c




c



Rabies is caused by a neurotropic virus of the genus À


of the family
Rhabdoviridaeand is transmissible to all mammals. As it is transmissible to humans by
inoculation or inhalation of infectious virus, all suspected infected material must be handled
under the appropriate safety conditions specified by the World Health Organisation (WHO)
(37).
Seven distinct genetic lineages can be distinguished within the genus À


by cross-
protection tests and molecular biological analysis (5, 14, 21), namely the classical rabies virus
itself (RABV, genotype 1, serotype 1), Lagos bat virus (LBV, genotype 2, serotype 2), Mokola
virus (MOKV, genotype 3, serotype 3), and Duvenhage virus (DUUV, genotype 4, serotype 4).
The European bat lyssaviruses (EBLV), subdivided into two biotypes (EBLV1, genotype 5 and
EBLV2, genotype 6) and the Australian bat lyssavirus (ABLV, genotype 7), recently isolated in
Australia (24), are also members of the À


genus, but are not yet classified into

 



Clinical observation may only lead to a suspicion of rabies because signs of the disease are
not characteristic and may vary greatly from one animal to another (36). The only way to
perform a reliable diagnosis of rabies is to identify the virus or some of its specific
components using laboratory tests.
As rabies virus is rapidly inactivated, refrigerated diagnostic specimens should be sent to
the laboratory by the fastest means available. Shipment conditions must be considered to be
part of the 'rabies diagnostic chain'.
Several laboratory techniques may be used, and have been detailed and standardised in
the fourth edition of the WHO'sÀ
 


 

(37). The methods vary in
serotypes. Viruses of serotypes 2-4, EBLV and ABLV are known as rabies-related viruses. The
use of monoclonal antibodies (MAbs) directed against viral nucleocapsid or glycoprotein
antigens, and the sequencing of defined genomic areas has made possible the definition of
numerous subtypes within each serotype. Lyssaviruses cause a clinical disease
indistinguishable from classical rabies. Conserved antigenic sites on the nucleocapsid
proteins permit recognition of all lyssaviruses with modern commercial preparations of anti-
rabies antibody conjugates used for diagnostic tests on brain tissue. For RABV, DUUV, EBLV
and ABLV, conserved antigenic sites on the surface glycoproteins allow cross-neutralisation
and cross-protective immunity to be elicited by rabies vaccination. Little or no cross-
protection against infection with MOKV or LBV is elicited by rabies vaccination and most anti-
rabies virus antisera do not neutralise these lyssaviruses.
their efficiency, specificity and reliability. They are classically applied to brain tissue, but they
can also be applied, though less effectively, to other organs (e.g. salivary glands). In the brain,
rabies virus is particularly abundant in the thalamus, pons and medulla. The hippocampus
(Ammon's horn), cerebellum and different parts of the cerebrum have been reported to be
negative in 3.9-11.1% of the positive brains. The structure of choice is the thalamus as it was
positive in all cases. It is recommended that a pool of brain tissues that includes the brain
stem should be collected and tested (12). To reach these parts of the brain, it is necessary to
remove the entire organ after having opened the skull in a necropsy room. Under some
conditions (e.g. in the field or when sampling for large epidemiological studies), a simplified
method of sampling through the occipital foramen (11), or through the orbital cavity (26),
can be used.

Humans working with suspect material must be vaccinated against lyssaviruses or other  

