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1、SARS病原与检测,中国疾病预防控制中心传染病预防控制所阚 飙,SARS病原与检测,中国疾病预防控制中心传染病预防控制所阚 飙,引起肺炎的病原体,细菌 肺炎链球菌、金黄色葡萄球菌、甲型溶血性链球菌、肺炎克雷白杆菌、流感嗜血杆菌、变形杆菌、铜绿假单胞菌、肠杆菌属一些细菌、变形杆 菌、军团菌、棒状杆菌、梭状杆菌等病毒 腺病毒、呼吸道合胞病毒、流感病毒、副流感病毒、麻疹病毒、巨细胞 病毒、单纯疱疹病毒、汉坦病毒、禽流感病毒、尼巴病毒、冠状病毒等支原体 肺炎支原体衣原体 肺炎衣原体、鹦鹉热衣原体真菌 白色念珠菌、曲菌、放线菌等其它 立克次体（Q热立克次体等）、弓形虫（鼠弓形虫等)、原虫（卡氏肺孢 子虫
2、等）、寄生虫类（肺包虫、肺吸虫、肺血吸虫等）,冠状病毒,冠状病毒科（Coronaviridae）单链RNA，包括冠状病毒属和环曲病毒属1937年从小鸡体内分离到传染性支气管炎病毒（IBV)1951年，鼠肝炎病毒（MHV)1965年，从人上呼吸道感染患者分离到B814病毒1966年，分离到229E病毒（人冠状病毒HCoV-229E）1968年，电镜观察病毒形态，命名冠状病毒1975年，ICTV命名冠状病毒科,冠状病毒属血清分学分组及所致疾病血清分组 病毒种和株 宿主 呼吸道疾病 肠道疾病 肝炎 神经系统感染 其它 HCoV-229E（人冠状病毒229E株）人？TGEV（猪传染性胃肠炎病毒）猪 I
3、 CCoV（犬冠状病毒）狗 FECoV（猫冠状病毒）猫 FIPV（猫传染性腹膜炎病毒）猫 RbCoV（兔冠状病毒）兔 HCoV-OC43（人冠状病毒OC43株）人？MHV（鼠肝炎病毒）小鼠 II BCoV（牛冠状病毒）牛 TCoV（火鸡冠状病毒）火鸡 HEV 猪 III TCoV（火鸡冠状病毒）火鸡 IBV 鸡 SARS-CoV,引自 贺福初主编.严重急性呼吸综合征.科学出版社，2003.,SARS病原体的发现与确证,2002年11月，广东出现SARS病人2003年3月15日，WHO组织国际研究网络实验室3月18日20日，观察到副粘病毒颗粒以及获得相关序列3月21日，猴肾细胞培养获得病毒分离物
4、，并排除甲乙型流感病毒病毒、呼吸道合胞病毒、副流感病毒1、2、3型、腺病毒、鼻病毒、肠道病毒、人间质肺炎病毒等，报告发现衣原体颗粒3月22日，发现冠状病毒样颗粒3月23日，明确发现冠状病毒颗粒和核酸片段，开始灵长类动物实验3月244月11日，获得更多的SARS病毒分离物、核酸片段序列，抗体检测4月12日、14日，SARS冠状病毒的全基因组序列公布4月16日，WHO宣布确认一种变异冠状病毒引起SARS4月17日，利用动物实验按科赫原则确定SARS病原体SARS coronavirus,“SARS的研究速度令人惊讶。由于全世界各国实验室之间非同寻常的合作，我们现在肯定地知道谁是SARS的元凶。”（
5、WHO传染病规划执行主任David Heymann 博士）,2003,348:1953-1966,SARS CoV基因组,2003,348:1967-1976.,冠状病毒颗粒结构,From N Engl J Med,2003,348:1948-1951.,2003,348:1953-1966,SARS病毒变异变异的意义？变异的方向？追踪变异的意义病原基本特征结合流行病学分析寻找传播链预测新的流行？疫苗设计,标本采集、保存与运输种类 最佳采集时间 采样 用途咽拭子与 发热早期 消毒的带塑料杆涤纶织物或人造纤维拭子签 鼻咽拭子 含2ml病毒保存液（或等渗盐溶液、组织 病毒分离、PCR 培养液等）的
6、旋 盖塑料冻存管，冷藏运输 鼻咽清洗物 发热早期 导管，无菌生理盐水 病毒分离、PCR漱口液 发热早期 5-10ml无菌盐水，置无菌旋 盖塑料离心管 病毒分离、PCR痰液 无时间限制 无菌平皿，拭子签刮取至含5ml运输液的 病毒分离、PCR 旋 盖塑料离心管下呼吸道标本 无时间限制 支气管灌洗、气管吸取液置旋 盖塑料离心管 病毒分离、PCR粪便 一周后检出率高 5-10g，旋 盖塑料离心管 病毒分离、PCR尿液 一周后检出率高 10-20ml 旋 盖塑料离心管 病毒分离、PCR血液 急性期和恢复期 抗凝管和析出血清用的血液采集管 急性期全血可作病毒 分离、PCR，血清作 抗体检测尸检标本 采集
7、肺、气管、肾、脾、肝、心脏、脑、病毒分离、PCR、淋巴结等，样品运输液为Hank氏液、Eagle液 电镜观察 等（加青、链霉素和制霉菌素），立即冻存；福尔马林固定者室温保存,标本运输与储存临床采集的标本，宜尽快送至专业检测实验室，在运送抵达前的24-48小时内，标本可4C冷藏（非福尔马林固定的尸检标本冷冻保存）。检测实验室内长期保存，血液标本-20 C冷冻保存，其它-70 C保存。,标本的生物安全操作采集与运输 标本管：螺旋盖、密封，贴生物危险性提示标签 运输过程中保证密封处理任何可能产生气溶胶的操作均应在生物安全柜内进行。操作者个人防护。二级生物安全（BSL-2）装备内进行的操作：血清和血标
8、本的各种常规诊断性检查。三级生物安全（BSL-2）室内进行的操作：SARS病原体分离培养、浓缩 标本中核酸提取 动物接种医务人员对SARS或疑似病人采样时的个人防护 口罩（符合N,R,P95/99/100,FFP2/3标准）、护目镜、手套、隔离衣在没有把握控制病毒泄漏（包括产生气溶胶）的情况下，不能进行标本的操作,SARS检测及结果解释检测方法抗体（IgG/IgM)：ELISA(Enzyme Linked ImmunoSorbant Assay)酶联免疫吸附实验 IFA(Indirect ImmunoFluorescence Assay)间接免疫荧光实验核酸（RNA）：RT-PCR（Rever
