Adjuvant activity of ethanol extract of Hippophae rhamnoides leaves with inactivated rabies virus antigen.

CONTEXT: Hippophae rhamnoides L. (Elaeagnaceae), commonly known as seabuckthorn (SBT), is known for its medicinal and nutritional properties. OBJECTIVE: Evaluation of in vivo adjuvant activity of SBT leaf extract (SBTE) with inactivated rabies virus antigen (Rb). MATERIALS AND METHODS: Swiss albino mice were immunized with aqueous-alcoholic SBTE (100 mg/kg body weight) or algel (aluminium hydroxide gel) with or without Rb (5% v/v). After priming, booster was administered on day 14. Rabies virus neutralizing antibody (RVNA) titers were estimated by rapid fluorescent focus inhibition test in sera samples collected on days 7, 14, 21, 28 and 35. Effect of adjuvant administration on cytotoxic T lymphocytes (CTLs), memory T cells, plasma and CD11c+ cells was studied by flow cytometry. In vitro hemolysis was assayed in human RBC. RESULTS: RVNA titers were significantly enhanced (p < 0.05) after booster administration in mice immunized with SBTE + Rb as compared to the controls. In combination, SBTE, algel and Rb, enhanced the RVNA titers. CTLs significantly increased (p < 0.05) in SBTE + Rb immunized mice. Memory T cells and plasma cells were 27.9 and 15.9%, respectively, in SBTE + Rb immunized mice as compared to that of 20.3 and 11.3%, respectively, in Rb immunized group. SBTE + Rb enhanced peritoneal CD11c+ cells (25.8%) as compared to 9.4% cells in Rb immunized mice, showed 3.2-fold increment in LPS induced IL-1ß. No RBC hemolysis was observed with SBTE. CONCLUSIONS: This study demonstrates the potential adjuvant activity of SBTE with Rb by increasing RVNA titers and CTL response.

Serological Evidence of Lyssavirus Infection among Bats in Nagaland, a North-Eastern State in India.

Bats are known to be reservoirs of several medically important viruses including lyssaviruses. However, no systematic surveillance for bat rabies has been carried out in India, a canine rabies endemic country with a high burden of human rabies. Surveillance for rabies virus (RABV) infection in bats was therefore carried out in Nagaland, a north-eastern state in India at sites with intense human-bat interfaces during traditional bat harvests. Brain tissues and sera from bats were tested for evidence of infection due to RABV. Brain tissues were subjected to the fluorescent antibody test for detection of viral antigen and real-time reverse transcriptase PCR for presence of viral RNA. Bat sera were tested for the presence of rabies neutralizing antibodies by the rapid fluorescent focus inhibition test. None of the bat brains tested (n = 164) were positive for viral antigen or viral RNA. However, rabies neutralizing antibodies were detected in 4/78 (5·1%) bat sera tested, suggesting prior exposure to RABV or related lyssaviruses. The serological evidence of lyssaviral infection in Indian bats may have important implications in disease transmission and rabies control measures, and warrant extensive bat surveillance to better define the prevalence of lyssaviral infection in bats.

Comparative study on the immunogenicity and safety of a purified chick embryo cell rabies vaccine (PCECV) administered according to two different simulated post exposure intramuscular regimens (Zagreb versus Essen).

Despite availability of effective rabies vaccines, India has the highest global mortality rate for rabies. Low socio-economic communities are most affected due to lack of awareness of the disease and poor compliance to post-exposure prophylactic regimens. Currently, the only approved intramuscular regimen for post-exposure prophylaxis (PEP) against rabies in India is the Essen regimen, which consists of 5 injections administered over 5 separate days in a period of one month. The high number of doses and clinical visits, however, are major reasons for non-compliance, and thus a shorter regimen would be beneficial. In a simulated PEP trial in healthy, adult subjects, this study evaluated whether purified chick embryo cell vaccine (PCECV), administered according to the WHO-recommended 4-dose/3 visit Zagreb vaccination regimen is of equal immunogenicity and safety as the standard Essen regimen in Indian subjects. Two hundred and 50 healthy adults were enrolled and randomized into a Zagreb or Essen group, each receiving PCECV according to their respective regimen. Blood samples were collected on Days 0, 7, 14 and 42 and analyzed using the rapid fluorescent focus inhibition test (RFFIT). By Day 14, all subjects across both groups attained rabies virus neutralizing antibody (RVNA) concentrations of ≥ 0.5IU/ml. The Zagreb regimen was then demonstrated to be immunologically non-inferior to the Essen regimen by Day 14, which was the primary endpoint of the study. No safety issues were noted and the occurrence of adverse events was similar in both groups (17% and 15%, respectively). NCT01365494. CTRI No.: CTRI/2011/07/001857.

