核酸扩增技术应用于儿童肠道及疱疹病毒脑炎病原学诊断

Application of PCR technique in etiological diagnosis of children with enterovirus and herpesvirus encephalitis

目的探究疑似病毒性脑炎患儿肠道病毒(EV)、疱疹病毒(HHV)检测阳性率。方法收集湖南省儿童医院2017年8月至2019年12月365份疑似病毒性脑炎患儿脑脊液、血清、痰、大便、尿标本于-80℃冻存,采用一步法巢式逆转录多聚酶链反应(RT-PCR)检测EV,采用实时荧光定量PCR法(qPCR)检测HHV,分析这两类病毒在临床疑似病毒性脑炎患儿中的感染率。其中132例患儿确诊为EV脑炎或HHV脑炎,比较分析病毒在各种标本中的检出时间。结果365例疑似病毒性脑炎患儿中20.5%(75/365)的患儿诊断为EV脑炎,15.6%(57/365)患儿诊断为HHV脑炎。75例EV脑炎患儿以埃可病毒6型(echo 6)感染为主,占52.0%(39/75);其次为EV71感染(30.7%,23/75),echo11(6.7%,5/75),柯萨奇病毒A组6型(CA6,4%,3/75)及echo30、echo9、echo4、柯萨奇病毒B组1型(CB1)、脊髓灰质炎病毒各1例(1.3%,1/75)。57例HHV脑炎主要以人疱疹病毒6型(HHV6)感染最多见,占35.1%(20/57)。其次为巨细胞病毒(CMV,31.6%,18/57),EB病毒(12.3%,7/57),单纯疱疹病毒1型(HSV1,10.5%,6/57),单纯疱疹病毒2型(HSV2,8.8%,5/57),水痘带状疱疹病毒(1.8%,1/57)。患儿入院后病毒在脑脊液中的检出时间明显早于血清,脑脊液在发病后2~7 d可检出病毒,血清为7~26 d。结论采用一步法巢式RT-PCR技术及实时荧光qPCR技术检测脑脊液病毒核酸极大程度提高了不明原因脑炎患儿病原体的检出率。肠道病毒Echo 6、EV71、疱疹病毒HHV6、CMV是导致本院病毒性脑炎的最主要的病原体,临床可根据发病时间合理选择标本类型进行测定。

Objective To examine the infection of the enterovirus and human herpes virus in children with suspected encephalitis.Methods A total number of 365 suspected encephalitis cases were included in this study from August 2017 to December 2019 in Hunan Children′s Hospital.The clinical samples,the cerebrospinal fluid(CSF),serum,sputum,stool and urine were collected and preserved at-80℃condition.The enterovirus(EV)and human herpesvirus(HHV)were examined by a one-step nested reverse transcription PCR(RT-PCR)and a real-time fluorescent quantitative PCR(qPCR),respectively.The positive rate of the two viruses in clinical specimens of children with suspected encephalitis was examined.Among all cases,132 cases were diagnosed with EV encephalitis or HHV encephalitis.Results the EV encephalitis were identified in 20.5%(75/365)children with suspected viral encephalitis;whereas HHV encephalitis infection was identified as 15.6%(57/365).Among the 75 cases of EV encephalitis,echo 6 was the main sub-type of these diseases 52.0%(39/75)and others were EV71(30.7%,23/75),echo11(6.7%,5/75),Coxsackie virus A group 6(CA6,4.0%,3/75),echo30(1.3%,1/75),echo9(1.3%,1/75),echo4(1.3%,1/75),Coxsackie virus B group 1(CB1,1.3%,1/75)and poliovirus(1.3%,1/75).Human herpes virus type 6(HHV6)was the most common pathogen in 57 cases of HHV encephalitis,accounting for 35.1%(20/57).The other pathogens were Cytomegalovirus(CMV,31.6%,18/57),Epstein-Barr virus(8.8%,7/57),Herpes simplex virus 1(HSV1,10.5%,6/57),HSV2(8.8%,5/57),and Varicella zoster virus(VZV,1.8%,1/57).The virus in CSF detected significantly earlier than that in serum after onset.Virus could be detected in CSF 2-7 days after onset,but 7-26 days in serum.Conclusions This study uses nested PCR and qPCR to detect pathogens in clinical specimens of children.This not only expands our understanding of the clinical examination and diagnosis of viral encephalitis in children,but also promotes the method of this study to benefit more children.