Investigation Of Viral Pathogens Of Lower Respiratory Infection Among Hospitalized Children In A Hospital Of Zhejiang Province

1.Respiratory viruses are the most commonly detected pathogens causing community-acquired pneumonia(CAP).Rapid identification of respiratory viruses is important for therapeutic approach and infection control.In the present study,we developed a multiplex quantitative real-time reverse transcriptase polymerase chain reaction(mqRT-PCR)assay panel consisting of 7 internally controled qRT-PCR assays to detect 16 different respiratory viruses and compared with a previously reported two-tube multiplex reverse transcription PCR assay(two-tube assay)as the reference method using clinical sputum specimens.Of 363 tested respiratory specimens selected from children hospitalized with CAP,340(93.66%)and 332(91.46%)were positive for one or more viruses by mqRT-PCR assay panel and two-tube assay respectively,with a concordance rate of 0.97 and a kappa coefficient of 0.89.Individual mqRT-PCR assays for human rhinovirus(HRV),parainfluenza virus 3(PIV3),humanmetapneumovirus(HMPV)and human coronavirus OC43(OC43)showed slightly lower sensitivities compared with two-tube assay,with most discrepancies occurring in specimens infected with HRV.Overall,mqRT-PCR assay performed comparably with two-tube assays for most viruses,offering the advantages of quantitation analysis,easier performance,less prone to contamination and shorter turn-around time in laboratories equipped with conventional real-time PCR instrumentation.Hence mqRT-PCR is a valuable tool for routine surveillance of respiratory virus infection in China.2.Respiratory viruses are among the major causes of human morbidity and mortality worldwide,especially for infants and young children.Most published reports on respiratory pathogen infection in China were short of a strict clinical diagnosis and were not performed systematically and thus detailed etiologic data were lacking.Here,we conducted active population-based surveillance to investigate respiratory viral pathogen infection among hospitalized children in Zhejiang Province from February 2017 through January 2019.Among 1926 children with respiratory infection and with specimens available for bacterial and viral testing,a viral or bacterial pathogen was detected in 1345(69.8%),one more viruses or bacterial pathogen in 443(23.0%),Respiratory syncytial virus(RSV)(416,21.6%)was more common among children younger than 3 years of age,followed by Human rhinovirus/Enterovirus(HRV/EV)(352,18.3%),PIV(258,13.4%),Mycoplasma(My)(191,9.9%),Influenza viruses(Flu)(104,5.4%)and HMPV(85,4.4%)The burden of hospitalization for children with respiratory pathogen infection is the highest among the very younger children.However,respiratory pathogens associated with respiratory infection are often detected in the respiratory samples of healthy children,making their contributions to respiratory infection difficult to determine.We aimed to determine the contribution of common viral pathogens to respiratory infections by comparing the detection rates in both symptomatic and asymptomatic groups to inform future diagnosis,treatment and preventive strategies.A case-control study was then conducted from February 2017 to February 2018.A total of 383 respiratory infection children in case group and 383 pediatric surgery patients without symptoms in control group were enrolled among children<18 years.We found RSV was most strongly associated with respiratory infection(adjusted odds ratio,aOR 16.41;95%CI:8.50-31.70).We estimated the aOR for HMPV,Flu,Human bocavirus(HBov)and PIV,HRV//EVwere 14.43(95%CI:4.23-49.24),10.1(95%CI:4.36-23.39),5.32(95%CI:2.90-9.76),5.31(95%CI:2.90-9.76 and 1.82(95%CI:1.21-2.73),respectively.We conluded that RSV,HMPV,PIV and Flu were major contributors to respiratory infection in Zhejiang children.3.Numerous protocols for viral enrichment and genome amplification have been developed.However,the direct identification of viral genomes from clinical specimens using next-generation sequencing(NGS)still has challenges.As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS,it is valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly.In this study,we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits:QIAamp Viral RNA Mini Kit(VRMK),QIAamp MinElute Virus Spin Kit(MVSK),RNeasy Mini Kit(RMK),and RNeasy Plus Micro Kit(RPMK),using a mixed respiratory clinical sample without any pre-treatment.This sample contained an adenovirus(ADV),Flu A,PIV3,OC43,and HMPV.The quantity and quality of the viral extracts were significantly different among these kits.The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK.The MVSK had the lowest Ct values for ADV and PIV3.The RMK revealed the lowest detectability for HMPV and PIV3.The most effective rate of NGS data at 67.47%was observed with the RPMK.The other three kits ranged between 12.1-26.79%effectiveness rates for the NGS data.Most importantly,compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK.The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV,while the RMK revealed the best extraction efficiency by NGS analysis.The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis.The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples.In addition,viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus.Our results highlighted the importance of nucleic acid extraction kit selection,which has a major impact on the yield and number of viral reads by NGS analysis.Therefore,the choice of extraction method for a given viral pathogen needs to be carefully considered.4.Based on the investigation of respiratory viral pathogen infection among hospitalized children in a hospital of Zhejiang Province,we observed that PIV3 was detected in both respiratory infection patients and pediatric surgery patients.To analysize the difference of PIV3,we collected clinical samples from both 9 respiratory infection patients and 9 pediatric surgery patients(PSP)with hospital-acquired PIV3 infection from March 2017 through December 2017.We obtained sufficient sequencing coverage to yield whole genomes for PIV3.We reconstructed the phylogenetic trees using the whole genomes of PIV3 and publicly available data.PIV3 was diverged into two clades and clade 1 from Viet Nam was predominant in 2017.Phylogenetic clustering suggested PIV3 might have been circulating in Zhejiang.However,we could not exclude the possibility of hospital-acquired infection.HRV/EV was most commonly found in combination with PIV and HRV-A was a predominant subtype.