Category: Biology

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Antigen Antibody Reactions and its types | Working principle, Applications

The interaction between antigen and antibody is called antigen-antibody reactions. It is abbreviated as Ag-Ab reaction. This reaction is the basis of humoral immunity. The antigen and the antibody react to form immune complex.

Ag + Ab …………….. Ag – Ab complex The reaction between antigen and antibody is highly specific. It is compared to the lock and key system. The part of the antigen that combines with the antibody is called epitope or antigenic determinant. The part of antibody which combines with the antigen is called paratope or antigen determining site. Most of the antibodies have two binding sites and IgM has 5-10 binding sites.

Immunofluorescence

When antibodies are mixed with the fluorescent dyes such as fluorescein or rhodamine, they emit radiation. This phenomenon of emitting radiation by antibodies labelled with fluorescent dye is called immuno fluorescence. This reaction is well observed under fluorescent microscope. It is used to locate and identify antigens in tissues.

Types of Immunofluorescence

• Direct method
• Indirect method

Direct Method

In this method, the antibody labelled with fluorescent dye is directly applied on the tissue section. The labelled antibody binds with specific antigen. This can be observed under the fluorescent microscope.

Indirect Method

In this method, unlabelled antibodies are directly applied on the tissue sectionswhich bind with the specific antigens. Then the antibody labelled with the fluorescent dye is added to the tissue. Anti-antibody specifically binds with already added or linked unlabelled antibody (Figure 11.1).

ELISA (Enzyme Linked Immuno Sorbent Assay)

ELISA (Enzyme-Linked Immuno Sorbent Assay) is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins, antibodies and hormones. It is also known as Enzyme Immuno Assay (EIA).

In 1971, after the descriptions of Peter Perlmann and Eva Engvall at Stockholm University in Sweden, ELISA has become the system of choice when assaying soluble antigens and antibodies. All assays for antibody production depend upon the measurement of interaction of elicited antibody with antigen.

Principle

The principle of ELISA is very simple. The test is generally conducted in micro titre plates. (Figure 11.2 Micro titre plate).

If the antigen is to be detected the antibody is fixed in the micro titre plate and vice versa. Test sample is added in the microtitre plate, if there is presence of Ag or Ab in the test sample, there will be Ag-Ab reactions (with immobilized Ab or Ag). Later enzyme labelled antibody is added in the reaction mixture, which will combine with either test antigen or Fc portion of test antibody.

The enzyme system consists of:

1. An enzyme:

Horse Radish Peroxidase(HRP), alkaline phosphatase which is labelled or linked, to a specific antibody.

2. A specific substrate:

• O-Phenyl-diaminedihydrochloride for peroxidase
• P nitrophenyl Phosphate – for Alkaline Phosphatase

Substrate is added after the antigenantibody reaction. The enzyme hydrolyses the substrate to give a yellow colour compound in case of alkaline phosphatase (Figure 11.3). The intensity of the colour is proportional to the amount of antibody or antigen present in the test sample, which can be quantified using ELISA reader
(Figure 11.4 ELISA reader)

Types

There are four kinds of ELISA assay tests. They are: Direct ELISA, Indirect ELISA, Sandwich ELISA and Competitive ELISA (Figure 11.5).

i. Direct ELISA

An antigen is immobilized in the well of an ELISA plate. The antigen is then detected by an antibody directly conjugated to an enzyme such as HRP. Direct ELISA detection is much faster than other ELISA techniques as fewer steps are required.

The assay is also less prone to error since fewer reagents and steps are needed, i.e. no potentially cross-reacting secondary antibody needed. Finally, the direct ELISA technique is typically used when the immune response to an antigen needs to be analyzed.

ii. Indirect ELISA

Indirect ELISA is used to detect antibody. A known antigen is coated on the micro titre plate. If the patient’s serum contains antibody specific to the antigen, the antibody will bind to the antigen.

After incubation the wells are washed and the enzyme labelled anti Human Gamma Globulin (HGG) is added to the well. AntiHGG can react with antigen antibody complex. The substrate for the enzyme is added finally which is hydrolysed by the enzyme which develops a colour.

iii. Sandwich ELISA

Sandwich ELISA is used to detect antigen. A known antibody is coated on the micro titre plate. A test antigen is added to each well and allowed to react with the bound antibody. If the patient’s serum contains antigen specific to the antibody, the antigen will bind to the antibody.

Specifically bound antigen and antibody will remain in the wells even after washing. The second antibody is added and allowed to react with bound antigen. Substrate is added to measure colour reaction.

iv. Competitive ELISA

It is used for the detection of antigens. Antibody is first incubated with a sample-containing antigen. The antigen and antibody complex is added to the antigen coated microtitre well. If more antigen present in the sample, the less free antibody will be available to bind to the antigen coated well.

Addition of an enzyme conjugated secondary antibody specific to the primary antibody can be used to determine the amount of primary antibody bound to the well. It is a quantitative test for the antigen detection.

Application

An ELISA test may be used to diagnose:

HIV, Lyme disease, pernicious anaemia, Rocky Mountain spotted fever, rotavirus, squamous cell carcinoma, syphilis, toxoplasmosis, varicella-zoster virus, which causes chickenpox and Zika virus.

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Overview of Arbo Virus and its Various Types

Arbo Viruses (arthropod – borne viruses) are viruses of vertebrates biologically transmitted by hematophagous insect vectors. They multiply in blood sucking insects and are transmitted by bite to vertebrate hosts. Arbo viruses are worldwide in distribution.

Arbo viruses have been named according to the disease caused (yellow fever), the place of isolation of the virus (kyasanur forest disease) or the local name for the disease (chikungunya). They are classified into Toga, Flavi, Bunya, Reo and Rhabdovirus families.

Arbo viruses have a very wide host range including many species of animals and birds. The most important arbo virus vectors are mosquitoes, followed by ticks. The virus enters the body through the bite of the insect vector.

After multiplication in the reticuloendothelial system, viremia of varying duration occurs, or the virus is transported to the target organs such as central nervous system in encephalitis, the liver in yellow fever and the capillary endothelium in hermorrhagic fever.

Clinical syndromes are fever with or without rash, encephalitis, hemorrhagic fever, systemic disease and yellow fever. Diagnosis may be established by virus isolation or serology.

Samples (Blood, CSF) are inoculated intra cerebrally into sucking mice. The animal develop fatal encephalitis. Viruses may be isolated in tissue cultures or in eggs. Isolates are identified by hemagglutination inhibition, complement fixation, gel precipitation, immunofluorescence and ELISA. Virus isolated from insect vectors and from reservoir animal.

Toga Viruses

Toga viruses are spherical enveloped viruses with a diameter of 50-70nm. Single stranded RNA genome. The virus replicates in the cyloplasm of the host cell and released by budding through host cell membranes. The name Toga Virus is derived from ‘toga’ meaning the Roman Mantle refers to the viral envelope.

The genus Alpha Virus was formerly classified as Group A arbo viruses which explains the name Alpha Virus. The genus Alpha Virus contains 32 species of which 13 infect humans. All are mosquito borne.

Chikungunya Virus

The virus was first isolated from human patients of Aedes aegypti mosquitoes (Figure 10.9) from Tanzania in 1952. The name Chikungunya is derived from the native word for the disease in which the patient lies ‘doubled up due to severe joint pains’. The virus first appeared in India in 1963 in Calcutta, Madras and
Other areas.

