Treatment of activeand latent T.B.. ⢠BCGvaccine containing live, attenuated Mycobacterium bovis organisms may prevent or limit extent of disease but does not ...
SIMPLIFIED MEDICAL MICROBIOLOGY AND IMMUNOLOGY FOR PHYSICIANS
DR Ayman A. Allam Professor of Microbiology and Immunology Faculty of Medicine Zagazig University
To the soul of my father, my mother and my family
Second Edition 2015/2016
الطبعة الثانية 2016/2015 رقم االيداع بدار الكتب 2015/1534 حقوف الطبع محفوظة للمؤلف
Preface This book represents a simplified and rapid review of medical microbiology and immunology. It is very suitable for medical undergraduate, post graduate students and physicians. Paramedical personnel can easily understand it. It is updated, concise and covers all the important topics of microbiology and immunology. I hope you will find this book interesting, relevant and useful for undergraduate studying microbiology and preparation for medical examination. The author Dr Ayman A. Allam
Contents General Bacteriology Bacterial structure--------------------------------------------------------------------------------------1 Bacterial physiology------------------------------------------------------------------------------------4 Bacterial genetics---------------------------------------------------------------------------------------5 The bacteriophage--------------------------------------------------------------------------------------6 Recombinant DNA technology-----------------------------------------------------------------------7 Sterilization ----------------------------------------------------------------------------------------------9 Disinfection---------------------------------------------------------------------------------------------10 Antibiotics-----------------------------------------------------------------------------------------------11 Antibiotic Resistance---------------------------------------------------------------------------------14 Host parasite relationship---------------------------------------------------------------------------15 Bacterial Virulence factors--------------------------------------------------------------------------16
Immunology Cells of immune response---------------------------------------------------------------------------18 Antigens--------------------------------------------------------------------------------------------------19 Innate immunity---------------------------------------------------------------------------------------21 Phagocytosis--------------------------------------------------------------------------------------------21 Acquired (Adaptive) immunity---------------------------------------------------------------------22 Humeral immunity------------------------------------------------------------------------------------23 Antibodies (Immunoglobulins)---------------------------------------------------------------------23 Monoclonal Antibodies ------------------------------------------------------------------------------26 Cell mediated immunity-----------------------------------------------------------------------------26 The complement -------------------------------------------------------------------------------------28 Cytokines------------------------------------------------------------------------------------------------30 Antigen antibody reactions(serological reactions)--------------------------------------------32 Hypersensitivity----------------------------------------------------------------------------------------36 Tolerance------------------------------------------------------------------------------------------------39 Auto-immunity-----------------------------------------------------------------------------------------40 Tumour immunology---------------------------------------------------------------------------------41 Transplantation----------------------------------------------------------------------------------------44 Immune-deficiency------------------------------------------------------------------------------------47 Assessment of immune function ------------------------------------------------------------------49 Immunization-------------------------------------------------------------------------------------------50
Systematic Bacteriology Gram positive cocci-----------------------------------------------------------------------------------55 Staphylococci---------------------------------------------------------------------------------55 Streptococci-----------------------------------------------------------------------------------56 Pneumococci----------------------------------------------------------------------------------58 Gram negative cocci (Neisseria)--------------------------------------------------------------------60 Gram positive rods (Bacilli)--------------------------------------------------------------------------62 Bacillus -----------------------------------------------------------------------------------------62 Clostridia---------------------------------------------------------------------------------------63 Corynebacterium diphtheriae-------------------------------------------------------------65 Listeria------------------------------------------------------------------------------------------66 I
Gram negative rods related to enteric system-------------------------------------------------68 E coli--------------------------------------------------------------------------------------------68 Salmonella Spp-------------------------------------------------------------------------------69 Vibrio cholerae-------------------------------------------------------------------------------72 H pylori-----------------------------------------------------------------------------------------74 Pseudomonas aerruginosa----------------------------------------------------------------74 Bacteroides fragilis--------------------------------------------------------------------------75 Gram negative rods related to respiratory system--------------------------------------------75 Haemophilus Influenzae--------------------------------------------------------------------75 Bodetella pertusis----------------------------------------------------------------------------76 Legionella pneumophila--------------------------------------------------------------------76 Gram negative rods related to animal sources-------------------------------------------------77 Brucella Spp-----------------------------------------------------------------------------------77 Yersinia pestis--------------------------------------------------------------------------------79 Mycobacteria------------------------------------------------------------------------------------------79 Actinomycetes and Nocardia ----------------------------------------------------------------------84 Mycoplasma--------------------------------------------------------------------------------------------84 Spirochetes----------------------------------------------------------------------------------------------85 Treponema pallidum------------------------------------------------------------------------85 Borrellia spp-----------------------------------------------------------------------------------87 Leptospira interrogans----------------------------------------------------------------------88 Chlamydiae----------------------------------------------------------------------------------------------88 Rickettsiae-----------------------------------------------------------------------------------------------89 Minor Bacterial pathogens -------------------------------------------------------------------------91
Mycology General mycology------------------------------------------------------------------------------------- 93 Laboratory diagnosis of fungal infection---------------------------------------------------------94 Antifungal therapy------------------------------------------------------------------------------------95 Opportunistic mycosis--------------------------------------------------------------------------------96 Cutaneous mycosis------------------------------------------------------------------------------------98 Subcutaneous mycosis-------------------------------------------------------------------------------99 Dimorphic fungi--------------------------------------------------------------------------------------100
General Virology Viral structure----------------------------------------------------------------------------------------101 Viral replication---------------------------------------------------------------------------------------103 Viral pathogenesis-----------------------------------------------------------------------------------104 Diagnosis of viral infections-----------------------------------------------------------------------106 Antiviral drugs----------------------------------------------------------------------------------------107
Systematic Virology Hepatitis viruses--------------------------------------------------------------------------------------109 DNA enveloped viruses-----------------------------------------------------------------------------115 Human Herpes viruses--------------------------------------------------------------------115 Pox viruses-----------------------------------------------------------------------------------117 DNA non- enveloped viruses----------------------------------------------------------------------118 Adeno viruses------------------------------------------------------------------------------118 Human papilloma viruses---------------------------------------------------------------118 parvo virus----------------------------------------------------------------------------------119 II
RNA enveloped viruses----------------------------------------------------------------------------119 RNA non-enveloped viruses----------------------------------------------------------------------125 Arbo-viruses------------------------------------------------------------------------------------------129 Oncogenic viruses-----------------------------------------------------------------------------------130 Human immune deficiency virus----------------------------------------------------------------132 Slow viruses and Prions-----------------------------------------------------------------------------134 Viral hemorrhagic fevers--------------------------------------------------------------------------135 Ebola virus---------------------------------------------------------------------------------------------136
Applied microbiology Normal flora-------------------------------------------------------------------------------------------138 Anaerobic infections--------------------------------------------------------------------------------139 Infectious causes of different clinical conditions -------------------------------------139-146 Nosocomial infection-------------------------------------------------------------------------------146 Infection control-------------------------------------------------------------------------------------147
III
General Bacteriology Eukaryotes & Prokaryotes Cells have evolved into two different types, eukaryotic and prokaryotic. Human, Fungi, protozoa and helminthes are eukaryotic, whereas bacteria are prokaryotic.Viruses are not cells and do not have a nucleus. Characteristics of Prokaryotic and Eukaryotic Cells Characteristic
Prokaryotic Eukaryotic Bacterial Cells Human Cells
DNA within a nuclear membrane
No
Yes
Mitotic division
No
Yes
DNA associated with histones
No
Yes
Chromosome number
One
More than one
Membrane-bound No organelles, such as mitochondria and lysosomes
Yes
Size of ribosome
70S
80S
Cell wall containing peptidoglycan Cell membrane contains sterols
Yes No except Mycoplasma
No Yes
Shape & Size Bacteria range in size from about 0.2 to 5 µm. Bacteria are classified by shape into three basic groups: cocci, bacilli, and spirochetes. The cocci are round, the bacilli are rods, and the spirochetes are spiral-shaped. Some bacteria are variable in shape and are said to be pleomorphic (many-shaped). The shape of a bacterium is determined by its rigid cell wall. Bacteria are arranged singly, in pairs (diplococci), some in chains (streptococci), and others in grapelike clusters (staphylococci). These arrangements are determined by the orientation and degree of attachment of the bacteria at the time of cell division. Bacterial Structure 1-Bacterial Cell Wall: -It is the outer most basic structure of the bacterial cell. -It is mainly formed of peptidoglycan polymers which maintain the rigidity and shape of the cells. 1. Cell Wall of Gram Positive Bacteria Consists of: thick layer of peptidoglycan 50:60% of the cell wall. It is the site of action of penicillins, cephalosporins and lysozyme enzyme. Techoic acid: surface fibers highly immunogenic (somatic O antigen). -1-
2. Cell Wall of Gram Negative Bacteria Consists of: Inner layer ofpeptidoglycan : thin layer (5-10% of the cell wall) outer membrane (OM): composed of lipopolysaccharide (LPS) containing : lipid A layer (endotoxin) & polysaccharide (somatic O antigen).The endotoxin causes the fever and hypotension and induce septic shock. periplasmic space laying between cytoplasmic membrane and outer membrane and filled with gel like substance of protein and enzymes. FUNCTIONS OF BACTERIAL CELL WALL: 1-It is a rigid structure that maintains the shape of bacteria. 2-It is osmotically insensitive; it protects the cytoplasmic membrane from bursting in hypotonic solutions. 3-It plays a role in cell division. 4-The cell wall is responsible for the staining affinity of the organism. 5-It contains somatic O antigen. 6-lipid A is the toxic component of endotoxin of gram negative bacteria.
CELLWALL-DEFICIENT BACTERIA Mycoplasmas: Bacterial species which are naturally devoid of cell wall.They are pleomorphic in shape, not destroyed by penicillin which acts on the bacterial wall, and cannot be stained with gram stain. Protoplasts, Spheroplasts and L-Forms: are bacteria which lost cell wall under the effect of certain environmental conditions e.g. treatment with penicillins and lysozyme . - L-Forms can revert to the parental form upon removal of the cell wall inhibitor. - They can cause chronic infection e.g. urinary tract infection. 2-Cytoplasmic Membrane: is composed of a phospholipid bilayer. It generally do not contain sterol (Except the genus Mycoplasma). The membrane has four important functions: (1) active transport of molecules into the cell, (2) energy generation by oxidative phosphorylation, (3) synthesis of precursors of the cell wall, and (4) secretion of enzymes and toxins. -Mesosomes: They are convoluted invaginations of the cytoplasmic membrane. They play a role in cell division and respiration. 3- Cytoplasm 1. An amorphous matrix that contains ribosomes, nutrient granules, metabolites, and plasmids. 2. An inner, nucleoid region composed of DNA. -2-
Ribosomes: are the site of protein synthesis. Bacterial ribosomes are 70S in size, with 50S and 30S subunits, whereas eukaryotic ribosomes are 80S in size, with 60S and 40S subunits. Granules: serve as storage areas for nutrients and stain characteristically with certain dyes. For example, Metachromatic volutin granules is a reserve of high energy stored in the form of polymerized metaphosphate. It appears as a "metachromatic" granule since it stains red with methylene blue dye instead of blue as one would expect. They are a characteristic of Corynebacterium diphtheria. 4-Nucleoid( The Nucleus):The nucleoid is the area of the cytoplasm in which DNA is located. The DNA of prokaryotes is a single, circular molecule that has a molecular weight (MW) of approximately 2 x 109 and contains about 2000 genes. The nucleoid contains no nuclear membrane, no nucleolus, no mitotic spindle, and no histones. The bacterial DNA has no introns, whereas eukaryotic DNA does. Plasmids: are extrachromosomal, double-stranded, circular DNA molecules that are capable of replicating independently of the bacterial chromosome. Plasmids occur in both gram-positive and gram-negative bacteria, and several different types of plasmids can exist in one cell: 1. Transmissible plasmids can be transferred from cell to cell by conjugation. 2. Non-transmissible Plasmids carry the genes for the following functions: 1. Resistance to antibiotics (R plasmid) heavy metals such as mercury. 2. Pili (fimbriae), which mediate the adherence of bacteria to epithelial cells. 3. Sex Pili (F Factor) responsible for congugation. 4. Exotoxins, including several enterotoxins. 5. Bacteriocins are toxic proteins produced by certain bacteria that are lethal for other bacteria. 6. Nitrogen fixation enzymes in Rhizobium in the root nodules of legumes. 7. Several antibiotics produced by Streptomyces. Transposons:Transposons are pieces of DNA that move readily from one site to another either within or between the DNAs of chromosome, plasmids, and bacteriophages. Because of their unusual ability to move, they are called jumping genes. Transposons can code for drug-resistant enzymes, toxins, or a variety of metabolic enzymes and can either cause mutations in the gene into which they insert or alter the expression of nearby genes. 5-Capsule The capsule is a gelatinous layer covering the entire bacterium. It is composed of polysaccharide, except in the anthrax bacillus, which has a capsule of polymerized D-glutamic acid. 1. It is a determinant of virulence of many bacteria since it limits the ability of phagocytes to engulf the bacteria as in pneumococci. 2. Specific identification of an organism can be made by using antiserum against the capsular polysaccharide. 3. Quellung reaction: In the presence of the homologous antibody, the capsule will swell greatly.
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4. are used as the antigens in certain vaccines because they are capable of eliciting protective antibodies. For example, the purified capsular polysaccharides of 23 types of S. pneumoniae are present in its current vaccine. Glycocalyx or biofilm (Slime Layer):The glycocalyx is loose polysaccharide network surrounding and secreted by many bacteria thus form biofilm. This biofilm covers surfaces and allows the bacteria to adhere firmly to various structures, e.g., skin, heart valves, and catheters. Bacteria become embedded in the biofilm protected from antibiotics ans antibodies. Examples, Staphylococcus epidermidis which cause endocarditis and Streptococcus mutanprodoce biofilm and adhere to the surface of teeth forming plaque, the precursor of dental caries. 6-Flagella: Flagella are long, whip like appendages that move the bacteria (Organ of motility). It is composed of flagellin protein. Responsible for H antigen 7-Pili (Fimbriae) Pili are hairlike filaments that extend from the cell surface. They are shorter and straighter than flagella and are composed of subunits of pilin protein. Ordinary pili mediate the attachment of bacteria to specific receptors on the human cell surfaceas in Neisseria gonorrhoeae. The sex pilus, forms the attachment between the male (donor) and the female (recipient) bacteria during conjugation. 7-Spores: 1. These highly resistant structures are formed in response to adverse conditions by two genera of medically important gram-positive rods: the genus Bacillus, which includes the agent of anthrax, and the genus Clostridium, which includes the agents of tetanus and botulism. Spore formation (sporulation) occurs when nutrients are depleted. 2. It is resistant to heat, dehydration, radiation, and chemicals. This resistance may be mediated by dipicolinic acid but it can be killed by autoclaving. Bacterial physiology Bacterial Reproduction: Bacteria reproduce by binary fission, a process by which one parent cell divides to form two progeny cells. Because one cell gives rise to two progeny cells, bacteria are said to undergo exponential growth (logarithmic growth). The doubling (generation) time of bacteria ranges from as little as 20 minutes for Escherichia coli to more than 24 hours for Mycobacterium tuberculosis. The Growth Curve: The growth cycle of bacteria has four major phases. If a small number of bacteria are inoculated into a liquid nutrient medium and the bacteria are counted at frequent intervals, the typical phases of a standard growth curve are: 1. The lag phase, the cells adapt to the new medium with no increase in number. It is represented by incubation period the disease. -4-
2. The log (logarithmic) phase is when rapid cell division occurs. It is represented by symptoms and signs of the disease. 3. The stationary phase occurs when nutrient depletion or toxic products cause growth to slow until the number of new cells produced balances the number of cells that die resulting in a steady state. 4. The decline (death) phase, which is marked by a decline in the number of viable bacteria. It is represented by convalescence and recovery from the disease. Oxygen Requirement of Bacteria: 1. Some bacteria, such as M. tuberculosis, are obligate aerobes;that require oxygen to grow. 2. Other bacteria, such as E. coli, are facultative anaerobes; they can grow in the presence or absence of oxygen. 3. The third group of bacteria consists of the obligate anaerobes, such as Clostridium tetani, which cannot grow in the presence of oxygen because they lack either superoxide dismutase or catalase, or both. These enzymes are needed to get the bacteria of oxygen radicals generated during bacterial metabolism. 4. Microaerophilic Bacteria: require oxygen in concentration less than its athospheric pressure. Nutritional requirement: 1. Heterotrophs: require preformed organic compounds ( Sugars, amino acids). Most pathologic bacteria are heterotrophs 2. Autotrophs: Can synthesize organic compounds from inorganic compounds (chemolithotrophs) or from CO2 and water by the light (photolithotrophs). Temperature: Most pathogenic bacteria require temperature of 37C for growth( mesophilic bacteria). Others need very high degrees (Thermophilic bacteria) or very low Temprature< 4 C (Psychrophilic bacteria). Bacterial Genetics Bacteria are haploid as they have a single chromosome and therefore a single copy of each gene. Eukaryotic cells (such as human cells) are diploid, which means they have a pair of each chromosome and therefore have two copies of each gene. -The Gene: is the basic unit of inheritance formed of a segment of DNA molecule that carries the genetic information for specific biochemical or physiological function. - The Genome (Genotype): refers to the entire collection of hereditary material (DNA) within a cell. It includes chromosomal and extra-chromosomal DNA (plasmid). -The Phenotype: refers to the observed properties exhibited by the microorganisms under the influence of the environment. The Nucleic acids are either DNA or RNA. DNA is polynucleotides formed of complementary double stranded molecule (Adenine-Thymine or Guanine-Cytosine base pairing). It carries the genes. RNA is single stranded molecule. Uracil substitutes thymine of DNA and ribose instead of Deoxy-ribose of DNA. It is found in three forms: 1. mRNA( messenger RNA) carries the genetic information from DNA to ribosome. 2. rRNA (Ribosomal RNA) is a constituent of ribosome. It reads the genetic code on mRNA and form protein according to it. -5-
3. tRNA (Transfer RNA): carries the amino acids from cytoplasm to ribosome. Gene expression: Two processes Transcription: the formation of mRNA from DNA by RNA polymerase enzyme. Translation: is the formation of protein by ribosome according to codons (base triplets on mRNA). DNA replication: Separation of the two strands occurs by helicase enzyme then each strand form a complimentary copy of it by DNA polymerase enzyme. Each new double-stranded DNA molecule contains one original and one new strand, so called; semi-conservative replication. Genetic variation Genetic variation: occur through Mutation or Gene transfer. 1-Mutation: is a permanent change in the DNA base sequence. It may occur without inducer (Spontaneous mutation) or under the effect of an inducer as radiation(induced mutation). -Types: a-Point mutations: an unexpected base is substituted for a normal base. A purine base can substitute for another purine or for a pyrimidine or vice- versa. b- Frame shift Mutation: It is a mutation in which nucleotides are either added or removed from a DNA molecule leading to alteration of the reading frame of the mRNA -This can occur by deletions, insertions or duplications of nucleotides. Also, frame shift mutations may arise from the insertion or removal of bacteriophages or transposons genes. -Effects of Mutations (for both point and frame shift mutations): a- Mis-sense mutation: the change in the DNA sequence leads to substitution of an amino acid in the protein by another. It may result in a totally non functional protein or a functional protein with reduced activity. b-Non-sense mutations: the change in the DNA sequence lead to the formation of a stop codon in the middle of the genetic message instead of at its the end. This will lead to premature termination of translation resulting in a truncated (nonfunctional) protein. c-Loss-of-function mutation: this mutation causes a gene to malfunction. Gene Transfer Gene Transfer: Transfer of genetic material between two bacterial cells(donor and recipient). It can occur through: Transformation: is the uptake of naked DNA by competent recipient cell from its medium. Competence is the ability of bacterial cell to uptake naked dna. It can be genetic as in pneumococci or acquired by electric shock. -6-
Congugation: the transfer of plasmid from donor F+ cell to recipient F- through contact of both cells by sex pilus. Recipient F- cell will acquire a copy of F+ plasmid thus converted to F+ cell. This process is important in transfer of antibiotic resistance between different bacteria. Transduction: It is the transfer of DNA from a donor to a recipient cell through bacteriophages. Transduction may be: Generalized Transduction: Any gene can be transferred between the donor to recipient bacteria by the lytic phage. Specialized Transduction: Specific gene (besides the prophage) can be transferred between the donor to recipient bacteria by the lysogenic (temperate phage)phage. The Bactriophage or Phage It is the virus which attack and infect bacteria. It is composed of DNA or RNA genetic material coated by a protein capsid with a tail and attachment fibrils. Life cycle: Attachment to bacteria thorough the fibrils on specific receptors on the bacteria followred by penetration of the genetic material inside the cell.Then according to the type of the phage: The lytic or virulent phage: the genetic material of the phage direct the host cell machinery to produce its essential components proteins ( enzumes and capsid proteins) . Replication of phage genetic materials, then assembly of the viruses with release of the progeny phages after lysis of the host bacterial cells. Lysogenic or temperate phage: The genetic material incorporates into the host genetic material and called prophage and remains dormant. Lysogenic conversion: This integrated prophage can be translated by the host bacterial cell giving it a new property eg Toxin production. The prophage can be replicated as a part of its genome. After induction (by UV rays), the prophage cuts itself, and formation of progeny phages with lysis of the bacterial cells. Medical importance of phage: 1. Phage typing of bacteria for epidemiological trace the source of infection. 2. Phage therapy. Recombinant DNA Technology (DNA Cloning) In vitro manipulationof DNA in a suitable host cell so as to produce many identical copies of this DNA( molecular clones) . Requirements for DNA cloning: 1. Target DNA :The DNA molecules containing the gene of interest. 2. Vector: to carry the DNA. There are 3 main cloning;plasmids, phages, and cosmids (formed of bacteriophage capsid and a plasmid inside it). They carry DNA sizes of 10, 20 and 45 kb, respectively. They contain a marker gene(as antibiotic resistance gene) for easy detection after incorporation into a host cell. 3. Enzymes:
-7-
Restriction endonucleases: recognize a specific, short nucleotide sequence on a double-stranded DNA molecule, called a restriction site, and cleave the DNA at this recognition site. DNA ligase: joins two pieces of DNA by forming phosphodiester bonds. 4. Competent host cells: For cloning the recombinant DNA molecules. They may be bacterial or yeast cells. -Steps of DNA Cloning : 1. Both the source and vector DNA are extracted. 2. Both the source and vector DNA are cut with the same restriction enzymes. 3. The vector and source DNA are mixed with a ligase enzyme and covalently bonded together. 4. The ligated DNA is transformed into a host cell. Usually the host cell is a competent bacterial or eukaryotic cell. Host cells may be either subjected to a heat shock or to a strong electric current to be competent. 5. After suitable growth of the bacterial cells has occurred, the host cells are tested for the presence of the source of cloned DNA in their cytoplasm. So cells can express the cloned gene a produce its products. Uses Of Cloned DNA 1. Sequencing: to determines the base pair sequence of a gene. 2. Site directed mutagenesis: To study the effects of single amino acid changes on the function of the gene product. 3. Gene therapy: replacement of a mutated form of the gene in the original host cell with a healthy form of the gene. In this process, cells are taken from the patient, altered by adding genes, and returned to the patient. If these cells become established, the expression of the normal genes may be able to cure the patient. It was used to correct a deficiency of the enzyme adenosine deaminase (ADA) associated with an immune deficiency disease. 4. Production of large quantities ofgene product for commercial purposes as human insulin, human growth hormone.
