杭州市手足口病医务人员隐性感染状况研究

目的分析传染科手足口病医务人员肠道病毒71型(EV71)和柯萨奇病毒A组16型(CA16)的隐性感染情况。方法 2016年6~9月杭州市儿童医院儿科医务人员63人(其中传染科医务人员23人),健康成人体检43人,采用荧光PCR法进行粪便EV71和CA16病毒核酸检测,利用酶联免疫分析(ELISA)间接法检测血清EV71和CA16的IgG抗体。结果医务人员与非医务人员粪便肠道病毒核酸均阴性。医务人员与非医务人员血清EV71抗体IgG阳性率分别为17.46%及18.60%,差异无统计学意义(χ~2=0.023,P=0.88);血清CA16抗体IgG阳性率分别为95.24%和53.49%,差异有统计学意义(χ~2=26.219,P=0.00)。传染科与非传染科医务人员比较,血清EV71抗体IgG阳性率分别为26.09%和12.50%,差异无统计学意义(χ~2=1.047,P=0.31);血清CA16抗体IgG阳性率分别为91.30%和97.50%,差异无统计学意义(χ~2=0.247,P=0.62);医生与护士比较,血清EV71抗体IgG阳性率分别为11.11%和35.71%,差异无统计学意义(χ~2=1.72,P=0.19);血清CA16抗体IgG阳性率分别为100.00%和92.86%,差异无统计学意义(χ~2=0.672,P=0.41)。结论儿科医务人员CA16隐性感染要高于健康人群。手足口病医务人员虽然存在着一定程度EV71和CA16隐性感染情况,但粪便均未能检测出EV71和CA16病毒核酸,说明传播风险系数较低。另手足口病病房与其他病房医务人员EV71和CA16隐性感染情况无差异。     

2016-2020年杭州地区单中心EB病毒感染患儿阳性检出率的统计与分析

目的 回顾性分析杭州地区2016年至2020年间儿童EB病毒的感染情况、构成、流行趋势等信息。方法 选取2016年至2020年期间,杭州市儿童医院病原体核酸检测的样本共72526例,将其中EB病毒核酸阳性的1391例纳入统计研究,并分析各年份杭州地区儿童EB病毒感染的人数、感染月份、性别比例及年龄构成等因素。结果 2016-2020年,报告EB病毒核酸阳性检出病例1391例,占全院儿童检测病原体包括肺炎支原体,沙眼支原体,解脲支原体,EBV和人巨细胞病毒等各类DNA病毒的1.92%,五年间EB病毒核酸阳性检出率分别为1.56%,1.81%,1.89%,1.63%和3.11%;性别方面,男性感染患儿的核酸阳性检出778例,女性感染核酸阳性检出613例,男女比例为1.27：1;年龄分布方面,1-6学龄前儿童EB病毒感染患儿核酸阳性检出1184例,占我院1-15岁患儿总检出病例数的85.12%。流行季节方面,夏秋季为感染高峰,冬春季不明显。结论 2016年-2019年,杭州地区EB病毒感染患儿比例相对平稳（x2趋势检验,P>0.05）,但在2020年,阳性检出率明显上升,这可能与新冠疫情有关;学龄前儿童为EB病毒的主要易感者,这与学龄前儿童的免疫水平以及淋巴细胞比例水平可能存在一定相关性,同时男童与女童的阳性检出率存在显著差异（P<0.05）且男童易感率更高,此外EB病毒传播还具有一定季节规律。     

儿童麻疹流行的临床特点及基因特征

目的探讨儿童麻疹流行的临床特点及基因特征,为临床治疗提供参考依据。方法回顾性分析2012年3-8月38例麻疹患儿的临床特点,用逆转录-聚合酶链反应(RT-PCR)检测麻疹患者的咽拭子病毒标本,对部分PCR扩增阳性的片段进行研究基因测序、并绘制系统进化树进行同源性分析。结果 38例麻疹患者咽拭子标本中经RT-PCR检测阳性32例,阳性率84.2%;随机选择8例阳性标本进行基因测序,均为H1a基因亚型;与该型参考株AF045205﹑AF045206核苷酸同源性为98.0%⁹9.0%。结论麻疹病毒流行优势株均是H1a基因亚型,无输入性的其他亚型出现。     

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目的 探讨新生儿化脓性脑膜炎(以下简称化脑)新的快速诊断方法.方法 2003年8至2006年2月浙江大学医学院附属儿童医院采用脑脊液细菌16S rRNA基因芯片技术对24例临床上疑似化脑患儿脑脊液(CSF)的细菌DNA进行测定,同期进行与CSF细菌培养的对照.结果 (1)49株细菌PCR扩增产物基因芯片杂交结果显示,33株C+菌其G+探针和通用探针均阳性,G-探针阴性;16株G-菌中,G-探针和通用探针均阳性,G+探针阴性,均能与相应特异的探针发生杂交.(2)大肠埃希菌DNA的PCR产物其16S rRNA基因芯片的最小检出量为1pg,约等于10个拷贝教,相当于2个细菌.(3)16S rRNA基因芯片检测24份脑脊液标本发现11份阳性,阳性率为45.83%(11/24),明显高于脑脊液培养的阳性率12.50%(3/24),差异有统计学意义(P<0.01).结论 脑脊液细菌16S rRNA基因芯片检测技术特异性强、敏感性高,需标本量少,是早期快速诊断儿童化脑的可靠方法,具有较大的应用价值.     

