Rabies

In this presentation you will find about the zoonotic potential of rabies virus, its impact in terms of DALYs.
Epidemiology and geographical distribution of rabies.
You will learn about the reservoir and source of rabies, transmission of rabies virus.
You will also learn about the virology of rabies virus inculding its family, genus, its structure, its different protein and the replication cycle of rabies virus.
It will also put light on the pathogenesis of rabies virus and different stages of rabies virus infection.
Then it will discuss about the laboratory diagnosis of rabies virus infection in humans as well as in animals including specimen collection, culturing, microscopy, animal inoculation and molecular diagnosis.
After that, you will get information about the prevention and control of rabies and different successful control strategies adopted by several countries of the world.
In the end it will discuss the status of rabies in Pakistan and specially WHO responses to rabies control in pakistan.

1. PRESENTED BY: HAFIZ MUHAMMAD MOAVIA UNIVERSITY OF VETERINARY AND ANIMAL SCIENCES LAHORE, PAKISTAN

2. INTRODUCTION Rabies is a viral zoonotic disease. Responsible for an estimated 59 000 human deaths and over 3.7 million disability-adjusted life years *(DALYs) lost every year. Rabies is the most fatal infection in humans and is almost invariably fatal once clinical signs occur. Most cases occur in Africa and Asia, with approximately 40% of cases in children aged <15 years. Rabies virus is the most important member of the Rhabdoviridae family. Rabies virus causes rabies, a viral infection of the central and peripheral nervous systems that causes encephalitis with or without paralysis. No specific antirabies agents are useful, once clinical signs or symptoms develop. * The overall burden of disease is assessed using the disability-adjusted life year (DALY), a  time-based measure  that combines years of life lost due to premature mortality and years of life lost due to time lived in states of less than full health, or years of healthy life lost due to disability (YLDs).

3. Sign and Symptoms

4. EPIDEMIOLOGY Geographical distribution: Rabies is prevalent throughout the world except in Islands. The risk of rabies is highest in countries with hyperendemic canine rabies, including most of Asia, Africa, and Latin America. According to World Health Organization (WHO), a country that has no record of indigenously acquired case of human or animal rabies within two years period due to surveillance and import regulations can claim rabies free status. The countries free from rabies in Asian subcontinent are Bahrain, Cyprus, Hong Kong, Japan, Malaysia, Maldives, Qatar, Singapore.

5. The RABV circulates with two epidemiological cycles, which are interrelated i.e. urban and sylvatic cycle. Urbans cycle having mainly pet dogs, cats and in sylvatic cycle wild mammals like fox, raccoon, jackal, wolf, badger, mongoose and bats, etc., as vectors/reservoirs, respectively. Both cycles may overlap in some geographical situations. Mainly community and stray dog population maintains the urban cycle and spill-over to pet dogs creates additional burden to human with a risk of rabies.

6. Risk factors Stray Dog Population Urbanization Deforestation Dog trade (catching, handling, loading, holding, transportation) Dog slaughterhouses (un-hygienic practices and unorganized slaughter houses---dissemination of body parts and by-products to far off places).

7. Reservoir and source of Rabies Dogs are the important reservoir of infection. Other animal reservoirs include bats, raccoons, skunks, wolves, jackals, foxes, cats, ferrets, opossums, fowl, etc. The virus is excreted in saliva of infected dogs, foxes, wolves, jackals, vampire bats, raccoons, and skunks. The virus is found in the salivary gland of these infected animals. Infected saliva or infected CNS tissue, including corneal transplants in humans, are the sources of infection.

8. Transmission of Rabies in humans The virus is transmitted to humans by following ways: ■ Bite of a rabid dog or other infected animal ■ Contact of saliva with broken skin or with mucous membranes, exposure to aerosolized secretions from an infected animal(e.g., bat). ■ Corneal transplants: The only documented cases of rabies caused by human-to-human transmission occurred in eight recipients of transplanted corneas. Currently, donated corneas are not accepted if the donor died from encephalitis that may be consistent with rabies.

