Communicable Diseases Rabies - General Aspects &amp Laboratory Diagnostic Techniques

RABIES
General Aspects & Laboratory Diagnostic Techniques
ZOONOSIS DIVISION
National Institute of Communicable Diseases

WHO Collaborating Centre for Rabies Epidemiology
(Directorate General of Health Services) 22-Sham Nath Marg, Delhi - 110 054
RABIES
General Aspects & Laboratory Diagnostic Techniques
2007
ZOONOSIS DIVISION National Institute of Communicable Diseases WHO Collaborating Centre for Rabies Epidemiology
(Directorate General of Health Services) 22-Sham Nath Marg, Delhi 110054
Contributors
Dr. Mala Chhabra Deputy Director Dr. Veena Mittal Joint Director & Head Zoonosis Division Dr. U.V.S. Rana Joint Director Dr. R.L. Ichhpujani Addl. Director & NPO IDSP
Dr. Dipesh Bhattacharya Joint Director
Dr. Shiv Lal Addl. Director General & Director
Designing & Printing:

New Concept Information Systems Pvt. Ltd. e-mail: nc.communication@gmail.com
CONTENTS
1. RABIES
1.1 1.2 1.3 Introduction Causative agent 1.2.1 Susceptibility to physical and chemical agents Epidemiology 1.3.1 Global status 1.3.2 Rabies in India 1.3.3 Mode of transmission 1.3.4 Pathogenesis 1.3.5 Incubation period Clinical features in humans Differential diagnosis Rabies in animals 1.6.1 Clinical features in dogs 1.6.2 Clinical features in cats 1.6.3 Clinical features in cattle Immune response in rabies infection and vaccination 1.7.1 Immunity in rabies infection 1.7.2 Immunity following vaccination Laboratory diagnosis 1.8.1 Laboratory tests Prevention of rabies in humans 1.9.1 Decision to treat 1.9.2 Management of wound 1.9.3 Passive immunisation 1.9.4 Active immunisation 1.9.4.1 Intra-muscular regimen 1.9.4.2 Intra-dermal regimens 1.9.5 Post exposure therapy for previously vaccinated persons 1 1 1 3 4 4 4 5 6 6 6 8 8 8 9 9 9 9 10 10 11 12 12 12 13 15 16 17 22 23 23 23 24
Contents
1.4 1.5 1.6
1.7
1.8 1.9
1.10 Pre-exposure prophylaxis 1.11 Prevention and control of rabies 1.11.1 Prevention of human deaths due to rabies 1.11.2 Reducing the transmission of the disease
iii
2.
2.1
LABORATORY DIAGNOSIS OF RABIES: TECHNIQUES

Collection, preservation, packing and transportation of specimens 2.1.1 Specimens from humans 2.1.2 Collection of specimens from suspected rabid animals 2.1.3 Preservation 2.1.4 Labelling 2.1.5 Packing 2.1.6 Transportation Laboratory tests Principles and procedures of commonly used techniques 2.3.1 Sellers staining for detection of Negri bodies 2.3.2 Fluorescent Antibody Test (FAT) 2.3.3 Mouse Inoculation Test (MIT)/Biological Test (BT) 2.3.4 Rabies Tissue Culture Infection Test (RTCIT) 2.3.5 Polymerase Chain Reaction (PCR) 2.3.6 Modified Counterimmuno Electrophoresis (CIEP) serum neutralisation 2.3.7 Enzyme Linked Immunosorbent Assay (ELISA) 2.3.8 Rapid Fluorescent Focus Inhibition Test (RFFIT)
26 26 26 27 28 28 28 28 28 29 29 31 33 34 36 38 40 42 50 50 51 51 51 51 51 51 52 52 52 52 52 52 53 53
2.2 2.3
3.
3.1 3.2 3.3 3.4
SAFETY IN RABIES LABORATORY

Laboratory design, operation and equipment Training of manpower Safe laboratory procedures Collection of specimens 3.4.1 Antemortem specimens 3.4.2 Postmortem specimens Equipment and glassware Aerosols Handling of experimental animals Safe handling of hazardous chemicals Disposal of equipment, glassware and protective clothing
3.5 3.6 3.7 3.8 3.9
3.10 Decontamination 3.11 Disposal 3.12 Pre-exposure immunoprophylaxis 3.13 Post-exposure management
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RABIES General Aspects & Laboratory Diagnostic Techniques
4.
4.1 4.2 4.3
QUALITY ASSURANCE IN RABIES DIAGNOSIS

Components of quality assurance Factors influencing quality External Quality Assessment Scheme (EQAS)
55 55 55 57 58 61 62
ANNEXURE 1: Stains, Buffers and Reagents ANNEXURE 2: WHO Collaborating Centres for Rabies in India Suggested further reading
Contents
PREFACE
Rabies is a disease of antiquity. It continues to persist as a major public health problem. It is perhaps the most gruesome and dreadful of all communicable diseases afflicting human beings. This disease is world wide in distribution, with the exception of a few areas and countries that have historically been free from it primarily due to their geographical locations. Rabies is endemic in India except for probably the water-locked islands of Lakshadweep and Andaman & Nicobar. This publication is a brief yet comprehensive document covering various aspects of the disease, aetiological agent, management of animal bites and laboratory diagnosis. Laboratory techniques have been described in detail to enable medical and veterinary laboratories to set up the diagnostic facilities. Due emphasis has been given to biosafety and quality assurance aspects which are an integral part of the laboratory system. It is sincerely hoped that the publication will help in improving the laboratory diagnosis of rabies and would provide updated information on animal bite management. The financial help received from WHO to bring out this publication is thankfully acknowledged.
Dr Shiv Lal Addl DG & Director NICD, Delhi
RABIES
1.1 INTRODUCTION
Rabies is an acute viral disease which causes encephalomyelitis in virtually all the warm blooded animals including man. The causative agent is found in domestic and wild animals, and is transmitted to other animals and to humans through close contacts with their saliva (i.e. bites, scratches, licks on broken skin and mucous membranes). Rabies is an important zoonotic infection in which man is dead end of the infection and hence does not play any role in its spread to new hosts. In most of the developing countries, dogs are the principle reservoirs of rabies (canine rabies) whereas sylvatic rabies involving animals such as foxes, racoons and coyotes are principle reservoirs of this disease in developed countries. Rabies has terrified man since antiquity. The fear is by no means unfounded since the disease is invariably fatal and perhaps the most painful and horrible of all communicable diseases in which the sick person is tormented at the same time with thirst and fear of water (hydrophobia). Till date no treatment has succeeded in curing hydrophobia and in spite of great strides in the prevention of rabies, with few exceptions, the disease is no less a global problem now than it was almost a century ago.
1.2 CAUSATIVE AGENT

Rabies virus belongs to the family Rhabdoviridae and genus Lyssavirus (Lyssa: Greek: rabies). The genus was at first divided into four serotypes (1 4) by antigenic cross-reactivity with sera and monoclonal antibodies, which correspond to the following species: serotype 1, rabies virus (RABV); 2, Lagos bat virus (LBV); 3, Mokola virus (MOKV); and 4, Duvenhage virus (DUVV). Further isolations of new bat lyssaviruses in Europe, then Australia and the progress in genetic characterisation of several genes (N, P, and G) supported the delineation of seven genotypes (17), confirming and expanding the
Rabies
antigenic data (Table 1): 1, RABV; 2, LBV; 3, MOKV; 4, DUVV; 5, European bat lyssavirus 1 (EBLV-1); 6, European bat lyssavirus 2 (EBLV-2); and 7, Australian bat lyssavirus (ABLV). Within each genotype, sublineages correspond to variants circulating in specific geographical regions and/or animal hosts. The genotypes further segregate in two phylogroups including genotypes 1, 4, 5, 6 and 7 (phylogroup I); and 2 and 3 (phylogroup II). Viruses of each phylogroup differ in their biological properties (pathogenicity, induction of apoptosis, cell receptor recognition, etc.)
Table 1: Classification of lyssaviruses

Phylogroup Isolates characterised I 1 Rabies virus RABV Worldwide (except several islands) Southern Africa Europe Europe Australia Carnivores (worldwide); bats (Americas) Insectivorous bats Insectivorous bats (Eptesicus serotinus) Insectivorous bats (Myotis sp.) Frugivorous/ insectivorous bats (Megachiroptera/ Microchiroptera) Frugivorous bats (Megachiroptera) Unknown Genotype Species Abbreviation Geographical Potential vector(s) (ICTV)a origin
I I I I
4 5 6 7
Duvenhage virus DUVV European bat lyssavirus type 1 European bat lyssavirus type 2 Australian bat lyssavirus EBLV-1 EBLV-2 ABLV
II II Isolates to be characterised as new genotypes
2 3
Lagos bat virus Mokola virus
LBV MOKV
Sub-Saharan Africa Sub-Saharan Africa
Aravan virus
ARAV
Central Asia
Insectivorous bats (isolated from Myotis blythi) Insectivorous bats (isolated from Myotis mystacinus) Insectivorous bats (isolated from Murina leucogaster) Insectivorous bats (isolated from Miniopterus schreibersi)
Khujand virus
KHUV
Central Asia
Irkut virus
IRKV
East Siberia
West Caucasian bat virus
WCBV
Caucasian region
Lyssaviruses have a 12 kb-long non-segmented RNA genome of negative polarity encoding five viral proteins (3 to 5): nucleoprotein N, phosphoprotein P, matrix protein M, glycoprotein G and polymerase L. The lyssavirus particle has a bullet-shaped form, 100300 nm in length and 75 nm in diameter (Fig 1). It is composed of two structural and functional units [Fig 2]: (i) the outer envelope covered with spike-like projections (10 nm in length) corresponding to G-protein trimers which recognise specific viral receptors on susceptible cell membranes; Hence pathogenicity of Lyssavirus is attributed to protein G. (ii) the internal helically packaged ribonucleocapsid, which is composed of the genomic RNA intimately associated with protein N, polymerase L and its cofactor protein P (formerly named M1). The ribonucleocapsid complex ensures genome transcription and replication in the cytoplasm.
Fig 1: Negatively stained rabies virus as seen by transmission microscopy
G glycoprotein SPIKES
M protein lipid bilayer membrane
helical nucleocapsid (RNA plus N protein) polymerase (2 proteins)
Fig 2: Structural and functional units of Rabies virus
Finally, protein M (formerly named M2) occupies an intermediate position between the ribonucleocapsid and the envelope, and is responsible for virus budding and the bullet-shaped morphology.
1.2.1 Susceptibility to physical and chemical agents

The rabies virus is highly resistant against cold, dryness and decay. However, it is susceptible to action of various physical and chemical agents. This knowledge is most useful in management of wounds for inactivating the virus (Table-2).
Rabies
Table 2: Susceptibility of rabies virus

Inactivation of rabies virus At 60C within 35 seconds (sensitive to pasteurisation and boiling) At pH < 4 or > 10 By action of oxidizing agents, most organic solvents, surface acting agents, quaternary ammonium compounds, proteolytic enzymes, ultraviolet rays and X-rays Soaps and detergents Alcohol Preservation of rabies virus By freeze drying At ultra low temperatures (< - 20C) Glycerine
1.3 EPIDEMIOLOGY
1.3.1 Global status
Rabies occurs in all continents with the exception of Australia and Antarctica. Several (>50) countries are currently free of rabies. Even in infected countries the disease is not uniformly distributed. Areas free of disease, of low and high endemicity and areas with epizootic outbreaks can be found in many countries. In Africa and Asia (with few important exceptions such as Japan and Singapore), rabies is prevalent in almost whole of the territory with a stable pattern. Most of the countries of Americas and Europe report occurrence of disease in limited or border areas (Fig-3:).
1.3.2 Rabies in India

Rabies is responsible for extensive morbidity and mortality in India. The CBHI reported around 301 deaths due to hydrophobia during 2005. However, multicentric rabies survey (2003) conducted by Association for Prevention and Control of Rabies in India for assessing the burden of rabies estimated annual human rabies deaths incidence to be around 20,000. The annual incidence of animal bites was estimated to be 1.7% (17.5 million per year). Based on vaccine utilisation it is estimated that almost 2.3 million people annually receive post exposure prophylaxis against rabies following bite or exposure to rabid or suspected rabid animal. With the exception of Andaman & Nicobar and Lakshadweep Islands, human cases of rabies are reported from all over the country. The cases occur throughout the year. About 96% of the mortality and morbidity is associated
Fig 3: Global status of rabies
with dog bites. Cats, wolf, jackal, mongoose and monkeys are other important reservoirs of rabies in India (Table-3). Bat rabies has not been conclusively reported from India.
Table 3 : Animals transmitting rabies in India

Frequent Dogs & cats Occasionally Monkeys Cows & buffaloes Mongoose Foxes, Wolves & jackals Sheep & goats Bears Pigs Donkeys Horses Camels Not reported Bats * Rodents * Birds Squirrel
Note: All exposures in wild are considered as Category III exposures. * Bite by bats or rodents do not ordinarily necessitate rabies vaccination in India. However, bites by bats or rodents in unusual circumstances may be considered for vaccination in consultation with an expert in the field of rabies.
1.3.3 Mode of transmission

Rabies virus is predominantly neurotropic and kills the host in short period after it has entered the neurones. Before death, virus reaches salivary glands and is excreted in saliva. The saliva gains entry into another host through a preexisting breach in skin when mere licking or contamination is adequate or the bite of the rabid animal creates a mechanical breach of skin through which the rabies virus gains entry (Table -4).
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Table 4: Mode of transmission

Common

Rare

Bites from infected animals Licks on broken skin and mucous membrane Scratches
Inhalation Organ transplantation Ingestion
1.3.4 Pathogenesis
On entering into human body through wounds or direct contact with mucosal surfaces, the rabies virus either multiplies at local site of inoculation in non-nervous tissues or directly enters peripheral nerves and travels by retrograde axoplasmic flow to the central nervous system prior to its spread towards brain via the nerves (Fig 4). Within the brain, virus spreads from infected to contagious cells. There may be regional differences in the intensity with which areas of brain become infected. The main areas affected are usually the cerebellum, hypothalamus, hippocampus and scattered neurons in the reticular formation. It may be that aggression in rabies is related to the presence of virus in mid brain raphe nuclei and medical hypothalamus, since these are the two inhibitory centres of aggressive behaviour. It may also be that the distribution of virus in the brain has a bearing as whether the disease becomes the manifest in dumb or classical furious rabies. It does not follow the haematogenous spread. The movement of the virus is extremely slow (15100 mm per day) which results into a long incubation period. The virus then moves from CNS via anterograde axoplasmic flow within peripheral nerves and reaches salivary glands and other organs. The virus is widely disseminated throughout the body at the time of clinical onset. This has practical implications as organ transplantation has resulted in transmission of the disease to the recipient.
1.3.5 Incubation period

