Lecture #: HYPERSENSITIVITY

Hypersensitivity in medical context is defined as heightened state of immune responsiveness. Typically, it is an exaggerated response to a harmless antigen that results in injury to the tissue, disease, or even death.

In Type I reactions, cell–bound antibody reacts with antigen to release physiologically active substances. Type II reactions are those in which free antibody reacts with antigen associated with cell surfaces. In Type III hypersensitivity, antibody reacts with soluble antigen to form complexes that precipitate in the tissues. In these latter two types, complement plays a major role in producing tissue damage. Type IV hypersensitivity differs from other three, because sensitized T cells rather than antibody are responsible for the symptoms that develop.

Although some disease manifestations may overlap among these categories, knowledge of the general characteristics of each will help in understanding the immune process that triggers such tissue damage.

Types I through III have previously been referred to as immediate hypersensitivity, because symptoms develop within a few minutes to a few hours. Type IV hypersensitivity has been called delayed hypersensitivity, because its manifestations are not seen until 24 to 48 hours after contact with antigen.

The Effector cells of Immediate Hypersensitivity

1.      Mast cells

Mast cells are the derived from precursors in the bone marrow that migrate to specific tissue sites to mature. Although they are found throughout the body, they are most prominent in connective tissue, the skin, the upper and lower respiratory tract, and the gastrointestinal tract. In most organs, mast cells tend to be concentrated around the small blood vessels, the lymphatics, the nerves, and the glandular tissue. These cells contain numerous cytoplasmic granules that are enclosed by a bilayer membrane. Histamine, which comprises approximately 10% of the total weight of granular constituents, is found in 10 times greater supply per cell than in basophils.

Mast cell population in different sites differs in the amounts of allergic mediators that they contain and in their sensitivity to activating stimuli and cytokines. All types of cells are triggered in the same manner; however, they release a variety of cytokines and other mediators that enhance the allergic response.

2.      Basophils

Basophils represent approximately 1% of the white blood cells in peripheral blood. They have half–life of about 3 days. They contain histamine–rich granules and high affinity receptors for IgE, just as in mast cells. They respond to chemotactic stimulation and tend to accumulate in the tissues during an inflammatory reaction. In the presence of IgE, the number of receptors has been found to increase, indicating a possible mechanism of upregulation during an allergic reaction.

Mediators of Immediate Hypersensitivity

1.      Preformed Mediators

These substances are responsible for early phase symptoms seen in allergic reactions, which occur within 30 to 60 minutes after exposure to the allergen. These mediators are released from the cytoplasmic granules after cross– linking of surface–bound IgE on basophils and mast cells by a specific allergen.

a.      Histamine

A vasoactive amine which is a major component of mast cell granules; its effect appear within 30 to 60 seconds after release. This is dependent on activation of four different types of tissue:

(1)   Activation of H₁ receptors results in contraction of smoot muscle in bronchioles, blood vessels and the intestines and generally induces proinflammatory activity. In addition, there is increased capillary permeability, altered cardiac contractility and increased mucous gland secretion in the upper respiratory tract.

(2)   Binding to H₂ receptors increases gastric acid secretion, airway mucus production, and permeability of capillaries and venules.

(3)   H₃ receptors are found on central and peripheral neural tissue.

(4)   H₄ receptors appear to be involved in immune regulation, including chemotaxis of mast cells. Bone marrow cells, mast cells and peripheral blood cells such as eosinophils, neutrophils and basophils all have H₄ receptors.

In the skin, histamine is responsible for local erythema or redness and wheal and flare formation. Contraction of the smooth muscle in the bronchioles may result in airflow obstruction. Increased vascular permeability may cause hypertension or shock. Depending on the route by which an individual is exposed to the triggering allergen, one or more of these effects may be seen.

b.     Heparin

Heparin inhibits allergen induced mast cell degranulation and prevents subsequent development of reaction cascade leading to inflammation, bronchial hyper reactivity and asthma. It also modulates migration of proinflammatory cells, eosinophils and neutrophils, into the site of allergic reaction.

c.       Eosinophil chemotactic factor of anaphylaxis (ECF–A)

This attracts eosinophils to the area and induces expression of eosinophil receptors for C3b. Eosinophils have granules that contain major basic protein, eosinophil cationic protein, eosinophil peroxidase, eosinophil derived neurotoxins, histaminases, and phospholipase D. all of these products are toxic to bronchial epithelial cells and to helminth parasites. Killing of parasites is thought to be the reason that IgE production evolved.

d.     Proteases

One of the proteolytic enzymes released is tryptase. Tryptase cleaves kininogen to generate bradykinin, which induces prolonged smooth muscle contraction and increases vascular permeability and secretory activity. Complement split products are also released when C3 is converted to C3a and C3b.