The disease presents as a sudden onset of fever, Crippling joint pains, lymphadenopathy and conjunctivitis. A maculopapular rash in common. The fever is typically biphasic with a period of remission after 1-6 days of fever. The vector is Aedes aegypti. No animal reservoir has been identified. Antibody to the virus has been demonstrated in horses, cattle and other domestic animals.

Flavi viruses

The family flaviviridae contains only one genus flavivirus. They are smaller than alpha viruses, being 40nm in diameter. There are over 60 arthropod borne flava viruses classified as mosquito-borne and tick borne viruses. Examples of mosquito borne group known as encephalitis viruses they are St.

Louis encephalitis Virus, Ilheus virus, west nile virus, murray valley encephalitis virus and Japanese encephalitis. Tick borne viruses are classified in to tick borne encephalitis viruses and tick borne hemorrhagic fevers.

Dengue

The name dengue is derived from the ‘Swahili ki denga pepo’, meaning a sudden seizure by a demon. Dengue fever is similar to the illness caused by chikungunya. Four types of dengue virus exist: DEN1, DEN2, DEN3 and DEN4.

Dengue presents after an incubation period of 3-14 days as fever of sudden onset with headache, retrobulbarpain, conjunctival injection, pain in the back and limbs (break bone fever), lymphadenopathy and maculopapular rash. The fever is typically biphasic (saddle back) and lasts for 5-7 days.

Dengue may be more serious forms with hemorrhagic manifestations (dengue Hemorrhagic fever) or with shock (dengue shock syndrome). Dengue virus is transmitted from person to person by Aedes aegypti mosquitoes. The Incubation period is 8-10days. All four types of dengue virus are identified. Demonstration of circulating IgM antibody provides early diagnosis. IgM ELISA test offers reliable diagnosis. Difference between Dengue and Chikungunya is given in Table 10.2.

Difference between Dengue and Chikungunya

Zika Virus

Zika virus is a mosquito-borne flavivirus that was identified in Uganda in 1947 in monkeys. Zika spreads by daytime-active Aedes mosquitoes, such as A. aegypti and A. albopictus. The infection is known as Zika fever or Zika virus disease. Zika is related to the dengue, yellow fever, Japanese encephalitis, and West Nile viruses.

Zika virus is enveloped and icosahedral and has a non segmented, single-stranded, positive-sense (+) RNA genome (Figure 10.10). A positive-sense RNA genome can be directly translated into viral proteins, the RNA genome encodes seven nonstructural proteins and three structural proteins. One of the structural proteins forms the envelope. The RNA genome forms a nucleocapsid along with copies of the 12-kDa capsid protein.

Viral genome replication depends on the making of double-stranded RNA from the single-stranded, positive-sense RNA (ssRNA(+)) genome followed by transcription and replication to provide viral mRNAs and new ssRNA(+) genomes.

Pathogenesis and Clinical features

Zika virus replicates in the mosquito’s mid gut epithelial cells and then its salivary gland cells. After 5-10 days, the virus can be found in the mosquito’s saliva. If the mosquito’s saliva is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the skin and the Langerhans cells. The pathogenesis of the virus is hypothesized to continue with a spread to lymph nodes and the bloodstream.

Zika virus is primarily transmitted by the bite of an infected mosquito from the Aedes genus, mainly Aedes aegypti. The mosquitoes usually bite during the day, peaking during early morning and late afternoon or evening. This is the same mosquito that transmits dengue, chikungunya and yellow fever.

Zika virus is also transmitted from mother to fetus during pregnancy, through sexual contact, transfusion of blood and blood products, and organ transplantation.

The incubation period of Zika virus disease is estimated to be 3-14 days. The majority of people infected with Zika virus do not develop symptoms. Symptoms are generally mild including fever, rash, conjunctivitis, muscle and joint pain, malaise, and headache, and usually last for 2-7 days.

Zika fever (also known as Zika virus disease) is an illness caused by the Zika virus. Zika virus infection during pregnancy is a cause of microcephaly and other congenital abnormalities in the developing fetus and newborn. Zika infection in pregnancy also results in pregnancy complications such as fetal loss, stillbirth, and preterm birth.

Laboratory diagnosis

Virus can be demonstrated from the blood or other body fluids, such as urine or semen. Zika virus grow well in a variety of mammalian and insect cell lines. Zika virus is identified by NAAT – Nucleic acid Amplification test, Zika Antigen is detected by ELISA and PCR. Zika Antibody IgM is detected by MAC ELISA, IgG by ELISA and by PRNT plaque reduction neutralization test.

Prevention and Treatment

Protection against mosquito bites during the day and early evening is a key measure to prevent Zika virus infection. It is important to eliminate these mosquito breeding sites, Health authorities may also advise use of larvicides and insecticides to reduce mosquito populations and disease spread.

There is no treatment available for Zika virus infection or its associated diseases. No vaccine is yet available for the prevention or treatment of Zika virus infection. Development of a Zika vaccine remains an active area of research.

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Human Immuno Deficiency Virus

Human Immuno Deficiency Virus (HIV), the etiological agent of AIDS, belongs to the lentivirus subgroup of the family Retroviridae.

Structure

HIV is a spherical enveloped Virus, about 90-120 nm in size. The nucleo capsid has an outer icosahedral shell and an inner cone shaped core, enclosing the ribonucleo proteins. The genome is diploid, composed of two identical single stranded, positive sense RNA copies.

When the virus infects a cell, the Viral RNA is transcribed by the reverse transcriptase enzyme, first into single stranded DNA and then to double stranded DNA (provirus) which is integrated into the host cell chromosome. The virus coded envelope proteins are the projecting knob like spikes which binds to the CD4 receptors on susceptible host cells (Figure 10.8)

Viral Genes and Antigens

The genome of HIV contains the three structural genes (gag, pol and env) as well as other nonstructural and regulatory genes specific for the virus. These products of these genes, both structural and non structural act as antigens.

Genes coding for structural proteins

1. The gag gene → Determines the core and shell of the Virus. Precursor protein, p⁵⁵ and it is cleaved into three proteins p¹⁵, p¹⁸ and p²⁴. Major core antigen p²⁴ can be detected in serum.

2. The env gene → Determines the synthesis of envelope glycoprotein gp¹⁶⁰. Cleaved in to gp¹²⁰ and gp⁴¹

3. The pol gene → Codes for the reverse transcriptase and other viral enzymes such as protease and endonucleases. It’s expressed as a precursor protein, which is cleaved into protein p31, p51 and p66.

Pathogenesis

Infection is transmitted when the Virus enters the blood or tissues of a person and comes into contact with a suitable host cell, principally the CD4 lymphocyte. The receptor for the virus is the CD4 antigen and therefore the virus may infect any cell bearing the CD4 antigen on the surface.

Specific binding of the virus to CD4 receptor is by the envelope glycoprotein gp¹²⁰. Cell fusion is brought about by transmembrane gp⁴¹. After fusion with the host cell membrane, the HIV genome is uncoated and internalized into the cell.

Viral reverse transcriptase mediate transcription of its RNA into double stranded DNA, which is integrated into the genome of the infected cell through the action of the viral enzyme integrase, causing a latent infection.