手足口病病原体流行特征分析及临床意义

Objective To investigate the epidemic characteristics of etiological agents in children with hand, foot and mouth disease (HFMD) and analyze the differences between the severe and mild cases with HFMD seen from 2008 to 2009 in the Children's Hospital Methods A total of 154 patients with HFMD were enrolled from May 2008 to September 2008 and from May 2009 to September 2009, including 28 severe HFMD patients. Data from 80 cases with suspected herpangina were collected as control. Enterovirus universal type, enterovirus type 71 (EV71) and coxsackie virus group A 16 (CA16) were detected by realtime RT-PCR respoctively. Results The positive rate of enterovirus universal type in the 154 patients with HFMD was 81.82% (126/154). EV71 positive rate in these 126 patients with enterovirus universal type infection was 57.14% (72/126). The positive rate of enterovirus universal type in the 80 cases with suspected herpangina was 68.75% (55/80). There was no EV71 infection in these 80 cases with suspected herpangina. EV71 infection was mainly popular in 2008. Both EV71 and CA16 were prevalent in 2009. The epidemic characteristics of enterovirus infection with HFMD between 2008 and 2009 had significant differences (X2 = 23.50, P = 0.000) ( P ＜ O.01 ). The epidemic characteristics of enterovirus infection between severe and mild HFMD patients also had significant differences (X2 = 29.85, P ＜ O. 01). There were 28 cases with severe HFMD, in whom the EV71 positive rate was 92.86% (26/28). EV71 positive rate in the mild HFMD was 36.51% (46/126) (X2 =29.22, P ＜0.01). There was no significant difference in the gender ( X2 = 0.135, P = 0.714) and virus load (t = 0.141, P = 0.889) between the mild and severe HFMD cases. But the age of mild and severe HFMD showed a significant difference ( t = 2.926, P =O.009). Patients who were less than 2 years of age had a proportion of 88.89% (8/9) with severe HFMD.The mean age of mild HFMD patients was 3.19 years. Conclusion HFMD showed different epidemic characteristics at different times of enterovirus infection. There was no significant difference in the gender and virus load between the mild and severe cases with HFMD. Children under 3 years of age with EV71 infection were at high risk for severe HFMD.     

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目的探讨儿童化脓性脑膜炎(化脑)新的快速诊断方法。方法2003-08—2005-12采用16SrRNA荧光定量法对浙江大学儿童医院49例临床疑似化脑患儿脑脊液(CSF)的细菌DNA进行测定;监测化脑患儿脑脊液细菌DNA拷贝数,同期进行CSF细菌培养的对照。结果(1)荧光定量PCR(FQ-PCR)检测49份脑脊液标本发现17份阳性,阳性率为34.7%(17/49),明显高于脑脊液培养的阳性率10.2%(5/49),差异具有显著性(P<0.01)。(2)对17份FQ-PCR阳性标本进一步测定细菌DNA的拷贝数,发现患儿病情与其DNA拷贝数呈正相关,与其Ct值(指每个反应管内的荧光信号到达设定的阈值时所经历的循环数)呈负相关,Ct值越低,脑脊液细菌DNA拷贝数越高,患儿的预后越差。(3)FQ-PCR、CSF细菌培养同时阳性的仅为5例。(4)对2例脑脊液FQ-PCR的产物测序,Ct值17.9的测序提示为大肠埃希菌,符合CSF细菌培养结果;Ct值31.8的,测序未果。结论荧光定量PCR特异性强、敏感性高,需标本量少,是早期快速诊断儿童化脑的可靠方法,具有较大的应用价值。     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

Objective Human herpesvirus 6(HHV-6)isolates are classified into two variants,HHV-6A and HHV-6B,based on distinct genetic,antigenic and biological characteristics.HHV-6 has been associated with encephalitis in children recently.This study aireed to estabhsh a real time PCR assay for simultaneous detection of the two subtypes of HHV-6,and apply this new assay to children with suspected encephalitis,then analyze the relationship between the infeetion with HHV-6 and encephalitis in children.Method The universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene(U38)of HHV-6.The 5'end of the probes for HHV-6A and HHV-6B was labeled with the fluoreseein reporter tetrachloro-6-carboxyfluorescein and 6-earboxyfluorescein(6-FAM),separately,while the 3'end were quenched with 6-carboxy-tetramethyl-rhedamine.The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established.Then,the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 109 to 10°copies/μl,together with controls were used for testing both sensitivity and specificity of the real time PCR assay.The cerebrospinal fluid(CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.Result Both HHV6A(strain ZJ-159)and HHV-6B (strain GS)were positive on the real time PCR assay.There were no cross-reaction with herpes simplex virus type 1,type 2(HSV-1,HSV-2),varicella-zoster virus(YZV),cytomegalovirus(CMV),EpsteinBarr virus(EBV),hepatitis B virus,Staphylococcus aureus,Mycoplasma pneumoniae and human DNA.A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6.The sensitivity threshold was 10 copies/μl for the real time PCR.HHV-6 positive rate by the real time PCR assay was 4.72%(21/445),including 4 ca8es with HHV-6A infection,16 cases of HHV-6B infeedon and l case with mixed HHV-6A and HHV-6B infeetion.The new PCR assay usually took 2 to 3 hours to provide results.Conclusion This new real time PCR assay call simultaneously detect both subtypes of HHV-6,and have high specificity and sensitivity.It will pmvide an early and sensitive diagnosis of HHV-6 encephalitis in children.     