2.      Newly synthesized Mediators

These newly formed mediators are responsible for a late phase reaction in 4 to 6 hours after exposure to allergen, during which numerous cells such as eosinophils, neutrophils, Th2 cells, mast cells, basophils, and macrophages exit the circulation and infiltrate the allergen – filled tissue. They release further mediators that prolong the hyperactivity and may lead to tissue damage.

a.      Prostaglandin (PG) D₂

PGD₂ is the major product of the cyclooxygenase pathway. When released by mast cells, it mimics the effects of histamine, causing bronchial constriction and vasodilation. It is much more potent than histamine, but it is released in smaller quantities. In skin reaction PGD2 triggers wheal and flare formation. Thus, the overall effect is to enhance and potentiate the action of histamine.

b.     Platelet activating factor (PAF)

PAF is a phospholipid released by monocytes, macrophages, neutrophils, eosinophils, mast cells and basophils. The effect of PAF includes platelet aggregation, chemotaxis of eosinophils and neutrophils, increased vascular permeability, and contraction of smooth muscle in the lungs and intestines.

c.       Leukotrienes (LT) B₄

Leukotrienes, resulting from the 5–lipoxygenase pathway of arachidonic acid metabolism, are also responsible for late–phase symptoms of immediate sensitivity. Leukotrienes C₄, D₄, and E₄ were originally collectively named the slow–reacting substances of anaphylaxis (SRS–A). LTC₄ and LTD₄ are 1000 times more potent than histamine in causing increased vascular permeability, bronchoconstriction and increased mucous secretion in small airways. In the intestines, leukotrienes induce smooth muscle contraction. Systematically, they may produce hypotension as a result of diminished cardiac muscle contractility and lessened blood flow.

LTB₄ is a potent chemotactic factor for neutrophils and eosinophils. The appearance of eosinophils is especially important as a negative feedback control mechanism. Eosinophils release histaminase, which degrades PAF. Additionally, superoxides created in both eosinophils and neutrophils cause the breakdown of leukotrienes. Eosinophil products can also have a damaging effect. Eosinophil cationic protein and eosinophil–derived neurotoxin contribute to extensive tissue destruction.

d.     Cytokines

Cytokines released from mast cells include IL–1, IL–3, IL–4, IL–5, IL–6, IL– 9, IL–13, IL–14, IL–16, GM–CSF and TNF–α. These cytokines alter the local microenvironment, leading to an increase in inflammatory cells such as neutrophils, eosinophils, and macrophages. IL–3 and IL–4 increase IgE production to further amplify the Th2 response. In addition, IL–3 is a growth factor for mast cells and basophils, and IL–4 recruits T cells, basophils, eosinophils, and monocytes. IL–5 also recruits eosinophils. A high concentration of TNF–α may contribute to the symptoms of shock seen in systemic anaphylaxis.

TYPE I HYPERSENSITIVITY

The distinguishing feature of type I hypersensitivity is the short time lag, usually seconds to minutes, between exposure to antigen and the onset of clinical symptoms. The key reactant present in type I, or immediate sensitivity reactions, is IgE. Antigens that trigger formation of IgE are called antopic antigens, or allergens. Atopy refers to an inherited tendency to respond to naturally occurring inhaled ad ingested allergens with continued production of IgE.

Typically, patients who exhibit allergic or immediate hypersensitivity reactions produce a large amount of IgE in response to a small concentration of antigen. IgE levels appear to depend on the interaction of both genetic and environmental factors.

