Human coronaviruses (HCoVs) were discovered in the 1960s. Six viruses, including HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-coronavirus (MERS-CoV), have been recognized as causative agents of a range of respiratory tract infections.
HCoV-NL63 and HCoV-HKU1 were described in 2004 and 2005, respectively, and together with HCoV-229E and HCoV-OC43 are responsible for up to 35% of upper respiratory tract infections, usually in epidemic outbreaks.1,2 The HCoV-OC43 is the most prevalent and is detected mainly in children <5 years of age. They are very frequently identified in coinfections with other respiratory viruses, which makes it difficult to understand their true role. In addition, they have been associated with more severe conditions, which often require hospitalization due to bronchospasm, especially in children with underlying diseases. Fatal cases have been described by HCoV-NL63 in immunosuppressed patients.3 Despite published series, there are no prospective studies investigating the role of HCoV infections in relation to other respiratory viruses in children.
SARS-CoV was described in 2003 in a unique outbreak in China, which caused >700 deaths with 20%–30% requiring mechanical ventilation and with a lethality of 10%, especially high in patients with comorbidities.4 The MERS-CoV was first detected in 2012 causing a similar clinical picture, but with a higher lethality (36%).5 This infection has not been extinguished, and sporadic cases persist. Both are zoonoses thought to originate in bats, transmitted to humans. MERS-CoV first transmitted to dromedaries and from camels to humans, although human-to-human contagion has been described, mainly between healthcare personnel with low transmissibility.6
Human SARS-CoV2 infection was first described in December 2019 in China and later around the world. Like other HCoV, SARS-CoV2 is an enveloped single-stranded RNA virus, with a diameter of 60–140 nm, and has a spherical or elliptical and pleomorphic shape.7 Previous studies reported that the novel virus shares (between 86.9% and 89%) the nucleotide sequences of the genome of a coronavirus similar to SARS found in bats (bat-SL-CoVZC45).7,8 The nucleotide sequence of the main envelope protein of the virus is also highly consistent with that of bat-SL-CoVZC45 (84%) and SARS-CoV (78%).
The objective of this work is to describe HCoV infections in hospitalized children in a prospective surveillance study of a 14-year period and compare these observations with those of other respiratory viruses.
MATERIALS AND METHODS
This is a substudy of an ongoing prospective investigation of respiratory tract infections in children, funded by FIS (Fondo de Investigaciones Sanitarias – Spanish Health Research Fund) Grants N°: PI06/0532, PS09/00246, PI12/0129, PI15CIII/00028 and PI18/0167 and approved by The Medical Ethics Committee (Carlos III).
The study population comprised children <14 years of age with acute respiratory infection admitted to the secondary public hospital Severo Ochoa (Leganés, Madrid), between October 2005 and June 2018. Informed consent was obtained from parents or legal guardians. The exclusion criterion was a refusal to participate in the study. All patients were evaluated by an attending physician. Clinical characteristics of patients were analyzed. During hospital stay, and as part of the study, a physician filled out a study questionnaire with the following variables: age, sex, month of admission, clinical diagnosis, history of prematurity and underlying chronic diseases, need for oxygen therapy—evaluated via transcutaneous oxygen saturation—fever and maximum axillary temperature, presence of infiltrates and/or atelectasis in chest radiographs, administration of antibiotic therapy, length of hospital stay, total white blood cell count, C-reactive protein serum values and blood culture results (for those cases where such tests had been performed) and results of virologic study. Asthma or recurrent wheezing was not considered an underlying chronic disease.
Upper respiratory tract infection was diagnosed in patients with the following symptoms: rhinorrhea and/or cough and no signs of wheezing, dyspnea, crackles or bronchodilator use, with or without fever. Asthma was diagnosed according to the National Asthma Education and Prevention Program guidelines.9 All other episodes of acute expiratory wheezing were considered to be recurrent wheezing. Acute expiratory wheezing was considered to be bronchiolitis when it occurred for the first time in children <2 years of age.10 Laryngotracheobronchitis was associated with inspiratory stridor and wheezing. Laryngitis was associated with inspiratory stridor without wheezing. Cases with both focal infiltrates and consolidation in chest radiographs were, in the absence of wheezing, classified as pneumonia. Nevertheless, if wheezing were present, the patient was included for an episode of wheezing.
Specimens consisted of nasopharyngeal aspirates (NPAs) that were taken from each patient at admission. NPA and nasopharyngeal swabs were sent for virologic investigation to the Influenza and Respiratory Viruses Laboratory at the National Center for Microbiology (Instintuo de Salud Carlos III), Madrid, Spain. Samples were stored at 4°C in the refrigerator and processed within 24 hours after collection. Upon reception, 3 aliquots were prepared and stored at −80°C. Both the reception and the NPA sample processing areas are separated from those defined as working areas.
RNA and DNA from 200-μL aliquots of NPA were extracted by using the QIAamp Mini Elute Virus spin kit in an automated extractor (QIAcube; Qiagen, Valencia, CA).
