1例特殊的疑似狂犬病死亡病例的实验室检测和确诊

目的采用实验室检测方法确诊一例特殊的疑似狂犬病病例。方法利用荧光抗体(FA)试验、双抗体夹心ELISA和逆转录-聚合酶链式反应(RT-PCR)方法对一例死亡的疑似狂犬病人的脑组织和此脑组织经乳鼠脑内接种传代后的鼠脑组织分别进行检测和分析。结果死亡病人脑组织的直接FA试验和双抗体夹心ELISA检测为阴性,但RT-PCR检测结果为阳性。将该病人脑组织经乳鼠传代后对鼠脑组织用上述三种方法检测的结果均为阳性。对该街毒株的RT-PCR产物中的核蛋白基因序列进行测定,用Mega3.1软件进行分子进化关系分析,证实所分离的街毒株为基因Ⅰ型狂犬病病毒,与以往在该地区所分离的街毒株有较近的遗传关系。结论该病例可确诊为狂犬病死亡病例,其主要和决定性的死因为狂犬病,但并发的肺部感染可能加速了死亡的进程。WHO认为FA技术是狂犬病诊断的金标准,但本病例说明,在某些复杂情况下,只有将多种实验室检测方法联合运用才能确诊狂犬病,而且病毒分离和RT-PCR可能比FA试验更敏感。     

广东首例苏里南输入性寨卡病毒感染病例的实验室检测

目的对从广州白云国际机场口岸入境的一例自苏里南归国发热人员进行寨卡病毒实验室检测。方法采集发热人员的血液和唾液样本,同时使用两种寨卡病毒实时荧光RT-PCR试剂进行寨卡病毒核酸检测。RT-PCR扩增寨卡病毒部分基因片段,并进行序列测定和同源性比较。基于扩增片段构建系统进化树,分析输入性病例的来源。结果实时荧光RT-PCR结果显示,该病例血清样本寨卡病毒核酸弱阳性、唾液样本寨卡病毒核酸阳性。RT-PCR扩增出预期大小约1.5Kb片段,测序结果表明该片段与巴西寨卡病毒株(GenBank号KX197250)相应序列的同源性为99%。系统进化树显示该病毒属亚洲谱系。结论根据患者流行病学史、临床表现和病例标本核酸检测情况,确诊该病例为广东首例从苏里南地区输入性寨卡病例。     

福建省1株登革Ⅱ型病毒的鉴定

目的对2005年福建省分离的1株登革病毒(DV)进行鉴定,并从分子水平追踪其可能的传染源。方法采用酶联免疫吸附试验(ELISA)检测疑似登革热患者血清中DV(IgM、IgG抗体;同时应用C6/36细胞、单克隆抗体间接免疫荧光(McAb-IFA)、逆转录(套式PCR法分别进行病毒的分离和鉴定,并对分离株的部分基因进行核苷酸序列分析。结果患者血清登革病毒特异性IgM抗体阳性、IgG抗体可疑,表明该患者在近期感染过登革病毒。患者血清接种C6/36细胞,观察到登革病毒特有的CPE。受感染的C6/36细胞能与登革病毒Ⅱ型单克隆抗体反应,表明分离的病毒株为登革Ⅱ型病毒。分离株的核酸提取物经RT(PCR扩增,登革病毒通用引物可扩增出511bp的特异性条带,型特异性引物扩增出119bp的特异性条带,进一步证实分离的病毒株为登革Ⅱ型病毒。分离株RT(PCR扩增产物的核苷酸序列与30株不同地域来源的登革Ⅱ型病毒相应序列构建的系统发生树表明,此毒株与东南亚地区的毒株比较接近。此次分离株的序列与1999年登革Ⅱ型病毒福建株的对应序列在亲缘关系上有一定程度距离。结合流行病学调查资料,进一步确定此病例为输入性感染病例。结论福建省首次从输入性登革热患者血清中分离出登革Ⅱ型病毒,该病毒来源于东南亚地区。     