Mechanism of action

a.      IgE is primarily synthesized in the lymphoid tissue of the respiratory and gastrointestinal tracts. Normal levels are in the range of approximately 150 ng/mL. The regulation of IgE production appears to be a function of a subset of T cells called type 2 helper cells (Th2).

b.      The normal immune response to microorganism and possible allergens is a function of type 1 helper cells (Th1), which produce interferon–gamma (IFN–Ɣ). IFN–Ɣ, along with interleukin–12 and interleukin–18, which are produced by macrophages, may actually suppress production of IgE type antibodies. However in people, with allergies, Th2 cells respond instead and produce interleukin–3 (IL –3), interleukin–4 (IL–4), interleukin–5 (IL–5), interleukin–9 (IL–9) and interleukin–13 (IL–13)

c.       IL–4 and IL–13 are responsible for the final differentiation that occurs in B cells, initiating the transcription of the gene that codes for the epsilon–heavy chain of immunoglobulin molecules belonging to the IgE class.

d.     IL–5 and IL–9 are involved in the development of eosinophils, while IL–4, IL–9 promote development of mast cells.

e.      IL–4, IL–9 and IL–13 all act to stimulate overproduction of mucus, a characteristic of most allergic reactions.

f.        This propensity to secrete cytokines  that promote production of IgE is linked to a gene locus on chromosome 5 that encodes cytokines IL–3, IL–4, IL–5, IL–9, IL–13 and granulocyte–monocyte colony stimulating factor (GM–CSF). All of these cytokines are key to a switch to a Th2 response. IL–4 and IL–13 activate transcription of the epsilon gene in B cells when they bind to specific receptors.

g.      Although actual antibody synthesis is regulated by the action of cytokines, the tendency to respond to specific allergens appears to be linked to inheritance of certain major histocompatibility complex (MHC) genes. Various human leukocyte antigen (HLA) class II molecules, especially HLA–DR2, DR4 and DR7, seem to be associated with a high response to individual allergens. HLA–D molecules are known to play a role in antigen presentation and thus individuals who possess particular HLA molecules are more likely to respond in certain allergens.

Clinical signs of Type I Hypersensitivity

1.      Clinical signs of anaphylaxis begin within minutes after antigenic challenge and may include bronchospasm and laryngeal edema, vascular congestion, skin manifestations such as urticarial (hives) and angioedema, diarrhea or vomiting and intractable shock because of the effect on blood vessels and smoot muscle of the circulatory system. The severity of the reaction depends on the number of previous exposures to the antigen with consequent buildup of IgE on mast cells and basophils. Massive release of reactants, especially histamine, from the granules is responsible for the ensuing symptoms. Death may result from asphyxiation due to upper–airway edema and congestion, irreversible shock, or a combination of these symptoms.

2.      Rhinitis is the most common form of atopy, or allergy. Symptoms include paroxysmal sneezing; rhinorrhea or runny nose; nasal congestion; and itching of the nose and eyes. Although the condition itself is merely annoying, complications such as sinusitis, otitis media (ear infection), Eustachian tube dysfunction and sleep disturbance may result. Pollen, mold spores, animal dander and particulate matter from house dust mites are examples of airborne foreign particles that act directly on the mast cells in the conjunctiva and respiratory mucous membranes to trigger rhinitis.

3.      Asthma is a recurrent airflow obstruction that leads to intermittent sneezing, breathlessness and, occasionally, a cough with sputum production. The airflow obstruction is due to bronchial smooth muscle contraction, mucosal edema and heavy mucous secretion. All of these changes lead to an increase in airway resistance, making it difficult for inspired air to leave the lungs. This trapped air creates the sense of breathlessness.

4.      Some of the most common food allergies involve cow’s milk, peanuts and eggs. Symptoms limited to the gastrointestinal tract include cramping, vomiting and diarrhea.

Test for Type I Hypersensitivity

1.      In Vivo Skin Test

Skin testing is relatively simple and inexpensive, and it lends itself to screening for a number of allergens. However, antihistamines must be stopped 24 to 72 hours before testing, and there is the danger that a systemic reaction can be triggered. In addition, skin testing is not recommended for children under 3 years of age.

a.      Cutaneous test

In cutaneous testing, or a prick test, a small drop of material is injected into the skin at a single point. After 15 minutes, the spot is examined, and the reaction is recorded. A positive reaction is formation of a wheal that is 3mm greater in diameter than the negative control.

b.     Intradermal test

A 1 mL tuberculin syringe is used to administer 0.01 to 0.05 of test solution between layers of the skin. The test allergen is diluted 100 to 1000 times more than the solution used for cutaneous testing. After 15 to 20 minutes, the site is inspected for erythema and wheal formation. A wheal 3 mm greater than then negative control is considered a positive test.