9. Family and Genus The Rabies virus belongs to Rhabdoviridae family of RNA viruses. The family Rhabdoviridae consists of more than 185 different viruses isolated from both plants and animals. Currently, animal rhabdoviruses include four genera: Lyssavirus Vesiculovirus Ephemerovirus Novirhabdovirus The rabies virus is the prototypical human Lyssavirus pathogen.

10. Structure Bullet-shaped virus It is a negative-sense Non-segmented genome Single-stranded RNA virus The nucleocapsid shows helical symmetry Approximately 100 to 430 nm long and 45 to 100 nm in diameter

11. Proteins It consists of an outer envelope (E Protein), a lipid-containing bilayer covered with transmembrane glycoprotein (G protein) spikes. It mediates the attachment of virus to the acetylcholine receptors of neural tissues. Viral matrix proteins (M Protein) are structural proteins linking the viral envelope with the virus core. They play a crucial role in virus assembly, and interact with the RNP complex.  An internal protein core or nucleoprotein(N Protein) encapsidate the nucleic acid with each molecule of N protein covering nine bases. L protein is responsible for the virion-associated RNA polymerase activity and associated to all activities related to RNA synthesis. P protein is responsible for binding L protein to the N protein–RNA template and inhibits host cell’s interferon production.

12. Replication

13. Pathogenesis The virus may enter the peripheral nervous system directly at the site of bite. In some cases, however, it may replicate in muscle tissue after entering the host. The virus infects the sensory neurons and moves rapidly by axonal transport centripetally to the central nervous system (CNS) for replication. During its transport within the neurons, it is protected from the host immune system. The virus travels along the axons at a rate of 12–24 mm in a day to enter the spinal ganglion. Its multiplication in the ganglion is indicated by the onset of pain or paresthesia at the site of the inoculum, which are the first clinical symptom and a hallmark finding. From here, the virus spreads quickly, into the CNS, and the spread is marked by rapidly progressive encephalitis. Thereafter, the virus spreads to the periphery and salivary glands. During the course of infection, encephalitis develops, associated with the death of neurons and demyelination. Acidophilic intracytoplasmic neuronal inclusion bodies are found in infected neurons, which is important for laboratory diagnosis

14. Stages of Rabies In general, four stages of rabies are recognized in humans. Incubation period Prodromal period Acute neurologic period Coma which subsequently lead to death

15. Incubation Period The average period of incubation is 20–90 days and can be as long as six months. During the incubation period; The virus travels from peripheral areas to the CNS. The patients remain asymptomatic during the period. Incubation period is influenced by various factors including host species, virus strain, the amount of inoculum and the site of introduction of the virus. The rabies virus is protected from the immune system during this period and no antibody response is observed.

16. Prodormal Period The virus enters the CNS during the prodromal period. The duration of this period is 2–10 days. The period is characterized by nonspecific symptoms and sign. Paresthesia or pain develops at the inoculation site and is pathognomonic for rabies. Other symptoms may include malaise, anorexia, headache, fever, chills, pharyngitis, nausea, emesis, diarrhea, anxiety, agitation, insomnia, and depression

17. Acute neurologic period This period is associated with objective signs of developing CNS disease. The duration is 2–7 days. Furthermore, it presents with the following conditions: Furious rabies: Patients develop agitation, hyperactivity, restlessness, thrashing, biting, confusion, or hallucinations. After several hours to days, this becomes episodic and interspersed with calm, cooperative, lucid periods. Furious episodes last for less than 5 minutes. Episodes may be triggered by visual, auditory, or tactile stimuli, or they may be spontaneous. Seizures may occur. Autonomic instability is observed, including fever, tachycardia, hypertension, hyperventilation, drooling, anisocoria, mydriasis, lacrimation, salivation, perspiration, and postural hypotension. Other neurologic signs include cranial nerve involvement with diplopia, facial palsy, and optic neuritis. This phase may either end in cardiorespiratory arrest or progress to paralysis.

18. Paralytic rabies: It is also known as dumb rabies or apathetic rabies, because the patient is relatively quiet compared to a person with the furious form. Paralysis develops from the outset. Fever, headache, and nuchal rigidity are prominent. Paralysis is symmetric and may be either generalized or ascending and may be mistaken for Guillain–Barré syndrome. Calmness and clarity gradually deteriorates to delirium, stupor, and then coma.