The primary pathogenic mechanism in HIV infection is the damage caused to the CD4+T lymphocyte. The T₄ cells decrease is numbers. Infected T₄ cells do not release normal amounts of interleukin, gamma interferon and other lymphokines, this is damping effect on cell mediated immune response.

Clinical Features

AIDS is only the last stage in the wide spectrum in HIV infection.

1. Acute HIV infection

3-6 weeks of infection, persons experience low grade fever, malaise, headache, lymphadenopathy, with rash. Antibodies are usually negative at the onset of the illness but become positive during its course called ‘Sero conversion illness’.

2. Asymptomatic or latent infection

All HIV infected persons, whether or not they experience Sero conversion illness, pass through a phase of symptomless infection which may last up to several years. The infection progresses in course of time through various stages, CD4 lymphocytopenia, minor opportunistic infections, AIDS-related complex (ARC),
ultimately terminating to AIDS.

3. Persistent generalized
lymphadenopathy (PGL)

It is defined as the presence of enlarged lymph nodes at least 1cm, in diameter in two or more non contiguous extrainguinal, sites that persists for at least three months.

4. AIDS related complex (ARC)

This group includes patients with considerable immunodeficiency, suffering from various symptoms or minor opportunistic infections. eg. Oral candidiasis, Salmonellosis or Tuberculosis.

5. AIDS

End-stage disease, poor immune defence mechanism leading to the opportunistic infection and malignancies.

a. Commonest symptoms

Drycough, dyspnea and fever. Pheumonia may be viral (cmv) or fungal (Cryptococcus, Histoplasma).

b. Gastrointestinal system

The mouth is often involved with thrush, stomatitis, gingivitis, hairy leukoplakia. Dysphagia due to esophageal Candidiasis. Intestinal pathogen in AIDS is cryptosporidium. Other pathogens are Salmonellae, Mycobacteria, CMV or adeno viruses. ‘Gay bowel syndrome’ is common among the male homosexuals.

c. Central nervous system

The typical CNS opportunistic infections are toxoplasmosis and cryptococcosis. Lymphomas of the CNS are Common.

d. Malignancies

Kaposi’s Sarcoma was the lesion seen in male homosexuals. The tumours commonly seen are lymphomas, both the Hodgkin and non Hodgkin types.

e. Cutaneous

Herpes lesions, Candidiasis, Dermatitis, impetigo are common cutaneous lesions.

6. Dementia

Direct cytopathogenic damage in the CNS. It cross the blood-brain barrier and cause encepthalopathy leading to dementia.

7. Pediatric AIDS

Viral transmission may occur to the fetus in pregnancy. Many of the infected children may not survive for a year. Children may also acquire the infection from blood transfusion or blood products.

Laboratory Diagnosis

Lab diagnosis of HIV infection include tests for immuno deficiency in HIV infection.

A. Immunological tests

• Total leukocyte and lymphocyte count to demonstrate leucopenia and a lymphocyte count usually below 2000/mm³.
• Platelet count will show thrombocytopenia.
• Raised IgG and IgA levels

B. Specific tests for HIV infection

1. Antigen detection

Single massive infection, as by blood transfusion, the virus antigens may be detectable in blood after about two weeks. The major core antigen p24 is the virus marker in blood.

2. Polymerase Chain reaction

It is the most senstitive and specific test.

3. Antibody detection

Demonstration of antibodies is the simplest and widely employed technique. It takes 2-8 weeks to months for antibodies to appear after infection, during this period, the individual may be highly infectious. This sero negative infective stage is known as the ‘window period’. Antibody can be detected by

1. ELISA
2. Western blot test.

Treatment

The treatment of AIDS include:

• The treatment and prophylaxis of infections and tumours
• General management
• Immunorestrorative measures
• Specific anti-HIV agents

Effective drugs are available, they are Zidovudine, Didanosine, Zalcitabine, Lamivudine and Protease inhibitors like Saquinavir, Ritonavir, Indinavir used as monotherapy or in various combination.

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Rabies Virus Types and its Laboratory Diagnosis

The Family Rhabdoviridae contains viruses that infects mammals, reptiles, birds, fishes, insects and plants. The disease in human being is called hydrophobia because the patient exhibits fear of water, being incapable of drinking though subject to intolerable thirst.

Pasteur established that the rabies virus was present in the brain of infected animals. By serial intracerebral passage in rabbits, he demonstrated fixed virus that could be rendered immune by a series of injections. Vaccine was prepared by drying pieces of spinal card from rabbits infected with the fixed virus.

Joseph Meister a nine year old boy, severely bitten by a rabid dog and in grave risk of developing rabies, was given a course of 13 inoculations of the infected cord vaccine by Pasteur. The boy survived. This dramatic event was a mile stone in the development of medicine.

Morphology

The rabies virus is bullet shaped, with one end rounded or conical and the other planar or concave. The lipoprotein envelope, carries knob like spikes, composed of glycoprotein G responsible for pathogenesis, virulence and immunity beneath the envelope is the matrix (M) protein layer which may be invaginated at the planar end. The membrane may project outwards forming a bleb. The genome is unsegmented linear RNA (Figure 10.6).

The rabies virus isolated from natural human or animal infection is termed ‘the street virus’. Rabies has been recognized from very ancient times as a disease transmitted to humans and animals by the bite of ‘mad dogs’. The name rabies comes from the Latin word rabidus, meaning ‘mad’, derived from the Sanskrit root rabhas, for frenzy.

Pathogenesis

Human infection is usually caused by the bite of rabid dogs or other animals. The virus present in the saliva of the animal is deposited in the wound (Figure 10.7). Rarely, infection can also occur following non-bite exposures such as licks or aerosols.

The virus appears to multiply in the muscles, connective tissue or nerves at the site of deposition for 48-72 hours. It penetrates the nerve endings and travels in the axoplasm towards the spinal cord and brain, at speed of about 3 mm per hour. The virus multiples and spreads centrifugally along the nerve trunks to
various parts of the body including the salivary glands. It multiplies in the salivary glands and is shed in the saliva.

The virus reaches every tissue in the body and dissemination may be interrupted at any stage by death. In humans the incubation period is usually from 1-3 months, short as 7 days or as long as three years. The incubation period is usually short in persons bitten on the face or head and long in those bitten on the legs. This may be related to the distance the virus has to travel to reach the brain. The incubation period is generally shorter in children than in adults.

The four stages of the disease are as follows, prodrome, acute encephalitic phase, coma and death. The onset is marked by symptoms such as fever, headache, malaise, fatigue and anorexia, anxiety, agitation, irritability, nervousness, insomnia or depression. The neurological phase begins with hyperactivity. Attempts to drink during such painful spasms of the pharynx and larynx produce choking or gagging that patients develop a dread of even the sight or sound of water (hydrophobia).

Animal Infection

In dogs, the incubation period is usually 3-6 weeks but it may range from 10 days to a year. The initial signs are an alert,troubled air and restlessness,snapping at imaginary objects, licking or gnawing at the site of the bite. After 2-3 days of this prodromal stage, the disease develops into either the furious or dumb types of rabies. In furious rabies, dog runs biting without provocation, the lower jaw droops and saliva drools from the mouth.

Paralysis convulsions and death follow. In dumb rabies, is the paralytic form, animals lies huddled, unable to feed. About 60% of rabid dogs shed the virus in saliva.
Rabid dogs usually die in 3-5days.