2.      In Vitro Test: Total IgE

a.      Total IgE

Total serum IgE testing is used clinically to aid in diagnosis of allergic rhinitis, asthma or other allergic conditions that may be indicated by patient’s symptoms. It serves as a screening test to determine if more specific allergy testing is indicated. It is also important in diagnosis of parasitic infections; bronchopulmonary aspergillosis; and hyper–IgE syndrome, a condition in which excessive amounts of IgE produced.

(1)   Radioimmunosorbent Test (RIST)

The RIST used radiolabeled IgE to compete with patient IgE for binding sites on a solid phase coated with anti–IgE.

(2)   Non–competitive solid–phase immunoassay

Anti–human IgE is bound to a solid phase such as cellulose, a paper disk, or a microtiter well. Patient serum is added to detect the bound patient IgE. This label is an enzyme or a fluorescent tag. The second anti–IgE antibody recognizes a different epitope than that recognized by the first antibody. The resulting “sandwich” of solid–phase anti– IgE, serum IgE, and labeled anti–IgE is washed, and the label is measured. In this case, the amount of label detected is directly proportional to the IgE content of the serum.

Total IgE values are reported in kilo international units (IU) per liter. One IU is equal to a concentration of 2.4 ng of protein per milliliter. IgE concentration varies with individual’s age, sex and smoking history.

In infants, normal serum levels of IgE are less than 2 kIU/L; increases of up to 10 kIU/L are indicative of allergic disease. After the age of 14 years, IgE levels in the range of 400 kIU/L are considered to be abnormally elevated. A cutoff point of 100 IU, however, is frequently used in testing to identify individuals with allergic tendencies.

           

b.     Antigen–specific IgE

(1)   Antigen–specific IgE testing is a noncompetitive solid–phase immunoassay in which the solid phase is coated with specific allergen and reacted with patient serum. A carbohydrate solid phase, such as paper cellulose disks, agarose beads, or microcrystalline cellulose particles, seems to work best. After washing to remove unbound antibody, a labeled anti–IgE is added.

Traditional Radioallergosorbent Test (RAST) uses radioactive label, while newer tests used an enzyme or fluorometric label. A second incubation occurs, and then, after a washing step, the amount of label in the sample is measured. The amount of label detected is directly proportional to the amount of specific IgE in the patient’s serum. Controls and standards are run in parallel with patient’s serum.

(2)   Microarray Testing

Microarrays can be attached to a standard microscope slide, using only a few pictograms of each allergen to be tested. In this manner, at least 5000 allergens can be tested for at once, using a minimal amount of serum, approximately 20 µL. The principle is the same as noncompetitive immunoassays, in that patient serum with possible IgE is allowed to react with the microarray of allergens, and then an anti– IgE with fluorescent tag is added. After careful washing, slides are read automatically. The presence of color indicates a positive test for that allergen.

TYPE II HYPERSENSITIVITY

The reactants responsible for type II hypersensitivity, or cytotoxic hypersensitivity, are IgG and IgM. They are triggered by antigens found on cell surfaces. These antigens may be altered self–antigens on cell surfaces. These antigens may be altered self–antigens or heteroantigens. Antibody coats cellular surfaces and promotes phagocytosis by both opsonization and activation of the complement cascade.

Macrophages, neutrophils and eosinophils have Fc receptors that bind to the Fc region of antibody on target cells, thus enhancing phagocytosis. Natural killer (NK) cells also have Fc receptors, and if these link to cellular antigens, cytotoxicity results.

If the complement cascade is activated, complement can trigger cellular destruction in two ways: (1) by coating cells with C3b, thus facilitating phagocytosis through interaction with specific receptors on phagocytic cells, or (2) by complement– generated lysis if the cascade goes to completion. Hence, complement plays a central role in the cellular damage that typifies type II reactions.

1.      Hemolytic Disease of the Newborn

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2.      Hemolytic Transfusion Reaction

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3.      Autoimmune Hemolytic Anemia

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TYPE III HYPERSENSITIVITY

Type III hypersensitivity reactions are similar to type II reactions in that IgG or IgM is involved and destruction is complement mediated. However, in the case of type III diseases, the antigen is soluble.