The average incubation period is between 30-90 days. It ranges between 2 weeks and 6 years. Because of the wide range of incubation period, post- exposure prophylaxis should be given as soon as possible, however, it should not be denied to persons reporting late. Factors which may influence the length of the incubation period include the site of bite, the amount of virus in saliva of the bitting animal, the virus strain, and the age and immune status of the victim. It is shorter in case the bite is closer to brain and massive dose of virus has been inoculated.
1.4 CLINICAL FEATURES IN HUMANS

The clinical features can be described under three broad groups (Table 5) Prodormal phase Excitation phase Paralytic phase
5. Brain infected
6. Virus travels from brain via nerves to other tissues such as eye, kidneys, salivary glands
4. Virus replicates in dorsal root ganglion and travels up spinal cord to brain
3. Virus infects nerve in peripheral nervous system Moves by retrograde transport 2. Virus replicates in muscle at site of bite
1. Virus enters via animal bite
Fig 4 Pathogenesis of rabies
The first symptom to appear may be pain and tingling in the affected limb, especially around the site of bite. This is seen in 35-65% cases. Hydrophobia is the best known symptom of this disease and is pathognomonic for rabies.
Table 5: Clinical features in humans

Prodormal phase Discomfort or pain at the site of bite Numbness and tingling in limb Elevated temperature Malaise Headache Sore throat Priapism Restlessness, tremors Pharyngeal and laryngeal spasms Fear of water (hydrophobia) Terror and excitement Respiratory arrythmias Cardiac arrythmias Hypertension Convulsions on exposure to light air sound Intense thirst and dehydration Change in tone of voice Death Restlessnes, convulsions Flaccid and limp muscles Unconsciousness Death Rabies
Excitation phase
Paralytic phase
Hydrophobia (fear of water) is pathognomonic feature of rabies which is erroneously considered synonymous with rabies. It is usually the only neurologic abnormality found in patients presenting with furious rabies. This is due to a violent jerky contraction of the diaphragm and accessory muscles of inspiration that is triggered by the patients attempts to swallow liquid and by a variety of other stimuli such as strong current of air, loud noise and bright light. Hydrophobia is usually not associated with pain in neck or throat. It is also not a conditioned reflex caused by aspiration of liquid into trachea. About 20% of the patients present with paralytic form of rabies do not have hydrophobia. In these patients diagnosis can only be established by laboratory confirmation. This is important as rabies has been documented from USA in cluster of human rabies cases associated with transplantation of solid organs from a misdiagnosed rabies patient. Rabies cases have also been documented following corneal transplantation.
1.5 DIFFERENTIAL DIAGNOSIS

Before the appearance of hydrophobia and in those cases where it does not manifest, rabies needs to be differentiated from other clinical conditions (Table-6).
Table 6: Differential diagnosis of rabies

Initial phase Lockjaw Encephalitis Hysteria
Paralytic phase
Acute polyneuritis Poliomyelitis Belladona poisoning Delerium tremens
Rabies post vaccinal encephalomyelitis
1.6 RABIES IN ANIMALS

All warm blooded animals are vulnerable to rabies infection, and susceptibility is affected by factors such as viral variant, quantity of virus inoculated, and the bite site. In addition, the degree of species susceptibility varies considerably. Younger animals are usually more susceptible to rabies infection than older ones.
1.6.1 Clinical features in dogs

After an incubation period of around three months (range 10 days to 6 months), dog may manifest one or more of the following clinical features. There may be
change in behavior of dog, change in bark tone, change in feeding habits, the animals may go off feed and eat abnormal objects. They may develop fever, vomiting, excessive salivation, paralysis of lower jaw, anxiety, restlessness, convulsions, paralysis leading to death with in 5-7 days on onset of disease. There is however no hydrophobia in animals. Rabies in dogs is also classified as dumb (predominantly paralytic manifestation with docile behavior of animal) or furious (mainly convulsions and aggressive behavior with greatly exaggerated biting tendencies). Clinical rabies in dogs has to be differentiated from other diseases which manifest with similar features. These include distemper, hepatitis, epilepsy, poisoning, brain tumours and head injury.
1.6.2 Clinical features in cats

Rabid cats show extreme aggressiveness, great sensitivity to touch/noise, profuse salivation and may attempt to attack dog or even man.
1.6.3 Clinical features in cattle

In cattle, rabies is manifested as abnormal movements of posterior extremity, foamy yellow froth and decrease in yield of milk. Milk of such animals has been shown to have viable rabies virus and its ingestion in raw form may require post exposure treatment in those individuals who have ulcers or abrasions in mouth or pharynx. Otherwise the gastric juice destroys the rabies virus. Pasteurisation, boiling and cooking also kills the virus.
1.7 IMMUNE RESPONSE IN RABIES INFECTION AND VACCINATION

1.7.1 Immunity in rabies infection
Since rabies virus is strictly neuroinvasive, little antigen is present in the early stages of the disease hence the humoral response to viral antigens in the infected host is negligible until late in the course of infection and remains low until the terminal phase of the disease. High levels of antibody appear in serum and in cerebrospinal fluid (CSF) only at death and in cases where illness is naturally or artificially prolonged. Virus neutralizing antibodies are produced in response to the massive amount of viral antigen that is generated through widespread infection of the CNS and made accessible to the hosts reticuloendothelial system. The critically important cell-mediated immune response to rabies virus infection is, perhaps the most puzzling of the hosts total immune response to rabies virus infection. TH Lymphocytes which play an essential role in supporting B cell production of antibodies and Cytotoxic T-Lymphocytes (CTLs) which function
Rabies
independently in cell mediated viral clearance are both key responses in the cell mediated immunity derived from viral antigens. The function of CTLs is to destroy target cells that display virus induced changes on their surfaces. However, in spite of their importance in viral clearance, T lymphocytes appear to be suppressed in animals infected by pathogenic street viruses. As a result of virus induced immunosuppression the disease increases in severity and mortality rises. It is clear from various studies that both B and T cells play an important role in virus clearance.
1.7.2 Immunity following vaccination

Immunisation with attenuated live and inactivated rabies virus vaccines induces humoral and cell mediated immune responses that develop to functional levels in 7 10 days. Vaccine induced antibody in animals and humans is regarded as a key factor in the prophylactic protection of animals and humans. Hence RIG, which provides immediate passive immunity with proven effectiveness over the initial few weeks after exposure in humans, is recommended in post exposure treatment of humans to cover up the initial period before development of antibodies due to active immunisation. The antibodies help in controlling the spread of rabies virus infection as they are capable of effectively neutralizing virus that is present in intercellular spaces or in body fluids, and it may bind to virus expressed on the cell surface, allowing complement or antibody dependent cellular cytotoxicity to mediate killing of infected cells. The efficacy of post exposure immunisation and long term effects of vaccine induced prophylaxis against rabies seems also to be linked to the stimulation of a strong CTL response. Induction of CTLs and other effector T cells during infection with live attenuated rabies virus vaccine strains and in response to immunisation with inactivated virus is consistent with the observation that T lymphocytes, and CTLs in particular are essential for protection against a lethal dose of rabies virus.
1.8 LABORATORY DIAGNOSIS

In presence of typical features (such as hydrophobia in man), diagnosis of rabies has been essentially clinical in both animals and man. There are, however, instances both in man and animals when clinical diagnosis becomes difficult. The definitive diagnosis of rabies can only be obtained by laboratory investigations, otherwise it may result into a rabid animal remaining undiagnosed and hence a greater risk to human beings. Similarly, persons bitten by animals who have died of diseases presenting with similar clinical features can be unnecessarily vaccinated against rabies unless diagnosis is established. Laboratory diagnosis may be required in human beings when the clinical features are not unambiguous. It is of utmost importance in the era of organ transplantation.
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Though antemortem as well as post-mortem diagnostic techniques are available, the former do not have a sensitivity of more than 25% in best of hands. Battery of tests should be performed. A positive test by one of the standard procedures overrides negative reaction in the others. Negative tests do not rule out the possibility of rabies (Table - 7).
Table 7: Interpretation of ante-mortem diagnostic tests

Difficult require battery of tests No single test has been positive in every case Distribution of rabies antigen in nuchal biopsy or corneal impression may be extremely irregular False positive results have been obtained in fluorescent examination of corneal epithelium Antibody response to infection on occasions may be absent It is necessary to test repeated samples Samples should be tested using all currently available tests Ante-mortem diagnosis should be attempted only by experienced laboratories A negative diagnosis does not rule out rabies
1.8.1 Laboratory tests

A number of tests can be employed for detection of rabies antigen and antibodies. Commonly employed tests for rabies diagnosis are described in Table 8.
Table 8: Common tests used for rabies diagnosis

Test Sellers Staining for inclusion bodies (Negri bodies) Advantages

Limitations
Simple Rapid (1 hour) Easy to perform No special equipment required Specific (near 100%) Sensitive (near 100%) Relatively Rapid (1 day) Easy to perform Can detect very small quantity of virus Confirmatory
Positive in 50 70% of cases
Fluorescent Antibody Test (FAT) for antigen detection
Expensive Good Fluorescent microscope Good quality conjugate Trained manpower Takes long time (21 days) More of academic value Use of laboratory animals (mice). Thus laboratories should have a well maintained animal house. Ethical issues involved in use of laboratory animals Special cell culture laboratory Expensive Trained manpower
Mouse Inoculation Test (MIT) for virus isolation
Rapid Tissue Culture Infection Test (RTCIT) for virus isolation
Rapid (4 days) Sensitive
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1.9 PREVENTION OF RABIES IN HUMANS

Because of long incubation period, which is typical of most cases of human rabies, it is possible to institute prophylactic post exposure treatment. This must be started at the earliest to ensure that the individual will be immunised before the rabies virus reaches the Central Nervous System.
1.9.1 Decision to treat

In a rabies endemic country like India, where every animal bite is potentially suspected as a rabid animal bite, the treatment should be started immediately. To bring out uniformity globally, the WHO recommended classification of animal bite for post-exposure treatment should be followed. (Table-9). Although unvaccinated animals are more likely to transmit rabies, vaccinated animals can also do so if the vaccination of the biting animal was ineffective for any reason. The risk of dog being infected with rabies is greatly reduced when it appears healthy and there is confirmed history of vaccination with minimum of two immunisations with potent rabies vaccine in last two years. The treatment should be started immediately after the bite. The treatment may be converted to pre exposure prophylaxis if animal involved (dog or cat) remains healthy throughout an observation period of 10 days. The observation period is valid
Table 9: WHO Guidelines for post exposure treatment against rabies

Category Type of contact with suspected animal* Touching or feeding of animals Licks on intact skin Nibbling of uncovered skin Minor scratches or abrasions without bleeding. Licks on broken skin Single or multiple transdermal bites or scratches. Contamination of mucous membrane with saliva Recommended treatment None, if reliable case history is available Administer vaccine immediately, and stop if 10 day observation or laboratory techniques confirm suspect animal to be rabies negative** Administer rabies Immunoglobulin and vaccine immediately with and stop if suspected animal confirmed as rabies negative***
i ii
iii.
* Exposure to rodents,rabbits and hares seldeom,if ever,requires specific anti rabies treatment. ** If an apparently healthy dog or cat in or from a low risk area is under observation the situation may warrant delaying the initiation of treatment. *** Observation period only applies to dogs and cats. Other domestic and wild animals (except threatened or endangered species) suspected as rabid should be killed humanely and their tissues examined using appropriate laboratory techniques.
Source: Guidelines for post-exposure treatment. WHO Expert Consultation on Rabies,2004, WHO Technical Series 931.
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for dogs and cats only. Bite by all wild animals should be treated as category III exposure. It should be noted that bites by rats, mice, squirrel, hare and rabbits seldom require treatment. Bat rabies has not been conclusively proved in India and hence exposure does not warrant treatment. It is re-emphasised that the treatment should be started as early as possible after exposure, but it should not be denied to person reporting late for treatment. There are three components of prevention of rabies in man. All three carry equal importance and one should not be given undue importance, or utter neglect, at the cost of other two components. Physician must attempt to provide the animal bite victim the benefit of all three of these. These components are: Management of wound Post-exposure immunisation Pre-exposure immunisation Advice to the patient
1.9.2 Management of wound

Wound toilet: Since the rabies virus enters the human body through a bite or scratch, it is imperative to remove as much saliva, and thereby the virus, from the wound as is possible by an efficient wound toilet that should not involve additional trauma. Since the rabies virus can persist and even multiply at the site of bite for a long time, wound toilet must be performed even if the patient reports late (Table 10). Injection of . tetanus toxoid should be given to the unimmunised individual. To prevent sepsis in the wound, a suitable course of an antibiotic may be recommended. Suturing of wound should be avoided as far as possible. If unavoidable, minimum loose sutures should be applied after adequate local treatment along with proper infiltration of anti rabies serum.
1.9.3 Passive immunisation

Equine Rabies Immunoglobulin (ERIG): The anti rabies serum provides passive immunity in the form of ready-made anti rabies antibody to tide over the initial phase of the infection. Anti rabies serum or RIG has the property of binding with the rabies virus, thereby resulting in the loss of infectivity of the virus. Human Rabies Immunoglobulins (HRIG): HRIG are free from the side effects encountered in a serum of heterologous origin, and because of their longer half life, are given in half the dose of equine anti rabies serum. The anti rabies sera should always be brought to room temperature (20 25C) before use.
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Table 10: Wound management

