Molecular Epidemiology and clinical Features Analysis of Respiratory Human Adenovirus Infections in Hospitalized Children: A Retrospective Study in Zhejiang

Background: HAdV is one of the common pathogens in hospitalized children with respiratory infection (RI). we aim to describe the epidemiological characteristics, clinical features, and genotype of inpatients with HAdV positive with RI. Methods: Respiratory samples were gathered from inpatients diagnosed RI in Children’s Hospital, Zhejiang University School of Medicine and were detected by using Direct Immunouorescence Assay (DFA) from 2018 to 2019. PCR amplication and sequencing of the hypervariable zone of hexon gene were used for genotyping. The clinical and laboratory features, and typing of HAdV, and epidemiological characteristic analysis were retrospectively performed. Results: Of 7072 samples collected, 488 cases were identied as HAdV-positive RI. The overall detection rate was 6.9%. The peaked detection rate was 14.1% in January 2019. HAdV-positive cases with RI mainly appeared in winter and summer. The detection rate was highest among children between 6 months and 2 years (8.7%, 123/1408). Clinical diagnosis included pneumonia (70.3%, 343/488), bronchitis (7.0%, 34/488) and acute upper respiratory tract infection (22.7%, 111/488). The common clinical manifestations were fever (93.4%, 456/488), cough (94.7%, 462/488), wheezing (26.2%,128/48), and shortness of breath (14.8%, 72/488). 213 (43.6%) cases had co-infection and 138 (28.3%) cases had extrapulmonary symptoms. 96(19.7%) cases had intrapulmonary and intrathoracic complications.78 (16.0%) had an underlying condition, the most of them were congenital heart diseases (20.5%,17/78). The proportions of hyperpyrexia, duration of fever more than 10 days, severe pneumonia, and proportion of wheezing in the co-infection group were remarkably higher than those in HAdV single-infection group (p<0.05). 4 HAdV species were successfully identied in 155 cases and represented by 8 genotypes. HAdV-B3 (56.1%,87/155) and HAdV -B7 (31.0%,48/155) were the most predominant detected types and occurred commonly in different severity groups (p=0.000), while, HAdV-B55 was detected only in the severe group. HAdV-B7’s detection rate in the severe group was signicantly higher than the non-severe infected group. Conclusion: HAdV detection rate is related to age and season. Bronchopneumonia accounts for about 70% HAdV -positive inpatients. The common manifestations include hyperpyrexia, cough, wheezing, and shortness of breath. HAdV-B3 and HAdV-B7 are the most common types in children diagnosed with RI.


Introduction
Human adenovirus (HAdV) comprises A to G species which is subdivided into over 90 genotypes currently [1,2] . About 5-10% of all respiratory infections (RI) in infants and children are associated with HAdV [3,4] . HAdV was regarded as one of the frequent microorganisms attributed to 15% of ARI in hospitalized children [3,5,6] . A previous study revealed that clinical presentation of HAdV infection is mostly dependent on the genotype. HAdV genotypes associated with ARI include B (types 3, 7, 11, 14, 16, and 21), C (types 1, 2, 5, and 6) and E (type 4) [7] . Although HAdV infections cause light to moderate ARI within immunocompetent children, a serious condition may occur in younger children [1] , immunocompromised patients, and those with underlying chronic diseases which can cause signi cant morbidity and mortality [8,9] . HAdV often causes respiratory adenovirus epidemics and outbreaks in different provinces of China [10,11] , as well as in North America, Malaysia, and Korea [12][13][14] . The detection rate of HAdV in respiratory infection increased signi cantly recently.
It is necessary to strengthen surveillance of HAdV and observe its epidemic and infection characteristics. identi ed as samples detected by D3 Ultra TM DFA Respiratory Virus Screening & ID Kit (Diagnostic Hybrids, Inc., USA). Respiratory syncytial virus (RSV), human adenovirus, in uenza A viruses (Flu A) and in uenza B viruses (Flu B), and parain uenza 1, 2, and 3 viruses were detected. All NPAs or nasopharyngeal swabs samples were collected within 24 hours after admission. The specimens were divided into two parts and preserved in standard transport media. Rapid antigen testing for specimens was performed based on the instruction of the manufacturer within 2 hours after collection. All the samples were stored immediately in a tube containing 1.5 ml of viral transport medium at -80℃ for further analysis. Meanwhile, bronchial alveolar lavage uids (BALF), pleural effusion (PE) (if available), and blood from HAdV-positive patients during hospital stay were used to identify other pathogens. Patient age was de ned according to the date of hospitalization. Case de nition for RI was any cases with fever ( >37.5℃) at initial presentation accompanied by cough, expectoration, nasal obstruction, sneeze, sore throat, dyspnea during the previous week. All participants must satisfy the inclusion criteria as follows: age under18 years old and more than 28 days, disease course on admission ≤1 week con rmed acute respiratory tract infection with fever at initial presentation, cough, nasal obstruction, expectoration, sneeze, dyspnea, tachypnea, dyspnea, or wheeling/rales upon auscultation.
HAdV positive in the testing of NPAs or nasopharyngeal swabs samples through direct immuno uorescence assay.

