The prevalence of local dominant viral etiologies is important for clinical management and prevention of common viral respiratory tract infections. Unfortunately, there is limited large-scale data about common viral respiratory infection in south China. To survey dominant viral etiology and seasonality of acute respiratory infections in hospitalized children, a 4-year consecutive study was conducted in Shenzhen, China. Nasopharyngeal swab specimens were obtained from 30,443 hospitalized children younger than 14 years with respiratory tract diseases in Shenzhen Children’s Hospital from January 2012 to December 2015. Nasopharyngeal swabs were routinely examined by direct immunofluorescence assay to detect respiratory agents including seven respiratory viruses. Data were analyzed to describe the frequency and seasonality. Of the 30,443 children enrolled in the study, 4428 (14.55 %) were positive for at least one viral pathogen, among whom 4110 (92.82 %) were ≤3 years of age. The predominant viruses were respiratory syncytial virus (RSV, 68.11 %), adenovirus (ADV, 16.01 %) and parainfluenza virus 3 (PIV-3, 11.0 %). The common respiratory viruses detected peaked in the spring (17.69 %), and were minimal in autumn (9.73 %), but PIVs detection peaked in November. The common virus detection rate in male subjects (15.40 %) was significantly higher than in female subjects (13.02 %). PIVs detection rates were complementary with RSV in autumn in each year. This study demonstrated common respiratory viruses were the major cause of hospitalized acute respiratory infection (ARI) in children in Shenzhen, China. RSV was the most common detected infection, while ADV was the predominant pathogen in hospitalized children. These findings provide a better understanding of virus distribution among children of different ages, infection stratification by gender, and seasonality, all of which will contribute to modification of therapeutic approaches and development of effective prevention strategies for each respiratory virus infection during peak seasons.

Pneumonia is one of the most important causes of morbidity and mortality in children. Identification and characterization of pathogens that cause infections are crucial for accurate treatment and accelerated recovery. However, in most cases, the causative agent cannot be identified, which is partly due to the limited spectrum of pathogens covered by current diagnostics based on nucleic acid amplification. Therefore, in this study, we explored the application of metagenomic next-generation sequencing (mNGS) for the diagnosis of children with severe pneumonia. From April to July 2017, 32 hospitalized children with severe nonresponding pneumonia in Shenzhen Children’s Hospital were included in this study. Blood tests were conducted immediately after hospitalization to assess cell counts and inflammatory markers, oropharyngeal swabs were collected to identify common pathogens by qPCR and culture. After bronchoscopy, bronchoalveolar lavage fluid (BALF) samples were collected for further pathogen identification using standardized diagnostic tests and mNGS. Blood tests were normal in 3 of the 32 children. In 9 oropharyngeal swabs, bacterial pathogens were detected, in 5 of these Mycoplasma pneumoniae was detected. Adenovirus was detected in 5 BALF samples, using the Direct Immunofluorescence Assay (DFA). In 15 cases, no common pathogens were found in BALF samples, using the current standard diagnostic tests, while in all 32 BALFs, pathogens were identified using mNGS, including adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, cytomegalovirus and bocavirus. This study shows that, with mNGS, the sensitivity of detection of the causative pathogens in children with severe nonresponding pneumonia is significantly improved. In addition, mNGS gives more strain specific information, helps to identify new pathogens and could potentially help to trace and control outbreaks. In this study, we have shown that it is possible to have the results within 24 hours, making the application of mNGS feasible for clinical diagnostics.

