[Analysis of respiratory syncytial virus nonstructural protein 1 amino acid variation and clinical characteristics].

Objective: To investigate the relationship between amino acid variations of respiratory syncytial virus (RSV) nonstructural protein (NS) 1 and the clinical characteristics. Method: A retrospective case review was conducted. From December 2018 to January 2020, a total of 81 cases of hospitalized children who were tested only positive for RSV by RT-PCR or PCR at the Department of Respiratory Medicine, Children's Hospital of Chongqing Medical University were included in the study. The NS1 genes of RSV subtype A and subtype B were amplified by PCR and sequenced. The amino acid sequences were analyzed. The Chi-square test and Mann-Whitney rank sum test were used to compare the clinical characteristics and type Ⅰ interferon levels of children with or without NS1 variation in the variation and non-variation groups. Results: Among 81 cases, there were 58 males and 23 females. There were 11 cases in the variation group, the age of onset was 2.0 (1.0, 11.0) months, included 4 cases of subtype A (variant sites were: 2 cases for Lys33Gln, one case for Gly2Asp, Pro67Ser, Leu137Phe, respectively) and 7 cases of subtype B (variant sites were: two cases for Val121Ile, one case for Tyr30Cys, Val65Met, Asn85Ser, Ser118Asn, Asp124Asn, respectively). These variant sites all appeared at a very low frequency 0.08 (0.04, 0.29) % in the NCBI PROTEIN database. There were 70 cases in non-variation group, the onset age was 3.5 (1.0, 7.0) months. The proportion of dyspnea in the variation group was higher than that in the non-variation group (10/11 vs. 47% (33/70), χ2=7.31, P<0.01). Conclusions: There are some variant sites in nonstructural protein NS1 of RSV. Children may be prone to have dyspnea with NS1 variations.

[Clinical characteristics and risk factors for bronchoscopic airway mucus hypersecretion in childhood pneumonia infected by different pathogens].

Objective: To investigate the risk factors for airway mucus hypersecretion in childhood pneumonia infected by different pathogens. Method: A retrospective cohort included 968 children who were hospitalized for Mycoplasma pneumoniae pneumonia (MPP), respiratory syncytial virus (RSV) pneumonia, adenovirus pneumonia and underwent bronchoscopy in Respiratory Department of Children's Hospital of Chongqing Medical University from January 2019 to December 2021 was conducted. The children were divided into two groups distinguished by airway mucus secretion according to the airway mucus hypersecretion score which were scored according to the mucus secretion under the bronchoscope. The demographic characteristics, clinical characteristics, laboratory tests and disease severity of the two groups were compared. And the risk factors for the development of airway mucus hypersecretion in two groups were analyzed. Chi square test, Mann-Whithey U test and Fisher exact test were used to analyze the differences between the two groups, and multivariate Logistic regression was used to analyze the influencing factors. Result: There were 559 males and 409 females in the 968 children, with an age of 4.0 (1.4, 6.0) years. Among the 642 children with MPP, 185 cases were in the hypersecretion group and 457 cases were in the non-hypersecretion group. There were 41 cases in the hypersecretion group and 160 cases in the non-hypersecretion group of 201 children with RSV pneumonia. In the 125 children with adenovirus pneumonia, there were 39 cases in the hypersecretion group and 86 cases in the non-hypersecretion group. In these children, the age of children in the hypersecretion group was older than that in the non-hypersecretion group (6.0 (4.0, 7.0) vs. 5.0 (3.0, 7.0) years old, 1.5 (0.5, 3.6) vs. 0.8 (0.4, 1.6) years old, 2.0 (1.2, 4.5) vs. 1.3 (0.8, 2.0) years old, U=35 295.00, 2 492.00, 1 101.00, all P<0.05). Through multivariate Logistic regression analysis it found that increased risk of airway mucus hypersecretion was present in childhood MPP with increase in peripheral blood white blood cell count (OR=3.30, 95%CI 1.51-7.93, P=0.004) or increase in neutrophil ratio (OR=2.24, 95%CI 1.16-4.33, P=0.016) or decrease in lymphocyte count (OR=3.22, 95%CI 1.66-6.31, P<0.001) or decrease in serum albumin (OR=2.00, 95%CI 1.01-3.98, P=0.047). The risk of airway mucus hypersecretion was increased in children with RSV pneumonia combined with elevated peripheral blood eosinophils (OR=3.04, 95%CI 1.02-8.93, P=0.043). Meanwhile, airway mucus hypersecretion was associated with severe pneumonia (OR=2.46, 95%CI 1.03-6.15, P=0.047) in children with RSV pneumonia. Older age was associated with increased risk of airway mucus hypersecretion in children with adenovirus pneumonia (OR=1.02, 95%CI 1.00-1.04, P=0.026). In these children with occurrence of pulmonary rales, wheezes or sputum sounds (OR=3.65, 95%CI 1.22-12.64, P=0.028) had an increased risk of airway mucus hypersecretion. Neutrophils in bronchoalveolar lavage fluid (BALF) demonstrated higher ratio in hypersecretion group from children with MPP (0.65 (0.43, 0.81) vs. 0.59 (0.34, 0.76), U=24 507.00, P<0.01), while the proportion of macrophages in BALF was lower (0.10 (0.05, 0.20) vs. 0.12 (0.06, 0.24), U=33 043.00, P<0.05). Nucleated cell count and neutrophil ratio in BALF were higher in hypersecretion group of children with RSV pneumonia (1 210 (442, 2 100)×106 vs. 490 (210, 1 510)×106/L, 0.43 (0.26, 0.62) vs. 0.30 (0.13, 0.52), U=2 043.00, 2 064.00, all P<0.05). Conclusions: The increase in peripheral blood white blood cell count, neutrophil ratio and decrease in lymphocyte count, serum albumin in children with MPP is related to the development of airway mucus hypersecretion. In children with RSV pneumonia, the abnormal increase of eosinophils in peripheral blood has relationship with hypersecretion. The appearance of lung rale, wheezing, and sputum rale are associated with airway mucus hypersecretion in children with adenovirus pneumonia. In addition, local neutrophil infiltration in the respiratory tract is closely related to the occurrence of airway mucus hypersecretion caused by Mycoplasma pneumoniae and RSV infection.

