Filariasis - Wuchereria bancrofti

• Filarial (filum (L)=thread) worms are slender thread like transmitted by the bite of blood-sucking insects.
• Filarial worms reside in the subcutaneous tissues, lymphatic system or body cavities of humans.
• Adult male worms are smaller, female worms are ovoviviparous, give birth to microfilarial larvae
• Microfilariae released by the female worm are seen in the peripheral blood or subcutaneous tissues
• Depending on the presence of largest number of microfilariae in blood, filarial worms can exhibit nocturnal, diurnal or no periodicity at all
• Lifecycle in 2 hosts- definitive host-man, intermediate host-blood sucking arthropods
• Eight species of filarial worms infect man, infection termed filariasis
• Traditionally, it refers to lymphatic filariasis caused by Wuchereria bancrofti or Brugia species

Wuchereria bancrofti

• Genus named after Wucherer, a Brazilian physician, who reported microfilaria in 1868.
• Manson in 1878 identified in China, Culex mosquito as the vector.
• Filariasis known from ancient times- if left untreated, the infectivity can expand into chronic disease called Lymphatic filariasis, it also causes asthmatic disease.
• Elephantiasis is a classic sign of late-stage disease. Also called Malabar leg
• No vaccine is commercially available, but various antifilarial medications reported to cure

Epidemiology

• W. bancrofti is distributed widely in the tropics and subtropics of sub-Saharan Africa, South East Asia, India and the Pacific islands.
• In India, the endemic areas are along the sea coast and along the banks of the rivers.

 Morphology

• W. bancrofti is a dioecious worm with sexual dimorphism.
• Adults are whitish, translucent, thread-like worms with smooth cuticle and tapering ends.
• The body is fragile, making removing it difficult from tissues.
• Males and females can be distinguished by structure and size of their tail tips.

• The male worm is smaller (40 mm x 0.1mm) wide, and shows a ventrally curved tail. The tip of the tail has 15 pairs of minute caudal papillae, which serve as sensory organs. 
• Contrary to above, the female is larger (60 x 0.25 mm). Its tail steadily tapers and is rounded at the tip. No extra sensory structures are seen. Its vulva is present towards the anterior side of the worm.
• Adult males and females are mainly coiled together in the abdominal and inguinal lymphatics and in the testicular tissues, are hard to separate.
• The adult worms live for many years. 

Microfilaria

    The juveniles or embryos or first-stage larvae are referred to as microfilariae. They are present in the circulation. They are released encased in the elongated egg shell, which persists as a sheath. The sheath is delicate and close fitting but  projects at the anterior and posterior ends of the microfilaria. It can actively move forward and backward within the sheath. 

This sheath, along with the area in which the worms reside, makes identification  in humans easier.

The microfilaria has a colourless, translucent body with a blunt head and pointed tail. It is a miniature adult, and is 250-300 μm long and 6-10 μm wide. When stained with Leishman or other Romanowsky stains, structural details can be made out.                          

Along the central axis of microfilaria, a column of granules can be seen, which are called somatic cells or nuclei. At the head end is a clear space devoid of granules, called the cephalic space. 

In the anterior half of the microfilaria is an oblique area devoid of granules, called the nerve ring. Approximately midway along the length of body is the anterior V-spot which represents the rudimentary excretory system. The posterior V-spot (tail spot) represents the cloaca or anal pore. The genital cells (G-cells) are situated anterior to the anal pore, in M. bancrofti, the tail tip is devoid of nuclei.

Microfilaria do not multiply or undergo any further development in human body. If they are not taken up by the female mosquito vector, they die. Lifespan is about 2-3 months. A microfilarial density of at least 15 per drop of blood is necessary for infecting mosquitoes.

 Periodicity

The microfilariae circulate in the blood stream. In most Asian countries, including India, they show a nocturnal periodicity in peripheral circulation of human. It is seen in large numbers in peripheral blood only at night, between 10 pm and 4 am. This correlates with the night biting habit of the vector (culex mosquitoes). During the day, they are present in the deep veins, and during the night, they migrate to the peripheral circulation.

