key: cord-1028270-5co8gtef authors: Lin, M.-R.; Yang, S.-L.; Gong, Y.-N.; Kuo, C.-C.; Chiu, C.-H.; Chen, C.-J.; Hsieh, Y.-C.; Kuo, C.-Y.; Fang, C.-W.; Tsao, K.-C.; Huang, Y.-C. title: Clinical and molecular features of adenovirus type 2, 3, and 7 infections in children in an outbreak in Taiwan, 2011 date: 2016-11-13 journal: Clin Microbiol Infect DOI: 10.1016/j.cmi.2016.11.004 sha: cab161ad72a6e459b8aa7fd3aa7904f14f40f8c6 doc_id: 1028270 cord_uid: 5co8gtef OBJECTIVES: We studied paediatric patients with human adenovirus (HAdV) infection during the 2011 outbreak in northern Taiwan to define the clinical features of different HAdV genotypes in children. METHODS: Between January and December 2011, 637 patients <19 years of age exhibited culture-confirmed adenoviral infection in Chang Gung Memorial Hospital, and provided specimens available for genotyping by multiplex real-time PCR. Clinical data were collected retrospectively. RESULTS: Excluding five cases with multiple genotypes, 632 cases were included for analysis. Three genotypes were identified, including HAdV-3 (429/632; 67.6%), HAdV-7 (144/632; 22.6%) and HAdV-2 (59/632; 9.8%). Median age was 4.58 years (range 2 months to 18 years), with children infected with HAdV-3 significantly older (82.9% >3 years; p <0.001). Of the 621 inpatients, 98.2% had fevers and all exhibited respiratory symptoms, 75 patients (12.1%) had lower respiratory tract infections, 20 (3.2%) required intensive care (HAdV-2: 1; HAdV-3: 8; and HAdV-7: 11), and three died (all HAdV-7-infected). HAdV-3-infected patients were significantly more likely to have upper respiratory symptoms and a high serum C-reactive protein level >100 mg/L, whereas leucocytosis (white blood cell count >15 000/mm(3)) was more common in HAdV-2-infected patients (p 0.007). HAdV-7 infections were significantly associated with a longer duration of fever, leucopenia (white blood cell count <5000/mm(3)), thrombocytopenia (platelet count <150 000/mm(3)), lower respiratory tract infections, a longer length of hospital stay, and requiring intensive care (all p <0.001). CONCLUSION: Childhood HAdV-2, HAdV-3 and HAdV-7 infections may exhibit different clinical manifestations. Although HAdV-3 was the most prevalent genotype observed during the 2011 Taiwan outbreak, HAdV-7 caused more severe disease characteristics and outcomes. Human adenovirus (HAdV) is a common pathogen in children that causes a variety of diseases, including respiratory disease, gastroenteritis, conjunctivitis and haemorrhagic cystitis [1e6] . HAdV is a double-stranded DNA virus that can be subdivided into >60 genotypes in seven species classified from A to G based on genomic homology [7] . Each genotype can cause different clinical manifestations, with genotypes 2, 3, 4, 7, 14, 21 and 55 resulting in more severe and disseminated diseases, including pneumonia and encephalitis [8e14] . According to previous studies of adenoviral epidemiology in Taiwan, HAdV-3 has circulated annually since 1999 and remains the dominant circulating genotype [15] as compared with the low prevalence of HAdV-7. A large community outbreak of adenoviral infections was detected by the nationwide surveillance system of the Centre for Disease Control-Taiwan (CDC-Taiwan) between weeks 11 and 41 in 2011 [16] , with the average HAdV-positivity rate increasing from 5.75% between 2008 and 2010 to 25.9% in 2011, along with an increase in the number of children admitted to the intensive care units of two medical centres in northern Taiwan owing to HAdV infection [17] . During this period, HAdV-7 re-emergence also occurred, with HAdV-7 proportions in Taiwan increasing significantly from 0.3% between 2008 and 2010 to 10% in 2011 [16] . Studies showed that HAdV-7 is associated with more severe clinical courses and complications [18e21]; however, there are no reports concerning the clinical features associated with large outbreaks of HAdV-7 infections in children. To better understand the clinical features and complications of different HAdV infections in children, we conducted a retrospective study to investigate the clinical features of children with HAdV infections during the 2011 outbreak in Taiwan. We conducted a retrospective study of children with adenoviral infections in Chang Gung Memorial Hospital during 2011. Children <19 years old with culture-confirmed adenoviral infections were enrolled, excluding those infected with more than two types of virus or with insufficient samples for further genotype stratification. Demographic data were collected and analysed from all patients, whereas clinical symptoms, laboratory results, images, complications and clinical outcomes were collected and analysed from inpatients only. This retrospective study was approved by the Institutional Review Board of Chang Gung Memorial Hospital (CGMH; No. 100-2518B). Clinical information was analysed anonymously so informed consent was waived. Procedures used for viral isolation and identification and designs of multiplex real-time PCR for HAdV genotyping are described in the