[Development of a Ranking Tool for Scientificity, Transparency and Applicability of Clinical Practice Guidelines].

Objective: To address the limitations of existing methods and tools for evaluating clinical practice guidelines, we aimed to develop a comprehensive instrument focusing on the three main dimensions of guideline development: scientificity, transparency, applicability. We will use it to rank the guidelines according to the scores. We abbreviated it as STAR, and its reliability, validity and usability were also tested. Methods: A multidisciplinary expert working group was set up, including methodologists, statisticians, journal editors, medical professionals, and others. Scoping review, Delphi methods and hierarchical analysis were used to determine the final checklist of STAR. Results: The new instrument contained 11 domains and 39 items. Intrinsic reliability of each domain was indicated by Cronbach's α coefficient, with a average value of 0.646. The Cohen's kappa coefficients for methodological evaluators and clinical evaluators were 0.783 and 0.618. The overall content validity index was 0.905. The R2 for the criterion validity analysis was 0.76. The average score for usability of the items was 4.6, and the mean time spent to evaluate each guideline was 20 minutes. Conclusion: The instrument has good reliability, validity and evaluating efficiency, and can be used for evaluating and ranking guidelines more comprehensively.

[Impact of Streptococcus pneumoniae colonization in upper airway on the clinical manifestations of children with respiratory syncytial virus infection].