猪狂犬病的确证

目的 了解广东省某一猪场发生狂犬病猪只的感染状况。方法 采集疑似狂犬病发病猪的大脑、小脑以及海马回,制成切片,用姬姆沙染色,在光学显微镜下检测狂犬病病毒包涵体。用抗狂犬病病毒N蛋白荧光抗体染色,在荧光显微镜下检测特异荧光。用蛋白酶K处理病料,抽提核酸,以狂犬病病毒N基因特异引物作RT-PCR检测狂犬病病毒特异性核酸。将病料接种小白鼠接毒,观察小鼠的发病。结果 病理学切片检测病猪脑海马神经细胞中有包涵体;脑组织印片经特异性抗狂犬病病毒核蛋白(NP)单克隆抗体免疫荧光染色后,荧光显微镜下可见大量的针尖状绿色荧光颗粒;狂犬病病毒NP特异性目的基因RT-PCR得到与预期结果大小一致的扩增产物;小白鼠接毒试验,3日龄以内乳鼠接毒后出现特异性的神经症状。结论 该猪场病死猪检测狂犬病病毒感染阳性,确定猪群中发生狂犬病。     

深圳市输入性寨卡病毒分离株的进化分析及生物学特性研究

目的 对从深圳宝安国际机场口岸入境的1例自柬埔寨归国发热人员进行寨卡病毒实验室检测,研究寨卡病毒分离株的遗传进化和生物学特征,为寨卡病毒病的预防控制提供参考依据。方法 采集患者血液、唾液和尿液样本,采用荧光RT-PCR方法进行寨卡病毒核酸检测;选用多种组织培养细胞分离病毒,分别观察病毒株的致病变效应、空斑形成及病毒滴度等,并对两株病毒基因序列进行分子遗传进化研究,分析输入性病例来源。结果 荧光RT-PCR结果显示,该病例血清、唾液以及尿液样本寨卡病毒核酸阳性并持续一段时间。首次成功从唾液和尿液标本中分离到寨卡病毒株,将其分别命名为ZIKV/S/SZ1901和ZIKV/U/SZ1901。两分离株均可引起Vero细胞病变,不引起BHK-21细胞病变;均可在Vero细胞中形成空斑,滴度分别为3.4×10⁵ pfu/mL、1.4×10³ pfu/mL。RT-PCR扩增出预期大小约10 272 bp片段,测序结果表明该片段与ZIKV/Hu/Thai/KngSG/17-D501株相应序列的同源性为99.6%。系统进化树显示该病毒属亚洲谱系,与输入至我国的其它寨卡病毒株位于不同的进化分支。寨卡病毒编码区氨基酸位点分析提示,SZ1901毒株在结构基因的氨基酸位点(D683E、V763M、T777M)和非结构基因NS1基因的(A188V)变异与近几年流行株完全相同。结论 根据患者流行病学史、临床表现和实验室检测结果,确诊该病例为深圳市从柬埔寨输入性寨卡病毒感染病例,且寨卡病毒分离株具备与2016年以来流行的寨卡病毒大部分相同的分子基础。     

福建省狂犬病毒街毒株分离鉴定及生物学特性研究

目的 分离福建省狂犬病毒街毒株,了解病毒株的生物学特性。方法 采集疑似狂犬的脑组织,通过细胞培养和乳鼠脑接种分离狂犬病毒,用直接免疫荧光和RT-PCR等方法进行鉴定,并对病毒的分子生物学特性进行研究。结果 从疑似狂犬的脑组织中分离出狂犬病毒,TCID₅₀的测定结果显示,细胞毒滴度不高,病毒株对BHK-21细胞的适应性不强,LD₅₀的测定结果显示,病毒株为狂犬病毒强毒株。结论 从福建省家犬中成功分离到狂犬病毒街毒株,为狂犬病的实验室研究奠定基础。     