Dos Physical
Wash with running tap water
Mechanical removal of virus from the wound
Chemical
Washing the wound with soap and water Apply disinfectants Povidone iodine Spirit Household antiseptics Infiltration of immunoglobulins in the depth and around the wound in Category III exposures Neutralisation of the virus
Biological
Donts

Touch the wound with bare hand Apply irritants like soil, chillies, oil, herbs, chalk, betel leaves etc. Suture Cauterise
Dose of Rabies Immunoglobulins (RIG): The dose of equine anti rabies serum is 40 i.u. per kg body weight of patient and is given after testing of sensitivity, upto a maximum of 3000 i.u. The ARS produced in India contains 300 i.u. per ml. The dose of the human rabies immunoglobulins (HRIG) is 20 i.u. per kg body weight (maximum 1500 i.u.). HRIG does not require any prior sensitivity testing. HRIG preparation is available in concentration of 150 i.u. per ml. Administration of Immunoglobulins: As much of the calculated dose of RIG as is anatomically feasible should be infiltrated into and around the wounds. Multiple needle injections into the wound should be avoided. Remaining, if any, after all wounds have been infiltrated, should be administered by deep intramuscular injection at an injection site distant from the vaccine injection site. Animal bite wounds inflicted can be severe and multiple, especially in small children. In such cases, the calculated dose of the rabies immunoglobulin may not be sufficient to infiltrate all wounds. In these circumstances, it is advisable to dilute the immunoglobulins in sterile normal saline 2 to 3 fold to be able to permit infiltration of all wounds. The total recommended dose of immunoglobulin must not be exceeded as it may reduce the efficacy of the vaccine. If immunoglobulin was not administered when vaccination was begun, it can be administered upto the seventh day after the administration of the first dose of
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vaccine. Beyond the seventh day, Rabies Immunoglobulin (RIG) is not indicated since an antibody response to anti rabies vaccine is presumed to have occurred. Immunoglobulin should never be administered in the same syringe or at the same anatomical site as vaccine. Sensitivity test before administration of heterologous serum: With antisera of equine origin, anaphylactic shock may occur and thus sensitivity testing is mandatory before giving ERIG. Skin test may be performed as per the manufacturers instructions given in the product insert. Otherwise, general guidelines are described in Table-11.
Table 11: Skin testing prior to administration of ARS/ERIG

Inject 0.1 ml ARS/ERIG diluted 1:10 in physiological saline intradermally into the flexor surface of the forearm to raise a bleb of about 3 mm diameter. Inject an equal amount of normal saline as a negative control on the flexor surface of the other forearm. After 15 minutes an increase in diameter to > 6 mm of wheal surrounded by flare is taken as positive skin test, provided the reaction on the saline test was negative.
A negative skin test must never reassure the physician that no anaphylactic reaction will occur. Those administering ERIG should always be ready to treat early anaphylactic reactions with adrenalin. The dose is 0.5 ml of 0.1% solution (1 in 1000, 1mg/ml) for adults and 0.01 ml/kg body weight for children, injected subcutaneously or IM. If patient is sensitive to ERIG, HRIG should be used. Approach to a patient requiring rabies immunoglobulins when none is available: In circumstances where no immunoglobulins are available greater emphasis should be given to proper wound toileting followed by Essen schedule of Tissue culture vaccine with double dose on day 0 at 2 different sites intramuscularly (0 day 2 doses on left and right deltoid, 3, 7, 14 and 28 days). It is emphasised that doubling the first dose of TCV is not a replacement to RIG. A full course of vaccine should follow thorough wound cleansing and passive immunisation. Tolerance and side effects: With RIG, there may be transient tenderness at the injection site and a brief rise in body temperature which do not require any treatment. Skin reactions are extremely rare. RIG must never be given intravenously since this could produce symptoms of shock, especially in patients with antibody deficiency syndromes.
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Serum sickness occurs in 1% to 6% of patients usually 7 to 10 days after injection of ERIG, but it has not been reported after treatment with HRIG.
1.9.4 Active Immunisation

Active immunisation is achieved by administration of safe and potent TCVs. In India, NTV was used for post exposure treatment in public sector which was being given free of cost. However, as this vaccine was reactogenic, the production was stopped in December, 2004. 1.9.4.1 Intra-muscular regimen (IM) The currently available vaccines in India for IM administration are described in Table 12.
Table 12: Anti rabies vaccines available in India

Name of the vaccine 1. Neural tissue vaccine BPL inactivated sheep brain vaccine (Semple type) 2. Cell Culture vaccines i) Human Diploid Cell Vaccine (HDCV) ii) Purified Chick Embryo Cell Vaccine (PCEC) iii) Purified Vero Cell Rabies Vaccine (PVRV) Fixed virus strain PV 11 Pitman Moore (PM) LEP-Flury MRC-5 Substrate Sheep brain Available Production stopped since December 2004 Produced locally in private. Sector Produced locally in private sector Imported + produced locally in public & private sector Produced locally in private Sector
Primary SPF chick embryo cells Vero Cells
Pitman Moore (PM) Pitman Moore (PM)
3.
Purified Duck Embryo Vaccine
Duck Embryo
Schedule
Essen Schedule : Five dose intramuscular regimen - The course for post exposure prophylaxis should consist of intramuscular administration of five injections on days 0, 3, 7, 14 and 28. The sixth injection (D90) should be considered as optional and should be given to those individuals who are immunologically deficient, are at the extremes of age and on steroid therapy. Day 0 indicates date of first injection. This schedule is followed in India. Zagreb schedule: Abbreviated multisite intramuscular regimen (2-1-1) One dose of vaccine administered intramuscularly in the left and one into right deltoid region on day 0 followed by one dose on days 7 and 21 in deltoid region. This schedule saves 2 clinic visits and one vaccine dose.
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Indications. All age groups of animal bite victims of Category II and III require the same number of injections and dose per injection. The Category III exposures, in addition require administration of rabies immunoglobulins as discussed earlier. Site of inoculation. The deltoid region is ideal for the inoculation of these vaccines. Gluteal region is not recommended because the fat present in this region retards the absorption of antigen and hence impairs the generation of optimal immune response. Storage and transportation. Though most tissue culture vaccines are marketed in freeze dried (lyophilised) form which is more tolerant of vagaries of temperature, yet it is recommended that these vaccines should be kept and transported at a temperature range of 2-8oC. Freezing does not damage the lyophilised vaccine but there are chances of breakage of ampoule containing the diluent. Liquid vaccines should never be frozen. Reconstitution and storage. The lyophilised vaccine should be reconstituted with the diluent provided with the vaccine immediately prior to use. However, in case of unforeseen delay it should not be used after 6-8 hours of reconstitution. Protective level of anti rabies antibody. Humoral antibodies are believed to play important role in protection against rabies and a titre of 0.5 i.u./ml or more in serum is considered as protective. Adverse effects with tissue culture vaccines. The tissue culture vaccines are widely accepted as the least reactogenic rabies vaccines available today. Various studies have now shown that adverse effects can be either general in nature or allergic in origin. The general adverse reactions include sore arm, headache, malaise, nausea, fever and localised oedema at the site of injection. Symptomatic treatment may be needed. Switch over from one brand of vaccine to the other. Shifting from one brand of TCV to other brand should not be encouraged as literature supports that good immunity is best achieved with same brand. 1.9.4.2 Intra-dermal (ID) regimens Concept of intra-dermal inoculation of anti rabies vaccines (IDRV) Intra-dermal regimens consist of the intra-dermal administration of a fraction of intramuscular dose of certain rabies vaccine on multiple sites. The vaccines used are same; however route, dose and site of administration differs. The use of intra-dermal route leads to considerable savings in terms of total amount of vaccine needed for full pre- or postexposure vaccination, thereby reducing the cost of active immunisation.
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Intra-muscular route (IM): Single bolus dose (1ml) of rabies vaccines/antigen when given by IM route gets deposited in the muscles. There after the antigen is absorbed by the blood vessels and is presented to antigen presenting cells which triggers immune response. Intra-dermal route (ID): Small amount (0.1ml) of Rabies vaccines/antigen is deposited in the layers of the skin at multiple sites. The antigen is directly presented to the antigen presenting cells (with out circulation/dilution in blood) at multiple sites triggering a stronger immune response. Mechanism of action of IDRV Intra-dermal inoculation is deposition of approved rabies vaccine (or antigen) in the layers of dermis of skin. Subsequently the antigen is carried by antigen presenting cells via the lymphatic drainage to the regional lymph nodes and later to the reticulo-endothelial system eliciting a prompt and highly protective antibody response. Immunity is believed to depend mainly upon the CD 4 + T- cell dependent neutralizing antibody response to the G protein. In addition, cell-mediated immunity has long been reported as an important part of the defense against rabies. Cells presenting the fragments of G protein are the targets of cytotoxic T- cells and the N protein induced T helper cells. The immune response induced by IDRV is adequate and protective against rabies. International Scenario Multiple clinical trials since early eighties proved that ID route of inoculation of Tissue Culture Anti rabies Vaccines (TCARV) is efficacious, safe and economical. It requires less quantity of vaccine which brings down the cost of immunisation and allows wider coverage in the existing quantity of vaccine. WHO recommended use of ID route of inoculation of TCARV in 1992. Thailand, Srilanka and Philippines have successfully adopted ID route of inoculation. Schedules approved by WHO for ID route 1. Updated Thai Red Cross Schedule (2-2-2-0-2): Vaccines approved for TRC schedule: Purified Vero cell Rabies Vaccine (PVRV) produced by Aventis Pasteur Purified Chick Embryo Cell rabies Vaccine (PCECV) produced by Chiron Vaccines Regimen: 2-2-2-0-2 i.e. one dose of vaccine, in a volume of 0.1ml is given intradermally at two different lymphatic drainage sites, usually the left and right upper arm, on days 0,3,7 and 28.
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2. Eight site intradermal regimen (8-0-4-0-1-1) Vaccines approved for eight site schedule: Human Diploid Cell Vaccine (HDCV) produced by Aventis Pasteur Purified Chick Embryo Cell rabies Vaccine (PCECV) produced by Chiron Vaccines Regimen: 8-0-4-0-1-1 i.e. one dose of 0.1 ml is administered intra-dermally at eight different sites (Fig.-5) (upper arms, lateral thighs, Fig 5: Sites for ID inoculation in suprascapular region and lower quadrant of 8-site ID regimen abdomen) on day 0. On day 7, four 0.1ml injections are administered intra-dermally into each upper arm (deltoid region) and each lateral thigh. Following these injections one additional 0.1ml dose is administered on days 28 and 90. General guidelines Vaccines to be applied by intradermal route of administration should meet the WHO requirements for production and control related to vaccines for intra-muscular use including an NIH potency test of at least 2.5IU per single dose (intramuscular). Immunogenicity and safety of vaccine in question should be demonstrated in appropriate human trials using WHO post-exposure regimens. In countries where national health authorities have approved the ID route for pre-post exposure prophylaxis, and for vaccines that can be used by that route, the vaccine package leaflet should include a statement indicating that the potency as well as immunogenicity and safety allow safe use of vaccine by ID pre- and post-exposure prophylaxis in addition to other relevant information as described in WHO requirements for vaccine production and control. The decision to implement economical ID post-exposure prophylaxis rests with government agencies that define rabies prevention and treatment policies in their own countries. Intradermal injections must be administered by staff trained in this technique. Vaccine vials must be stored at 2 to 8C after reconstitution The total content of reconstitute vial should be used as soon as possible, but atleast with in 8 hours. All the reconstitute vaccines should be discarded after 8 hours of reconstitution and at the end of the day Rabies vaccines formulated with an adjuvant should not be administered intra-dermally. Vaccine when given intra-dermally should raise a visible and palpable bleb in the skin. In the event that the dose is inadvertently given subcutaneously or intramuscularly, a new dose should be given intradermally.
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IDRV Vaccines and regimen approved for use in the country The following vaccines have been approved by National Authorities in first phase for use by intradermal route after assessing the safety, efficacy and feasibility data as recommended by WHO: Vaccines PVRV Verorab, Aventis Pasteur (Sanofi Pasteur) India Pvt. Ltd. PCEC Rabipur, Chiron Behring Vaccines Pvt. Ltd. PVRV Pasteur Institute of India, Coonoor PVRV Abhayrab, Human Biologicals Institute. Potency of approved vaccines: The vaccines should have stated potency of > 2.5 IU per IM dose, irrespective of reconstituted volume. The same vaccine is used for ID administration as per stated schedule. 0.1ml of vaccine, irrespective of reconstituted volume, is administered per ID site as per schedule below. Schedule Post exposure Prophylaxis Updated Thai Red Cross schedule, (2-2-2-0-2). This involves injection of 0.1ml of reconstituted vaccine per ID site and on two such ID sites per visit (one on each deltoid area, an inch above the insertion of deltoid muscle) on days 0, 3, 7 and 28. The day 0 is the day of first dose administration of IDRV and may not be the day of rabies exposure/animal bite. Pre-exposure prophylaxis: This involves injection of 0.1 ml of reconstituted vaccine per ID site on days 0,7 and 21 or 28. Maintenance of vaccine vial in use Use aseptic technique to with draw the dose Store in a refrigerator at 2C to 8C Reconstituted vaccines should be used as soon as possible or within 6 to 8 hours if kept at 2C to 8C. All unused reconstituted vaccine at the end of 6-8 hours must be discarded. Materials required A vial of rabies vaccine approved for IDRV Fig 6: 1ml syringe with hypodermic and its diluent. needle (Insulin syringe) 2 ml disposable syringe with 24 G needle for reconstitution of vaccine. Disposable 1 ml (insulin) syringe (with graduations upto 100 or 40 units) with a fixed (28 G) needle (Fig.6) Disinfectant swabs (e.g. 70% ethanol, isopropyl alcohol) for cleaning the top of the vial and the patients skin.
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Fig.7 Insertion of needle for ID inoculation
Fig. 8 Bleb raised on ID inoculation
ID injection technique Using aseptic technique, reconstitute the vial of freeze-dried vaccine with the diluent supplied by the manufacturer. With 1 ml syringe draw 0.2 ml (up to 20 units if a 100 units syringe is used or upto 8 units if a 40 units syringe is used) of vaccine needed for one patient (i.e. 0.1 ml per ID site X 2 sites 0.2 ml) and expel the air bubbles carefully from the syringe thereby removing any dead space in the syringe. Using the technique of BCG inoculation, stretch the surface of the skin and insert the tip of the needle with bevel upwards, almost parallel to the skin surface (Fig.7) and slowly inject half the volume of vaccine in the syringe (i.e. 0.1ml; either 10 or 4 units) into the uppermost dermal layer of skin, over the deltoid area, preferably an inch above the insertion of deltoid muscle. If the needle is correctly placed inside the dermis, considerable resistance is felt while injecting the vaccine. A raised papule should begin to appear immediately causing a peau d orange (orange peel) appearance (Fig.8). Inject the remaining half the volume of vaccine (i.e. 0.1ml; either 10 or 4 units) on the opposite deltoid area. If the vaccine is injected too deeply into the skin (subcutaneous), papule is not seen. Then the needle should be withdrawn and reinserted at an adjacent site and the ID vaccine given once more. Anti rabies treatment centres which meet the following criteria may use ID administration: Have adequately trained staff to give ID inoculation of anti rabies vaccine Can maintain cold chain for vaccine storage Ensure adequate supply of suitable syringes and needles for ID administration Are adequately well versed in management of open vial and safe storage practices.
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General Considerations for Implementation
The vaccine which is being used for IM administration can be used for ID administration after the vaccine manufacturers amend the label and package insert to indicate that the vaccine is fit for IM and ID route in designated centres. The manufactures should get the same approved by DCGI. A copy of protocol to generate Post Marketing Surveillance (PMS) data should be prepared and submitted by vaccine manufacturers for approval from DCGIs office. PMS should be maintained for minimum of two years. Since inclusion in Indian Pharmacopeia takes a long time, with the approval of vaccine from DCGI office and undertaking from the Pharmaceutical firms that the vaccine is fit for use by IM and ID route, the State health authorities may start use of ID route of inoculation of anti rabies vaccines.
Considering the recommendations on intradermal application of rabies by WHO and results of safety, efficacy and feasibility trials conducted in India, Drug Controller General of India (DCGI) approved the use of reduced dosage intradermal vaccination regimen for rabies post-exposure prophylaxis. The use of this route leads to considerable savings in terms of the total amount of vaccine needed for a full post-exposure vaccination, thereby reducing the cost of active immunisation. Precautions while using ID regimen: When the intradermal route is used, precautions include staff training, conditions and duration of vaccine storage after reconstitution, use of appropriate 1 ml syringe and short hypodermic needles. Vaccines to be applied by intradermal route of administration should meet WHO requirements for production and control related to vaccines for intramuscular use, including an NIH test potency of at least 2.5 IU per single (intramuscular) dose. In addition, immunogenicity and safety of the vaccine in question should be demonstrated in appropriate human trials using WHO/national post-exposure prophylaxis regimens. The vaccine package leaflet should include a statement indicating that the potency as well as immunogenicity and safety allow safe use of the vaccine for intradermal post-exposure prophylaxis, in addition to other relevant information as described in the WHO requirements for vaccine production and control.
1.9.5 Post exposure therapy for previously vaccinated persons