9、sed transcription-Ploymerase Chain Reaction）Nested PCR real-time PCR gene chip病毒分离 细胞培养,SARS检测及结果解释解释（Interpretation of test results）阳性结果-对于PCR和细胞培养：SARS病人正在或近期被SARS病毒感染。-对于ELISA和IFA：正在或近期被SARS病毒感染，或既往感染（需注意不同检测方法的特异性）阴性结果-病人没有被SARS病毒感染，而是由其它传染性（病毒、细菌、真菌）或非传染性因素。-实验结果不正确（“假阴性”）。目前的方法需要提高灵敏度。-对于PCR和细
10、胞培养：病人标本并没有在病毒或其遗传物质出现的时段内采集。病毒及其遗传物质仅存在一段较短的时期（依赖于不同标本种类）。-对于ELISA和IFA：标本收集过早，抗体还没有产生。(检测SARS病毒结果为阴性并不就表示病人没有感染SARS病毒),Laboratory case definition of SARS A person with symptoms and signs that are clinically suggestive of SARS AND with positive laboratory findings for SARS-CoV based on one or more o
11、f the following diagnostic criteria:a)PCR positive for SARS-CoV PCR positive using a validated method from:At least two different clinical specimens(eg nasopharyngeal and stool)OR The same clinical specimen collected on two or more occasions during the course of the illness(eg sequential nasopharyng
12、eal aspirates)OR Two different assays or repeat PCR using a new RNA extract from the original clinical sample on each occasion of testing.b)Seroconversion by ELISA or IFA Negative antibody test on acute serum followed by positive antibody test on convalescent phase serum tested in parallel OR Fourfo
13、ld or greater rise in antibody titre between acute and convalescent phase sera tested in parallel.c)Virus isolationIsolation in cell culture of SARS-CoV from any specimen AND PCR confirmation using a validated method.Testing should only be undertaken in a national or regional reference laboratory as
14、 per WHO recommendations(Use of laboratory methods for SARS diagnosis).WHO will assist resource poor countries to confirm their first cases of SARS through laboratory collaboration.Alert,verification and public health management of SARS in the post-outbreak period-WHO,Recommendations on interpretati
15、on of laboratory results Positive SARS diagnostic test findings a)Confirmed positive PCR for SARS virus:-at least 2 different clinical specimens(eg nasopharyngeal and stool)OR-the same clinical specimen collected on 2 or more days during the course of the illness(eg 2 or more nasopharyngeal aspirate
16、s)OR-2 different assays or repeat PCR using the original clinical sample on each occasion of testing b)Seroconversion by ELISA or IFA:-negative antibody test on acute serum followed by positive antibody test on convalescent serum OR-four-fold or greater rise in antibody titre between acute and conva
17、lescent phase sera tested in parallel c)Virus isolation:-Isolation in cell culture of SARS-CoV from any specimen;plus PCR confirmation using a validated method.Confirmation of positive PCR-The PCR procedure should include appropriate negative and positive controls in each run,which should yield the
