MONOCLONAL ANTIBODIES

Antigens, by their nature as macromolecules having primary, secondary, tertiary, and quaternary structures, constitute a “mosaic” of antigenic determinants. An outgrowth of basic serologic principles and techniques has been the attempt to “purify” antigens to reduce the heterogeneity of antibodies developed against them. Antigen molecules with only a single epitope are rarely encountered; rather, hundreds or even thousands of potential antigenic determinants may exist on a cell surface or within the mix of other substances. When these mixed antigens are injected into an animal, an equal number of lymphocyte clones are stimulated. Even though each clone produces a specific antibody, the final result is a highly heterogeneous mixture of antibody molecules, the specificity and affinity of which are often unknown and difficult to control from batch to batch. When these polyclonal antisera are used in immunologic test systems involving infectious agents, cross-reactivity may be noted either because antigenic determinants are shared by different species or because mutations may have led to the evolution of epitopes sufficiently close in specificity to produce detectable cross-reactions. Attempts to produce pure antibodies through absorption with cross-reacting antigens or to prepare “clonal” from “polyclonal” antisera via techniques such as affinity column chromatography have only been partially successful.

As the science of serologic testing evolved, the view was held that the availability of an antibody having a high degree of molecular homogeneity and with specificity for a single, antigenic epitope without cross-reactivity would solve many of the problems encountered in the use of polyclonal antibodies. Highly specific monoclonal antibodies, the product of a single clone of lymphoid cells, gradually emerged as a by-product of the investigations in cell fusion and hybridoma technology conducted by Kohler and Milstein. Because of their discovery, it is now possible to isolate cloned lines of individual lymphocytes that produce unique, monospecific antibody molecules. Monoclonal antibodies refer to a uniform, homogeneous, molecular species of Ig, rather than to a heterogeneous array of Igs as is produced during the usual immune response. The principal feature of this technology was not that a single line of monoclonal antibody (MAB)-producing cells could be isolated, but rather that these mouse lymphocytes could be “fused” with mouse myeloma cells to produce hybrid cells having two inherent properties: (1) the capability of producing monospecific antibodies (acquired from the parent lymphocytes) and (2) the ability to grow permanently in culture (the characteristic “immortality” acquired from the transformed myeloma cells). Thus, individual monoclonal antibodies can be produced in a continuous and almost endless supply.

Monoclonal antibodies have been developed against clinically relevant antigens of many bacterial, viral, fungal, and parasitic agents, and reagents prepared with these antibodies are used in many commercially available EIA and immunofluorescence test kits. In addition to the direct detection of microbial structural antigens (e.g., capsular polysaccharides, outer membrane protein antigens), monoclonal antibodies have also been developed for the detection of microbial virulence factors, such as toxins produced by enterohemorrhagic E. coli (e.g., Shiga and Shiga-like toxins). This approach introduces a new way of evaluating the relationship of microorganisms and infectious diseases. Instead of the conventional focus on detection and identification of the organisms themselves, these reagents allow specific detection of microbial virulence factors that may be shared by several bacterial species associated with a given symptom complex. For example, it may be more important to know that an enteric toxin is the cause of hyperosmotic diarrhoea, rather than to receive the information that the patient is infected with Shigella species or an enterotoxigenic E. coli strain.

Procedure for Production of Monoclonal Antibodies.

1. Selection of Antigen

Monoclonal antibodies can be produced against any substance recognized as an antigen by the immune system of the animal being injected. Using a pure antigen is ideal. In fact, certain antigens, such as chemically purified drugs used for assays (e.g., digoxin), may be homogeneous. Even so, one can never guarantee that an antigenic determinant will consist of only one epitope. The fact that impure antigens can be used in MAB production is a chief advantage over conventional methods used to produce polyclonal antibodies.

2. Animal Immunization

The chief objectives in the immunization procedure are to prime the immune system of the animal to avidly recognize all antigens injected, to maximally stimulate B-lymphocyte clones, and to have the spleen cells divide at a high rate. In the production of monoclonal antibodies, the BALB/c mouse strain is most commonly used. The antigen is injected subcutaneously or intraperitoneally, with the simultaneous injection of Freund’s adjuvant. Injections are repeated at weekly intervals, and a final “booster” injection is given intravenously approximately 3 days prior to harvesting the spleen cells. At the end of the injection schedule, the animal is killed and the spleen is aseptically removed.

3. Fusion of Splenic Lymphocytes and Myeloma Cells

The animal spleen is placed in sterile culture medium containing antibiotics. The splenic tissue is teased to release cells and to form a slurry. This material is passed through a mesh to obtain single cells. Ficoll is added, and the slurry is centrifuged to remove RBCs. Polyethylene glycol (PEG) is added to the slurry to reduce cell-to-cell surface tension; this brings the cells into close proximity to one another, allowing their membranes to fuse. Dimethylsulfoxide (DMSO) is added to the fusion mixture to maximize cell contact even more. Finally, the cells are packed into a pellet by gently centrifuging the mixture for 5 minutes. Thus, at the end of these steps, the preparation consists of unfused myeloma cells, unfused lymphocytes, and a few fused hybrid lymphocyte-myeloma cells (It should be recognized that splenic lymphocytes and myeloma cells fuse with a frequency of only about 1 per 105 or 106 cells.).

4. Selection of Hybrid Lymphocyte-Myeloma Cells

Unfused myeloma cells rapidly outgrow the hybrids and must be removed in some manner. The myeloma cells used for fusion are grown in the presence of 8-azaguanine, a drug that causes the cells to permanently switch off the production of hypoxanthine phosphoribosyl transferase (HPRT), an enzyme that is needed to continue growth. If these HPRT-negative cells are suspended in a medium containing hypoxanthine, aminopterin, and thymidine (HAT medium), only the hybridoma cells will grow successfully. The hybridoma cells inherit HRPT from the splenic lymphocytes with which they have fused and will survive. The unfused myeloma cells, unable to synthesize DNA because of inability to produce HPRT, will be killed by the aminopterin in the selective HAT medium. It should also be remembered that unfused splenic lymphocytes do not survive beyond a few days in culture medium; therefore, the fused hybrid lymphocyte-myeloma cells alone survive in the HAT medium.

