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.     

一例暴露因素不明的狂犬病病例的分子流行病学溯源

目的 对一例暴露因素不明的人狂犬病病例进行实验室确诊,通过分子流行病学分析探索其可能的感染来源。方法 通过对存活疑似人狂犬病病例的唾液、脑脊液、血清标本采用直接免疫荧光试验、反转录-聚合酶链式反应和快速荧光灶抑制试验进行实验室确诊;通过时空进化分析探索该病例可能的感染来源。结果 病例唾液标本直接免疫荧光试验结果呈阳性,病例血清标本经快速荧光灶抑制试验检测抗狂犬病病毒中和抗体呈阳性。病例唾液标本经反转录-聚合酶链式反应获得狂犬病病毒核蛋白基因预期扩增片段,序列测定进一步证实为狂犬病病毒。该狂犬病病毒株与2011年安徽省一株犬源狂犬病病毒株核苷酸序列同源性最高,为98.4%。结论 该疑似狂犬病病例可确诊为狂犬病病例。其发病源于至少2011年以来的某次不自觉的狂犬病病毒感染。     

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.     

WHO recommended pre-exposure prophylaxis for rabies using Japanese rabies vaccine

After severe exposure to suspected rabid animal, WHO recommends a complete vaccine series using a potent effective vaccine that meets WHO criteria, and administration of rabies immunoglobulin (RIG). RIG is not available globally, and is not marketed in Japan. If pre-exposure prophylaxis for rabies is given, RIG is unnecessary even after severe exposure. It is thus important to give pre-exposure prophylaxis for rabies to people who plan to go to rabies-endemic areas. In Japan, pre-exposure prophylaxis for rabies consists of 3 doses of cell-culture rabies vaccine. The first two doses are given 4 weeks apart, and the third dose is given 6-12 months after the first dose, all of which are injected subcutaneously (standard regimen). People who plan to travel abroad to rabies-endemic areas may know of their destinations only 1 or 2 months in advance at best. Therefore, it is virtually impossible to complete the 3 dose regimen for rabies in Japan. Pre-exposure prophylaxis recommended by WHO consists of 3 doses given intramuscularly on days 0, 7, and 28, making it possible to complete pre-exposure prophylaxis in one month. This WHO recommended pre-exposure prophylaxis using Japanese cell-cultured rabies vaccine (PCEC-K) has not been studied, so we elected to fill the gap using PCEC-K, administered based on the WHO recommendation and examined its efficacy and safety. Subjects were 26 healthy volunteers with no previous rabies vaccination giving oral and written consent. Vaccine was administered on days 0, 7, and 28, and rabies antibody levels were tested on days 7, 28, and 42. On day 7, every antibody level was negative. On day 28, antibody levels were between 0.7-3.5 EU/ mL, with the exception of 3 cases still negative. On day 42, all cases, including the 3 negative cases, exceeded 1.6 EU/mL, providing sufficient protection against rabies. This result was not inferior compared to the standard regimen. Local adverse effects such as erythema and pain were noted, but none were serious. In conclusion, WHO recommended pre-exposure prophylaxis for rabies using PCEC-K is considered effective and safe.     

Knowledge and practices about rabies among travel medicine consultants in Greece

The number of travellers returning with animal bites from rabies enzootic areas has increased in Greece. The aim of this study was to assess the knowledge of travel-associated risk and preventive measures for rabies. A questionnaire was sent to Travel Medicine consultants in all prefectures. Of 100 Travel Medicine consultants, advice about rabies was given to long-term travellers, business travellers, travellers to rural areas, and travellers engaged in animal activities in rabies enzootic countries by 44%, 22%, 58%, and 75% of them respectively. Avoidance of animals, post-exposure medical assistance, return back to their country, and special caution about children was recommended by 89%, 95%, 8%, and 65% of them, respectively. Rabies pre-exposure vaccination was recommended for travellers to rural areas, long-term travellers, and travellers engaged in animal activities by 61%, 35%, and 81% of them, respectively. Regarding post-exposure vaccination, 78% and 37% answered correctly with regards to travellers with no pre-exposure prophylaxis and travellers with pre-exposure prophylaxis, respectively. Counselling about rabies and management of risk exposure needs to be improved. Our findings indicate the need to promote continuous training in Travel Medicine in Greece and provide practical information about rabies prophylaxis.     

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.     