Laboratory Diagnosis

Human Rabies

The specimens tested are corneal smears and skin biopsy. Commonly used method for diagnosis is the demonstration of rabies virus antigens by immuno fluorescence. Direct immunofluorescence is done using antirabies serum tagged with fluorescein isothiocyanate.

Negri bodies in the brain, are demonstrated, Isolation of the virus by intracerebral inoculation in mice can be attempted from the brain, CSF, saliva and urine. The mice are examined for signs of illness, and their brains are examined after death.

Animal Rabies

The whole carcassof the animal suspended to have died of rabies may be sent to the laboratory. The brain may be removed sent for biological test and microscopy respectively. The portion of brain sent should include the hippocampus and cerebellum as negri bodies are most abundant. The following tests are done in the laboratory.

1. Demonstration of rabies virus antigen by immuno fluorescence
2. Demonstration of inclusion bodies – Negri bodies are seen as intracytoplasmic, round or oval purplish pink with characteristic basophilic inner granules. Negribodies vary in size from 3.27 Mm.

Antirabic Vaccines

Antirabic vaccines fall into two main categories neural and non-neural.

Neural Vaccines

Suspension’s of nervous tissues of animals infected with the fixed rabies virus. Following are the modified forms.

1. Semple Vaccine:

Vaccine developed by semple (1911). It is a 5% suspension of sheep brain infected with fixed virus and inactivated with phenol at 37°C leaving noresidual live virus.

2. Beta propiolactone (BPL) Vaccine:

Beta propiolactone is used as the inactivating agent instead of Phenol.

3. Infant Brain Vaccine:

The enceptalitogenic factor in brain tissue is a basic protein associated with myelin. Vaccines were developed using infant mouse, rat or rabbit brain. Infant brain vaccine is impractical in India.

Non-neural Vaccines

Non-neural vaccines includes

1. Egg Vaccines
2. Tissue Culture Vaccines
3. Subunit Vaccine

Passive Immunisation

Human rabies immune globulin (HRIG) is free from the danger of sensitization but should be ensured free from HIV and hepatitis viruses.

Vaccines for Animals

Antirabies immunization in animals is to be done as pre-exposure prophylaxis concentrated cell culture vaccines – inactivated virus gives good protection after a single Intramuscular injection. Injections are given at 12 weeks of age and repeated at 1-3 years intervals.

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Hepatitis Viruses | Hepatitis Viruses Types, Laboratory Diagnosis

The term viral hepatitis refers to a primary infection of the liver, hepatitis viruses consists of types A, B, C, D, E and G. Except for type B which is a DNA virus all the others are RNA viruses. Two types of viral hepatitis had been recognised. Type one affects mainly children and young adults and transmitted by the fecal-oral route called as infective or infectious hepatitits or type A hepatitis.

Second type transmitted mainly by receiving serum inoculation or blood transfusion named as homologous serum jaundice, serum hepatitis transfusion hepatitis or type B hepatitis.

Type A Hepatitis (HAV)

HAV is a 27nm non enveloped RNA virus belonging to the picorna virus family. It is designated as ‘entero virus 72’, HAV is recognised as new genus ‘Hepatovirus’. It can be grown in human and simian cell cultures and is the only human hepatitis virus which can be cultivated in vitro.

HAV transmission is by the fecal oral route. Infection is by ingestion. The virus multiplies in the intestinal epithelium and reaches the liver by hematogenous spread. Once jaundice develops, it is rarely detectable in feces. The incubation period is 2-6 weeks. The clinical disease consists of two stages the prodromal and
the icteric stage. The onset may be acute with fever, malaise, anorexia, nausea, vomiting and liver tenderness.

These usually subside with the onset of jaundice. Recovery is slow, over a period of 4-6 weeks. The disease is milder in children. Type A hepatitis caused by contaminated food, water or milk. Over crowding and poor sanitation favour its spread.

Laboratory Diagnosis

Diagnosis of type A hepatitis may be made by demonstration of the virus or its antibody. Virus can be visualized by Immunelectron Microscopy (IEM) in fecal extracts during the late incubation period.

IgM anti-HAV antibody appears during the late incubation period disappears after 3-4 months. IgG peaks in 3-4 months and persists much longer for life. ELISA kits for detection of IgM and IgG antibodies are available.

A safe and effective formalin inactivated, alum conjugaged vaccine containing HAV grown in human diploid cell culture is used. Course consists of two intra muscular injections of the vaccine. Protection begins 4 weeks after injection and lasts for 10 to 20 years. No specific antiviral drug is available.

Type B Hepatitis (HBV)

HBV is a 42nm DNA virus with an outer envelope and an inner core 27nm in diameter. Enclosing the viral genome and a DNA polymerase. It belongs to the family Hepadna Viridae HBV is ‘Hepadna Virus type 1’. Australia antigen was found to be associated with serum hepatitis. It was the surface component of HBV, so named as hepatitis B surface antigen (HBsAg).

3 types of particles are visualized, most abundant form is a spherical particle, 22nm in diameter. The second type of particle is filamentous or tabular with a diameter of 22nm both are antigenically identical. Third type of particle are fewer in number, is a double walled spherical structure 42 nm in diameter. This particle is the complete hepatitis B virus, known as Dane particle.

The envelope proteins expressed on the surface contains hepatitis B surface antigen (HBsAg). HBsAg consists of two major polypeptides, one of which is glycosylated. The nucleocapsid or core contains hepatitis B core antigen (HBcAg A) (Figure 10.5). Third antigen called the hepatitis B e antigen (HBeAg)
is a soluble non particulate nucleocapsid protein.

The nucleocapsid encloses the viral genome consisting of two linear strands of DNA held in a circular configuration. One of the strands is incomplete (+ strand) DNA appears partially double stranded and partially single stranded. Associated with the + strand is a viral DNA polymerase (has both DNA dependent
DNA polymerase and RNA dependent reverse transcriptase functions).

This polymerase can repair the gap in the plus strand and render the genome fully double stranded. Natural infection occurs only in humans. The virus is maintained in carriers whose blood contains circulating virus for long periods. Carriers are of two categories, the highly infectious super carriers and the simple carriers. Former have high titre HBsAg along with HBsAg, DNA polymerase and HBV in ciruculation.

Simple carriers have low infectivity and low titre HBsAg in blood. HBV is a blood borne virus and the infection is transmitted by parenteral, sexual and perinatal models. The virus may also be present in other body fluids and excretions such as saliva, breast milk, semen, vaginal secretions, urine bile and feces of these semen and saliva are known to transmit the infection very commonly.

Transfusion of carrier blood once, the most widely known mode of infection has largely been eliminated by donor screening that is strictly enforced. Infection by direct contact with open skin lesions such as pyoderma, eczema, cuts and scratches is very common among young children in developing countries.

Certain groups and occupations carry a high risk of infection. These include medical and paramedical staff of blood banks, dialysis units, barbers, sex workers. The incubation period is long about 1-6 months. The onset is insidious and fever is not prominent.

Extra hepatic complications like arthralgia, urticaria and glomerulonephritis may occur. About 90-95% of adults with acute hepatitis B infection recover within 1-2months of onset and eliminate the virus from the body. They may be Asymptomatic carriers or may progress to recurrent or chronic liver disease.