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STERILIZATION AND DISINFECTION
o o
Decontamination: Removal or destruction of microorganisms present on animate (living) or inanimate (non-living) surfaces to render it safe for the intended use. Sterilization is the killing of all forms of microbial life including bacterial spores. Disinfection: is reducing the number of bacteria including large number of spores to a level low enough that disease is unlikely to occur. For example, disinfection of the water supply is achieved by treatment with chlorine. Cleaning: Removal of foreign materials, e. g., dirt, dust, organic matter and microorganisms from surfaces or items. Sepsis: infection Aseptic: without introducing microorganisms. Antisepsis: any procedure that inhibits the growth and multiplication of microorganisms. Methods of sterilization: Physical Methods -Heat -Filtration -radiation Sterilization by Heat: Dry Heat: a) Incineration: as for Hospital Biowast in the incinerator Hot air oven المحرقة. b) Hot flaming: as bacteriological loop. c) Hot air oven: 160 -180 C for 1-2 hrs for metal and glass equipments. Moist heat: Below Temp 100 C: Pasteurization for milk 63C for 30 min or 73 C for 30 seconds. It destroys milk borne bacteria as Brucellae & Salmonella. At Temp 100 C: a) Boiling For 10 minutes. It destroys HBV, HCV and HIV and many spores. It is considered amethod of heat disinfection. b) Steaming at 100 C: As Tyndalization: Exposure to steam at 1normal atmospheric pressure on 3 successive days. For sugar media in Kokh sterilizer. Simple autoclave o At Temp over 100 C: Steaming over 100C (Autoclaving ): Autoclaving: sterilization by exposure to steam under pressure. The best method of sterilization. It uses steam under pressure. The usual temperatures used are: At 2 atmoapheric pressure at 121 C for 20 min At 3 atmoapheric pressure at 134 C for 3-4 min Sterilization monitoring tests: Physical tests: the most important. Monitor the functions of the sterilizers (temperature records and pressure records). Chemical indicators: undergo change in colour upon correct exposure. -9-
Biological indicators: preparations of dried bacterial spores placed within the sterilizer and after completion of the sterilization process are tested for viability. For steam sterilizers Bacillus stearothermophilus are used. o Sterilisation of Heat Sensitive Items: 1. Low temperature with gas: Low-temperature steam with formaldehyde gas sterilizer Ethylene oxide sterilizer Gas plasma sterilization: The plasma state of matter can be produced through the action of powerful electric or magnetic fields on matters. The free radicals in the plasma destroy the micobial components. 2. Chemical sterilization (cold sterilization): -Glutaraldehyde solution (2% or 10%) for a minimum of three hours. - Peracetic acid.
- Hydrogen peroxide
3. Tyndalization 4. Filtration: Applied for serum, plasma and vitamins. As Seitz filter made of asbestos 5. Radiation: ionizing radiations used for syringes and rubber gloves. Disinfectants High-level disinfection: killing many spores, tubercle bacilli, vegetative cells, fungi, and a wide range of viruses. Intermediate-level disinfection: killing little number of spores, tubercle bacilli, vegetative cells, fungi, and intermediate range of viruses (including hepatitis viruses and HIV). Low-level disinfection: killing vegetative cells, some fungi, and a narrow range of viruses. Disinfectant: chemical compound used for disinfection of inanimate surfaces, e.g. floor. Antiseptic: chemical compound used for disinfection of animate surfaces, e.g. skin. The efficiency of disinfectants is measured by Phenol coefficient test. Available Disinfectants: 1. Disruption of Cell Membranes Alcohol: Ethanol is widely used to clean the skin before immunization or venipuncture. Ethanol requires the presence of water for maximal activity; i.e., it is far more effective at 70% than at 100%. Detergents "surface-active" agents: Quaternary ammonium compounds, e.g., benzalkonium chloride, are cationic detergents widely used for skin antisepsis. Phenols:Phenol was the first disinfectant used in the operating room but it is rarely used as a disinfectant today because it is too caustic. Hexachlorophene,is used in germicidal soaps. 2. Modification of Proteins Chlorine: Chlorine is used as a disinfectant to purify the water supply and to treat - 10 -
swimming pools. It is also the active component of sodium hypochlorite (bleach, Clorox (5% )), which is used as a disinfectant in the home and in hospitals (1:4 dilution of clorox). Iodine: Iodine is the most effective skin antiseptic used in medical practice. Iodine is supplied in two forms: 1. Tincture of iodine (2% solution of iodine and potassium iodide in ethanol) is used to prepare the skin prior to blood culture. 2. Iodophors as betadine (povidone-Iodine) are complexes of iodine with detergents that are used to prepare the skin prior to surgery because they are less irritating than tincture of iodine. Heavy Metals: Mercurochrome is used as skin antiseptics. Silver nitrate drops are useful in preventing gonococcal ophthalmia neonatorum. Hydrogen Peroxide: is used as an antiseptic to clean wounds and to disinfect contact lenses. It is an oxidizing agent that attacks sulfhydryl groups, inhibiting enzymatic activity. Formaldehyde: denatures proteins and nucleic acids. Glutaraldehyde(Cidex): In hospitals, it is used to sterilize respiratory therapy equipment and fiberoptic endoscopes. It kills bacteria, fungi, its spores, mycobacteria and viruses. Ethylene Oxide: Ethylene oxide gas is used in hospitals for the sterilization of heat-sensitive materials such as surgical instruments and plastics. It should be used with caution as it is explosive. Acids & Alkalis: Weak acids, such as benzoic, propionic, and citric acids, are frequently used as food preservatives because they are bacteriostatic. 3. Modification of Nucleic Acids crystal violet (gentian violet), which is used as a skin antiseptic
ANTIBIOTICS Antibiotics: are substances produced by microorganisms that kill, or prevent the growth, of other microorganisms. Chemotherapeutic Agents: synthetically produced compounds that kill, or prevent the growth of microorganisms. Bactericidal antibiotics: kill the susceptible bacteria Bacteriostatic antibiotic: an antibiotic that reversibly inhibit the growth of bacteria. Bactericidal antibiotics as Beta-lactam antibiotics (as penicillins and cephalosporins), aminoglycosides and bacteriostatic antibiotics as tetracycline, erythromycin Post-antibiotic effect: bacterial re-growth is inhibited for several hours after the antibiotic concentration has fallen below the MIC. Narrow spectrum : act against few bacterial groups as erythromycin, vancomycin Broad spectrum : act against a wider number of bacterial groups as levofloxacin and, tetracycline. MECHANISMS OF ACTION OF ANTIMICROBES: 1-Inhibition of Cell Wall Synthesis: as Beta lactam antibiotics: inhibit transpeptidase thus inhibiting peptidydoglcan synthesis as Penicillins and Cepalosporin. - 11 -
2-Inhibition of Cytoplasmic Membrane Functions: as Polymyxin E, Daptomycin and amphotericin B. 3-Inhibition of Protein Synthesis by - Binding to the 30S ribosomal subunit as tetracycline and aminoglycosides - Binding to the 50S ribosomal subunit as chloramphenicol, clindamycin, macrolides, linezolids, andstreptogramin. 4-Inhibitors of RNA synthesis and functione.g rifampin inhibits to DNAdependent RNA polymerase. 5-Inhibitors of DNA synthesis and function as quinolones. 6-Anti-metabolite Antimicrobials as Sulfonamides compete with para- amino benzoic acid (PABA) for the active site of an enzyme involved in folic acid synthesis thus inhibiting its formation.
ADVERSE REACTIONS OF ANTIMICROBIAL DRUGS 1-Toxicity: Bone marrow failure: eg with chloramphenicol. Ototoxicity and vestibular toxicity as aminoglycosides. Renal toxicity as in aminoglycosides. Liver toxicity as in isoniazide and tetracycline. 2-Hypersensitivity Reactions:( especially with penicillin): Type I: Anaphylaxis and atopy Type II: Haemolytic anaemia Type III: Serum sickness Type IV: Drug rash 3-Drug interactions: rifampicin stimulates liver enzymes, so decrease effect of many drugs while erythromycin inhibits liver enzymes so increase effect of many drugs. - 12 -
1.
2.
3. 4.
4-Super infection: use of broad spectrum antibiotics kill most flora of the body leaving resistant organisms causing super-infection as use of clindamycin causes pseudomembranous colitis due to overgrowth of Clostridium difficile. 5-Development of antibiotic resistance. 6- Masking the diagnosis:by interfering with antibody rise. ANTIBIOTIC SUSCEPTIBILITY TESTS Testing a group of antibiotics against certain organism to choose the most effective on this organism. The most common methods are: Disk diffusion test: The bacterial isolate is inoculated uniformly onto the surface of an agar plate. A filter disk impregnated with a standard amount of an antibiotic is applied to the surface of the plate. The plates are incubated for 24 hr at 37C. Following incubation, zones of inhibition of bacterial growth form around the Disk diffusion Ab sensitivity test antibiotic disk. Broth dilution method: inoculating the organism in two fold dilutions of antimicrobial placed in tubes or cups. After incubation at 35Cº for 18 hours, we can measure minimal inhibitory concentration (MIC) MIC: The MIC is the lowest concentration of the antibiotic that results in inhibition of visible growth under standard conditions. MBC: The MBC (minimal bactericidal concentration) is the lowest concentration of the antibiotic that kills 99.9% of the original inoculum in a given time. E -test Automated systems: e. g., Vitek, Microscan and Pheonex.
Rules for antibiotic prescription: 1. After antibiotic sensitivity testing. 2. Best guess: a- According to the site of infection eg 1st generation cephalosporin for tonsillitis and norfloxacin for urinary tract infection. b- According to organism eg ciprofloxacin, pipracillin or 3rd generation cephalosporin for Pseudomonas infection. Clindamycin or metronidazole for anaerobes. c- According to age, some antibiotics are prohibited in children as quinolones and tetracyclines. 3-Avoiding unnecessary antibiotic prescription as for viral infections. 4-Parenteral (IV or IM) antibiotic prescription and starting treatment as soon as possible is better in life threatening infections as sepsis. 5-Antibiotic combination is used only if indicated. 6- Adequate duration: most infections need a duration of 5-10 days. Some infections need prolonged courses as brucellosis and endocarditis. - 13 -
Antibiotics combinations Antibiotics combinations: Two or more antibiotics are used. Why? a) to treat life-threatening infections before the cause has been identified b) to prevent the emergence of resistant bacteria during prolonged treatment regimens c) to achieve a synergistic (augmented) effect. A synergistic effect: the effect of two drugs given together is much greater than the sum of the effect of the two drugs given individually as the combination of a penicillin and an aminoglycoside on enterococci. Antagonism: The combination is less efficacious than one of the two components alone e.g combination of tetracycline and beta lactame drugs. ANTIBIOTIC RESISTANCE Mechanisms of Resistance: 1. Altered permeability of the antimicrobial agent: due to the inability of the antimicrobial agent to enter the bacterial cell, e.g., mutation in porin proteins cause resistance to aminoglycosides. 2. Active export of the antibiotic agent from the cell, e.g, multi-drug resistance pump cause efflux of quinolones. 3. Enzymatic Inactivation of the antimicrobial agent: as result of the production of an enzyme that is capable of inactivating the antimicrobial agent, e.g., beta- lactamases destroy beta- lactam ring of penicillins. 4. Altered target site of antibiotic action:e.g., altered PBP in MRSA. 5. Development of an altered metabolic pathway: Resistance to sulfonamides by the use of pre- formed folic acid without the need of PABA. Origin of drug resistance: A) Genetic Basis of Antimicrobial Resistance 1-Chromosomal mediated resistance: due to mutation in the genes coding for the target of the drug. -Less frequent so clinically less important. 2-Extrachromosomal mediated resistance (plasmid and transposons mediated):mediated through resistance plasmid (R factor). It is clinically important as plasmid and transposons can be easily transferred and spread the resistance between bacterial species and genera. B) Non Genetic Basis of Resistance: 1-Selection: In any bacterial population there is few resistant strains. With the use of an antimicrobial, all microbes are either killed except resistant strain which survive and replicate dominating the microbial population. 2- Walling off the organism: Antibiotic can not reach the organism as the bacteria is present in an abscess cavity with thick wall. 3-Dormant bacteria as M. tuberculosis are insensitive to antibiotics 4-Inappropriate use: the use of antimicrobial in case of a viral infection. 5-Hospital use: the extensive use of antimicrobials in hospitals especially in ICU. 6-Agricultural use: adding antibiotics to agricultural feed promotes drug resistance. CHEMOPROPHYLAXIS: The use of antimicrobials to prevent infection. 1. Surgical Indication: few hours before the operation and never be continued for longer than 24hours. - 14 -
2. Medical Indications: a) Immuno-compromised person as pneutropenia b) Long acting penicillin to prevent rheumatic fever c) Ciproflxacin and Rifampicin to prevent meningitis d) 4-Isoniazide to prevent progression of M tuberculosis.
General lines of Diagnosis of bacterial infection: 1. Specimens: according to the type of lesion; wound swab, pus aspiration, CSF, sputum, stools…etc 2. Direct smear: to show the motility of the organism 3. Stained smear: to show the staining affinity, shape and arrangement of bacteria 1)Gram stain: differentiate bacteria into gram negative or positive organisms.2)Ziehl-Neelsen stain: show Acid Fast Bacilli. 4. Culture: on the suitable medium usually for 24 h at 37°C aerobically for aerobic organisms and anaerobically for anaerobic organisms. The colonies identified by: 1) microscopically 2) macroscopic shape of the colony 3) Biochemical reactions as catalase, oxidase..etc 4) serologically 5. Antibiotic sensitivity testing: to know the antibiotics effective for the causative organisms. 6. Serology: to detect specific anti-organism IgM or rising titer of specific anti organism IgG. 7. Genetic diagnosis: Genetic probes or PCR A) Genetic probes: are labeled RNA or DNA segment complementary to unique genes in the target organism. B) Polymerase chain reaction (PCR): to amplify a DNA sequence unique to the organism to be easily detected.
Host Parasite relationship
Colonization: establishment of micro-organisms, pathogenic or non-pathogenic, in large numbers, on a host without tissue damage or clinical disease. Infection: invasion of the host by a microorganism associated with host response (e.g. inflammation) with or without clinical manifestations. Infectious Disease (communicable disease): is an infection that can be transmitted between humans (or organisms/animals, etc.). The organism is able to spread from one host to another and is therefore infectious. A contagious disease: is a subset category of transmissible diseases which are transmitted to other persons either by physical contact with the person suffering the disease, or by casual contact with their secretions or objects touched by them or airborne route. Clinical infection: is one associated with the presence of overt signs and symptoms of disease. Subclinical Infection: infection with no signs or symptoms. TYPES OF PATHOGENS: The pathogens: are microorganisms which are capable of causing disease. Two main types of pathogens: 1-True pathogens: those who are capable of causing disease in healthy persons. - 15 -
2-Opportunistic pathogen : rarely cause disease in immune-competent people but can cause serious infections in immune-compromised patients. These opportunists are frequently members of the body's normal flora. STAGES OFBACTERIAL PATHOGENESIS 1- Transmission from an external source into the portal of entry. 2-Evasion of primary host defences such as skin or stomach acid. 3- Adherence to mucous membranes, usually by bacterialpili. 4- Colonization by growth of the bacteria at the site of adherence. 5-Disease symptoms caused by toxin production or invasion accompanied by inflammation. 6- Host responses, both nonspecific and specific (immunity) during steps 3, 4 and 5. 7- Progression or resolution of the disease. Chain of Infection:The components needed for infection to occur. (1)The organism (2)The reservoir (3)Portal of exit (3) Vector (4)Mode of Transmission (4)Portal of entry (5)Vulnerable population Virulence: is a quantitative measure of pathogenicity and is measured by the number of organisms required to cause disease, it is the degree of pathogenicity. Infectious dose: The dose of an organism required to cause disease varies greatly among the pathogenic bacteria. For example the infectious dose of Shigella is less than 100 organisms whereas 100,000 organisms for Salmonella. Virulence Factors: Certain structures or products that help microorganisms to overcome body defence mechanisms and cause disease. Bacteria produce disease through invasion or toxin production. Invasion is facilitated through capsule and degradative enzymes. Invasive bacteria, on the other hand, grow to large numbers locally and induce an inflammatory response consisting of erythema, edema, warmth, and pain. 1. Capsule: capsulated bacteria as Streptococcus pneumoniae, and Neisseria meningitidis resist phagocytosis. 2. Pili: pili help bacteria to adhere to the surface of host cells as in gonococci. 3. Ezymes : Collagenase and hyaluronidase, which degrade collagen and hyaluronic acid, respectively, thereby allowing the bacteria to spread through subcutaneous tissues; they are especially important in cellulitis caused by Streptococcus pyogenes. Coagulase produced by Staphylococcus aureus produce fibrin from its precursor fibrinogen (this clot may protect the bacteria from phagocytosis by walling off the infected area and by coating the organisms with a layer of fibrin). Immunoglobulin A (lgA) protease, which degrades lgA, allowing the organism to adhere to mucous membranes, and is produced chiefly by Neisseria gonorrhoeae, Haemophilus influenzae, and Streptococcus pneumoniae. Leukocidins, which can destroy both neutrophilic leukocytes and macrophages. 4- Bacterial toxins: There are two types of toxins.
