摘要
目的:采用病毒分离和RT-PCR法对临床诊断为手足口病(Hand-foot-and-mouth disease,HFMD)标本进行系列检测,以对该地区手足口病的病原进行鉴定;对经微量中和试验确证的EV71分离株VP1区进行核苷酸序列测定,并将测定结果与国内外EV71毒株进行同源性分析,绘制出进化树,为手足口病防治提供科学依据,为EV71毒株基因库及分子流行病学提供可靠资料。
方法:(1)采集临床诊断为HFMD患儿咽拭子和疱疹液标本共22份,采用病毒分离和RT-PCR两种方法进行检测。将待检标本按常规要求处理后接种RD细胞和HEp-2细胞,观察到特征性细胞病变后,收获病毒,采用微量中和试验和RT-PCR对分离物进行鉴定。在进行病毒分离的同时,将标本离心后,收集沉淀物进行RT-PCR。以每份标本核酸抽提物作为模板,分别用肠道病毒通用引物和EV71特异性引物进行RT-PCR扩增检测。采用统计学方法对两种标本和两种检测方法阳性率进行比较。
(2)将经微量中和试验和RT-PCR扩增并测序为EV71阳性的4株分离株VP1基因进行全基因测序,所得序列与网上公布的我国深圳、武汉、台湾、阜阳的EV71流行株,以及美国、韩国、新加坡、马来西亚等国家的EV71病毒株共计15株标准基因序列的VP1蛋白基因核苷酸序列进行比对分析,构建种系进化树,并对该基因特征与分型进行分析。
(3)对病毒分离和RT-PCR检测阳性患儿的年龄、性别和发病季节等一般情况,以及发热、皮疹、发热和皮疹的先后关系,有无神经系统并发症等临床表现进行了较详细的调查和分析。
结果:(1)从22份咽拭子和疱疹液标本中分离出可疑肠道病毒6株,阳性率为27.3%,经微量中和试验和RT-PCR检测,以及VP1全基因测序证实分离株均为EV71病毒。VP1蛋白基因全长891bp,编码一个含297个氨基酸的蛋白,其中4株分别命名为1-4-Luan-CHN-08、14-1-Luan-CHN-08、6-6-Luan-CHN-08和19-1-Luan-CHN- 08,VP1区全基因序列与检索到的15株EV71基因相关序列进行比对,构建种系进化树。结果显示:4株分离株与EV71病毒A基因型代表株BrCr的VP1区核苷酸序列同源性为89%~96%,较为接近,差异率小于12%;与EV71病毒B、C基因型代表株比较,同源性在74%~82%,差异率在18%~26%。表明六安地区这次手足口病主要病毒流行株属于A基因型,与中国大陆其他地区流行的EV71病毒株不属于同一基因型,至今国内未见报道。
(2)本次22份标本中,咽拭子和疱疹液分别为14份和8份,其中RT-PCR检测为EV71阳性的10份,阳性率为62.5%(10/16);22份标本全部进行了病毒分离,EV71阳性的6份,占27.3%(6/22),两种检测方法的阳性率有明显差别(χ2=6.35,P<0.05),RT-PCR检测阳性率远高于病毒分离阳性率。
(3)经检测确证为EV71感染所致HFMD患儿8名,占57.1%(8/14),男女性别比为5:3;从年龄来看,RT-PCR检测阳性的病例除两例年龄大于4岁(分别为5岁和7岁)外,其余均为4岁以下,占75%(6/8)。HFMD发病主要集中出现在5月上旬。8例EV71感染者中,手足口臀膝皮肤黏膜表面都有不同程度的皮疹或疱疹,其中1例有轻度的脑炎症状,6例还伴有发热症状,且发现这6例热患儿中有3例表现为先出疹后发热,1例发热和出疹同时出现,2例先发热后出疹。
结论:(1)本研究显示EV71是2008年六安地区HFMD流行的主要病原体,分离株属于A基因型,与中国大陆其他地区流行的EV71病毒株不属于同一基因型。而A型EV71已多年未在全球流行,必须引起高度关注。
(2)咽拭子标本在用RT-PCR和病毒分离进行检测EV71时均具有较高的检出率,推荐咽拭子应作为临床标本采集时的首选标本。
(3)EV71不仅可致手足口病,同时也可引起中枢神经系统症状,本研究确证为EV71感染8例患儿中,手足口臀膝皮肤黏膜表面都有不同程度的皮疹或疱疹,其中1例有轻度的脑炎症状,6例还伴有发热症状,且发现这6例热患儿中有3例表现为先出疹后发热,1例发热和出疹同时出现,2例先发热后出疹。
Objectives Throat swabs and vesicle swabs will be taken from HFMD in epidemic area, they were detected by RT-PCR and virus isolation; Compare with the results of different samples and methods, it will provide data of electing fit samples and methods; To sequence the VP1 gene of EV71 isolated, it’ll be analyzed the nucleotide identity with other strains at home and abroad. At the same time it will provide information on molecular epidemiology of EV71 local strain in Lu’an.