Rabid fox bites and human rabies in a village community in southern India: epidemiological and laboratory investigations, management and follow-up.

Human rabies transmitted from wild animals is rarely reported in endemic countries like India, where nearly 95% deaths occur due to bites from rabid dogs. In this paper, we report an incidence of rabid fox bites in a village in southern part of India involving 18 individuals, including 4 children. All people had category III exposures, including bites on the face and neck. The attacking fox was killed by the forest department and buried immediately. The victims of the fox bite did not receive appropriate and adequate postexposure treatment. Thirteen days after the bite, one of the bite victims developed typical symptoms of furious rabies and died 2 days later in a local hospital. His brain tissue, obtained at autopsy, was strongly positive for rabies by fluorescent antibody technique (FAT) and virus isolation. Panic prevailed in the community and the rest of the 17 cases were referred to our institute for advice and further management. Only 35% of them had protective levels of rabies virus neutralizing antibodies (RVNA). All of the patients were administered with an 8-site intradermal regimen with purified chick embryo cell (PCEC) vaccine and were followed up regularly. All of them developed adequate titers (>0.5 IU/mL) of RVNA 7 days later. They were under regular follow-up and after nearly 2 years none have developed rabies. The partial Nucleoprotein (N) gene sequencing of the virus isolate from the patient who died of rabies had close homology with species I (prototype rabies) sequences available in GenBank and our own past isolates from dogs and humans, thus confirming that virus spillover from wildlife to domestic dogs continues to occur. This episode should prompt health authorities to focus more attention on training rural medical practitioners in state-of-the-art modern prophylactic measures.

Rabies viral encephalitis with proable 25 year incubation period!

We report a case of rabies viral encephalitis in a 48-year-old male with an unusually long incubation period, historically suspected to be more than 20 years. The case was referred for histological diagnosis following alleged medical negligence to the forensic department. The histology and immunocytochemical demonstration of rabies viral antigen established the diagnosis unequivocally. The case manifested initially with hydrophobia and aggressive behavior, although he suddenly went to the bathroom and drank a small amount of water. History of dog bite 25 years back was elicited retrospectively following clinical suspicion. There was no subsequent history to suggest nonbite exposure to a rabid dog to consider recent event causing the disease, although this cannot be totally excluded.

Combination of N-methylisatin-ß-thiosemicarbazone derivative (SCH16) with ribavirin and mycophenolic acid potentiates the antiviral activity of SCH16 against Japanese encephalitis virus in vitro.

AIM: To investigate the drug to drug interaction of N-methylisatin-ß-thiosemicarbazone (MIBT) derivative (SCH16) with ribavirin, mycophenolic acid and pentoxifylline against Japanese encephalitis virus in vitro. Our earlier studies have reported significant antiviral activity of these compounds against Japanese encephalitis virus in vitro and in vivo. METHODS AND RESULTS: An in vitro drug to drug combination analysis was carried out to investigate whether or not the direct antiviral effect shown by the individual MIBT derivative could be effectively increased when lower concentrations of two compounds in combination were used. The results of this study showed that the combination of MIBT derivative (SCH16) with ribavirin or mycophenolic acid significantly enhanced the antiviral activity of SCH16 against JEV in vitro. In contrast, the combination of SCH16 and pentoxifylline resulted in antagonism. CONCLUSION: The antiviral activity showed by SCH16 was enhanced in the presence of ribavirin and mycophenolic acid. SIGNIFICANCE AND IMPACT OF THE STUDY: Studying the synergistic/additive interaction of the compounds in combination would help in lowering the effective concentration so as to overcome the concern of toxicity.