When soluble antigen combines with antibody, complexes are formed that precipitate out of the serum. Normally such complexes are cleared by phagocytic cells, but if the immune system is overwhelmed, these complexes deposit in the tissues. There they bind complement, causing damage to the particular tissue. Deposition of antigen–antibody complexes is influenced by the relative concentration of both components.

If a large excess of antigen is present, sites on antibody molecules become filled before cross–links can be formed. In antibody excess, a lattice cannot be formed because of the relative scarcity of antigenic determinant sites. The small complexes that result in either of the preceding cases remain suspended or may pass directly into the urine. Precipitating complexes, on the other hand, occur in mild antigen excess, and these are the one most likely to deposit in the tissues. Sites in which this typically occurs include the glomerular basement membrane, vascular endothelium, joint linings, and pulmonary alveolar membranes.

Complement binds to these complexes in the tissues, causing the release of mediators that increase vasodilation and vasopermeability , attract macrophages and neutrophils and enhance binding of phagocytic cells by means of C3b deposited in the tissues. If the target cells are large and cannot be engulfed for phagocytosis to take place, granule and lysosome contents are released by a process known as exocytosis. This results in the damage to host tissue that is typified by type III reactions. Long–term changes include loss of tissue elements that cannot regenerate and accumulation of scar tissue.

A more general method of determining immune complex diseases is by measuring complement levels. Decreased levels of individual components or decreased functioning of the pathway may be indicative of antigen–antibody combination.

1.      Arthus Reaction

Though rare in humans, Arthus reaction is the deposition of antigen–antibody complexes mainly in vascular walls, serosa (pleura, pericardium, synovium) and glomeruli. This is not an acute, immediately overwhelming condition. It generally develops 6 to 12 hours if antibody levels are already high, or it can develop over several days (e.g., in serum sickness) as antibody levels increase and antigen persists. In this reaction, immune complexes in the walls of blood vessels initiate and inflammatory reaction involving complement and leukocytes, particularly neutrophils.

Neutrophils release toxic produces such as oxygen–containing free radicals and proteolytic enzyme. Activation of complement is essential for the Arthus reaction, because the C3a and C5a generated activate mast cells to release permeability factors; consequently, immune complexes localize along the endothelial cell basement membrane.

2.      Serum Sickness

Serum sickness results from passive immunization with animal serum, usually horse or bovine, used to treat such infections as diphtheria, tetanus and gangrene. Vaccines and bee stings may also trigger this type of reaction. Generalized symptoms appear in 7 to 21 days after injection of the animal serum and include headache, fever, nausea, vomiting, joint pain, rashes, and lymphadenopathy.

In this disease, the sensitizing and the shocking dose of antigen are one and the same, because antibodies develop while antigen is still present. High levels of antibody form immune complexes that deposit in the tissues. Usually this is a benign and self–limiting disease, but previous exposure to animal serum can cause cardiovascular collapse or reexposure. These symptoms have occurred with monocolonal antibody treatment using mouse antibodies to human cells. Now, however, monoclonal antibodies are genetically engineered human antibodies, and such reactions do not occur.

3.      Autoimmune Diseases

Ehrlich used the term autoimmunity to signify an immune response against self and introduce the phrase horror autotoxicus, suggesting that there are mechanisms to protect against autoimmunity. Over the years, autoimmunity has been recognized as not uncommon and not necessarily detrimental. Thus, an important distinction must be drawn between autoimmunity, which may be asymptomatic, and autoimmune disease, which occurs when autoimmunity leads to an inflammatory response, resulting in tissue injury. An autoimmune response does not necessarily imply the existence of autoimmune disease.

TYPE IV HYPERSENSITIVITY

Type IV hypersensitivity differs from the other three types of hypersensitivity in that sensitized T cells, usually a subpopulation of Th1 cells, plays the major role in its manifestations. Antibody and complement are not directly involved. There is an initial sensitization phase of 1 to 2 weeks that takes place after the first contact with antigen. Then upon subsequent exposure to the antigen, symptoms typically take several hours to develop and reach a peak 48 to 72 hours after exposure to antigen.