Objective: To investigate the effects of Streptococcus pneumoniae (Spn) colonization and the change of upper airway microbiome on the clinical manifestations in children with respiratory syncytial virus (RSV) infection. Methods: A retrospective cohort included 508 RSV-infected children with pneumonia and hospitalized in Respiratory Department of Children's Hospital of Chongqing Medical University from July 2009 to July 2018. A total of 508 cases of RSV-infected children (RSV non-sequencing group) were divided into 2 groups: children with Spn airway colonization (RSV+Spn group) and children without with Spn airway colonization (RSV group) according to the detection for virus and bacteria in nasopharyngeal aspirate, and these 2 groups were compared in terms of clinical manifestations by chi-square test in different age groups. In addition, in RSV pandemic season from November 2018 to February 2020, nasopharyngeal aspirates were collected from 20 children hospitalized in Respiratory Department of Children's Hospital of Chongqing Medical University and infected with RSV but without any positive detection of bacteria (RSV 16 S-sequencing group) and from children undergoing surgery without any sign of respiratory infection (control group). The difference of microbiome detected by 16 S RNA sequencing was compared using rank sum test between RSV 16 S-sequencing group and control group, and also between children with severe and mild pneumonia in RSV 16 S-sequencing group. Results: A total of 508 RSV non-sequencing group included 346 males and 162 females, and the visiting age was 6 (2, 12)  months. RSV group included 443 cases and RSV+Spn group included 65 cases. In the study 244 cases were aged <6 months and 264 cases were aged ≥6 months. In children aged ≥6 months of RSV non-sequencing group, the proportion of cases presenting fever over 38 ℃ and cases with severe pneumonia in RSV+Spn group were higher than those in RSV group (53.2% (25/47) vs. 34.6% (72/217), 38.3% (18/47) vs. 21.2% (46/217), χ²=5.70,6.15, both P<0.05). RSV 16 S-sequencing group included 16 males and 4 females and the visiting age was 3.0 (1.9, 8.0) months. Airway microbiome diversity in RSV 16 S-sequencing group was lower than that in control group (alpha index: 0.93 (0.42, 2.51) vs. 3.05 (2.88, 3.61), U=60.00, P=0.001). Conclusions: RSV infection is associated with the changes of the upper airway microbiome. When the balance of airway microbiome is broken and the presence of the dominant colonization of Spn follows, it may aggravate the severity of RSV infection in children aged ≥6 months.

[The correlation study on human respiratory syncytial virus daily incidence and meteorological parameters in the main urban area of Chongqing from 2009 to 2019].

Objective: To explore the relationship between the daily incidence of human respiratory syncytial virus (HRSV) and meteorological parameters in the main urban area of Chongqing. Methods: This study took 3 107 children hospitalized with acute lower respiratory tract infections from June 2009 to June 2019 in department of Respiratory medicine, Children's Hospital of Chongqing Medical University (CHCMU). Nasopharyngeal aspirate (NPA) was collected on the day of admission to detect HRSV and common respiratory virus; combined with the meteorological data of the main urban area of ​​Chongqing during the same period, the correlation and distribution lag nonlinear model analysis of the daily incidence of HRSV and meteorological parameters were carried out. Results: Among 3 107 children, HRSV positive accounted for 34.53% (1 073 cases), the age was 6 (3, 13) months, and males accounted for 64.31% (690 cases). The daily incidence of HRSV was negatively correlated with minimum temperature (r=-0.220, P<0.001), maximum temperature (r=-0.221, P<0.001), average temperature (r=-0.221, P<0.001) and precipitation (r=-0.052, P<0.001), and positively correlated with sunshine time (r=0.011, P<0.001) and average relative humidity (r=0.095, P<0.001). Compared with the reference temperature (20 ℃), when the lowest temperature of 6-10 ℃ lags for 4-8 d, the RR value of HRSV was 1.11-1.14, and when the lowest temperature of 5-19 ℃ lags for 5 d and 2-19 ℃ lags for 10 d, the RR values were 1.02-1.14 and 1.00-1.03. When the cumulative lag is 5, 10, 15 and 21 d, compared with the reference temperature (20 ℃), the RR (95%CI) values at the lowest temperature of 10.4 ℃ were 1.93 (1.08-3.46), 3.49 (1.64-7.45), 5.00 (2.01-12.46) and 6.69 (2.18-20.48); the RR (95%CI) values of the lowest temperature of 22.1 ℃ were 0.87 (0.77-0.98), 0.77 (0.66-0.90), 0.74 (0.62-0.89) and 0.68 (0.55-0.85). In the cumulative effect, compared with the reference temperature (20 ℃), the gender stratification showed that the maximum RR (95%CI) values of the lowest temperature for boys and girls under different lag days were 7.24 (1.84-28.51) and 2.19 (1.07-4.46), the age stratification showed that the maximum RR (95%CI) values of the lowest temperature for children<6 months old and children ≥6 months old under different lag days were 4.72 (1.05-21.23) and 11.98 (1.70-84.35). Conclusions: In the main urban area of Chongqing, the daily incidence of HRSV in children is correlated with climatic parameters. Among them, the lowest temperature has a delayed and cumulative effect on HRSV infection. 6-10 ℃ has a greater impact on the incidence of HRSV when the lag is 4-8 days. The effect has a more obvious impact on the incidence of HRSV in boys and children ≥ 6 months.