上海地区2004年疑似狂犬的病毒检测分析及其应用

目的对2004年上海地区疑似狂犬进行病毒检测,分析应用于狂犬伤多人应急事件的处理。方法收集126份疑似狂犬病的犬脑标本,用ELISA法快速检测狂犬病毒抗原、小鼠感染法(MIT)分离病毒和免疫荧光法(IF)鉴定病毒型别。对疑似狂犬进行地区和时间分布调查。结果3种方法完全一致。确诊狂犬的阳性率为22.2%(28/126),鉴定为1型狂犬病毒。6-8月检出率最高,为全年的75%(21/28)。病毒检测阳性的地区进行紧急防治措施。结论狂犬病毒检测3种方法,既可快速诊断,又可获得定型的毒株。便于对狂犬地区采取应急防治措施,利于控制预防狂犬病。     

3例SARS患者的RT-PCR与ELISA确认

目的 为了对SARS疑似患者进行快速早期诊断和进行血清学确认。方法 从SARS疑似患者含漱液与细胞病毒分离上清液中提取病毒RNA，进行逆转录套式PCR反应，扩增SARS病毒特异性核酸片段，进行序列测定与序列比对。并对患者不同发病日期采集的血清标本进行SARS病毒ELISA抗体测定。结果 SARS患者含漱液标本和细胞病毒分离上清液中均能扩增出特异性核酸片段，经测序证实来自SARS冠状病毒。患者血清标本中SARS病毒抗体在发病后18d开始呈阳性。以后随发病日期增加而升高。结论 逆转录套式PCR是一种早期、敏感、特异、快速检测传染性非典型肺炎疑似患者临床样本中SARS冠状病毒的理想方法。浙江省3例SARS临床诊断病例的含漱液和血清标本经SARS病毒核酸和抗体检测。证实为SARS病例。     

一起由诺如病毒和肠道腺病毒混合感染引起的急性胃肠炎聚集性疫情的病原鉴定及进化分析

目的?对一起儿童急性胃肠炎聚集性疫情的病原进行鉴定、分型及进化分析。 方法?收集本次疫情患儿粪便标本，提取核酸，采用实时荧光PCR初步进行病原鉴定。通过反转录-聚合酶链反应对诺如病毒阳性标本的聚合酶区和衣壳VP1区部分基因序列进行扩增；通过PCR对腺病毒阳性标本的六邻体区部分基因序列进行扩增。对扩增产物进行测序，利用生物信息学软件及网站对病毒进行型别鉴定及进化分析。结果?共收集到3例患儿粪便标本，诺如病毒和腺病毒均为阳性。3株诺如病毒毒株均为诺如病毒GII.P12-GII.3型，其聚合酶区部分基因序列（206 bp）与2015─2018年我国GII.P12-GII.3型诺如病毒参考序列的同源性为98%，且位于同一进化树分支上；3株腺病毒毒株均为腺病毒F亚属41型（F41型），其六邻体区部分基因序列（420 bp）与2015─2019年我国F41型腺病毒参考序列的同源性为100%，且位于同一进化树分支上。 结论?本次儿童急性胃肠炎聚集性疫情是由GII.P12-GII.3型诺如病毒和F41型腺病毒混合感染引起，毒株序列分别与近年来我国流行的GII.P12-GII.3型诺如病毒及F41型腺病毒序列高度同源。本研究为混合感染急性胃肠炎疫情病原的快速鉴定及溯源分析提供了参考。     

Diagnosis and analysis of a recent case of human rabies in Canada.