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exotoxin is a polypeptides secreted by an organism. Exotoxins are very toxic. A very small amount can be fatal to a host organism and even though the immune system can often identify and attack the toxin, the toxin spreads so quickly that the host does not have an opportunity to mount a defense. Endotoxins are lipopolysaccharides (LPS), which form an integral part of the cell wall. Endotoxins occur only in gram-negative rods and cocci, are not actively released from the cell, and cause fever, hypotension, and other generalized symptoms even septic shock. Effect of endotoxin is due to: a) Production of cytokines, including IL-1, IL-6, IL-8, tumor necrosis factor (TNF) and platelet-activating factor. b) Activation of the complement cascade. c) Activation of the coagulation cascade. Difference between Exotoxins and Endotoxins Property
Exotoxin
Endotoxin
Source
Certain species of gram-positive and gram-negative bacteria
Cell wall of gram-negative bacteria
Secreted from Yes cell
No
Chemistry
Polypeptide
Lipopolysaccharide
Location of genes
Plasmid or bacteriophage
Bacterial chromosome
Toxicity
High
Low (fatal dose on the order of hundreds of micrograms)
Clinical effects
Various effects
Fever, shock
Mode of action
Various modes
Includes TNF and interleukin-1
Antigenicity
Induces high-titer antibodies called antitoxins
Poorly antigenic
Vaccines
Toxoids used as vaccines
No toxoids formed and no vaccine available
Heat stability
Destroyed rapidly at 60°C (except Stable at 100°C for 1 hour staphylococcal enterotoxin)
Typical diseases
Tetanus, botulism, diphtheria
- 17 -
Meningococcemia, sepsis by gram-negative rods
IMMUNOLOGY Immunology is a branch of biomedicalscience that covers the study of all aspects of the immune system. It deals with the physiological functioning of the immune system in states of both health and diseases. Immunity: is enhanced state of protection against foreign organisms or substances (antigens). The immune system consists of : Central lymphoid organs: Bone marrow and thymus. They are the locations of maturation of lymphoid cells. Peripheral lymphoid organs:as the lymph nodes, spleen, tonsils and payer's patches. They are the sites of where lymphoid cells meets its antigens to react against it. The immune system Cells of the immune system: Antigen presenting cells It engulfs the antigen, processes it and presents it to immune cell in the groove of MHC II to be recognized by immune cells. o Monocyte-Macrophage:Its functions are: a)Phagocytic cell b)Antigen presenting cells c)Secretes IL-1, IL-8and TNF o Dendritic cell: as Langerhans cells found in the epidermis and mucous membrane and interstitial cells which populate most organs, e.g. heart, lung, liver and kidney. o B Lymphocytes: responsible for antigen presentation, and humeral immunity. Cells of specific immune response: o B Lymphocytes: a) B cells are responsible for antibody mediated immunity. b) B cells are derived from pre-B cells and mature in the bone marrow. c) It has B cell receptor formed of membrane Ig M which is responsible for binding to the antigen An associated Igα and Igβ chains which initiate signaling events inside the cell. o T Lymphocytes: T cells are responsible for cell mediated immunity. They mature in the thymus under influence of thymic hormones. It has a T cell receptor(TCR) composed of two transmembrane polypeptide chains designated α and β,each containing one variable (V) domain, one constant (C) domain, a transmembrane region and a short cytoplasmic region. The TCR is associated with CD3 and ζ chains to form T T cell receptor - 18 -
cell receptor complex. These accessory molecules are vital to propagate the signal from the TCR into the cell There are two subpopulations of T cells: CD4+ T helper cells (Th): Th cells are subset of T cells that express a unique antigen on their surface called CD4. They constitute 65% of all T cells. They are divided into different subsets into Th1, Th2, Th17 and T reg cells. The different subsets are involved in both humoral and cellular mediated immune response. CD8+ T cytotoxic cells (Tc): Tc cells are subset of T cells that express a unique antigen on their surface called CD8. They constitute 35% of all T cells. They kill infected and transformed cells and thereby protect the host from viral infections and cancer. Cells of Non Specific Immune response: o Phagocytic cells: macrophage and neutrophils. o Natural Killer (NK) cells: NK cells are large granular lymphocytes. They do not require thymus to mature. Its main function is to patrol the body looking for cells that lost expression of the normal class I MHC molecule; a situation called (missing self recognition). Thus they have non-specific cytotoxic activity on virally infected cells and tumor cells. They are important in innate immunity. They kill the cells by release of perforin molecules and by inducing apoptosis. Antibody dependent cytotoxicity (ADCC). o Mast cell and Basophil: responsible for type I hypersensitivity o Esinophils: increase in allergy and parasitic infestation. A. Major histocompatibility complex (MHC) molecules: o A membrane protein that is important for antigen recognition by by T lymphocytes(MHC Restriction).It is encoded in MHC gene complex. o Two structurally distinct types of MHC molecules exist. Class I MHC molecules: They are present on all nucleated cells. They bind peptides derived from cytosolic proteins (endogenous Ag) and are recognized by CD8+ T cells. Class II MHC molecules: They are molecules presented largely on professional APCS, macrophages and В lymphocytes. They bind peptides derived from exogenous Ag, and are recognized by CD4+ T cells.
ANTIGENS
Immunogen: A substance that induces a specific immune response (humoral or cell mediated immune response). - 19 -
Antigen (Ag): A substance that reacts with the products of a specific immune response (antibody molecule or T cell receptor). Hapten: Haptens are small molecules which can't induce an immune response by themselves but can produce an immune response when coupled to a carrier molecule. Haptens have the property of antigenicity but not immunogenicity, e.g. drugs, peptide hormones and steroid hormones. Antibody (Ab): A specific glycoprotein which is produced in response to an immunogen and which reacts with an antigen. Epitope or Antigenic determinant:are small chemical groups (8-15 amino acids) on the antigen molecule that can elicit and react with antibody. Mitogen:An agent that induces mitosis and activates T cells and or B cells without help from APCs, e.g. concanavalin A (ConA), phytohemagglutinin (PHA) and LPS (lipopolysaccharide). Paratope: is the part of the antibody molecule that reacts with the epitope. T dependent antigens: Most of the antigens are T- dependent antigens that need T cell help for the the production of antibody by B cell. Different proteins are T dependent antigens, e.g. microbial proteins. T independent antigens: are antigens which can directly stimulate the B cells to produce antibody without the requirement for T cell help. As Polysaccharides, e.g. pneumococcal polysaccharide, LPS and flagella. They are characterized by: (1)Polymeric structure (2)Polyclonal activation of B cell (3)No immunological memory Superantigens:as staphylococcal enterotoxin and toxic shoch syndrome toxin. are a class of antigens which cause non-specific activation of T cells resulting in polyclonal T cell activation and massive cytokine release (IL-1, IL-2 and TNF) producing a cytokine storm similar to toxic shock syndrome and septic shock. They interact with the MHC molecule outside the peptide binding groove. They bind to the variable segment of the β chain (Vβ) only but not the chain of the TCR. This antigenic stimulation is not specific for the pathogen and there is no memory.
Factors that affects the immunogenicity: Foreignness:In general, molecules recognized as "self" are not immunogenic, To be immunogenic, molecules must be recognized as "nonself," i.e., foreign. Molecular Size: The most potent immunogens are proteins with high molecular weights, i.e., above 100,000. While very small ones, e.g., an amino acid, are nonimmunogenic.. Chemical-StructuralComplexity: More complex substance is more immunogenic e.g., amino acid homopolymers are less immunogenic than heteropolymers containing two or three different amino acids. Dosage:very high or very low dode of immunogen cause anergy. Route of Antigen Administration:Sc route is better than ingestion The genetic constitution of the host : Different strains of the same species respond differently to the same antigen. Age :Immunity is less than optimal in the newborn and the elderly.
- 20 -
Adjuvants: are substances that enhance the immune response when introduced with an immunogen e.g. Freund's adjuvant, Aluminum hydroxide. Its action is through: Slow release of immunogen, i.e. Depot effect prolonging exposure to Ag. Enhance uptake of immunogen by APCs. Induce costimulatory molecules. Stimulate macrophages which enhance the response of T cells and B cells.
INNATE IMMUNITY Immunity includes both innate and adaptive (acquired)immunity. Type of Immunity
Specificity
Effective Immediately After Exposure to Microbe
Improves After Has Exposure Memory
Innate
Nonspecific
Yes—acts within minutes
No
No
Adaptive
Highly specific
No—requires several days before becoming effective
Yes
Yes
Innate Immunity: Non specific immunity born with every subject. Its components are summarized in the underlying table. Factor
Mode of Action
I. Factors that limit entry of microorganisms into the body Keratin layer of intact skin Lysozyme in tears and other secretions Respiratory cilia Low pH in stomach and vagina; fatty acids in skin
Acts as mechanical barrier Degrades peptidoglycan in bacteria cell wall Elevate mucus-containing trapped organisms Retards growth of microbes
Surface phagocytes (e.g., alveolar Ingest and destroy microbes macrophages) Defensins (cationic peptides)
Create pores in microbial membrane
Normal flora of throat, colon, and Occupy receptors, which prevents colonization by vagina pathogens II. Factors that limit growth of microorganisms within the body Natural killer cells
Kill virus-infected and tumor cells
Neutrophils
Phagocytosis
Macrophages and dendritic cells
Phagocytosis, and Ag presentation to helper T cells
Interferons
Inhibit viral replication
Complement
C3b is an opsonin; membrane attack complex creates holes in bacterial membranes
Transferrin and lactoferrin
Sequester iron required for bacterial growth
Fever
Elevated temperature retards bacterial growth
Inflammatory response
Limits spread of microbes
Phagocytosis The predominant phagocytic cells in inflammation are neutrophils and macrophages. Neutrophils are seen in the pyogenic inflammatory response - 21 -
to bacteria such as Staphylococcus aureus and Streptococcus pyogenes, whereas macrophages are seen in the granulomatous inflammatory response to bacteria such as Mycobacterium tuberculosis. Chemtaxis:Neutrophils and macrophages are attracted to the site of infection by chemokines, which are small polypeptides produced by cells at the infected site. Interleukin-8 and C5a are important chemokines for neutrophils. In response to most bacterial infections, there is an increase in the number of neutrophils in the blood. This increase is caused by the production of Granulocyte-stimulating factors by macrophages. Phagocytosis After neutrophils are attracted to the infected site by chemokines, they attach to the endothelium first using selectins on the endothelium, then by the interaction of integrins (LFA proteins) on the neutrophils with ICAM proteins on the endothelium. Diapedesis: Neutrophils then migrate through the endothelium to site of inflammation. Endocytosis: engulfment of the organism through pseudopodia to enter the cytoplasic vesicle called phagosome. Opsonisation:Facilitation of phagocytosis of organism through use of opsonins.IgG and C3b are opsonins, which enhance ingestion of the bacteria. Killing of the bacteria within the neutrophil is caused by hypochlorite, hydrogen peroxide, and superoxides(Oxygen-dependant). Lysosomes contain various degradative enzymes (Oxygen-independant) and fuse with the phagosome to form a phagolysosome within which the killing occurs. Exocytosis of cellular debris and digestive enzymes are released outside the phagocyte.
ACQUIRED (ADAPTIVE) IMMUNITY
Passive immunity refers to protection based on the transfer of preformed antibody from one person (or animal) to another person. Passive immunity provides immediate but short-lived protection (lasting a few months). Examples of passive immunity include: a)administration of antitoxin as tetanus antitoxin b) passage of IgG from mother to fetus across the placenta c)passage of IgA from mother to newborn through breast milk Active immunity refers to protection based on the formation of both antibodies and cell-mediated immunity after exposure either to: the microbe itself (clinical or subclinical infection) the antigens of the microbe in a vaccine. Active immunity develops more slowly but provides long-term protection. Acquired (adaptive) immunity: consists of humeral and cell mediated immunity. - 22 -
Humeral adaptive immunity Humeral adaptive immunity is through Antibodies. The main functions of antibodies are 1) to neutralize bacterial toxins and viruses 2) opsonize bacteria 3) activate complement to form a membrane-attack complex that can kill bacteria, 4) interfere with attachment to mucosal surfaces. IgG is the main opsonizing antibody, IgG and IgM activate complement, and IgA interferes with attachment to the mucosa. 5) Allergy (hypersensitivity), e.g., hay fever, anaphylactic shock 6) Autoimmunity The Primary immune Response When an antigen is first encountered, antibodies are detectable in the serum after a longer lag period than occurs in the secondary response. The lag period is typically 7 to 10 days. A small clone of B cells and plasma cells specific for the antigen is formed. The serum antibody concentration continues to rise for several weeks, then declines and may drop to very low levels. The firstand main antibodies to appear are IgM, followed by IgG or IgA. IgM levels decline earlier than IgG levels. The Secondary immune Response When there is a second encounter with the same antigen , months or years after the primary response, there is a rapid antibody response (the lag period is typically only 3–5 days, to higher levels than the primary response. This is attributed to the persistence of antigen-specific "memory cells" after the first contact. These memory cells proliferate to form a large clone of specific B cells and plasma cells, which mediate the secondary antibody response. During the secondary response, the amount of IgM produced is similar to that after the first contact with antigen. However, the main Ig is IgG which is produced in a larger amount and the levels tend to persist much longer than in the primary response.
ANTIBODIES Introduction: Antibodies are globulin proteins (immunoglobulins) that react specifically with the antigen that stimulated their production. They make up about 20% of the protein in blood plasma. Blood contains three types of globulins, alpha, beta, and gamma, based on their electrophoretic migration rate. Antibodies are gamma globulins. There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE. Antibodies are subdivided into these five classes based on differences in their heavy chains. Ig structure Immunoglobulin Structure The simplest antibody molecule has a Y shape and consists of four polypeptide chains: two H (Heavy) chains and two L (Light) chains. The four chains are linked by disulfide bonds. An individual antibody molecule always consists of identical H chains and identical L chains. - 23 -
light chains have a molecular weight of about 25,000, whereas heavy chains have a molecular weight of 50,000 to 70,000. L and H chains are subdivided into variable and constant regions. The variable region is present in the amino-terminal of the chain has variable amino acid sequence while the constant region is present in the carboxylterminal of the chain and has constant amino acid sequence. The variable regions of both the light and heavy chain are responsible for antigen-binding, whereas the constant region of the heavy chain is responsible for various biologic functions, e.g., complement activation and binding to cell surface receptors. The complement binding site is in the C H2 domain. The constant region of the light chain has no known biologic function. The variable regions of both L and H chains have three extremely variable (hypervariable) amino acid sequences at the amino-terminal end that form the antigen-binding site(the Paratope). Only 5 to 10 amino acids in each hypervariable region form the antigen-binding site. Antigen–antibody binding involves electrostatic and van der Waals' forces and hydrogen and hydrophobic bonds rather than covalent bonds. The remarkable specificity of antibodies is due to these hypervariable regions. Domains: are composed of three-dimensionally folded (globular), repeating segments. Each domain is approximately 110 amino acids long. An L chain consists of one variable (VL) and one constant (CL) domain. Most H chains consist of one variable (VH) and three constant (C H) domains. (IgG and IgA have three CH domains, whereas IgM and IgE have four.) Fragments of the Ig: -If an antibody molecule is treated with theproteolytic enzyme papain, peptide bonds in the "hinge" region are broken, producing: 1. two identical Fab fragments, which carry the antigen-binding sites 2. one Fc fragment, which is involved in Effector (biologic) functions: a) placental transfer Ig D b) complement fixation Ig M & G c) attachment site for various cells as mast cell binding Ig E e)Mucosal attachment Ig A -Digestion of Ig by pepsin gives the divalent fragment F(ab')2 without FC fragment
Immunoglobulin Genes 1) To produce the very large number of different immunoglobulin molecules (10 6– 109) without requiring excessive numbers of genes, special genetic mechanisms, e.g., DNA rearrangement and RNA splicing, are used. The DNA rearrangements are performed by recombinases encoded in two important genes RAG-1 and RAG-2. Mutations in these genes arrest the development of lymphocytes and result in severe combined immunodeficiency. 2)For each type of immunoglobulin chain, there is a separate pool of gene segments located on different chromosomes. Each pool contains a set of different V (variable) gene segments widely separated from the D (diversity, seen only in H chains), J (joining), and C gene segments. Immunoglobulin Class Switching (Isotype Switching) Initially, all B cells carry IgM specific for an antigen and produce IgM antibody in response to exposure to that antigen. Later, gene rearrangement permits the - 24 -
elaboration of antibodies of the same antigenic specificity but of different immunoglobulin classes. Note that the antigenic specificity remains the same for the lifetime of the B cell and plasma cell because the specificity is determined by the variable region genes (V, D, and J genes on the heavy chain and V and J genes on the light chain) no matter which heavy-chain constant region is being utilized. Properties of Human Immunoglobulins Property
lgA
lgM
lgD
lgE
Percentage of total immunoglobulin 75 % in serum
15%
9%
0.2%
0.004 %
Serum concentration (mg/dL)
200
120
3
0.05
19S
7S
8S
900
180
190
Sedimentation coefficient
lgG
1000
1
7S
7S or 11S
1
Molecular weight (x1000)
150
170 or 400
Structure
Monomer
Monomer or dimer
Monomer Monomer Monomer or pentamer
H chain symbol
γ
α
µ
δ
Ε
Complement fixation
+
–
+
–
–
Transplacental passage
+
–
–
–
–
Mediation of allergic responses
–
–
–
–
+
Found in secretions
–
+
–
–
–
Opsonization
+
–
–
–
–
Antigen receptor on B cell
–
–
+
?
–
Polymeric form contains J chain
–
+
+
–
–
2
Isotypes, Allotypes, &Idiotypes Because immunoglobulins are proteins, they are antigenic. That property allows them to be subdivided into isotypes, allotypes, and idiotypes. 1. Isotypes are defined by antigenic (amino acid) differences in their constant regions. Although different antigenically, all isotypes are found in all normal humans. a) The five immunoglobulin classes—IgG, IgM, IgA, IgD, and IgE—are different isotypes; their H chains are antigenically different. b) The IgG isotype is subdivided into four subtypes, IgG1, IgG2, IgG3, and IgG4, based on antigenic differences of their heavy chains. c) Similarly, IgA1 and IgA2 are different isotypes (the antigenicity of the constant region of their H chains is different. d) anti-isotypic antibodyis used in detecting and quantitating Ig in serum 2. Allotypes, on the other hand, are antigenic features of immunoglobulins that vary among individuals of the same species. They vary because the genes that code for the L and H chains are polymorphic, and individuals can have different alleles. Anti-allotypic antibody is used in genetic studies & determining paternity. 3. Idiotypes are due to the antigenic difference the hypervariable regions. Each idiotype is unique for the immunoglobulin produced by a specific clone of antibody-producing cells. Anti-idiotypic antibody is used in tumor immunotherapy. - 25 -
Allelic Exclusion A single B cell expresses only one L chain gene (either κor λ) and one H chain gene. Only one set of genes is expressed, either maternal or paternal, and the other set is silent, i.e., it is excluded. This is called allelic exclusion. Each individual contains a mixture of B cells, some expressing the paternal genes and others the maternal ones. The mechanism of this exclusion is unknown. Monoclonal Antibodies Polyclonal antibodies : that arise in an animal in response to typical antigens are heterogeneous, because they are formed by several different clones of plasma cells, i.e., they are polyclonal. Monoclonal antibodies: antibodies that arise from a single clone of cells, e.g., in a plasma cell tumor (myeloma), are homogeneous, i.e., they are monoclonal. Monoclonal antibodies also can be made in the laboratory from hybridoma cell. Such hybridomas produce virtually unlimited quantities of monoclonal antibodies that are useful in diagnostic tests and in research Hybridoma cells are made in the following manner: (1) An animal, e.g., a mouse, is immunized with the antigen of interest. (2) Spleen cells from this animal are grown in a culture dish in the presence of mouse myeloma cells. The myeloma cells have two important attributes: they grow indefinitely in culture and they do not produce immunoglobulins. (3) Fusion of the cells is encouraged by adding certain chemicals, e.g., polyethylene glycol. (4) The cells are grown in a special culture medium (HAT medium) that supports the growth of the fused, hybrid cells but not of the "parental" cells. (5) The resulting clones of cells are screened for the production of antibody to the antigen of interest. Uses of monoclonal antibodies: A. Diagnostic uses: o Diagnosis of bacterial and viral infections. o Detection of tumor antigens. o Identification of cell surface markers, e.g. of T & B lymphocytes. o Tissue typing (HLA antigens). o Hormonal assay. B. Therapeutic uses: o Anti-tumor therapy by monoclonal antibodies against tumor antigens either alone or combined with cytotoxic drugs "magic bullet therapy". o Passive immunization against viral infections, e.g. hepatitis B, varicella zoster and cytomegalovirus. o Anti Rh (D) produced to prevent Rh incompatibility. o Neutralize drug toxicity. o Immunosuppressive therapy, e.g. anti-CD3 to prevent graft rejection. o Treatment of atopy by a monoclonal antibody that binds to IgE and prevents it from binding to mast cells. Cell Mediated Immunity (CMI) CMI is mediated by T lymphocytes and involves the activation of macrophages, antigen specific cytotoxicT lymphocytes and the release of various cytokines in response to an antigen. - 26 -
The main functions of cell-mediated immunity are a) to protect against intracellular bacteria b) to kill virus-infected cells c) To kill tumor cells. Helper T cells (and macrophages) protect against intracellular bacteria, whereas cytotoxic T cells kill virus-infected cells and tumor cells. d) Allergy (hypersensitivity), e.g., poison oak e) Graft and tumor rejection f) Regulation of antibody response (help and suppression) Steps of Cell mediated Immunity: Antigen recognition: - Exogenous antigens are engulfed by antigen presenting cells and process it then present it as peptides in the groove of MHC II molecules to CD4 to be recognized by its T cell receptor. -Endogenous antigens as viral antigens or tumor antigens are presented in the groove of MHC I to be recognized by CD8. MHC restriction: T lymphocytes can't recognize naïve antigen except after its processing and presentation in the groove of MHCII to CD4 cells or MHC I to CD8 cells. Activation of Th cells: Two signals are required for full activation of Th cells: The first (initial) signal: The interaction of an antigenic peptide In MHC II groove with the TCR complex. The second (co-stimulatory) signal: This interaction occurs between: B7 (CD 80) on APCs bind to CD28 on T cells. CD40 on APCs binds to CD40 ligand (CD40L) on T cells. In absence of co-stimulatory signal, No stimulation of the naive T cells occur ( clonal anergy) which is T cell inactivation and inability to proliferate in response to a peptide MHC complex. Clonal Expansion of T cells:The stimulated T cell begin to proliferate giving antigen specific clones and secretes IL 2 which further stimulates the T cells. Th cell differentiation: CD4+ T helper cells (Th cells): Cytokines produced at the site of antigen recognition determine Th subset. Naive T cells can differentiate into at least four major lineages, Th1, Th2, Th17 and Treg cells that participate in different types of immune responses. Th1 subset: Th0 cells differentiate into Th1 cells under the influence of IL-12 secreted from macrophages. - Th1 cells control cell mediated immunity by secreting: - IL-2 which activates CD8+ T cytotoxic cells and natural killer cells. Both cells will destroy the target cells. - IFN-γ which causes activation and recruitment of macrophages. It also suppresses development of Th2 cells. - Tumor necrosis factor beta (TNF-β). Th2 subset: Th0 cells differentiate into Th2 cells under the influence of IL-4 secreted from mast cells.