Emerging Viral Infections in India.

Despite an elaborate armamentarium to tackle microbes, emerging infectious diseases remain a crucial global challenge. Emerging infections can be defined as "infections that have newly appeared in a population or have existed previously but are rapidly increasing in incidence or geographic range." Several factors like increase in international travel and trade, human encroachment on wild-life habitats, changes in agricultural practices and wild-life trade have contributed to the emergence of pathogens. Emergence/re-emergence of several viral infections has been reported from India in the past few decades; some of the important emerging viral infections are discussed in this review. They include infection due to Nipah, Hantaviruses, Chikungunya, Human Enterovirus-71, Influenza, Chandipura, Crimean Congo, SARS Coronavirus, Buffalopox, Dengue and Japanese Encephalitis viruses. Creating increased awareness and training of clinical microbiologists/virologists for identification of new/emerging pathogens, and prompt reporting and management of outbreaks is essential to tackle the threat posed by emerging/re-emerging infections.

Role of apoptosis in rabies viral encephalitis: a comparative study in mice, canine, and human brain with a review of literature.

To evaluate the role of apoptosis in rabies encephalitis in humans and canines infected with wild-type street virus, in comparison with rodent model infected with street and laboratory passaged CVS strain, we studied postmortem brain tissue from nine humans, six canines infected with street rabies virus, and Swiss albino mice inoculated intramuscularly (IM) and intracerebrally (IC) with street and CVS strains. Encephalitis and high rabies antigen load were prominent in canine and human brains compared to rodents inoculated with street virus. Neuronal apoptosis was detectable only in sucking mice inoculated with CVS strain and minimal in street virus inoculated mice. In a time point study in suckling mice, DNA laddering was noted only terminally (7 days p.i.) following IC inoculation with CVS strain but not with street virus. In weanling and adult mice, apoptosis was restricted to inflammatory cells and absent in neurons similar to human and canine rabies-infected brains. Absence of neuronal apoptosis in wild-type rabies may facilitate intraneuronal survival and replication while apoptosis in inflammatory cells prevents elimination of the virus by abrogation of host inflammatory response.

Rabies viral encephalitis: clinical determinants in diagnosis with special reference to paralytic form.

BACKGROUND: Rabies is an important public health problem in developing countries such as India where an alarmingly high incidence of the infection is reported every year despite the availability of highly effective, potent and safe vaccines. In clinical practice, diagnosis of the furious (encephalitic) form of rabies poses little difficulty. In contrast, the paralytic form poses a diagnostic dilemma, to distinguish it from Guillain-Barré syndrome. The problem is further compounded in the absence of a history of dog bite, clinical features resembling a psychiatric syndrome. METHOD: The present study analysed the spectrum of neurological manifestations in 47 cases of rabies encephalitis (34 paralytic, six encephalitic, and seven psychiatric manifestations) from two hospitals in south India, confirmed at post-mortem by demonstration of a viral antigen in the brain. A history of dog bite was elicited in 33 patients and fox bite in one. Twenty-two patients received postexposure prophylaxis. The incubation period ranged from 7 days to 4 years. Clinical features were analysed, looking for any clinical pointers that provide clues to a diagnosis of paralytic rabies. RESULTS AND DISCUSSION: Fever, distal paresthaesias, fasciculation, alteration in sensorium, rapid progression of symptoms and pleocytosis in cerebrospinal fluid should alert the neurologist to consider rabies encephalomyelitis. Detection of the viral antigen in the corneal smear and a skin biopsy from the nape of the neck had limited usefulness in the ante-mortem diagnosis. Although a few clinical signs may help indicate rabies encephalomyelitis antemortem, confirmation requires neuropathological/neurovirological assistance. The preponderance of atypical/paralytic cases in this series suggests that neurologists and psychiatrists need to have a high index of clinical suspicion, particularly in the absence of a history of dog bite.