5. Cloning the Hybridoma Cells

The single hybrid cells producing the desired antibody must be isolated and grown as a clone. Two techniques can be used: (1) limiting dilution and (2) growth in an agar gel medium. In the limiting or doubling dilution technique, the suspension of hybrids (after maximum growth) is diluted and distributed into a series of sterile wells in a microtiter plate. The dilutions are so calculated that each well contains an average of only one cell that can then be replaced as a single antibody-producing clone. In the alternative method, using agarose gel supplemented with serum, amino acids, and antibiotics, the dividing hybrid cells form tiny, sphere-like clusters. These spheres can be selected with a Pasteur pipette and transferred to microtube wells for further culture and ultimately for assay to determine whether the desired antibody is being produced.

6. Screening for Desired Antibodies

In the fusion step of the procedure of producing monoclonal antibodies, many lymphocytes other than those producing the desired monoclonal antibodies may have fused. In fact, less than 5% of the hybrid cells out of those selected actually produce the desired specific antibodies. Thus, assays of the selected cell lines are required to determine if the desired antibody is being produced. Radioimmunoassays, enzyme-linked immunosorbent assay (ELISA), precipitin techniques, and blotting techniques can be used for this phase of the procedure.

7. Mass Production of Monoclonal Antibodies

Once the desired clone of hybrid cells has been selected, the next step is the production of large quantities of monoclonal antibodies. The peritoneal cavity of mice, preferably the same strain that was used for the initial immunization step, can be used to grow the selected hybrid cell clone. First, the peritoneal cavity is injected with an organic irritant, such as pristane, to produce a chemical peritonitis. Next, the selected hybrid cell line is injected into the peritoneal cavity. Within days, a tumor known as a hybridoma develops. This tumor produces large quantities of monoclonal antibodies that can be harvested by aspirating the ascitic fluid from the mouse’s peritoneal cavity. A tumor-bearing mouse will survive for 4 to 6 weeks, during which time large quantities of antibody can be harvested. Hybridomas can also be grown in tissue cultures in which highly purified antibodies are produced without the potential for contamination from serum, nonspecific interference from ascites proteins, or cross-reactivity of histoincompatibility antibodies derived from the mouse tissues.

  

VACCINES

BIOMEDICAL WASTE MANAGEMENT

WHAT IS FOOD INTOLERANCE?

Food intolerance is a broad term that is used to describe a wide range of adverse reactions to foods, that cause symptoms after eating some foods. These include stomach pain, bloating, gas/flatulence, diarrhoea, irritable bowel syndrome (IBS), rashes, hives (urticaria), recurrent mouth ulcers or headaches. If food intolerances are not properly managed, these symptoms can adversely affect general health and wellbeing.

Food intolerances are sometimes confused with, or mislabelled as food allergies. Food intolerances involve the digestive system, whilst food allergies involve the immune system. Unlike Immunoglobulin E (IgE) antibody mediated food allergy, food intolerances (except for sulphite and benzoate reactions) do not cause anaphylaxis (severe allergic reactions), that can be life threatening.

What are the symptoms of a food intolerance?

Symptoms of a food intolerance include:

• Abdominal (belly) pain.
• Diarrhoea.
• Gas and bloating.
• Headaches or migraines.
• Heartburn.
• Nausea.
• Upset stomach.

Natural substances in foods can cause food intolerances

Foods are composed of proteins, carbohydrates, fats, nutrients and several natural chemicals. The following naturally occurring substances often add flavour and smell to food, but they can trigger symptoms in some people:

• Lactose intolerance is an example of an enzyme deficiency, which occurs when people are born with, or develop, insufficient lactase enzymes to digest lactose in cow’s milk and other dairy products. This can result in bloating, gas/flatulence, stomach upset and diarrhoea after having dairy products. This condition is uncomfortable but not dangerous and does not cause rashes or anaphylaxis. Diagnosis is by temporary elimination of lactose and reintroduction.

• Monosodium glutamate -Glutamates also occur naturally in foods such as camembert cheese, Parmesan cheese, tomatoes, soy sauce, and mushrooms. MSG stimulates nerve endings, which may be why it is used as a flavour enhancer when it is added to food.

• Vasoactive amines such as tyramine, serotonin and histamine are well known triggers of migraines in some people. They are naturally present in pineapples, bananas, baked meat, vegetables, red wine, wood-matured white wine, avocados, chocolate, citrus fruits, and mature cheese. Amines can act directly on small blood vessels to expand their capacity. This may be why they can trigger flushing, migraines, and nasal congestion in some people.

• Salicylates are natural aspirin like compounds that are present in a wide variety of herbs, spices, fruit and vegetables. Reactions to salicylates may be even more common than reactions to artificial colours and preservatives. Aspirin can trigger hives, by acting directly on skin mast cells, and therefore salicylates can also worsen hives in some people.

• Toxins can cause severe symptoms. Contamination of food with micro-organisms (such as bacteria) or their products (due to spoilage) can cause food poisoning due to toxins. For example, if some types of fish are stored poorly, their gut bacteria can convert histidine to histamine, resulting in allergy like symptoms.

• Irritants such as caffeine and curry are gut irritants that can trigger indigestion in some people. It is important to realise that reactions to these substances are not due to allergy.

How is a food intolerance managed or treated?

You may need to change your diet to limit or eliminate problem foods. Many people with food intolerances find that consuming small amounts of food causes few symptoms if any. When symptoms occur, over-the-counter medicines like antacids or antidiarrheal can help.

People who are lactose intolerant can consume lactose-free milk and dairy products. You can also buy lactase enzymes at drugstores. You can take lactase pills before consuming dairy products or add lactase drops directly to milk to break down the lactose.

 

THYROID FUNCTION TEST

What is the thyroid gland?