Laboratory Diagnosis

Serology

Diagnosis of hepatitis B depends on the serological demonstration of the viral markers. HBsAg is the first marker to appear in blood after infection, being detectable. It remains in circulation throughout the symptomatic course of the disease (2-6months). Anti HBs is the protective antibody.

HBcAg is not demonstrable in circulation because it’s enclosed within the HBsAg coat but its antibody, anti HBc appears in serum a week or two after the appearance of HBsAg.

As anti HBc remains life long, it serves as a useful indicator of prior infection with HBV. HBeAg appears in blood concurrently with HBsAg, indicating the high infectivity. Molecular methods such as DNA: DNA hybridization and PCR at present used for HBV DNA testing are highly sensitive and quantitative.

Immunization

Both passive and active methods of immunization are available. Active immunization is more effective. The currently preferred vaccine is genetically engineered by cloning the S gene for HBV in Baker’s yeast. A special vaccine containing all antigenic components of HBsAg (Pre-S1, Pre-S2 and s) has been developed. No specific antiviral treatment is available for acute HBV infection.

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Herpes Viruses | Herpes Viruses Structure, Classification, Clinical Features, Laboratory Diagnosis

The herpes virus family contains more than a hundred species of enveloped DNA viruses that affect humans and animals.

Structure

The herpes virus capsid is icosahedral, composed of 162 capsomers and enclosing the core containing the linear double stranded DNA genome. The nucleocapsid is surrounded by the lipid envelope derived from the host cell. The envelope carries surface spikes (Figure 10.4). Teguments are present in between the envelope and capsid. The enveloped virion measures about 200nm and the naked virion about 100 nm in diameter.

Classification

Herpes virus belongs to the family Herpesviridae.

i. Alpha herpes viruses

They have relatively short replicative cycle (12-18 hours) and a variable host range. They cause latent infection in sensory ganglia. Example: Herpes simplex virus and varicella zoster virus.

ii. Beta herpes viruses

They replicate slowly (more than 24 hours) and have a narrow host range, grow well in fibroblasts. They cause latent infection of salivary gland and other organs. Example: Cytomegalovirus.

iii. Gamma herpes viruses

They have a narrow host range and replicate in lymphoblastoid cells. They are specific for either B or T lymphocytes and causes latent infection in lymphoid tissue Example: Epstein – Barr Virus. Eight different types of herpes viruses are known whose primary hosts are humans. They have been designated as Human herpes virus type 1-8.

1. Herpes Simplex

The herpes simplex virus (HSV) occurs naturally only in humans, but it can produce experimental infection in laboratory animals. There are two types of the herpes simplex virus. HSV type 1 (Human herpes virus type 1) is isolated from lesions in and around the mouth and is transmitted by direct contact or droplet spread from carrier. HSV type 2 (Human herpes virus type 2 or HHV type 2) is responsible for the genital herpes infections transmitted venereally.

Pathogenesis

Herpes simplex is one of the most common viral infection in humans, the sources of infection are saliva, skin lesions or respiratory secretions. In type 2, transmission occurs by close contact and may be veneral in genital herpes. The virus enters through defects in the skin or mucous membranes and multiples
locally, with cell to cell spread.

The herpes lesions are thin walled, umbilicated vesicles, the roof of which breaks down, leaving tiny superficial ulcers. They heal without scarring.

Clinical features

The clinical manifestations depend on the site of infection, age and immune status of the host and the antigenic type of the virus. They are

• Cutaneous infections
• Mucosal infections
• Ophthalmic infections
• Nervous system infections
• Visceral infections
• Genital infections

Laboratory diagnosis

Microscopy

Smears are prepared from the lesions, from the vesicles and stained with 1% aqueous solution of toluidine blue ‘O’ for 15 seconds. Multinucleated giant cells with faceted nuclei with ground glass chromatin (Tzanck cells) are observed.

Virus isolation

Inoculation in mice and on chick embryo CAM is insensitive. Primary human embryonic kidney, human amnion cells are susceptible, but human diploid fibroblasts are preferred. Vesicle fluid, spinal fluid, saliva and swabs may be used. Cytopathic changes may appear as early as 24-48 hrs.

Serology

Antibodies develop within a few days of infection and rise in titre of antibodies may be demonstrated by ELISA, neutralization or complement fixation tests. Chemotherapy Indoxyuridine used topically in eye and skin infection, acyclovir and vidarabine are given for deep and systemic infections.

2. Varicella Zoster

In 1889, Von Bokay had suggested that varicella (Chicken pox) and herpes zoster are different manifestations of the same virus infection. The virus is therefore called Varicella zoster virus (VZV). The chicken pox follows primary infection in a non immune individual, while herpes zoster is a reaction of the latent virus when the immunity has fallen to infective levels.

VZV is similar to the herpes simplex virus in its morphology. It can be grown in cultures of human fibroblasts human amnion or HeLa cells. Chicken pox is one of the mildest and most common of child hood infections. The disease may, occur at any age.

3. Cytomegaloviruses

Cytomegaloviruses (CMV) formerly known as salivary gland viruses are a group of ubiquitous herpes viruses of humans and animals. They are characterized by enlargement of infected cells and intranuclear inclusions.

In 1926, cytomegalia presumed to be due to viral infection was reported in the salivary glands of guinea pigs and children and the viral agent was called the ‘salivary gland virus’.

4. Epstein – Barr Virus

A number of different viruses apparently ‘Passenger Viruses’ were isolated from cultured lymphoma cells. Epstein, Barr and Achong in 1964 observed a new type of herpes virus and named it has ‘EB Virus’ affecting B lymphocytes of only human and some sub human primate B cells have receptors (CD21 molecules)
for the virus.

The source of infection is usually the saliva of infected persons who shed the virus in oropharyngeal secretions. Intimate oral contact,as in kissing, appears to be the predominant mode of transmission. This accounts for infectious mononucleosis being called as ‘The kissing disease’.

5. Human Herpes Virus Types 6, 7, 8

A herpes virus, first isolated in 1986 from the peripheral blood of patients with lympho proliforative disease called as human B lymphotropic virus, renamed as HHV – 6. HHV – 7 was isolated in 1990 from peripheral CD4 cells of a healthy person appears to be widely distributed and transmitted through saliva.

In 1994, DNA sequences presumed to represent a new herpes virus from tissues of Kaposi’s sarcoma from AIDS patients was named as HHV8. Later Kaposi’s sarcoma was identified in persons not infected with HIV and referred to as Kaposi’s Sarcomaassociated Herpes Virus (KSHV).