Protecting Indian schoolchildren against rabies: pre-exposure vaccination with purified chick embryo cell vaccine (PCECV) or purified verocell rabies vaccine (PVRV).

Although rabies can be effectively prevented by means of preexposure or post-exposure prophylaxis, in India, an estimated 17,000 to 20,000 human rabies deaths occur annually. Tragically, 50% of these victims are children under the age of 15. In addition to immediate post-exposure prophylaxis measures, including active and passive immunization, pre-exposure vaccination using tissue culture vaccines is a safe and effective but highly underutilized method of preventing rabies in humans living or working in areas at risk. This study assessed the safety and immunogenicity of Purified Chick Embryo Cell Vaccine (PCECV) and Purified Verocell Rabies Vaccine (PVRV), administered as a three-dose intramuscular pre-exposure regimen on days 0, 7 and 28 in 175 healthy schoolchildren. PCECV was administered after reconstitution using either 1.0 mL or 0.5 mL (half the diluent volume) and PVRV was given after reconstitution with 0.5 mL. Vaccine safety was assessed observer-blind, including pain assessment with a validated visual analogue scale for children. Rabies virus neutralizing antibody (RVNA) concentrations were measured on day 49 by RFFIT. All children developed adequate RVNA concentrations above 0.5 IU/mL. Solicited local and systemic reactions were within the range expected, pain after vaccination was reported in 2 to 12% of study subjects, fever was reported in 2 to 5%. There was no statistical difference by vaccination group or vaccination day. No unexpected or serious adverse event was reported during the study. In conclusion, PCECV and PVRV are safe and immunogenic when administered intramuscularly for pre-exposure prophylaxis of rabies in children. A 1.0 mL dilution volume for PCECV was as well tolerated as PVRV or PCECV reconstituted in half the volume.

Rabies encephalitis.

A 12-year-old boy presented with fever and sore throat of 6 days duration followed by vomiting and altered sensorium. He had received 4 doses of antirabies vaccine following a dog bite 4 weeks back. Rabies immunoglobulin was not given. History of hydrophobia and aerophobia were strikingly absent. The possibilities of rabies encephalitis and vaccine induced acute disseminated encephalomyelitis (ADEM) were considered. MRI brain showed exclusive grey matter changes characteristic of rabies. The diagnosis was further confirmed by serological tests.

Assessing the burden of human rabies in India: results of a national multi-center epidemiological survey.

OBJECTIVE: Human rabies has been endemic in India since time immemorial, and the true incidence of the disease and nationwide epidemiological factors have never been studied. The main objectives of the present study were to estimate the annual incidence of human rabies in India based on a community survey and to describe its salient epidemiological features. METHODS: The Association for Prevention and Control of Rabies in India (APCRI) conducted a national multi-center survey with the help of 21 medical schools during the period February-August 2003. This community-based survey covered a representative population of 10.8 million in mainland India. Hospital-based data were also obtained from the 22 infectious diseases hospitals. A separate survey of the islands of Andaman, Nicobar, and Lakshadweep, reported to be free from rabies, was also undertaken. RESULTS: The annual incidence of human rabies was estimated to be 17,137 (95% CI 14,109-20,165). Based on expert group advice, an additional 20% was added to this to include paralytic/atypical forms of rabies, providing an estimate of 20,565 or about 2 per 100000 population. The majority of the victims were male, adult, from rural areas, and unvaccinated. The main animals responsible for bites were dogs (96.2%), most of which were stray. The most common bite sites were the extremities. The disease incubation period ranged from two weeks to six months. Hydrophobia was the predominant clinical feature. Many of the victims had resorted to indigenous forms of treatment following animal bite, and only about half of them had sought hospital attention. Approximately 10% of these patients had taken a partial course of either Semple or a cell culture vaccine. The islands of Andaman, Nicobar, and Lakshadweep were found to be free of rabies. CONCLUSION: Human rabies continues to be endemic in India except for the islands of Andaman, Nicobar, and Lakshadweep. Dogs continue to be the principal reservoir. The disease is taking its toll on adult men and children, the majority from rural areas, due to lack of awareness about proper post-exposure immunization. The keys to success in the further reduction of rabies in India lies in improved coverage with modern rabies vaccines, canine rabies control, and intensifying public education about the disease.