The cause of this periodicity remains unknown, but the advantages of the microfilariae being in the peripheral blood during these hours may ensure the vector, the night time mosquito, will have a higher chance of transmitting them elsewhere. Physiological changes also are associated with sleeping, such as lowered body temperature, oxygen tension, and adrenal activity, and an increased carbon dioxide tension, among other physical alterations, any of which could be the signals for the rhythmic behavior of microfilarial parasites.

If the hosts sleep by day and are awake at night, their periodicity is reversed.

Life cycle

W. bancrofti carries out its lifecycle in two hosts. Humans serve as the definitive host and mosquitos as the intermediate host. The adult parasites reside in the lymphatics of the human host. They are found mostly in the afferent lymphatic channels of the lymph glands in the lower part of the body.

Definitive host - Humans serve         Intermediate host- mosquitoes

Infective form -Actively motile third stage filarifom larvae

Mode of transmission- Bite of infected vector/ mosquito carrying filariform larvae

During a blood meal, an infected mosquito introduces third-stage filarial larvae onto the skin of the human host, where they penetrate into the bite wound. They develop into adults that commonly reside in the lymphatics. Adults produce microfilariae, which are sheathed and have nocturnal periodicity, except the South Pacific microfilariae which have the absence of marked periodicity. The microfilariae migrate into lymph and blood channels moving actively through lymph and blood. A mosquito ingests the microfilariae during a blood meal. After ingestion, the microfilariae lose their sheaths and they migrate to the thoracic muscles. There the microfilariae develop into first-stage larvae and subsequently into third-stage infective larvae. The third-stage infective larvae migrate through the hemocoel to the mosquito’s prosbocis and can infect another human when the mosquito takes a blood meal.

     The life cycle of Wuchereria bancrofti commences, when a male and a female worm, mate within lymphatic vessels of an infected human being. The female discharges thousands of microfilariae (prelarval eggs) into the blood flow. When the host is aware and awake, the microfilariae are likely to stay in deep blood vessels. During the sleep they reach the peripheral/marginal blood vessels. This enables them to get picked up by the night biting mosquito.

In Mosquito:  In the stomach of the mosquito, ex-sheathing occurs, they lose their sheaths, penetrate the stomach wall and migrate to the thoracic muscles where further development occurs. First stage larvae, sausage shaped with a spiky tail and second stage larvae develop then into the elongated actively motile third stage filariform larva which is the infective form. It travels through the hemocoel to the mosquito's proboscis. 

There is no multiplication of microfilaria in the mosquito and one microfilaria develops into one infective larva only. 

The time for development of microfilaria in the mosquito from the bite till the development of infective third stage filariform larva is the extrinsic incubation period. Its duration varies with environmental factors such as temperature and humidity, as well as with the vector species and is around 10-20 days, under optimal conditions.

In Humans: When the mosquito bites another person, the larvae are injected into the skin of another human. Large number of infective mosquito bites are required to ensure transmission- as many as 15,000 infective bites per person. Many larvae fail to penetrate the skin itself and many are destroyed by immunological and defense mechanisms.

 After penetrating the skin, they travel to the lymph vessels and in abdominal or inguinal lymph nodes, they mature into fully adult forms within six months. Fully developed females can live up to seven years. The gravid female releases large numbers of microfilariae, as many as 50,000 per day, which pass through thoracic duct and pulmonary capillaries to enter into peripheric circulation. They are ingested with the blood meal by mosquito and the cycle is repeated.

 Prepatent period

The period from the entry of the infective third stage larvae into the human host till the first appearance of microfilaria in circulation is the biological incubation period or the prepatent period. This is usually about 8-12 months.

Clinical incubation period

The period from the entry of the infective third stage larvae into the human host till the first appearance of earliest clinical manifestation is called the clinical incubation period. This is variable, but usually about 8-16 months.