Detection of respiratory virus was performed by 4 independent multiplex real-time polymerase chain reaction (RT-PCR) assays. The first assay detected influenza A, B and C viruses, the second was used to detect parainfluenza viruses 1–4, rhinoviruses (RVs) and enteroviruses and the third detected the presence of respiratory syncytial virus (RSV) A and B types, human metapneumovirus, human bocavirus and adenoviruses. These 3 assays were real-time multiplex RT-PCRs and used the SuperScript III Platinum One-Step Quantitative RT-PCR System (Invitrogen, Carlsbad, CA). A fourth multiplex RT-PCR was used for investigation of HCoV, using generic primers which were able to detect both α and β coronavirus. Typing of HCoV was made using a reverse-specific primer for detection of HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1. Primers and probes of three independent multiplex RT-PCRs are based on previously published designed by our group11 and HCoV primers are available under request.
Descriptive data were expressed as mean, SD, median and first and third quartile (1st, 3rd Q) for continuous variables and through counts and percentages for categorical variables.
Continuous variables that followed a normal distribution were compared using one-way analysis of variance with Bonferroni correction, or through t tests. When the distribution was not normal, we used Mann-Whitney U test or Kruskal-Wallis test with Dunn correction. Categorical variables were compared using χ2 test or Fisher exact test, and results were expressed as odds ratio (OR).
To control for potentially confounding variables, we examined differences between groups using logistic regression models.
P values <0.05 were considered statistically significant, and confidence intervals (CIs) were calculated at 95% for all the estimations. Analyses were carried out using SPSS software (version 21; SPSS Inc, Chicago, IL).
The study population consisted of 5131 hospitalized cases with a respiratory infection diagnosis in children <14 years of age. A total of 3901 cases (75.9%) had a positive respiratory viral identification (1207; 29.5% were coinfections with >1 virus). A total of 205 cases (4.1%) had positive HCoV. Only 42 patients had single HCoV detection (0.8% of hospitalizations). Other identified viruses were RV as the most prevalent (1639; 31.9%), followed by RSV (1607; 31.3%). Influenza was identified in 229 cases (4.5%).
HCoV Infections Characteristics
A total of the 205 cases were detected. Only 55 of them were typed, corresponding to 26 (47%) OC43, 18 (32%) NL63, 9 (16%) 229E and 2 (3.6%) HKU1. Only 42 cases (20%) of HCoV infection were detected as single infections. The most frequent coinfection was with RV (84 cases; 41%) followed by RSV (57; 27%). Clinical data of HCoV cases are shown in Table 1. A total of 109 (53%) had fever (defined as >38°C). Recurrent wheezing was the most common diagnosis, and 112 children (54%) had hypoxia, of which 33 cases (16%) were treated with high-flow oxygen therapy. Fifty children presented an infiltrate in chest radiograph (24%). In 28 children (13.7%), the hospital stay was longer than 7 days.
Seasonality of HCoV infections presented a peak of higher incidence in December-January-February (42%) although they were present throughout the year. Additionally, we found significant differences (P = 0.05) between HCoV monthly circulations and each of the other viruses studied (Fig. 1).
Clinical Differences Between HCoV Single Infections and HCoV Coinfections
Patients with HCoV single infections (N = 42) were younger than those with viral coinfections (N = 163) (P < 0.001). The clinical diagnosis was also significantly different between both groups, as pneumonia was more frequent in HCoV coinfections, whereas laryngitis was more prevalent in HCoV single infections (P = 0.01).
Some differences could also be observed regarding clinical severity with a higher proportion of hypoxia among HCoV coinfections (P < 0.001) and a tendency of longer duration of hypoxia, a value near statistical significance (P = 0.065). After multivariate analysis, the length of hypoxia maintained its independent association with HCoV coinfections (P = 0.030).
No difference regarding the monthly circulation was observed between HCoV single infections and HCoV coinfections.
Clinical Differences Between HCoV-OC43 and HCoV-NL63 Infections
The 2 most frequent subtypes, HCoV-OC43 (N = 26) and HCoV-NL63 (N = 18), were compared. Both patient groups were similar regarding age, sex and prematurity history. However, some differences were found; although most patients in both groups were diagnosed with asthma attack or bronchiolitis, pneumonia was more frequent in the HCoV-NL63 group (P = 0.03). In this group, patients were 11 times more likely to need high-flow oxygen therapy (P = 0.03) and a longer hospital stay (P = 0.04) (Table 2).
No differences regarding the monthly circulation profile were observed between both groups.