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- Th2 cells control humoral immunity by secreting: IL-2, IL-4, IL-5, IL-6, IL-10, IL-13 Treg cells: They represent 5-10% of CD4+ T cells. 1. They differentiate under the influence of Il-2 and transforming growth factor beta (TGF-β). 2. When activated, they secrete IL-10 and TGF-β. They function to down-regulate the immune response. 1. CD8+ T cytotoxic cells (Tc cells): Tc cells become activated under the influence of two signals: a. Recognition of peptide-MHC molecules class I on the surface of target cells. b. IL-2 which is produced from Th1 cells. Activated Tc cells kill the target cells by: a. They release perforins to create pores in the cell wall of target cells through which granzymes will enter in the cytoplasm to induce apoptosis of target cells. b. On activation, Tc cells will express Fas ligand (FasL) that binds Fas protein on target cells. The result is degradation of target cell DNA. 2. Memory cells: As the infection is cleared and the stimuli for lymphocyte activation disappear, these cells die by a process of apoptosis (programmed cell death). A fraction of antigen activated T cells differentiates into long lived memory T cells. These memory T cells can be found in lymphoid tissues, in mucosal barriers and in the circulation. Memory T cells can restore defense rapidly on subsequent exposure to their specific antigen. The Complement 1. The complement system consists of approximately 30 proteins that are present in normal human (and other animal) serum. Complement is an important component of our innate host defenses. 2. Complement is heat-labile, i.e., it is inactivated by heating serum at 56°C for 30 minutes. 3. Several complement components are proenzymes, which must be cleaved to form active enzymes. Activation of the complement system can be initiated either by antigen–antibody complexes or by a variety of nonimmunologic molecules, e.g., endotoxin. 4. All three pathways lead to the production of C3b, the central molecule of the complement cascade. The presence of C3b on the surface of a microbe marks it as foreign and targets it for destruction. C3b has two important functions: (1) It combines with other complement components to generate C5 convertase, the enzyme that leads to the production of the membrane attack complex. (2) It opsonizes bacteria because phagocytes have receptors for C3b on their surface. In the classic pathway: A specific antibody is needed; antigen–antibody complexes activate C1 to form a protease, which cleaves C2 and C4 forming C3 convertase, which cleaves C3 molecules into two fragments, C3a and C3b.C5 convertase formed while C3a, an anaphylatoxin. a) Only IgM and IgG fix complement. One molecule of IgM can activate complement; however, activation by IgG requires two cross-linked IgG molecules. - 28 -
b) C5 is cleaved by C5 intoC5aand C5b.C5a is an anaphylatoxin and a chemotactic factor. c) C5b binds to C6, C7, C8 and C9 to produce the membrane attack complex (C5b,6,7,8,9), which causes cytolysis. In the lectin The complement pathways pathway, mannan-binding lectin (MBL) binds to the surface of microbes bearing mannan. This activates proteases to cleave C2 and C4 components of complement and activate the classic pathway. In the alternative pathway, many cell surface substances, e.g., bacterial LPS (endotoxin), fungal cell walls, and viral envelopes, can initiate the process by binding C3 and factor B. This complex is cleaved by a factor D, to produce C3b,Bb. This acts as a C3 convertase to generate more C3b. Regulation of the complement proteins: a) C1 inhibitor is an important regulator of the classic pathway. It inactivates the protease activity of C1 b) Regulation of the alternative pathway is mediated by the binding of factor H to C3b and cleavage of this complex by factor I, a protease. This reduces the amount of C5 convertase available. c) Protection of human cells from lysis by the membrane attack complex of complement is mediated by decay-accelerating factor (DAF, CD55)—a glycoprotein located on the surface of human cells. This prevents the formation of the membrane attack complex. Biologic Effects of the complement: a) Opsonization: C3b is an opsonin facilitates phagocytosis. b) Chemotaxis: C3a and C5a attract phagocytes to site of infection. c) Anaphylatoxin: C3a, C4a, and C5a cause degranulation of mast cells with release of mediators, e.g., histamine, leading to increased vascular permeability and smooth muscle contraction. d) Cytolysis:membrane attack complex C5b,6,7,8,9 non enzymatically destroy the cell membrane leading to lysis of many types of cells as erythrocytes, bacteria, and tumor cells. e) Enhancement of antibody production: Binding of C3b to its receptor on activated B cells promotes the antibody production. - 29 -
Clinical Aspects of Complement 1. Inherited (or acquired) deficiency of complement components, especially C5–C8, enhances susceptibility to Neisseria bacteremia and other infections. 2. A deficiency of C3 leads to severe, recurrent pyogenic sinus and respiratory tract infections. 3. Inherited deficiency of C1 esterase inhibitor results in angioedema. When the amount of inhibitor is reduced, an overproduction of anaphylatoxins, which cause capillary permeability and edema. 4. Acquired deficiency of decay-accelerating factor on the surface of cells results in an increase in complement-mediated hemolysis. Clinically, this appears as the disorder paroxysmal nocturnal hemoglobinuria 5. In transfusion mismatches, e.g., when type A blood is given by mistake to a person who has type B blood, antibody to the A antigen in the recipient binds to A antigen on the donor red cells, complement is activated, and large amounts of anaphylatoxins and membrane attack complexes are generated. The anaphylatoxins cause shock, and the membrane attack complexes cause red cell hemolysis. 6. Immune complexes bind complement, and thus complement levels are low in immune complex diseases, e.g., acute glomerulonephritis and systemic lupus erythematosus. 7. Patients with severe liver disease have cannot synthesize sufficient complement proteins, are predisposed to infections caused by pyogenic bacteria.
CYTOKINES
They are a diverse group of low MW non-antibody proteins that act as mediators between cells. They are products mainly of immune cells that act as mediators and regulators of immune process. Monokines: cytokines produced by mononuclear phagocytic cells. Lymphokines: cytokines produced by activated lymphocytes, especially Th cells. Interleukins: cytokines that act as mediators between leukocytes. Chemokines: cytokines primarily responsible for leukocyte migration. Cytokines Affecting Lymphocytes 1. IL-1 is produced mainly by macrophages. It is a proinflammatory cytokine, i.e., plays an important role, along with tumor necrosis factor (TNF), in inducing inflammation. In addition, IL-1 is endogenous pyrogen, which acts on the hypothalamus to cause the fever associated with infections (Exogenous pyrogen is endotoxin). 2. IL-2 is produced mainly by helper T cells. It stimulates both helper and cytotoxic T cells to grow. IL-2 is T-cell growth factor 3. IL-4 is produced by the Th-2. IL-4 stimulates the development of Th-2 cells from Naïve T cells that have been activated by exposure to antigen. It also induces class switching to IgE. IL-4 is the "signature" (most characteristic) cytokine produced by Th-2 cells. 4. IL-5 is produced by the Th-2 class of helper T cells. It induces class switching to IgA thereby increasing mucosal immunity. It also increases the number and activity of eosinophils.. - 30 -
5.
IL-6 is produced mainly by macrophages. It is a proinflammatory cytokine that induces fever by affecting the hypothalamus and induces the production of acutephase proteins by the liver. 6. IL-10 and IL-12 regulate the production of Th-1 cells, the cells that mediate delayed hypersensitivity . IL-12 is produced by macrophages and promotes the development of Th-1 cells, whereas IL-10 is produced by Th-2 cells and inhibits the development of Th-1 cells. The relative amounts of IL-4, IL-10, and IL-12 drive the differentiation of Th-1 and Th-2 cells and therefore enhance either cellmediated or humoral immunity, respectively. 7. The IL-12–gamma interferon axis is very important in the ability of our host defenses to control infections by intracellular pathogens, such as M. tuberculosis and L. monocytogenes. IL-12 increases the number of Th-1 cells, and Th-1 cells produce the gamma interferon that activates the macrophages that phagocytose and kill the intracellular bacterial pathogens mentioned above. 8. IL-13 is implicated as the mediator of allergic airway disease (asthma). 9. The main function of transforming growth factor- β (TGF-β) is to inhibit the growth and activation of T cells. It is an "anti- inflammatory" cytokine. It is produced by many types of cells, including T cells, B cells, and macrophages. Cytokines Affecting Macrophages & Monocytes Chemokines are a group of cytokines that can attract either macrophages or neutrophils to the site of infection. Chemokines are produced by various cells in the infected area, such as endothelial cells and resident macrophages. The circulating neutrophils and macrophages (monocytes) are attracted to the site. Approximately 50 chemokines have been identified. IL-8 is a very important member of this group. Cytokines Affecting Polymorphonuclear Leukocytes 1. TNF activates the phagocytic and killing activities of neutrophils and increases the synthesis of adhesion molecules by endothelial cells. 2. Chemotactic factors for neutrophils, basophils, and eosinophils selectively attract each cell type. Interleukin-8 and complement component C5a are important attractants for neutrophils. 3. IL-17 plays an important role in mucosal immunity, especially in the GI tract. Cytokines Affecting Stem Cells 1. IL-3 is made by activated helper T cells and supports the growth and differentiation of bone marrow stem cells. 2. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is made by T lymphocytes and macrophages. It stimulates the growth of granulocytes and macrophages and enhances the antimicrobial activity of macrophages. It is used clinically to improve regeneration of these cells after bone marrow transplantation. 3. Granulocyte colony-stimulating factor (G-CSF, filgrastim) enhances the development of neutrophils from stem cells and is used clinically to prevent infections in patients who have received cancer chemotherapy. Cytokines Produced by Macrophages that Affect other Cells 1. TNF- α (cachectin)is a proinflammatory cytokine produced primarily by macrophages. TNF-α inhibits lipoprotein lipase in adipose tissue, resulting in - 31 -
cachexia. It also causes the death and necrosis of certain tumors in experimental animals. 2. Nitric oxide (NO) is made by macrophages in response to endotoxin. NO causes vasodilation, 3. Macrophage migration inhibitory factor (MIF) made by macrophages in response to endotoxin. The function of MIF is to retain the macrophages at the site of infection. Cytokines with Other Effects 1. Interferons (IFN) are glycoproteins that block virus replication and exert many immune-modulating functions. a. Type I IFN: Alpha interferon (from leukocytes) and beta interferon (from fibroblasts) are induced by viruses (or double-stranded RNA). These interferons exert antiviral activity by inhibiting viral replication. They also activate NK. b. Type II IFN:Gamma interferon is a lymphokine produced by the Th-1 and is the "signature" cytokine involved in the inflammation mediated by those cells. It is one of the most potent activators of thephagocytic activity of macrophages, NK cells, and neutrophils, thereby enhancing their ability to kill microorganisms and tumor cells. It also increases the synthesis of class I and II MHC proteins in a variety of cell types. This enhances antigen presentation by these cells. 2. Lymphotoxin (also known as TNF-β) is made by activated T lymphocytes and causes effects similar to those of TNF-α.
ANTIGEN ANTIBODY REACTION (SEROLOGICAL REACTION) Serology: is the use of in vitro antigen antibody interaction for diagnosis. Affinity: Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody. Avidity: Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies. Zone phenomena: Ag-Ab interaction gives Visible complexes only when the there is optimum concentration of the Ag and Ab. The complexes are invisible in case of inappropriate high concentration of the Ab( prozone effect) egin Brucella agglutination test, seum can gives false negative result wheras its dilution gives positive results. Antibody Titer: the maximum dilution (lowest concentration) that gives visible Ag-Ab reaction. Importance: A rise in titer of an antibody to a particular bacterium indicates an infection with that bacterial type. N.B. a fourfold rise in titer is generally taken as a significant rise in antibody titer. Agglutination: Ag-Ab reaction when one of the Ag or Ab is particulate as RBC, Bacteria, Latex particles. Examples: ABO Blood typing: mixing RBCs group B with Anti-B Ab gives agglutination Bacterial infections: Widal test for diagnosis of Typhoid fever Coombs'antiglobulin test: Coomb's Test (Antiglobulin Test) Direct Coomb's Test: Incomplete antibodies:a functional definition of some antibodies that bind to but do not cause agglutination of red blood cells. - 32 -
Direct Coombs Test: Detects incomplete antibodies on erythrocytes In order to detect the presence of nonDirect CoombTest agglutinating antibodies on red blood cells, one simply adds a second antibody directed against the immunoglobulin (antibody) coating the red cells. This antiimmunoglobulin can now cross link the red blood cells and result in agglutination. Indirect Coomb's Test: Detects Indirect comb test anti-erythrocyte antibodies in serum. This test is done by incubating the red blood cells with the serum sample, washing out any unbound antibodies and then adding a second anti-immunoglobulin reagent to cross link the cells. Application of comb test: - Detection of anti-rhesus factor (Rh) antibodies. Antibodies to the Rh factor generally do not agglutinate red blood cells. Thus, red cells from Rh+ children born to Rh- mothers, who have anti-Rh antibodies, may be coated with these antibodies. To check for this, a direct Coombs test is performed. To see if the mother has antiRh antibodies in her serum an Indirect Coombs test is performed. -Autoimmune hemolytic anemia Precipitation: Ag-Ab reaction when both of the Ag and Abare soluble. Single Radial Immunodiffusion: antibody is incorporated into the agar gel as it is poured and different dilutions of the antigen are placed in holes punched into the agar. As the antigen diffuses Single radial immunodiffusion into the gel, it reacts with the antibody and when the equivalence point is reached a ring of precipitation is formed. The diameter of the ring is proportional to the log of the concentration of antigen since the amount of antibody is constant. Double Radial Immunodiffusion (ouchterlony): Solutions of Ag and AB are placed in adjacent wells cut in agar plate. Diffusion of both ag and Ab until they meat forming line of precipitation. This reaction can give idea of identity of Antigens. Immunoassays Is Ag-Ab reaction in which one of them is labeled Double radial immunodiffusion with amrker molecule. The label is a radioactive compound in the RIA, an enzyme that causes a change of color in a solution in the ELISA (Enzyme linked immunosorbent Assay) and fluorescent dye in immunofluorescence (IF). A. Enzyme linked immunosorbent Assay (ELISA): Indirect ELISA: To detect and measure antibody concentration in a sample. o The antigen is coating the surface of microtiter plates. - 33 -
o Suspect serum is added and unbound proteins are washed off. Then second labeled antibodies are added followed by the chromogenic substrate. o The color developed is directly proportional to the antibody present in the serum. Sandwich ELISA technique: o To detect and measure antigen concentration in a sample. o The well is coated with the antibody then specimen (Ag) is added and allowed to react. o The wells are then washed to remove unbound Ag. Then a labeled Ab of the Ag is added. Unbound Ab is removed by washing and this is followed by adding the substrate and the colored reaction is measured. o The intensity of color is directly proportional to the concentration of the Ag in the sample. B. Immunofluorescence: Ag-Ab reaction in which an antibody is labeled with a fluorescent dye as fluorescein to permit the visualization of antigen in cells or tissues by the fluorescence emitted by the bound antibody. Direct immunofluorescence: The antibody specific to the antigen is directly tagged with the fluorochrome as in detection of Rabiesvirus in brain biopsy. -Specimen (Ag) is fixed to slide. Fluorescein labeled Anti RabiesAb is added to react with rabies virus. Washing then examination with UV microscope. Indirect immunofluorescence:The antibody specific for the antigen is unlabeled and a second anti-immunoglobulin antibody directed toward the first antibody is tagged with the fluorochromeeg detection of ANA in SLE.
- 34 Direct immune-fluorescence
Indirect immune-fluorescence
Flow Cytometry: Cells in suspension are labeled with a fluorescent dye. The cells are then passed as single cell row and are illuminated with a laser beam. The amount of laser light that is scattered off the cells as they passes through the laser can be measured, which gives information concerning the size of the cells. In addition, the laser can excite the fluorochrome on the cells and the fluorescent light emitted by the cells can be measured by one or more detectors. Flow cytometry is commonly used in the clinical laboratory to identify and enumerate cells bearing a particular antigen. It can be used to measure the amount of DNA in cells, identify the stage of growth cycle and detect gene abnormality Comlement Fixation Test: Ag-Ab reaction in which complement is consumed. Ag mixed with test serum to be assayed for Ab (test system). Standard amount of complement is added. Erythrocytes coated with Abs is added (Indicator system). Amount of erythrocyte lysis is determined. Examples: Wasserman reaction to diagnose syphilis.