An assessment of rabies free status of the Island of Andaman, Nicobar and Lakshadweep: results of the WHO sponsored national multicentric rabies survey.

The Islands of Andaman and Nicobar and Lakshadweep have been reported to be rabies free from time immemorial. Recently, a survey of the islands of Andaman and Nicobar & Lakshadweep was done between July and August 2003 to assess their rabies free status. It was revealed that these islands continue to remain rabies free. However, in Andamans the increasing dog population, poor vigil on import of dogs and lack of laboratory surveillance for rabies posed a threat to this status. The Lakshadweep islands, which are free of dogs, however, faced a threat from the lack of vigil on the entry and presence of cats and poor surveillance for rabies in them.

Boosting effect of purified chick embryo cell rabies vaccine using the intradermal route in persons previously immunized by the intramuscular route or vice versa.

BACKGROUND: At present, in the event of re-exposure to rabies, 2 booster doses are recommended for people who have been previously vaccinated with cell culture rabies vaccines by the conventional intramuscular route. As the intradermal route of vaccination is likely to be introduced in the future, we investigated the immune response to a cell culture rabies vaccine after crossing over from the intramuscular to the intradermal route and vice versa. METHODS: Twenty healthy adult volunteers who had received a primary course of rabies vaccination with purified chick embryo cell rabies vaccine by either the intramuscular (n = 10) or intradermal (n = 10) route received booster vaccination with the same vaccine by the alternative route. The regimen used was 0.1 ml of vaccine by the intradermal route at two sites (deltoid area) for the intramuscular group, or 1 ml of vaccine by the intramuscular route (deltoid muscle) to the intradermal group on days 0 and 3. RESULTS: There was a 15-fold rise in the rabies virus neutralizing antibody response both by the intradermal and intramuscular routes of booster vaccination (p < 0.0001). Thus, the change of route of purified chick embryo cell booster vaccination did not alter the anamnestic immune response to the vaccine. No side-effects were observed after vaccination with either of the routes. CONCLUSION: Purified chick embryo cell vaccine was found to be safe and immunologically efficacious following booster vaccination after cross-over from the intradermal to the intramuscular route and vice versa.

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.

Ante mortem diagnosis of human rabies using saliva samples: comparison of real time and conventional RT-PCR techniques.

BACKGROUND: Rabies is an enzootic and fatal disease and is still a major problem in developing countries. Ante mortem diagnosis in human cases is necessary for medical management of the patient and to ensure appropriate post-exposure treatment of contacts. Both conventional RT-PCR and Real time PCR (TaqMan) have been described for the detection of rabies virus RNA from saliva and tissue respectively, however to date, there have been no studies comparing conventional and real time PCR assays for detection of rabies virus nucleic acid using saliva samples for ante mortem diagnosis. OBJECTIVES: In this study, we evaluated the utility of conventional RT-PCR and SYBR Green I Real time PCR in the ante mortem diagnosis of rabies using saliva samples. STUDY DESIGN: Saliva samples collected from twenty-four patients presenting with typical clinical manifestations of rabies were tested in the two assays. RESULTS: Amongst the 24 samples tested, 21 samples (87.5%) were positive by either of the two molecular methods. Of these 21, rabies virus RNA was detected in 6/21 in the conventional RT-PCR assay while SYBR Green I Real time PCR could detect RNA in 18/21 samples. CONCLUSION: Real time PCR assay was more sensitive than conventional RT-PCR assay (sensitivity 75% versus 37%, p=0.0189). This study highlights the utility of molecular diagnostic tests in establishing ante mortem diagnosis of rabies using saliva samples within a few hours.