[Effect of interferon-γ on airway inflammation following respiratory syncytial virus reinfection in mice].

Objective: To identify the role of interferon (IFN)-γ during respiratory syncytial virus (RSV) re-infection in mice. Method: Female wild type C57BL/6 mice and IFN-γ knockout mice (IFN-γ(-/-) mice) at the age of 6 to 8 weeks were randomly divided into two groups: control group and RSV group, according to random number table.Each group was further divided into primary infection group and re-infection group.There were 8 groups.Mice were sacrificed on days 5, 7, 14 to collect samples.There were 5-8 mice in each group at each time point.And experiment was repeated twice. Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, left lung tissues were stained with HE and histopathological scoring (HPS) was performed.The concentrations of IFN-γ, IL-5, IL-13 were determined with ELISA.T test or single factor analysis of variance was used to compare between groups. Result: (1) Mice infected or reinfected with RSV showed pale hair, weight loss, decreased activity and anorexia.(2) IFN-γ levels significantly increased on days 5 and 7 following RSV primary infection and reinfection as compared to control groups in wild type mice ((192±44) vs.(36±8) and (531±161) vs.(23±4) pg/ml on day 5, (100±23) vs.(36±8) and (862±186) vs.(23±4) pg/ml on day 7, t=2.654, 2.513, 2.654, 3.968, all P<0.05). (3) Compared to the RSV-reinfected IFN-γ(-/-) mice, RSV-reinfected wild type mice had less body weight loss ((13.6±2.6)% vs.(22.7±2.9)% on day 5, (18.0±3.1)% vs.(26.5±1.8)% on day 7, t=2.314, 2.308, both P<0.05), lower lung tissue histopathological score ((1.50±0.09) vs.(2.07±0.11) on day 5, (1.53±0.11) vs.(2.08±0.09) on day 7, (1.10±0.06) vs.(1.59±0.08) on day 14, t=3.916, 3.890, 4.837, all P<0.01), less BALF inflammatory cells count ((11.6±2.0) vs.(44.2±10.6)×10(5)/ml on day 5, (18.2±3.9) vs.(38.3±2.2)×10(5)/ml on day 7, t=2.818, 4.786, both P<0.05), and lower levels of IL-5 and IL-13 ((24±3) vs.(148±23), (23±4) vs.(169±26) pg/ml on day 5, (30±8) vs.(233±44), (20±5) vs.(182±19) pg/ml on day 7, (91±6) vs.(129±19), (62±8) vs.(132±5) pg/ml on day 14, t=5.252, 5.445, 4.517, 7.326, 3.816, 7.577, all P<0.01). Conclusion: IFN-γ can alleviate airway inflammation following RSV reinfection in mice.

ORMDL3 variants associated with bronchiolitis susceptibility in a Chinese population.

Recent studies revealed common genetic risks for both viral bronchiolitis and asthma. Genome-wide association studies revealed that rs7216389 in the ORMDL3 gene is associated with childhood asthma. We conducted a case-control study examining the associations between ORMDL3 polymorphisms (rs7216389, rs12603332, and rs11650680) and bronchiolitis susceptibility/viral findings among 247 infant bronchiolitis cases and 190 healthy controls. We genotyped single nucleotide polymorphisms by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and detected respiratory viruses with multiplex reverse transcriptase-polymerase chain reaction. Only the genotype and allele frequencies of rs7216389 significantly differed between bronchiolitis and controls. The frequencies of the TT homozygote and the T allele of rs7216389 were significantly higher in the bronchiolitis patients (P = 0.0325; P = 0.0089, respectively). Polymorphisms were not associated with bronchiolitis severity. Cases were further stratified by viral infection, but no significant differences in the ORMDL3 genotype between the virus-detected group (e.g., respiratory syncytial virus alone, respiratory virus alone, virus detected) and no-virus-detected group were observed. Bronchiolitis is associated with the ORMDL3 gene in Chinese children, and there were no significant associations between genetic variations and disease severity or respiratory viruses. The TT homozygote and the T allele of rs7216389 in ORMDL3 increased bronchiolitis risk. The rs7216389 polymorphism may be a predictor for identifying infants with predisposition to virus-induced wheezing to persistent asthma.