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Cultivation of Viruses

Viruses areobligate intracellularparasites; they cannot be grown on any inanimate culture medium. Three methods are employed for the cultivation of viruses – inoculation into animals, embryonated eggs and tissue culture or cell culture.

i. Animal Inoculation

The earliest method for the cultivation of viruses causing human diseases was inoculation into human volunteers. Monkeys were used for the isolation of the polio virus by Landsteiner and Popper (1909). The embryonated hen’s egg was first used for cultivation of viruses by Good pasture (1931). The embryonated egg offers several sites for the cultivation of viruses. Non human primates provide the only method for virus cultivation. Mice are most widely employed animals in Virology.

ii. Embryonated Eggs

a. Chorioallantonic Membrane (CAM)

Inoculation on the chorioallantonic membrane produces visible lesions (pocks). Different viruses have different pock morphology. Example: variola or vaccinia

b. Allantonic Cavity

Inoculation on the allantonic cavity provides a rich yield of influenza and some paramyxo viruses.

c. Amniotic Sac

Inoculation into the amniotic sac is for the primary isolation of the influenza virus.

d. Yolk Sac

Inoculation into the yolk sac is for the cultivation of some viruses like Chlamydiae and Rickettsiae. Allantonic inoculation is employed for growing influenza virus for vaccine production (Figure 10.3).

iii. Tissue Culture

First tissue culture in Virology was maintained by Steinhardt and colleagues (1913) for the vaccinia virus in fragments of rabbit cornea. Bacterial contamination was the major limitation. Different types of culture used are:

a. Organ culture

Small bits of organs can be maintained, used for the isolation of some viruses.
Example: Corona virus (respiratory pathogen) cultured on tracheal ring organ culture.

b. Explant culture

Fragments of minced tissue are grown as ‘explants’. This is also known as tissue culture.
Example: Adeno virus cultured on Adenoid tissue explants.

iv. Cell Culture

Tissues are dissociated into the component cells by the action of enzymes (trypsin) or by mechanical process and are suspended in a growth medium (amino acids, vitamins, salts, glucose) supplemented with fetal calf serum of antibiotics and indicator (Phenol red).

This media is dispensed in bottles, tubes or petridishes. The cells adhere to the glass surface and on incubation divides to form a confluent monolayer sheet of cells covering the surface within about a week. The cell culture is classified into three types.

a. Primary cell cultures

In this culture, normal cells are taken from the body and cultured. They are capable of only limited growth in culture. Example: Monkey kidney, Human embryonic kidney, Chick embryo cell culture.

b. Diploid cell strains

These are cells of a single type that retain the original diploid chromosome number and serotype during serial sub cultivation for limited number of times. Example: Human fibroblast.

c. Continuous cell lines

These are single type, derived from cancer cells that are capable of continuous serial cultivation.
Example: Cells derived from cancers, such as Hela, Hep-2 and KB cell lines.

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Evolutionary Origin of viruses

The origin of viruses is not known, but two theories of vital origin can be summarized as follows;

1. Viruses may be derived from (DNA or RNA) nucleic acid components of host cells to replicate and evolve independently.
2. Viruses may be degenerate forms of intracellular parasites.

Morphology

Size

Viruses are smaller than bacteria, known as filterable viruses vary widely in size. The largest among them is the Pox virus measuring about 300nm. The smallest virus is Parvo virus measuring about 20 nm.

Structure and Shape

The virion consists of nucleic acid surrounded by a protein coat, the capsid. The capsid with the enclosed nucleic acids is known as the nucleo capsid. The capsid is composed of a large number of capsomers. The functions of the capsid are to protect the nucleic acid from the deleterious agents and also to introduce
viral genome into host cells by adsorbing readily to cell surfaces (Figure 10.1).

Two kinds of symmetry encountered in the virus are icosahedral and helical. Virions may be enveloped or non enveloped (naked). The envelope or outer covering of viruses is derived from the host cell membrane when the progeny virus is released by budding. The envelope is lipoprotein in nature. The lipid is of host cell origin while the protein is virus coded. Protein subunits may be seen as projecting spikes on the surface of the envelope and are known as Peplomers.

Overall shape of the virus particle varies; mostly animal viruses are roughly spherical. Some are irregular and pleomorphic. The rabies virus is bullet shaped, Ebola virus is filamentous and pox viruses are brick shaped.

Chemical Properties

Viral protein determines the antigenic specificity of the virus. Some viruses contain small amounts of carbohydrates. Most Viruses do not possess any enzymes but retro virus has a unique enzyme, such as RNA dependent DNA polymerase or transcriptase which can synthesise DNA from RNA.

Resistance

Viruses are inactivated by sunlight, UV rays and ionizing radiations. The most active antiviral disinfectants are oxidizing agents such as hydrogen peroxide, potassium permanganate and hypochlorites. Organic iodine compounds are actively virucidal.

Chlorination of drinking water kills most viruses but its efficacy is influenced by the presence of organic matter. Some viruses such as hepatitis virus, polio viruses are relatively resistant to chlorination.

Viral Multiplication

The genetic information necessary for viral replication is contained in the viral nucleic acid, and also depends on the synthetic machinery of the host cell for replication.

The Viral replication cycle can be divided into six steps and they are as follows,

1. Adsorption or attachment,
2. Penetration,
3. Uncoating
4. Bio synthesis
5. Maturation and
6. Release.

1. Adsorption

Virions may come into contact with cells by random collision but adsorption takes place only if there is an affinity between the virus and the host. The cell surface should contain specific receptor site for the virus to attach on to it.

2. Penetration

Bacteria possess rigid cell walls, only the viral nucleic acid is introduced intracellularly by a complex mechanism. Animal cells do not have rigid cell walls and the whole virus can enter and virus particles may be engulfed by a mechanism resembling phagocytosis, a process known as ‘Viropexis’. In case of the enveloped viruses, the viral envelope may fuse with the plasma membrane of the host cell and release the nucleocapsid into the cytoplasm.

3. Uncoating

Release of the viral nucleic acid from the capsid into the host cell. With most viruses, uncoating is affected by the action of lysosomal enzymes of the host cell.

4. Biosynthesis

Virus can synthesise viral nucleic acid, capsid protein and also the enzymes necessary in the various stages of viral synthesis, assembly and release. In addition certain regulator proteins are also synthesized. Most DNA viruses synthesise their nucleic acid in the host cell nucleus. Most RNA viruses synthesise all their components in the cytoplasm.

5. Maturation

Assembly of daughter virions follows the synthesis of viral nucleic acid and proteins. Virions assembly may take place in the host cell nucleus or cytoplasm. Herpes and adeno viruses are assembled in the nucleus, while picorna and pox viruses are assembled in the cytoplasm.

6. Release

In case of bacterial viruses, the release of progeny virions takes place by the lysis of the infected bacterium. However, in the case of animal viruses, release usually occurs without cell lysis. Eclipse phase is from the stage of penetration till the appearance of mature daughter virions. The virus cannot be demonstrated inside
the host cell. The virus seems to disappear (Figure 10.2).

Viroids

Viriods are small, single stranded covalently closed circular RNA molecules existing as highly base paired rod like structure. The viroid depends on the host for replication. These are responsible for some of the transmissible plant diseases.

Prion

Prions are small proteinaceous infectious agents without genetic material. These are responsible for a number of degenerative brain diseases (Example: Creutzfeldt) and hereditary dementia.

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Medical Opportunistic Mycosis

The opportunistic systemic mycoses are infections found in patients with underlying pre disposing conditions. It is produced by non pathogenic or contaminant fungi in a host, where the immunological defense mechanisms are weakened by endogenous causes like cancer, leukemia or exogenous causes
like immunosuppressive therapy and AIDS. The examples of opportunistic mycoses are Candidiasis, Cryptococcosis, Aspergillosis and zygomycosis.

Candidiasis

Candidiasis is the commonest fungal disease found in humans affecting mucosa, skin, nails and internal organs of the body. It is caused by yeast like fungi called Candida albicans. The infection may be acute or chronic, superficial or deep and found mainly as secondary infection in individuals with immune compromised condition.

Pathogenesis and Pathology

Some of the virulence factors contributing to pathogenicity are toxins, enzymes and adhesion. The organism adheres to the epithelial and endothelial cells by proteinase production.