 
pathogens that may be present in diagnostic samples. The laboratory must comply with
national biocontainment and biosafety regulations to protect staff from contact with During the shipment of suspect material for diagnosis (animal heads, brain or other
pathogens; it should also comply with the guidelines in Chapter I.1.6. Human safety in the tissue samples), no risk of human contamination should arise: brains must be placed in a
veterinary microbiology laboratory. leak-proof rigid container (animal heads will be wrapped in absorbent material) as prescribed
in the International Air Transport Association (IATA) Dangerous Goods Regulations must be
WHO recommends the preventive immunisation of exposed staff. The immunisation protocol followed. These regulations are summarised in Chapter I.1.1. Sampling methods.
includes three injections, e.g. at days 0, 7, and 28. The serological evaluation of immunisation
is made 1-3 weeks after the last injection, and checked every 6 months in the case of When it is not possible to send refrigerated samples, other preservation techniques
laboratory workers or every 2 years for other diagnosticians. Booster vaccination must be may be used. The choice of the preservative is closely linked to the tests to be used for
given when the titre falls below 0.5 International Units (IU) per ml. In the absence of diagnosis:
serological monitoring, the vaccination regimen should consist of a booster vaccination at 1
year and thereafter every 1-3 years. Formalin inactivates the virus, thus the isolation tests cannot be used and diagnosis
depends on using a modified and less sensitive direct fluorescent antibody test (FAT),
As no clinical sign or gross post-mortem lesion can be considered pathognomonic in domestic immunohistochemistry or histology (33, 37);
or wild animals, the diagnosis of rabies has to rely on laboratory testing. Serological evidence
of infection is rarely useful because of late seroconversion and the high mortality rate of host Infectivity at room temperature may be extended for several days if brain material is
species, although such data may be used in some epidemiological surveys. kept in a mixture of 50% glycerol in phosphate buffered saline (PBS). Glycerol/PBS slows
bacterial action and therefore protects against the chemical and biological effects of

putrefaction. It does not protect against titre decline due to thermal conditions and

therefore, because rabies is thermo-labile, the virus titre will decline during glycerol/PBS

storage. Under normal transport conditions in the tropics, this protection may only be

effective for a matter of several days. Therefore, whenever possible samples in
glycerol/saline should be kept refrigerated. As the virus is not inactivated by glycerol/PBS, all
laboratory tests can be used on these samples.
à  

  been inoculated for diagnosis. The FAT gives reliable results on fresh specimens within a few
Usually the brain is collected following the opening of the skull in a necropsy room, and
the appropriate samples are collected. This step may be hazardous if laboratory technicians
are not fully trained, or under field conditions. In such cases, there are two possible methods
of collecting some brain samples without opening the skull:

!
!"
!
à!
 
A 5 mm drinking straw (11) or a 2 ml disposable plastic pipette (16) is introduced into
the occipital foramen in the direction of an eye. Samples can be collected from the rachidian
bulb, the base of the cerebellum, hippocampus, cortex, and medulla oblongata. Bovine
spongiform encephalopathy (BSE) should be considered in the differential diagnosis of most
cattle that are considered to be 'rabies suspect'. Sampling of brain specimens for both
diseases can be done using the 'brain scoop or tool' developed for BSE tissue sampling rather
than a straw or pipette. The resulting samples are relatively easily recognised as to the area
of brain sampled.
!#!à
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à!
 
In this technique (26), a trocar is used to make a hole in the posterior wall of the eye

 



socket, and a plastic pipette is then introduced through this hole. The sampled parts of the
brain are the same as in the former technique, but they are taken in the opposite direction.
 "
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 same sensitivity as FAT (22), but attention should be paid to the risk of nonspecific false-
Laboratory diagnosis can be performed by using three kinds of procedure.
 


!!


Negri bodies correspond to the aggregation of viral proteins, but the classical staining
techniques detect only an affinity of these structures for acidophilic stains.
Immunohistochemical tests are the only histological test specific to rabies.
An unfixed tissue smear may be stained by the Seller's method, diagnosis is then
obtained in under 1 hour. Generally, histological tests, such as Mann's test, are performed on
fixed material after a paraffin-embedding step, and the result of the test is obtained within
3days. These techniques have the advantage that the laboratory equipment needed to
perform them is inexpensive and any need to keep specimens cold after fixation is avoided.
Whichever staining method is used, the evidence of infection is provided by intracytoplasmic
acidophilic bodies. These histological methods, especially the Seller's method, can no longer
be recommended because they have very low sensitivity and should be abandoned.



 



"


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i)  




 

