Rabies is an invariably fatal disease in human and animals due to acute encephalomyelitis. The disease is caused by the neurotropic viruses of Lyssavirus genus. It is a zoonosis of all mammalian species around the world. Human infections occur through the bite of the infected animals. The average estimated number of human deaths due to rabies is more than 60000 per year globally.
VIRUS
Rabies virus belongs to the order Mononegavirales, family Rhabdoviridae and genus Lyssavirus. According to the 2015 release of International Committee on Taxonomy of Viruses (ICTV), Lyssavirus genus includes 14 virus-species based on genetic distance, antigenic pattern, host range and geographic distribution (Table 1). Rabies virus is the type species and is responsible for almost all the cases of human rabies.
Table1. List of virus species of Lyssavirus genus
Rabies virus is a bullet-shaped virus, the average dimension of which is around 200 nm length and 75 nm diameter. The genome is a single stranded, non-segmented RNA of negative polarity of 12 kilobase pairs in length. It codes for five viral proteins; nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA dependednt RNA polymerase (L or large protein). The RNA genome along with N, P and L proteins form the ribonucleoprotein complex. The nucleocapsid is present in a helical symmetry which is surrounded by the matrix protein (M protein).
The outermost layer is the envelop which contains surface projections of glycoproteins (G protein).
EPIDEMIOLOGY
Rabies is still a huge public health problem in canine infested developing countries which together contribute the near total deaths due to rabies in humans. Human rabies is present all over the world, in all continents including 150 countries and territories excepting Antarctica.
Canine rabies is still endemic in major parts of Asia, Africa, and Latin American countries. According to the estimation in 2010, the average numbers of annual death due to rabies is 61000. Of this, near one-fourth is contributed by India only (largest number by any single country) and near 60% by all Asian countries. Most of these deaths (near 85%) occur in rural areas and in children.
Several countries in the world have been declared free of canine rabies, such as the United States of America (USA), Canada, Western Europe, Japan, Malaysia and a few Latin American countries. Australia has been free from carnivore rabies. Whereas, Pacific islands are free from rabies and rabies-related viruses.
Rabies virus is transmitted by bite of rabid dog or other mammals. Dogs are the primary vector, contributing to more than 99% of all human rabies cases. Transmission through inhalation has been associated with laboratory accidents during vaccine preparation and exposure to insectivorous bats in the cave. Ingestion of raw milk from rabid animal possesses risk of infection, but there is so far no documented report available. Humans are the dead-end host, but rare transmission of virus from human-to-human has been reported through transplantation of infected organs, human bite, kissing, transplacental route, and through human breast milk.
PATHOGENESIS
Rabies virus enters the human body through bite of infected animals and is inoculated in the skin and muscle. It cannot enter through intact skin and enters only through broken skin with intact mucosa. The source of virus is the salivary fluid of the rabid animal. The initial replication of the virus occurs in the muscle and then the virus binds to the nicotinic acetylcholine receptor present at the postsynaptic neuromuscular junction. Along the peripheral nerves, the virus spreads centripetally via fast retrograde transport along motor axons towards the spinal cord and brainstem. The rate of travel through peripheral nerve is approximately 50–100 mm per day. Once it reaches the CNS, extensive virus replication occurs in the neurons and clinical disease develops. Then the virus disseminates through the axonal transport to all over the CNS. From CNS, the virus then transverses centrifugally along sensory and autonomic nerves to other tissues of the body such as salivary gland, adrenal gland, heart, cornea and skin (Fig. 1). By the time patient develops the symptoms, virus is widely distributed throughout the CNS and possibly to extraneural organs.
Fig1. Pathogenesis of rabies virus
The average incubation period is 2–3 months, which ranges from 5 days to more than 1 year depending on the amount of virus inoculums, density of the motor end plate at the site of animal bite and the proximity of site of virus inoculation to the CNS.
PATHOLOGY
Mild inflammatory changes occur with mononuclear cell infiltration in the leptomeninges, perivascular regions and brain parenchyma. Negri body is the most pathognomonic finding of rabies. These are intracytoplasmic eosinophilic inclusion bodies present in the neuronal cells and composed of viral RNA and proteins. The common sites of negri bodies are pyramidal neurons of hippocampus and Purkinje cells of the cerebellum. However, negri bodies are seen only in small number of rabies cases. Unlike other viral encephalitis, neuronophagia and neuronal apoptosis are not common in rabies encephalitis.