Epidemiology and prevention of animal bite and human rabies in a rural community-One health experiment

ObjectiveTo estimate the incidence of human rabies and animal bite/exposure; to describe the post exposure prophylaxis received by animal bite/exposure cases; to assess the safety and immunogenicity of rabies vaccine (purified chick embryo cell vaccine) administered as pre-exposure vaccination for school children and risk groups by intradermal route in the rural community and to demonstrate a decrease in the incidence of human rabies and animal bite/exposures through implementation of one health experiment.MethodsThis prospective interventional study was conducted over a period of 2 years (December 2009-November 2011) in a rural area near Bangalore, Karnataka, South India and consisted of six villages (project villages), three villages were identified as study villages with active interventions (Implementation of rabies awareness activities, post exposure prophylaxis, pre-exposure intradermal rabies vaccine) and three villages as control villages without any active interventions.ResultsA majority of the animal bite cases were category III exposures and all of them had received rabies immunoglobulin and anti-rabies vaccine as per WHO recommendation. A majority received 3 to 5 doses of vaccine. Three hundred and sixty eight subjects had received pre-exposure intradermal rabies vaccination thrice on days 0, 7 and 28 d.ConclusionsNo human rabies case was reported during the study period and there was 30% decrease in animal bite/exposure cases in study villages after the one health experiment project was implemented. Pre-exposure vaccination was safe and immunogenic.     

Current rabies vaccines and prophylaxis schedules: preventing rabies before and after exposure

Travellers are probably the largest group in the general population to receive rabies pre-exposure prophylaxis. The dangerous consequences of the unavailability of rabies immune globulin in many countries could be ameliorated if pre-exposure rabies vaccination were practised more widely, especially in children, living in dog rabies enzootic countries. The WHO has recommended several different regimens for post-exposure prophylaxis, while individual countries decide on protocols for local use. Intramuscular regimens are expensive and waste vaccine. Although failure to receive vaccine is usually the due to the cost, the economical potential of intradermal vaccination has still not been realised 19 years after its introduction. The currently recommended 2-site intradermal post-exposure regimen is not economical for use in rural areas where 80% of Indian rabies deaths occur. Most countries using it demand higher potency vaccine, indicating that they do not have complete confidence in the method. This intradermal regimen has only been used where immunoglobulin is likely to be available for severely bitten patients. Increased intradermal doses are sometimes used for selected patients. Provision of economical rabies prophylaxis can be improved. Decisions to change recommendations should take account of the immunological, financial, practical and logistical aspects of dog bite treatment in remote areas.     

我国首次从犬中检出Negri小体

温州市 1999年前无狂犬病疫情报告 ,2 0 0 0年 12月以来该市的部分县 (市 )相继发生狂犬病病人。为了解新的狂犬病流行区动物狂犬病感染情况 ,对当地犬、猫等动物的脑组织进行Negri小体的病理学检测。共检查 13只犬和 2只猫。先将这些动物处死 ,获取其脑组织 ,用福尔马林固定。然后进行染色 ,检查Negri小体。结果显示 ,13只犬中有 1只检出Negri小体 ,而 2只猫则未检出。从犬中检出Negri小体在我国为首次。检测动物Negri小体有助于人狂犬病疫情监测 ,并可为疫情控制提供依据。     

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.     

Nucleic-acid sequence based amplification in the rapid diagnosis of rabies

Current serological tests do not reliably diagnose rabies. We describe a technique based on amplification of nucleic-acid sequences to detect rabies-specific RNA in the saliva and cerebrospinal fluid (CSF) of four living patients with rabies. Rabies RNA could be detected in either saliva or CSF, or both, in all patients and as early as day 2 after onset of symptoms. Both saliva and CSF should be serially tested because not every sample can be expected to be positive. The whole process, including automated extraction, isothermal amplification, and detection can be done within 4 h.     

Rabies post-exposure prophylaxis after being bitten by a ferret suspected to have rabies, case report

A woman bought two ferrets from a pet shop. These ferrets became brutal soon, excreted a large amount of saliva and then died. One of these ferrets had bitten the owner's hand before it died. As for the ferret, rabies and distemper were suspected. To receive rabies post-exposure prophylaxis the woman visited our vaccine clinic. To clarify the ferret's cause of death virological examinations were requested to Tokyo Metropolitan Institute of Public Health. The rabies examination of the ferret that had bitten the owner was performed according the law. However the tests of the other ferret that had not bitten the owner was not done though it was suspected to die from rabies, because the law does not mention such a case like the latter ferret. The virological tests concerning distemper were not accepted by the Institute, so the examination was done at a private laboratory. These two ferrets were diagnosed to have had distemper from the results. It is clearly shown from this bite-accident that the legal system where the cause of the animal's death required is extremely incomplete in Japan.     