Managing re-exposure following post-exposure treatment with TCV: If reexposed, persons who have previously received full post-exposure treatment (either by IM or ID route) with a potent cell-culture vaccine should be given only two booster doses, intramuscularly/intra-dermally (0.1 ml at two sites) on days 0 and 3, but no rabies immunoglobulin.
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Managing exposure following pre-exposure prophylaxis with TCV: If after recommended pre-exposure prophylaxis, a vaccinated person is exposed to rabies, a proper wound toileting should be done and two IM/ID (0.1 ml at two sites) doses of Tissue Culture Vaccine be given on days 0 and 3. Treatment with RIG is not necessary. Managing re-exposure following post exposure treatment with NTV: Persons who have previously received full post-exposure treatment with NTV should be treated as fresh case and may be given treatment as per merits of the case.
1.10 PRE-EXPOSURE PROPHYLAXIS

Pre-exposure prophylaxis may be offered to high risk group like laboratory staff handling the virus and infected material, clinicians and para-medicals attending to hydrophobia cases, veterinarians, animal handlers and catchers, wildlife wardens, quarantine officers and travellers from rabies free areas to rabies endemic areas. Pre-exposure vaccination is administered as one full dose of vaccine intramuscularly or 0.1 ml intradermally on days 0, 7 and either day 21 or 28. Laboratory staff and others at high continuing risk of exposure should have their neutralizing antibody titres checked every six months. If it is less than 0.5 i.u./ml a booster dose of vaccine should be given. Such individuals on getting exposed to rabies virus after successful pre-exposure immunisation require only two booster injections of vaccine given on days 0 and 3 without any anti rabies serum.
1.11 PREVENTION AND CONTROL OF RABIES

Prevention and control of rabies includes three major components: Prevention of human deaths due to rabies Reducing the transmission of disease in animals Maintaining Rabies free areas free of the disease
1.11.1 Prevention of human deaths due to rabies

Prevention of disease in humans can be achieved by providing timely and appropriate post exposure prophylaxis to all exposed. Availability of safe and effective tissue culture vaccines, and safe immunoglobulins nearly assure cent percent protection from the disease. Health professionals should be trained regarding rabies and appropriate animal bite management. Awareness should be created amongst the general community to seek timely and appropriate post exposure treatment. In endemic country like India, all animal bite victims
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should receive post exposure prophylaxis as per merits of the exposure without waiting for laboratory results or time period of observation of the biting animal.
1.11.2 Reducing the transmission of the disease

Control of canine rabies poses a major challenge to public health experts throughout the world. However, the impact that vaccines have had in dramatically reducing in the incidence of canine rabies in North America, Western Europe and Japan clearly holds promise for prevention and control of rabies. Canine rabies control should incorporate three basic elements : Epidemiological surveillance Mass vaccination Dog population management Epidemiological surveillance: Surveillance of rabies is the basis for programme of rabies control. Epidemiological data should be collected, analysed and disseminated between sectors and different administrative levels. Emphasis of surveillance should be on laboratory confirmation and effective reporting of human and animal rabies cases. Attempts should be made to isolate viruses for characterization of prevalent strains. The number of people seeking and receiving post-exposure prophylaxis should be reported in order to provide epidemiological information on disease burden and to evaluate effectiveness and cost benefit of rabies control programme. Canine mass parentral vaccination campaigns: Mass parentral vaccination campaigns have been the most effective measure for controlling canine rabies. At least 70% of the dog population in each community should be vaccinated in areas where canine rabies is endemic. High vaccination coverage can be attained through strategies consisting of educational campaigns, intersectoral co-operation, community participation and availability of quality vaccine. Dogs of all age groups should be immunised. The campaign should be conducted annually. For mass parenteral vaccination campaigns only inactivated and adjuvenated rabies vaccines should be used. Registration and identification of vaccinated dogs should be done. The use of colour tags or plastic collars may be used for identification of vaccinated dogs. This helps in evaluation of vaccination coverage rate and identify unvaccinated dogs for supplementary follow up measures. Vaccination campaigns can be organised by any of the following mentioned approaches: House to house visits Fixed vaccination pets Temporary vaccination posts set up by mobile teams
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Supplementary measures - Oral Vaccination of Dogs (OVDs): Oral vaccines are live attenuated vaccine strain (SAD stain) which are incorporated in the bait. The bait can be made species specific. Although the preferential vaccines for dog immunisation is parenteral vaccines, oral vaccines can be used whenever there is high population of free roaming and poorly supervised dogs which are difficult to access. Since these are live attenuated vaccines, they should be distributed under strict supervision in countries like India where animals and children of poor socio-economic status share similar ecological niche. These vaccines are expensive as compared to parenteral vaccines. Hence strategies for vaccine bait distribution must be carefully worked out. Dog population management and animal birth control (ABC) programme: Killing or removal of dogs alone does not have any significant effect on dog population density or spread of rabies. This may be because of high turnover of dogs and increased survival rate. However, three practical methods of dog population management have been recommended by WHO. These are movement restriction, habital control and reproduction control. Animal birth control programmes when conducted in a defined locality covering sizeable population of dogs has shown encouraging results with reduction in size of street dog population.
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LABORATORY DIAGNOSIS OF RABIES: TECHNIQUES

2.1 COLLECTION, PRESEVATION, PACKING AND TRANSPORTATION OF SPECIMENS
Acute infectious nature of rabies does not require further elaboration. Therefore, in the collection of specimens from suspected cases of rabies-human or animal, it must be borne in mind that highly dangerous material is being handled. It is imperative to follow all precautionary measures during collection, packing, transportatioin and handling of specimens to avoid any serious mishap.
2.1.1 Specimens from humans

From a clinical case (hydrophobia) the antemortem specimens that may be collected include saliva, corneal smear, skin biopsy, hair follicles, blood and cerebrospinal fluid. Postmortem specimens shall be usually of brain or spinal cord and sometimes other organs too. The specimens are collected as follows: Saliva/Sputum Saliva is collected from under the tongue: a) Wet a sterile cotton swab with tissue culture medium or physiological saline and remove excess medium by squeezing on the sides of the vial. b) Swab under the tongue, rinse in the tissue culture medium or physiological saline containing 2% normal horse serum (NHS). c) Take another swab similarly and make two smears each on clean labelled glass slides. d) Air dry the glass slides for 10 minutes. e) Discard the swabs in suitable disinfectant. f) Treat the slides immediately with chilled acetone and process or wrap in paper and despatch to the laboratory. Often due to dehydration there is very little saliva in the mouth. The patient, if responsive, may be asked to cough and spit in petri dish or beaker. Mix the
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sputum with few ml of tissue culture medium or 2% NHS in physiological saline and transfer to screw capped vial. Corneal Smear a) Retract the eye lids with thumb and one finger and press a clean marked slide against the cornea. b) Prepare two smears on each slide taking care to apply sufficient pressure to get the smear. c) Avoid exerting too much pressure as it may damage the eye. d) Air dry the smears for 10-15 mins at room temperature. e) Treat with chilled acetone and process further Skin Biopsy With very fine sharp scissors collect small pieces of skin from the site of bite and the face near the mandible. Preserve in vial containing 50% glycerol saline (prepared by mixing equal volume of glycerol and physiological saline and sterilised by autoclaving). Cerebrospinal Fluid (CSF) The CSF in acute phase of the disease is processed for isolation of the virus and in the later phase for antibodies. It is collected by lumbar puncture. Usually no preservative is used but, if required, 50% glycerol saline may be used. Blood Acute phase venous blood specimen is collected as soon as possible with the usual aseptic precautions. If the patient survives for several days, a second sample is taken. In case the patient recovers another blood sample is taken before discharging the patient. Brain The brain is collected at autopsy. Many times the relatives do not agree for a full postmortem. In such cases Vim-Silverman needle may be used to collect a small piece of brain sample which is then put in 50% glycerol saline.
2.1.2 Collection of specimens from suspected rabid animals

The specimen useful for proper diagnosis of rabies in animals are mainly brain and salivary glands. Though it is risky to collect antemortem specimen, if required, saliva and corneal smears may be collected as already described. The better course, however, is to permit the animal to die natural death unless otherwise required (see microscopic examination). Facilities for removal of the animal brain and salivary glands are not available in the laboratory and hence the whole brain or salivary glands should be sent to the laboratory after postmortem.
Laboratory Diagnosis of Rabies: Techniques
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If it is not possible to send the whole brain, pieces from Ammons horn of hippocampus, cerebrum, cerebellum, pons and medulla may be included.
2.1.3 Preservation
If possible the samples of brain and salivary glands may be sent in widemouth leakproof containers preserved on ice. However, if the samples are to be sent long distance these may be preserved by use of following: a) 10% formal saline/Zenkers fluid for half of the brain b) 50% glycerol saline for other half of the brain and salivary glands. c) Tissue culture medium 2% NHS saline for saliva, CSF,urine etc.
2.1.4 Labelling
All the specimens e.g. slides,vials must be labelled with number of specimens, name of the patient, or species of the animal,type of preservative used etc. Permanent markers should be used. The parcels should also be labelled properly. Information to be enclosed a) Hydrophobia: Name, Age, Gender, Treatment taken, Exposure to animal etc. may be enclosed. b) Animal: The species and breed of animal, contact with other animal, symptoms, mode and date of death, vaccination status etc.
2.1.5 Packing
a) b) c) d) It should preferably be wide mouth leakproof plastic containers. Seal the mouth of the container with tape or sealing paraffin. Pack in plastic bags and put in thermocol box with sufficient ice. If sending by post pack in sturdy wooden boxes with sufficient packing material (preferably absorbent cotton/saw dust/paddy husk).
2.1.6 Transportation
a) b) By courier By air/by post.
Utmost urgency should be exhibited in transportation of these specimens because any undue delay, especially in tropical climates, shall wither away the cooling effect of ice and result into putrefaction of the sample making it unsuitable for the diagnosis.
2.2 LABORATORY TESTS

Many tests have been reported from time to time for the detection of rabies antigen/ virus, assessment of rabies antibodies, study for the morphology and growth characterisitics of rabies virus. The use of these tests in a diagnostic laboratory
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depends on the availability of appropriate facilities. The tests may be for detection of rabies antigen in ante-mortem specimens like corneal smear, CSF or saliva etc. or postmortem specimens which include brain and salivary glands.
2.3 PRINCIPLES AND PROCEDURES OF COMMONLY USED TECHNIQUES

