Early diagnosis of rabies virus infection by RPA-CRISPR techniques in a rat model.

Rabies, which is caused by rabies virus (RABV), poses an ever-present threat to public health in most countries of the world. Once clinical signs appear, the mortality of rabies approaches 100%. To date, no effective method for early rabies diagnosis has been developed. In this study, an RPA-CRISPR nucleic-acid-based assay was developed for early rabies diagnosis by detecting viral RNA shedding in the cerebrospinal fluid (CSF) of rats. This method can detect a single copy of RABV genomic RNA in 1 µL of liquid. RABV genomic RNA released from viral particles in the CSF could be detected via RPA-CRISPR as early as 3 days postinfection in a rat model. This study provides an RPA-CRISPR technique for early detection of RABV with potential application in the clinical diagnosis of human rabies.

Phylogenetic and epidemiologic evidence of multiyear incubation in human rabies.

Eight years after emigrating from Brazil, an otherwise healthy man developed rabies. An exposure prior to immigration was reported. Genetic analysis revealed a canine rabies virus variant found only in the patient's home country, and the patient had not traveled internationally since immigrating to the United States. We describe how epidemiological, phylogenetic, and viral sequencing data provided confirmation that rabies encephalomyelitis may present after a long, multiyear incubation period, a consideration that previously has been hypothesized without the ability to exclude a more recent exposure. Accordingly, rabies should be considered in the diagnosis of any acute encephalitis, myelitis, or encephalomyelitis.

Metabolomics of cerebrospinal fluid from humans treated for rabies.

Rabies is a rapidly progressive lyssavirus encephalitis that is statistically 100% fatal. There are no clinically effective antiviral drugs for rabies. An immunologically naïve teenager survived rabies in 2004 through improvised supportive care; since then, 5 additional survivors have been associated with use of the so-called Milwaukee Protocol (MP). The MP applies critical care focused on the altered metabolic and physiologic states associated with rabies. The aim of this study was to examine the metabolic profile of cerebrospinal fluid (CSF) from rabies patients during clinical progression of rabies encephalitis in survivors and nonsurvivors and to compare these samples with control CSF samples. Unsupervised clustering algorithms distinguished three stages of rabies disease and identified several metabolites that differentiated rabies survivors from those who subsequently died, in particular, metabolites related to energy metabolism and cell volume control. Moreover, for those patients who survived, the trajectory of their metabolic profile tracked toward the control profile and away from the rabies profile. NMR metabolomics of human rabies CSF provide new insights into the mechanisms of rabies pathogenesis, which may guide future therapy of this disease.

Role of reverse transcriptase polymerase chain reaction for the diagnosis of human rabies.

BACKGROUND & OBJECTIVES: Traditionally, rabies diagnosis is made by demonstration of rabies viral antigen by direct immunofluorescence (DIF) and mouse inoculation test (MIT). The present study was carried out to evaluate the role of reverse transcriptase-polymerase chain reaction (RT-PCR) in comparison with these conventional techniques for the diagnosis of rabies. METHODS: Skin biopsies, corneal impression smears and saliva sample were collected ante-mortem and brain tissue and CSF were collected post-mortem from ten clinically suspected rabies patients. DIF, Seller staining, MIT and RT-PCR were performed on the patients' samples for the diagnosis of rabies. The ability of RT-PCR to detect rabies virus earlier as compared to other assays was tested both for reference virus as well as clinical isolates. RESULTS: All samples taken ante-mortem were negative for DIF test. Six of 10 post-mortem brain tissues of the clinically suspected patients were positive both by RT-PCR and MIT, of these six, five were positive by DIF test and four were positive by Seller stain. RT-PCR could detect the rabies virus earlier as compared to DIF, both from clinical isolates and fixed rabies virus. INTERPRETATION & CONCLUSIONS: The present results showed 100 per cent sensitivity and specificity of RT-PCR as compared to 83.3 per cent of DIF and 66.7 per cent of Sellers stain for diagnosis of rabies. RT-PCR also detected rabies viral infection earlier as compared to conventional tests and can also be used on ante-mortem samples. Thus, the present study shows the usefulness of RT-PCR as an alternative to MIT for the confirmation of rabies diagnosis.

Presumptive abortive human rabies - Texas, 2009.

Rabies is a serious zoonotic disease. Recovery has been well documented in only six human patients worldwide. Five of those patients had received rabies vaccinations before illness; one had not received rabies vaccination but survived infection after prolonged intensive care. In most of these survivors, moderate to profound neurologic sequelae occurred. In all six survivors, rabies was diagnosed based on exposure history, compatible clinical symptoms, and detection of rabies virus-neutralizing antibodies (VNA). This report describes the clinical course and laboratory findings of an adolescent girl with encephalitis who had not had rabies vaccination and who had been exposed to bats 2 months before illness. Antibodies to rabies virus were detected in specimens of the girl's serum and cerebrospinal fluid (CSF) by indirect fluorescent antibody test (IFA). However, the presence of rabies VNA was not detected until after she had received single doses of rabies vaccine and human rabies immune globulin (HRIG). Although the patient required multiple hospitalizations and follow-up visits for recurrent neurologic symptoms, she survived without intensive care. No alternate etiology was determined, and abortive human rabies (defined in this report as recovery from rabies without intensive care) was diagnosed. Public education should emphasize avoiding exposure to bats and other potentially rabid wildlife and seeking prompt medical attention after exposure to such animals. Rabies is preventable if rabies immune globulin and vaccine are administered soon after an exposure; however, this case also suggests the rare possibility that abortive rabies can occur in humans and might go unrecognized.