provision of informed consent
The exclusion criteria were: disease course on admission more than one week infection occurred in the neonatal period acquired respiratory infection in hospital died or discharged within 48 hours after admission children with immunode ciency including neurological diseases, inherited metabolic diseases, asplenia, cerebral palsy, and/or complex-conditions. cases with substantial missing data.

Data collection
Patient general demographics (including age, sex, and sample collection time), social, clinical characteristics, laboratory results, radiological ndings, and disease severity of HAdV positive respiratory infection children admitted to our hospital were collected from the medical records after the patients' discharge from the hospital. Patient characteristics, symptoms, signs, diagnosis, and treatment were recorded. All the data were entered into a standard electronic database. Underlying medical diseases included congenital or acquired heart disease, asthma, epilepsy, liver, and gastrointestinal diseases, kidney disease, diabetes, or other endocrine diseases.

De nition of clinical severity
Patients were categorized into upper respiratory infection (URI) and lower respiratory infection (LRI) according to clinical diagnosis. Severe pneumonia was diagnosed according to American Thoracic Society's guideline for the management of community-acquired pneumonia [15] .

Detection of HAdV and molecular genotyping
Viral nucleic acids were extracted from specimens of the HAdV-positive NPAs or swabs using QIAamp MinElute Virus Spin Kit (QIAGEN, German) based on instructions of the manufacturer. Nested-PCR ampli cation that targeted highly variable regions of the hexon gene was proceeded to screen HAdV infection. The primers of nested-PCR were: PCR1-F 5′-GATGCCGCAGTGGKCKTACATG-3′, PCR1-R 5′-GCTTACAAYTCNCTSGCT-3′, and PCR2-F 5′-GACGCYTCGGAGTACCTGAG-3′, PCR2-R 5′-GGCYAGCACNTACTTTGACATYCG −3′. The following reaction conditions were: initial denaturation at 94°C for 10 min and then 35 cycles of1 min at 94 °C, 1 min at 55 °C, and 2 min at 72 °C, and an extension at 72 °C for 7 min. PCR ampli cation products were puri ed by 1% agarose gel electrophoresis and then sent to Beijing Genomics Institute (BGI) for Sanger sequencing of hypervariable regions of hexon gene which were con rmed as true. Samples are de ned as untyped which are unable to amplify. Sequencing results were sequenced on GenBank for typing.

Statistical Analysis
The results were analyzed utilizing SPSS software (version 19.0). Continuous data were expressed by means and standard deviations (SDs) for normal distribution, and median (range) for non-normal distribution. Categorical data were expressed in frequency and percentage. χ2 test and Wilcoxon's ranksum test were used to analyze statistical difference which P-value was set at less than 0.05 with statistical signi cance.

Clinical diagnosis and Clinical
Characteristics of the HAdV-positive inpatients. As

