Human rabies in India: an audit from a rabies diagnostic laboratory.

OBJECTIVES: Rabies, an acute progressive encephalomyelitis, continues to be a serious public health problem in India and many other countries in Asia and Africa. The low level of commitment to rabies control is partly attributable to challenges in laboratory diagnosis and lack of adequate surveillance to indicate the disease burden. A laboratory audit of human rabies cases was undertaken to disseminate information on the clinical, demographic, prophylactic and most importantly the laboratory diagnostic aspects of rabies. METHODS: A retrospective analysis of all clinically suspected human rabies cases, whose samples were received at a rabies diagnostic laboratory in South India in the last 3 years, was performed. Clinical and demographic details of patients were obtained. The clinical samples included cerebrospinal fluid (CSF), serum, saliva and nuchal skin biopsy collected antemortem, and brain tissue obtained post-mortem. Various laboratory tests were performed for diagnosis. RESULTS: Clinical samples from 128 patients with suspected rabies, from 11 states in India, were received for diagnostic confirmation. About 94% of the victims reported dog-bites, more than a third of them were children and most of the victims did not receive adequate post-exposure prophylaxis. Antemortem confirmation of rabies by a combination of laboratory diagnostic assays (detection of viral RNA in CSF, skin and saliva, and neutralising antibodies in CSF) could be achieved in 40.6% cases. CONCLUSIONS: Increasing awareness about adequate post-exposure prophylaxis, additional rabies diagnostic facilities, and enhanced human and animal rabies surveillance to indicate the true disease burden are essential to control this fatal disease.

Diagnostic difficulties in human rabies: A case report and review of the literature.

Rabies is a zoonotic disease with the highest fatality rate of any infectious disease. The clinical features of rabies encephalopathy are highly nonspecific at the onset and clinicians from low endemic areas usually face difficulties in recognizing cases during the early stages. The need for establishing a rapid and accurate test to identify rabies during the ante-mortem period is important. However, in actual clinical practice, the latter may remain difficult for various reasons. In human rabies, positively identifying the antigen, antibody or genetic material by various diagnostic methods during the symptomatic period is affected by the unpredictable nature of viremia, levels of antibody immune response of the host, and the virulence of the infecting strain. Also, more advanced testing with greater sensitivity may not be readily available at all centers. Here we describe a case of a young male who was bitten by a rabid dog and developed progressive encephalopathy with a fatal outcome, with negative antibodies in the cerebrospinal fluid (CSF). A review of the literature on the clinical features, diagnostic tests, treatment and prevention of rabies is also presented.

Utility of real-time Taqman PCR for antemortem and postmortem diagnosis of human rabies.

Rabies, a fatal zoonotic viral encephalitis remains a neglected disease in India despite a high disease burden. Laboratory confirmation is essential, especially in patients with paralytic rabies who pose a diagnostic dilemma. However, conventional tests for diagnosis of rabies have several limitations. In the present study the utility of a real-time TaqMan PCR assay was evaluated for antemortem/postmortem diagnosis of rabies. Human clinical samples received for antemortem rabies diagnosis (CSF, saliva, nuchal skin biopsy, serum), and samples obtained postmortem from laboratory confirmed rabies in humans (brain tissue, CSF, serum) and animals (brain tissue) were included in the study. All CSF and sera were tested for rabies viral neutralizing antibodies (RVNA) by rapid fluorescent focus inhibition test (RFFIT) and all samples (except sera) were processed for detection of rabies viral RNA by real-time TaqMan PCR. All the 29 (100%) brain tissues from confirmed cases of human and animal rabies, and 11/14 (78.5%) CSF samples obtained postmortem from confirmed human rabies cases were positive by real-time TaqMan PCR. Rabies viral RNA was detected in 5/11 (45.4%) CSF samples, 6/10 (60%) nuchal skin biopsies, and 6/7 (85.7%) saliva samples received for antemortem diagnosis. Real-time TaqMan PCR alone could achieve antemortem rabies diagnosis in 11/13 (84.6%) cases; combined with RVNA detection in CSF antemortem rabies diagnosis could be achieved in all 13 (100%) cases. Real-time TaqMan PCR should be made available widely as an adjunctive test for diagnosis of human rabies in high disease burden countries like India.

A comparative study of direct fluorescent antibody, mouse inoculation, and tissue culture infection testing for rabies diagnoses.