Utility of rabies neutralizing antibody detection in cerebrospinal fluid and serum for ante-mortem diagnosis of human rabies.

BACKGROUND: Early ante-mortem laboratory confirmation of human rabies is essential to aid patient management and institute public health measures. Few studies have highlighted the diagnostic value of antibody detection in CSF/serum in rabies, and its utility is usually undermined owing to the late seroconversion and short survival in infected patients. This study was undertaken to examine the ante-mortem diagnostic utility and prognostic value of antibody detection by rapid fluorescent focus inhibition test (RFFIT) in cerebrospinal fluid (CSF)/serum samples received from clinically suspected human rabies cases from January 2015 to December 2017. METHODOLOGY/PRINCIPAL FINDINGS: Samples collected ante-mortem and post-mortem from 130 and 6 patients with clinically suspected rabies respectively, were received in the laboratory during the study period. Ante-mortem laboratory confirmation was achieved in 55/130 (42.3%) cases. Real time PCR for detection of viral nucleic acid performed on saliva, nuchal skin, brain tissue and CSF samples could confirm the diagnosis in 15 (27.2%) of the 55 laboratory confirmed cases. Ante-mortem diagnosis could be achieved by RFFIT (in CSF and/or serum) in 45 (34.6%) of the 130 clinically suspected cases, accounting for 81.8% of the total 55 laboratory confirmed cases. The sensitivity of CSF RFFIT increased with the day of sample collection (post-onset of symptoms) and was found to be 100% after 12 days of illness. Patients who had received prior vaccination had an increased probability of a positive RFFIT and negative PCR result. Patients who were positive by RFFIT alone at initial diagnosis had longer survival (albeit with neurological sequelae) than patients who were positive by PCR alone or both RFFIT and PCR. CONCLUSIONS/SIGNIFICANCE: Detection of antibodies in the CSF/serum is a valuable ante-mortem diagnostic tool in human rabies, especially in patients who survive beyond a week. It was also found to have a limited role as a prognostic marker to predict outcomes in patients.

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.

Presence of rabies specific immune complexes in cerebro-spinal fluid can help in ante-mortem diagnosis of human paralytic rabies.

BACKGROUND: Human rabies presents in two clinical forms, viz. furious or encephalitic and paralytic. Clinical diagnosis of paralytic form is difficult and requires laboratory confirmation. Presently available diagnostic techniques are not very sensitive for ante-mortem confirmation of rabies. OBJECTIVE: In the present study, we investigated whether presence of rabies specific immune complexes in cerebro-spinal fluid (CSF) of paralytic rabies patients could help in ante-mortem diagnosis of rabies. STUDY DESIGN: A capture ELISA based on monoclonal antibodies to rabies nucleoprotein (N) and glycoprotein (G) was developed to detect immune complexes to rabies N and G proteins. We studied CSF samples collected ante-mortem from 30 suspected paralytic rabies patients in whom diagnosis was later confirmed by autopsy. We included 30 CSF samples from people undergoing spinal anesthesia as negative controls and 30 CSF samples from other viral encephalitis as disease controls. RESULTS: Twenty-three out of 30 CSF samples (76.6%) showed presence of immune complexes to both rabies N and G proteins. None of the negative controls and CSFs from other confirmed viral infections were positive. Thus, the results were 100% specific and the sensitivity of this test was 76.6%. CONCLUSIONS: Detection of immune complexes to rabies antigens may be used as one of the techniques for rapid ante-mortem diagnosis of human rabies.

Rabies encephalomyelitis: clinical, neuroradiological, and pathological findings in 4 transplant recipients.

BACKGROUND: Three patients received solid organ transplants from a common donor and were subsequently discharged from the hospital following an uneventful hospital course. Within 30 days, all 3 organ recipients returned to the hospital with varying symptoms that progressed to rapid neurological deterioration, coma, and death. OBJECTIVE: To describe the clinical, neuroradiological, and pathological findings of rabies virus infection in organ transplant recipients infected from a common donor. DESIGN: Case series involving a common donor and 3 organ recipients ascertained through review of clinical course and autopsy findings. A fourth case was determined by review of pending autopsy cases in which death occurred within the same time interval. Portions of postmortem central nervous system and organ tissues were frozen and formalin-fixed. Fluids and tissues were also collected for cultures, serology, and molecular studies. Postmortem fluids and tissues and antemortem fluids and tissues from all 4 transplant recipients and serum and banked lymphocyte or spleen cells from the donors were sent to the Centers for Disease Control and Prevention for further evaluation. SETTING: Transplant unit of an urban teaching hospital. RESULTS: Antemortem cerebrospinal fluid analysis for 3 of the 4 recipients was consistent with a viral etiology. Neuroimaging and electroencephalogram studies were suggestive of an infectious encephalitis or a toxic encephalopathy. Initial laboratory testing did not demonstrate an infectious etiology. Postmortem histologic analysis, immunohistochemistry, electron microscopy, and direct fluorescence antibody testing revealed rabies virus infection. Serological testing done postmortem confirmed rabies virus infection in the common donor. CONCLUSIONS: These cases demonstrate a risk for transmitting rabies virus infection through solid organ and tissue transplantation, and this diagnosis should be considered in any rapidly progressing neurological disease.