BACKGROUND: On September 30, 2000, staff at the Canadian Food Inspection Agency's Centre of Expertise for Rabies, located at the Animal Diseases Research Institute in Ottawa, Ontario, diagnosed rabies in a child from Quebec. This was the first case of rabies in a human in Canada in 15 years and in 36 years in the province of Quebec. After spending a week in intensive care in a Montreal hospital, the nine-year-old boy succumbed to this nearly always fatal disease. The boy had been exposed to a bat in late August 2000, while vacationing with his family in the Quebec countryside. METHODS: Antemortem specimens taken from the patient were sent to the Canadian Food Inspection Agency laboratory for rabies diagnosis. Samples included saliva, eye secretions, corneal impressions, cerebral spinal fluid and skin. Specimens were examined by direct immunofluorescence microscopy, and results were confirmed using molecular biological techniques. Virus strain identification was performed by both genetic methods and phenotypic analysis with monoclonal antibodies. RESULTS: Initial results from direct immunofluorescence staining indicated that rabies virus was present in the skin biopsy specimen but not in the corneal impressions. This diagnosis of rabies was confirmed by polymerase chain reaction product analysis from several of the submitted specimens. Virus isolation from postmortem samples was not possible because fresh brain tissue was not available. Rabies virus was isolated from saliva and was determined to be similar to a variant that circulates in populations of silver-haired bats. INTERPRETATION: Intravitam diagnosis of rabies in humans is very dependent on the samples submitted for diagnosis and the method used for testing. Upon first examination, only skin specimens were positive for rabies virus antigen; using polymerase chain reaction analysis, only saliva yielded positive results for rabies virus genome. Extensive testing and retesting of specimens submitted for rabies diagnosis in humans must be done to avoid false negative results.     

Diagnosis and analysis of a recent case of human rabies in Canada

BACKGROUND:

On September 30, 2000, staff at the Canadian Food Inspection Agency''s Centre of Expertise for Rabies, located at the Animal Diseases Research Institute in Ottawa, Ontario, diagnosed rabies in a child from Quebec. This was the first case of rabies in a human in Canada in 15 years and in 36 years in the province of Quebec. After spending a week in intensive care in a Montreal hospital, the nine-year-old boy succumbed to this nearly always fatal disease. The boy had been exposed to a bat in late August 2000, while vacationing with his family in the Quebec countryside.

METHODS:

Antemortem specimens taken from the patient were sent to the Canadian Food Inspection Agency laboratory for rabies diagnosis. Samples included saliva, eye secretions, corneal impressions, cerebral spinal fluid and skin. Specimens were examined by direct immunofluorescence microscopy, and results were confirmed using molecular biological techniques. Virus strain identification was performed by both genetic methods and phenotypic analysis with monoclonal antibodies.

RESULTS:

Initial results from direct immunofluorescence staining indicated that rabies virus was present in the skin biopsy specimen but not in the corneal impressions. This diagnosis of rabies was confirmed by polymerase chain reaction product analysis from several of the submitted specimens. Virus isolation from postmortem samples was not possible because fresh brain tissue was not available. Rabies virus was isolated from saliva and was determined to be similar to a variant that circulates in populations of silver-haired bats.

INTERPRETATION:

Intravitam diagnosis of rabies in humans is very dependent on the samples submitted for diagnosis and the method used for testing. Upon first examination, only skin specimens were positive for rabies virus antigen; using polymerase chain reaction analysis, only saliva yielded positive results for rabies virus genome. Extensive testing and retesting of specimens submitted for rabies diagnosis in humans must be done to avoid false negative results.Key Words: Bat rabies, RabiesRabies in humans is a disease rarely seen in Canada. The last reported case of human rabies in Canada occurred in 1985 in British Columbia, after a man was bitten by a bat (1), and it has been over 36 years since a human died of rabies in Quebec (2). Because human rabies is seen infrequently in Canada, physicians may not always suspect it when presented with encephalitides of unknown origin.In Canada, rabies is a reportable disease that falls under federal jurisdiction. All diagnoses of rabies are made by the staff of the Canadian Food Inspection Agency''s Centre of Expertise for Rabies at the Animal Diseases Research Institutes in Ottawa, Ontario and Lethbridge, Alberta. Diagnosis of rabies is a relatively straightforward procedure in animals, where only postmortem diagnosis on brain tissues is performed (3). Direct immunofluorescent (IF) microscopic examination of brain impressions is the only method used in Canada for rabies diagnosis using fresh animal tissues. The IF test is very sensitive with 100% specificity (3).Diagnosis of rabies in humans by examination of biopsy specimens and bodily fluids is performed only at the laboratory in Ottawa. This type of diagnosis is much more complicated than rabies diagnosis in animals because brain material is not available for testing. Attending physicians who suspect that a patient may have rabies usually telephone the Centre of Expertise for Rabies in Ottawa for instructions on which samples to submit. Samples for submission usually include saliva, eye secretions, corneal impressions and a plug of highly enervated skin from the back of the neck. Cerebral spinal fluid (CSF) and serum samples may also be sent to the laboratory if they are available. Prediction of which specimens will be positive if a rabies virus infection is present is very difficult; therefore, physicians should always try to send all of the above listed specimens to aid in a rabies diagnosis.In the present report, we describe the laboratory diagnosis of rabies in a nine-year-old boy from Quebec. A case report describing the clinical findings in this case was published previously (4,5). Briefly, a young boy vacationing with his parents in the Laurentian mountains of western Quebec was believed to have been exposed to a rabid bat on or about August 28, 2000. The patient was admitted to hospital on September 27, from which time his condition rapidly deteriorated. On September 29, a diagnosis of rabies was considered, and biopsy material was collected and submitted to the Canadian Food Inspection Agency for rabies diagnosis.     