The laboratory diagnosis of rabies is of fundamental importance to the evaluation of suspected cases of rabies virus (RABV) infection. Confirmation of direct fluorescent antibody test (DFAT) results via viral isolation (VI) is recommended, and the mouse inoculation test (MIT) is being replaced by the rabies tissue culture infection (RTCIT) test for ethical reasons. We evaluated 6.514 results from central nervous system (CNS) samples of different animals analyzed at the Pasteur Institute between 2008 and 2016 using the DFAT, RTCIT and MIT techniques and evaluated their concordance, sensitivity, specificity, and accuracy indices. The DFAT technique presented the best sensitivity (93.58 %), specificity (95.90 %), and accuracy (95.67 %) results. The RTCIT values of sensitivity, specificity and accuracy (70.42 %, 86.16 % and 84.62 % respectively) were lower than those of DFAT. The concordance between RTCIT and DFAT was moderate, with a kappa quotient k = 0.341. The MIT values of sensitivity, specificity, and accuracy were 89.58 %, 100 % and 98.97 % respectively. The concordance between MIT and DFAT was substantial, with a k value of 0.720. DFAT, considered the "gold standard", was effective in all animals except horses. Our analyses evidenced that DFAT presents satisfactory results, although RTCIT did not appear favorable as a confirmatory technique.

Challenges to improved animal rabies surveillance: Experiences from pilot implementation of decentralized diagnostic units in Chad.

Better surveillance is desperately needed to guide rabies prevention and control to achieve the goal of zero dog-mediated human rabies by 2030, defined by the World Health Organization (WHO) and partners in 2015. With the help of funding from the Vaccine Alliance (GAVI) learning agenda, we implemented animal rabies surveillance based on One Health communication, improved accessibility of diagnostic testing and facilitated sample transport to increase case detection in three regions of Chad. Through the project, rabies surveillance, previously only available in N'Djaména, was extended to selected provincial rural and urban areas. Nine decentralized diagnostic units (DDU) were established, hosted by veterinary district agencies (VDA) in four different administrative regions. Four additional VDAs in the study area were reinforced with facilitation of sample collection and transport. Staff from all these 13 veterinary facilities were trained in sample collection and diagnostics. DDUs performed Rapid Immunodiagnostic Tests (RIDT) providing a preliminary result before samples were sent to the central laboratory in N'Djamena for confirmation with the standard Florescent Antibody Test (FAT). Within the project period from June 2016 to March 2018, 115 samples were reported by veterinary facilities in the study areas compared to 63 samples received from outside the study area, the vast majority of them originating from the capital city N'Djaména (N=61). Eighty nine percent of all 178 samples reported to IRED during the project period tested positive. Most of the samples originated from dogs (92%). Other confirmed rabies positive animals observed were cats, a donkey and a pig. Although surveillance of animal rabies was the focus, four human saliva samples were also submitted for diagnosis. We observed high differences in reporting rates between the four study regions. This could be attributable to differences in rabies epidemiology but are also influenced by the distance to the central laboratory in N'Djaména, the cultural background and the level of public awareness. The possibility for local testing through RIDT was very welcomed by local veterinary staff and preliminary insights suggest a positive influence on One Health communication and PEP initiation. However, these aspects as well as the relative impact of local testing on sample collection in comparison to reinforcement of sample collection and transport alone, need to be further investigated. Challenges encountered related to poor infrastructure (buildings, appliances, materials) and low logistic capacity (lacking means and material for transport and communication) of veterinary services in Chad. In addition, veterinary personnel lack experience in data management. Together with staff turnover, this leads to a need for repeated training. Major shortcoming of the approach was the high cost per sample and limited sustainability beyond the project timeframe.

Child survivor of rabies in India: a case report.

A 4-year-old boy was admitted with an acute onset fever for 4 days and drowsiness for 3 days, followed by progressive flaccid weakness of both lower limbs and encephalopathy soon after admission. He had sustained a WHO Class III stray dog bite 2 weeks previously and had received three doses of post-exposure rabies vaccination with purified vero cell vaccine but not rabies immunoglobulin. He was diagnosed with rabies based on the presence of rabies virus neutralising antibody in CSF (Day 1 1:128 and Day 26 1:2048) and typical findings on neuro-imaging. Rabies viral RNA was not detected in CSF, in saliva or on nuchal skin. The child survived with supportive treatment alone but he has extensive neurological sequelae. This report demonstrates the detailed clinico-investigative profile of a child who survived rabies following inadequate post-exposure prophylaxis and adds to the sparse knowledge of this usually fatal condition. ABBREVIATIONS: ADEM, acute disseminated encephalomyelitis; CBNAAT, cartridge-based nucleic acid amplification test; CSF, cerebrospinal fluid; EEG, electroencephalogram; GCS, Glasgow coma scale; EVM, eye opening, best verbal response, best motor response; IM, intramuscular; IVIg, intravenous immunoglobulin; MRC, Medical Research Council; MRI/FLAIR, magnetic resonance imaging/fluid attenuation inversion recovery; PCR, polymerase chain reaction; RFFIT, rapid fluorescent focus inhibition test; RIg, rabies immunoglobulin; RNA, ribonucleic acid; WBC, white blood cells; WHO, World Health Organization.