 inoculated intracerebrally. It is recommended, though not strictly essential, to use specific
The most widely used test for rabies diagnosis is the FAT, which is recommended by
both WHO and OIE. This test may be used directly on a smear, and can also be used to
confirm the presence of rabies antigen in cell culture or in brain tissue of mice that have
hours in more than 95-99% of cases. The sensitivity of the FAT depends on the specimen (the
degree of autolysis and how comprehensively the brain is sampled, see Section B.1.) (1, 9),
on the type of lyssavirus and on the proficiency of the diagnostic staff. Sensitivity may be
lower in samples from vaccinated animals due to localisation of antigen, which is confined to
the brainstem. For direct rabies diagnosis, smears prepared from a composite sample of
brain tissue, that includes the brain stem, are fixed in high-grade cold acetone and then
stained with a drop of specific conjugate. Anti-rabies fluorescent conjugates may be
prepared in the laboratory. Those available commercially are either polyclonal conjugates
specific to the entire virus or specific to the rabies nucleocapisid protein, or they may be
prepared from a mix of different MAbs. In the FAT, the specific aggregates of nucleocapsid
protein are identified by their fluorescence. The specificity and sensitivity of these anti-rabies
fluorescent conjugates for locally predominant virus variants should be checked before use.
The FAT may be applied to glycerol-preserved specimens. If the specimen has been
preserved in a formalin solution, the FAT may be used only after the specimen has been
treated with a proteolytic enzyme (6, 7, 32, 33). However, the FAT on formalin-fixed and
digested samples is always less reliable and more cumbersome than when performed on
fresh tissue.
ii) 
The antibody may be conjugated to an enzyme such as peroxidase instead of
fluorescein isothiocyanate (FITC). This conjugate may be used for direct diagnosis with the
positive results. This risk is considerably reduced by the thorough training of the technicians.
It must also be emphasised that this technique needs one incubation step more than the
FAT.
Peroxidase conjugate may be used on sections of formalin-fixed tissue for
immunohistochemical tests.
An enzyme-linked immunosorbent assay (ELISA) that detects rabies antigen is one
variation of the immunochemical test. This rapid rabies enzyme immunodiagnosis test
(RREID) is available commercially (28). The correlation between the FAT and the RREID
ranges between 96% and 99% (8, 15). The 'routine' version of this test is not sensitive to
rabies-related viruses as RREID only detects genotype 1 lyssaviruses.
. 


! 

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These tests detect the infectivity of a tissue suspension in cell cultures or in laboratory
animals. They should be used if the FAT gives an uncertain result or when the FAT is negative
in the case of known human exposure.
i) u
Five-to-ten mice, 3-4 weeks old (12-14 g), or a litter of 2-day-old newborn mice, are
pathogen free (SPF) mice. The inoculum is the clarified supernatant of a 20% (w/v)
homogenate of brain material (cortex, Ammon's horn, cerebellum, medulla oblongata) in an samples, the use of an indirect ELISA with rabies glycoprotein-coated plates has been shown
isotonic buffered solution containing antibiotics. To reduce animal pain, mice should be to be as sensitive and specific as the VN test on cells (19).
anaesthetised when inoculated. The young adult mice are observed daily for 28 days, and
every dead mouse is examined for rabies using the FAT. For street fox rabies strains, deaths  '!"
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due to rabies generally begin 9 days post-inoculation. For faster results in newborn mice, it is )
!!à

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possible to check one baby mouse by FAT on days 5, 7, 9 and 11 post-inoculation.
The principle of the fluorescent antibody virus neutralisation (FAVN) test (18) is the
This 
test is quite expensive, particularly if SPF mice are used, and should be neutralisation 
of a constant amount of rabies virus ('challenge virus standard' [CVS]
avoided where possible. It does not give rapid results (compared with 
inoculation strain adapted to cell culture) before inoculating cells susceptible to rabies virus: BHK-21 C13
tests), but when the test is positive, a large amount of virus can be isolated from a single cells.
mouse brain for strain identification purposes. Another advantage of this low-tech test is that
it can be easily and practicably be applied in situations where skills and facilities for other The serum titre is the dilution at which 100% of the virus is neutralised in 50% of the
tests (e.g. cell culture) are not available. wells. This titre is expressed in IU/ml by comparing it with the neutralising dilution of a
standard serum under the same experimental conditions (OIE serum of dog origin or WHO
ii) 
 


 standard for rabies immunoglobulin [human] No. 2, or both). An internal control calibrated
Neuroblastoma cell lines, e.g. CCL-131 in the American Type Culture Collection against the international control may be used.
(ATCC: American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia
20110-2209, United States of America), is used for routine diagnosis of rabies. The cells are This microplate method uses 96-well plates, and is an adaptation of the technique of
grown in Dulbecco's modified Eagle's medium (DMEM) with 5% fetal calf serum (FCS), Smith