Antemortem diagnosis of human rabies in a veterinarian infected when handling a herbivore in Minas Gerais, Brazil

The Ministry of Health's National Human Rabies Control Program advocates pre-exposure prophylaxis (PEP) for professionals involved with animals that are at risk of contracting rabies. We report an antemortem and postmortem diagnosis of rabies in a veterinarian who became infected when handling herbivores with rabies. The antemortem diagnosis was carried out with a saliva sample and a biopsy of hair follicles using molecular biology techniques, while the postmortem diagnosis used a brain sample and conventional techniques. The veterinarian had collected samples to diagnose rabies in suspect herbivores (bovines and caprines) that were subsequently confirmed to be positive in laboratory tests. After onset of classic rabies symptoms, saliva and hair follicles were collected and used for antemortem diagnostic tests and found to be positive by RT-PCR. Genetic sequencing showed that the infection was caused by variant 3 (Desmodus rotundus), a finding confirmed by tests on the brain sample. It is essential that professionals who are at risk of infection by the rabies virus undergo pre-exposure prophylaxis. This study also confirms that molecular biology techniques were used successfully for antemortem diagnosis and therefore not only allow therapeutic methods to be developed, but also enable the source of infection in human rabies cases to be identified accurately and quickly.     

Real-time PCR analysis of dog cerebrospinal fluid and saliva samples for ante-mortem diagnosis of rabies

The use of a 10-day observation to determine whether a dog is rabid is standard practice. This study was conducted in order to look for evidence of rabies vius in saliva and cerebrospinal fluid (CSF) of suspected live rabid dogs at the time of quarantine by using a SYBR Green real-time RT-PCR based assay for the detection of rabies virus RNA. Saliva and CSF of dogs were collected once on the day of admission for the 10-day quarantine. All test dogs were or became ill and died of rabies within the observation period. Thirteen of 15 dogs (87%) had saliva samples that were positive for rabies RNA. Two dogs with furious rabies had negative saliva samples. Positive CSF samples were found in 4 of 15 dogs (27%) whose saliva samples were positive. The time from sample collection to result was less than 5 hours. Because virus may be absent or present at very low level in both clinical fluids, samples taken for ante-mortem diagnosis cannot definitively rule out rabies.     

我国各地区动物狂犬病街毒的检测及分析

目的 检测我国不同地区动物中狂犬病毒带毒率并分析糖蛋白编码基因序列.方法 El.ISA、免疫荧光法分别检测586份采集自中国不同地区的犬、猫,蝙蝠和野鼠脑标本和16份犬唾液中狂犬病街毒,阳性标本乳鼠颅内接种.并测序.结果 ELISA、免疫荧光法均在犬脑中分离到10株狂犬病街毒,其中贵州省113份犬脑中分离到2株病毒,湖南省62份犬脑中分离到2株病毒,武汉市70份犬脑中分离到2株病毒,江苏省85份犬脑中分离到4株病毒,沈阳市69份犬脑中未分离到狂犬病毒;79份猫脑、100份蝙蝠脑及8份鼠脑中未检出狂犬病街毒,16份犬唾液标本未检出狂犬病毒.在贵州省和武汉市,冬季采集的112份犬脑末检出狂犬病毒.春夏季采集的40份犬脑中,4份阳性.分离的10株狂犬病毒阳性株颅内接种乳鼠后.均发病死亡.所有分离株均属狂犬病毒基因1型,可分为4个亚组.结论 同一地区的狂犬病毒分离株以及相邻省份的狂犬病毒分离株的同源性十分接近,动物样本采集时间与狂犬病毒阳检率有关.     