19. Coma The patient may go into coma within 10 days of onset; but duration is variable. Coma leads to respiratory failure within a week of neurologic symptoms. Hypoventilation and metabolic acidosis predominate. Acute respiratory distress syndrome is common. Without intensive supportive care, respiratory depression, arrest, and death occur shortly after coma. A few reports indicate that those patients who survived had pre-exposure or postexposure prophylaxis supported by most advanced life-support system.

20. Laboratory Diagnosis in Humans

21. Laboratory Diagnosis in Humans Specimens: Saliva, serum, cerebrospinal fluid (CSF), blood, urine, and skin and brain biopsy are the frequently used specimens for diagnosis of rabies. Microscopy: Demonstration of Negri bodies by microscopy is the characteristic histopathological feature of rabies. Negri bodies are demonstrated in 80% of human cases of rabies. Therefore, failure to demonstrate Negri bodies in neural tissue does not rule out the diagnosis of rabies. Viral antigens can be demonstrated in the corneal smear, skin biopsy collected from the face or neck, and saliva (antemortem) or in the brain tissue (postmortem) for diagnosis of rabies

22. Direct fluorescent antibody technique(DFAT) test using specific monoclonal antibodies is a specific method to demonstrate rabies antigen in clinical specimens. Isolation of Virus: Viruses can be isolated from brain tissue, CSF, saliva, and urine by culture in cell lines or in animals. Culture : Isolation of virus in cell lines (WI-38, BHK-21, and CER) is a sensitive method. demonstration of viral antigen within 2–4 days. The DFA test using monoclonal rabies antibodies tagged with fluorescein isothiocyanate. Animal inoculation: Mouse is the animal of choice. CSF, saliva, and urine are inoculated intracerebrally. After death of the mice or after 28 days of inoculation, the brain tissues are examined for the presence of Negri bodies by microscopy or for viral antigen by the DFA test.

23. Molecular Diagnosis: Polymerase chain reaction is being increasingly evaluated for the diagnosis of rabies. The nucleic acid sequence-based amplification (NASBA) on saliva and CSF can be used for rapid diagnosis as early as 2 days after symptom onset

24. Laboratory Diagnosis in Animals

25. Laboratory Diagnosis in Animals In endemic areas, suspect domestic carnivores which have bitten humans should be isolated and observed for up to 14 days. The brains of animals which develop clinical signs should be examined for the presence of virus. Rapid laboratory confirmation is essential for the implementation of appropriate treatment of human patients. Non-suppurative encephalitis characterized by perivascular lymphoid cuffing and intracytoplasmic inclusions (Negri bodies) may be demonstrable histologically. The direct fluorescent antibody test (FAT), which provides a rapid and specific diagnosis, may yield false- negative results with autolysed brain specimens. The conjugated antisera usually used for diagnosis are specific for rabies virus (serotype 1). Rabies virus can be cultured in neuroblastoma cells or in baby hamster kidney cells. Rabies virus, which is non-cytopathic, can be detected in tissue culture using conjugated antisera. Reverse transcriptase polymerase chain reaction (RT- PCR) has been used to detect viral RNA in brain samples. The sensitivity of RT- PCR can be enhanced by combining the technique with ELISA which aids detection of amplified product

26. Prevention and Control

27. Prevention and Control Pre-exposure prophylaxis: PrEP is recommended for individuals at higher risk due to occupation, such as dog handlers, other animal handlers, and veterinarians. PrEP should be considered in sub-populations living in remote, rabies endemic areas, where access to PEP is difficult, the dog bite incidence is >5% per year or vampire bat rabies is known to be present. For immunologically naive individuals of all age groups WHO recommends the following PrEP schedules: a 2 sites ID or a 1-site IM vaccine administration on days 0 and 7. A routine PrEP booster or serology for neutralizing antibody titres is recommended only if a continued, high risk of rabies exposure remains. Individuals with documented immunodeficiency should be evaluated on a case-by-case basis and best receive an ID or IM PrEP schedule as above, plus a third vaccine administration between days 21 to 28. Additionally, in the event of an exposure, a complete PEP course, including RIG, is recommended.

