[Value of anti-neutrophil cytoplasmic antibody in assessing the severity of bronchiolitis obliterans in children].

OBJECTIVE: To study the value of anti-neutrophil cytoplasmic antibody (ANCA) in assessing the severity of bronchiolitis obliterans (BO) in children. METHODS: A prospective analysis was performed on 59 children who were diagnosed with BO from June 2009 to October 2014. ELISA was used to measure the concentrations of myeloperoxidase (MPO)-ANCA and proteinase 3 (PR3)-ANCA in serum. According to the results of ELISA, the children were divided into three groups: double-negative ANCA (n=22), single-positive ANCA (n=17), and double-positive ANCA (n=20). The three groups were compared in terms of the scores of BO risk factors, clinical symptoms, chest high-resolution computed tomography (HRCT), and lung pathology on admission, as well as the changes in the expression level of ANCA and the scores of clinical symptoms and chest HRCT over time. RESULTS: Compared with the double-negative ANCA group, the double-positive ANCA group had a significantly higher score of BO risk factors (P<0.05), and the single-positive ANCA group and the double-positive ANCA group had significantly higher scores of clinical symptoms, chest HRCT, and lung pathology (P<0.05). The children were followed up for 6 months after discharge, and there were significant reductions in MPO-ANCA and PR3-ANCA titers from admission and discharge to the end of follow-up (P<0.05), as well as a significant reduction in the score of clinical symptoms from admission to the end of follow-up (P<0.05), while there was no significant change in the score of chest HRCT from admission to the end of follow-up (P>0.05). The single-positive ANCA and double-positive ANCA groups still had a significantly higher score of clinical symptoms than the double-negative ANCA group (P<0.05). CONCLUSIONS: The expression level of ANCA is correlated with the severity of BO in children and thus has certain clinical significance in disease evaluation.

Protein profile of well-differentiated versus un-differentiated human bronchial/tracheal epithelial cells.

Un-differentiated (UD) and well-differentiated (WD) normal human primary bronchial/tracheal epithelial cells are important respiratory cell models. Mature, WD cells which can be derived by culturing UD cells at an air-liquid interface represent a good surrogate for in vivo human airway epithelium. The overall protein profile of WD cells is poorly understood; therefore, the current study evaluated the proteomic characteristics of WD and UD cells using label-free LC-MS/MS and LC-PRM/MS. A total of 3,579 proteins were identified in WD and UD cells. Of these, 198 proteins were identified as differentially expressed, with 121 proteins upregulated and 77 proteins downregulated in WD cells compared with UD cells. Differentially expressed proteins were mostly enriched in categories related to epithelial structure formation, cell cycle, and immunity. Fifteen KEGG pathways and protein interaction networks were enriched and identified. The current study provides a global protein profile of WD cells, and contributes to understanding the function of human airway epithelium.

Paramyxoviruses respiratory syncytial virus, parainfluenza virus, and human metapneumovirus infection in pediatric hospitalized patients and climate correlation in a subtropical region of southern China: a 7-year survey.

To investigate the features of paramyxovirus respiratory syncytial virus (RSV), parainfluenza virus (PIV), and human metapneumovirus (HMPV) infection and determine the effect of meteorological conditions in Guangzhou, a subtropical region of southern China. We collected 11,398 respiratory samples from hospitalized pediatric patients with acute respiratory illness between July 2009 and June 2016 in Guangzhou. The samples were tested simultaneously for 18 respiratory pathogens using real-time PCR. Local meteorological data were also collected for correlation analysis. Of 11,398 patients tested, 5606 (49.2%) patients tested positive for one or more pathogens; RSV, PIV, and HMPV were the first, sixth, and ninth most frequently detected pathogens, in 1690 (14.8%), 502 (4.4%), and 321 (2.8%) patients, respectively. A total 17.9% (4605/5606) of patients with positive results had coinfection with other pathogens. Significant differences were found in the prevalence of RSV, PIV, and HMPV among all age groups (p < 0.001). RSV and HMPV had similar seasonal patterns, with two prevalence peaks every year. PIV appeared alternatively with RSV and HMPV. Multiple linear regression models were established for RSV, PIV, and HMPV prevalence and meteorological factors (p < 0.05). RSV and PIV incidence was negatively correlated with monthly mean relative humidity; RSV and HMPV incidence was negatively correlated with sunshine duration; PIV incidence was positively correlated with mean temperature. We described the features of paramyxovirus infection in a subtropical region of China and highlighted the correlation with meteorological factors. These findings will assist public health authorities and clinicians in improving strategies for controlling paramyxovirus infection.