Then the yeast cells of Candida encounter a particular host tissue and colonization takes place at the local site or they invade deeper into the host tissue and induce various clinical symptoms.

Clinical Features

The Candida species are found as commensal on mucosal surfaces of the body. They cause disease as and when conditions are favourable. This yeast like fungi colonizes mucocutaneous surfaces, which can be portals of entry into deeper tissues when the host defenses are compromised. They may cause a simple lesion to event the life threatening systemic infection.

The clinical manifestations of Candidiasis are divided into two broad categories. They are:

1. Infectious Diseases

a. Mucocutaneous Involvement

i. Oral Candidiasis:

Most common form of Candida colonizes on the oral cavity. Oral thrush is infection of the buccal mucosa, gums, tongue. Reddening of the mucous membrane gives dry, smooth metallic taste and burning at the local site (Figure 9.9).

ii. Alimentary Candidiasis:

Candida colonizes on the oesophagus causing oesophagitis. It is mostly asymptomatic or it may cause burning pain in the epigastrium or throat.

b. Cutaneous Dermatitis

i. Diaper Dermatitis:

Candida that colonize on the cutaneous layer causes cutaneous Candidiasis, leading to maculopapules vesicles with erythematous rash. This is common among infants and known as Diaper rash.

ii. Intertrigo:

This is an inflammatory lesion of the skin folds due to candidal infection.

c. Systemic Involvement:

The Candida colonizes in various organs and causes various manifestations through the blood stream. Clinical features are found to be Urinary tract Candidiasis, Candiduria, Endocarditis, Pulmonary Candidiasis, Arthritis, Osteomyelitis, Meningitis, Candidemia and Septicemia.

2. Allergic Diseases

Allergic manifestation is caused due to the metabolites of Candida. The cutaneous allergies are urticaria and eczema, and bronchial asthma.

Laboratory Diagnosis

i. Samples

Specimens collected are mucous membrane from the mouth, vagina, skin and sputum based on the site of involvement.

a. Direct Examination

Gram staining LPCB, and KOH wet mount are used to visualize the yeast cells. Presence of yeast cells approximately 4.8 µm with budding and pseudo hyphae are observed. Other stains like periodic acid – Schiff stain and Gomori’s methylamine silver stain are also used to observe the fungal elements in tissue.

b. Fungal culture

The clinical specimens can be cultured on Sabouraud dextrose agar (SDA) with antibiotics and incubated at 25°C and 37°C (Figure 9.10). The colonies appear in 3-4 days as cream coloured, smooth and pasty. Some of the species of Candida are Candida albicans, Candida tropicalis, Candida krusei and Candida glabrata.

ii. Special Test

Germ tube test The culture of Candida species is treated with sheep or normal human serum and inoculated at 37°C for 2 to 4 hours. A drop of suspension is examined on the slide. The germ tubes are seen as long tube-like projections extending from the yeast cells. The demonstration of the germ tube is known as Reynolds – Braude phenomenon.

Biochemical tests

Sugar fermentation and assimilation tests are used for the identification of Candidal species. C.albicans ferments Glucose and Maltose and assimilates Glucose, Maltose, Sucrose, Lactose and Galactose.

Chlamydospores formation

Candida isolates are grown on corn meal, agar (CHN) or rice starch agar (RSA) and incubated at 25°C for 2-3 days. The formation of large, thick walled terminal chlamydospores is demonstrated in C.albicans and C. dubliniensis.

iii. Treatment

• 1% gentian violet is locally applied to the affected areas.
• The azole creams like Clotrimazole, Miconazole, Ketoconazole and Econazole are also used.

Cryptococcosis

Cryptococcosis is an acute, sub acute or chronic fungal disease caused by encapsulated yeast called Cryptococcus neoformans. It is pathogenic to man and animals. It causes opportunistic infection, involving the lungs and disseminates to extra pulmonary sites through circulation to different body organs particularly to central nervous system causing Meningoencephalitis.

Pathogenesis and Pathology

Cryptococcal infection occurs through inhalation of small forms or basidiospores. The fungus may remain dormant in the lungs until the immune system weakens and then can disseminate to the central nervous system and other body sites.

Clinical Features

The clinical features of Cryptococcosis depend upon the anatomical sites.

i. Pulmonary Cryptococcosis

The respiratory route is usually the portal of entry for propagules in Pulmonary Cryptococcosis that subsequently disseminate to extra pulmonary sites. The symptoms are dry cough, dull chest pain and milder or no fever with small gelatinous granules all over the lungs.

ii. CNS Cryptococcosis

This is an infection of brain and meninges leading to Meningoencephalitis. Nitrogenous source such as asparagines and creatinine present in cerebrospinal fluid enrich the yeast. The symptoms are nausea, dizziness, impaired memory, blurred vision and photophobia. The enlarged granulomatous cerebral lesions
are called cryptococcoma.

iii. Visceral Cryptococcosis

This infection usually spreads from a primary focus to invade the optic nerve and meninges. Visual loss in patients is due to intra cranial pressure. There are two distinct patterns of visual loss namely; rapid visual loss (within 12 hrs) and slow visual loss (within weeks to months).

Laboratory Diagnosis

i. Samples

Specimens collected are mainly serum, CSF and other body fluids.

a. Direct Examination

10% Nigrosin or India ink staining, Gram staining and LPCB are used to visualize the yeast cell.

Biopsy material is stained with periodic acid – Schiff and Gomoris’s methylamine silver stain to observe the fungal cells in the tissue. Round budding yeast cells with a distinct halo gelatinous capsule can be seen (Figure 9.11a). Gram positive budding yeast cells are demonstrated by Gram staining.

b. Fungal Culture

The clinical specimens can be cultured on Sabouraud dextrose agar, Bird Seed agar and incubated at 37°C. The colonies are mucoid, cream to buff – colored in SDA (Figure 9.11b), whereas brown colored due to conversion of the substrate into melanin by Phenoloxidase in BSA (Figure 9.11c).

ii. Treatment

• Amphotericin B, Flucytosine is given together as induction and maintenance therapy.
• Fluconazole is also recommended.

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Systemic Mycosis

Systemic mycoses are caused by dimorphic fungi; these infections are acquired by inhalation of spores. These primarily involve the respiratory system and are self-limiting and asymptomatic. If symptomatic, it spreads to other parts of body through circulation. These infections are caused by true fungal pathogens.

Systemic and opportunistic infections together cause Deep mycoses. The organisms have a mycelial form when grown on fungal culture and have yeast form in the tissue. The examples of systemic mycoses are Histoplasmosis, Blastomycosis.

Histoplasmosis

Histoplasmosis is caused by dimorphic fungus Histoplasma capsulatum. The fungi live inside the cells of the reticuloendothelial system, where they grow within macrophages and giant cells. This infection is also known as Darling’s disease.

Pathogenesis and Pathology

The infection with H. capsulatum develops when conidia or mycelial fragments are inhaled and converted into yeasts in alveolar macrophages in the lungs. The oval yeast cells parasitize macrophages, which are activated by T lymphocytes resulting in localized granulomatous inflammation.

Clinical Features

The disease is mostly asymptomatic. The development of symptom or symptomatic disease appears to depend on the intensity of exposure to conidia and cellular immune response of the host. The disease may be classified as follows.