Methods (1) Collecting 22 clinical samples of throat and vesicle swabs from patients of HFMD or them associated with central nervous system involvement. Throat and vesicle swabs were inoculated into the RD and HEp-2 cell, we got virus identified by microneutralization test and RT-PCR when CPE occurred. At the same time the samples were centrifugaled, the sediments were detected by RT-PCR. The use of statistical methods for the two specimens and two methods to compare the positive rate, give us provide reference for the selection of methods and specimen in practical work.
(2) As well as the complete VP1 gene sequences for 4 strains of EV71 isolated in Lu’an were determined and analyzed. A phylogenetic was constructed by comparison of the sequences with other EV71 strains isolated from Shenzhen, Wuhan, Taiwan, Fuyang, as well as Korea,Singpore, Malaysia and the United States. Genotype and subgenotype of EV71 isolated Lu’an were analyzed.
(3) Positive patients of virus isolation and RT-PCR were analyzed by children's age, sex, time of onset, fever, skin rash, with or without clinical manifestations of neurological complications.
Results (1) Six strains of enteroviruses were isolated from 22 specimens of throat swabs and vesicular swabs, the positive rate was 27.3%. The enteroviruses confirmed by neutralization test, RT-PCR and the VP1 sequence determination are EV71. Molecular analyses of 4 strains of EV71 by sequencing the VP1 differed from BrCr-ts by 89%~96%. In contrast, it exhibited a nucleotide variability of 74%~ 82% when compared with EV71 isolated respectively genotype B and C. It shows epidemic strains of HFMD in Lu’an belong to genotype A, differed from genotypes of EV71 isolated in the other regions of our mainland. Genotype A wasn’t reported till the date in our country.
(2) We collected 22 clinical samples including throat and vesicular swabs from 16 HFMD patients, but 16 specimens were determined by RT-PCR and 22 were done by viral culture. The positive rates of RT-PCR and viral culture were 62.5% (10/16) and 27.3% (6 / 22) respectively, the two methods have significant difference(χ2=6.35,P<0.05). Because RT-PCR method has its own limitations, can not rule out the possibility of false positive, and viral culture is "gold standard" for detection of EV71, so we selected detection method of EV71 in accordance with the actual conditions.
(3)Through patients confirmed as EV71 infection by RT-PCR and virus isolation, accounting for 57.1% (8 / 14), male and female ratio was 5:3. From the age point of view, RT-PCR detection of positive cases in addition to two cases of age over 4 years (separately for the 5-year-old and 7 years old), the rest of children are all less than 4 years old, accounting for 75% (6 / 8). Children with HFMD mainly appear in early May. 8 cases of EV71 infection were caused HFMD with hand, foot, hip and knee in skin rash or herpes varying degrees, one of cases of encephalitis have mild symptoms. 6 cases had be fever, and 3 cases of patients were fever prior to rash, and 2 were rash before fever, but 1 was fever and rash simultaneously. Conclusion (1)This study showed that EV71 are pathogens of HFMD in Lu’an region in 2008, and nucleotide sequences of VP1 of EV71 were amplificated by RT-PCR before the sequences were determined. This showed epidemic strains of EV71 were genotype A in Lu’an. As genotype A of EV71 occurred in the mainland of China, and it had not prevailed all over world for many years, we must pay close attention to. (2) Because throat swab specimens have higher detectable rate than other specimen in RT-PCR and virus isolation for detection of EV71, it requires carrying out RT-PCR and virus isolation, at least throat swab should be collected in order to improve detection rate.
(3) EV71 can cause not only HFMD but also complication of central nervous system. Genotype A of EV71 were isolated in Lu’an, it hasn’t been isolated since 1970. EV71 strains isolated in the other mainland of China belonged to genotype C, which put forward a new topic in prevention of HFMD in our country. 8 cases of EV71 infection were caused HFMD with hand, foot, hip and knee in skin rash or herpes varying degrees, one of cases of encephalitis have mild symptoms. 6 cases had be fever, and 3 cases of patients were fever prior to rash, and 2 were rash before fever, but 1 was fever and rash simultaneously.
引文
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