Supplementary material (Data S1). HAdV sequencing was performed on full-length hexon genes as previously described [22] . PCR amplification was performed using specific primers (see Supplementary material, Table S1 ). We defined cases with lower respiratory tract infection (LRTI) as those having at least a pneumonia patch according to chest plain films or those without a pneumonia patch but with severe respiratory distress requiring oxygen supplementation and intensive care. In Taiwan, Mycoplasma pneumoniae caused frequent LRTIs in children >3 years old, and is usually screened for in this age group when pneumonia is suspected. Despite possible positivity for urinary pneumococcal antigen owing to asymptomatic carriage, some paediatricians still preferred using it as a screening tool, because Streptococcus pneumoniae is a common pneumonia pathogen in children <5 years old. For possible M. pneumoniae co-infection, we considered three situations as positive results (summarized in the Supplementary material, Data S1). Clinical laboratory data on admission and peak or nadir values during hospitalization were collected. White blood cell count >15 000/mL was defined as leucocytosis but a count of <5000/mL was defined as leucopenia. Platelet count >450 000/mL was defined as thrombocytosis and <150 000/mL as thrombocytopenia. Serum creatinine levels >1.0 mg/dL were considered evidence of impaired renal function, whereas a three-fold elevation in normal aspartate aminotransferase and alanine aminotransferase levels was considered evidence of impaired liver function. All 107 complete hexon sequences were downloaded from GenBank and aligned using CLUSTALO [23] against 25 sequences provided in this study. The evolutionary history of these sequences was inferred using the maximum likelihood method based on the HasegawaeKishinoeYano model [24] . Evolutionary analyses were conducted using MEGA7 [25] and evaluated using 1000 bootstrap replicates. We used the chi-square test or Fisher's exact test to compare categorical variables. Non-categorical variables were compared by one-way independent analysis of variance with post-hoc analysis. We looked for factors associated with LRTI and intensive care admission, using a multivariate logistic regression analysis (variables with p values <0.20 in univariate analysis were entered in the model). Statistical significance was set at p <0.05. Of 1598 children with positive virus cultures, adenovirus alone was identified in 661 specimens (41.4%). Other viruses identified are listed in the Supplementary material (Table S2 ). Further genotyping by PCR was possible in 637 patients who had adequate specimens, most of which were obtained by throat swab (616/632; 97.5%; see Supplementary material, Table S3 ). After excluding five cases with multiple genotypes, a total of 632 cases were included for further analysis. Only three genotypes were identified, with HAdV-3 (429/632; 67.6%) being the most common, followed by HAdV-7 (144/632; 22.6%) and HAdV-2 (59/632; 9.8%). Of the 621 patients (98.3%) hospitalized, 75 (12%) had defined LRTIs, 20 (3.2%) required intensive care, and three (0.5%) eventually died (Fig. 1 ). Fig. 1 . Flow chart describing case selection. a Patients excluded due to insufficient samples. b HAdV-2, -3 and -7, human adenovirus type 2, 3, and 7. c LRTI, lower respiratory tract infection; PICU, paediatric intensive care unit. d LRTI patients (14/75) exhibiting severe respiratory distress. e These patients included three with neurological dysfunction, two suffering from shock, and one with massive bleeding in the gastrointestinal tract. The median age was 4.58 years (range: 2 months to 18 years), with HAdV-3-infected children being significantly older than children harbouring other infections (Table 1) . Detailed clinical characteristics of hospitalized children are shown in Table 1 . HAdV-3infected children were more likely to experience symptoms of upper respiratory tract infection, including cough, rhinorrhoea, nasal congestion and exudative tonsillitis. HAdV-7-infected children experienced longer duration of fever and were more likely to be less active. Skin rash accompanied with oral ulcers (6.9%) mimicking clinical symptoms of enterovirus infection was also observed, particularly in HAdV-7-infected patients. All clinical diagnoses are listed in the Supplementary material (Table S4) . Detailed laboratory data are shown in Table 2 . Leucocytosis was noted in 163 (26.2%) children, and a serum C-reactive protein (CRP) level 40 mg/L was identified in 423 (68.1%) children. HAdV-2infected children showed leucocytosis and thrombocytosis, whereas HAdV-3-infected children were characterized by high serum CRP levels. By contrast, HAdV-7-infected children were significantly more likely to show leucopenia, thrombocytopenia, and impaired liver and renal function. LRT involvement and radiological findings LRTI was diagnosed in 75 cases, including 63 with a pneumonia patch observed on chest plain films and 12 with respiratory distress Twenty (3.2%) patients were