(30), modified by Zalan

(38) and by Perrin

(27). Several publications
incubated at 36°C with 5% CO2. Its sensitivity has been compared with that of baby hamster (17, 18) have shown that the FAVN test and the rapid fluorescent focus inhibition test (RFFIT)
kidney (BHK-21) cells (29). This cell line is sensitive to street isolates without any adaptation give equivalent results.
step, but should be checked for susceptibility to locally predominant virus variants before
use. Presence of rabies virus in the cells is revealed by the FAT. The result of the test is

*"
obtained after at least 18 hours (one replication cycle of virus in the cells); generally
incubation continues for 48 hours (10) or in some laboratories up to 4 days. Humidified incubator at 37°C with 5% CO2; dry incubator at 37°C; biocontainment
cabinet; fluorescence microscope suitable for FITC fluorescence equipped with x10 eye-piece
This test is as sensitive as the mouse inoculation test. Once a cell culture unit exists and x10 objective. The global magnification of the microscope ranges between x100 and
in the laboratory, this test should replace the mouse inoculation test as it avoids the use of x125 due to the extra magnification of some epi-fluorescence systems.
live animals, is less expensive and gives more rapid results.


It is often advisable to carry out more than one type of test on each sample, at least !à+ also called HYDROPHOBIA or LYSSA, acute, ordinarily fatal, %!
 of the !

when there has been human exposure. !%"
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a bite. All warm-blooded animals, including humans, are susceptible to rabies infection.
 !


The virus, a !à%!", is often present in the  %!$
 s of rabid animals and is
The tests above may be completed in specialised laboratories (such as OIE or WHO excreted in the  %; thus, the bite of the infected animal introduces the virus into a fresh
Reference Laboratories) using MAbs, nucleic acid probes, or the polymerase chain reaction wound. Under favourable conditions, the virus propagates along !%
" from the
(PCR), followed by DNA sequencing of genomic areas for typing the virus (16). This enables a wound to the à! and becomes established in the central

distinction to be made between vaccine virus and a field strain of virus, and possibly the
geographical origin of the latter. 




& ! 

Serological tests are rarely used in epidemiological surveys, due to late seroconversion
and the low percentage of animals surviving the disease and therefore having post-infection
antibodies. Oral immunisation of rabies reservoirs is the method of choice for wildlife rabies
control. For follow-up investigations in oral vaccination campaigns, virus neutralisation (VN)
tests in cell culture are preferred. However, if poor quality sera are submitted, the VN tests in
cell culture are sensitive to cytotoxicity, which could lead to false-positive results. For such
)à,


Because rabies is fatal unless treated promptly, always suspect rabies in any person who
suffers an unprovoked 
à, until you can prove otherwise.
Virus isolation from the patient͛s saliva or throat and examination of his blood for fluorescent
rabies antibody (FRA) provide the most evidence for a definitive diagnosis. Other results
 

.
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typically include an elevated white à 

", with increased polymorphonuclear and
O IM only
large mononuclear cells, and elevated urinary glucose, acetone, and protein levels. @ into the deltoid muscle (vaccine failure has occurred when administered
 of the suspected animal for 10 days of observation by a veterinarian may also into the gluteal area)
@ administer at an anatomical site distant from rabies immune globulin
provide evidence to support this diagnosis. If the animal appears rabid, it should be killed and


'

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its brain tissue tested for FRA and !
à (oval or round masses that conclusively

confirm rabies).

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Read more at http://www.wrongdiagnosis.com/r/rabies/diagnosis.htm?ktrack=kcplink Requires prior approval from Medical Health Officer

 

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The list of complications that have been mentioned in various sources for à includes:
O Pre-exposure Immunization
O Brain infection- @ Primary Series
Any infection of the brain is usually called encephalitis, though meningitis ± 1 mL IM x 3 doses on days 0, 7, and 21
O Cardiac failure @ Reinforcement:
± 1 mL IM (for persons with ongoing risk of exposure when rabies
O Respiratory failure antibody titre drops below 1:32 for Imovax and 1:5 for
O Death - about 80% of cases RabAvert
O Coma
O Post-exposure Immunization

@ No previous immunization against rabies:
± 1 mL IM x 5 doses on days 0, 3, 7, 14, and 28
!à
% an inactivated virus vaccine used for pre- and postexposure immunization
± administer on the day of exposure or as soon as possible after
against rabies; it may be prepared from virus grown in human diploid cell culture J exposure; the vaccine has been given as late as 6 months post