The imbalanced respiratory microbiota observed in pneumonia causes high morbidity and mortality in childhood. Respiratory metagenomic analysis demands a comprehensive microbial gene catalogue, which will significantly advance our understanding of host–microorganism interactions. We collected 334 respiratory microbial samples from 171 healthy children and 76 children with pneumonia. The respiratory microbial gene catalogue we established comprised 2.25 million non-redundant microbial genes, covering 90.52% of prevalent genes. The major oropharyngeal microbial species found in healthy children were Prevotella and Streptococcus. In children with Mycoplasma pneumoniae pneumonia (MPP), oropharyngeal microbial diversity and associated gene numbers decreased compared with those of healthy children. The concurrence network of oropharyngeal microorganisms in patients predominantly featured Staphylococcus spp. and M. pneumoniae. Functional orthologues, which are associated with the metabolism of various lipids, membrane transport, and signal transduction, accumulated in the oropharyngeal microbiome of children with pneumonia. Several antibiotic resistance genes and virulence factor genes were identified in the genomes of M. pneumoniae and 13 other microorganisms reconstructed via metagenomic data. Although the common macrolide/β-lactam resistance genes were not identified in the assembled M. pneumoniae genome, a single-nucleotide polymorphism (A2063G) related to macrolide resistance was identified in a 23S ribosomal RNA gene. The results of this study will facilitate exploration of unknown microbial components and host–microorganism interactions in studies of the respiratory microbiome. They will also yield further insights into the microbial aetiology of MPP.

Background: In recent years, the morbidity of Mycoplasma pneumoniae pneumonia (MPP) has increased significantly in China. A growing number of studies indicate that imbalanced respiratory microbiota is associated with various respiratory diseases. Methods: We enrolled 119 children, including 60 pneumonia patients and 59 healthy children. Nasopharyngeal (NP) and oropharyngeal (OP) sampling was performed for 16S ribosomal RNA (16S rRNA) gene analysis of all children. Sputum and OP swabs were obtained from patients for pathogen detection. Results: Both the NP and OP microbiota of patients differ significantly from that of healthy children. Diseased children harbor lower microbial diversity and a simpler co-occurrence network in NP and OP. In pneumonia patients, NP and OP microbiota showed greater similarities between each other, suggesting transmission of NP microbiota to the OP. Aside from clinically detected pathogens, NP and OP microbiota analysis has also identified possible pathogens in seven cases with unknown infections. Conclusion: NP and OP microbiota in MPP and non-MPP are definitely similar. Respiratory infection generates imbalanced NP microbiota, which has the potential to transmit to OP. Microbiota analysis also promises to compliment the present means of detecting respiratory pathogens.

Oseltamivir is a widely used influenza virus neuraminidase (NA) inhibitor that prevents the release of new virus particles from host cells. However, oseltamivir-resistant strains have emerged, but effective drugs against them have not yet been developed. Elucidating the binding mechanisms between NA and oseltamivir may provide valuable information for the design of new drugs against NA mutants resistant to oseltamivir. Here, we conducted large-scale (353.4 μs) free-binding molecular dynamics simulations, together with a Markov State Model and an importance-sampling algorithm, to reveal the binding process of oseltamivir and NA. Ten metastable states and five major binding pathways were identified that validated and complemented previously discovered binding pathways, including the hypothesis that oseltamivir can be transferred from the secondary sialic acid binding site to the catalytic site. The discovery of multiple new metastable states, especially the stable bound state containing a water-mediated hydrogen bond between Arg118 and oseltamivir, may provide new insights into the improvement of NA inhibitors. We anticipated the findings presented here will facilitate the development of drugs capable of combating NA mutations.

Non-typeable Haemophilus influenzae (NTHi) is a common pathogen causing respiratory infections, including pneumonia in children, and can also be found in the upper respiratory tracts of asymptomatic individuals. This study examines genomic variations between NTHi strains from healthy children and those from children with acute or chronic community-acquired pneumonia (CAP). Using bacterial genome-wide association studies (bGWAS), we compared these strains to identify key differences. Our analysis revealed that approximately 32% of genes exhibit variations between commensal and pathogenic states. Notably, we identified changes in peptidoglycan biosynthesis pathways and significant virulence factors associated with pneumonia. Furthermore, we observed a significant difference in β-lactam resistance due to PBP3 mutations between the healthy and pneumonia groups, confirmed by the ampicillin susceptibility test and characterized by the mutation pattern D350N, S357N, S385T, L389F. These findings contribute valuable insights into the genomic basis of NTHi pathogenicity and may inform more targeted clinical diagnostics and treatments.