Analysis Of Clinical And Molecular Characteristics Of Neonates Infection With Echovirus 11 And Establishment Of A Molecular Detection Method

BackgroundInfection is an important cause of neonatal death.Hospitalized children are prone to hospital-acquired infections due to factors such as immature immune system,use of broad-spectrum antibiotics,and invasive surgical procedures,especially in neonatal intensive care units(NICU),hospital-acquired infections are significantly associated with the morbidity and mortality of neonatal.Our laboratory has been monitoring the pathogenic spectrum of fever respiratory tract syndrome,fever rash syndrome and diarrhea syndrome for patients in our hospital for a long time,80%of the patients are children,including neonate.It was found that from May to July 2019,7 cases of positive Echovirus 11(EchoV 11)positive samples from the NICU of our hospital were detected.EchoV 11 belongs to group B enterovirus,and most lethal enterovirus infections are caused by EchoV 11.Since 1977,many outbreaks of EchoV 11 in neonatal wards have been reported abroad,with a high mortality rate.At present,most of them are sporadic cases.In recent years,hidden dangers of neonatal nosocomial infections have also begun to appear,but they have not attracted widespread attention.At present,the commonly used EchoV 11 detection method is to use polymerase chain reaction(PCR)to amplify the sequence of VP1 region for sequencing and identification.The detection period of this method is longer(about 2?3 days).There are no reports of rapid molecular detection methods for EchoV 11 at home and abroad.ObjectivesTo analyze the clinical characteristics and genetic characteristics of EchoV 11 VP1 region in children with positive EchoV 11 infection detected from NICU in our hospital,explore the correlation between cases and the evolutionary source of the virus,and provide a reference for the prevalence of EchoV 11 different genotypes.On the basis of the above research,try to establish a highly sensitive and specific fluorescent quantitative PCR(Quantitative PCR,qPCR)method for EchoV 11 VP1 region for rapid detection of EchoV 11 instead of the time-consuming traditional VP1 region amplification and sequencing,in order to achieve quick and easy screening and monitoring of infections in hospitals and communities caused by the pathogen.Methods1.Collecting anal swab samples from the children in NICU of our hospital with suspected enterovirus infections between January 1,2013 and December 31,2019.Non-EV-A71/CV-A16 enterovirus-positive samples were screened,identification of enterovirus serotypes using 5'-UTR and VP1 RT-PCR combined with sequencing.Samples identified as EchoV 11 positive were isolated and cultured,using RT-PCR amplifies partial or complete sequence of VP1 Region.Analyzing the clinical characteristics of EchoV 11-positive children and downloading the complete sequence of EchoV 11 VP1 region from GenBank at home and abroad to construct a phylogenetic tree.Comparing the evolutionary distance between the isolates of this study and other domestic and foreign strains.2.'Aligning the sequences of EchoV 11 VP1 regions that have been popular in China recent years and establishing a highly sensitive and specific qPCR method for EchoV 11 VP1 region,and constructing a recombinant plasmid as qPCR standard sample to analyze the sensitivity,specificity,repeatability and detection limit of the method.Result1.From May to July 2019,7 EchoV 11 positive samples(9.7%)from 6 patients in NICU of our hospital were detected.All 6 children had clinical manifestations such as shortness of breath and dyspnea.Four EchoV 11 strains were isolated and cultured,with nucleotide homology between 97.9%?100.0%and amino acid homology between 99.0%?100.0%,all of them are D5 subtypes,and have high homology with the Yunfu and Shunde strains isolated by the Guangdong Provincial Center for Disease Control and Prevention in 2019.Phylogenetic analysis are close to the evolutionary distance of the D5 subtype strains that have been popular in other regions in China since 2016;and foreign isolates:AY121374.1(Tunisia,1999),AY121375.1(Netherlands,2010),HG793702.1(France,2010)and MN896926.1(US A,2019)belong to the same branch.2.This study successfully established the EchoV 11 SYBR Green qPCR detection method suitable for domestic popularization,with an amplification efficiency of 84.0%,sensitivity and specificity of 100.0%(95%CI:56.1%?100.0%)and 98.7%(95%CI:91.8%?99.9%),the lower limit of detection is 104?105 copies/ml,and the coefficient of variation of the test results ranged from 1.60%to 4.73%.Conclusions1.The EchoV 11 isolated in this study are all D5 gene subtypes,which have high homology with other D5 subtype isolates in China in recent years.This subtype may be imported from the Netherlands,France,etc.,and has become popular in China in recent years.It can invade the respiratory system and nervous system and cause corresponding respiratory and neurological symptoms.The future trend of EchoV 11 needs further analysis by follow-up research.2.This study has successfully established the EchoV 11 SYBR Green qPCR detection method.The sensitivity and specificity of the initial exploration are good.The detection performance,application prospects of molecular diagnostic methods,and whether it can effectively improve the clinical diagnosis efficiency of infected patients,need to be further evaluated or improved by subsequent related research.