, that grown in cultures of chicken fibroblasts J

 


 or exposure
that grown in cultures of fetal rhesus lung and concentrated by adsorption to aluminum ± one dose of rabies immune globulin should also be given on day
phosphate J 






 0

@ Previous immunization against rabies:
c '

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± 1 mL IM x 2 doses on days 0 and 3
-c. c
± it is not necessary to administer rabies immune globulin
1
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.
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Vaccine - active immunizing agent
c-
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O hypersensitivity reactions including anaphylaxis in patients allergic to neomycin or
IMOVAX Rabies, RabAvert
other components of the vaccine

O local pain, redness, swelling or itching at site of injection
 c

O active immunization against rabies for both: /1c
/1- c

O pre-exposure immunization (primary series and periodic reinforcement) O transient headache, nausea, abdominal pain, muscle aches and dizziness may
occur; once initiated rabies vaccination should not be interrupted or discontinued
O post-exposure immunization
because of local or mild systemic adverse reactions; such reactions can be


c
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-0

successfully managed with anti-inflammatory and antipyretic agents (e.g. aspirin)
O stable in refrigerator; do not freeze
O pre-exposure vaccination should be deferred in persons with acute illness or fever
O reconstitute vaccine with 1mL diluent provided (sterile water for injection); gently
swirl the contents until completely dissolved
O persons taking chloroquine, corticosteroids, or immunosupressants may not
develop an adequate antibody response to the vaccine
O use immediately after reconstitution
/1c
-0

O persons with continuing high risk of exposure such as veterinarians should have
serum tested for rabies antibodies every 2 years; those working with live rabies
O incompatible in same syringe or at same site as Rabies Immune Globulin virus in laboratories should be tested every 6 months; an acceptable antibody titre
O do not mix with other drugs is 1:32 (Imovax) or 1:5 (RabAvert)
Encounters, especially bites or scratches from bats, skunks, coyotes, foxes, bobcats and 3. Active immunisation with the vaccine
raccoons are considered very high risk for rabies infection. However, bites or scratches from is the recommended protocol for all severe exposures (see below)
dogs or cats also can be dangerous as unvaccinated pets are often the most common link
-
!


4"
" 
 "(
between wildlife rabies and humans.
O Scrubbing with soap and water for 5 minutes
O Removal of foreign material
"The biggest thing we can do to avoid exposure is to make sure that people have their pets
O Application of a virucidal agent such as povidone iodine or aqueous iodine, if
vaccinated against rabies," Warner said. "Do your best to counsel your children not to pet
possible
strange dogs and cats under any circumstances."
O Do not suture the wound
O Anti-tetanus treatment and/or antibiotics where indicated
Symptoms of rabies can vary, Warner said. Watch for signs that may include: 




Rabies immunoglobulin is indicated for all persons known or suspected to have been exposed
-- Abnormal behavior or confusion. For example, nocturnal animals such as coyotes or to the rabies virus and is used in conjunction with the rabies vaccine (active immunisation
raccoons should not be seen during daylight hours. administered at the same time, but at a different anatomical site). Rabies immunoglobulin
-- Excessive salivation and aggression. However, not all animals with rabies will foam at the must be given for any mucous membrane exposure to saliva i.e. licks, and all single and
mouth. In some cases, animals will become lethargic. multiple bites or scratches inflicted by a suspected rabid animal, especially if associated with
any signs of bleeding, irrespective of the interval between exposure and initiation of
If you or a loved one is bitten or scratched by an animal follow these steps:
treatment. Individuals previously immunised with rabies vaccine and whose antibody titre
-- Wash the wounds thoroughly with plenty of soap and water and consult a physician as has been recently confirmed as adequate should be treated with vaccine only.
soon as possible.
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4

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!
-- Secure the animal for observation or testing if possible. a
!à

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4 

!