Epidemiology of HBoV1 infection and relationship with meteorological conditions in hospitalized pediatric patients with acute respiratory illness: a 7-year study in a subtropical region.

BACKGROUND: Human bocavirus 1 (HBoV1) is an important cause of acute respiratory illness (ARI), yet the epidemiology and effect of meteorological conditions on infection is not fully understood. To investigate the distribution of HBoV1 and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of China. METHODS: Samples from 11,399 hospitalized pediatric patients (≤14 years old), with ARI were tested for HBoV1 and other common respiratory pathogens using real-time PCR, between July 2009 and June 2016. In addition, local meteorological data were collected. RESULTS: Of the 11,399 patients tested, 5606 (49.2%) were positive for at least one respiratory pathogen. Two hundred forty-eight of 11,399 (2.2%) were positive for HBoV1 infection. Co-infection was common in HBoV1-positive patients (45.2%, 112/248). A significant difference in the prevalence of HBoV1 was found in patients in different age groups (p < 0.001), and the peak prevalence was found in patients aged 7-12 months (4.7%, 56/1203). Two HBoV1 prevalence peaks were found in summer (between June and September) and winter (between November and December). The prevalence of HBoV1 was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. CONCLUSIONS: This study provides a better understanding of the characteristics of HBoV1 infection in children in subtropical regions. Data from this study provide useful information for the future control and prevention of HBoV1 infections.

Epidemiology and clinical characteristics of human coronaviruses OC43, 229E, NL63, and HKU1: a study of hospitalized children with acute respiratory tract infection in Guangzhou, China.

Human coronaviruses (HCoV) OC43, 229E, NL63, and HKU1 are common respiratory viruses which cause various respiratory diseases, including pneumonia. There is a paucity of evidence on the epidemiology and clinical manifestations of these four HCoV strains worldwide. We collected 11,399 throat swabs from hospitalized children with acute respiratory tract infection from July 2009 to June 2016 in Guangzhou, China. These were tested for four strains of HCoV infection using real-time polymerase chain reaction (PCR). HCoV-positive patients were then tested for 11 other respiratory pathogens. 4.3% (489/11399) of patients were positive for HCoV, of which 3.0% were positive for OC43 (346/11399), 0.6% for 229E (65/11399), 0.5% for NL63 (60/11399), and 0.3% for HKU1 (38/11399). Patients aged 7-12 months had the highest prevalence of HCoV and OC43 when compared with other age groups (p < 0.001). The peak seasons of infection varied depending on the HCoV strain. Patients infected with a single strain of HCoV infection were less likely to present fever (≥ 38 °C) (p = 0.014) and more likely to present pulmonary rales (p = 0.043) than those co-infected with more than one HCoV strain or other respiratory pathogens. There were also significant differences in the prevalence of certain symptoms, including coughing (p = 0.032), pneumonia (p = 0.026), and abnormal pulmonary rales (p = 0.002) according to the strain of HCoV detected. This retrospective study of the prevalence of four HCoV strains and clinical signs among a large population of pediatric patients in a subtropical region of China provides further insight into the epidemiology and clinical features of HCoV.

Clinical, virological and immunological features from patients infected with re-emergent avian-origin human H7N9 influenza disease of varying severity in Guangdong province.