CLINICAL FEATURES
Clinical symptoms of rabies start with the prodromal symptoms which are usually manifest with febrile illness with change in mood. The common prodromal symptoms are fever, headache, nausea and vomiting. The mood change usually manifests in the form of anxiety, irritability, restlessness, and agitation. The first specific symptom is pain, pruritus, itching or tingling sensation at the site of bite, which occurs in around half of the patients.
The acute neurologic form of human rabies can be manifested in one of the two forms— furious or encephalitic form and dumb or paralytic form. The former type is more common and seen in around 80% of patients, whereas the later is seen in only 20% of cases. This is probably because of the predominant involvement of brainstem, cranial nerves and limbic system in encephalitic form and medulla, spinal cord and spinal nerves in the paralytic form.
The encephalitic form is rapidly progressive in nature. The general symptoms of encephalitis such as fever, hallucination, seizure are same as that of other encephalitis. The rabies specific symptoms are autonomic dysfunction which manifest as hypersalivation, hydrophobia, aerophobia, foaming at the mouth, and period of hyperexcitability alternating with lucidity. The duration of lucidity gradually decreases. The term hydrophobia means “dread of water” which is painful, involuntary contractions of the diaphragm and other accessory respiratory muscles that is provoked by attempt to drink water or liquids. Similar reflex caused by stimulation of air. Finally patient develops complete paralysis and multiorgan failure and death ensues within days of development of classic rabies symptoms.
The dumb or paralytic form of rabies has a slow and protracted course. The neurological manifestations start as flaccid muscle weakness in the bitten limb which ascends gradually and leads to quadriparesis and ultimately affecting the respiratory and deglutition muscles. Involvement of sphincter is common leading to urinary retention and constipation. The classic features of encephalitic form are usually not seen in paralytic form and it can be confused with other neurological disorders like Guillain-Barré syndrome.
Some of the other members of Lyssavirus genus, most of which infect bat, can produce disease in humans indistinguishable from human rabies. These are: Australian bat Lyssa virus, European bat Lyssavirus 1 and 2, Irkut virus and Duvenhage virus and Mokola virus (which has been so far isolated from shrews). These viruses are called “rabies related viruses”.
LAB DIAGNOSIS
Samples for confirmation of rabies are collected either during life or postmortem. Antemortem diagnosis is difficult as collection of samples is difficult in practical situation from a furious case of rabies and also because the negative result of the diagnostic test does not rule out the possibility of infection.
For intra-vitam diagnosis, saliva, cerebrospinal fluid (CSF), skin biopsy and serum are commonly collected. Collection of multiple saliva samples (3 no.) at the interval of 3 6 hours increases the sensitivity. Brain tissue though considered as the best sample, is usually not recommended for intra-vitam diagnosis. Samples are stored at –20°C.
For confirmation of infection postmortem, brain tissue is the most preferred sample. The tissue can be collected through cribriform plate at the bedside or in the field situation. Skin biopsy from nape of the neck also can be collected. Tissue samples can be preserved in glycerol and stored at –20°C. When tissue samples have been preserved in formalin, they should be taken out from formalin and kept in absolute ethanol for subsequent molecular testing and antigen detection.
Rabies viral infection is commonly diagnosed by detection of virus or its components (i.e. antigen detection, viral RNA detection and virus isolation), or by detection of rabies virus antibody in the serum. Demonstration of negri body (intracytoplasmic rabies virus inclusion body in neuronal cells) in neurons by Seller’s stain is not considered these days because of its low sensitivity and availability of better alternative diagnostic tests.
Detection of rabies antigen: This can be done by various methods.
Direct fluorescent antibody testing (DFAT): The test is considered as the gold standard for rabies diagnosis. It is used for detection of rabies virus antigen in the brain tissue of the patient using polyclonal or monoclonal antibody. The fixed tissue or impression smear made on slide is incubated with the antirabies polyclonal antibody or broadly reactive monoclonal antibody tagged to fluorescein isothiocyanate. The test has 99% sensitivity and specificity, but the performance depends largely on the quality of the antirabies conjugate and expertise of the observer and not reliable in degraded sample.
Other antigen detection tests based on enzyme immune assay, rapid immunodiagnostic test and immunohistochemistry have been shown to be comparable with DFAT. These tests are rapid and does not require fluorescence microscope. But most of these tests are not available commercially and still under evaluation.