The reaction cannot be transferred from one animal to another by means of serum, but only through transfer of T lymphocytes. Langerhans cells in the skin and macrophages in the tissue capture and present antigen to T helper cells of the Th1 subclass. Th1 cells are activated and release cytokines, including IL–3, interferon gamma (IFN–Ɣ), tumor necrosis factor–beta (TNF–β), and tumor necrosis factor– alpha (TNF–α), that recruit macrophages and neutrophils, produce edema, promote fibrin deposition, and generally enhance an inflammatory response. Cytotoxic T cells are also recruited, and they bind with antigen – coated target cells to cause tissue destruction. Allergic skin reactions to bacteria, viruses, fungi, and environmental antigens such as poison ivy typify this type of hypersensitivity.

1.      Contact Dermatitis

Contact dermatitis is a form of delayed hypersensitivity that accounts for a significant number of all occupationally acquired illnesses. Reactions are usually due to low–molecular–weight compounds that touch the skin. The most common causes include poison ivy, poison oak and poison sumac, all of which give off urushiol in the plant sap and on the leaves.

Other common compounds that produce allergic skin manifestation include, nickel, rubber, formaldehyde, hair dyes and fabric finishes, cosmetics and medications applied to the skin, such as topical anesthetics, antiseptics and antibiotics. Most of these substances probably function as haptens that bind to glycoprotein on skin cells. The Langerhans cells, a skin macrophage, functions as the antigen–presenting cell at the site of antigen contact. It appears that Langerhans cells may migrate to regional lymph nodes and generate sensitized Th1 cells there. This sensitization process takes several days, but once it occurs, its effects last for years.

After repeat exposure to the antigen, cytokine production causes macrophages to accumulate. A skin eruption characterized by erythema, swelling and the formation of papules appears anywhere from 6 hours to several days after the exposure. The papules may become vesicular, with blistering, peeling and weeping. There is usually itching at the site.

The dermatitis is first limited to skin sites exposed to the antigen, but then it spreads out to adjoining areas. The duration of the reaction depends upon the degree of sensitization and the concentration of antigen absorbed. Dermatitis can last for 3 to 4 weeks after the antigen has been removed.

2.      Hypersensitivity Pneumonitis

Evidence shows that hypersensitivity pneumonitis is mediated predominantly by sensitized T lymphocytes that respond to inhaled allergens. IgG and IgM antibodies are formed, but these are thought to play a minor part. This is an allergic disease of the lung parenchyma, characterized by inflammation of the alveoli and interstitial spaces. It is caused by chronic inhalation of a wide variety of antigens, and it is most often seen in men between the ages of 30 and 50 years.

Depending on the occupation and particular antigen, the disease goes by several names, including farmer’s lung, pigeon breeder’s disease and humidifier lung disease.

Alveolar macrophages and lymphocytes trigger a chronic condition characterized by interstitial fibrosis with alveolar inflammation.

3.      Tuberculin–type Hypersensitivity

This is based on the principle that soluble antigens from Mycobacterium tuberculosis induce a reaction in people who have or have had tuberculosis. When challenged with antigen intradermally, previously sensitized individuals develop an area of erythema and induration at the injection site. This is the result of infiltration of T lymphocytes and macrophages into the area. The blood vessels become lined with mononuclear cells, and the reaction reaches a peak by 72 hours after exposure.

The tuberculin skin test uses a Mycobacterium tuberculosis antigen prepared by making a purified filtrate from the cell wall of the organism. This purified protein derivative (PPD) is injected under the skin, and the reaction is read at 48 to 72 hours. A positive test indicates that the individual has previously been exposed to Mycobacterium tuberculosis or a related organism, but it does not necessarily mean there is a presently active case.

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Test for Type IV Hypersensitivity

1.      Patch Test

The patch test is considered the gold standard in testing for contact dermatitis. This must be done when the patient is free of symptoms of when he or she has a clear test site. A nonabsorbent adhesive patch containing the suspected allergen is applied on the patient’s back, and the skin is checked for a reaction over the next 48 hours. Redness with papules or tiny blisters is considered a positive test. Final evaluation is conducted at 96 to 120 hours. All readings should be done by a skilled evaluator.

2.      Mantoux method

0.1 mL of the antigen is injected intradermally, using a syringe and a fine needle. The test site is read at 48 to 72 hours for the presence of induration. An induration of 5 mm or more is considered a positive test. Antigens typically used for testing are Candida albicans, tetanus toxoid, tuberculin and fungal antigens such Trichophyton and histoplasmin.