2.3.1 Sellers Staining for detection of Negri Bodies
Principle Many virus infections are associated with presence of inclusion bodies in the infected cells. These inclusion bodies may be intranuclear or intracytoplasmic acidophilic or basophilic in nature depending on their reaction with the stains used. The presence of rabies infection can be demonstrated by the presence of intracytoplasmic inclusion bodies commonly known as Negri bodies. The intracellular nature of Negri bodies may be altered in impression smears due to the rupture of cells when these may be found both intracellular and extracellular. The Negri bodies are acidophilic in staining reaction and are seen as pink to purplish-pink in colour in differential stains that use basic fuchsin or eosin with methylene blue as their base. Reagents and Equipment a) Sellers Stain b) Light microscope c) Glass slide (5 x 7.5 cm and 2.5 x 7.5 cm) d) Scissors and forceps e) Filter paper Preparation and Staining of Slides The brain sample received in the laboratory may be unpreserved or preserved in 50% glycerol saline or 10% formol saline. For demonstration of Negri bodies the unpreserved specimen can be processed as such. The formaline preserved specimen can be used for histopathology. The glycerinated specimen can be used for making impression smears and biological test like unpreserved specimen but the presence of glycerine interferes with the adherence of tissue to the glass slide. Therefore, the glycerine must be washed off by immersing the tissue pieces in physiological saline for 30 minutes or more during which at least three changes of physiological saline are required to wash off the glycerine. The procedure for preparation of smear and its staining is as follows: a) With a pair of scissors make a longitudinal incision about 3-5 cms from the occipital pole into the dorsal surface of cerebral hemisphere approximately 2 cm lateral to midline of the brain.
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b) c) d)
Cut through to lateral ventricle. Widen the opening to expose the hippocampus on ventricle floor. The hippocampus can be seen as white, glistening, semi cylindrical and curved body. e) Cut out a small piece of hippocampus (0.5-1 cm) and place it on a spatula/ filter paper with cut surface facing upwards. f) Place the filter paper on glass slide (5 x 7.5cm) g) Lightly sponge the cut surface with the edge of a filter paper to remove blood. h) Press a clean microscope slide on the tissue piece on spatula/filter paper to get an impression smear. i) Make at least 3 smears on each slide. j) While the smears are wet, flood the smear with working stain. k) Stain for 2-3 seconds. l) Quickly wash with tap water by gently flushing the slide. m) Air dry the smear. n) Examine under oil immersion. Observations Following may be observed (Fig. 9) Nerve cells Blue cytoplasm and dark blue nucleus Stroma Pink Erythrocytes Copper coloured Negri bodies Magenta to dark red with dark blue or black inner granules. Similarly smears can be prepared from cortex and cerebellum and examined for Negri bodies.
Fig 9: Sellers stain: Negri Bodies
Negri Bodies
Other Inclusion Bodies Some of the inclusion bodies in normal brains of cats, foxes, mouse etc. may appear similar to the Negri bodies to an inexperienced worker. The inclusion body due to some other diseases in dogs e.g. distemper may also stain similar to Negri bodies. However, these can be differentiated from Negri bodies on the basis of following features:
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Negri bodies a) b) c) d) e) Oval, round or elongated Size 3-20m Presence of basophilic inner granules and heterogenous matrix Less refractile Mostly abundant in hippocampus
Other Inclusion bodies Oval or round Small to large Homogenous matrix without granules More refractile Rare in hippocampus
2.3.2 Fluorescent Antibody Test (FAT)

Principle Rabies specific antibodies and antigen when mixed and kept under optimum conditions combine to form an antigen-antibody complex. This complex is not visible to naked eye. To detect this complex use is made of dyes like fluorescein isothiocynate (FITC) which emit fluorescence when exposed to ultraviolet rays. The FITC is tagged with anti rabies antibody to form the conjugate. If the detection of antigen is a one step procedure, it is called a direct fluorescent antibody test (FAT). If more than two steps of combination of antigen and antibodies are required for detection, it is known as indirect immunofluorescent test (IFAT) and is employed for detection of rabies antibodies. Reagents and Equipment a) Fluorescent Microscope (Box; Fig 10 & 11) b) Incubator c) Anti rabies FITC conjugate (Pretitrated) (Lyophilised Absorbed Rabies antinucelocapsid conjugate), Bio-Rad, Cat. No. 72112. Lyophilised: 4 x 3ml vials. d) Phosphate buffer saline pH 7.5
Fluorescent microscope is a light microscope used to study properties of organic or inorganic substances using the phenomena of fluorescenceand phosphorescence instead of, or in addition to, reflection and absorption. Principle: A component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore (such as Green fluorescent protein, fluorescein or DyLight 488). The specimen is illuminated with light of a specific wavelength (or wavelengths) which is absorbed by the fluorophores, causing them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is separated from the much weaker emitted fluorescence through the use of an emission filter. Typical components of a fluorescence microscope are the light source (Xenon or Mercury arc-discharge lamp), the excitation filter, the dichroic mirror (or dichromatic beamsplitter), and the emission filter (see figure below). The filters and the dichroic are chosen to match the spectral excitation and emission characteristics of the fluorophore used to label the specimen. Most fluorescence microscopes in use are epi-fluorescence microscopes (ie : excitation and observation of the fluorescence are from above (epi) the specimen). The cost of Fluorescent Microscope ranges between Rs 500000/- to 600000/Laboratory Diagnosis of Rabies: Techniques
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Detector Emission Filter
Dichroic Mirror
Excitation Filter
Obj
Specimen
Fig 10: Fluorescent Microscope
Fig 11: Ray diagram to show the working of a Fluorescent Microscope
Procedure a) Prepare two impression smears of approximately 1 cm diameter about 1.5 cm from each end of the labelled slides. Prepare at least 4 slides each from the cerebrum, hippocampus, midbrain, cerebellum and medulla of brain. b) Air dry the smears for 25-35 minutes at room temperature. c) Immerse the slides in coplin jars containing chilled acetone in a deep freeze at 20C to 25C. d) Keep for 4 hrs to overnight. e) Drain off the acetone and store slides at 20C till stained. f) Take out the impression smear slides from deep freeze and dry at room temperature for 20 minutes. Include known positive and negative smears also. g) Mark the outline of the smears with grease marking pencil or wax pen. h) Flood the smear with ready to use conjugate with the help of a pasteur pipette. i) Place the slides in a chamber with moist filter paper at the bottom. j) Cover the chamber and keep at 37C for 30 minutes. k) Wash the slides in 0.01 M phosphate buffered saline (pH 7.5) for 5 minutes. l) Repeat washing. m) Remove from buffer and dip in a jar of distilled water for 5 minutes with gentle shaking (Washing can also be performed by gentle agitation using a magnetic stirrer). n) Remove from distilled water and dry the slides at room temperature. o) Mount in 90% buffered glycerol (pH 9.0). p) Examine under a microscope with ultraviolet source of light (fluorescent microscope), the known positive, known negative and test slides. A dark ground condenser is useful.
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Observations Rabies antigen is seen as fine dusty particles emitting bright to dull yellowish green fluorescence. (Fig 12)
2.3.3 Mouse Inoculation Test (MIT)/Biological Test (BT)

Introduction The growth of rabies virus, just like any other virus takes place only in living tissues. These may be animals, developing chick embryo or tissue culture. Rabies virus has been found to be pathogenic to all mammals when given by intracerebral route. However, the animals commonly used for experimental studies of rabies are the mouse, rat, guinea pigs, hamster, rabbit and dog. The white laboratory mice are susceptible to infection and are easy to handle in routine laboratory examination of specimens. Suckling mice are more susceptible than the adult but a regular supply of these may not be possible in all the laboratories. The presence of rabies virus in any specimen can be detected by intracerebral inoculation of specimen into mice and observing the animals for sufficient period for the development of sickness and death due to rabies. Reagents and Equipment a) 2% Normal horse serum (NHS) in distilled water. b) 3-4 weeks old mice and mice cages. c) Centrifuge.
Fig 12: Fluorescent Antibody Test showing apple green fluorescence of rabies antigen in brain tissue of rabid animals.
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d) e) f)
Centrifuge tubes. Pestle mortar, tissue grinder or omni mixer. Scissors and forceps.
Procedure Brain a) Aseptically collect 3-4 gm or pieces Fig 13: Intra cerebral inoculation of mice of brain tissue of approximately 1 cm diameter cut out from different areas of the brain, i.e. hippocampus, cerebrum, cerebellum, medulla, pons etc. b) By weighing calculate the exact weight of the tissue pieces. c) Homogenise these pieces to make a fine paste with the help of a pestle mortar/tissue grinder/ electric grinder/omnimixer. d) Add enough chilled distilled water containing 2% inactivated normal horse serum (serum should be collected from horses not vaccinated against rabies) to make a 10% suspension. Mix thoroughly while adding the diluent. e) Transfer to a sterile 15 ml centrifuge tube. f) Centrifuge at 1000-1500 rpm for 5 minutes. g) Collect the supernatant with the help of pipette in a Bijou bottle/half tube kept in ice bath. h) Add 100 units/ml penicillin and 50g/ml of streptomycin and keep for 30 minutes. i) Inoculate into 3-4 weeks old white mice by intracerebral route with long 26/27 gauze needle in 0.03 ml dose/mice. (Fig 13) Use at least eight mice for each inoculum. j) Observe for 21 days for signs like roughening and loss of lustre of the fur, tremor, hyperexicitability, arching of the back, convulsions, paralysis of the hind legs and death. k) Check any mouse dying after 72 hours for presence of Negri bodies in brain by Sellers staining or antigen by FAT. Salivary Gland Grind the salivary glands with the help of sterile sand or sterile coarse glass powder in a pestle and mortar and repeat the processes e to k as described above.
2.3.4 Rabies Tissue Culture Infection Test (RTCIT)

RTCIT is performed for isolation of rabies virus in cell lines (Murine Neuroblastoma 2A).
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Reagents and Equipment a) Murine Neuroblastoma 2A cell lines (Fig 14) b) Culture tubes c) MEM Medium + 10% FCS d) TPVG solution Brain tissue preparation a) Take approximately 0.5 gm of tissue from the hippocampus of the brain. b) To this add 5 ml of PBS containing antibiotic to make a 10% w/v suspension. c) Vortex mix vigorously and allow to settle for at least 1 hour at 4C. d) Withdraw the upper clear layer and dilute 10-fold with MEM medium supplemented with 10% fetal calf serum. e) Add 100 units/ml penicillin and 50g/ml of streptomycin. Shaken or vortex mix vigorously and allow to settle for at least 1 hour at 4C. CSF Can be inoculated directly after treatment with antibiotics. Cell suspension Murine Neuroblastoma 2A cells are grown for maintenance at 35 37C in 25 cm2 plastic culture bottles/tubes containing 5 ml of MEM-10 media. For inoculation of samples, the subculture is done in 25 cm 2 culture tube with 5 ml MEM supplemented with 10% FCS media.
Fig 14 : Murine Neuroblastoma 2A Cell lines (10x 40 magnification) Laboratory Diagnosis of Rabies: Techniques
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Procedure a) For each specimen take 2 ml of cell suspension in a culture bottle. b) Add 4 ml of the 1% suspension (in MEM + 10% FCS) to each of the tubes containing cells. c) With each batch use a known positive control and negative control. d) Incubate the tubes at 37C for four days. e) The tubes should be checked daily for contamination. f) After four days of growth, take Teflon slides and keep in the acetone for 2 5 minutes at room temperature. g) Air dry the Teflon slides in laminar flow. h) The fluid is separated in sterile tubes from infected cell culture tubes and stored at 70C in liquid Nitrogen. Approximately 0.5 ml fluid is left in the cell culture tube. i) Gently tap the tubes and by repeated forceful pipetting attempt to detach the cells. j) Inoculate the resuspended cells with the help of the Pasteur pipettes in the wells of Teflon coated slides including the test samples cells, positive control and negative control. k) Air dry the drops for 2 3 hours in the laminar flow. l) Immerse the slides in coplin jar containing chilled acetone in a deep freeze at 20C to 25C for 4 hours to overnight. m) Take out the slides from chilled acetone and dry at room temperature for 20 minutes. n) Pour 1 2 drops of rabies antinucleocapsid FITC conjugate with the help of Pasteur pipette. o) Place the slides in moist chamber at 37C for 30 minutes. p) Wash the slide in 0.01 M phosphate buffer saline (pH 7.5) twice for 5 minutes each. q) After this give two washings with distilled water for 5 minutes each. r) Take out the slides from distilled water and dry at room temperature. s) Mount in 90% buffered glycerol. t) Examine the slides under a fluorescent microscope.
2.3.5 Polymerase Chain Reaction (PCR)

Principle Rabies virus is an RNA virus and hence before amplification the target RNA segment is converted to c-DNA with the help of enzyme reverse transcriptase (RT). c-DNA is then amplified using Taq polymerase. This is called RT-PCR. Primers For diagnosis of rabies and rabies related isolates, regions on N gene are selected as primers since N gene is highly conserved region on the viral genome and it is transcribed in the highest amounts during multiplication.
36
N1 (+) sense : (587) 5-TTT GAG ACT GCT CCT TTT G-3 (605) N2 (-) sense : (1029) 5 CC CAT ATA GCA TCC TAC 3 (1013) For typing and molecular studies the highly variable area, rabies pseudogene is selected. It is a non protein-coding region and is highly susceptible to mutation and therefore is most suitable for detecting strain variation and closely related isolates. G (+) sense : (4665) 5-GAC TTG GGT CTC CCG AAC TGG GG-3 (4687) L (-) sense : (5543) 5 CAA AGG AGA GTT GAG ATT GTA GTC3 (5520) Procedure RNA extraction c-DNA synthesis c-DNA amplification Detection of PCR products RNA extraction Homogenise the infected brain tissue with lysine buffer to lyse the cells, cell membranes and nuclear membrane. This can be done by manual method, 1-phenol, 2 phenol/chloroform (1:1) and 1-chloroform as described by Tordo et al or by RNA extraction kits (trizol, Life technologies) c-DNA synthesis Anneal the RNA (1g) with the N1 primer (100g) at 65C for 3 minutes. After chilling on ice, add murine leukemia virus reverse transcriptase and incubate for 90 minutes at 42C. c-DNA amplification by PCR To the c-DNA reaction, add PCR buffer containing 100g of N2 primer and 2 units Taq polymerase and subject it to five initial cycles of denaturation (D) 60 seconds at 94C, annealing (A) 90 seconds at 45C and elongation (E) 90 seconds at 72C followed by 30 additional cycles of D for 30 seconds at 94C, A for 20 seconds at 50C and E for 60 seconds at 72C. Carry out the final elongation at 72C for 10 minutes. Detection of PCR product by polyacralmide gel electrophoresis To 1.2% agrose gel add 0.5g/ml. of ethidium bromide and pour in trough with combs to make wells. Add approximately 250 ml of 1X electrophoresis buffer to the reservoir tank. Mix 20l of PCR product with 2l of loading dye (containing bromophenol blue, xylene cynol and glycerol) and load in the well. Add 100 bp DNA ladder to the first lane. Switch on the power supply, adjust to 100V and leave it for electrophoresis. Switch off the power supply when the dye front is about 0.5 cm from the positive end. View the gel in UV trans illumination. DNA segment can be seen at 443 bp.
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While using G & L primers for typing and moleculer studies DNA segment can be seen at 880 bp. (Fig 15)
Lane I II III IV V VI VII
Precautions Always wear gloves while handling the samples and Fig 15: reagents Agarose Gel showing amplicons from RT-PCR of Frequently change gloves Rabies Street Strain Virus RNA. while handling RNA A. Lane I - Positive sample showing band of 880 b.p. Carry out high-speed b. Lane II - Negative sample c. Lane III - Positive sample showing band of 880 b.p. mixing and centrifugation d. Lane IV - Negative sample procedure in tightly e. Lane V - Positive control (Paw RNA) closed container and in a f. Lane VI - Negative control biological safety cabinet. g. Lane VII - 100 b.p. DNA ladder Avoid mouth pipetting Always include a positive control (infected mouse brain) a negative control (uninfected mouse brain) and a negative PCR control (Add water instead of the template) in each run of PCR. Uses PCR is probably not a very practical diagnostic tool, its main use is for strain differentiation while using it as an epidemiological marker.
2.3.6 Modified Counterimmuno Electrophoresis (CIEP) Serum Neutralisation