The thyroid gland is a butterfly-shaped endocrine gland that is normally located in the lower front of the neck. The thyroid’s job is to make thyroid hormones, which are secreted into the blood and then carried to every tissue in the body. Thyroid hormones help the body use energy, stay warm and keep the brain, heart, muscles, and other organs working as they should.

How does the thyroid gland function?

The major thyroid hormone secreted by the thyroid gland is thyroxine, also called T4 because it contains four iodine atoms. To exert its effects, T4 is converted to triiodothyronine (T3) by the removal of an iodine atom. This occurs mainly in the liver and in certain tissues where T3 acts, such as in the brain. The amount of T4 produced by the thyroid gland is controlled by another hormone, which is made in the pituitary gland located at the base of the brain, called thyroid stimulating hormone (abbreviated TSH). The amount of TSH that the pituitary sends into the bloodstream depends on the amount of T4 that the pituitary sees. If the pituitary sees very little T4, then it produces more TSH to tell the thyroid gland to produce more T4. Once the T4 in the bloodstream goes above a certain level, the pituitary’s production of TSH is shut off. In fact, the thyroid and pituitary act in many ways like a heater and a thermostat. When the heater is off and it becomes cold, the thermostat reads the temperature and turns on the heater. When the heat rises to an appropriate level, the thermostat senses this and turns off the heater.

What is thyroid function test (TFT)

Thyroid function tests are usually done to find out whether the thyroid gland is working properly. This is mainly to diagnose underactive thyroid gland (hypothyroidism) and an overactive thyroid gland (hyperthyroidism).

TSH (Thyroid stimulating hormone) Test: The TSH test is often done first. If the thyroid hormone levels in blood are too low, pituitary gland makes larger amounts of TSH to stimulate production of thyroid hormone and vice versa.

T4 (Thyroxine) Test: A high level of T4 indicates hyperthyroidism. Most of the T4 in body is found to protein and a small percentage is free, together called Total T4. Need to monitor T4 levels if taking thyroid replacement therapy (medication), to check underactive thyroid in new-borns, evaluate conditions like goitre, thyroid nodules and issues with pituitary gland or hypothalamus.

Free T4 Test: A small portion of T4 is not bound to proteins and are called free T4, which are readily available for our body to use. High free T4 above normal range could mean you have an overactive thyroid also seen in Grave’s disease, an autoimmune disorder. Abnormally low free T4 levels may signal hypothyroidism.

T3 (Triiodothyronine)Test: T3 mostly exists as a bound form with proteins. Total T3 refers to the collection of both bound and unbound forms of T3 circulating in the blood. T3 test is most often used to diagnose hyperthyroidism.

Free T3 test: Free T3 are unbound T3 that enters one’s body tissues where its needed. Free T3 is in a small percentage compared to Total T3. However, measuring free T3 is more accurate than measuring Total T3 as it represents the immediately available thyroid hormone which can be used.

 

Thyroid function test Interpretation
TSH	Free T4	Free T3	Condition
Normal	Normal	Normal	None
Low	High	High	Hyperthyroidism
Low	Normal	Normal	Subclinical hyperthyroidism
Low	Normal	High	T3 toxicosis
Low	High	Normal	Thyroiditis T4 ingestion Hyperthyroidism in the elderly or with comorbid illness
Low	Low	Low	Euthyroid sick syndrome Central hypothyroidism
High	Normal	Normal	Subclinical hypothyroidism Recover from euthyroid sick syndrome
High	Low	Low	Primary hypothyroidism
High	High	High	TSH producing pituitary adenoma

 

 

 

RABIES PROPHYLAXIS

Rabies is an acute viral disease that causes fatal encephalomyelitis in most of the warm blooded animals including man. The virus is found in wild and some domestic animals and is transmitted to other animals and humans through their saliva (following bites, scratches, licks on broken skin and mucus membrane). In India, dogs are responsible for about 95% of human rabies, followed by cats (2%), jackals, mongoose and others (1%). Therefore, the disease is mainly transmitted by the bite of a rabid dog. 

Rabies is caused by a RNA virus that is present in saliva of rabid animal, which is being neurotropic (high affinity for nerves). The virus enters the peripheral nerve through a free nerve ending via the neuromuscular junction and moves internally through axoplasm slowly at a speed of 3mm per hour to reach the spinal cord and brain. 

The incubation period for rabies is typically 2-3 months but may vary form 1 week to 1 year, depending upon factors such as the location of virus entry and viral load. Initial symptoms of rabies include a fever with pain and unusual tingling, pricking, or burning sensation at the wound site. As the virus spreads to the central nervous system, progressive and fatal inflammation of the brain and spinal cord develops.

There are two forms of disease;

1. Furious rabies: results in signs of hyperactivity, excitable behavior, hydrophobia (fear of water) and sometimes aerophobia. Death occurs after a few days due to cardiorespiratory arrest.
2. Paralytic rabies: accounts for 20% cases, less dramatic and usually longer course than the furious form. Muscles gradually become paralyzed, starting at the site of bite or scratch. A comma slowly develops and eventually death occurs.    

POST EXPOSURE PROPHYLAXIS (PEP)

When to seek medical care ?

In countries like India, the disease is endemic with sustained dog to dog transmission, every animal bite is suspected and treatment should be started immediately after exposure. Post exposure prophylaxis should be considered in the following conditions.

• Bites by all warm blooded animals
• Exposure to wild animals
• Rodent bites (especially in forest areas)
• Exposure to bats
• Human to human transmission: Risk is minimal and there are no well documented cases. 

Observation of biting dog/cat:

The PEP should be administered immediately after the exposure. The observation period of 10 days is valid for dogs and cats only. The treatment may be modified if the suspected animal is healthy after a 10 day observation period and PEP can be converted to Pre-exposure prophylaxis.

Vaccination status of biting animal:

Animals vaccinated against rabies do not suffer and transmit the disease. However, animal vaccine failures may occur because of improper administration, inadequate doses, poor quality of vaccines or poor health status of the animal. Hence, irrespective of the vaccination status of the biting animal, the PEP should be given. 

The following World Health Organization (WHO) classification is used for grading the exposure to rabies and guide to provide rabies prophylaxis.