人疱疹病毒6型荧光定量分型方法的建立和临床应用

Objective Human herpesvirus 6(HHV-6)isolates are classified into two variants,HHV-6A and HHV-6B,based on distinct genetic,antigenic and biological characteristics.HHV-6 has been associated with encephalitis in children recently.This study aireed to estabhsh a real time PCR assay for simultaneous detection of the two subtypes of HHV-6,and apply this new assay to children with suspected encephalitis,then analyze the relationship between the infeetion with HHV-6 and encephalitis in children.Method The universal primers and variant-specific TaqMan probes were designed based on the highly conserved sequences of the DNA polymerase gene(U38)of HHV-6.The 5'end of the probes for HHV-6A and HHV-6B was labeled with the fluoreseein reporter tetrachloro-6-carboxyfluorescein and 6-earboxyfluorescein(6-FAM),separately,while the 3'end were quenched with 6-carboxy-tetramethyl-rhedamine.The real time PCR assay for simultaneous detection of HHV-6A and HHV-6B was established.Then,the plasmids of HHV-6A and -6B which were diluted by a 10-fold series from 109 to 10°copies/μl,together with controls were used for testing both sensitivity and specificity of the real time PCR assay.The cerebrospinal fluid(CSF) specimens from 445 cases of suspected encephalitis were tested with this real time PCR and positive samples were then sequenced.Result Both HHV6A(strain ZJ-159)and HHV-6B (strain GS)were positive on the real time PCR assay.There were no cross-reaction with herpes simplex virus type 1,type 2(HSV-1,HSV-2),varicella-zoster virus(YZV),cytomegalovirus(CMV),EpsteinBarr virus(EBV),hepatitis B virus,Staphylococcus aureus,Mycoplasma pneumoniae and human DNA.A linear regression curve was obtained when plotting Ct values against the log10 of the viral DNA input for both subtypes of HHV-6.The sensitivity threshold was 10 copies/μl for the real time PCR.HHV-6 positive rate by the real time PCR assay was 4.72%(21/445),including 4 ca8es with HHV-6A infection,16 cases of HHV-6B infeedon and l case with mixed HHV-6A and HHV-6B infeetion.The new PCR assay usually took 2 to 3 hours to provide results.Conclusion This new real time PCR assay call simultaneously detect both subtypes of HHV-6,and have high specificity and sensitivity.It will pmvide an early and sensitive diagnosis of HHV-6 encephalitis in children.     

杭州市重症手足口病患儿的临床症状及脑脊液检查

目的研究杭州市重症手足口病(HFMD)流行特征,比较不同肠道病毒感染后的差异,寻求早期预判重症HFMD关键指标。方法对2014年杭州市儿童医院收治的498例重症HFMD患儿进行统计分析。采集重症患儿的脑脊液进行常规检测,并比较不同病原体感染后临床症状的差别。结果 498例重症HFMD患儿中,男女患儿比例为1.6∶1,1岁~3岁患儿占76.7%。重症组患儿平均年龄为(2.6±1.2)岁,重症HFMD患儿以EV71感染为主,占76.5%(381/498)。EV71感染的患儿肢体抖动和呕吐的发生率高于其他肠道病毒感染(P0.05)。EV71感染脑脊液有核细胞计数和蛋白质定量明显高于其他肠道病毒感染(P0.05)。结论年龄小、EV71感染、脑脊液有核细胞计数和蛋白质含量增高,出现肢体抖动和呕吐等临床症状,是预判重症发展为危重症HFMD的重要因素。     

手足口病病原体流行特征分析及护理

目的：对2013年3—6月手足口( HFMD)流行期间的449例HFMD患儿进行病原学流行特征分析,探讨其对临床护理实践的启示,为制定 HFMD 患儿的护理措施提供参考。方法收集2013年3—6月期间449例手足口病患儿的资料,用荧光定量RT-PCR法进行肠道病毒通用型、肠道病毒71型(EV71)和柯萨奇病毒A组16型(CA16)定量分型。结果449例HFMD患儿肠道病毒总阳性率为69.5%(331/476),其中EV71型203例、CA16型109例,重症组的患儿EV71阳性率高于轻症组,两组比较差异有统计学意义(χ2=52.981,P<0.05)。结论 HFMD患儿以EV71型流行为主,重症手足口病多发生于年龄<3岁的儿童,且基本为EV71型感染。重视肠道隔离仍是手足口病防治的重点。