18、expected results:1 negative control for the extraction procedure and 1 water control for the PCR run 1 positive control for extraction and PCR run the patient sample spiked with a weak positive control to detect PCR inhibitory substances(inhibition control)-If a positive PCR result has been obtained
19、,it should be confirmed by:repeating the PCR using the original sample ORhaving the same sample tested in a second laboratory.Amplifying a second genome region could further increase test specificityUse of laboratory methods for SARS diagnosis-WHORecommendations on interpretation of laboratory resul
20、ts Recommendations for laboratories testing for SARS,Recommendations for laboratories testing for SARS Reference laboratories should be identified at national level.PCR testing Laboratories testing for SARS by PCR should already have experience with PCR testing.They should adopt quality control proc
21、edures and identify a partner laboratory in their country or among the WHO collaborating research laboratories listed in Multi-centre Collaborative Network:Laboratories testing for SARS to cross-check their positive findings.Laboratories performing SARS specific PCR tests should adopt strict criteri
22、a for confirmation of positive results,especially in low prevalence areas,where the positive predictive value might be lower.A PCR-kit for SARS is commercially available,including internal controls.PCR primers and procedures have been published and can be adapted by laboratories.Positive control RNA
23、 is available from the Bernhard-Nocht Institute in Hamburg,Germany.The sensitivity of PCR tests for SARS depends on the specimen and the time of testing during the course of the illness.This may result in real cases of SARS testing negative by PCR(false negative results).Sensitivity can be increased
24、 if multiple specimens/multiple body sites are tested.The specificity of PCR tests for SARS is excellent if technical procedures used follow quality control guidelines.False positive results may arise as a result of technical problems(e.g.laboratory contamination),so every positive PCR test should b
25、e verified.Antibody testing ELISA and IFA tests are being developed by research laboratories.Because SARS a new disease in humans,SARS-CoV antibodies are not found in populations that have not been exposed to the virus.An antibody rise between acute and convalescent phase sera tested in parallel is very specific.,