Categories of contact with suspect rabid animal	Post-exposure prophylaxis measures
Category I - touching or feeding animals, animal licks on intact skin (no exposure)	Washing of exposed skin surfaces, no PEP
Category II - nibbling of uncovered skin, minor scratches or abrasions without bleeding (exposure)	Wound washing and immediate vaccination
Category III - single or multiple transdermal bites or scratches, contamination of mucous membrane or broken skin with saliva from animal licks, exposures due to direct contact with bats (severe exposure)	Wound washing, immediate vaccination and administration of rabies immunoglobulin

Post exposure prophylaxis include;

1. Management of animal bite wound
2. Passive immunization with Rabies Immunoglobulin (RIG)
3. Active immunization with Anti-Rabies vaccines (Rabies vaccines)

1. Management of animal bite wound

Prompt local treatment of all bite wounds and scratches is important. The recommended first aid procedures include immediate, thorough flushing and washing of all wounds with soap and water and application of Povidone iodine or Antiseptic having virucidal activity. Washing of wounds is desirable up to 15 minutes and should be carried out as soon as possible with soap and water. If soap or a virucidal agent is not available, the wounds should be thoroughly and extensively rinsed with water.

A bleeding wound at any site indicates severe exposure and should be infiltrated with RIG. Severe bite wounds are best treated by daily dressing, followed by secondary suturing when necessary. 

Tetanus prophylaxis should be given to prevent sepsis in the wounds. 

2. Rabies Immunoglobulin (RIG)

The anti-rabies serum/RIG provides passive immunity in the form of readymade neutralizing antibodies at the site of exposure before the patient to produce his/her own antibodies following anti-rabies vaccination. RIG should be administered to all patients with category III exposure.

There are two types of RIG- Equine RIG (ERIG) and Human RIG (HRIG)

• Equine RIG (ERIG): ERIG is produced by hyper immunization of horses. It carries a small risk of anaphylaxis due to heterogenous origin. (Dosage : 40 IU per kg body weight)
• Human RIG (HRIG): Is of homologous origin and relatively free form the side effects encountered in ERIG. (Dosage : 20 IU per kg body weight).

RIG is administered only once, preferably at or as soon as possible after initiation of post exposure vaccination. It is not recommended beyond the seventh day after the first dose of rabies vaccine. The entire immunoglobulin dose or as much as possible should be infiltrated carefully into or as close as possible to the wounds or exposure sites. RIG must never be given intravenously.

3. Rabies vaccines

Active immunization is achieved by administration of potent cell culture vaccines (CCVs). Currently available CCVs should be administered by IM regimen and CCVs approved for ID use shall be administered by ID regimen.

All animal bite victims of Category II and III exposures, irrespective of their age and body weight require, the same number of injections and dose per injection. 

Rabies vaccines can be administered by intradermal or intramuscular route. 

• Intradermal route: Regimen for PEP (Updated Thai Red Cross Schedule 2-2-2-0-2): This involves the injection of 0.1 ml of reconstituted vaccine per ID site and on two sites per visit (one on each deltoid area, an inch above the insertion of deltoid muscle) on days 0,3,7, and 28.
• Intra muscular route: Regimen for PEP (Essen regimen 1-1-1-1-1) : Intramuscular administration at deltoid region of five injections, one dose each given on days 0,3,7,14 and 28.

Management of Re-exposure in in previously vaccinated individuals

• Proper wound management should be done
• No need for administration of RIG
• One-site intradermal vaccine administration on days 0 and 3 or
• One-site intramuscular administration of an entire vaccine vial on days 0 and 3.

PRE-EXPOSURE PROPHYLAXIS

Pre-exposure vaccination may be offered to High Risk groups such as

• Laboratory staff handling the virus and infected material, clinicians and individuals attending to human rabies case
• Veterinarians, animal handlers and dog catchers
• Wildlife wardens, quarantine officers, etc
• Travelers from rabies free areas to rabies endemic areas

Vaccination: Total three doses are recommended for pre-exposure prophylaxis.

• IM route: 1 full vial to be given on days 0,7 and booster on either day 21 or 28
• ID route: 0.1 ml on one site to be given on days 0,7 and booster dose on either day 21 or 28

High risk groups should check their antibody titers on every 6 months during the initial two year period after the primary vaccination. If it is less than 0.5 IU.ml, a booster dose of vaccine should be given.

Hand, Foot and Mouth Disease (HFMD)

Hand, foot and mouth disease (HFMD) is a viral infection caused by agents of Enterovirus family. Common causes of HFMD are Coxsackievirus A16, Coxsackievirus A6, and Enterovirus 71 (EV-A71). It is a common viral illness of infants and children and uncommon in adults.

Transmission

HFMD is moderately contagious. Infection is spread from person to person by direct contact with nose and throat discharges, saliva, fluid from blisters, or the stool of infected persons. Rarely, infection by swallowing recreational water, such as water in swimming pools. This can happen if the water is not properly treated with chlorine and becomes contaminated with feces from a person who has HFMD.

Clinical features

HFMD is mucocutaneous manifestation that usually affects persons in their preteen and teenage years. Serotypes CVA16 and EV71 are responsible for most epidemic cases of HFMD, but occasionally may be associated with CVA4-CVA7, CV9, CVA10, CVB1-CVB3, CVB5, and echovirus 4. 

After an incubation period of 3-6 days, a prodrome characterized by low fever, malaise, and abdominal or respiratory symptoms precedes the mucosal lesions by 12-24 hours. Adult cases have been associated with severe symptoms and occasional onychomadesis.

Oral lesions typically appear first and are most common on the hard palate, tongue, and  buccal mucosa. The lesions can vary in number from 1 to 10 and typically begin as macules that rapidly progress to 2 to 3 mm vesicles and then to shallow, yellow-grey painful ulcers with an erythematous halo.