BACKGROUND: The second wave of avian influenza H7N9 virus outbreak in humans spread to the Guangdong province of China by August of 2013 and this virus is now endemic in poultry in this region. METHODS: Five patients with H7N9 virus infection admitted to our hospital during August 2013 to February 2014 were intensively investigated. Viral load in the respiratory tract was determined by quantitative polymerase chain reaction (Q-PCR) and cytokine levels were measured by bead-based flow cytometery. RESULTS: Four patients survived and one died. Viral load in different clinical specimens was correlated with cytokine levels in plasma and broncho-alveolar fluid (BALF), therapeutic modalities used and clinical outcome. Intravenous zanamivir appeared to be better than peramivir as salvage therapy in patients who failed to respond to oseltamivir. Higher and more prolonged viral load was found in the sputum or endotracheal aspirates compared to throat swabs. Upregulation of proinflammatory cytokines IP-10, MCP-1, MIG, MIP-1α/ß, IL-1ß and IL-8 was found in the plasma and BALF samples. The levels of cytokines in the plasma and viral load were correlated with disease severity. Reactivation of herpes simplex virus type 1(HSV-1) was found in three out of five patients (60%). CONCLUSION: Expectorated sputum or endotracheal aspirate specimens are preferable to throat swabs for detecting and monitoring H7N9 virus. Severity of the disease was correlated to the viral load in the respiratory tract as well as the extents of cytokinemia. Reactivation of HSV-1 may contribute to clinical outcome.

Epidemiology of acute respiratory infections in children in Guangzhou: a three-year study.

Acute Respiratory Infections (ARI) are some of the most common human diseases worldwide. However, they have a complex and diverse etiology, and the characteristics of the pathogens involved in respiratory infections in developing countries are not well understood. In this work, we analyzed the characteristics of 17 common respiratory pathogens in children (≤14 years old) with ARI in Guangzhou, southern China over a 3-year period using real-time polymerase chain reaction. Pathogens were identified in 2361/4242 (55.7%) patients, and the positivity rate varied seasonally. Ten of the 17 pathogens investigated showed positivity rates of more than 5%. The most frequently detected pathogens were respiratory syncytial virus (768/2361, 32.5%), influenza A virus (428/2361, 18.1%), enterovirus (138/2361, 13.3%), Mycoplasma pneumoniae (267/2361, 11.3%) and adenovirus (213/2361, 9.0%). Co-pathogens were common and found in 503 of 2361 (21.3%) positive samples. When ranked according to frequency of occurrence, the pattern of co-pathogens was similar to that of the primary pathogens, with the exception of human bocavirus, human coronavirus and human metapneumovirus. Significant differences were found in age prevalence in 10 of the 17 pathogens (p≤0.009): four basic patterns were observed, A: detection rates increased with age, B: detection rates declined with age, C: the detection rate showed distinct peaks or D: numbers of patients were too low to detect a trend or showed no significant difference among age groups (p>0.05). These data will be useful for planning vaccine research and control strategies and for studies predicting pathogen prevalence.

Epidemiology and clinical presentation of the four human parainfluenza virus types.

BACKGROUND: Human parainfluenza viruses (HPIVs) are important causes of upper respiratory tract illness (URTI) and lower respiratory tract illness (LRTI). To analyse epidemiologic and clinical characteristics of the four types of human parainfluenza viruses (HPIVs), patients with acute respiratory tract illness (ARTI) were studied in Guangzhou, southern China. METHODS: Throat swabs (n=4755) were collected and tested from children and adults with ARTI over a 26-month period, and 4447 of 4755 (93.5%) patients' clinical presentations were recorded for further analysis. RESULTS: Of 4755 patients tested, 178 (3.7%) were positive for HPIV. Ninety-nine (2.1%) samples were positive for HPIV-3, 58 (1.2%) for HPIV-1, 19 (0.4%) for HPIV-2 and 8 (0.2%) for HPIV-4. 160/178 (88.9%) HPIV-positive samples were from paediatric patients younger than 5 years old, but no infant under one month of age was HPIV positive. Seasonal peaks of HPIV-3 and HPIV-1 occurred as autumn turned to winter and summer turned to autumn. HPIV-2 and HPIV-4 were detected less frequently, and their frequency of isolation increased when the frequency of HPIV-3 and HPIV-1 declined. HPIV infection led to a wide spectrum of symptoms, and more "hoarseness" (p=0.015), "abnormal pulmonary breathing sound" (p<0.001), "dyspnoea" (p<0.001), "pneumonia" (p=0.01), and "diarrhoea" (p<0.001) presented in HPIV-positive patients than HPIV-negative patients. 10/10 (100%) HPIV-positive adult patients (≥14 years old) presented with systemic influenza-like symptoms, while 90/164 (54.9%) HPIV-positive paediatric patients (<14 years old) presented with these symptoms (p=0.005). The only significant difference in clinical presentation between HPIV types was "Expectoration" (p<0.001). Co-infections were common, with 33.3%-63.2% of samples positive for the four HPIV types also testing positive for other respiratory pathogens. However, no significant differences were seen in clinical presentation between patients solely infected with HPIV and patients co-infected with HPIV and other respiratory pathogens. CONCLUSIONS: HPIV infection led to a wide spectrum of symptoms, and similar clinical manifestations were found in the patients with four different types of HPIVs. The study suggested pathogenic activity of HPIV in gastrointestinal illness. The clinical presentation of HPIV infection may differ by patient age.