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Hypersensitivity (Allergy) Hypersensitivity is the term used when an immune response results in exaggerated or inappropriate reactions harmful to the host. The term "allergy" is often equated with hypersensitivity but more accurately should be limited to the IgE-mediated reactions "Type I: Immediate (Anaphylactic) Hypersensitivity. Hypersensitivity reactions can be subdivided into four main types. Types I, II, and III are antibody-mediated, whereas type IV is cell-mediated. Type I reactions are mediated by IgE, whereas types II and III are mediated by IgG. Type I (Immediate) hypersensitivity Allergen: is an antigen which induces Ig E in allergic patients. Allergens may be : 1)Ingestants as banana, fish, egg and milk 2) Inhalants: as pollens, dust, mite and spores 3) Contactants: as cosmetics 4)Injectants: drugs like penicillin, insect venom Pathogenesis:The process begins when an allergens induces the formation of IgE antibody, which binds firmly by its Fc portion to receptors on the surface of basophils and mast cells. Reexposure to the same allergens results in cross-linking of the cell-bound IgE, degranulation, and release of pharmacologically active mediators within minutes (immediate phase). Symptoms such as edema and erythema ("wheal and flare") and itching appear rapidly because these mediators, e.g., histamine, are preformed. The late phase of IgE-mediated inflammation occurs approximately 6 hours after exposure to the antigen and is due to other mediators, e.g., leukotrienes (SRS-A), that are synthesized after the cell degranulates. The increased IgE is the result of increased class switching to IgE in B cells caused by large amounts of IL-4 produced by Th-2 cells. Non-allergic individuals respond to the same antigen by producing IgG, which does not cause the release of mediators from mast cells and basophils. There is a genetic predisposition to immediate hypersensitivity reactions. There are two clinical types: Systemic form (anaphylaxis): common allergen is drug like penicillin or serum injection like anti-tetanic serum. Manifestations include: itching, respiratory distress due to, bronchospasm, urticaria, shock due to hypotension Local form (atopy):as bronchial asthma, allergic rhinitis, diarrhea, urticaria, and conjunctivitis. The tendency to develop atopy is inherited among families. Diagnosis: 1)History taking. 2)Skin test: Intradermal injection of a panel of different allergens. Erythema and wheal develop at the site of allergen to which the patient is allergic within 15-20 minutes. 3) Determination of serum IgE level (both total and specific By RAST): Treatment: 1) Avoid exposure to the allergen. 2) Drugs: Epinephrine : The main therapy as it reverse the effect of histamine. Antihistamines: Block effect of histamine on histamine receptors. Corticosteroids: prevent mediator synthesis Sodium chromoglycate which stabilizes the mast cells. - 36 -
Montelukast (Singular) which blocks the effect of leukotrienes. Omalizumab which is monoclonal antibody against IgE. It binds to IgE and prevents it from binding to mast cells. 3) Hyposensitization: Long term weekly administration of the allergen by subcutaneous injection of gradually increasing doses of the allergen extract. It acts by stimulating the production of IgG blocking antibody. Induces a switch from Th2 to Th1 response. Type II: Cytotoxic Hypersensitivity Cytotoxic hypersensitivity occurs when antibody directed at antigens of the cell membrane activates complement. This generates a membrane attack complex , which damages the cell membrane. The antibody (IgG or IgM) attaches to the antigen via its Fab region and acts as a bridge to complement via its Fc region. As a result, there is complement-mediated lysis Clinical examples: A. Mismatched blood transfusion. B. Hemolytic disease of the newborn. C. Autoimmune diseases like SLE, autoimmune hemolytic anemia and idiopathic thrombocytopenic purpura. D. Hyperacute graft rejection. E. Drug reaction:Penicillin may attach as haptens to RBCs and induce antibodies which are cytotoxic for the cell drug complex leading to hemolysis. N.B: Non cytotoxic type II hypersensitivity. Example: Graves' disease: A form of thyroiditis in which antibodies are produced against TSH receptors. These antibodies do not lead to destruction of the cells but stimulate the thyroid cells similar to TSH action Type III: Immune Complex Hypersensitivity Immune complex hypersensitivity occurs when antigen–antibody complexes induce an inflammatory response in tissues. Normally, immune complexes are promptly removed by the reticuloendothelial system, but occasionally they persist and are deposited in tissues, resulting in several disorders. In persistent microbial or viral infections, immune complexes may be deposited in organs, e.g., the kidneys, resulting in damage. In autoimmune disorders, "self" antigens may elicit antibodies that bind to organ antigens or deposit in organs as complexes, especially in joints (arthritis), kidneys (nephritis), or blood vessels (vasculitis). Wherever immune complexes are deposited, they activate the complement system. Polymorphonuclear cells are attracted to the site, and inflammation and tissue injury occur. Two typical type III hypersensitivity reactions are the arthus reaction and serum sickness. 1. Arthus Reaction: is the localized inflammation caused by the deposition of immune complexes at a localized site. Antigen, antibody, and complement are deposited in vessel walls; polymorphonuclear cell infiltration and intravascular clumping of platelets then occur. These reactions can lead to vascular occlusion and necrosis e.g.: an arthus reaction can occur at the site of insulin injection. 2. Serum Sickness: is a systemic inflammatory response to the presence of immune complexes deposited in many areas of the body. - 37 -
-After the injection of foreign serum, i.e., antitetanic serum (or, more commonly these days, exposure to certain drugs), the antigen is excreted slowly. During this time, antibody production starts. The simultaneous presence of antigen and antibody leads to the formation of immune complexes, which may circulate or be deposited at various sites. -Typical serum sickness results in fever, urticaria, arthralgia, lymphadenopathy, splenomegaly, and eosinophilia a few days to 2 weeks after injection of the foreign serum or drug. Diseases caused by type III hypersensitivity: 1. Post streptococcal glomerulonephritis. 2. Certain infections in which the antigens are persistently produced in the body such as viral hepatitis. 3. Farmer lung (hypersensitivity pneumonitis):It follows repeated inhalation of dust, mold spores or dried animal products. 4. Autoimmune diseases such as SLE and rheumatoid arthritis. Diagnosis:Detection of immune complexes: In tissues:By using fluorescein labeled anti-Ig or anti-C3 to detect the presence of Ig or C3 of the immune complexes fixed to the tissues. In serum by: Measurement of serum levels of C3 and C4. Their levels are usually decreased due to their exhaustion in the immune complexes. Precipitation of immune complexes by polyethylene glycol and estimation of the amount of Ig or C3 in the precipitate. Treatment: o Withdrawal of the allergenic drugs. o Suppression of the immune response by corticosteroids or cyclosporine. o Plasmapheresis to remove the circulating immune complexes Type IV: Delayed (Cell-Mediated) Hypersensitivity Delayed hypersensitivity is a function of cells (T lymphocytes), not antibody. It can be transferred by immunologically committed (sensitized) T cells, not by serum. The response is "delayed," i.e., it starts hours (or days) after contact with the antigen and often lasts for days. Clinically Important Delayed Hypersensitivity Reactions 1. Contact Hypersensitivity -This manifestation of cell-mediated hypersensitivity occurs after sensitization with simple chemicals (e.g., nickel, formaldehyde), plant materials (e.g., poison ivy, poison oak), topically applied drugs (e.g., sulfonamides, neomycin), some cosmetics, soaps, and other substances. -In all cases, the small molecules acting as haptens enter the skin, attach to body proteins, and become complete antigens -Cell-mediated hypersensitivity is induced, particularly in the skin. Upon a later skin contact with the offending agent, the sensitized person develops contact dermatitis characterized by erythema, itching, vesicles, eczema, or necrosis of skin within 12 to 48 hours caused by the attack of cytotoxic T cells. -Patch testing on a small area of skin can sometimes identify the offending antigen. Subsequent avoidance of the material will prevent recurrences. - 38 -
2. Tuberculin skin test (TST): 4) Intradermal injection of purified protein derivative (PPD) of tubercle bacilli in a sensitized person, i.e. previously exposed to tuberculosis results in induration and erythema that develop 48-72 hours after injection.Induration occurs as a result of cellular infiltration of the injected site with macrophages andCD4 lymphocytes. 3. Tuberculin like reactions: 5) Lepromin test for diagnosis of leprosy. 6) Frei test for diagnosis of lymphogranulomavenereum. 7) Casoni test for diagnosis of hydatid disease. 4. Granulomatous diseases: 8) Delayed hypersensitivity is responsible for granuloma formation in chronic diseases caused by intracellular infections such as leprosy, tuberculosis. 5. Graft rejection. Tolerance Tolerance is specific immunologic unresponsiveness, i.e., an immune response to a certain antigen (or epitope) does not occur, although the immune system is otherwise functioning normally. Autotolerance: unresponsiveness to self antigens usually acquired during embryonic life. Induced tolerance: Tolerance which is acquired later in life to specific foreign antigen. Immunosuppression : is loss of immune response to all antigens. Although both B cells and T cells participate in tolerance, it is T-cell tolerance that plays the primary role. T-Cell Tolerance Tolerance to self antigens acquired within the thymus is called central tolerance, whereas tolerance acquired outside the thymus is called peripheral tolerance. Central tolerance (also called clonal deletion) :This process involves the killing of T cells ("negative selection") that react against antigens (primarily self MHC proteins) present in the fetus at that time. Exogenous substances injected into the fetus early in development are treated as self. The self-reactive cells die by a process of programmed cell death called apoptosis. A transcriptional regulator called the "autoimmune regulator" (AIRE) is needed for central tolerance. Mutations in the gene encoding the AIRE protein result in the development of an autoimmune disease called autoimmune polyendocrinopathy. Peripheral tolerance: is necessary because some antigens are not expressed in the thymus and therefore some self-reactive T cells are not killed in the thymus. It occurs by: a) Clonal anergy is the term used to describe self-reactive T cells that are not activated because proper co-stimulation does not occur. b) Clonal ignorance refers to self-reactive T cells that ignore self antigens. These selfreactive T cells are either kept ignorant by physical separation from the target antigens, e.g., the blood–brain barrier, or ignore self antigens because the antigens are present in such small amounts. c) Some T cells are suppressed by regulatory T cells producing inhibitory cytokines. - 39 -
B-Cell Tolerance B cells also become tolerant to self by two mechanisms: (1) clonal deletion, probably while the B-cell precursors are in the bone marrow and (2) clonal anergy of B cells in the periphery. However, tolerance in B cells is less complete than in T cells, an observation supported by the finding that most autoimmune diseases are mediated by antibodies. Factors influencing the induction of tolerance: 1. Immunologic maturity of the host: Tolerance is readily induced in infants as they are immunologically immature. 2. The structure of the antigen: TheSimple molecules induce tolerance more than complex molecule. 3. The dose of the antigen: Very low or very high doses of an antigen may result in immunologic anergy. 4. Persistence of the antigen: persistence of an antigen in small dose is required. 5. Immunosuppressive drugs enhance tolerance. Clinical importance of tolerance: 1. Organ transplantation: Induction of tolerance can be aline of treatment to to prevent graft rejection. 2. Tumor development: Tolerance to tumor antigen results in growth of the tumor. 3. Autoimmune diseases: loss of autotolerance leads to autoimmune diseases. Auto-Immunity Autoimmune disease: is disease caused due to loss of auto-tolerance (self antigen is recognizes as non self antigen and stimulate the immune system). Auto-immune disease is classified as: Organ specific diseases: immune response against specific tissue such as autoimmune hemolytic anemia and autoimmune thyroiditis. Systemic diseases :which show wide spread distribution of lesions in the connective tissues and vascular system as rheumatoid arthritis and SLE Aetiology: Genetic predisposition: Many autoimmune diseases exhibit a marked familial incidence and there is a strong association of some diseases with certain human leukocyte antigen (HLA) specificities, especially the class II genes. Rheumatoid arthritis occurs predominantly in individuals of the HLA-DR4. Ankylosing spondylitis is 100 times more likely to occur in people of HLA-B27. Hormonal Factors: 90% of autoimmune diseases occur in women. Molecular Mimicry (Cross reactivity):The environmental trigger(various bacteria and viruses) resembles a component of the body sufficiently that an immune attack is directed against the cross-reacting body component. e.g Antibodies against certain M proteins of S. pyogenes cross-react with cardiac myosin, leading to rheumatic fever. Alteration of Normal Proteins: Drugs can bind to normal proteins and make them immunogenic. Procainamide-induced systemic lupus erythematosus is an example of this mechanism. Release of Sequestered Antigens: Certain tissues, e.g., sperm, central nervous system, and the lens and uveal tract of the eye, are sequestered so that their antigens - 40 -
are not exposed to the immune system. These are known as immunologically privileged sites When such antigens enter the circulation accidentally, e.g., after damage, they elicit both humoral and cellular responses, producing aspermatogenesis, encephalitis, or endophthalmitis, respectively.. Epitope Spreading: Epitope spreading is the term used to describe the new exposure of sequestered autoantigens as a result of damage to cells caused by viral infection leading to stimulate autoreactive T cells, and autoimmune disease results. Failure of Regulatory T Cells: leads to over stimulation of immune system. Mechanisms of tissue damage in autoimmune diseases: Type II hypersensitivity (cytotoxic antibody mediated mechanism). Examples are: Autoimmune hemolytic anemia, Idiopathic thrombocytopenic purpura (ITP). Grave's disease (the autoantibodies against TSH receptors will stimulate rather than destroy the thyroid gland). Type III hypersensitivity (immune complex mediated mechanism). Examples are: Rheumatoid arthritis, and SLE. Type IV hypersensitivity (cell mediated mechanism). Examples are: Ulcerative colitis Diagnosis of autoimmunity: Elevated serum immunoglobulins. Autoantibodies can be detected in the serum, e.g. anti-nuclear Ab (ANA in SLE), anti-smooth muscles, anti-mitochondrial antibodies. Testing for antibodies specific to the particular antigen involved in organ specific diseases, e.g. anti-thyroid antibodies in Haishomoto's thyroiditis. Complement level may be decreased. Detection of immune complexes in serum or tissue biopsies. Treatment of autoimmunity: Anti-inflammatory nonsteroidal drugs as aspirin. Corticosteroids. Immunosuppressive cytotoxic drugs such as methotrexate. Plasmapheresis (plasma exchange therapy): Patient’s plasma is removed and replaced with normal serum albumin or fresh frozen plasma to remove the causative antibodies or immune complexes such as Guillain-Barré syndrome and myasthenia gravis. Specific measures to the special condition: as splenectomy in autoimmune hemolytic anemia and ITP. Antibody to TNF and soluble receptor for TNF that acts as a decoy. Both infliximab and adalimumab (antibody to TNF) as well as etanercept (TNF receptor) have been shown to ameliorate the joint inflammation of rheumatoid arthritis and the skin lesions of psoriasis. However, these anti-TNF therapies increase the risk of infections, such as activating latent tuberculosis and skin and soft tissue infections caused by pyogenic bacteria. TUMOR IMMUNOLOGY MALIGNANT TRANSFORMATION The proliferation of normal cells is carefully regulated. However, such cells when exposed to chemical carcinogens, irradiation and certain viruses may undergo - 41 -
mutations leading to their transformation into cells that are capable of uncontrolled growth, producing a tumor or neoplasm. TUMOR ASSOCIATED ANTIGENS A number of alterations in gene expression occur in cells during tumorigenesis. Tumorigenesis may lead to expression of new antigens (neoantigens) or alteration in existing antigens that are found on normal cells. There are 2 main types of tumor antigens: Tumor-specific transplantation antigens (TSTA) which are unique to tumor cells and not expressed on normal cells. They are responsible for rejection of the tumor. Tumor associated transplantation antigens (TATA) that are expressed by tumor cells and normal cells. Onco-fetal antigens :Oncofetal antigens are proteins expressed at high levels in normal developing fetal tissue but not in normal adult tissue. However they may reappear in cancer cells. These include alpha-fetoprotein (AFP) in patients with hepatocellular carcinoma whereas carcinoembryonic antigen (CEA) is found in colon cancer. Antigens of oncogenic viruses: The products of oncogenic viruses are displayed on the cells infected by the oncogenic virus e.g Human papilloma virus (HPV) E6 and E7 proteins are associated with cervical carcinoma. Antigens on chemically-induced tumors: They are extremely heterogeneous in their antigenic characteristics. Thus, any two tumors induced by the same chemical, even in the same animal, rarely share common tumor specific antigens .These unique antigens on chemically-induced tumors are referred to as tumor specific transplantation antigens (TSTA). Products of mutated oncogenes and tumor suppressor genes: Examples: Human breast cancer cells express elevated levels of the oncogene encoded Her2/Neu protein. P53 is a tumor suppressor gene whose mutations is the most common seen in tumors and stimulates an immune response by T cytotoxic lymphocytes. Immune response mechanisms to destroy tumor : 1. Immune surveillance: It is a physiologic function of the immune system to recognize and destroy transformed cells before they grow into tumors and to kill tumors after they are formed. 2. Natural Killer (NK) cells: They kill many types of tumor cells especially those with reduced or lost class I MHC expression because class I molecules deliver inhibitory signals to NK cells. The tumoricidal capacity of NK cells is increased by cytokines as IL-2 to give the lymphokine activated killer (LAK) cells and also by IFN-γ. 3. Macrophages: They can kill tumor cells through the release of hydrogen peroxide, proteolytic enzymes and TNF, also by antibody dependent cellular cytotoxicity (ADCC). 4. CD8+ T cytotoxic cells: a. Recognition of tumor antigens associated with class I MHC molecules. b. Interaction with B7 costimulator. - 42 -
c. IL-2 secreted from Th cells mainly Th1 cells. 5. CD4+ T helper cells: Activated CD4 cells secrete the following lymphokines: IL-2 that activates Tc cells, NK cells and macrophages against tumor cells. IFN-γ that activates macrophages, NK cells and increases the expression of class I MHC molecules on tumor cells. Humoral immunity: Antibodies play a minor role against tumors. Antibodies against tumors bind the tumor antigens and kill tumor cells by: Activation of complement and cell lysis. Opsonization of tumor cells by macrophages. ADCC. Tumor Escape phenomena: Many malignant tumors possess mechanisms that enable them to evade or resist the host immune responses against the tumor Antigen loss variant Tumor cells: the tumor stops expression of antigens that stimulate the immune response. Absence or reduced level of class I MHC molecules on tumor cells, so they are not recognized by the Tc cells. Lake of co-stimulatory signals as B7 so they fail to stimulate Th cells. Tumor cells encountered by the immune system during early development may induce specific immunological tolerance. Blocking antibodies, which are non-complement fixing antitumor antibodies, bind to the tumor cell antigens and make them inaccessible to cell mediated destruction. Masking of the tumor by: Shedding of soluble tumor antigens which bind induced antibodies and form immune complexes to mask the antigen recognition sites of T cells. Tumor cells secrete sialic acid containing mucopolysaccarides which act as a physiological barrier and mask the tumor antigens. Some tumors produce immunosuppressive products as transforming growth factorβ (TGF-β) which has an inhibitory action on lymphocytes Other tumor cells express Fas ligand (FasL) which induces apoptotic death of lymphocytes. Tumour markers: These are tumor antigens or tumor products (enzymes or hormones) secreted in the blood of the patient and used to confirm the diagnosis and follow up during treatment. Examples: Alpha-feto proteins (AFP) in hepatoma. Carcino-embryonic antigen (CEA) in gastrointestinal tumors. Prostatic specific antigen (PSA) and prostatic acid phosphatase (PAP) in prostatic tumors. Human chorionic gonadotrophins (HCG) in choriocarcinoma. CA 125 in ovarian cancer. Immunotherapy Both active and passive means of stimulating the non-specific and specific immune systems have been employed, in some cases with significant success. - 43 -
Active Immunotherapy:In this, the host actively participates in mounting an immune response Specific activation is achieved by using vaccines: e.g. Hepatitis B vaccine and Human Papilloma virus (HPV) vaccine Non-specific activation is achieved by immunization with, for example, Bacillus Calmette-Guerin (BCG). Passive Immunotherapy:This involves transfer of preformed antibodies, immune cells and cytokines into the hosts. Antibodies: Antibodies against tumor antigens (e.g. Her2/Neu for treatment of breast cancer) Antibodies against CD20 expressed on non-Hodgkin’s B cell lymphoma. These antibodies bind to tumor antigens on the cell surface and activate complement to mediate tumor cell lysis and ADCC. Magic Bullet:Antibodies conjugated to toxins, and anti-cancer drugs have also been used. These enter the cells and kill them. lymphocytes:Lymphokine-activated killer (LAK) cells which are T and NK cells activated by IL-2. Cytokines: IL-2: Activates T cells/NK cells expressing IL-2 receptors. This is used in the treatment of renal cell carcinoma and melanoma IFN-alpha: This induces MHC expression on tumors and used in the treatment of hairy B cell leukemia. Transplantation Immunology Definition: Transfer of graft from donor to recipient. Major Histocompatibility Complex (MHC): MHC : are a group of genes that encodes HLA antigens. MHC genes are located on the short arm of chromosome 6 and encodes the MHC (HLA) antigens. Regions A, B and C code for class Ι MHCAntigens namely HLA-A, HLA-B and HLA-C, respectively. The D region comprises 3 loci DP, DQ and DR which code for class ΙΙ MHCAntigens namely HLA-DP, HLA-DQ and HLA-DR, respectively. Both class Ι and class ΙΙ genes are highly polymorphic. This means that multiple alleles exist for each gene. Expression of these genes is codominant, i.e., the proteins encoded by both the paternal and maternal genes are produced. A third region between Iand II regions codes for class ΙΙΙ MHC molecules. A. Human Leucocytes Antigens (HLA) or Major histocompatibility complex (MHC) molecules: o A membrane protein encoded in the MHC locus that is important for antigen recognition by T lymphocytes (MHC Restriction). o Two structurally distinct types of MHC molecules exist. 1. Class I MHC molecules: They are present on all nucleated cells. They are formed of heavy polypeptide chain (α) which is non-covalently linked to a β2 microglobulin light chain (encoded on chromosome15. - 44 -
They bind peptides derived from cytosolic proteins (endogenous Ag) and are recognized by CD8+ T cells. Typing by Lymphocytotoxicity assay. 2. Class II MHC molecules: They are molecules presented largely on professional APCS, macrophages and В lymphocytes. Each class ΙΙ molecule is formed of non-covalently linked 2 polypeptide chains (one α and one β). Both chains are encoded by chromosome 6. They bind peptides derived from exogenous Ag, and are recognized by CD4+ T cells. Typing byLymphocytotoxicity assay or Mixed lymphocyte reaction (MLR). 3. Class III MHC molecules:are some complement components C2, C4, factor B and 2 cytokines TNF-α and β and has no effect on transplantation. Biological significance of MHC antigens: 1. Organ transplantation and graft rejection. 2. MHC restriction: This requirement to recognize antigen in association with a "self" MHC protein is called MHC restriction. Tc (CD8) cells are activated when they recognize the foreign peptide in association with class Ι MHC antigen and Th(CD4) cells are activated when they recognize the foreign peptide in association with class ΙΙ MHC antigen. 3. Paternity testing. 4. Association with certain diseases as ankylosing spondylitis is associated with HLA-B27. I. Types of Grafts: Autograft: Graft transplanted from one individual to the same individual, permanently accepted. Isograft (syngeneic graft): Graft transplanted between two genetically identical individuals (twins), permanently accepted. Allograft (homograft):Graft transplanted between two genetically different individuals of the same species. This graft is rejected unless the recipient is given immunosuppressive drugs.The severity and rapidity of rejection vary depending on the degree of mismatching between the donor and recipient regarding mainly MHC molecules. Xenograft (heterograft):Graft transplanted between two different species, always rejected by immunocompetent recipients. Orthotopic graft: Graft placed into the normal location. Heterotopic graft: Graft placed into a different anatomical location. Immunoprivileged sites:There are certain locations in the body in which allografts are not rejected such as brain, anterior chamber of the eye, testis, and uterus. This is because: These sites lack good lymphatic drainage. Such sites express molecules such as Fas ligand that kills any immune cells that may come in contact with these tissues. Immunoprivileged tissues:Tssues that can be transplanted without matching and without being rejected as corneal graft. This is because avascularity of the - 45 -
graft bed prevents the corneal alloantigens from reaching regional lymphatic tissue. Graft versus host reaction (GVHR):This occurs when an immunocompetent tissue like bone marrow or thymus gland is transplanted into an immunosuppressed recipient.The grafted cells recognize the host tissues as being foreign and react against them. Manifestations are Fever, anemia, weight loss, skin rash, diarrhea, and hepato-splenomegaly. Types of Graft rejection: Hyper acute rejection: when the recipient has preformed antibodies. It occurs within hours due to vascular spasm, occlusion and failure of organ perfusion. The preformed antibodies are due to previous pregnancies or blood transfusion. Acute rejection: Occurs within 10-30 days after transplantation due to sensitized T lymphocytes and a cell mediated immune response. It is mainly due to T cell mediated response evidenced by infiltration of small lymphocytes and mononuclear cells which destroy the graft. Chronic rejection: Slow loss of tissue over months or years. It is probably a chronic delayed type hypersensitivity (DTH) reaction to alloantigens in the vessel wall, an antibody response or both. Mechanism of graft rejection: Allograft HLA are recognized by recipient T cell either directly or indirectly: Directly: Donor MHC presented on donor APCs to recipient T cell Indirectly: Donor MHC are picked up by recipient APC, processed and presented to recipient T cell. Tcare activated and destroy graft cells by performs, granzyme secretion and apoptosis. Th secrets cytokines that attract and activate macrophage, NK cells and polymorph infiltrating and destroying the graft (type IV hypersensitivity) B cell recognizes foreign on the graft and produce antibodies which bind to graftcells and destroy it by - complement activation - opsonisation by macrophages and neutrophils - Antibody dependantcyto-toxicity by macrophages, NK cells and neutrophils. Immune-complex depositionon the basement membrane with complement activation inducing platelet aggregation and microthrombi formation leading to ischemia (Type III hypersensitivity). II. Procedures to Enhance Graft Survival: i.