Clinical Differences Between HCoV Single Infections and Other Respiratory Viruses
HCoV single infections (42 episodes) were compared with single infections of 894 RV, 993 RSV and 134 influenza cases detected in the same period (Table 3). Clinical data for infections caused by HCoV were similar to infections associated with the other respiratory viruses, but some differences were observed. Compared with RV, in the univariate analysis, HCoV patients were younger |(P = 0.001), had hypoxia less frequently (P < 0.001), but needed more often intensive care unit (ICU) admission (P = 0.001). After logistic regression, both, hypoxia (OR: 0.28; 95% CI: 0.13–0.61; P = 0.01) and ICU admissions (OR: 12.60; 95% CI: 4.67–33.95; P < 0.001), were independently associated with HCoV infections.
In the univariate analysis regarding RSV, HCoV patients presented less frequently with hypoxia (P = 0.001) and a diagnosis of bronchiolitis (P = 0.001), but also needed more often ICU admission (P = 0.001). After multivariate analysis, the independent association of HCoV infections with less hypoxia (OR: 0.11; 95% CI: 0.05–0.24; P < 0.01) but more ICU admissions (OR: 10.66; 95% CI: 3.88–29.29; P < 0.001) was confirmed.
Finally, compared with influenza patients, HCoV ones had less fever (P > 0.001) and more ICU admissions (P = 0.001). Both variables maintained their independent association with HCoV infections (OR: 0.15; 95% CI: 0.03–0.35; P < 0.01; and OR: 12.73; 95% CI: 2.81–57.61; P = 0.001, respectively).
This prospective 14-year surveillance study shows that HCoV detections in children hospitalized for respiratory infections are frequent, accounting for 4% of viral identifications. However, in two thirds of cases, these respiratory infections are coinfections with other viruses. HCoV circulated throughout the year and its most frequent types were OC43 and NL63, with higher severity in NL63 type. Single HCoV infections were only 1% of hospitalized cases, but when this group was analyzed, the following valuable information could be concluded. These were young children, most <1 year of age, with episodes of wheezing and a significant percentage of them required admission to the pediatric intensive care unit, although none of the children died. In short, these clinical characteristics do not differ much from other respiratory viruses. The main differences are seen with RSV infection, which causes bronchiolitis much more frequently and is associated with hypoxia at a higher percentage, and with influenza, which is more frequently associated with fever.
Other authors, such as Jean et al,2 also found that children with HCoV-OC43 infection are often young, approximately 1 year of age. However, in their study with 68 cases, the patients presented mostly mild symptoms without requiring hospitalization. They detected coinfections up to 32% of cases. Our series focuses on hospitalized children, so we cannot confirm if mild symptoms are detected in ambulatory children. Lee et al1 found HCoV-OC43 in 4% of viral respiratory infections and NL63 in 2% of cases, with around 35% of coinfections in children <5 years of age. The pediatric intensive care unit admission was 14% in single HCoV infections but lower than other viruses such as picornaviruses or RSV. Our higher proportion of coinfections could be explained by the fact that we tested 16 different viruses. In a Norwegian cohort study, results also demonstrated a high proportion of coinfections, up to 68%, and HCoV detections were associated with lower respiratory tract infections in hospitalized children. In this study, 13 other respiratory viruses were tested.12 In a study conducted in Israel,13 HCoV infections were frequent in infants and young children and, as in our series, fever was more common in influenza, whereas dyspnea was more frequent in RSV infections.
In a Chinese study on respiratory infections, HCoV accounted for 2.2% of the samples analyzed, and the pediatric population <3 years of age was the most frequently affected.14 Although there is little information on risk groups, it seems that children with previous pulmonary pathology have a higher risk for more serious lower respiratory tract infections, without great differences observed between immunocompromised and healthy children.15
A comparison of HCoV infections in children can also be made with other coronaviruses such as SARS-CoV or MERS and also with the new SARS-CoV2. A total of 11 children with MERS-CoV infection were described by Memish et al.16 Despite the high mortality in adults, children were asymptomatic, and only 2 presented respiratory symptoms, one of whom died. A similar situation has been described in children with SARS-CoV infection.17 The clinical course is usually mild; it is characterized by fever and symptoms of upper-tract infection, and may present as a lower-tract infection indistinguishable from other viral infections, without any mortality rate described so far.18 Finally, SARS-CoV2 infections in children have been identified in around 1%–2% of cases in China15 although cases are mostly mild, even in infants.19 Critical patients comprise 0.6% of children, but 50% of them are <1 year.20 Deaths are scarcely reported. In the Wuhan series, a 10-month-old child with intussusception presented with multiorgan failure and died 4 weeks after admission.21
In short, respiratory infections in children due to the different coronaviruses behave in a similar way and generally have a mild–moderate evolution with a favorable clinical outcome.
The present study has strengths in its prospective design, the long study duration and comparison with other respiratory viruses. As for limitations, the small number of cases with single HCoV infection makes it difficult to draw firm conclusions. Additionally, we do not have our own cases of SARS-CoV or MERS infections, and thus, we cannot make a real comparison with these 2 types of HCoV.
The authors thank Kinga Amália Sándor-Bajusz for the revision of the English language.
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