Laboratory investigation of human deaths from vampire bat rabies in Peru.

In the spring of 1996, multiple cases of an acute febrile illness resulting in several deaths in remote locations in Peru were reported to the Centers for Disease Control and Prevention (CDC). The clinical syndromes for these cases included dysphagia and encephalitis. Because bat bites were a common occurrence in the affected areas, the initial clinical diagnosis was rabies. However, rabies was discounted primarily because of reported patient recovery. Samples of brain tissue from two of the fatal cases were received at CDC for laboratory confirmation of the rabies diagnosis. An extensive array of tests on the formalin-fixed tissues confirmed the presence of both rabies viral antigen and nucleic acid. The virus was shown to be most closely related to a vampire bat rabies isolate. These results indicate the importance of maintaining rabies in the differential diagnosis of acute febrile encephalitis, particularly in areas where exposure to vampire bats may occur.     

The role of postmortem studies in pneumonia etiology research

The diagnosis of etiology in severe pneumonia remains a challenging area. Postmortem lung tissue potentially increases the sensitivity of investigations for identification of causative pathogens in fatal cases of pneumonia and can confirm antemortem microbiological diagnoses. Tissue sampling allows assessment of histological patterns of disease and ancillary immunohistochemical or molecular diagnostic techniques. It may also enhance the recognition of noninfectious conditions that clinically simulate acute pneumonia. Biobanking of lung tissue or postmortem culture isolates offers opportunities for new pathogen discovery and research into host-pathogen interactions. The Pneumonia Etiology Research for Child Health study proposes a percutaneous needle biopsy approach to obtain postmortem samples, rather than a full open autopsy. This has the advantage of greater acceptability to relatives, but risks greater sampling error. Both approaches may be susceptible to microbiological contamination or pathogen degradation. However, previous autopsy studies have confirmed the value of histological examination in revealing unsuspected pathogens and influencing clinical guidelines for the diagnosis and treatment of future pneumonia cases.     

Polymorphism of a Class 3 Aldehyde Dehydrogenase Present in Human Saliva and in Hair Roots

The types of aldehyde dehydrogenases (ALDH) present in human hair roots and in saliva were investigated. ALDH was detected by activity staining following separation of crude extracts by isoelectric focusing. Hair roots were found to express ALDH1, ALDH2, ALDH3, and ALDH4, whereas saliva expressed ALDH3. Two different patterns of ALDH3 were detected in hair roots collected from 42 donors, 40 expressed one pattern (variant I) and two another pattern (variant II) of activity staining. The variant I pattern of hair root ALDH3 changed with repetitive freezing and thawing of the sample, whereas the variant II pattern was stable. In contrast to hair root ALDH3, all patterns of ALDH3 activity in saliva were stable. The patterns of ALDH3 activity present in human hair roots that had been frozen and thawed twice matched those present in saliva collected from the same individual. Three polymorphisms of ALDH3 (variants I, II, and III) were detected in the 33 saliva samples analyzed. Variants I and II were inherited in each of three generations of a 10-member family.     

Rabies pre-exposure prophylaxis using intradermal human diploid cell vaccine: immunologic efficacy and cost-effectiveness in a university medical center and a review of selected literature

The authors studied the antigenicity of intradermal human diploid cell rabies vaccine administered to 40 laboratory workers considered to be at-risk at the University of Virginia Medical Center. A 1-year postvaccination serology was determined for 20 of those 40, all of whom demonstrated an antirabies titer greater than or equal to 1:50 by the raped fluorescent focus inhibition test. By 2 years' postvaccination, 5 of 40 subjects had "unprotective levels" (less than 1:5), whereas 35 had titers greater than or equal to 1:5, and none had a titer greater than or equal to 1:50. Booster doses given to four subjects whose titers had declined produced a 1-month postvaccination antirabies titer greater than or equal to 1:50 in all cases. Vaccine administration by the intradermal rather than the intramuscular route resulted in a cost savings of $120 (U.S.) per employee. This data indicate that the intradermal administration of human diploid cell vaccine for rabies pre-exposure prophylaxis achieves an immunologic response thought to be protective while providing a substantial cost savings when compared with the intramuscular route of administration. Those who receive primary pre-exposure rabies vaccination should have serologic confirmation of immunologic protection every 2 years with a booster dose given to subjects demonstrating a titer less than 1:5.     

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。