admitted to the intensive care unit, including 14 with respiratory distress, three with shock, and one each with acute encephalitis and status epilepticus with hydrocephalus, respectively. Among these, six (33%) had underlying chronic systemic diseases, including neurological diseases (cerebral palsy) for three cases, tracheo-oesophageal fistula for two, and cystic fibrosis for one. These 20 patients experienced a longer duration of fever and length of hospital stay, and were more likely to have pneumonia involving multiple lobes (61.5% versus 25.5%; p 0.012) and receive antibiotics treatment (100% versus 60.1%; p <0.001) ( Table 3) . Compared with those infected with non-HAdV-7 HAdV, patients infected with HAdV-7 were more likely to require intensive care (OR 4.43, 95% CI 1.78e10.91) and have a longer hospital stay. Three HAdV-7-infected patients eventually died, and extracorporeal-membrane oxygenation therapy was performed on two of these three patients. These two cases exhibited an underlying disease of cerebral palsy. Multivariate regression analysis revealed that underlying diseases, fever duration >7 days and HAdV-7 infection were predictive risk factors for severe infection requiring intensive care (see Supplementary material, Table S5 ). Phylogenetic tree of the hexon gene HAdV-2, -3 and -7 were the main causative agents of HAdV infections in the 2011 outbreak in Taiwan. Although HAdV-3 was the dominant circulating genotype, HAdV-7 caused more severe clinical symptoms, and HAdV-7-infected children were more likely to exhibit lower respiratory complications and higher mortality rates, especially in those with underlying neurological diseases. HAdV is a common pathogen associated with acute respiratory infection in children. Previous epidemiological and molecular studies [26, 27] indicated that respiratory syncytial virus was the most common pathogen in paediatric inpatients with acute respiratory infection and, compared with respiratory syncytial virus, human rhinovirus and bocavirus, they also found that HAdV tended to infect older children and was more likely to cause leucocytosis, high CRP levels, longer hospital stays, pneumonia and more frequent antibiotics prescriptions [26] . Among our studied cohort, 41.4% of paediatric patients with acute respiratory infections exhibited HAdV infection, with~3.4% exhibiting co-infection identified by viral culture instead of real-time PCR. This might explain the underestimated rate of co-infection; however, our findings might reflect a community outbreak of HAdV, whereas most cases admitted to the hospital were due to more severe clinical presentations. Notably, none of our cases involved a diagnosis of bronchiolitis. This finding might be explained by 75% of the patients being >3 years old, as well as diagnoses by clinical physicians of bacterial infection rather than viral infection due to leucocytosis and high CRP levels. HAdV-7 was involved in a community outbreak in southern Taiwan from November 1999 to March 2000 [15] . However, over the previous decade, no HAdV-7 outbreaks and only a few HAdV-7 cases during 2008 and 2010 were observed according to surveillance data from CDC-Taiwan [16] . Studies indicated that HAdV-7 was more likely to cause lower respiratory tract involvement and lead to long-term pulmonary sequelae compared with HAdV-3 [28] . This study revealed HAdV-7 re-emergence during the 2011 community outbreak in northern Taiwan. Compared with those infected with HAdV-2 or HAdV-3, HAdV-7-infected children experienced longer durations of fever and hospital stay and were significantly associated with leucopenia, thrombocytopenia, and impaired liver and renal function. By contrast, HAdV-3 usually leads to leucocytosis and a high serum CRP level [29, 30] . A LRTI was the most common complication associated with HAdV infection during this outbreak. Compared with HAdV-2 and HAdV-3, HAdV-7 was significantly associated with pulmonary complications and intensive care requirements, even in otherwise healthy children. Underlying medical problems, especially neurological diseases, were important risk factors associated with development of respiratory failure and subsequent higher mortality rates. Life-threatening complications were also reported in children with disabilities during an HAdV-7 outbreak in a residential facility for severely disabled children [31] . Lai et al. [17] also reported a total of 45 paediatric patients with HAdV infection that required intensive care in two medical centres, including our hospital, in northern Taiwan in 2010e2011 (16 patients overlapped with this study). Among these, HAdV-7 was the main causative agent (49%) and resulted in higher mortality rates, particularly in those with underlying neurological diseases. Pneumonia was also the most common clinical diagnosis (89%), with half of these patients subsequently developing respiratory failure. A previous study suggested that HAdV-7 is strongly related to severe infections, pneumonia and underlying neurological diseases and is a risk factor for