Rabies- Lab diagnosis & Prophylaxis

 Lab diagnosis of Rabies:

Human Rabies

-Was of little practical importance till recently- death was considered inevitable.

-Survival was shown possible in rare instances

Diagnosis

A clinical diagnosis of hydrophobia can be made on the basis of history of bite by a rabid animal and characteristic signs and symptoms.

Laboratory diagnosis

Rabies can be confirmed in patients early in the illness by antigen detection using immunofluorescence of skin biopsy, and by virus isolation from saliva and other secretions

1. Demonstration of rabies virus antigens by Immunofluorescence

Specimens: Corneal smears, skin biopsy, (face/neck), saliva -(antemortem) and brain - salivary gland, brain stem, hippocampus, cerebellum - (post mortem)

Direct IF done using anti-rabies serum tagged with fluorescence isothiocyanate.

2. Post-mortem diagnosis by demonstration of Negri bodies in the brain (may be absent in 20% cases)

3. Isolation of Virus by intracerebral inoculation in mice- from brain, CSF, saliva and urine- more chance of isolation early in disease- few days after onset, neutralsiing antibodies appear- inoculated mice examined for signs of illness- brains checked for Negri bodies or by IF

4. Isolation of Virus in tissue culture cell lines (WI 38, BHK 21, CER) – Minimal CPE- virus identified by IF, +ve IF obtained as early as 2-4 days after inoculation

5. High titre antibodies present in CSF in rabies, but not after immunisation- important for diagnosis

6. Detection of rabies virus RNA by RT-PCR- sensitive method, particularly when the sample is small (e.g., saliva) or when large numbers of samples must be tested in an outbreak or epidemiological survey.

Other tests to detect the virus include immunohistochemistry and enzyme-linked immunosorbent assays (ELISAs).

Animal Rabies

Lab diagnosis of rabies in dogs and other biting animals important- to determine risk of infection and to decide post exposure treatment.

In Rabies endemic area, animals captured should be sent for laboratory confirmation of Rabies but without any delay, post exposure treatment of the bitten person should be done.

Domesticated dogs and cats, particularly if previously vaccinated against Rabies should be observed in isolation for upto 10-14 days. If they survive for that time, it is unlikely they were incubating rabies virus at the time of incident. If they succumb or die, anti-rabies treatment of bitten person should be started.

1.     Microscopy/ Histological examination:

This involves the examination of tissue infected with rabies virus rapidly and accurately.

Whole carcass/severed head sent to lab. Brain removed and made into two portions- in 50% glycerol saline (biological test) and 1 in Zenker’s fixative (microscopy). Should include Hippocampus and cerebellum- contain abundant number of Negri bodies.

A definite pathological diagnosis is based on the finding of Negri bodies in the brain or spinal cord. Negri bodies are found in impression preparation or histological sections.

Impression smear- a sample of cells, microorganisms or fluids obtained by pressing against the surface of a specimen, which may be excised tissue or in situ

-Demonstration of Inclusion Bodies-

• Impression preparation of brain and cornea tissue is often used.

- Brain impression smears stained by Seller’s technique (Basic fuchsin and Methylene Blue in Methanol) – Negri bodies seen as intracytoplasmic, round/oval, purplish pink structures with characteristic basophilic inner granules- vary in size.

·       If impression smears are negative, tissue should be sectioned and stained by Giemsa or Mann’s method.

2. Demonstration of Rabies virus antigen by IF- more sensitive

3. Isolation of Rabies virus- as in human rabies diagnosis

Treatment

There is no specific treatment for rabies, once the clinical signs appear.

Case management includes the following procedure:

(a) The patient should be isolated in a quiet room protected as far as possible from external stimuli such as bright light, noise or cold draughts which may precipitate spasms or convulsions.

(b) Relieve anxiety and pain by liberal use of sedatives.

(d) Ensure hydration and diuresis.

(e) Intensive therapy in the form of respiratory and cardiac support may be given.

 • Patients with rabies are potentially infectious because the virus may be present in the saliva, vomits, tears, urine or other body fluids.

• Nursing personnel attending rabid patients should be warned against possible risk of contamination and should wear face masks, gloves, goggles and aprons to protect themselves. 

• Persons having cuts or open wounds should not look after the patient.

•Where human cases of rabies are encountered frequently, pre-exposure prophylaxis is recommended.

 Prevention

1. Animal Rabies

Rabies can be prevented in domesticated animals by vaccination and by the avoidance of contact with rabid wild animals. Rabies vaccines are available for dogs, cats, cattle, sheep and horses.

Both inactivated and modified live vaccines are effective, but rare cases of post-vaccinal rabies have been reported with the modified live vaccines in dogs and cats.

Preventing animals from roaming will reduce the risk of exposure to rabid wild animals. To protect pet rabbits and rodents, they should be housed indoors, and watched closely if they are allowed outside to exercise.

 2. Human Rabies

a. Post-exposure prophylaxis. b. Pre-exposure prophylaxis.

 Post-Exposure Prophylaxis

The aim of post-exposure prophylaxis is to neutralize the inoculated virus before it can enter the nervous system.

1. Local treatment of wound: The purpose of local treatment is to remove as much virus as possible from the site of inoculation before it can be absorbed on nerve endings.

