in previously healthy children: 5/11 (45.4%) vs 6/26 (23.1%), respectively [11]. In children with an underlying disease, the 229E was more often detected as a single infection than as a coinfection, whereas the HKU1 was more often detected as a coinfection.

Our study also contains several limitations. Due to the retrospective nature of the study, clinical data were not available for all included patients due to the noncomprehensive or missing clinical documentation. The reasons for the missing clinical data were that children were hospitalized due to the respiratory tract infection but were not stationed in pulmonary department and were distributed to other departments of the children's hospital because of the overcrowding. For these patients the clinical records were not available to investigators. Another limitation of this study is the fact that different samples were taken in some of the patients i.e. oropharyngeal instead of nasopharyngeal swab) and this could influence the yields of coronavirus (as well as other respiratory viruses). However due to the retrospective nature of the study we couldn't influence the sampling descisions made at the clinic. The politics of sample collection in place was that nasopharyngeal swab is preferred as the optimal sample but in cases where the physical or psychological conditions of the patient would not allow for safe nasopharyngeal swab collection, the laboratory would accept the oropharyngeal swab. The samples such as tracheal aspirates or BAL samples were collected from the severly affected children. All samples originated from hospitalized patients and that can also be considered as source of selection bias. Samples from outpatients and healthy control group children would be desirable in the study to determine HCoV prevalence and clinical features more accurately and also to exclude the possibility that viruses are detected as “contaminants” or “normal flora”. Due to the retrospective nature of the study, follow up testing was not performed. At the time of the study, detection of respiratory viruses was routinely performed by DFA instead of PCR which might underestimate the true prevalence of infections. However, we believe that these differences in sensitivity are not very prominent, especially in regard to detection of RSV virus and the fact that the study population represents young children in which DFA has best sensitivity.

In conclusion, we found that all four non-SARS coronavirus were detected with ARTIs in hospitalized children in Slovenia and that their identification with routine diagnostic techniques is feasible. An association between age and the viral load was found. The highest viral load was detected in children approximately 10 months of age. Further investigation of HCoV, with the inclusion of a control group of healthy children, is required to better understand the clinical importance of HCoV in respiratory infections.