Rapid diagnosis of rabies in humans and animals by a dot blot enzyme immunoassay.

OBJECTIVES: The presently advocated tests for rapid diagnosis of rabies, such as the fluorescent antibody test (FAT) are expensive and require expertise to carry out and interpret the results. In this study, a simple direct dot blot enzyme immunoassay (DIA) has been developed and evaluated to detect the rabies antigen in brain specimens of animals and humans. The utility of this test in the ante-mortem diagnosis of human rabies has also been evaluated. METHODS: Brain homogenates of suspected rabid animals (n = 250), humans (n = 16) and clinical samples like saliva (n = 12) and cerebrospinal fluid (CSF) (n = 12) were directly spotted on polyvinylidene difluoride membrane (PVDF) and the absorbed rabies nucleoprotein antigen was detected using biotinylated antinucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and color development with diamino benzedine (DAB). Rabies-infected and normal mouse brain homogenates were used as positive and negative controls, respectively. The results of this test were evaluated with fluorescent antibody technique (for brain samples) and mouse inoculation test (for saliva and CSF samples). RESULTS: A distinct dark brown color was seen in the positive control and all positive samples, while there was no color development with either the negative control or the negative samples. The concordance between the fluorescent antibody test (FAT) and dot immunoassay was 98.4% for brain samples, 83.3% for saliva and 91.6% for CSF samples. The specificity of the test was found to be 100%. CONCLUSIONS: The dot blot enzyme immunoassay (DIA) test described here is a sensitive, specific and rapid test for the post-mortem diagnosis of rabies in animals and humans. The utility of this test for the ante-mortem diagnosis of rabies needs to be further evaluated.     

云南省狂犬病病人和犬类感染狂犬病病毒及M基因序列分析

目的 调查云南省部分地区狂犬病病人和疫点犬群携带狂犬病病毒(rabies virus, RABV)状况及RABV膜基质蛋白(matrixprotein, M)基因序列分析,为狂犬病防控提供科学依据。方法 2008-2009年在云南省采集犬脑组织标本606份,狂犬病病人唾液8份,脑脊液1份,用直接免疫荧光试验检测RABV抗原,用RT-PCR检测RABV核酸,对阳性标本进行M基因序列测定和分析。结果 所有标本经检测,RABV抗原和/或核酸阳性16份,其中疫点扑杀的貌似健康犬脑组织3.10%(10/323),狂犬病病人唾液5份、脑脊液1份。狗肉餐馆屠宰犬的脑组织283份均为阴性。云南16株RABV的M基因核苷酸和氨基酸同源性分别为88.5%~100%和85.2%~99.5%。它们与中国人用疫苗株aG核苷酸和氨基酸同源性分别为83.9%~85.7%和82.3%~93.6%;与人用疫苗株CTN181核苷酸和氨基酸同源性分别为99%~99.7%和98.5%~99%。系统进化分析表明,云南16株RABV均属基因Ⅰ型并可分为进化Ⅰ和Ⅱ群并分别与泰国等东南亚国家和相邻省份流行株具有较近的亲缘关系。结论 云南省狂犬病流行与周边省份和东南亚地区的狂犬病传播扩散有一定关系;狂犬病疫点部分貌似健康犬携带RABV并具有传染源意义;云南狂犬病病毒株M基因与我国人用狂犬病疫苗CTN株的同源性和亲缘关系较近,但与aG株同源性存在。