Cutaneous vesicles appear concomitantly with or soon after the oral lesions and are most prevalent on the hands and feet, including the palms and soles, but can appear on the face, legs, and buttocks. The lesions can vary in number from a few to over 100. Cutaneous lesions also begin as erythematous macules, but are larger (3-7mm) and develop into cloudy, white oval vesicles with a red halo. Both oral and cutaneous lesions are usually tender or painful and resolve in 5-10 days without treatment or scarring.

HFMD can cause neurologic manifestations that range from aseptic meningitis to acute flaccid paralysis and brainstem encephalitis, which can be associated with systemic features such as severe pulmonary edema and shock. 

Laboratory diagnosis

In mild cases of HFMD, particularly in patients with a high probability of having the disease based on their clinical characteristics and sick contacts, laboratory testing is not necessary. Testing is usually reserved for severe cases and epidemiological studies.

Samples collected : Throat and vesicle specimens are considered to be the most useful sources for diagnostic purpose. Other samples collected include blood, rectal swabs, stool. 

Laboratory tests include: Viral culture, Polymerase chain reaction and ELISA.

Prevention

• Frequent washing of hands with soap and water after changing diapers, after using toilets, after using blowing nose, coughing or sneezing , and before and after caring for someone who is sick.
• Clean and disinfect frequently touched surfaces and shred items including toys and doorknobs.
• Avoid touching eyes, nose and mouth with unwashed hands
• Avoid close contact with sick people

Treatment

No proven antiviral treatment exists for HFMD. Thus, the goal of treatment are typically supportive, as for any self limited viral syndrome.

PHAGE LYSINS

Viruses that specifically infect bacteria are called bacteriophage or phage. After replicating inside the bacterial cells, phages needs to efficiently exit the bacterium to disseminate its progeny to begin a new cycle. For this purpose, double stranded deoxyribonucleic acid phages have evolved a lytic system to weaken the bacterial cell wall resulting in hypotonic bacterial lysis or lysis from within.

Phage lytic enzymes or lysins are highly efficient molecules to release it progeny phage. These enzymes target the integrity of the cell wall, and are designed to attack one of the five major bonds in the peptidoglycan. The lysins acts along with a translocase system called holin. The holin molecules are inserted in the cytoplasmic membrane forming patches, resulting in a hole through which the lysins access the peptidoglycan to cleave the specific bonds, thereby causing immediate cell lysis and release of progeny phage. 

Structure of lysins (Endolysins)

Lysins consists of two separate functional domains , one is enzymatically catalytic domain (EAD) which is N-terminal and the second is the C-terminal cell wall binding domain (CBD). EAD is responsible for for the catalytic activity and works by cleaving various bonds in the peptidoglycan, where as the CBD recognizes and specifically binds to its target bacterial cell wall receptors. Gram positive and Gram negative lysins are structurally different. Unlike Gran positive lysins that exhibit the classical structure with both the domains, Gram negative lysins only possesses EADs , they mostly lack a modular structure.

 Mechanism of action

Several types of EADs are identified which works at different sites in the peptidoglycan layer. N-acetyl muramidases, N-acetylglucosaminidases and transglycosilases work on the sugar backbone of the peptidoglycan layer whereas the other class called endopeptidases attack on the peptide bonds of the cross-linking and interpeptide bridges, N-acetylmuramoyl-L-alanine amidases work by attacking the amide bond between N-acetylmuramic acid and L-alanine causing rapid cleavage. In addition, Lysin-Holin system seen in many Gram positive infecting phages that direct the lysins to gain easy access to the cell wall from within. Finally, the bacterial cell loses rigidity leading to cell lysis and death. 

These cell wall breaching enzymes represent a step ahead in the antibacterial campaign acting as active killing mechanisms with high specificity. Moreover, phage lysins are direct, kill instantly, lacking the issues of associated resistance with no off-target effects as peptidoglycan does not exist in mammalian tissue which is a clear advantage over antibiotics and chemical preservatives. Also the biodegradable nature of these enzymes fits perfectly into its application in/on food stuffs as well as use as therapeutics.  

Lysins as Antibacterial agents

Due to the emergence of antibiotic resistance in organisms, many infectious agents have become life threatening agents in humans. To overcome theses issues, research has focused on phage derived endolysins for both topical and systemic infections in humans.

One particular pathogen that has received significant focus is Staphylococcus aureus, due to its involvement in topical skin or tissue infections, as well as systemic blood poisoning, bone, and cardiac infections. Furthermore, the rise in multidrug resistant and methicillin resistant S. aureus (MRSA), has reduced the availability of effective antibiotics. Commercially available recombinant endolysins, Staphefekt SA.100, and XDR.300 have been implemented in patients with chronic skin disease caused by S. aureus. Another endolysin, CHAPk has the potential reduce S. aureus colonization in the skin.

In contrast, Gram negative pathogens such as Acinetobacter baumannii and Pseudomonas aeruginosa have considered major opportunistic pathogens in burn wounds. To treat such drug resistant pathogens, novel engineered endolysin named artilysins have been suggested.  

Use of lysins in veterinary medicine

Food animals such as cattle, poultry, and swine have been found to be a major reservoir of antibiotic resistant bacteria and its specific gene that can move to people directly or indirectly by the food chain. As a consequence, the use of antibiotics in animal feed has been banned in many countries. Therefore, there is significant need for antibiotic alternatives such as endolysins for veterinary use.

Endolysins have been recommended to treat most farm animal related pathogens such as Clostridium perfringens, Streptococcus suis, Paenibacillus larvae and Salomonella species. 

Examples include;

• Endolysin phiSM101 against Clostridium perfringens
• amidase endolysin, CP25L against Lactobacillus johnsonii
• amidase endolysin PlyG against Bacillus anthracis
• amidase endolysin PlyC Streptococcus equi

Use of lysins in Agriculture

The prevalence of antibiotic resistance in the food chain process within agriculture and crop culture has led to the cause of bacterial infection in humans. For example, Multidrug resistant leaf blight rice can cause nosocomial infections in immunocompromised individuals. Thus endolysin therapy has been suggested to ensure safety of plants. 

Examples include;

• Transgenic tomato plant with CMP1 phage to prevent infection of Clavibacter michiganensis, responsible for canker.
• Transgenic potato plant with T4 phage lysin to Erwinia carotovora. 