28. Postexposure Prophylaxis Postexposure prophylaxis is started immediately after exposure to infection. After exposure to possibly infected dog or other rabid animals, immediate preventive actions are taken up, which consist of Local treatment Confirmation whether or not the animal is rabid Administration of hyperimmune serum Anti-rabies vaccine

29. Local treatment: This involves prompt cleaning of the wound. The wound should be immediately scrubbed with soap and water followed by treatment with quaternary ammonium compounds, tincture, or aqueous solution of iodine or alcohol. Confirmation whether or not the animal is rabid: This can be made by clinical observation of suspected dog. If dog is still healthy 10 days after biting human, rabies is extremely unlikely. Diagnosis can also be made by demonstration of the Negri bodies in brain tissues at autopsy or viral antigens in the brain tissue or saliva.

30. Administration of hyperimmune serum: RIG provides passive immunization and is administered only once, as soon as possible after the initiation of PEP and not beyond day 7 after the first dose of vaccine. It is carried out by administering purified equine rabies immune globulin (ERIG) and human rabies immune globulin (HRIG). WHO recommends the following: The maximum dose is 20 IU (HRIG) and 40 IU (ERIG) per kg body weight. Infiltrate as much as possible into the wound; the remainder of the calculated dose of RIG does not need to be injected IM at a distance from the wound. Antirabies vaccine: Rabies is the only disease where postexposure vaccination is employed extensively and successfully. This is due to long incubation period of the disease. The chances of preventing the rabies are more when vaccination is given to humans as early as possible after exposure.

31. WHO Rabies Exposure Categories Category I touching or feeding animals, animal licks on intact skin (no exposure) Category II nibbling of uncovered skin, minor scratches or abrasions without bleeding (exposure) Category III single or multiple transdermal bites or scratches, contamination of mucous membrane or broken skin with saliva from animal licks, exposures due to direct contact with bats (severe exposure).

32. PEP Recommendations By Category Of Exposure

33. How different countries have been able to control rabies successfully America have reduced canine rabies by over 95% in humans and 98% in dogs. Australia and UK have also been able to eradicate canine rabies through: Dog vaccination campaigns Raising public awareness Widespread availability of post exposure prophylaxis (PEP). Strict continuous monitoring. Quarantine of imported animals. Regulations to avoid the entrance of virus particularly with the import or introduction of infected animals.

34. Rabies in Pakistan In 2010, more than 97 000 recorded cases of dog bites were reported by basic health units alone. Those managed by secondary and tertiary care facilities, private practitioners and spiritual healers and  hakims  are not recorded. Research has revealed that most of the population are either unaware of the risk of rabies when bitten by rabid dogs, or do not seek the right treatment for its prevention. Other weaknesses in the control of rabies in Pakistan include: Lack of a surveillance system Limited access to up-to-date rabies vaccines and immunoglobulin Inadequate resources and political support Weak collaboration between different government departments and sectors including the health authorities, livestock and veterinary science authorities and local government.

35. WHO response to rabies in Pakistan The main features of WHO's strategy for rabies control are: Mass awareness on rabies transmission, prevention and self-protection using cost-effective methods such as local government and community communication structures Establishment of rabies treatment centres at each district headquarters hospital where health-care providers will be trained on management and treatment of dog bites with anti-rabies vaccine Ensuring the most cost-effective and efficacious anti-rabies vaccines in designated rabies treatment centres in all districts Enactment and enforcement of laws relating to vaccination of pet animals , such as dogs and cats, in order to decrease rabies incidence among them Creation of a mechanism to decrease the stray dog population in districts through the use of modern methods such as bait vaccination and dog elimination. Development of  a surveillance system to monitor dog bites, dog rabies and human rabies through the use of the existing death reporting system under local government structures Close collaboration between the three government departments involved in the control of rabies Research on animal rabies and the development of animal and human rabies vaccines by the university of veterinary sciences in lahore .