Principle Antigen-antibody when permitted to diffuse through gels have a property of forming precipitin lines at the point where these meet in optimal concentrations and this reaction is specific. The reaction can be enhanced and expedited by use of electrophoresis. The immunoelectrophoresis technique reduces the time required for formation of these precipitin lines. Anti rabies antibodies can also be assessed by use of counter immunoelectrophoresis serum neutralisation test where residual amount of antigen is detected by using a known positive anti rabies serum as indicator serum. Reagents and Equipment a) Electrophoresis apparatus b) Glass slides 5 cm x 7.5 cm c) Barbitone buffer pH 8.2 d) Phosphate buffer saline (PBS) pH 7.2
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e) f) g) h) i)
Beta-propiolactone (BPL) Merthiolate Agarose Mice/suckling rabbit Waterbath
Procedure Preparation of Agar gel slides a) Prepare 0.9% agarose in 0.5 M Barbitone buffer (pH 8.2) containing 1:10,000 methiolate b) Apply a thin coat of the agarose on to clean 5 x 7.5 cm glass slides c) Dry the slides at room temperature d) Add 8 ml of melted agarose on to each slide e) Allow to gel at room temperature f) Place in a petridish and keep at +4C for 30 minutes. g) Cut out wells of 6 and 3 mm each in parallel rows at 8 mm distance. The distance between wells of same row being 10 mm Preparation of Titration of Antigen a) Prepare a 40% (w/v) suspension of Challenge Virus Strain (CVS) infected brain mouse/suckling rabbit in 0.01M PBS (pH 7.2) b) Add BPL to get a final concentration of 1:3000 c) Incubate for 2 hours at 37C in a waterbath d) Keep at +4C overnight e) Next day remove an aliquot (1.0 ml) for checking inactivation by inoculating intracerebrally in six mice. f) Centrifuge at 17,000 g for 10 minutes (14000 rpm) g) Collect and supernate and discard the deposit h) Add merthiolate in final concentration of 1:10000 i) Heat in a waterbath at 56C for 30 minutes. j) Distribute in small volumes i.e. 1 ml/vial and store at +4C. It can be kept for six months. Titration a) Make 5 fold dilution of the antigen starting from 1:5 in 0.01 M PBS b) Add respective dilutions to 6 mm wells on 4 slides c) Make 1:5, 1:10, 1:20 and 1:40 dilution of equine hyperimmune anti rabies serum (200 IU/ml) in 0.01M PBS d) Add 1:5 dilution of serum to all the 3 mm wells of slide No 1. e) Add similarly 1:10, 1:20 and 1:40 dilutions of serum to wells of slide No. 2 to 4. f) Place in the electrophoresis chamber and run the current for 2 hours at 15mA/slide
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g) h)
Remove slides, read and note dilutions giving sharp preciptin band for finding out the optimum dilutions. Store optimum dilution of equine hyperimmune anti rabies serum at -25C for use as indicator serum
Test procedure a) Inactivate the sera at 48 - 50C for 30 minutes in a waterbath b) Dilute each serum in 0.01 M PBS (pH 7.2) to make neat, 1:5, 1:10, 1:20 and 1:40 dilutions c) Mix each dilution of serum with equal volume of optimal dilution of antigen (0.1 ml + 0.1 ml) in dilution tubes d) Shake well and incubate at 37C for one hour in a waterbath e) Cut out 6 mm wells in the agar gel slide f) Add serial final dilutions i.e. 1:2, 1:10, 1:20, 1:40 and 1:80 of serum to 6 mm wells. Care should be taken not to overflow the well g) Place in the electrophoresis chamber and run for 45 minutes at 10V/slide h) After the run remove agar from 3 mm wells and add indicator serum to these wells i) Continue electrophoresis for another 120 minutes at a constant current of 15mA/slide j) If the electrophoresis apparatus is not fitted with cooling device maintain the temperature of gel by periodically moistening the connecting filter paper k) After the run read the slides against light using a hand lens l) The slides may be washed and stained by using a portein slain Observations Fig 16: Immunoprecipitation lines Presence of bands against a particular (Amido black staining) dilution shows residual amount of antigen i.e. absence of anti rabies antibodies. (Fig 16)
2.3.7 Enzyme Linked Immunosorbent Assay (ELISA)

Principle ELISA is performed for detection and titration of IgG and rabies virus glycoprotein in human and animal (dogs, cats, foxes and raccoons) serum and plasma. It is based on the use of a solid phase enzyme immunoassay technique by indirect ELISA. A micro-plate is coated with rabies glycoprotein extracted from the inactivated and purified virus membrane. The enzymatic conjugate consists of a protein A from Staphylococcus aureus coupled with
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peroxidase. Positive controls, calibrated against WHO standards, allow the qualitative or quantitative determination of anti rabies antibody titre in the serum ofrplasma. The test is carried out using commercially available kit (PLATELIATM RABIES II kit, BIO-RAD, Ref: 3551180). Implementation of the test comprises the following reaction steps: The unknown sera as well as the calibrated positive controls or the quantification standards are distributed in the glycoprotein coated wells of the microplates. During incubation of one hour at 37C anti rabies antibodies present in the sample bind the glycoprotein coated to the microplate wells. After incubation, unbound antibodies and other serum proteins are removed by washings.
The conjugate (protein A labeled with peroxidase) is added to the microplate wells. During a second incubation of one hour at 37C, the labeled protein A binds to the anti rabies-antibody-antigen complexes attached to the microplate wells. The unbound conjugate is removed by washings. The presence of immune complex is demonstrated by the addition of a solution containing a peroxidase substrate and a chromogen including a colour development reaction. After 30 minutes incubation at room temperature, the enzymatic reaction is stopped by addition of a solution H2SO4 1N. The optical density reading obtained with a spectrophotometer set at 450 620 nm is proportional to the amount of anti rabies antibodies present in the samples. A standard curve is constructed out of the quantification standards obtained by serial dilutions of the calibrated positive controls. The optical density values for the unknown samples are compared with the positive controls. Sera titres in quantification tests are obtained after a direct reading on the standard curve and are expressed as equivalent units per ml (EU/ ml), unit equivalent to the international units defined by seroneutralisation). Reagents and Equipment a) Sprectrophotometer or ELISA reader b) Patient serum c) Microplate: 12 strips of 8 wells sensitised with the rabies virus glycoprotein. d) Wash solution: 10-fold concentrated Tris NaCl buffer. e) Negative control: Non reactive control TRIS EDTA. f) 0.5 EU/ml positive control (human): 0.5 EU/ml calibrated positive control. Glycerine buffer containing human anti rabies IgG and BSA. g) 4.0 EU/ml positive control (human): 4 EU/ml calibrated positive control. Glycerine buffer containing human anti rabies IgG and BSA.
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h) i) j) k) l)
Sample diluent: Ready to use TRIS-EDTA buffer for sample dilution. Conjugate: Solution containing Protein A-Peroxidase and purified bovine protein. Peroxydase substrate buffer: solution of citric acid and sodium acetate containing 0.015% H2O2 and 4% dimethylsulfoxide (DMSO). Chromogen: Tetramethylbenzidine (TMB) solution 0.25%. Stop solution: 1N sulphuric acid solution.
Procedure Strictly to be followed as per manufacturers instructions in the kit insert.
2.3.8 Rapid Fluorescent Focus Inhibition Test (RFFIT)