• Local treatment of wounds is to be done immediately after exposure; local wound treatment can reduce the chances of developing rabies by up to 80%.

 The local treatment comprises the following measures:

(a) Cleansing: Immediate flushing and washing the wound(s), scratches and the adjoining areas with plenty of soap and water, preferably under a running tap, for at least 15 minutes

• If soap is not available, simple flushing of the wounds with plenty of water should be done as first-aid.

(b) Chemical treatment: Residual virus should be inactivated by irrigation with virucidal agents either alcohol (400-700 ml/litre), tincture or 0.01 % aqueous solution of iodine or Betadine.

(c) Suturing: Bite wounds should not be immediately sutured to prevent additional trauma which may help spread the virus into deeper tissues. If suturing is necessary, it should done 24-48 hours later, applying minimum possible stitches, under the cover of rabies immunoglobulin locally.

(d)Antibiotics and anti-tetanus measure: The application of antibiotics and anti-tetanus procedures when indicated should follow the local treatment recommended above. The use of any local applicant or irritant like turmeric, red chilli, lime etc. should be discouraged and avoided.

 

2. Active Immunisation

• Tissue Culture Vaccines

Human Diploid Cell (HDC) Vaccine

1.     First cell culture vaccine- developed by Koprowski, Wiktor and Plotkin- purified and concentrated preparation of rabies virus (Pitman- Moore strain) grown on human dipoid cells (WI 38 or MRC 5), inactivated with beta propiolactone or tri-n-butyl phosphate- highly antigenic- free from side effects; but has high cost.

2.     Continuous cell culture vaccines grown on the Vero cell line from monkey kidneys

In India, the rabies vaccines available are,

1)     Purified Vero cell rabies vaccine (PVRV)

2)     Chromatographically purified Rabies Vaccine (CPRV)

3)     Human Diploid Cell Vaccine (HDC)

4)     Purified Chick Embryo Cell Vaccine (PCECV) 

5)     Purified Duck Embryo Vaccine (PDEV) 

• Subunit Vaccine

The Glycoprotein subunit on the virus surface (protective antigen), cloned and recombinant vaccines produced.

     

Neural vaccines are poor immunogens, contain mostly nucleocapsid antigen- may contain infectious agents and can be encephalitogenic. They are abandoned in most places now, because of the availability of tissue culture vaccines, at affordable price.

 

Vaccination Schedules

Antirabic vaccine is administered when a person is bitten, scratched or licked by a rabid animal. The animal should be observed for 10 days, if possible. Virus may be present in the saliva for 3-4 days, before the onset of symptoms and the animal usually dies within 5-6 days of developing the disease.

If the animal remains healthy after this period, there is no risk of rabies or vaccination, if already started it may be discontinued.  

WHO guidelines on post-exposure prophylaxis are based on the risk category to which the patient belongs.

 Categories of contact and recommended post-exposure prophylaxis

 

Categories	Type of contact with suspect rabid animal	Type of exposure	Post–exposure measures
Category I	Touching or feeding animals, licks on intact skin	None	None
Category II	Nibbling of uncovered skin, minor scratches or abrasions without bleeding	Minor	Immediate vaccination (ARV) and local treatment of the wound. Stop treatment if animal remains healthy throughout an observation period of 10 days or if proven negative for rabies by a reliable laboratory.
Category III	Single or multiple transdermal bites or scratches, licks on broken skin, contamination of mucus membrane with saliva from licks, contact with bats	Severe exposure	Immediate vaccination (ARV) and administration of rabies immunoglobulin, local treatment of the wound.

 

All three cell culture vaccines available in India (HDC- Human diploid cell vaccine, PCECV - Purified chick embryo cell vaccine, PVRV- Purified vero-cell rabies vaccine) have the same dosage schedule, both for children and adults.

It involves the injection of 0.1 ml of reconstituted vaccine per site and on two sites per visit (one on each deltoid area) on days 0, 3, 7 and 28. Day 0 is the date of the first dose of administration and not the date of exposure/ animal bite.

The vaccine is to be given IM or SC in the deltoid region, or, in the children, on the anterolateral aspect of thigh.

 

3.     Passive Immunization with antirabies serum

Done by administration of human rabies immunoglobulin (HRIG) pooled from the sera of immunized human donors. Rabies immunoglobulin from the horses (ERIG), was also used, but not generally preferred now due to hypersensitivity reactions. Purified ERIG is much safer, but not completely free from risk. HRIG, though limited in availability and more expensive, is preferred over ERIG, but should be free from HIV and hepatitis viruses.

Administration of Immunoglobulins

All of the rabies immunoglobulin (calculated dose), or as much as anatomically possible should be administered into and around the wound site or sites. The remaining immunoglobulin, if any, after all wound infiltrated, should be administered by deep i/m injection at an injection site distant from the vaccine injection site. Rabies immunoglobulin may be diluted to a volume sufficient (2-3 fold) for all wounds to be effectively and safely infiltrated.

Rabies immunoglobulin for passive immunization is administered only once. Beyond the seventh day after the first dose of ARV, rabies immunoglobulin is not indicated.

Pre exposure prophylaxis:

PEP is recommended for anyone who is at continual, frequent or increased risk of exposure to the rabies virus for example laboratory worker dealing with rabies virus, animal handlers, veterinarians.