Use of lysins in food technology

Lysins can be safely used in the food industry as a potential alternative to the chemical preservatives and antibiotics. The advantages are;

• Phages are ubiquitous in nature and are natural commensals in of humans and animal body. Their ubiquitous nature strongly support the fact that they are part of our foods and are completely safe and harmless entities.
• High specificity in their action allows attacking target bacterial cells only while not affecting the normal microbial flora unlike most of the preservatives and antibiotics do.
• Phage treatment of foods does not lead to any change in sensory, taste, organoleptic properties which may may discourage the final consumer acceptance.

Examples include;

• LinM-Ag8- immobilized phage active against  growth of Listeria monocytogenes in cantaloupe and RTE meat
• Team1, P68,LH1-MUT- phage cocktail eradicated S. aureus load after 14 days of cheddar cheese curd ripening at 4°C
• SE07-pahge application bought significant reduction of Salmonella enteritidis population in fruit juice and fresh eggs, beef, and chicken meat samples after incubation at 4°C for 48 hour.

 Use of Endolysin in Biofilm eradication

Bacteria are universally found in nature attached to surfaces such as living tissues, medical devices, industrial equipment or food. During attachment some bacteria produce extracellular polymeric substances forming a complex cluster of bacterial cells, known as biofilm.    

In clinical and food settings, biofilms are major concerns as they form on critical locations causing contamination that affects the efficacy of the established procedures; for example, the bacterial colonization on the outer surfaces of catheters. Also they cause treatment failure in surgeries and chronic wounds due to antibiotic-resistant bacteria housed within the biofilm network.

Examples include;

• endolysin LysPA26 to eliminate Psudomonas aeruginosa, Acinetobacter baumannii, Klebsiella pneumoniae and Escherichia coli  in the biofilm formation.
• LySMP, to treat Streptococcus suis biofilm.

 

 

SCRUB TYPHUS

Scrub typhus (Tsutsugamushi fever or Chigger borne typhus) is a chigger-borne zoonosis caused by Orientia tsutsugamushi. Humans are accidental hosts who acquire the disease by intruding into often sharply localized foci colonized by infected larval trombiculid mites (chigger mites).

The family Rickettsiaeceae currently comprises of three genera-Rickettsia, Orientia and Ehrlichia. These organisms are primarily parasites of arthropods such as lice, fleas, ticks and mites. 

Causative agent

Orientia tsutsugamushi is an obligate intracellular parasite. The organism are rods approximately 0.5 µm in width and from 1.2 to 3.0 µm in length. It differs from other members of the family in its genetic make up and in the composition of its cell wall structure since it lacks lipopolysaccharides and peptidoglycan and does not have an outer slime layer. 

Orientia tsutsugamushi invade host cells by induced phagocytosis and escapes from the phagosome to the cytosol. Once free in the host cytoplasm, the bacteria replicate by transverse binary fission in the perinuclear area. Organisms are released from the cell by pushing out the host cytoplasmic membrane from inside, and budding organisms accumulate at high density on the host cell from infected cells. The organism enters the cytosol of the new cell by lysing intervening host cell membranes. Organisms stain deep purple by Giemsa and characteristically are grouped in perinuclear clusters. 

 Antigenic structure

The major surface protein antigen of Orientia tsutsugamushi is the variable 56-kDa protein, which accounts for 10 to 15 % of its total protein. This protein is an immunodominant antigen, is reactive with group and strain specific monoclonal antibodies, and is recognized by sera from most scrub typhus patients. The other major surface proteins are 110, 47 and 25 kDa. 

Habitat and Ecology

Transmission often occurs in zones where primary forest has been cleared and replaced by secondary, or scrub, vegetation, hence the name scrub typhus.  

Humans are accidental hosts, acquiring Orientia tsutsugamushi during feeding of a larval trombiculid mite of the genus Leptotrombidium. These chiggers only feed on mammalian tissue fluid once in their lifetime and constitute the reservoir of infection through transovarial transmission. Mites are normally maintained in nature by feeding on a variety of wild rodents. Rodents are the key to the population density of chiggers but are not a reservoir of Orientia tsutsugamushi. Only a low proportion of chiggers acquire Orientia tsutsugamushi acquire from infected rats, and chiggers infected by feeding neither develop a generalized infection or transmit the organisms transovarially to their offspring. 

Humans become infected when the accidentally encroach on a zone where the rodent-chigger cycle is taking place. Transmission depends on the seasonal activities of both chiggers and humans. Chigger activity is determined by temperature and humidity, both of which are relatively stable in tropics. In much of southeast Asia where climatic conditions are favorable throughout the year, populations of mites and maintaining rodent hosts may be high and endemic areas extensive. 

Leptotrombidium deliense is the most important vector species in Southeast Asia and Southern China, where as L. akamushi, L. scutellare and L. pallidum are the main vectors in Korea and Japan. 

Pathogenesis and pathology

Heparin sulfate proteoglycans contribute to the attachment of Orientia tsutsugamushi to cells, but a specific cellular receptor has not been identified. Scrub typhus bacteria have been demonstrated in a variety of cells in humans, including monocytes, macrophages, kuppfer cells, cardiac myocytes, hepatocytes and endothelial cells. 

In fatal cases, the histopathology is chiefly disseminated focal vasculitis and perivasculitis, particularly in vessels of the skin, lungs, heart, and brain. Endovasculitis and focal hemorrhage may be present but are less prominent than in Rocky Mountain spotted fever and epidemic typhus. Pathologic abnormalities often correlate poorly with the clinical picture. Several series report consistent vasculitic lesions in the kidney and heart, but neither primary myocarditis nor renal failure are often seen clinically. The basic histopathological lesions, disseminated perivasculitis, and focal interstitial mononuclear infiltrates associated with edema suggest that macrophages are a more important target cell than the endothelium. Thrombotic lesions are rare, and little histologically evident vascular damage was seen in the most important histopathologic study of Orientia tsutsugamushi infection. The most important lesions are interstitial pneumonia with alveolar edema, hemorrhage, occasionally hyaline membranes, interlobular septal edema, and meningoencephalitis.