-- If an animal is killed, get it processed for shipment as soon as possible to reduce the 
"% à 
!
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chances of a "decomposed" test result.
I Touching or feeding of animals None, if reliable case history is
-- To reduce the chance of a "destroyed" test result, do not damage the animal's brain.
Licks on intact skin available
-- If an animal cannot be captured, but it remains in the area and can be observed for a 10-
b
day period, that is an acceptable alternative to treatment. II Nibbling of uncovered skin Administer vaccine immediately
"The treatment for rabies is expensive and long," Warner said. Once a patient receiving Minor scratches or abrasions Stop treatment if animal remains
treatment is given Rabies Immune Globulin Human (RIGH), a series of 5 shots is without bleeding healthy throughout an
c
Licks on broken skin observation period of 10 days
administered.
or if animal is killed humanely
"Once a person is diagnosed with rabies, the virus is almost always fatal," Warner said. and found to be negative for
rabies by appropriate laboratory
1c'

'c 

techniques
Rabies is caused by a rhabdovirus often present in the saliva of rabid animals that attacks the
III Single or multiple transdermal bites Administer rabies
central nervous system. Transmission is by inoculation into a wound, usually introduced
or scratches immunoglobulin and vaccine
through the bite of a rabid animal, and rarely by exposure of a mucous membrane or fresh b
immediately .
skin abrasion to infected saliva. Treatment is largely symptomatic and includes vigorous
supportive treatment. Contamination of mucous Stop treatment if animal remains
Rabies is an entirely preventable disease. Prevention includes: membrane with saliva (i.e. licks) healthy throughout an
c
O Pre-exposure immunisation of all persons at risk e.g. vets, animal handlers observation period of 10 days
O Immunisation of dogs or if animal is killed humanely
O Correct post-exposure prophylaxis for all non-immune persons. and found to be negative for
Post-exposure treatment: rabies by appropriate laboratory
technique
The combination of: a. Exposure to rodents, rabbits and hares seldom, if ever, requires specific anti-rabies
1. Prompt local treatment of the wound treatment.
2. Passive immunisation with rabies immunoglobulin
 b. If an apparently healthy dog or cat in or from a low-risk area is placed under
observation, the situation may warrant delaying initiation of treatment.
c. This observation period apples only to dogs and cats. Except in the case of
threatened or endangered species, other domestic and wild animals suspected as
rabid should be killed humanely and their tissues examined using appropriate
laboratory technique
Rabies immunoglobulin can only be obtained with a doctor's prescription. Discuss with your
doctor why it has been prescribed for you and the benefits and risks of this medicine.

use. Although rabies vaccines are usually administered under qualified medical supervision,
"
!
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!$ 5

The recommendations given here are intended as a general guide. It is recognized that, in
certain situations, modifications of the procedures laid down may be warranted. Such
situations include exposure of infants or mentally disabled persons and other circumstances
where a reliable history cannot be obtained, particularly in areas where rabies is enzootic,
even though the animal is considered to be healthy at the time of exposure. Such cases may
be treated as category II or III.
Post-exposure treatment, which consists of local treatment of the wound, followed by
vaccine therapy (with or without rabies immunoglobulin) should be initiated immediately
with contacts of categories II and III. Treatment may be discontinued if the animal involved
(dog or cat) remains healthy throughout an observation period of 10 days; or if the animal is
killed humanely and found to be negative for rabies by laboratory examination. Any biting
animal suspected of being rabid should be immediately killed humanely and tissues
examined using appropriate laboratory technique(s). Modification of the recommended
procedures would be indicated in a rabies-free area where animal bites are encountered. In
areas where canine or wildlife rabies is epizootic, adequate laboratory and field experience,
indicating that there is no infection in the species involved, may justify local health
authorities in not recommending specific anti-rabies treatment.
The indication for post-exposure vaccination with or without rabies immune globulin
depends on the type of contact with the rabid animal.