Diagnosis of human rabies by the cornea test.

The second death due to rabies encephalitis occurring among American servicemen stationed in the Republic of Vietnam is reported. The clinical diagnosis was confirmed by performing the direct fluorescent antibody test for rabies on smears of corneal epithelial cells obtained on the second day of hospitalization. The reliability of various laboratory procedures in confirming the diagnosis of human rabies early in the course of clinical illness is discussed. The cornea test is a useful and rapid method of diagnosing human rabies prior to the development of significant serum antibody titers.

Cerebrospinal fluid analysis of rabid and vaccinia-rabies glycoprotein recombinant, orally vaccinated raccoons (Procyon lotor).

Cerebrospinal fluid obtained from clinically normal free-ranging raccoons was analyzed and compared with CSF obtained from raccoons vaccinated orally with vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine and subsequently challenged peripherally with street rabies virus, and CSF from naive, rabies virus challenge-exposed control raccoons. Significant differences were not found in CSF of free-ranging or V-RG recombinant virus vaccine recipient raccoons, and there was no evidence of CNS invasion by V-RG virus. The CSF of naive, rabies challenge-exposed control raccoons contained high numbers of lymphocytes and monocytes, compatible with rabies virus encephalitis. Although V-RG orally vaccinated challenge-exposed raccoons were protected from lethal rabies virus infection, a mild lymphocytic pleocytosis was evident at 90 days after challenge exposure.

Rabies in horses: 21 cases (1970-1990).

The records of 21 horses with rabies were reviewed. Results of fluorescent antibody testing for rabies antigen in brain tissue were positive in each case. According to the histories, 5 of the horses had been vaccinated for rabies between 4 to 24 months prior to the onset of the clinical signs. Bite wounds were not observed on any of the horses, and exposure to a suspected rabid animal was witnessed in only 5 cases. Clinical signs of disease at the time of initial examination included ataxia and paresis of the hindquarters (9/21, 43%), lameness (5/21, 24%), recumbency (3/21, 14%), pharyngeal paralysis (2/21, 10%), and colic (2/21, 10%). The major clinical signs observed over the course of hospitalization included recumbency (21/21; 100%), hyperesthesia (17/21; 81%), loss of tail and anal sphincter tone (12/21; 57%), fever (11/21; 52%), and ataxia and paresis of the hindquarters (11/21; 52%). Mean survival time after the onset of clinical signs was 4.47 days (range, 1 to 7 days). Supportive treatment, given to 9 horses, had no effect on survival time and did not correlate with the detection of negri bodies at necropsy. Cerebrospinal fluid (CSF) was obtained from 6 horses and was determined to be abnormal in 5. The most common abnormality was a slightly high total cell count (5/6), with a predominance of lymphocytes (4/6). The CSF total protein concentration was high in only 2 horses. At necropsy, there was gross evidence of diffuse brain edema, meningeal congestion, and focal areas of hemorrhage in 5 horses (24%).(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of interferon alfa and tribavirin in rabies encephalitis.

OBJECTIVE: To test the effect of interferon alfa and tribavirin (ribavirin) in patients with rabies encephalitis. DESIGN: An open trial of chemotherapy and intensive care in patients with early rabies. SETTING: The intensive care unit of a Bangkok hospital. PATIENTS: Four conscious men with clinical rabies encephalitis. INTERVENTIONS: Rapid virological diagnosis of rabies. Treatment with intravenous and intraventricular injections of high doses of lymphoblastoid interferon alfa in three patients and tribavirin in one patient. Intensive care was given throughout. MAIN OUTCOME MEASURES: Rabies infection confirmed by antigen detection and virus isolation. Rabies neutralising antibody and specific IgM sought in serum and cerebrospinal fluid. Interferon concentrations monitored before and during treatment in three patients. RESULTS: Interferon alfa treatment produced high concentrations in serum and cerebrospinal fluid. All four patients died after 5 1/2 to 12 1/2 days of treatment with no evidence of virostatic or clinically beneficial effects from either treatment. CONCLUSION: Interferon alfa treatment is not effective in rabies encephalitis. The use of tribavirin warrants further study, possibly combined with new therapeutic methods.

[Difficulties in diagnosing rabies--on the basis of observing a patient]. / Trudnosci w rozpoznaniu wscieklizny--na podstawie obserwacji chorego.

The aim of this paper was to pay attention diagnostic and therapeutic difficulties in patient with Rabies suspicion. The severe course of disease with necessary of dialysis therapy and successful treatment, underline value of this case report.