Clinical features

The chigger bite can occur on any part of the body, is painless, and is not usually remembered by the patient. An eschar forms at the bite site in about half of primary infection and in a lesser proportion of secondary infections. However eschars are often located in hard to examine areas such as the genital region or under the axilla and are often missed. 

The eschar develops during the 6 to 20 day (average 10 days) incubation period and is usually well developed by the time fever appears. It begins as a small papule, enlarges, undergoes central necrosis, and acquires a blackened crust to form a lesion resembling a cigarette burn. Regional lymph node are enlarged and sometimes tender, and generalized lymphadenopathy and splenomegaly are not uncommon.

Fever and headache begin abruptly and are frequently accompanied by myalgias and malaise. Muscle tenderness is either absent or mild. A transient muscular rash may appear at the end of the first week of illness. The rash appears on the trunk, becomes maculopapular, and spreads peripherally. Hearing loss concurrent with the onset of fever occurs in about one-third of cases and is a very useful diagnostic clue. Acoustic nerve damage caused by scrub typhus has been well documented pathologically.

Cough, sometimes accompanied by infiltrates on the chest radiograph, is one of the most common presentations of scrub typhus infection. In severe cases, tachypnea progress to dyspnea, the patient become cyanotic, and full-blown ARDS may develop. Respiratory failure is the most common cause of death in severe scrub typhus infection.

Laboratory diagnosis

Diagnostic methods include

• Isolation of organism
• Serology
• Molecular methods (PCR)

Isolation of organism

As rickettsiae are highly infectious and comes under Group 3 organisms. isolation should be in laboratories equipped with appropriate safety provisions preferably Biosafety level-3 laboratory.

Rickettsia may be isolated in male guinea pigs or mice, yolk sac of chick embryos, vero cell line or MRC 5 cell lines from patients in early phase of the disease. Rickettsia grow well in 3-5 days on vero cell and MRC 5 cell coverslip cultures and can be identified by immunofluorescence using group and strain specific monoclonal antibodies.

Serological diagnosis

Several diagnostic tests are currently available for the demonstration of significant rise in titer of antibodies in the serum of patient during the course of infection and convalescence . Tests include Weil-Felix Test (WFT), Indirect Immunofluorescence (IIF), Enzyme Linked Immunosorbent Assay (ELISA), etc. 

Weil-Felix Test : Test detects antibodies produced during Orientia tsutsugamushi infection that cross-react by agglutination with the OX-K antigen of an unrelated bacteria, Proteus mirabilis.

Indirect Immunofluorescence (IIF): Which use yolk sac propagated or cell culture derived Orientia tsutsugamushi antigens

Molecular methods

For PCR, blood sample is collected in tubes containing EDTA or sodium citrate. Organism can be demonstrated by standard and nested PCR. Recently, detection of Orientia tsutsugamushi quantitative real time PCR has been reported.

Treatment

Antibiotic therapy is the most effective measure of treatment. Tetracyclines and chloramphenicol are  used for therapy. Doxycycline in a dose of 100 mg twice daily for 7-15 days or chloramphenicol 500 mg four times a day PO for 7-15 days (for children 150 mg/kg/day for 5 days) is recommended.

Prevention and control 

Chemoprophylaxis : Should be considered for persons with anticipated intense but transient exposure to Orientia tsutsugamushi. Weekly dose of 200 mg of doxycycline can prevent infection. 

Reduction in Chigger mite: Contact with chiggers can be reduced by applying repellent to the tops of boots, socks, and trouser legs and by not sitting or lying directly on the ground. 

Scrub typhus vaccine: An effective vaccine for humans has not been developed till now, mainly due to serotypic heterogencity of the organism.

 

DISCOVERED WORLD'S LARGEST BACTERIUM (Thiomargarita magnifica)

By definition, a microorganism or microbe is an organism of  microscopic size (they can only be seen with a microscope), which may exist as single celled form or as colonies.

But the newly described bacterium (Thiomargarita magnifica) living in Caribbean mangroves are different from known properties of microorganisms. Its threadlike single cell is visible to naked eye, growing up to 2 centimeters. This bacterium has a large genome which is not free floating inside the cell as in other bacteria, but instead encased in a membrane ( like the characteristics of complex cells).

Basically the living organisms can be divided into two broad categories: One is prokaryotes-which include bacteria and single celled organisms called Archaea and second is eukaryotes, which include either single celled or multicellular organisms that contain nucleus and other membrane bound organelles. There is a wide range of Eukaryotic organisms, including all animals, plants, fungi and protists. Prokaryotes are organisms that lacks a distinct nucleus and other organelles due to the absence of internal membranes. The newly described organism blurs the line between prokaryotes and eukaryotes.

Thiomargarita namebiensis was discoverd in Oceanic sediments off the Namibian coast in April 1997 and currently holds the world record for second largest bacterium. This microbe ranges from 100 to 300 micrometers in length with the largest reported to be 750 micrometers. In comparison, E.coli and other normal sized bacterium are an average of 2 micrometers, approximately 0.7% the size of Thimargarita namibiensis.

Like Thiomargarita namibiensis, found in Namibia, the new mangrove bacterium also has a huge sac presumably of water- that takes up 73% of its total volume. That similarity and a genetic analysis led the research team to place it in the same genus and propose calling it Thiomarita magnifica.

The genetic analysis with labelling the DNA with fluorescent tags shows the bacterial genome was so big because there are more than 500,000 copies of the same stretches of DNA. Ribosomes were also observed inside the DNA-filled sac. 

MARBURG VIRUS DISEASE (MVD)

Marburg virus disease formerly known as Marburg hemorrhagic fever, is a severe, often fatal illness in humans. MVD is caused by the Marburg virus, RNA virus of the Filovirus family.

Marburg was first recognized in 1967, when outbreaks of hemorrhagic fever occurred simultaneously in laboratories in Marburg and Frankfurt, Germany and in Belgrade, Serbia. The first people infected had been exposed to Ugandan imported African green monkeys or their tissues while conducting research.