Pathogenesis

Infection caused by W. bancrofti is termed Wuchereriasis or Bancroftian filariasis. It can present as

Ø  Classical filariasis

Ø  Occult filariasis

Classical filariasis

Lymphatic filariasis, considered globally as a neglected tropical disease (NTD), is a parasitic disease caused by microscopic, thread-like worms which live in the human lymphatic system. The lymphatic system maintains the body’s fluid balance and fights infections. Blockage of lymph vessels and lymph nodes by adult worms causes filariasis. Blockage could be due to mechanical factors or allergic inflammatory reaction to worm antigens and secretions. The affected lymph nodes and vessels are infiltrated with macrophages, eosinophils, lymphocytes and plasma cells. The worms inside lymph nodes can cause granuloma formation, with subsequent scarring and calcification. Recurrent secondary bacterial infections cause further damage.

Clinical manifestations

Wuchereria bancrofti infection is usually asymptomatic but with microscopic hematuria or proteinuria, dilated lymphatics and in men, scrotal infection.

ADL, acute adenolymphangitis, is characterised by high fever, lymphatic inflammation (lymphangitis, lymphadenitis) and transient local edema. High grade, sudden onset fever associated with rigors lasts for 2-3 days.  Lymphangitis (inflamed lymph vessels) and lymphadenitis (inflammation of lymph nodes) occur followed by lymphedema, which starts as swelling around the ankle, spreading to the back of foot and leg, arms, breasts, scrotum, or other parts of the body. Lymphoangiovarix which is the dilation of lymph vessels is also seen.

In men, swelling of the scrotum, called hydrocele is seen. Here, accumulation of fluid occurs due to obstruction of lymph vessels. Rupture of lymph vessels leading to the release of lymph results in lymphorrhagia.

The dysfunction of lymphatic vessels leading to inflammation and decreased current of the lymph fluid result in skin and lymph system infections. Affected limbs become grossly swollen; the skin may become thick and secondary infections are frequent due to lymphatic dysfunction. 

Over time, the disease causes thickening and hardening of the skin, a condition called elephantiasis which can be lethal and incurable. Commonly seen in leg, it is characterised by edema, thickened skin, cracks, fissures with secondary bacterial and fungal infections.

Occult filariasis

It occurs as a result of hypersensitivity reaction to microfilarial antigens, not directly due to lymphatic involvement. Microfilaria are absent in blood since they are destroyed by tissues.

Clinical manifestations

Massive eosinophilia, hepatosplenomegaly, pulmonary symptoms like dry nocturnal cough, dyspnea and asthmatic wheezing. Classical features of lymphatic filariasis absent. Arthritis, glomerulonephritis, thrombophlebitis etc. may be seen.

Occult filariasis also called Meyers Kouwenaar syndrome.

Tropical pulmonary eosinophilia

Occult filarial infection might also cause pulmonary tropical eosinophilia, which is typically present in patients living in Asia. Pulmonary tropical eosinophilia syndrome can cause: shortness of breath, cough, wheezing, chest pain, hyper eosinophilia, splenomegaly, and bloody sputum. There might be high levels of antifilarial antibodies and IgE (Immunoglobulin E). Children and young adults commonly affected in areas of endemic filariasis.

 Diagnosis 

The standard method for diagnosing active infection is the identification of microfilariae in a blood smear by microscopic examination. The microfilariae that cause lymphatic filariasis circulate in the blood at night (called nocturnal periodicity). Blood collection should be done at night to coincide with the appearance of the microfilariae, and a thick smear should be made and stained with Giemsa or hematoxylin and eosin. For increased sensitivity, concentration techniques can be used.

Adult filarial worms can be seen in sections of biopsied lymph nodes, but not routinely employed in diagnosis.

Intradermal injections of filarial antigens induce an immediate hypersensitivity reaction.  Not of diagnostic importance, because of high false positive and negative reactions.

Imaging techniques such as high frequency ultrasonography (USG) of scrotum and breast along with Doppler imaging may result in identification of motile adult worm (filaria dance sign) within the lymphatics.

X-ray used to detect dead and calcified worms. In Tropical pulmonary eosinophilia, chest x-ray helpful in detecting worms.

Serologic techniques provide an alternative to microscopic detection of microfilariae for the diagnosis of lymphatic filariasis. Patients with active filarial infection typically have elevated levels of antifilarial IgG4 in the blood that the human body develops to battle in opposition to antigens excreted by adult female Wuchereria bancrofti worms can be detected using routine assays like complement fixation, indirect haemagglutination (IHT), indirect fluorescent antibody (IFA) etc

Molecular diagnosis by polymerase chain reaction (PCR) is achievable, too.

