| BackgroundIn addition to fever and rash symptoms,respiratory infections,myocardial damage,meningitis,encephalitis and acute flaccid paralysis can be seen in enterovirus(EV)infections,and some infections can develop into systemic diseases.The serotypes of EV that are prevalent at different times in different regions may vary,and localized epidemics of certain serotypes of EV may occur in some areas.Serotype identification is mainly used for epidemiological surveillance,but less used in clinical laboratories.The EV genome is a single-stranded positive-stranded RNA in which the 5’-UTR sequence is highly conserved and is a universal target for detection and is suitable for screening.The capsid protein VP1 region is the most accurate typing target,but detection sensitivity of that is low and the operation is cumbersome.The 5’-UTR is not preferred for serotyping,but some scholars believe that it is valuable for serotyping and has high detection sensitivity.For the serotypes that are mainly prevalent in recent years in China,whether the 5’-UTR is applicable or not,there is currently no systematic evaluation.Except the hand,foot and mouth disease that has become a legal infectious disease,accurate epidemiological information on diseases such as respiratory infections and viral encephalitis caused by EV is obviously insufficient.The prevalence of EV serotypes varies by time and region,and there is a lack of systematic reporting of the association of EV with respiratory infections and the comparative study of the clinical features of respiratory infections caused by each serotype.EVD68,which previously caused severe respiratory and neurological diseases in Europe and USA,has also been reported in some parts of Asia in these years,but the true incidence of EVD68 is unclear.The clinical laboratory has not popularized the method for EV serotyping or EVD68,the current EVD68 monitoring data is limited by time and region,and there are few reports in China.First,we evaluated the performance of method based on the 5’-UTR region amplification combined sequencing for EV serotyping directly from clinical specimens.Second,after determining that the method can be used for EV serotyping of respiratory specimens,we investigated the EV serotypes of children with respiratory infection in our hospital from 2014 to 2018 and compered the demographic and clinical characteristics of children with different serotypes.At the same time,the intestinal EV serotypes and the carrying of EV in children with no respiratory symptoms were investigated.Finally,clinical characteristics and viral phylogenetic analysis of EVD68,which is the most important EV serotype in children respiratory infections,were performed.Method1.The patients with suspected respiratory or intestinal EV infection in our hospital were enrolled in the study.518 rectal swab and 148 nasal swab were detected EVpositive by pan-EV real-time PCR.Nucleic acids were directly extracted from clinical specimens,5’-UTR and the VP1 gene fragments were amplified and sequenced separately for serotyping.The inconsistent results by 5’-UTR and VP 1 serotyping were further determined by using the serotype-specific RT-PCR.Using VP1 serotyping combined with serotype-specific RT-PCR as a reference method,the effect of performance of 5’-UTR serotyping on diagnosis was evaluated.2.The hospitalized children with fever or respiratory symptoms,such cough,runny nose,dyspnea,etc.,that is,suspected respiratory EV infection were studied.EV were serotyped by VP1 and 5’-UTR method.The result of VP1 method was first choice and supplemented by the 5’-UTR method.Those who failed to be serotyped by two methods were excluded in the follow-up study.3.The EVD68-positive cases screened by the 5’-UTR method were confirmed by nested PCR of partial sequence of VP1 region.Clinical characteristics analysis was performed on patients who confirmed infection.Complete 927 bp sequence of VP1 region were amplified and cloned.The sequences of VP1 region of EVD68 from other districts or countries were downloaded from NCBI,and the phylogenetic tree was established to compare the differences of EVD68 between Guangdong and other districts.Results1.The 5’-UTR method has a higher serotyping rate for EV than the VP1 method.Compared with the VP1 method,the sensitivity and specificity of 5’-UTR method were 57.1%～100%and 67.4%～98.1%respectively,with varied consistence with serotypes(kappa value 0.214～0.283).For the nasal swabs,the most frequently detected serotype was EVD68,with the sensitivity of 100%,the specificity of 91.1%,and the poor consistence(kappa value 0.217).The inconsistent results were further confirmed by serotype-specific RT-PCR.Using VPI serotyping combined with serotype-specific RT-PCR as a reference method,the effect of performance of 5’-UTR method on diagnosis was increased.2.In recent years,respiratory EV infections were frequently occurred from April to August in Guangdong.Infected children are younger,and the proportion of males is generally higher than that of females.There were differences in serotypes distribution between respiratory and intestinal infection.Common serotypes in respiratory infections were some serotypes of rhinovirus C/A/B,EVD68 and partial serotypes of coxsackievirus A/B.A rare rash occurs in respiratory infections,and there are no significant differences in demographic and clinical characteristics between species(serotypes)of respiratory infections.In addition,EV can also be detected in healthy children,which is common with rhinovirus C/A.3.Since the outbreak in Europe and the United States in 2014,EVD68 has been prevalent in Guangdong,with a positive rate of respiratory infection of 0.68%,characterized by asthma-like dyspnea.EVD68 detected in our study were all B3 subtypes,which were close to the most strains detected in China.The phylogenetic characteristics of EVD68 had a relationship with the geographical location.ConclusionsThe diagnostic performance of 5’-UTR method is acceptable for the EV serotypes in recent years.5’-UTR method has the advantage of high sensitivity,but the specificity varies with serotypes,and the application needs to consider its adaptability and limitations.In addition to rhinovirus,EVD68 is the most important EV serotype in the respiratory infection.There are no significant differences in demographic and clinical characteristics between species(serotypes)of respiratory infections,and the importance of rhinovirus in respiratory infections needs to be further evaluated.The epidemic strains of EVD68 in China are mainly B3 subtype,which can cause respiratory and nervous system infections,characterized by asthma-like dyspnea... |
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