1. Acute pulmonary Histoplasmosis – Fever, headache, chills, sweating, chest pain, cough and dyspnoea.
2. Chronic pulmonary Histoplasmosis – Ulcerative lesions of the lips, mouth, nose, larynx and intestines.
3. Cutaneous, mucocutaneous Histoplasmosis – Mucous lesions on skin, abdomen wall and thorax.
4. Disseminated Histoplasmosis – Fever, anoxia, anemia, leucopenia constant hepatosplenomegaly and multiple lymphadenopathies.

Laboratory Diagnosis

i. Samples

Specimens collected are sputum, bone marrow and lymph nodes, cutaneous and mucosal lesions and peripheral blood film.

a. Direct Examination

Thick and thin smears should be prepared from peripheral blood, bone marrow and stained with Calcofluor white, Giemsa or Wright stains. The fungus is small, oval yeast like cells, 2-4 µm in diameter, within the mononuclear or polymorpho nuclear cells and occasionally in giant cells.

b. Fungal culture

The clinical samples is inoculated on Sabouraud dextrose agar (SDA) and Brain-heart infusion (BHI) agar with antibiotics and actidione at 25°C and 37°C. On Sabourad dextrose agar the colonies appear albino or brown.

The albino type consists of white, fine aerial hyphae and brown type consists of flat colonies with light tan or dark brown in color in seven days. At 37°C the colonies grow as granular to rough, mucoid and cream-colored turning tan to brown in 14 days.

ii. Treatment

Amphotericin B is given for the treatment of disseminated and other severe forms of Histoplasmosis.

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Subcutaneous Mycoses

Dermatophytoses are the most common cutaneous fungal infection seen in man and animals affecting skin, hair and nails. The fungi can invade the keratinized tissues of skin and its appendages and they are collectively known as Dermatophytes or Tinea or ring worm infection.

The dermatophytes are hyaline septate molds. They are divided into three main anamorphic genera depending on their morphological characteristics.

• Trichophyton [Cause infection in skin, hair and nails]
• Microsporum [Cause infection in skin and hair]
• Epidermophyton [cause infection in skin and nail]

The fungal species affecting humans are known as anthropophilic. Those inhabitating domestic and wild animals as well as birds are called zoophilic. Fungi species from soil are known as geophilic dermatophytes.

Pathogenesis and Pathology

The dermatophytes grow within dead keratinized tissue and produce keratinolytic proteases, which provide means of entry into living cells. Fungal metabolic products cause erythema, vesicles and pustule on the site of infection. Some dermatophytes species like soil saprobes digest the keratinaceous debris in soil and are capable of parasitizing keratinous tissues of animals.

Clinical Features

The clinical manifestations of Dermatophytoses are also called Tinea or Ringworm depending on the anatomical site involved. Following are the common clinical conditions produced by dermatophytes:

1. Tinea Capitis:

This is an infection of the shaft of scalp hairs. It can be inflammatory (eg. Kerion, Favus) or non – inflammatory (Black dot, Seborrheic dermatitis). The infected hairs appear dull and grey (Figure 9.5 a). Breakage of hair at follicular orifice which creates patches of alopecia with black dots of broken hair. It is caused by Trichophyton species.

2. Tinea Corporis:

This is an infection on the glabrous (non – hairy) skin of body. Erythematous scaly lesions with sharply marginated raised border appear on the infected areas (Figure 9.5 b). It is caused by Trichophyton rubrum.

3. Tinea Imbricata:

It forms concentric rings of scaling on the glabrous skin, leading to lichenification. It is caused by Trichophyton concentricum.

4. Tinea Gladiatorum:

This infection is common among wrestlers and athletes. Lesions are seen on arms, trunk or head and neck. It is caused by Trichophyton tonsurans.

5. Tinea Incognito:

It is steroid modified Tinea caused as a result of misuse of corticosteroids in combination with topical antimycotic drugs.

6. Tinea Faciei:

This is an infection of skin of face except beard. Erythematous annular plaques are formed. It is one of the forms of Tinea incognito.

7. Tinea Barbae:

This is the infection of the beard and moustache areas of the face. This is also called barber’s itch. It is caused by Trichophyton mentagrophytes, Trichophyton rubrum and Microsporum canis. Erythematous patches on the face with scaling appear and these develop folliculitis.

8. Tinea Pedis:

This is an infection of the foot, toes and interdigital web spaces. This is seen among the individuals wearing shoes for long hours and known as Athlete’s foot (Figure 9.5 c). Erythema and scaling associated with itching and burning sensation appear with thin fluid discharging from small vesicles. It is caused by Trichophyton mentagrophytes, Trichophyton rubrum and Epidermophyton floccosum.

9. Tinea Cruris:

This is an infection of the groin in men who use long term tight fitting garments. Erythematous sharp margin lesions known as Jock itch. It is caused by Trichophyton rubrum and Epidermophyton floccosum.

10. Tinea Manuum:

This is an infection of the skin of palmar aspect of hands. It causes hyperkeratosis of the palms and fingers. It is caused by Trichophyton mentagrophytes, Trichophyton rubrum and Epidermophyton floccosum.

11. Tinea Unguium:

This is an infection of the nail plates. The infection spreads on the entire nail plate infecting the nail bed. It results in opaque, chalky or yellowish thick ended nail. It is caused by Trichophyton mentagrophytes, Trichophyton rubrum and Epidermophyton floccosum. Figure 9.6 shows the microscopic view of major determatophytes.

Laboratory Diagnosis

i. Samples

Skin scrapings, hair and nail samples were collected.

a. Direct Examination

Samples are subjected to KOH (10%) wet mount, the affected site were disinfected with alcohol before collecting the clinical specimen.

b. Fungal culture

The samples are inoculated on Sabouraud dextrose agar (SDA) with antibiotics and cycloheximide and are incubated at 25°C-35°C. The colony morphology can be identified.

The three genera of dermatophytes are Trichophyton, Microsporum and Epidermophyton (Table 9.1). They are identified based on morphology of the macro conidia, micro conidia, their shape, position on the spore bearing hyphae such as spiral hypha, racquet hypha, nodular pectinate body.

Table 9.1: Microscopic and macroscopic characteristics of Dermatophytes.

ii. Special Techniques

1. Wood’s Lamp Examination

Clinical samples are exposed to Wood’s lamp. Wood’s glass consists of Barium silicate containing 9% Nickel oxide. It transmits long wave ultra violet light with a peak of 365 nm that shows a characteristic fluorescence produced by the samples. The patterns of fluorescence are bright green, golden yellow and coral red. Microsporum species and Trichophyton species are differentiated using this technique.

2. Hair brush sampling Technique

It involves brushing the scalp with a sterile plastic hair brush, which is then inoculated into an appropriate culture medium by plates, is incubated at 25°C-35°C. The colony morphology can be identified.

3. Hair perforation Test

It is used to differentiate T. mentagrophytes and T. rubrum. Wedge-shaped perforations in the hair shaft are observed in hair infected with T. mentagrophytes.

4. Urease Test

It is used to differentiate between T. mentagrophytes and T. rubrum. T. mentagrophytes hydrolyzes urea and becomes deep red, showing positive result.

iii. Treatment

Whitfield’s ointment is used for all Tinea infections. Oral griseofulvin is the drug of choice for nails and scalp infections. Itraconazole and terbinafine may be given as pulse therapy.