Detection of human bocavirus from children and adults with acute respiratory tract illness in Guangzhou, southern China.

BACKGROUND: Human bocavirus (HBoV) is a newly discovered parvovirus associated with acute respiratory tract illness (ARTI) and gastrointestinal illness. Our study is the first to analyze the characteristics of HBoV-positive samples from ARTI patients with a wide age distribution from Guangzhou, southern China. METHODS: Throat swabs (n=2811) were collected and analyzed from children and adults with ARTI over a 13-month period. The HBoV complete genome from a 60 year-old female patient isolate was also determined. RESULTS: HBoV DNA was detected in 65/2811 (2.3%) samples, of which 61/1797 were from children (<18 years old) and 4/1014 from adults (≥18 years old). Seasonal peaks of 4.8% and 7.7% were detected in May and June, respectively. 28 of 65 (43.1%) HBoV-positive samples were co-detected with 11/16 other potential pathogens. Mycoplasma pneumoniae had the highest frequency of 16.9% (11/65). Upper and lower respiratory tract illness were common symptoms, with 19/65 (29.2%) patients diagnosed with pneumonia by chest radiography. All four adult patients had systemic influenza-like symptoms. Phylogenetic analysis of the complete genome revealed a close relationship with other HBoVs, and a more distant relationship with HBoV2 and HBoV3. CONCLUSIONS: HBoV was detected from children and adults with ARTI from Guangzhou, southern China. Elderly people were also susceptive to HBoV. A single lineage of HBoV was detected among a wide age distribution of patients with ARTI.

[Development and characterization of a stable cell line expressing respiratory syncytial virus non-structural protein NS1].

To develop a stable cell line that could express the RSV NS1, the full-length RSV NS1 gene was generated by RT-PCR amplification from respiratory syncytial virus. NS1 gene was ligated with pBABE-puro to construct the recombinant retroviral expression plasmid pBABE-NS1, which was cotransfected into 293FT packaging cells with PIK packaging plasmid by calcium phosphate co-precipitation. The supernatant of 293FT was collected to infect HEp-2 cells, the resulting cell clones stably expressing NS1 were screened by puromycin. Using QPCR, CPE staining method and indirect immunofluorescence assay, the expression of NS1 at both gene and protein levels was identified. The recombinant plasmid pBABE-NS1 was identified by EcoRI and BamHI endonuclease digestion and the sequence analysis. QPCR results showed that the NS1 gene amplification in HEp-2-NS1 cells was 8483 fold higher than that in HEp-2 cells. Although the exogenous interferon was added, all cells were destroyed after 48 hours post infection using CPE staining method, showing that HEp-2-NS1 cells remained sensitive to the VSV virus. The results of RT-PCR and indirect immunofluorescence assay showed that the NS1 gene in HEp-2 cells could not only transcribe mRNA, but also express NS1 protein steadily. We had successfully established HEp-2-NS1 cell lines with stable expression of respiratory syncytial virus non-structural protein NS1.