Detection of viral RNA: Presently the method is recommended for intra-vitam diagnosis. The test is applicable in all types of samples inclu ding tissues and fluids. Reverse transcriptase polymerase chain reaction (RT-PCR) and real time RT-PCR are the commonly used methods for viral RNA detection. Other molecular amplification techniques have also been developed. The methods are highly sensitive and specific for viral RNA detection in a stringent quality control lab.
Virus isolation: Rabies virus can be isolated in cell culture or animal inoculation methods. Mice inoculation test (MIT) is done by intracerebral inoculation into 3–4 weeks old mice which are observed for 28 days for development of symptoms. Suckling mice of < 3 days old can also be used when rapid results are required. Mice brain is harvested for detection of viral antigen or viral RNA detection. The sensitivity of MIT is comparable to that of cell culture. However, due to animal protection issues, it is recommended by World Organization for animal health to replace the method of animal inoculation by cell culture wherever possible.
The cell culture method uses mice neuro blastoma cell line. Unlike MIT which takes around 28 days, cell culture is a rapid and inexpensive method where result is available in 24–48 hours. The method is as sensitive as MIT.
Detection of antibody: Presence of rabies antibody in serum or CSF in a non-vaccinated individual gives an indirect evidence of infection. Antibody appears in serum or CSF only after 8–10 days of symptoms. The test is, therefore, not helpful for patient’s diagnosis as majority of the patients dies within 6–7 days of onset of symptoms.
PREVENTION
Rabies is a 100% preventable disease when vaccination and immunoglobulin are administered as per the recommendations of world health authorities such as WHO or CDC.
Types of vaccine: There are two types of vaccines available for rabies, nerve tissue vaccine and cell culture or embryonated egg derived vaccine.
Cell culture or embryonated egg-based vaccine: Rabies virus is propagated in different cell substrates, such as human diploid cell line (HDCV), Vero cells, primary chick embryo cell or embryonated duck eggs. After propagation, virus is harvested and then undergoes further steps of processing, such as concentration, purification, inactivation and lyophilization. All preparations are highly safe and effective, and chick embryo cell and Vero cell preparations are less expensive than HDCV.
Nerve tissue vaccine: These vaccines are prepared by propagating the virus in the nerve tissues of different animals (sheep, suckling mice) followed by different methods of inactivation. These vaccines are less immunogenic, and associated with severe adverse effects, require multiple doses. Because of its association with serious adverse reaction, since 1984 WHO has advised to stop the preparation and use of neural vaccine and to replace them with cell culture or embryonated egg-based vaccines.
Types of vaccination: Rabies vaccination is indicated in two types of situations—pre exposure prophylaxis and post-exposure prophylaxis.
Pre-exposure prophylaxis: Pre-exposure vaccination is indicated in laboratory personnel working with rabies virus, animal handlers or persons coming in contact with animals, individuals travelling to or living in the area at risk.
Post-exposure prophylaxis: Post-exposure vaccination is indicated in persons who are bitten by any suspected rabid animal. Vaccination is advised when there is breach in the skin at the site of animal bite (category II and III of animal exposure), whereas in category I there is no breach in the skin and vaccination is not recommended. Category II which includes minor abrasion is advised for vaccination and category III which includes single or multiple bites is advised for vaccination along with immunoglobulin.
Vaccination schedule: The types of vaccine and route of administration are same for both pre-exposure and post-exposure prophylaxis. The numbers of doses are more with post exposure than pre-exposure prophylaxis (4/ 5 as compared to 3).
Vaccine can be given either intramuscularly or intradermal route following the same schedule. The dose of vaccine by IM route is 0.5 mL or 1 mL, and 0.1 mL by ID (Table 2).
Table2. Dose schedule of rabies prophylaxis
Immunoglobulin: Rabies immunoglobulin (RIG) is recommended in category III exposure and also in severely immunocompromised patients. RIG is available in 3 preparations— human RIG (HRIG), equine RIG (ERIG) and highly purified F(ab¢) 2 fragments of equine RIG.
RIG is administered just before or shortly after the first dose of vaccination and given at the site of animal bite and can be given within 7 days after the initiation of the primary series of vaccination.
The dose is 20 IU/kg body weight for HRIG and 40 IU/kg body weight for ERIG or F(ab¢) 2 fragments products. Maximum possible amount of RIG should be administered into or around the wound site and the remaining amount is given intramuscularly at the site distant from the vaccination site.
Novel approaches like genetically engineered live attenuated vaccines and cocktails of monoclonal antibodies are under different phases of trial.
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