severe HAdV infection [32] . During the 2011 outbreak, HAdV-3 remained the dominant circulating genotype in Taiwan, with >80% of HAdV-3-infected children >3 years old. These children were significantly more likely to manifest upper respiratory tract symptoms. A higher serum CRP level (>40 mg/L) was also noted in nearly 80% of HAdV-3-infected children and mimicked bacterial infection, subsequently resulting in antibiotics prescription for two-thirds of the patients. These findings were consistent with those reported for the 2004 and 2005 HAdV-3 outbreaks in Taiwan [29] . HAdV-7 hexon sequences [25] from Taiwanese patients with clinical symptoms of varying severity were nearly identical with other Taiwanese strains from 2011 and Chinese strains from 2009 and 2015 (Fig. 2) . Although one of those Chinese strains caused an outbreak of severe lower respiratory tract disease [13] , hexon sequences from 2011 cases were highly conserved [18] and contained only two non-synonymous mutations (S429Y and Q440L) as compared with the Taiwanese strains from 2002 and 2004. This study has several limitations. First, as a retrospective study, not all clinical information and laboratory data could be collected. Second, nearly all cases (98%) in this study were inpatients, which might result in overestimation of the proportion of HAdV-7 infection and disease severity. Third, co-infection rates might have been underestimated. Virus isolation by tissue culture was used in this study; however, viruses, including human bocavirus, coronavirus and human metapneumovirus, cannot present cytopathogenic effects in selected cells. KCT received two grants from Chang Gung Memorial Hospital, Taoyuan, Taiwan (Nos. CLRPG3B0044 and CLRPG3B0045; http:// www.cgmh.org.tw), and these two grants partially supported this work. The funders had no role in study design, data collection and analysis, decision to publish, or manuscript preparation. The authors report no conflicts of interest. Adenovirus associated haematuria The seattle virus watch. Vii. Observations of adenovirus infections Epidemiological and virological features of epidemic keratoconjunctivitis due to new human adenovirus type 54 in Japan Clinical features of adenovirus enteritis: a review of 127 cases Viral lower urinary tract infections Importance of enteric adenoviruses 40 and 41 in acute gastroenteritis in infants and young children Molecular evolution of human adenoviruses Adenovirus serotype 14 infection Lower respiratory tract infections due to adenovirus in hospitalized korean children: epidemiology, clinical features, and prognosis Adenovirus infection associated with central nervous system dysfunction in children A community-based outbreak of severe respiratory illness caused by human adenovirus serotype 14 Disseminated adenovirus disease in immunocompromised and immunocompetent children Adenovirus serotype 7 associated with a severe lower respiratory tract disease outbreak in infants in Shaanxi Province Fatal pneumonia cases caused by human adenovirus 55 in immunocompetent adults A two decade survey of respiratory adenovirus in Taiwan: the reemergence of adenovirus types 7 and 4 Community outbreak of adenovirus Adenovirus serotype 3 and 7 infection with acute respiratory failure in children in Taiwan Adenovirus pneumonia complicated with acute respiratory distress syndrome: a case report Quantitative real-time PCR assay panel for detection and type-specific identification of epidemic respiratory human adenoviruses Detection of a broad range of human adenoviruses in respiratory tract samples using a sensitive multiplex real-time PCR assay Impact of human adenovirus serotype 7 in hospitalized children with severe fatal pneumonia in the Philippines Molecular typing of human adenoviruses by PCR and sequencing of a partial region of the hexon gene Fast, scalable generation of high-quality protein multiple sequence alignments using CLUSTAL omega Dating of the human-ape splitting by a molecular clock of mitochondrial DNA Mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets Eight year prospective study of adenoviruses infections in hospitalized children. Comparison with other respiratory viruses Molecular identification and epidemiological features of human adenoviruses associated with acute respiratory infections in hospitalized children in southern China Adenovirus infection with serious pulmonary sequelae in korean children A communityderived outbreak of adenovirus type 3 in children in Taiwan between A cluster of adenovirus serotype 3 infections in children in northern Taiwan: clinical features and laboratory findings An outbreak of adenovirus type 7 in a residential facility for severely disabled children Risk factor analysis and molecular epidemiology of respiratory adenovirus infections among children in northern Taiwan The authors thank Dr Enzo Emanuele of 2E Science, Italy, for his expert editorial assistance. Additional Supporting Information may be found in the online version of this article at http://dx.doi.org/10.1016/j.cmi.2016.11.004.