Methods to reduce the immunogenicity of the allograft: These methods include: 1. ABO blood group compatibility must be fulfilled. 2. Tissue typing (HLA typing): This is done to determine the closest MHC match between the donor and recipient. Matching of HLA-DR, HLA-B and HLA-A is the most important. It is done by: Lymphocytotoxicity test: for class Ι and ΙΙ MHC antigens. - 46 -
Mixed lymphocyte reaction (MLR): for class ΙΙ MHC antigens. By mixing lymphocytes from both donor and recipient. The more the lymphobloid reaction, the more the incombitability. Molecular typing: amplification of MHC genes by PCR followed by detection of the amplified product by probes or sequencing. 3. Cross matching: Test the recipient serum for the presence of performed antibodies against donor MHC antigens. ii. Immunosuppressive therapy to prevent or treat graft rejection:non specifically decrease the immune response. However, immune-suppressed patients are susceptible to viral infections and virus associated tumors. Immunosuppressive drugs include: Drugs that inhibit T lymphocytes: cyclosporine, tacrolimus (FK-506). They inhibit the release of IL2. 1. Antimitotic agents that kill proliferating T cells: as azathioprine and mycophenolate mofetil (MMF). 2. Antibodies that react with T cell surface structures: Examples: OKT3 and anti-IL-2 receptor antibody. OKT3 is monoclonal antibody against CD3 of T lymphocytes. It is now widely used in postoperative prophylaxis and to reverse acute rejection. Corticosteriods: They inhibit production of cytokines. iii. Induction of Tolerance to graft antigen: IMMUNODEFICIENCY Immunodeficiency can occur in any of the four major components of the immune system: (1) B cells (antibody), (2) T cells, (3) complement, and (4) phagocytes. The deficiencies can be either congenital or acquired. Clinically, recurrent or opportunistic infections are commonly seen. Recurrent infections with pyogenic bacteria, e.g., staphylococci, indicate a B-cell deficiency, whereas recurrent infections with certain fungi, viruses, or protozoa indicate a T-cell deficiency. 1. Secondary (Acquired) Immunodeficiency Disorders A. Infections: immunodeficiency is usually mild in these cases. o Viral, e.g. AIDS, measles, rubella, cytomegalovirus and infectious mononucleosis. o Bacterial, e.g. tuberculosis and leprosy. o Fungal, e.g. coccidioidomycosis. o Parasitic, e.g. schistosomiasis. B. Malignant diseases, e.g. leukaemias and myeloma. C. Autoimmune diseases, e.g. SLE. D. Splenectomy. E. Drugs, e.g. immunosuppressive drugs, cytotoxic drugs, antibiotics which may lead to agranulocytosis or which on prolonged use may lead disturbed flora and colonization with serious infections. F. Other conditions which are chronic debilitating or lead to loss proteins, e.g. uraemia, diabetes, nephrotic syndrome, burns, old age, protein calorie malnutrition, etc.
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2. Primary (Congenital) Immunodeficiency Disorders: Deficient Component and Name of Disease
Specific Deficiency
Molecular Defect
Clinical Features
X-linked (Bruton's)
Absence of B cells; very low Ig levels
Mutant tyrosine kinase
Recurrent bacterial infections, especially of respiratory tract, caused by pyogenic bacteria such as pneumococci
Selective IgA
Very low IgA levels
Failure of heavyRecurrent infections, especially chain gene switching of the sinuses and lung, caused by pyogenic bacteria
B cell
Transient hypogammaglobulinemia of infancy.
Delayed onset of IgG synthesis more than 6 Onset of infections after 6 months months
Commonvariable hypogammaglobulinemia
Poor antibody Onset in adulthood response to infections
T cell Thymic aplasia (DiGeorge's)
Absence of T cells
Defective development of pharyngeal pouches; not a genetic disease
Chronic mucocutaneous Deficient T-cell Unknown candidiasis response to Candida
Viral, fungal, and protozoal infections; tetany
Skin and mucous membrane infections with Candida
Combined B and T cell Severe combined immunodeficiency (SCID)
Ataxia telangectasia Wiskott Aldrich syndrom
Deficiency of Either defective IL-2 both B-cell and receptor, defective T-cell function recombinases, defective kinases, absence of class II MHC proteins, or ADA or PNP deficiency Autosomal recessive X-linked
- 48 -
Bacterial, viral, fungal, and protozoal infections
Combined ID + Ataxia+ Telangectiasis Combined ID + Eczema+
thrombocytopenia
Complement Hereditary angioedema
Deficiency of C1 protease inhibitor
Too much C3a, C4a, and C5a generated
C3b
Insufficient C3 Unknown
Pyogenic infections, especially with Sta. aureus
C6,7,8
Insufficient C6,7,8
Neisseria infections
Unknown
Edema, especially laryngeal edema
Phagocytes Chronic granulomatous disease
Chediak-Higashi syndrome
Leucocyte adhesion deficiency
Defective Deficient NADPH bactericidal oxidase activity activity because no oxidative burst
Pyogenic infections, especially with Sta. aureus and Aspergillus
The lysosome The neutrophil fuse with the phagosome but contains giant lysosome impaired release of their contents resulting in delayed killing Mutation of gene Defect in encoding migration and integrins(CD18) adhesion of phagocytic cells
A. Assessment of T cell competence: 1. Enumeration of T cells: a. Enumeration of total T cells by using monoclonal antibodies to CD3 conjugated with fluorescent dyes. The cells are counted by fluorescent microscope or by flow cytometry. b. Enumeration of T cell subsets by using monoclonal antibodies to CD4 or CD8 or to other T cell surface markers. 2. Evaluation of T cell function: a. Delayed hypersensitivity skin tests: To detect delayed hypersensitivity to a variety of antigens to which the patient is supposed to be exposed during his - 49 -
life, e.g. tuberculin (PPD), candidin, mumps antigens, streptokinase, streptodornase, trichophytin. Absence of reactions to several of these skin tests suggests impairment of cell mediated immunity. b. Assessment of cytokines production and cytokine receptors. c. Test for lymphocyte transformation: Normally functioning T cells transform to large blast cells with increase in DNA synthesis when exposed to the mitogens such as phytohaemagglutinin (PHA), concanavalin A (Con A) or specific antigens such as PPD. d. Lymph node biopsy to study T cell area. B. Assessment of B cell competence: 1. Enumeration of B cells: by immunofluorescent techniques using fluroescein labelled polyclonal anti-human immunogloblin or by flow cytometry. 2. Evaluation of B cell function: a. Determination of the level of gamma globulins by protein electrophoresis: A decrease or increase in gamma globulins can point to an abnormality in antibody production. b. Quantitation of total and different types of immunoglobulins by different methods, e.g. single radial immunodiffusion, ELISA. c. B cell transformation: Normally functioning B cell can be stimulated to transform to large blast cells by using B cell mitogens, e.g. lipopolysaccharides (LPS). d. Active immunization for stimulation of production of specific antibodies to killed vaccines or toxoids. e. Lymph node biopsy to study B cell areas. C. Assessment of phagocytic functions: a) Assessment of chemotaxis: Done by testing the ability of phagocytic cells to migrate through membranes towards a chemotactic substance. b) Assessment of ingestion or phagocytosis: Done by direct visual counting of ingested particles (yeast cells) using a microscope or by fluorescent beads using flow cytometry or fluorescent microscope. c) Assessment of the ingestion and/or intracellular killing: Normal phagocytic cells when stimulated, (e.g. by endotoxin) ingest the nitrobluetetrazolium (NBT) dye and reduce it (it changes from yellow to blue).Abnormalities in intracellular killing abilities are associated with decreased NBT reduction. D. Assessment of complement disorders: 1. Estimation of total haemolytic activity of complement: The assay is based on the ability of the intact complement system to rupture sheep RBCs coated with their antibody. 2. Estimation of individual complement components: by RIA, ELISA or radial immunodiffusion. Immunizations Immunizations: is the process by which an individual's immune system becomes fortified against an agent (known as the immunogen).Two artificial methods to make an individual immune to a disease • Active immunization-administration of a vaccine - 50 -
• Passive immunization- acquiring immunity through the transfer of antibodies formed by an immune individual or animal. Types of vaccines: 1)Attenuated (live) 2) Killed (inactivated) 3) Toxoid Attenuated Vaccines: Using live pathogens but have reduced virulence so they don’t cause disease but can induce immune system. • Attenuation is the process of reducing virulence • Viruses often attenuated by raising them in tissue culture cells for which they aren’t adapted until they lose the ability to produce disease • Bacteria can be made avirulent by culturing under unusual conditions or through genetic manipulation • Can result in mild infections but no disease • Contain replicating microbes that can stimulate a strong immune response due to the large number of antigen molecules • Vaccinated individuals can infect those around them, providing herd immunity Problems with Attenuated Vaccines: • Attenuated microbes may retain enough virulence to cause disease, especially in immunosuppressed individuals • Pregnant women should not receive live vaccines due to the risk of the modified pathogen crossing the placenta • Modified viruses may occasionally revert to wild type. Inactivated Vaccines: • Can be either whole agent vaccines produced with deactivated but whole microbes, or subunit vaccines • safer than live vaccines • Formaldehyde is commonly used to inactivate microbes Problems with Inactivated Vaccines: • Do not stimulate herd immunity • Whole agent vaccines may stimulate a inflammatory response due to non antigenic portions of the microbe • Antigenically weak since the microbes don’t reproduce and don’t provide many antigenic molecules to stimulate the immune response • Administration in high or multiple doses may be required • High and multiple vaccine doses may produce allergic reactions Toxoid Vaccines: • Chemically or thermally modified toxins used to stimulate active immunity • Useful for some bacterial diseases • Stimulate antibody-mediated immunity Subunit vaccines: Composed of the fragments of microorganism that can create an immune response. Examples: a) the subunit vaccine against Hepatitis B virus that is composed of only the surface proteins of the virus (previously extracted from the blood serum of chronically infected patients, but now produced by recombination of the viral genes into yeast). B)thevirus-like particle (VLP) vaccine against human papillomavirus (HPV) that is composed of the viral major capsid protein. C)The hemagglutinin and neuraminidase subunits of the influenza virus. - 51 -
Conjugate vaccine: Certain bacteria have polysaccharide outer coats that are poorly immunogenic. By linking these outer coats to proteins (e.g., toxins), the immune system can be led to recognize the polysaccharide as if it were a protein antigen. This approach is used in the Haemophilus influenzae type B vaccine. Experimental vaccines: A number of innovative vaccines are also in development and in use: Dendritic cell vaccines combine dendritic cells with antigens in order to present the antigens to the body's white blood cells, thus stimulating an immune reaction. Recombinant Vector – by combining the physiology of one micro-organism and the DNA of the other, immunity can be created against diseases that have complex infection processes DNA vaccination – It works by insertion (and expression, enhanced by the use of electroporation, triggering immune system recognition) of viral or bacterial DNA into human or animal cells. Some cells of the immune system that recognize the proteins expressed will mount an attack against these proteins and cells expressing them. Because these cells live for a very long time, if the pathogen that normally expresses these proteins is encountered at a later time, they will be attacked instantly by the immune system. One advantage of DNA vaccines is that they are very easy to produce and store. T-cell receptor peptide vaccines are under development for several diseases using models of Valley Fever, stomatitis, and atopic dermatitis. These peptides have been shown to modulate cytokine production and improve cell mediated immunity. Valence of vaccines Vaccines may be monovalent (also called univalent) or multivalent (also called polyvalent). A monovalent vaccine is designed to immunize against a single antigen or single microorganism. A multivalent or polyvalent vaccine is designed to immunize against two or more strains of the same microorganism, or against two or more microorganisms. In certain cases a monovalent vaccine may be preferable for rapidly developing a strong immune response. Heterotypic vaccine Also known as Heterologous or "Jennerian" vaccines these are vaccines that are pathogens of other animals that either do not cause disease or cause mild disease in the organism being treated. The classic example is Jenner's use of cowpox to protect against smallpox. A current example is the use of BCG vaccine made from Mycobacterium bovis to protect against human tuberculosis. Contra -indications to vaccination:A)acutely ill patients B)Avoid giving live vaccines to pregnant women C) in the first trimester of pregnancy D) ive vaccines to immunosupressed patients or pregnants. Side effects: 1)May cause pain at the injection site 2)can cause general malaise or fever high enough to induce seizures 3)Anaphylactic shock Is an allergic reaction that may develop to a component of the vaccine 4)Residual virulence: Attenuated viruses occasionally cause disease in healthy children or adults 5)Allegations that certain vaccines against childhood diseases cause or trigger autism, diabetes, and asthma but research has not substantiated these allegations
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Active immunization schedule in Egypt Sabin (OPV) Vaccines
BCG
DPT
Salk
-0.1 ml ID
-0.5ml deepSC/IM
with DPT
-3 drops on tounge
Age
DT
HBV 0.5ml IM
Measles MMR -0.5ml SC
-0.5 ml + vit A 200k IU
Meningococcal -0.5ml deep SC
At 0 dose birth 3d
a. Infants & preschool children
-no tuberculin test
2m 1st dose
1st dose
1st dose
4m 2nd dose
2nd dose
1st dos e
2nd dose
6m 3rd dose
3rd dose
2nd dos e
3rd dose
9m Booster dose
1 dose
18m Booster dose 5-6y Booster dose
Booster dose Revaccination
1 dose Booster dose
8y
1 dose
Booster dose
b. School
-for susceptibl e contact
children Any age
Booster dose
At risk, when cases appear
-for injury c. int traveler Yellow fever vaccine - travel from/to endemic area ande receptive area [aedesegypti as arthropod present but no reservoir] d. occupational groups
-eg health care provider meningococcal meningitis , influenza, TB, HBV
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Passive vaccination: o Administration of preformed antibodies o Used when protection against a recent infection or an ongoing disease o The serum from human or animal donors that have been infected or immunized o Serum used for passive immunizations is called antiserum, examples: Antitetanic serum Rabies immunoglobulin Diphtheria antiserum Botulism antiserum Varicella Zoster
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Gram positive organisms
GRAM POSITIVE COCCI Catalase
-ve
+ve
Streptococcus (Chains)
Staphylococcus (Clusters) Coagulase
+ve S. aureus
-ve
Hemolysis
BETA: Bacitracin
+ve
S. pyogenes(group A)
CAMP/Hippurate +ve S.agalactiae (group B) S. epidermidis β hemolytic nonhemolytic mannitol mannitol ALPHA: Optochin /Bile Solubility +ve S. pneumoniae yellow white Note: S. viridans 6.5% NaCl +ve GAMMA: Bile Esculin +ve Group D is Is alpha Enterococcus hemolytic and negative for all the 6.5% NaCl -ve Non Enterococcus tests below (*can also be alpha hemolytic)
Gram-Positive Cocci Staphylococcus aureus Gram-positive cocci in clusters. Caagulase positive, catalase positive. Habitat and Transmission: Main habitat is human nose; also found on human skin. Transmission is via the hands. Virulence factors and Pathogenesis: Staphylococci -Abscess is the most common lesion; it is localized lesion due to coagulase production. Coagulase converts fibrinogen into fibrin. -Three exotoxins are also made. 1) Toxic shock syndrome toxin is a superantigen and causes toxic shock syndrome by stimulating many helper T cells to release large amounts of lymphokines, especially IL-2 and TNF. 2)Enterotoxin, which causes food poisoning, is also a superantigen. 3)Scalded skin syndrome toxin causes Scalded skin syndrome with peeling of skin in children. -Protein A is an important virulence factor because it binds to the heavy chain of IgG and prevents the activation of complement. -Staph aureus produce endopigment, haemolysin, catalase and leukocidin. Diseases (1)Local: Abscess, wound infection (2) general: endocarditis, hospital-acquired pneumonia, Sepsis. (3) Toxic diseases: food poisoning, toxic shock syndrome, and Scalded skin syndrome. Staph food poisoning: It is due to eating food containing staph enterotoxin. Incubation period is short 4-6 hours. Vomiting, diarrhea, colic but no fever. The staph enterotoxins are superantigens. Toxic shock syndrome: usually occur in young menstruating female using contaminated tampons. It is due to production of toxic shock syndrome toxin. It acts - 55 -
as superantigen stimulating many helper T cells to release large amounts of cytokines, especially IL-2 and TNF. Manifestations: fever, vomiting, rash, diarrhea, splenomegaly, hypotension and shock. Laboratory Diagnosis -Gram-stained smear shows Gram-positive cocci in clusters. -Culture: Yellow or golden yellow colonies on nutrient agar, complete haemolysis on blood agar, yellow colonies on Selective Mannitol salt agar medium Biochemical Reactions: Staph aureus is coagulase-positive, Catalase-positive. -Phage typing for epidemiological tracing the source of infection. Treatment Penicillin G for sensitive isolates; β-lactamase–resistant penicillins such as nafcillin for resistant isolates; vancomycin for isolates resistant to nafcillin. About 85% are resistant to penicillin G. Plasmid-encoded β-lactamase mediates most resistance. Resistance to nafcillin is caused by changes in binding proteins. Some isolates are tolerant to penicillin. Rare vancomycin-resistant strains have emerged. Methicillin Resistant Staphaureus (MRSA): are Staphaureus that are resistant to penicillinase resistant penicillis as oxacillin, fluxacillin..etc. The resistance due to change in the penicillin binding proteins (PBP) which are the target of beta lactamase. It is classified into: 1) Community acquired MRSA 2) Hospital acquired MRSA Hospital acquired MRSA is more resistant to antibiotics and is an important cause of hospital acquired infections. Treatment by: vancomycin, linezolid, daptomycin. Staphylococcus epidermidis Gram-positive cocci in clusters. Coagulase-negative. Catalase-positive. Habitat and Transmission: Normal flora of the human skin and mucous membranes. Diseases: -It is a low-virulence organism that causes disease primarily in immunecompromised patients and in those with implants. -Endocarditis on prosthetic heart valves, prosthetic hip infection, intravascular catheter infection, cerebrospinal fluid shunt infection, neonatal sepsis. Laboratory Diagnosis : Gram-stained smear and culture. Whitish, nonhemolytic colonies on blood agar. It is coagulase-negative. S. epidermidis is sensitive to novobiocin, whereas the other coagulase-negative staphylococcus, S. saprophyticus, is resistant. Serologic tests are not useful. Treatment: Vancomycin plus either rifampin or an aminoglycoside. It produces βlactamases and is resistant to many antibiotics. Staphylococcus saprophyticus Coagulase-negative. Resistant to novobiocin in contrast to S. epidermidis, which is sensitive. Causes community-acquired urinary tract infections in young women (but Escherichia coli is a much more common cause). Streptococci Gram-positive cocci arranged in chains. It is classified according to: 1)Haemolysis on blood agar: α (Incomplete) haemolysis: greenish colour surrounds colonies as pneumococci and streptococcus viridians. - 56 -
β (Complete) haemolysis: clearance around the colonies as streptococcus pyogenes. γ ( No) haemolysis: some strains of enterococci. 2) Cell wall carbohydrate antigen (Lancefield classification): groups A, B, C . Group A : is Streptococcus pyogenes & Group B is Streptococcus agalactiae Streptococcus pyogenes (Group A Streptococcus) Characteristics Gram-positive cocci in chains. β-hemolytic. Catalase-negative. Bacitracin-sensitive. β-hemolytic streptococci. Virulence and Pathogenesis Habitat is the human throat and skin. Transmission is via respiratory droplets. For suppurative infections, hyaluronidase ("spreading factor") mediates subcutaneous spread seen in cellulitis; other enzymes: streptokinase and streptodornase help to spread the infection. erythrogenic toxin (a superantigen) causes the rash of scarlet fever. M protein impedes phagocytosis. Two streptolysin toxin, O antigenic and S cause haemolysis on blood agar. Pyrogenic exotoxins A, B, and C are directly toxic and tend to be produced by strains causing necrotizing fasciitis. They are also super antigens. Clinically: A)Suppurative (pus-producing) diseases, Local as Tonsillitis, pharyngitis, scarlet fever, impetigo, erysipelas, cellulitis or necrotizing fascitis or Generalized disease as puerperal sepsis, endocarditis, pneumonia. B) Non-suppurative (immunologic) diseases, Rrheumatic fever is caused by immunologic cross-reaction between bacterial antigen and human heart and joint tissue (i.e., antibody against streptococcal M protein reacts with myosin in cardiac muscle). Clinically: arthritis, carditis, chorea, erythema …etc Acute glomerulonephritis is caused by immune complexes formed between streptococcal antigens and antibody to those antigens. The immune complexes are trapped by glomeruli, complement is activated, neutrophils are attracted to the site by C5a, and proteases produced by neutrophils damage glomeruli. Clinically: smoky urine, oliguria, hypertension and RBC cast in urine. -Tonsillitis: The main lesion for Streptococcus pyogenes. Characterized by fever, sore throat, malaise …etc. – Scarlet fever: sore throat, fever, maculopapular rash due to production of erythrogenic toxin. –puerperal sepsis: infection of the uterus after labour or septic abortion; fever, septicaemia and septic shock. Laboratory Diagnosis: The diagnosis of suppurative infections, e.g., cellulitis, differs from immunologic diseases, e.g., rheumatic fever. 1)Gram-stained smear: gram positive cocci in chains 2)Culture: β-hemolytic colonies on blood agar. If isolate is Streptococci sensitive to bacitracin, it is identified as Streptococcus pyogenes. 3)Antigen Detection: Rapid ELISA to detect group A streptococcal antigens in throat swabs. 4) blood culture for endocarditis and puerperal sepsis. -If rheumatic fever is suspected, patient's antistreptolysin O (ASO) antibody titer is tested to determine whether previous exposure to S. pyogenes has occurred. ESR - 57 -