Because lymphedema may develop many years after infection, lab tests are most likely to be negative with these patients.

Indirect evidences include eosinophilia or elevated serum IgE levels.

Treatment

Diethylcarbamazine (DEC) is the drug of choice. The drug kills the microfilariae in the bloodstream and occasionally adult worms in the lymphatic vessels. It has few side effects which include: fever, headache, dizziness, nausea and joint-muscle pain.

DEC should only be consumed, if Wuchereria bancrofti has been recognized. This is, since many people with lymphedema are not contaminated with parasites.

DEC can depreciate Onchocerciasis (an eye disease caused by Onchocerca volvulus) and can cause encephalopathy (brain disease) and demise in people who are contaminated with Loa-loa.

Another drug, ivermectin, can also be used, however it kills microfilariae only.

Tetracyclines inhibit endosymbiotic bacteria that are essential for the fertility of the worm, thus have an effect in the treatment of filariasis.

Supportive treatment

Antifilarial drugs may not cure chronic conditions. Other measures like elevation of affected limb, use of elastic bandage and local foot care are beneficial to reduce some symptoms of elephantiasis.

The swollen skin is susceptible to bacterial infections because immune defenses cannot work correctly due to the impaired stream of fluids. Therefore, the skin must be kept hygienic.

Surgery is required for hydrocele.  

Prophylaxis

To avert new infections

1)     Eradication of vector mosquitoes-elimination of breeding places, use of antilarval chemicals, using mosquito net and mosquito repellents

2)     Detection and treatment of carriers- use of DEC

Differences between Archaebacteria and Eubacteria

American microbiologist and biophysicist Carl Richard Woese proposed three kingdom classification system in 1990. This classification system divides the life forms into three domains and six kingdoms.

The three domains are archaea, bacteria, eukaryote, and six kingdoms are Archaebacteria (ancient bacteria), Eubacteria (true bacteria), Protista, Fungi, Plantae, Animalia.

Woese classified them based on their differences in the 16S ribosomal RNA (rRNA) structure. 16S rRNA can be used for comparative analysis between prokaryotic and eukaryotic species. 

Carl Woese used the rRNA as an “Evolutionary Chronometer” – an evolutionary time clock.

The Archaea (Archaebacteria)

• Archaea are ancient bacteria - believed to have evolved just after the evolution of first life on earth.
• Archaea are prokaryotic cells. The cell walls of Archaea contain no peptidoglycan, hence Archaea are not sensitive to some antibiotics that affect the Bacteria.
• Archaea have membranes composed of branched hydrocarbon chains (many also containing rings within the hydrocarbon chains) attached to glycerol by ether linkages. 
• The ether-containing linkages in the Archaea membranes is more stable than the ester-containing linkages in the Eubacteria  and are better able to withstand higher temperatures and stronger acid concentrations. 
• Archaea often live in extreme environments and include methanogens, extreme halophiles, and hyperthermophiles.
• Archaea contain rRNA that is unique to the Archaea, distinctly different from the rRNA of Bacteria and Eukarya.

  Archaea are found in Volcanic hot springs- Grand Prismatic Spring of Yellowstone National Park

                            The very cold and ultra-salty Deep Lake in East Antarctica is home to haloarchaea.

Hydrothermal vents on the ocean floor, where the surrounding water can reach over 300° Celsius, are home  for some archaeal species.

The Bacteria (Eubacteria)

Bacteria (also known as eubacteria or "true bacteria") are prokaryotic cells that are common in human daily life. Eubacteria can be found almost everywhere and serve as antibiotic producers and food digesters, pathogens etc. 

Bacteria are prokaryotic cells. They have membranes composed of unbranched fatty acid chains attached to glycerol by ester linkages. 

The cell walls of Bacteria contain peptidoglycan. Bacteria are sensitive to  antibacterial antibiotics.

Bacteria contain rRNA that is unique.

Bacteria include mycoplasmas, cyanobacteria, Gram-positive bacteria, and Gram-negative bacteria.