Principle The Rapid Fluorescent Focus Inhibition Test (RFFIT) is a rabies virus neutralisation test carried out in cell culture to determine the rabies virus antibody level in human or animal sera. Immunofluorescence staining is used as an indicator of viral growth. The RFFIT takes 20 hours and is sensitive and accurate in the hands of well-trained laboratory personnel. The RFFIT is performed by mixing dilutions of test sera with a constant amount of rabies virus (50-50% Fluorescing Foci Doses (50 FFD50/0.1 ml) in a multi chambered slide. After allowing the mixture to react in a CO2 incubator at 37C for 90 minutes, mouse neuroblastoma (MNA) cells in Eagles minimum essential medium with 10% fetal bovine serum (MEM-10) are added to each serum-virus mixture. The serum-virus-cell cultures are incubated for 20 hours in a CO2 incubator at 37C. The cultures are removed from the incubator, washed, fixed and then stained with a rabies conjugate and observed under a fluorescence microscope for the presence of fluorescing cells; 20 microscopic fields (160X 200X) are read for each serum dilution and compared against the virus control (50 FFD50/0/1 ml) which should contain 18 to 20 field with fluorescing cells. A reduction of 50% or more in the number of fields containing fluorescing cells indicates that there was enough antibody present to neutralise the virus. Reagent and Equipment A. a) b) c) d) Reagents Conjugate Anti rabies nucleocapsid conjugate (FITC) Acetone Gelatin Cell Culture Media : Minimum Essential Medium containing Earles salts but no L. Glutamine or sodium bicarbonate
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e) f) g) h) i) j) k) l) m) n)
o) B. a) b) c) d)
FBS Distilled water Fungizone Hepes Buffer L. Glutamine Antibiotic Antimycotic solution Sodium bicarbonate Trypsin Versene Phosphate Buffered Saline (PBS) Formula number 4550; 0.01M, pH 7.4 + 0.5 Formula number 4588; 0.01M, pH 7.4 7.6 Dimethylsulfonide (DMSO) Glassware and plastic ware Cell culture flask (Fig 16) Lab-Tek Chamber slides with cover slip (Fig 17) Disposable sterile plastic pipettes Centrifuge tube
Fig 17 Culture flasks
Fig 18 Inoculation in tissue culture chambered slides
e) f) g) C. a) b) c)
Cryovials Millipore disposable filter (pore size 0.22) 250 ml; 500 ml Hemacytometer Equipment Biological safety cabinet CO2 incubator Freezer
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d) e) f) g) D. a) b) c)
Inverted microscope Fluorescent Microscope Refrigerated centrifuge (0 8000 rpm) Waterbath Standards and references Mouse Neuroblastoma Cells (MNA) Rabies Challenge Virus Strain (CVS) Reference serum standards diluted 2 I.U./ml.
Procedure Preparation of seed virus a) Trypsinise a three day old 150 ml culture flask of MNA cells b) Resuspend cells in 3 ml of 10% MEM with FCS (30 x 106 cells) c) Add 1 ml of virus (CVS) 10 x 106 (Infectious units) d) Incubate for 15 minutes at 37C e) Add 10 ml of MEM with 10% FCS f) Centrifuge at 1000 rpm for 10 minutes, discard the supernatant g) Add 30 ml of MEM 10 h) Transfer to 150 ml flask i) Gently rock this flask j) Take 6 ml of suspension from the flask k) Put 0.2 ml suspension in each well of 3 lab - tek chamber slides for 20 hours, 40 hours, 64 hours l) Incubate the flask and slides at 37C and 38C in a humidified incubator with 0.5% CO2. m) Stain the 1st slide after 24 hours and observe n) Stain the 2nd slide after 40 hours and observe o) Stain the 3rd slide after 64 hours and observe p) Harvest the supernatant after 70% infectivity q) Centrifuge in 50 ml tube at 4000 g or 8800 rpm for 10 minutes r) Aliquot supernatant in 0.5 ml volume s) Store at 80C Titration of seed virus suspension a) Thaw one aliquot of seed virus b) Prepare 8 serial to fold dilutions (10-1 to 10-8) in MEM 10 c) Distribute 0.1 ml of each virus dilution into one well of an 8 well tissue culture chamber slide d) Add 0.2 ml of MNA cells suspended in MEM 10 to each well e) Mix the cells and virus by gentle rocking the slide f) Incubate at 37C in humidified 0.5% CO2 incubator for 40 hours g) Acetone fix and stain the slide using an immunofluorescence technique
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h)
Evidence of virus infection should be observed at 10-6 dilution of virus (so that virus stock suspension containing 1 x 10-6 infectious units per 0.1 ml)
Preparation of stock virus suspension a) Infect 30 x 106 cells MNA cells with 10 x 106 infectious units of seed virus b) Harvest the supernatant (24 hours) after the cells reach 100% infectivity c) Distribute he supernatant into 0.5 ml aliquots d) Store at 80C Titration of stock suspension a) Thaw one aliquot of the seed virus b) Prepare 6 serial to fold dilutions (10-1 to 10-6) in MEM 10 c) Distribute 0.1 ml of each virus dilution into one well of an 8 well tissue culture chamber slide d) Add 0.2 ml of MNA cells suspended in MEM 10 (concentration 1 x 105 cells per 0.2 ml) to each well e) Mix the cells and virus suspension by gently rocking the slide f) Incubate at 37C in humidified 0.5% CO2 incubator for 20 hours g) Acetone fix and stain the slide using immunofluorescence technique Procedure for performing RFFIT a) Heat inactivate (HI) human serum at 56C for 45 minutes. Animal serum is HI at 56C for 30 minutes b) Dilute the test sera in MEM 10. Serum end-point titration are routinely tested at 8 serial 5 fold dilutions (1:5 to 1:390625). Using an 8-well Tissue-Tek slide, use one slide per test sample. Alternately, serum can be screened per slide End-point titration a) Label slides with corresponding test sera number. Use 1 slide per test serum when determining end-point titer. The dilution factor is 8-serial five-fold. b) Add 0.075 ml of MEM 10 to the first well using a microtiter pipette. This will be the 1:5 dilution of test serum. c) Add 0.1 ml of MEM 10 to the seven other wells of the slide. d) Add 0.05 ml of test serum to the first well (1:5 dilution). Mix the serum and MEM 10 several times trying not to create bubbles. e) Transfer 0.025 ml of the 1:5 dilution well (serum MEM 10) to the second well (1:25 dilution) and continue to transfer 0.025 ml to each consecutive well up to the final dilution (1:390625), discarding 0.025 ml at the end. f) Prepare all serum samples as described above. Preparation of the control slide a) Prepare a control slide with a reference serum control, a virus back titration and a cell control.
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b) c)
d) e)
Add 0.75 ml of MEM 10 to the first well of the reference serum dilution on the left of the slide. This will be the 1:5 dilution of reference serum. Add 0.1 ml of MEM 10 to the remaining wells of the reference serum dilution wells (1:25 1:625) and to the 3 wells of the virus back titration. The cell control well receives 0.2 ml of MEM 10. Add 0.05 ml of reference serum containing 2IU/ml to the 1:5 dilution well on the bottom left of the slide (well 1). Mix several times and transfer 0.025 ml of the 1:5 dilution of reference serum (well 1) to the 1:25 dilution well (well 2) and continue through to the 1:625 dilution well (well 4) discarding 0.025 ml at the end.
Preparation of the challenge virus and back titration a) The amount of virus to be used in the test should be 50 FFD50/0.1 ml b) Using MEM 10 as diluent for CVS 11, prepare a 50ml centrifuge tube containing 50 FFD50/0.1ml. Allow 1.0 ml of the FFD50/0.1ml for each slide in the test. For example, if running 10-test serum, prepare 11 ml of CVS 11 in MEM 10 (10-test serum plus 1 control slide). c) Make 2-serial 10-fold dilutions (1:10 and 1:100) of CVS-11 from the FFD50/0.1 tube to give 5.0 FFD50 and 0.5 FFD50 and 0.5 FFD50/0.1 ml, respectively. Lable 2 vials 1 (1:10) and 2 (1:100) dilutions, add 0.9 ml of MEM 10 to each. Use a pipetman and to transfer 0.1 ml of 50 FFD50 to the 1:10 vial, mix several times and discard tip. Transfer 0.1 ml from the 1 vial to the 2 vial, mixing several times. d) Add 0.1 ml of the 0.5 FFD50, 5.0 FFD50,and 50 FFD50 of virus to sequential chambers e) Add 0.1 ml of the virus preparation containing 50 FFD50/0.1 ml to all chambers of the test sera and reference serum dilutions. f) Incubate all slides for 90 minutes at 37C in a CO2 incubator with 0.5% CO2.` Preparation of the Murine Neuroblastoma (MNA) Cells a) Prepare the cells just prior to use, 20 to 30 minutes before the end of the 90 minutes, even suspension of MNA cells in 10 ml of MEM 10%. b) Adjust the cell count to give 5.0 x 105 cell/ml. c) Add 0.2 ml of the 5.0 x 105 cell/ml to each chamber of the slides, starting with the cell control well on the bottom right corner of the control slide. d) Incubate the slides for 20 hours at 37C in a 0.5% CO2 incubator. Fixation of slides a) Decant the supernatant and remove the chamber slides from the slides. b) Dip rinse in phosphate buffered saline (PBS), formula number 4588. c) Dip rinse in cold acetone (-20C). d) Fix for a longer 30 minute incubation in fresh 20C acetone in a freezer. e) Following 30 minute fixation. Remove slides from acetone and allow them to air dry at room temperature for 10 minutes.
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Staining of slides a) Add rabies conjugate (working dilution previously determined) to each chamber monolayer sufficient to cover the entire monolayer (approximately 100l per well). b) Incubate the slide in a humidity chamber (e.g. place slides on a tray with a moistened paper towel and covered with the lid of a 96 well microtiter plate) at 37C for 30 minutes. c) Following 30 minute incubation, decant conjugate from slide and rinse 2X in PBS (4588) for 10 minutes each rinse. d) Dip rinse in distilled water and place slides in a slide holder on cold packs for reading using a fluorescent microscope. Interpretation Each of the 8-well Tissue Tek slide chambers contains 25 to 50 distinct microscopic fields when observed at 160 to 200 times magnification. Observe 20 low-power (160-200X) microscope fields in each chamber, and count the number of fields which contain fluorescing cells. Begin to count fields starting in one of the corners of the well. Read the control slide first. The cell control should have no fields with fluorescing cells, i.e. no virus infected cells. Of the 20 fields inspected in each well of the virus back titration, the 50 FFD50/0/1 ml should have at least 18 to 20 positive fields. The 50 FFD50/0/1 ml should have 10 to 20 positive fields, and the 0.5 FFD50/0/1 ml should have less than 10 positive fields. An example of the number of positive fields observed on a reference and test serum slide are given below: Control slide: 20 fields are observed per well and the number of positive fields (fields with fluorescing foci) are recorded on the RFFIT data sheet. Test serum: The test serum slides are observed and the number of positive fields are recorded on the RFFIT data result sheet. Calculations: Using the control slide and test serum values from the previous example, the test serum end-point titre and international units can be calculated. Determination of 50% end point titres of serum The serum neutralisation end-point titer is defined as the dilution factor of the highest serum dilution in which there is a 50% reduction in the number of fluorescing foci.
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The 50% end point titre of serum can be made by determining the number of fluorescing foci at each dilution, and then use the cumulative totals in the ReedMeunch formula: a) Calculate the percentage of fields containing infected cells
Serum dilution No.of fields containing infected cells 0/20 0/20 0/20 0/20 12/20 20/20 20/20 20/20 Fields containing infected cells 0 0 0 0 12 32 52 72 Field containing no infected cells 88 68 48 28 8 0 0 0 % of fields containing infected cells 0/88 = 0 0/68 = 0 0/48 = 0 0/28 = 0 12/20 = 60 32/32 = 100 52/52 = 100 52/52 = 100
1:5 1:25 1:125 1:625 1:3125 1:15625 1:79125 1:390625
b)
Using the method of Reed & Muench, calculate the difference between the logarithm of the starting dilution and the logarithm of the 50% end-point dilution (difference of logarithms) from the formula: 50% - (infectivity next below 50%) x logarithm of (infectivity next above 50%) - (infectivity next below 50%) dilution factor
In this example the starting point dilution (the dilution showing an infectivity next below 50%) is 625, the dilution factor is 5. Hence, the difference in logarithm is: 50 0 x 0.69897 = 0.582475 60 0 c) Since the infectivity is increasing as the dilution increases, the 50% end point dilution is higher than the starting point dilution and is calculated by adding the difference of logarithms as follows: Log (reciprocal of 50% end point dilution) Log (reciprocal of the starting point dilution) + difference of logarithms = 2.79588 + 0.582475 = 3.38 (approx.) Hence, log (50% end point dilution) = - 3.38 and the 50% end point dilution = 10-3.38 = (1:2399) Determination of the potency of test serum in international units (IU) per ml The results of the RFFIT can be expressed as a serum titer or in International Units (IU) of antibody. In either case, a reference serum of known titer is required. When the results are to be reported as a titer, the reference serum is used as a
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control to insure the sensitivity of the test. It should demonstrate approximately the same titer determined in previous tests. When the test serum results are expressed in IU, the calculation is dependent on the number of IU in the reference serum. The reference serum is diluted to contain 2 IU/ml and tittered along with the test serum. The ED50 titers of the reference serum and the test serum are then related in the following formula for calculation of IU/ml in the test serum: Number of IU/ml = End-point titer of the test serum x End-point titer of the reference 2 IU/ml in reference serum
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SAFETY IN RABIES LABORATORY

Safety in rabies laboratory demands utmost importance because of the lethality of the disease and the property of the virus to gain entry into the body through a discontinuity in the skin howsoever minute it may be or through the mucous membrane of the worker. Extreme caution has to be exercised in the laboratory to prevent contracting infection with rabies virus which is present in high titres in various organs/tissues e.g. brain, salivary glands, spinal cord, as well as in almost all body fluid e.g. saliva, urine, tears etc. all of which can come in close contact with the laboratory worker. Though transmission through aerosols is possible it is not a usual mode of transmission of infection, yet its probability should always be kept in mind in the laboratory where high concentration of virus can result due to handling and processing of large number of specimens. Various components of laboratory safety are discussed here under in brief.
3.1 LABORATORY DESIGN, OPERATION AND EQUIPMENT

Laboratory should be exclusively utilised for rabies diagnosis and entry to it should be restricted to only those persons who are properly vaccinated. Restricted area and Entry for Vaccinated Persons only signboards should be prominently displayed at the entry of the laboratory. Within the laboratory, areas should be clearly demarcated for different procedures. No eatables should be allowed in the laboratory. If possible, handling of infectious material should be done within the biosafety hoods instead of open benches. The table tops should preferably be of the material which can easily be cleaned otherwise before starting the work it should be covered with disposable paper. It would be advantageous if one side of this paper is polythene coated. An alternative is covering of table top with lint piece soaked in suitable disinfectant. After completion of the work, the working area should be cleaned with suitable disinfectant.
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3.2 TRAINING OF MANPOWER

The technical persons working in the laboratories should be sufficiently trained in the handling of pathogens. These persons should receive training either in an already established rabies laboratory or under direct supervision of the trainer. The supporting staff should also be made well conversant with the handling and proper disposal of infected material.
3.3 SAFE LABORATORY PROCEDURES

Human error, poor laboratory practices and misuse of equipment cause the majority of laboratory accidents and related infections. It is emphasised that good laboratory practice is fundamental to laboratory safety and cannot be replaced by specialised equipment which can only supplement it. There are various stages where the laboratory worker has to be extra cautious while dealing with any material in rabies laboratory where specimens should be considered as potentially having rabies virus unless proved otherwise.
3.4 COLLECTION OF SPECIMENS

3.4.1 Antemortem specimens
a) Hydrophobia case: The person collecting the samples like saliva and corneal smears should stand on the side or back of the patient. The cerebrospinal fluid should also be collected with precaution. Rabid Animals: The animals must be restrained before collection of antemortem specimens to avoid any chances of a bite.
b)
3.4.2 Postmortem specimens

These include brain, spinal cord, salivary glands etc. - all of which should be handled carefully and treated as highly infected material. Protective clothes like laboratory coats, rubber aprons, thick rubber gloves, mask and plain goggles should be used at the time of collection of samples, autopsy as well as handling of infectious material.
3.5 EQUIPMENT AND GLASSWARE

Instruments like scissors, forceps etc. used for collection and processing of specimens should be sharp to avoid undue pressure on gloves. No niched glassware is permitted. Any leaking mixer should be rectified.
Safety in Rabies Laboratory
51
3.6 AEROSOLS
Air borne rabies infection has been demonstrated hence all procedures which generate aerosols (high speed mixing, centrifugation, pipetting etc.) should be carried out in hoods with laminar flow of air under negative pressure. Centrifugation and mixing should be carried out in tightly closed containers. Laminar hoods should have UV light for disinfection. When not in use mouth pipetting should be strictly avoided.
3.7 HANDLING OF EXPERIMENTAL ANIMALS

The worker must use thick rubber gloves for handling of animals. For daily examination of small animals forceps should be used and at no stage the animals are to be handled with bare hands.
3.8 SAFE HANDLING OF HAZARDOUS CHEMICALS

Hazardous chemicals like Beta-propiolactone used for inactivation of rabies virus etc. which are highly carcinogenic should always be handled with care under hood. Transfer should be carried out by use of propipettes.
3.9 DISPOSAL OF EQUIPMENT, GLASSWARE AND PROTECTIVE CLOTHING

The used equipment, glassware etc. should be placed in the properly marked autoclavable containers containing suitable disinfectants. The used gloves before taking off are dipped several times in quarternary ammonium compound. It any doubt of spillage on to apron coat is apprehended it is packed separately for decontamiantion. The used disposable plastic material is also placed in specific marked container.
3.10 DECONTAMINATION
It is done by boiling for sufficient in time in steriliser or preferably autoclaving. The small animals may be put in formalin jar pending disposal.
3.11 DISPOSAL
The carcass of the small animals should be placed in plastic leakproof bags, sealed and incinerated. The bedding of animals and disposable plastic ware may also be incinerated.
52
3.12 PRE-EXPOSURE IMMUNOPROPHYLAXIS

All the persons working in rabies laboratory and handling rabies virus or rabid animals should be administered pre exposure immunoprophylaxis to protect them against contracting the disease. The availability of safer anti rabies vaccines has made this task very easy. The details of pre exposure immunisation have been discussed.
3.13 POST-EXPOSURE MANAGEMENT