Development of a quantitative real-time RT-PCR assay for detecting Taiwan ferret badger rabies virus in ear tissue of ferret badgers and mice.

In 2013, the first case of Taiwan ferret badger rabies virus (RABV-TWFB) infection was reported in Formosan ferret badgers, and two genetic groups of the virus were distinguished through phylogenetic analysis. To detect RABV-TWFB using a sensitive nucleic acid-based method, a quantitative real-time reverse transcription polymerase chain reaction targeting the conserved region of both genetic groups of RABV-TWFB was developed. This method had a limit of detection (LOD) of 40 RNA copies/reaction and detected viral RNA in brain and ear tissue specimens of infected and dead Formosan ferret badgers and mice with 100% sensitivity and specificity. The mean viral RNA load detected in the ear tissue specimens of ferret badgers ranged from 3.89 × 108 to 9.73 × 108 RNA copies/g-organ, which was 111-fold to 2,220-fold lower than the concentration detected in the brain specimens, but 2,000-fold to 5,000-fold higher than the LOD of the assay. This highly sensitive technique does not require facilities or instruments complying with strict biosafety criteria. Furthermore, it is efficient, safe, and labor-saving as only ear specimens need be sampled. Therefore, it is a promising technique for epidemiological screening of Taiwan ferret badger rabies.

Seroprevalence of rabies virus antibodies in bats from high risk areas in Brazilian Amazonia between 2013 and 2015.

Background: The outbreaks of human rabies that occurred between 2004 and 2005 in the Brazilian Amazon highlight the importance of bats in the transmission of this zoonosis. Ten years after, this region is still considered to be a risk area. Methods: Serum and brain tissue samples were obtained from bats captured between 2013 and 2015. The sera were tested for the presence of rabies antibodies, using the rapid fluorescent focus inhibition test, and the brain tissue samples were tested for the presence of the rabies antigen by the direct immunofluorescence method and intracerebral inoculation in mice. Results: A total of 64% (148/230) of the serum samples were seropositive, although none of the brain samples were positive for rabies infection. The seroprevalence was significantly higher in the second year of the study (p<0.001). This figure was detected in all variables (sex, age, season) and in most of the bat species. Conclusions: Our results indicate the possible occurrence of a recent peak in infection by the rabies virus in these bat populations, which represents an important alert, given that attacks by hematophagous bats are a constant threat in the study area, contributing to the probability of the occurrence of new cases of rabies.

Susceptibility of neuroblastoma cells to rabies virus may be affected by passage number.

Maintaining a healthy, continuous immortalized cell line is essential for rabies laboratories that perform virus isolation assays and test for the presence of viral neutralizing antibodies. Individuals who routinely work with rabies virus, such as rabies laboratory employees, or those who may have a high potential for exposure to rabies virus, including veterinarians, should be tested for the presence of anti-rabies viral neutralizing antibodies (VNA) every 6-24 months, depending on potential exposure level. The gold standard for serum neutralization assays require the use of live rabies virus and cells that are sensitive to rabies virus infection. Additionally, virus isolation assays are routinely performed in rabies laboratories as a back-up for the direct fluorescent antibody test (dFAT). Currently there are no guidelines or publications recommending the use of low, intermediate, or high passage cell lines in rabies assays. In this study, we compared the sensitivity of intermediate, high, and extremely high passaged neuroblastomas to rabies virus using virus isolation, serum neutralization, and real time RT-PCR techniques. Additionally, cells were examined microscopically to determine changes in morphology and dissemination of rabies virus antigen between intermediate, high, and extremely high passage cells. No significant difference was found between cell passage numbers and viral susceptibility between intermediate and high passaged cells. However, extremely high passaged cells (≥1200 passages) were less susceptible to viral infection and/or produced less virus following inoculation. As a result, rabies laboratories that use viral isolation and serum neutralization assays should regularly assess cell susceptibility to ensure the integrity and repeatability of the test.

Atypical rabies encephalitis in a six-year-old boy: clinical, radiological, and laboratory findings.

A 6-year-old boy from India developed an atypical form of rabies following a stray dog bite and as a consequence of not receiving the standard World Health Organization recommended post-exposure prophylaxis for category III wounds. Serial rising rabies virus neutralizing antibody titres in serum and cerebrospinal fluid by rapid fluorescent focus inhibition test helped confirm the diagnosis of rabies. The child has survived for 4 months since the onset of illness, albeit with neurological sequelae.