and CRP are positive. -If acute glomerulonephritis is suspected, antibody to streptococcal DNase B is used as evidence of a previous skin infection by S. pyogenes. Treatment: Penicillin G (no significant resistance) or macrolide Prevention: Penicillin is used in patients with rheumatic fever to prevent recurrent S. pyogenes pharyngitis. This prevents additional damage to heart valves. Streptococcus agalactiae (Group B Streptococcus) Pathogenesis: Gram-positive cocci in chains. β-hemolytic. Catalase-negative. Bacitracin-resistant. Main habitat is the human vagina. Transmission occurs during birth. It is pyogenic organism. Predisposing factors to neonatal infection include rupture of membranes more than 18 hours before delivery, labor prior to 37 weeks (infant is premature), absence of maternal antibody, and heavy colonization of the genital tract by the organism. It causes Neonatal meningitis and sepsis. It also can cause female endometritis. Laboratory Diagnosis Gram-stained smear and culture. β-hemolytic (narrow zone) colonies on blood agar that are resistant to bacitracin. It hydrolyzes hippurate and are CAMP test– positive. Treatment:Penicillin G or erythromycin . Prevention: IV Ampicillin should be given to mothers if prolonged rupture of membranes occurs, if mother has a fever, or if the neonate is premature. Enterococcus faecalis Characteristics: Gram-positive cocci single or in chains. Catalase-negative. Pathogenesis:Habitat is the human colon; urethra and female genital tract can be colonized. May enter blood stream during gastrointestinal (GI) or genitourinary tract procedures as endoscopy or operations and cause endocarditis. Main diseases are:1)Urinary tract and biliary tract infections are most frequent. 2)Endocarditis life-threatening. Laboratory Diagnosis Gram-stained smear and culture on blood agar. α, β-, γ hemolytic colonies on blood agar. Grows in 6.5% NaCl and hydrolyzes esculin in the presence of 40% bile. Treatment Penicillin or vancomycin plus an aminoglycoside such as gentamicin is bactericidal. Organism is resistant to either drug given individually, but given together they have a synergistic effect. Aminoglycoside alone is ineffective because it cannot penetrate. Penicillin or vancomycin weakens the cell wall, allowing the aminoglycoside to penetrate. Vancomycin-resistant enterococci (VRE) are important causes of nosocomial (hospital-acquired) infections. Linezolid can be used to treat VRE. Prevention: Penicillin and gentamicin should be given to patients with damaged heart valves prior to intestinal or urinary tract procedures. Streptococcus pneumoniae (Pneumococcus) Characteristics: Gram positive "lancet-shaped" cocci in pairs (diplococci) . α-hemolytic. Catalase-negative. Sensitive to bile and optochin in contrast to viridans - 58 -
streptococci, which are resistant. It has prominent polysaccharide capsule. 85 serotypes based on antigenicity of polysaccharide capsule. Virulence and Pathogenesis: 1. Habitat is the human upper respiratory tract. Transmission is via respiratory droplets. 2. Induces inflammatory response. Its Pneumococci polysaccharide capsule resists phagocytosis. Antipolysaccharide antibody opsonizes the organism and provides type-specific immunity. 3. IgA protease degrades secretory IgA on respiratory mucosa, allowing colonization. 4. Viral respiratory infection predisposes to pneumococcal pneumonia by damaging mucociliary elevator; splenectomy predisposes to sepsis. Skull fracture with spinal fluid leakage from nose predisposes to meningitis. 5. The most common diseases are lobar pneumonia and meningitis in adults and otitis media and sinusitis in children. Laboratory Diagnosis 1. Gram-stained smear: Gram-positive "lancet-shaped" cocci in pairs (diplococci) or short chains 2. Culture. α-hemolytic colonies on blood agar. 3. Growth inhibited by bile and optochin. 4. Quellung reaction occurs (swelling of capsule with type-specific antiserum). 5. Latex agglutination test for capsular antigen in spinal fluid can be diagnostic. Treatment: Ceftriaxone (1St choice) or macrolide or Penicillin G. Prevention: Two vaccines are available. The one used in adults contains capsular polysaccharide of the 23 serotypes that cause bacteremia most frequently. The other, which is used primarily in children under the age of 2 years, contains capsular polysaccharide of 7 serotypes coupled to carrier protein (diphtheria toxoid). Oral penicillin is used in immunocompromised children. Viridans Group Streptococci (e.g., S. sanguis, S. mutans) Diseases: Endocarditis is the most important. Also brain abscess, especially in mixed infections with mouth anaerobes. S. mutans implicated in dental caries. Characteristics:Gram-positive cocci in short chains. α-hemolytic. Catalase-negative. Resistant to bile and optochin in contrast to pneumococci, which are sensitive. Pathogenesis: Habitat is the human oropharynx. Organism enters bloodstream during dental procedures. Bacteremia from dental procedures spreads organism to damaged heart valves. Laboratory Diagnosis Gram-stained smear Gram-positive cocci in short chains. Culture:α-hemolytic colonies on blood agar. Growth not inhibited by bile or optochin, in contrast to pneumococci. Treatment: vancomycin or Penicillin G with an aminoglycoside. Prevention:Ampicillin to prevent endocarditis in patients with damaged or prosthetic heart valves who undergo dental procedures.
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Gram-Negative Cocci Neisseria meningitidis (Meningococcus) It causes Meningitis and meningococcemia. The most severe form of meningococcemia is Waterhouse-Friderichsen syndrome, which is characterized by high fever, shock, widespread purpura, disseminated intravascular coagulation, thrombocytopenia, and adrenal insufficiency. Bacteremia can result in the seeding of Meningococci intra & extracellularly many organs, especially the meninges. The symptoms of meningococcal meningitis are fever, headache, stiff neck, and an increased level of PMNs in spinal fluid. Characteristics: Gram-negative capsulated "kidney-bean shaped" diplococci intra and extracellularly. Oxidase-positive. Large polysaccharide capsule. It is one of the three classic encapsulated pyogenic bacteria causing meningitis (Streptococcus pneumoniae and Haemophilus influenzae are the other two). Virulence and pathogenesis: Habitat is the human upper respiratory tract; transmission is via respiratory droplets. After colonizing the upper respiratory tract, the organism reaches the meanings via the bloodstream. 1)Endotoxin in cell wall causes symptoms of septic shock seen in meningococcemia. 2)IgA protease produced destroy secretory Ig A. 3)Capsule is antiphagocytic. 4)Meningococci is divided into 13 serogroups.; most important groups A, B, C, Y, W135. Group A and C cause epidemics while B causes sporadic cases. 5)Deficiency in late complement components predisposes to recurrent meningococcal infections. Laboratory Diagnosis 1. Specimen: CSF by lumber puncture.CSF is under Tension and turbid with large number of pus cells >2000/cmm. Chemically protein elevated and reduced glucose. 2. Gram-stained smear: Gram-negative capsulated "kidney-bean shaped" diplococci intra and extra-cellularly. 3. Culture on chocolate agar or Thayer-Martin medium in 5-10% CO2. 4. Biochemically: Oxidase-positive colonies, Ferments glucose and maltose with acid production. 5. Blood culture usually positive. 6. Serology to detect capsular Ag in CSF. Treatment: Penicillin G (no significant resistance). Prevention: Vaccine contains capsular polysaccharide of strains A, C, Y, and W135. One form of the vaccine contains the polysaccharides coupled to a carrier protein (diphtheria toxoid) and one contains only the polysaccharides. - chemo prophylaxis: Rifampin or ciprofloxacin given to close contacts to decrease oropharyngeal carriage. Neisseria gonorrhoeae (Gonococcus) Disease: Gonorrhea. Also neonatal conjunctivitis (ophthalmia neonatorum) and pelvic inflammatory disease. Characteristics: Gram-negative "kidney-bean shaped" diplococci. Oxidase+ve. Pathogenesis: Habitat is the human genital tract. Transmission in adults is by - 60 -
sexual contact. Transmission to neonates is during birth. 1) Organism adheres to mucous membranes by pili, invades and causes inflammation. 2)Endotoxin present but weaker than that of meningococcus, so less severe disease when bacteremia occurs. 3)IgA protease and pili are virulence factors. 4) Repeated infection can occur due to antigenic variation of pili -superficial nature of infection. - IgA protease 1. Gonorrhea in men is characterized primarily by urethritis accompanied by dysuria and a purulent discharge. Epididymitis can occur. 2. In women, infection is located primarily in the endocervix, causing a purulent vaginal discharge and intermenstrual bleeding (cervicitis). The most frequent complication in women is an ascending infection of the uterine tubes (salpingitis, PID), which can result in sterility or ectopic pregnancy as a result of scarring of the tubes. Disseminated gonococcal infections (DGI) commonly manifest as arthritis, tenosynovitis, or pustules in the skin. Laboratory Diagnosis: Specimen: Male; urethral discharge. Females; urthral and cervical discharge. 1. Acute gonorrhea in male: Gram-stained smear of the urethral discharge is diagnostic if it shows Gram-negative "kidney-bean" diplococci intra and extra cellularly. In females the smear is not diagnostic because of the presence of commensal Neisseria and low number of pathogen. 2. Culture on chocolate agar or Thayer-Martin medium in 5-10% CO2. 3. Biochemically: Oxidase-positive colonies, Ferments glucose with acid production. 4. Florescent antibody staining. 5. PCR. Treatment: Ceftriaxone for uncomplicated cases. Tetracycline added for urethritis caused by Chlamydia trachomatis. Prevention: Condoms offer protection. Trace contacts and treat to interrupt transmission. Treat eyes of newborns with erythromycin ointment or silver nitrate to prevent conjunctivitis.
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Gram-Positive Bacilli (Rods) Non Spore Forming: (1)Corynebacterium diphtheria (2) Listeria monocytogenes Spore forming: 1. Aerobic----Bacillus spp 2. Anaerobic: Clostridium spp. Bacillus anthracis Aerobic, gram-positive, spore-forming rods with Bacillus anthracis square ends, frequently found in chains. Non motile. Positive McFadean Reaction: staining by polychrome methylene blue, it appear blue with pink capsule. Pathogenesis 1. Habitat is soil. Transmission is by contact with infected animals or inhalation of spores from animal hair and wool. Gastrointestinal anthrax occurs when contaminated meat is ingested. 2. Anthrax toxin consists of three proteins: edema factor, which is an adenylatecyclase; lethal factor, which kills cells and protective antigen, which mediates the entry of the other two components into the cell. 3. It has capsule composed of poly-D-glutamate. The capsule is antiphagocytic. 4. B. anthracis causes anthrax, which is common in animals but rare in humans. Human disease occurs in three main forms: cutaneous, pulmonary (inhalation), and gastrointestinal. 5. The typical lesion (malignant pustule) of cutaneous anthrax is a painless ulcer with a black eschar (crust, scab). Local edema is striking. Untreated cases progress to bacteremia and death. 6. Pulmonary (inhalation) anthrax, also known as "woolsorter's disease," begins with nonspecific respiratory tract symptoms, especially a dry cough and substernal pressure. This rapidly progresses tofatalhemorrhagic mediastinitis, bloody pleural effusions, septic shock, and death. The symptoms of gastrointestinal anthrax include vomiting, abdominal pain, and bloody diarrhea. Laboratory Diagnosis Gram-stained smear shows gram-positive, spore-forming rods with square ends, in chains. Positive McFadean Reaction. Aerobic culture: Non hemolytic colonies form on blood agar aerobically. Rise in antibody titer in indirect hemagglutination test is diagnostic. Treatment: Ciprofloxacin is the drug of choice or doxycycline. Prevention:-Vaccine consisting of protective antigen is given to individuals in highrisk occupations. -Incinerating animals that die of anthrax, rather than burying them, will prevent the soil from becoming contaminated with spores. Bacillus cereus Disease:Food poisoning. Pathogenesis:Habitat is grains, such as rice. Spores survive boiling during preparation of rice, and then germinate when rice held at warm temperature. Clinically :1) emetic type: with short incubation period 5 hours due to enterotoin act like superantigen. Usually associated with rice dish. - 62 -
2) Diarrhial type: with longer incubation period 24 hours due to enterotoin act like cholera toxin, i.e., cyclic AMP is increased within enterocytes. Usually associated with meat dish. Laboratory Diagnosis:Not done. -Treatment:Symptomatic only. Clostridium tetani Disease: Tetanus. Anaerobic, gram-positive, spore-forming rods. Spore is at one end ("terminal spore") so organism looks like a "tennis racket or drum sick appearance." Pathogenesis:Habitat is the soil. Organism enters through traumatic breaks in the skin. It can enter through a contaminated umbilicus or circumcision wound causing (neonatal tetanus). Spores germinate under anaerobic conditions in the wound. Organism produces exotoxin (tetanospasmin), which blocks release of inhibitory neurotransmitters (glycine and GABA) from spinal neurons. Excitatory neurons are unopposed, and extreme muscle spasm (tetanus, spastic paralysis) results. Trismus or Lock-jaw, hyperextension of the spine(opisthotonus) and facial spasm "risussardonicus" are two examples of the muscle spasms. Clostridium tetani Laboratory Diagnosis Primarily a clinical diagnosis. Organism is rarely isolated by anaerobic cultivation on anaerobic media or on blood agar with anaerobic incubation. Gram stain: grampositive with terminal spore (drum stick appearance). Treatment: Anti titanic serum or Hyperimmune human globulin to neutralize toxin. Also penicillin G and spasmolytic drugs (e.g., Valium). Wound Debridement. Prevention: Active Immunization:Toxoid vaccine (toxoid is formaldehyde-treated toxin). Usually given to children in combination with diphtheria toxoid and pertussis vaccine (DTP) at age of 2, 4, 6 months and booster at18 months and 4 years. If patient is injured and has not been immunized, give Anti-tetanic serum plus toxoid (passive–active immunization). Give tetanus toxoid booster every 10 years. Clostridium botulinum It is anaerobic, gram-positive, spore-forming rods.It causes botulism (a type of food poisoning). Pathogenesis: Habitat is the soil. Organism and botulinumtoxinis transmitted in improperly preserved food. Failure to sterilize food during preservation allows spores to survive. Spores germinate in anaerobic environment and produce toxin. The toxin is heat-labile; therefore, foods eaten without proper cooking are usually implicated. Botulinum toxin is absorbed from the gut and carried via the blood to peripheral nerve synapses, where it blocks release of acetylcholine. There are eight immunologic types of toxin; types A, B, and E are the most common in human illness. Incubation period more than 72 hrs. Neurological manifestations with no GIT manifestations. Descending weakness and paralysis, including diplopia, dysphagia, and respiratory muscle failure, is seen. No fever is present. N.B. - Other clinical forms can occur: (1) wound botulism, in which spores - 63 -
contaminate a wound, germinate, and produce toxin at the site and (2) infant botulism, in which the organisms grow in the gut and produce the toxin there. - Botox is a commercial preparation of exotoxin A used to remove wrinkles on the face Laboratory Diagnosis The organism is usually not cultured. Botulinum toxin is demonstrable in uneaten food and the patient's serum by ELISA or mouse protection tests. Mice are inoculated with a sample of the clinical specimen and will die unless protected by antitoxin. Treatment:Antitoxin to types A, B, and E made in horses. Respiratory support may be required. Prevention: proper food preservation techniques, cooking all home-cannedfood, and discarding bulging cans. Clostridium perfringens Anaerobic, gram-positive, spore-forming large rods. ItcausesGas gangrene (myonecrosis) and food poisoning. Pathogenesis:Habitat is soil and human colon. Myonecrosis results from contamination of wound with soil or feces. The wound is usually deep and macerated as car accidents or war wounds. The presence of foreign bodies favors infection. Gas gangrene in wounds is caused by germination of spores under anaerobic conditions and the production of several cytotoxic factors, especially alpha toxin, a lecithinase that cleaves cell membranes. Gas in tissue (CO2 and H2) is produced by organism's anaerobic metabolism. The gas compresses the blood vessels increasing the anerobic condition. Spread of infection occurs by hyaluronidases, collagenases and DNases. Food poisoning is caused by production of enterotoxin within the gut. Enterotoxin acts as a superantigen, similar to that of S. aureus. Laboratory Diagnosis - Gram-stained smear plus anaerobic culture. Spores not usually seen in clinical specimens as the organism is growing and nutrients are not restricted. - Culture on blood agar with anaerobic incubation gives haemolytic colonies. - Nagler reaction:Production of lecithinase is detected by opalescenceon egg yolk agar and identified by enzyme inhibition with specific antiserum. - Stormy clot: culture in litmus milk cause fermentation of lactose and production of large amount of gas splits the clot. Treatment: Penicillin G plus debridement of the wound in gas gangrene (no significant resistance to penicillin). Prevention: Extensive debridement of the wound plus administration of penicillin decreases probability of gas gangrene. Food Poisoningcaused by Clostridium perfringens type A Pathogenesis -Spores are located in soil and can contaminate food. The heat-resistant spores survive cooking and germinate. The organisms grow to large numbers in reheated foods, especially meat dishes. It produces enterotoxin in gut when it sporulates. The enterotoxin acts as a superantigen. - 64 -
-The disease has an 8- to 16-hour incubation period and is characterized by watery diarrhea with cramps and little vomiting. It resolves in 24 hours. Laboratory Diagnosis:Large numbers of the organisms can be isolated from uneaten food. Treatment: Symptomatic treatment is given; no antimicrobial drugs are administered. Clostridium difficile It is anaerobic, gram-positive, spore-forming rods. It causes pseudomembranous colitis. Pathogenesis: Habitat is the human colonin 3 % of the general population and 30% of hospitalized patients. Transmission is fecal–oral. Antibiotics suppress normal flora of colon, allowing C. difficile to overgrow and produce large amounts of exotoxins. Exotoxins A is enterotoxin casing outpouring of fluids with watery diarrhea. Exotoxin B is cytotoxinleads to death of enterocytescausing the pseudo membrane seen in the colon. Laboratory Diagnosis 1. Exotoxin in stool detected by cytopathic effect on cultured cells. 2. Exotoxin in the stool can also be detected by using an ELISA test. Treatment 1s choice oral Metronidazole. 2nd choice is oral Vancomycin( should not be used because it may select for vancomycin-resistant enterococci). Corynebacterium diphtheriae it is club-shaped gram-positive rods arranged in Chinese letter appearance. It has a beaded appearance due to granules of highly polymerized polyphosphate. The granules stain metachromatically, i.e., a dye that stains the rest of the cell blue will stain the granules red. Aerobic, non–spore-forming organism. It causes diphtheria Habitat is the human throat. Transmission is via respiratory droplets. Organism secretes an exotoxin that inhibits protein synthesis by adding ADP-ribose to elongation factor-2 (EF-2). Toxin has two components: subunit A, which has the ADP-ribosylating activity, and subunit B, which binds the toxin to cell surface receptors. Pseudomembrane in throat caused by death of mucosal epithelial cells. Clinically: Nonspecific: fever, sore throat, and cervical adenopathy. Later on : 1. Extension of the membrane into the larynx and trachea, causing airway obstruction. 2. Myocarditis accompanied by arrhythmias and circulatory collapse. 3. Nerve weakness or paralysis, especially of the cranial nerves. 4. Paralysis of the muscles of the soft palate and pharynx can lead C.diphtheriae to regurgitation Chinese letter appearance of fluids through the nose. Peripheral neuritis affecting the muscles of the extremities also occurs. Laboratory Diagnosis of case or carrier Specimen: Throat swab of case or nasopharyngeal swab of carrier. Gram-stained smear: club-shaped gram-positive rods arranged in Chinese letter appearance - 65 -
Culture:- on Loffler serum -Black colonies on K tellurite plate. Document toxin production with -Eleks test: Agar diffusion test in which filter paper impregnated in antitoxin is put perpendicular to streak of isolated strain. Precipitation lines form where toxin antitoxin reaction occurs (Toxic strain). -Animal inoculationproduce disease in laboratory animals -PCR to detect the toxin gene. Treatment: Antitoxin made in horses neutralizes the toxin. Penicillin G kills the organism. Prevention: Toxoid vaccine usually given to children in combination with tetanus toxoid and pertussis vaccine (DTP). Diphtheria toxoid is prepared by treating the exotoxin with formaldehyde. This treatment inactivates the toxic effect but leaves the antigenicity intact. Immunization consists of three doses given at 2, 4, and 6 months of age, with boosters at 1 and 6 years of age. Because immunity wanes, a booster every 10 years is recommended. Immunization does not prevent nasopharyngeal carriage of the organism. Listeria monocytogenes Pathogenesis: Small gram-positive rods. Aerobic, non–spore-forming organism.It causes meningitis and sepsis in newborns and immunocompromised adults. Gastroenteritis. Organism colonizes the GI and female genital tracts; in nature it is widespread in animals, plants, and soil. Transmission is across the placenta or by contact during delivery. Outbreaks of sepsis in neonates and gastroenteritis in the general population are related to ingestion of unpasteurized milk products, e.g., cheese. Listeriolysin is an exotoxin that degrades cell membranes. Reduced cell-mediated immunity and immunologic immaturity as in neonates predispose to disease. Intracellular pathogen that moves from cell-to-cell via "actin rockets." Laboratory Diagnosis: Gram-stained smear: Small gram-positive rods Culture: Small, β-hemolytic colonies on blood agar. Tumbling motility. Treatment: Ampicillin with or without gentamicin. Prevention: Pregnant women and immune-compromised patients should not ingest unpasteurized milk products or raw vegetables. Trimethoprim-sulfamethoxazole given to immunocompromised patients to prevent listeriosis.