Epidemic and molecular evolution of human bocavirus in hospitalized children with acute respiratory tract infection.

Human bocavirus (HBoV) is a novel parvovirus, often associated with respiratory tract diseases in children. This study explored the epidemiological characteristics and molecular evolution of HBoV-1 in southeastern China. Nasopharyngeal aspirates were collected from children admitted to hospital with acute respiratory tract infections. HBoV-1 was detected using real-time reverse transcription polymerase chain reaction and further characterized by complete genome sequences analysis. Among the 3,022 recruited children, 386 (12.77%) were HBoV-1-positive and 300 (77.72%) had co-detection with other respiratory viruses. Seasonal prevalence peaked in summer. HBoV-1 presence was significantly associated with asthma attack [odds ratio = 1.74; 95 % confidence interval: 1.30, 2.31; p < 0.001]. Similar results were obtained when either single detection or co-detection of HBoV-1 was considered, demonstrating the minor impact of co-detection on the clinical characteristics or epidemic pattern. Phylogenetic analysis based on the complete genome sequences showed that all the HBoV-1 sequences clustered together and no branch was formed that was supported by bootstrap value ≥ 750. The overall evolutionary rate of the complete genome of HBoV-1 was estimated at 1.08 × 10(-4) nucleotide substitutions per site per year (s/s/y) [95% highest probability density: (0.40-1.86) × 10(-4) s/s/y]. Selective pressure analysis showed that all the ω-values were less than 1, suggesting that HBoV-1 was under negative selective pressure. Site-by-site analysis identified the codon site 40 of the VP1 gene under positive selection. In conclusion, our study disclosed the epidemiological and genetic dynamics of HBoV-1 epidemics in southeastern China in the most recent 3 years, the information of which might help to further improve our understanding of HBoV-1 infection and guide better surveillance and control strategies in the future.

Clinical characteristics and molecular analysis of 21 Chinese children with congenital agammaglobulinemia.

Congenital agammaglobulinemia is a humoral primary immunodeficiency and affected patients have extremely low levels of peripheral B cells and profound deficiency of all immunoglobulin isotypes. Mutations of the Bruton's tyrosine kinase (BTK) gene are responsible for most of the congenital agammaglobulinemia. In this study, the phenotypes of congenital agammaglobulinemia were investigated in 21 male children from 21 unrelated Chinese families. Sixteen different mutations of BTK gene were identified in 18 patients, and three patients did not have BTK gene mutations. Nine mutations had been reported previously including one gross deletion (c.722_2041del), one missense mutation (c.1764G>T), three non-sense mutations (c.194C>A, c.895C>T and c.1821G>A) and four invariant splice-site mutations (c.971+2T>C, c.1481+2T>A, c.1482-2A>G, c.1699-2A>G). Seven novel mutations were identified (c.373_441del, c. 504delG, c.537delC, c.851delA, c.1637G>A, c.1879T>C and c. 1482_1882 del). Ten of the eighteen mutations of BTK gene were located in the TK domain, four in the PH domain, three in the SH3 domain and one spanned the TH, SH3, SH2 and TK domain. Candidate genes of autosomal-recessive agammaglobulinemia, including IGHM, CD79a, CD79b and IGLL1, were screened in three patients without mutations in the BTK gene. A compound heterozygosity mutation in the IGHM gene (c.1956G>A, c.175_176insC) was identified in one patient. The results of our study further support that molecular genetic testing represents an important tool for early confirmed diagnosis of congenital agammaglobulinemia and may allow accurate carrier detection and prenatal diagnosis.