Every effort should be made to avoid occurring of mishaps in the laboratory due to negligence by strictly following the safety precautions. However, in case of accident where the spillage or inoculation of virus occurs following action may be initiated: a) Working area should be immediately and meticulously cleaned by using proper disinfectants. b) The broken glass pieces should be picked up with the help of forceps and discarded in appropriate containers for disinfection. One must be cautious about picking of fine pieces of glass which can sometimes penetrate even thick rubber gloves. c) Any accidental inoculation or even suspicion of the same onto a person must be immediately reported to the appropriate authorities. d) The soiled area on the body must be immediately washed with plenty of soap and water to wash off as much of the virus as is possible. Care must be taken not to increase the existing trauma. However, if the spillage is only in the eyes, wash the eyes thoroughly and repeatedly with plenty of water. e) On exposed sites other than eyes, apply an antiseptic which is readily available in the laboratory. These include any quaternary ammonium compound, spirit, ethanol, tincture, iodine, savlon, dettol etc. f) As far as possible suturing of the wound be avoided. If it is inevitable, minimum number of stitches should be applied to bring the skin in apposition. Local and parentral administration of antibiotics shall depend upon the condition of the wound. g) The post exposure immunisation shall depend upon the immunisation status of the person. Combined serum and vaccine therapy shall be indicated in persons who are not immunised or who have not responded satisfactorily to previous immunisation against rabies. Two booster doses of anti rabies vaccine shall be needed by those who have been previously immunised satisfactorily. The primary aim of a rabies laboratory is to assist in reducing morbidity and mortality due to rabies and no death should occur due to acquisition of infection
Safety in Rabies Laboratory
53
in laboratory. If proper attention is given to prevention, personnel can be protected. Laboratory workers should be aware of the dangers of working with virus and should also know how to tackle the emergencies originating from accidents in laboratories. This occupational risk can be negated through safe laboratory design, safe equipment, protective clothing, thorough and repeated training, appropriate pre and post exposure immunisation and above all good laboratory practices which remains the fundamental of safety and even now, irreplaceable.
54
QUALITY ASSURANCE IN RABIES DIAGNOSIS

Quality assurance is the sum of all those activities that ensure reliability and accuracy of the emergent results or data. This automatically ensures a good credibility of the testing laboratory and enhances users confidence in the laboratory results.
4.1 COMPONENTS OF QUALITY ASSURANCE

Quality assurance has two components: Internal Quality Control (IQC) and External Quality Assessment (EQA).
4.2 FACTORS INFLUENCING QUALITY

Many factors can influence the quality of the laboratory results. These can be grouped into pre-analytical, analytical and post-analytical. i) Pre-analytical factors Of the various pre - analytical factors that influence the quality of the laboratory results, the most important is selecting the right investigation to perform. Subsequent to this is collection of the right clinical specimen in adequate quantities using right technique. If the sample cannot be immediately processed it should be stored and transported using recommended methods. Needless to mention that right labelling of the specimen and sending it to the right laboratory which performs that particular investigation is very essential. ii) Analytical factors The staff in the rabies laboratory should be trained & committed. Good quality reagents and reliable equipment should be used including positive and negative controls with each test run (Table 13). Standard operating procedure should be followed for all the tests. Record keeping of all that is done in the laboratory should be given due attention. The biosafety measures to make the working
Quality Assurance in Rabies Diagnosis
55
environment safe enhance the commitment and performance of the laboratory workers.
Table 13: Internal quality control in rabies laboratory tests

Test Sellers stain FAT Virus isolation MIT RTCIT Challenge virus strain Normal mouse brain Positive control Impression smear of mouse brain infected with CVS Impression smear of mouse brain infected with CVS Negative control Normal mouse brain impression smear Normal mouse brain impression smear
ELISA
Positive controls provided in the kit One sample with known titre
Negative control provided in the kit -
CIEP RFFIT
One sample with known titre

Control slide Tittered reference serum control Virus reference serum control Virus back titration Cell control
Standard operating procedures manual (SOP) should include all encompassing information such as instructions about collection, storage and transport of clinical samples, inoculation/test procedures, biosafety guidelines, quality assurance procedures, reporting and interpretation of results. Top management should regularly authenticate it. It should not only be available to all on the laboratory work benches, but instructions given there in should also be strictly adhered to, to assure uniformity. Essential features of SOPs are summarised here. iii) Post-analytical factors Of the post - analytical factors that can influence quality, right recording of the results is most important. Right interpretation of the results should be written on the report. Reporting of the laboratory results is the most crucial aspect in quality. Test reports should be given on a pre-designed, pre-tested format, written legibly or typed, highlighting the abnormal findings and interpreted in a simple language. The report given to the patient about a laboratory test should be short, clear, unambigous and should not leave any room for making errors in its interpretation.
56
4.3 EXTERNAL QUALITY ASSESSMENT SCHEME (EQAS)

The purposes of a quality assessment programme are: To provide assurance to both physicians and the general public that laboratory diagnosis is of good quality To assess and compare the reliability of laboratory performance on a national scale To identify common errors To encourage the use of uniform procedures To encourage the use of standard reagents To stimulate the implementation of internal quality control programme To network the laboratories performing or intending to perform rabies diagnosis Voluntary participation in an EQAS is key to success. An EQAS organizing laboratory can run the programme successfully by gaining the confidence of participating laboratories, by maintaining confidentiality of the individual reports, avoiding provocative statements and identifying unsatisfactory performers in groups. In addition, it should provide educational opportunities to participating laboratories. It should act as adviser and guide and not a fault finding agency. EQAS in Sellers stain NICD which is a WHO Collaborative Centre for Rabies Epidemiology has been running an External Quality Assessment Scheme in Sellers stained smears for rabies diagnosis. A set of five coded slides is sent to the participating laboratories and the necessary feedback given after receiving the reports from these laboratories. Strict confidentiality is maintained in the reports. The laboratories are coded for purpose of documentation. Persons reporting positive slides as negative need more to spend more time on slides and persons who report negative slides as positive need more training. 23 laboratories are currently participating in this programme. Additional laboratories are welcome to join this scheme.
Quality Assurance in Rabies Diagnosis
57
ANNEXURE 1: STAINS, BUFFERS AND REAGENTS

Sellers Stain
a) Composition i. Stock Solution A Methylene blue (BDH or E. Merck) CI - 52015 Methanol (acetone free) Mix by using a pestle & mortar and store in a tightly stoppered bottle at + 4C ii. Stock Solution B Basic fuchsin (BDH or E.Merk) CI - 42510 Methanol (acetone free) 1.0 gm. 100 ml.
1.0 gm. 100 ml.
Mix by using a pestle & mortar and store in a tightly stoppered bottle at + 4C. Any other make of methylene blue and basic fuchsin may be used with dye content not less than 85% and 92% respectively. iii. Working stain 1. Mix thoroughly and store in properly stoppered container Stock solution A -2 vol. Stock solution B -1 vol. 2. Keep for 24 hours before use 3. Can be kept for long period if protected from evaporation. b) Adjustment of stain With properly prepared stain usually no adjustment is required. However, sometimes the smear may stain too red or blue. Adjustment of the stain may be made by adding methylene blud or basic fuchsin solution. It may be standardised by staining slides for known positive brain. 10% Formal saline Formaline Physiological saline Prepare just before use Zenkers Fluid Potassium Dichromate Mercuric chloride Distilled water Bouins Fixative Saturated solution of picric acid (1.2%)
One part Nine parts
2.5 gm. 5.0 gm. 100 ml.
70 ml.
58
Formalin (40%) Glacial acetic acid Phosphate Buffered Saline (pH 7.5) NaH2PO4 Na2HPO4 NaCl Dissolved in 1000 ml. of distilled water 90% Buffered Glycerol 0.2M sodium phosphate buffer (pH 8.5) Glycerol neutral (Analar)
25 ml. 5 ml.
0.4 gm. 2.5 gm. 8.5 gm.
10 ml. 90 ml.
2% Normal Horse Serum Distilled Water Normal horse serum (Sterile) 10 ml. Distilled water (sterile) 490 ml. Mix and add Penicillin G sodium 1,25,000 units Streptomycin Sulphate 50 mg. Distribute in 50 ml. quantity in sterile screw capped vials, store at + 4C. 0.05 M Barbitone Buffer (pH 8.2) A. Stock solution a) 5,5 Diethyl Sodium Barbiturate Distilled water Dissolve by shaking b) 5,5 Diethyl Barbituric Acid Distilled water 95C Dissolve by shaking c) NaOH Distilled water to make Store in glass stoppered bottles d) Methiolate Dissolve by shaking B. Working buffer a) Mix a and b b) Adjust pH to 8.2 with c c) Add 0.15 g merthiolate d) Adjust volume to 1000 ml. 0.01 PBS pH 7.2 A. Stock M/2 PBS a) Na2HPO4 (Anhydrous) Distilled water
36.084 gm. 3500 ml. 6.925 gm. 750 ml. 10 gm. 50 ml. 0.15 gm.
109.6 gm. 1544 ml.

Stains, Buffers and Reagents
59
b) NaH2PO4.H2O Distilled water
31.5 gm. 456 ml.
Mix A + B and add 2 litres of distilled water. Add a few drops of chloroform for preservation. Stopper and store at room temperature. B. Working PBS M/2 Stock Phosphate Buffer NaCl Distilled water to make Staining solution for Electrophoresis slides A. Destainer Solution Acetic acid glacial Methanol Mix and store in airtight container B. Staining Solution Destainer solution (A) Amido black - 10B (Colour index No. 20470) Mix and store in airtight container
640 ml. 136 gm. 16 litres
200 ml. 400 ml.
100 ml. 2 gm.
C. Stain the slides in B for 90 minutes and destain in A for 1-2 minutes in 2-3 changes of destainer as required. Phosphate Buffered saline pH 7.2 - 7.4 A. Material required Sodium chloride Disodium hydrogen phosphate Sodium dihydrogen phosphate Distilled water
8.5 gm. 2.5 gm. 0.4 gm. 1000 ml.
B. Procedure Dissolve the above ingredients in 1000 ml. of distilled water and adjust pH to 7.2 - 7.4. Dissolve 0.25 ml of Tween 20 in 500 ml. of Phosphate buffer saline and shake well.
60
ANNEXURE II: WHO COLLABORATING CENTRES FOR RABIES IN INDIA

1. National Institute of Communicable Diseases 22-Sham Nath Marg, Delhi 110054 Ph: 23971272 FAX: 23922677 e-mail: dirnicd@bol.net.in 2. National Institute of Mental Health and Neuro Sciences Hosur Road, Bangalore 560 029 Ph: 080-6995128/9 FAX: 080-6562121
WHO Collaborating Centres for Rabies in India
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SUGGESTED FURTHER READING

1. Laboratory techniques in Rabies; WHO Monograph; 4th edition, 1996 2. WHO Expert Consultation on Rabies, Geneva, WHO Technical Report Series No. 931, 2004. 3. George M. Baer. The Natural History of Rabies: Second Edition, Centers for disease Control, Atlanta, GA, 4. Alan Jackson. Rabies, Academic Press, 2007. Web Sites www.who.int www.who.int/rabies/rabnet/en www.apcri.org www.rabiescontrol.org
62
Rabies is probably the most feared of all human diseases. It has plagued man since ancient times and is believed to be as old as our civilization. The most dreadful and gruesome of all the communicable diseases, it is one of the most important zoonotic diseases which confronts us today. It is fatal in virtually cent per cent of cases, if no treatment is administered. However, timely and appropriate treatment of animal bites nearly always prevents the occurrence of disease. The laboratory occupies an important place in efforts to prevent and control the disease. It also provides assurance that vaccines used for treatment have been effective. Laboratory plays a central role while testing for newer vaccine, schedule and route of administration.
National Institute of Communicable Diseases

WHO Collaborating Centre on Rabies Epidemiology
(Directorate General of Health Services) 22-Sham Nath Marg, Delhi - 110 054
ZOONOSIS DIVISION

Communicable Diseases Rabies - General Aspects &amp Laboratory Diagnostic Techniques

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RABIES

General Aspects & Laboratory Diagnostic Techniques

National Institute of Communicable Diseases

Dr. Dipesh Bhattacharya Joint Director

Dr. Shiv Lal Addl. Director General & Director

Designing & Printing:

1.4 1.5 1.6

LABORATORY DIAGNOSIS OF RABIES: TECHNIQUES

SAFETY IN RABIES LABORATORY

3.5 3.6 3.7 3.8 3.9

RABIES General Aspects & Laboratory Diagnostic Techniques

QUALITY ASSURANCE IN RABIES DIAGNOSIS

Dr Shiv Lal Addl DG & Director NICD, Delhi

1.2 CAUSATIVE AGENT

Table 1: Classification of lyssaviruses

II II Isolates to be characterised as new genotypes

Lagos bat virus Mokola virus

Sub-Saharan Africa Sub-Saharan Africa

West Caucasian bat virus

RABIES General Aspects & Laboratory Diagnostic Techniques

M protein lipid bilayer membrane

helical nucleocapsid (RNA plus N protein) polymerase (2 proteins)

Fig 2: Structural and functional units of Rabies virus

1.2.1 Susceptibility to physical and chemical agents

Table 2: Susceptibility of rabies virus

1.3.2 Rabies in India

RABIES General Aspects & Laboratory Diagnostic Techniques

Fig 3: Global status of rabies

Table 3 : Animals transmitting rabies in India

1.3.3 Mode of transmission

Table 4: Mode of transmission

Inhalation Organ transplantation Ingestion

1.3.5 Incubation period

1.4 CLINICAL FEATURES IN HUMANS

RABIES General Aspects & Laboratory Diagnostic Techniques

1. Virus enters via animal bite

Fig 4 Pathogenesis of rabies

Table 5: Clinical features in humans

1.5 DIFFERENTIAL DIAGNOSIS

Table 6: Differential diagnosis of rabies

Acute polyneuritis Poliomyelitis Belladona poisoning Delerium tremens

Rabies post vaccinal encephalomyelitis

1.6 RABIES IN ANIMALS

1.6.1 Clinical features in dogs

RABIES General Aspects & Laboratory Diagnostic Techniques

1.6.2 Clinical features in cats

1.6.3 Clinical features in cattle

1.7 IMMUNE RESPONSE IN RABIES INFECTION AND VACCINATION

1.7.2 Immunity following vaccination

1.8 LABORATORY DIAGNOSIS

RABIES General Aspects & Laboratory Diagnostic Techniques

Table 7: Interpretation of ante-mortem diagnostic tests

1.8.1 Laboratory tests

Table 8: Common tests used for rabies diagnosis

Positive in 50 70% of cases

Fluorescent Antibody Test (FAT) for antigen detection

Mouse Inoculation Test (MIT) for virus isolation

Rapid Tissue Culture Infection Test (RTCIT) for virus isolation

Rapid (4 days) Sensitive

1.9 PREVENTION OF RABIES IN HUMANS

1.9.1 Decision to treat

Table 9: WHO Guidelines for post exposure treatment against rabies

RABIES General Aspects & Laboratory Diagnostic Techniques