Thus, in short, Archaebacteria are called ancient bacteria whereas the eubacteria are called true bacteria. Eubacteria are usually found in soil, water, living in and on of large organisms. Eubacteria are divided into two groups known as gram positive and gram negative bacteria. Archaebacteria are found in salt brines, ocean depths and hot springs.  Three types of archaebacteria are found: methanogens, halophiles and thermoacidophiles.

 Eu Bacteria and Archaea – The Major Differences

  

	ArchaeaBacteria	EuBacteria
	-Ancient bacteria-	-True bacteria-
Complexity	Simple in their organization	Complex than archaebacteria
Habitat	Can sustain in extremely harsh environment such as oceans, hot springs, marshlands, hot springs and gut of animals	Found everywhere - soil, organic matter, earth’s crust, water, bodies of animals and plants, radioactive wastes, hot springs
Size	0.1-15 μm in diameter	0.5-5 μm in diameter
Shape	spheres, rods, plates, spiral, flat or square-shaped	cocci, bacilli, vibrio, rods, filaments or spiral in shape
Cell wall	Pseudopeptidoglycan	Lipopolysaccharide/ Peptidoglycan with muramic acid
Membrane lipids	Ether-linked, branched, aliphatic chains, containing D-glycerol phosphate	Ester-linked, straight chains of fatty acids, containing L-glycerol phosphates
RNA	Consists of single RNA	Three types of RNA
RNA polymerase	Complex subunit pattern	Simple subunit pattern
Introns (a long stretch of noncoding DNA found between exons (or coding regions) in a gene)	Present in archaebacteria	Absent in eubacteria
Metabolism	Methanogenesis- exhibit neither glycolysis nor Kreb’s cycle	Autotrophy, Aerobic and Anaerobic Respiration, Fermentation and Photosynthesis-exhibit both glycolysis and Kreb’s cycle
Reproduction and Growth	Asexual Reproduction, by fragmentation, budding and binary fission	Other than binary fission, budding and fragmentation, eubacteria can produce spores in order to remain dormant during unfavorable conditions
Types	Methanogens, halophiles and thermophiles	Gram positive and Gram negative
Examples	Halobacterium, Thermoproteus, Pyrobaculum, Thermoplasma and Ferroplasma	Mycobacteria, Bacillus, E. coli, Pseudomonas, Clostridium etc

 Interesting Read

https://www.ck12.org/c/biology/archaea/lesson/Introduction-to-Archaea-Advanced-BIO-ADV/

https://www.science.org.au/curious/earth-environment/what-are-archaea

Rabies- Lab diagnosis & Prophylaxis

 Lab diagnosis of Rabies:

Human Rabies

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

-Survival was shown possible in rare instances

Diagnosis

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

Laboratory diagnosis

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

1. Demonstration of rabies virus antigens by Immunofluorescence

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

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

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

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

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

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

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

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

Animal Rabies

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

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

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

1.     Microscopy/ Histological examination:

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

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

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

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

-Demonstration of Inclusion Bodies-

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

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

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

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

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

Treatment

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

Case management includes the following procedure:

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

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

(d) Ensure hydration and diuresis.

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

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

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

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

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

 Prevention

1. Animal Rabies

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

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

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

 2. Human Rabies

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

 Post-Exposure Prophylaxis

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

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

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

 The local treatment comprises the following measures:

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

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

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

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

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

 

2. Active Immunisation

• Tissue Culture Vaccines

Human Diploid Cell (HDC) Vaccine

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

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

In India, the rabies vaccines available are,

1)     Purified Vero cell rabies vaccine (PVRV)

2)     Chromatographically purified Rabies Vaccine (CPRV)

3)     Human Diploid Cell Vaccine (HDC)

4)     Purified Chick Embryo Cell Vaccine (PCECV) 

5)     Purified Duck Embryo Vaccine (PDEV) 

• Subunit Vaccine

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

     

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

 

Vaccination Schedules

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

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

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

 Categories of contact and recommended post-exposure prophylaxis

 

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

 

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

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

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

 

3.     Passive Immunization with antirabies serum

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

Administration of Immunoglobulins

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

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

Pre exposure prophylaxis:

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