The reservoir host of Marburg virus is the African fruit bat, Rousettus aegyptiacus. Fruit bats infected with virus do not show obvious symptoms. This Rousettus bat is sighted, cave-dwelling bat widely distributed across Africa.

The virus

Marburg viruses are filamentous, enveloped, single stranded, negative sense RNA viruses that belong to the family Filoviridae, genus Marburgvirus. There is only a single species Marburg marburgvirus, that include two viruses: Marburg and Ravn virus. Both viruses cause clinically indistinguishable disease.

The MARV genome encodes seven structural proteins with different role in pathogenesis. Viral RNA is associated with the nucleoprotein (NP), viral protein 30 (VP30), VP35 and the L-polymerase (L) which form the nucleocapsid. A matrix (composed of  VP40), VP 24 and lipid envelope with surface glycoprotein (GP) spikes surrounds the ribonucleoprotein. Cell and tissue tropism and virus-cell membrane fusion are determined by MARV GP. In addition, GP may play a role in immune evasion by counteracting the antiviral effects of tetherin, an antiviral interferon stimulated protein that inhibits viral spread. Function of VP40 is to suppress host cell response to IFN signaling. VP35 is virulence factor that facilitates immune evasion by impairing IFN response and is important in viral RNA synthesis. The L protein mediates genome replication and transcription.

MARV is classified as risk group 4 (RG-4) pathogen.

Transmission

Natural reservoir for the virus is R. aegyptiacus bats, but it is not clear how MARV transmission from bat to humans occurs.

• Once an individual is infected, interhuman transmission occurs via direct contact (broken skin or mucus membranes) with the blood and other body fluids (urine, saliva, faeces, vomit, breast milk, amniotic fluid, and semen) of infected people or 
• Indirect contact with contaminated surfaces and materials such as clothing, bedding, and medical equipment. 
• Infection may occur in relation to the burial of infected individuals.
• Contact with dead or living infected animals including bushmeat (eg. monkeys, chimpanzees, forest antelopes and bats) 

Filovirus can survive in liquid and dried material for many days. They are inactivated by gamma irradiations , heating for 60-75 minutes at 60 Degree Celsius or boiling for five minutes, and are sensitive to liquid solvents, sodium hypochlorite and other disinfectants.  

Clinical features

The incubation period last from 5 to 10 days (range 3-21 days), and is most likely related to the infectious dose and the route of infection. Transmission does not occur during the incubation period.

The clinical course can be divided into three phases, 

• Generalized phase (days 1-4)
• Early organ phase (days 5-13)
• Late organ or convalescent phase (days 13+)

The onset of  MVD is abrupt, with non-specific, flu like symptoms such as high fever, severe headache, chills, myalgia, prostration and malaise. In 50-70 % of patients , rapid debilitation, marked by gastrointestinal symptoms such as anorexia, abdominal discomfort, severe nausea, vomiting and diarrhoea, occurs within 2-5 days. The intensity of the disease increases on days 5-7, with a maculopapular rash and symptoms of haemorrhagic fever such as petechiae, mucosal and gastrointestinal bleeding. Fresh blood in vomitus and faeces is often accompanied by bleeding from the nose, gums, and vagina. Spontaneous bleeding at venipuncture sites (where intravenous access is obtained to give fluids or obtain blood sample) is particularly troublesome. Neurological symptoms (disorientation , agitation, seizures, and coma) can occur in later stages of the disease. Joint pain , uveitis, orchitis, recurrent hepatitis, pericarditis and mental dysfunction have been documented as complications during convalescence.

Disseminated intravascular coagulation, lymphopenia and thrombocytopenia typically appear within a week after the disease onset. Patients either recover with supportive therapy or die from dehydration , internal bleeding and multiorgan failure 8-16 days after symptom onset. MVD survivors have experienced various complications including exhaustion, myalgia, hyperhidrosis, skin desquamation and hair loss.

Diagnosis

Clinical diagnosis is difficult because many of the signs and symptoms are comparable to other infectious diseases such as malaria, typhoid fever and dengue as well as other viral hemorrhagic fevers.

Diagnostic methods include;

• Virus isolation
• Reverse transcription polymerase chain reaction (RT-PCR)
• Antigen detection
• Serology and
• Immunohistochemistry.

Virus isolation: Virus propagation in different cell lines (especially Vero cells and Vero E6).

Should be performed in a BSL-4 laboratory.

Molecular methods: (RT-PCR, nested PCR , real-time quantitative RT-PCR) targeting NP,L and GP genes are sensitive and specific.

Antigen detection: Since high virus titers are present in blood and tissues, antigen detection is suitable for diagnosis in early stages of MVD. The antigen capture ELISA targets the proteins NP, VP40 and GP.

Serology: Methods include ELISA & IFA. The recombinant proteins rGP and rNP are used for detection of MARV IgM & IgG antibodies. IgM capture ELISA has been shown to be very useful for detection MARV IgM antibodies which indicate recent infection and can be detected as early as 2-4 days after symptom onset. MARV IgG antibodies can be detected 8-10 days after symptom onset and persist up to 2 years. 

Histological technique: Antigen detection by immunohistochemistry (for post mortem diagnosis).

Treatment 

Currently there are no vaccines or antivirals treatments approved for MVD. However, supportive care-rehydration with oral or intravenous fluids- and treatment of specific symptoms improves survival.

Prevention and control

Raising awareness of risk factors for Marburg infection and protective measures that individuals can take is an effective way to reduce the human transmission.

Transmission from wildlife to people remains unclear, however avoiding fruit bats and sick non-human primates is one way to protect against infection.

Measures for prevention of secondary or person to person transmission are like those used for other hemorrhagic fevers. If a patient is either suspected or confirmed to have MVD, infection prevention and control measures should be used to prevent direct physical contact with the patient. These precautions include wearing protective gowns, gloves, and mask; placing the infected individual in strict isolation ands sterilization and proper disposal of needles, equipment's and patient excretions.