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!(

O category I ʹ touching or feeding animals, licks on the skin
O category II - nibbling of uncovered skin, minor scratches or abrasions without
bleeding, licks on broken skin
O category III ʹ single or multiple transdermal bites or scratches, contamination of
mucous membrane with saliva from licks; exposure to bat bites or scratches
For category I no treatment is required, whereas for category II immediate vaccination and
for category III immediate vaccination and administration of rabies immune globulin are
recommended in addition to immediate washing and flushing of all bite wounds and
scratches. Depending on vaccine type, the post-exposure schedule prescribes intramuscular
doses of 1 ml or 0.5 ml given as four to five doses over four weeks. For rabies-exposed
patients who have previously undergone complete pre-exposure vaccination or post-
exposure treatment with cell-derived rabies vaccines, two intramuscular doses of a cell-
derived vaccine separated by three days are sufficient. Rabies immune globulin treatment is
not necessary in such cases. The same rules apply to persons vaccinated against rabies who
have demonstrated neutralizing antibody titres of at least 0.5 IU/ml.
In order to reduce the cost of post-exposure treatment, intradermal multi-site regimens
using a fraction of the intramuscular volume per intradermal inoculation site have been
developed. Purified Vero cell vaccine has been given intradermally to more than 70 000
recipients in Thailand, where it has been in routine use for several years. Intradermal rabies
vaccination is also recommended by the ministries of health of Sri Lanka (since 1995) and the
Philippines (since 1997). In each of these countries the introduction of this route for post-
exposure treatment has permitted the discontinuation of the local production of vaccines
prepared on brain tissue. Only the cell-derived vaccines that meet the WHO requirements
regarding safety, potency and efficacy for this application may be considered for intradermal
field experience from routine infant immunization programmes with other intradermally
injected vaccines highlights the potential difficulties in assuring proper delivery. This
emphasizes the need for appropriate staff training to ensure correct storage, reconstitution
and injection. Provided that a correct sterile technique is used, the remaining doses may be
kept in the vial at 2ʹ8°C and used for another patient within six hours after reconstitution.
Tissue-culture or purified duck-embryo vaccines of potency at least 2.5 IU per single
intramuscular immunizing dose should be applied according to 
 4
" .


!"" !
" 

One dose of the vaccine should be administered on days 0, 3, 7, 14 and 30. All intramuscular
injections must be given into the deltoid region or, in small children, into the anterolateral
area of the thigh muscle. Vaccine should never be administered in the gluteal region.


càà!%
" 
" 

In the abbreviated multisite schedule, the 2-1-1 regimen, one dose is given in the right arm
and one dose in the left arm at day 0, and one dose applied in the deltoid muscle on days 7
and 21. The 2-1-1 schedule induces an early antibody response and may be particularly
effective when post-exposure treatment does not include administration of rabies
immunoglobulin.


WHO recommended the following intradermal regimens and vaccines for use by the
intradermal route:
O 8-site intradermal method (8-0-4-0-1-1) for use with HDC (Rabivac TM) and PCEVC
(Rabipur TM)
The 8 sites regimen should be particularly considered in emergency situations
when no RIG is available
O 2-site intradermal method (2-2-2-0-1-1) for use with PVRV (Verorab TM, Imovax
TM, Rabies vero TM, TRC Verorab TM) and PCECV (Rabipur TM)


.!
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For use with:
O Human diploid cell vaccine (HDCV) (Rabivac TM) and
O Purified chick embryo cell vaccine (PCECV) (Rabipur TM) both vaccines at 0.1 ml per
intradermal site


.!
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)&#&#&#7##

The volume per intradermal site is:
 O 0.1 ml for PVRV (Verorab TM, Imovax TM, Rabies vero TM, TRC Verorab TM)
O 0.1 ml for PCECV (Rabipur TM)

!#"
%

The use of brain-tissue vaccines should be discontinued. WHO does not recommend any
schedule using brain-tissue vaccine. National authorities should recommend a schedule of
immunization that has been shown to induce an adequate level of protection when brain
tissue vaccines are available in that country.


à
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Combined immunoglobulin-vaccine treatment was considered in the eighth report of the
WHO Expert Committee as the best specific systemic treatment available at that time for the
post-exposure prophylaxis of rabies in humans, although experience indicated that vaccine
alone was sufficient for minor exposures (category II). Immunoglobulin should be given in a
single dose of 20 IU per kg of body weight for human anti-rabies immunoglobulin, and 40 IU
per kg of body weight for heterologous (equine) immunoglobulin; the first dose of vaccine
should be inoculated at the same time as the immunoglobulin, but in a different part of the
body. Sensitivity to heterologous immunoglobulin must be determined before it is
administered. The physician should be prepared to deal with anaphylactic shock reactions.
Administration of rabies immunoglobulin (RIG) should be infiltrated into the depth of the
wound and around the wound as much as anatomically feasible. Any remainder should be
injected at an intramuscular site distant from that of vaccine inoculation e.g. into the anterior
thigh.
Treatment should be started as early as possible after exposure, but in no case should it be
denied to exposed persons whatever time interval has elapsed.


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Local treatment of wounds involving possible exposure to rabies ʹ recommended in all
exposures.