Discussion
HAdV was known as the most frequent cause of ARI in children which is a highly contagious pathogen. It is reported that the detection rate of HADV infection varied in different months, seasons, ages and regions, and circulated all year round, with epidemic outbreaks and clusters of severe cases occurring in individual regions [4,16,17] . Some HAdV types are common causes of severe respiratory tract infection in children. The majority of adenovirus respiratory infections are light to moderate and self-limited; however, sometimes they may cause life-threatening conditions, comorbidities, and serious sequelae [18][19][20] . The prognosis of severe HAdV infection in pediatric patients is poor, and many sequelae may be left, such as, bronchiectasis, interstitial brosis, etc., which can be life-threatening in severe cases [4,7] . Edmond K reported that association between adenovirus infection and a higher risk (55 %) for long-term sequelae compared to other causes of pneumonia in a meta-analysis of pneumonia in children younger than 5 years [18] .
The present study retrospectively analyzed the epidemiology, clinical features, and genotype of 488 hospitalized children with adenovirus positive for acute respiratory tract infection in our hospital from September 2018 to August 2019. We reported the clinical features, the recent molecular epidemiological detected results of circulated HAdV strains in hospitalized children with RI in Zhejiang China. Our study showed the overall detection rate was 6.9% in HAdV-positive hospitalized children with ARIs and highlighted that the peak detection rate was in children between 6 months and ≤2 years old; and suggesting that young children may be sensitive to HAdV in particular. Due to the protection of maternal antibodies, the detection rate was only 1.2% in HAdV-positive cases less than 6 months. We noted that 81.7% of 488 HAdV-positive cases were under 5 years which was consistent with other studies [21] . Our results were consistent with previous studies [21,22] . Pereira reported that children over 5 years old may have strong immunity to adenovirus infection [23] . December and January, February, July in the following year were discovered to be a high-detection month of HAdV respiratory infection, suggesting seasonality in HAdV respiratory infection. These results were consistent with Guangzhou reports in 2012 [24] . The season of high detection of respiratory adenovirus is different in different regions due to different climates which showed HAdV infection are associated with climatic factors (temperature and sunshine duration).
The clinical presentation of HAdV-positive ARIs is associated with age, genotype, and host immune function status. Previous studies have shown that age < 5 years, underlying diseases, immunocompromised status were more likely to be infected and developed severe HAdV-positive infection [25] . We noted that fever and cough (presented by 93.4% and 94.7%) were the most frequent symptoms observed in HAdV-positive hospitalized cases with ARIs, and the majority of children were less than 5 years old which is similar to the Italian study [26] . Wheezing and shortness of breath were more frequent in the severe group. Patients with severe HAdV infections have prolonged fever and required longer hospitalization. Of 488 hospitalized HAdV-positive cases, we noted that lower respiratory tract infections (70.3% in pneumonia and 7.0% in bronchitis), upper respiratory tract infection were found in 77.3%, death occurred in 0.4% of cases. Of the 343 cases of pneumonia, 17.6% (86/488) were serious. It is different in severity in HAdV infection under the age, environmental status, and immunological status of the patient. It is hard to clinical diagnose HAdV-positive ARI from other pathogens infections (such as mycoplasma pneumonia, in uenza, etc.) because the clinical presentation seemed to be alike. Therefore, we require further study on coinfections with HAdV and other respiratory pathogens.
Many studies have reported co-infection with other respiratory pathogens. In our study, the co-infections rate was 43.6% (213/488) that was in accord with previous reports [27] . Co-infection with other respiratory viruses was frequent ( 43.6%, 213/488, especially co-infection with in uenza virus, parain uenza virus human respiratory syncytial virus) in the study. And 39.1% (191/ 488) were double infected, 4.5% (22/488) were multiply infected. Previous clinical research reported that co-infected patients generally have a prolonged fever time, severe pneumonia, wheezing, and duration of hospitalization which was consistent with our study.
According to previous studies, HAdV-B (3,7,21), HAdV-C (1,2,5,6) and HAdV-E (4)were the most frequent HAdV species which caused ARIs in children worldwide [28] . HAdV-3 and HAdV-7 were the most frequent genotypes in China [29] . HAdV-7 is closely related to severe pneumonia and high mortality compared with other genotypes in infants and children [30,31] . Although patients from every age group may be susceptible to HAdV infections, more cases of HAdV infections were observed among less than 4 years old children -in this study. HAdV-B (90.3%, 140/155) was the most commonly identi ed genotype compared with other HAdV types, suggesting that young children with ARIs may be particularly susceptible to species B. HAdV-B3(56.1%, 87/155) was the dominant genotype in cases with HAdV respiratory infection. Over 60% of severe HAdV pneumonia tend to attribute to HAdV-B7. It also shows that the severity of HAdV pneumonia is related to HAdV genotypes and it is important to identify HAdV genotypes to correlate clinical disease and symptoms with all genotypes.
A nationwide epidemiological surveillance program for HAdV infection has not yet been built in China currently. HAdV identi cation is usually conducted for laboratory research and it is hard to be applied to the routine clinical diagnosis. We need long-time surveillance and rapid diagnostic methods of HAdV infection. Rapid detection is important to avoid unnecessary antibiotic consumption for HAdV respiratory infections.
This research had certain limitations. Firstly, this is a one-year single-center study and these results didn't generalize to other children across China; Secondly, we were unable to proceed with comparative analyses between genotypes owing to small sample size; thirdly, due to the retrospective analysis, we were unable to record all the symptoms and signs which may cause exposure misclassi cation.

Conclusion
HAdV detection rate is related to age and season. Bronchopneumonia accounts for about 70% HAdVpositive inpatients. The common manifestations include hyperpyrexia, cough, wheezing, and shortness of breath. HAdV-B3 and HAdV-B7 are the most common types in children diagnosed with RI.