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Gram negative Bacilli Categories of Gram Negative Rods Source of Site of Infection
Genus
Enteric tract 1. Both within and Escherichia, Salmonella outside 2. Primarily within Shigella, Vibrio, Campylobacter, Helicobacter 3. Outside only
Klebsiella–Enterobacter–Serratia group, Proteus–Providencia– Morganella group, Pseudomonas, Bacteroides
Respiratory tract
Haemophilus, Legionella, Bordetella
Animal sources
Brucella, Francisella, Pasteurella, Yersinia
Gram-Negative Rods Associated with the Respiratory Tract Species
Major Diseases
Laboratory Diagnosis
Factors X Vaccine Prophylaxis and V Available for Contacts Required for Growth
Haemophilus Meningitis, influenzae otitis media, sinusitis, pneumonia, epiglottitis
Culture; capsular + polysaccharide in serum or spinal fluid
+
Rifampin
Bordetella pertussis
Whooping cough (pertussis)
Fluorescent – antibody on secretions; culture
+
Erythromycin
Legionella Pneumonia pneumophila
Serology; urinary – antigen; culture
–
None
Gram-Negative Rods Associated with Animal Sources Species
Disease
Source of Human Inflection
Mode of Transmission from Animal to Human
Brucella species
Brucellosis Pigs, cattle, goats, sheep
Dairy products; contact Serology or culture with animal tissues
Francisella tularensis
Tularemia Rabbits, deer, ticks
Contact with animal tissues; ticks
Serology
Yersinia pestis
Plague
Rodents
Flea bite
Immunofluorescence or culture
Pasteurella multocida
Cellulitis
Cats, dogs
Cat or dog bite
Wound culture
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Diagnosis
Enterobacteriaceae : The Enterobacteriaceae is a large family of gram-negative rods found primarily in the colon of humans and other animals, many as part of the normal flora. These organisms are the major facultative anaerobes in the large intestine. They cause a variety of diseases with different pathogenetic mechanisms. All members of the Enterobacteriaceae, being gram-negative, contain endotoxin in their cell walls. In addition, several exotoxins are produced, e.g., E. coli and Vibrio cholerae secrete exotoxins, called enterotoxins. The antigens of the Enterobacteriaceae, especially Salmonella and Shigella, are important as they are used for identification purposes both in the clinical laboratory and in epidemiologic investigations. The three surface antigens are as follows: 1. The cell wall antigen (also known as the somatic, or O antigen) is the outer polysaccharide portion of the lipopolysaccharide. The O antigen is the basis for the serologic typing of many enteric rods. The number of different O antigens is very large, e.g., there are approximately 1500 types of Salmonella and 150 types of E. coli. 2. The H antigen is on the flagellar protein. Only flagellated organisms, such as Escherichia and Salmonella, have H antigens, whereas the non motile ones, such as Klebsiella and Shigella, do not. 3. The capsular or K polysaccharide antigen is particularly prominent in heavily encapsulated organisms such as Klebsiella. In Sal. Typhi, capsular Ag is called the Vi (or virulence) antigen. Specimens suspected of containing members of the Enterobacteriaceae and related organisms are usually inoculated onto a selective differential medium such as MacConkey's agar. The differential ability of the media is based on lactose fermentation and the selective effect of the media in suppressing unwanted grampositive organisms is exerted by bile salts or bacteriostatic dyes in the agar. On MacConkey's agar: Enterobacteria are divided into: The non–lactose fermenters, e.g., Salmonella, Shigella, and proteus form colorless colonies. The lactose fermenters, e.g., E. coli and Klebsiella form rose pink colonies. Gram-Negative Rods Related to the Enteric Tract Escherichia coli Pathogenesis: Facultative gram-negative mortile rods; lactose fermenter. Indole positive. -Habitat is the human colon; it colonizes the vagina and urethra. From the urethra, it ascends and causes urinary tract infection. Acquired during birth in neonatal meningitis and by the fecal–oral route in diarrhea. -It is the most common cause of urinary tract infection. Indwelling urinary catheters and intravenous lines predispose to UTI and sepsis, respectively. Endotoxin in cell wall causes septic shock. -Other diseases: pneumonia, nosocomial infection, wound infection and other pyogenic infections. Diarrheagenic E coli: 1. Enterotoxigenic E coli: causes traveler 's diarrhea Two enterotoxins are produced. The heat-labile toxin (LT) stimulates adenylate - 68 -
cyclase by ADP-ribosylation. Increased cyclic AMP causes outflow of chloride ions and water, resulting in diarrhea. The heat-stable toxin (ST) causes diarrhea, perhaps by stimulating guanylate cyclase. Other virulence factors include pili for attachment to mucosal surfaces and a capsule that impedes phagocytosis. 2. Entero-haemorrhagic E coli: Verotoxin (Shiga-like toxin) is an enterotoxin produced by E. coli strains with the O157:H7 serotype. It causes bloody diarrhea and hemolytic-uremic syndrome associated with eating undercooked meat. Verotoxin inhibits protein synthesis. 3. Enteropathogenic E coli: causes diarrhea in infants. It binds tightly to intestinal mucosa resulting in loss of microvilli. 4. Enteroinvasive E coli: causes dysentery like disease. 5. Entero-aggregative E coli. Laboratory Diagnosis -Gram-stained smear: gram-negative rods. -Culture on MacConkey's agar: Lactose-fermenting colonies. -Triple Sugar Iron agar shows acid slant and acid butt with gas but no H2S. Differentiate from other lactose-positive organisms by biochemical reactions (ferment glucose, lactose, maltose, mannite and sucrose) and indole positive. .-For epidemiologic studies, type organism by O and H antigens by using known antisera. Treatment: Ampicillin or sulfonamides for urinary tract infections. Third-generation cephalosporins for meningitis and sepsis. Rehydration is effective in traveler's diarrhea Antibiotic resistance mediated by plasmid-encoded enzymes, e.g., β-lactamase and aminoglycoside-modifying enzymes. Klebsiella spp Facultative gram-negative non motile rods; ferment lactose. Indole negative. They are frequently found in the large intestine but are also present in soil and water. K. pneumoniae has a very large polysaccharide capsule, which gives its colonies a striking mucoid appearance Urinary tract infections and pneumonia are the usual diseases associated with these bacteria, but bacteremia and secondary spread to other areas such as the meninges and liver occur. Laboratory Diagnosis: Organisms of this group produce lactose-fermenting mucoid colonies on differential agar such as MacConkey's Salmonella spp 1. Salmonellae are gram-negative rods, lactose non fermenter 2. The O antigens, which are the outer polysaccharides of the cell wall, are used to subdivide the salmonellae into groups A–I. 3. There are two forms of the H antigens, phases 1 and 2. Only one of the two H proteins is synthesized at any one time. 4. The Vi antigens (capsular polysaccharides) are antiphagocytic and are an important virulence factor for Sal. typhi, the agent of typhoid fever. The Vi antigens are also used for the serotyping of Sal. typhi in the clinical laboratory. 5. The typhoidal species are Sal. typhi and Sal. Paratyph A, B and C. 6. The non typhoidal species are the many strains of Sal. enteritidis. 7. Sal. choleraesuis is the species most often involved in metastatic infections. - 69 -
Salmonella typhi and S. paratyphi A, B, and C Motile, facultative gram-negative rods. Non–lactose-fermenting. Produces H2S. Pathogenesis: Typhoid fever is caused by Salmonella typhi (sero-group D). ParaTyphoid is caused by Salmonella para-typhi A, B and C (Serogroups A, B and C respectively) Habitat is the human colon only, in contrast to other salmonellae, which are found in the colon of animals as well. Transmission is by the fecal–oral route. Infects the cells of the reticulo-endothelial system, especially in the liver and spleen. Endotoxin in cell wall causes fever. Capsule (Vi antigen) of Salmonella typhi is a virulence factor. Decreased stomach acid resulting from ingestion of antacids or gastrectomy predisposes to Salmonella infections. Chronic carrier state established in gallbladder and urinary ladder. Organism excreted in bile results in fecal–oral spread to others. Clinically: the onset of illness is slow, with fever and constipation. Diarrhea may occur early but usually disappears by the time the fever and bacteremia occur. After the first week, as the bacteremia becomes sustained, high fever, delirium, tender abdomen, and enlarged spleen occur. Rose spots, i.e., rose-colored macules on the abdomen, are associated with typhoid fever but occur only rarely. Leucopenia and anemia are often seen. Liver function tests are often abnormal, indicating hepatic involvement. The disease begins to resolve by the third week Complications: intestinal hemorrhage or perforation can occur. About 3% of typhoid fever patients become chronic carriers. The carrier rate is higher among women, especially those with previous gallbladder disease and gallstones. Laboratory Diagnosis of case: 1st week of fever ------Blood culture. 5-10 ml blood from adults or 0.5-2 ml blood from children is inoculated in 50 ml bile broth medium (Blood culture bottle) then subculture is done on MacConkey's agar. Any non lactose fermenting colony is identified. 2nd week of fever ----stools and urine culture or widal test. The stool is inoculated 1st on enrichment medium as Selenite broth or tetrathionate broth for 37 hrs to inhibit other stool flora then subculture on MacConkey's agar. The urine is centrifuged and directly plated on MacConkey's agar. Any non lactose fermenting colony is identified : 1)Microscopically: gram-negative rods 2)Serologically with known antisera 3)Biochemically: Indole –ve, H2S +ve and fermentation of Glucose, maltose and sucrose. Widal test (agglutination test) detects agglutinating antibodies to O and H antigens in patient's serum. It is done by mixing serial dilution of patient serum with known O and H antigens for Salmonella. -It is positive on the 2nd week onwards. Titer >1/160 is positive. Rising titer is more important to diagnose active infection. Rising titer is 2-4 fold rise of antibody test after repetition of the test 7-10 days. -Anti –O antibody means recent infection while Anti H Ab means remote infection. Laboratory Diagnosis of Carrier: by isolating the organism from the stools or urine in case of intestinal or urinary carriers respectively. The specimen should be repeated as bacilli excretion is intermittent in carriers. - 70 -
Treatment: Most effective drug is ceftriaxone and quinolones for cases. Prevention: 1)Public health measures, e.g., sewage disposal, chlorination of the water supply, stool cultures for food handlers, and hand washing prior to food handling. 2) Vaccinesused:-one vaccine contains purified Vi polysaccharide capsule as the immunogen - The second contains live, attenuated S. typhi as the immunogen. - The third is TAB vaccine contains the killed salmonella and taken in two doses Sc gives partial protection. Salmonella food poisoning (Enterocolitis): Pathogenesis Habitat is the enteric tract of humans and animals, e.g., chickens and domestic livestock. Transmission is by the fecal–oral route. Many salmonella spp especially Salmonella enteritidis and Salmonella cholerasuis can cause food poisoning. They are animal pathogens. The most frequent animal source is poultry and eggs, but meat products that are inadequately cooked can cause the disease. Dogs and other pets, including turtles, snakes, are additional sources. The organism invades the mucosa of the small and large intestines. It can enter blood, causing sepsis. Infectious dose is at least 105 organisms. Infectious dose is high because organism is inactivated by stomach acid. Sickle cell anemia predisposes to Salmonella osteomyelitis. Clinically: After an incubation period of 12 to 48 hours, enterocolitis begins with nausea and vomiting, fever and then progresses to abdominal pain and diarrhea, which can vary from mild to severe, with or without blood. Usually the disease lasts a few days, is self-limited Laboratory Diagnosis: Specimen: Stools. Isolation of the organism by culture of stools as in typhoid fever. Treatment: it is usually a self-limited disease that resolves without treatment. Fluid and electrolyte replacement may be required. Antimicrobial agents are indicated only immune-compromised persons who are at risk for septicemia and disseminated abscesses. Prevention: Proper cooking of raw eggs or meat. Shigella Species (e.g., S. dysenteriae, S. sonnei) Shigella dysentriae is the most virulent. Other spp as S. sonnei, S. boydii, and S. flexnerie is less virulent. It causes acute bacillary dysentery. Pathogenesis:Facultative gram-negative rods. Non–lactose-fermenting. Non-motile organism. Its Habitat is the human colon only. Transmission is by the fecal–oral route.Infectious dose is very low (1–10 organisms). The organism invades the mucosa of the ileum and colon but does not penetrate farther. It secretes exotoxin (Shigatoxin) which is neurotoxic, enterotoxic and cytotoxic,. Endotoxin in cell wall irritates the mucosa causing tenesmus with pus in stools. Clinically: After an incubation period of 1 to 4 days, symptoms begin with fever and abdominal cramps, followed by diarrhea, which may be watery at first but later contains blood and mucus with tenesmus. Dehydration may occur. Laboratory Diagnosis The stool is inoculated 1st on enrichment medium Selenite broth for 37 hrs to inhibit other stool flora then subculture on MacConkey's agar. Any non lactose fermenting - 71 -
colony is identified: 1) Microscopically: gram-negative rods 2)Serologically with known antisera. 3)Biochemically: Indole –ve Shigella dysentrae-- Fermentation of Glucose with acid production. Other Shigella spp ---- Fermentation of Glucose and mannitol with acid production. Treatment In most cases, fluid and electrolyte replacement only. In severe cases, ciprofloxacin 1stchoice. Trimethoprim-sulfamethoxazole is an alternative choice. Prevention:Public health measures, e.g., sewage disposal, chlorination of the water supply, and hand washing prior to food handling. No vaccine is available. Proteus Spp 1) These organisms primarily cause urinary tract infections, both community- and hospital-acquired, wound infection, sepsis. 2)These gram-negative rods produce lactose non fermenting colonies on MacConkey's agar. And produce a striking swarming effect on blood agar, characterized by expanding rings (waves) of organisms. 3)urease positive. 4) The cell wall O antigens of certain strains of Proteus, such as OX-2, OX-19, and OX-K, cross-react with antigens of several species of rickettsiae; This test, called the Weil-Felix reaction. Vibrio cholerae Vibrios are curved, comma-shaped gram-negative rods. Oxidase-positive, which distinguishes them from Enterobacteriaceae. V. cholerae is divided into two groups according to the nature of its O cell wall antigen. Members of the O1 & O139 sero-groups cause epidemic disease, whereas other nonO1 nonO139 organisms either cause sporadic disease or are non-pathogens. The O1 organisms have two biotypes, called classical and El Tor. Each biotype has three serotypes, called Ogawa, Inaba, and Hikojima. Virulence and Pathogenesis: Habitat is the human colon. Transmission is by the fecal–oral route. Massive, watery diarrhea (Rice water stools) caused by enterotoxin (choleragen) that activates adenylate cyclase by adding ADP-ribose to the stimulatory G protein. Increase in cyclic AMP causes outflow of chloride ions and water. Toxin has two components: subunit A, which has the ADP-ribosylating activity; and subunit B, which binds the toxin to cell surface receptors. Organism produces mucinase, which enhances attachment to the intestinal mucosa. Infectious dose is high (>107 organisms). Carrier state rare. Clinically: Watery diarrhea (Rice-water stool) in large volumes is the hallmark of cholera. There are no red blood cells or white blood cells in the stool. Marked dehydration, acidosis, and fluid and electrolytes imbalance, leads to cardiac and renal failure. Laboratory Diagnosis: Diagnosis of Cholera case in a non-endemic area: - Diagnosis of the first case : Comma shaped V cholera 1- Specimen: rice water stools. 2- Culture: Cultural characters; Highly aerobic. Growth is favored by alkaline pH (8.59.5). First, the stool is inoculated in alkaline peptone water for 6-8h. Then, subculture is done on thio-sulphate citrate bile sucrose agar (TCBS). If yellow colonies appear, - 72 -
they are further identified by: 1) Wet mount film: darting motility 2)Gram stained smears: Gram negative comma shaped bacilli 3)Biochemical reactions: sugar fermentation (glucose, maltose, mannite & surcose), positive oxidase and indole tests, cholera red reaction and string test positive and TSI inoculation. Cholera red reaction positive: when Vibrio cholerae is inoculated in nitrate peptone medium, nitrate is reduced to nitrite and indole is produced from decomposition of tryptophan in peptone. Nitrite combines with indole producing nitroso-indole compound which gives red color on addition of few drops of strong sulphuric acid (H2SO4). -String test positive: when a colony is emulsified in a drop of 0.5% sodium deoxycholate in distilled water, within one minute, the cells lyse and DNA strings when a loopful is lifted from the slide. This test differentiates Vibrio cholerae form Aeromonas hydrophila which is string test negative. 3- Agglutination tests with specific anti-O1 and 139 sera. 4-Direct methods:immune-fluorescence and PCR for detection of cholera toxin gene. -Diagnosis of secondary cases during an epidemic: Cases are diagnosed just by microscopic examination of stools for comma shaped bacilli with darting motility which can be immobilized by specific anti-O sera. B-Diagnosis of a carrier state: Rectal Swab is the specimen of choice which is cultured on the same lines of a case. Treatment Treatment of choice is fluid and electrolyte replacement. Tetracycline is not necessary but shortens duration and reduces carriage. Prevention Public health measures, e.g., sewage disposal, chlorination of the water supply, examination of food handlers, and hand washing prior to food handling. Vaccine containing killed cells (Koll vaccine) given in 2 intramuscular doses with one week interval. It usually gives low level of protection, about 50%, that lasts only for 6 months. Disadvantages are: it induces formation of antibacterial not antitoxin antibodies. It has limited effectiveness. Tetracycline used for close contacts. Vibrio parahaemolyticus Comma-shaped gram-negative rod found in warm sea water. Causes watery diarrhea. Acquired by eating contaminated raw seafood. Diarrhea is mediated by enterotoxin similar to cholera toxin. Vibrio vulnificus Comma-shaped gram-negative rod found in warm sea water. Causes cellulitis and lifethreatening sepsis with hemorrhagic bullae. Acquired either by trauma to skin, especially in shellfish handlers, or by ingestion of raw shellfish, especially in patients who are immune-compromised or have liver damage. Campylobacter jejuni Comma-shaped gram-negative rods. Microaerophilic. Grows well at 42°C. Pathogenesis: Habitat is human and animal feces. Transmission is by the fecal–oral route.It causes enterocolitis. Invades mucosa of the colon but does not penetrate. No enterotoxin known. Laboratory Diagnosis Gram-stained smear plus culture on special agar, e.g., Skirrow's agar, at 42°C in high- 73 -
CO2, low-O2 atmosphere. Treatment: Usually symptomatic treatment only; erythromycin for severe disease. Prevention:Public health measures, and handwashing prior to food handling. No preventive vaccine. Helicobacter pylori Curved gram-negative rod. It causes gastritis and peptic ulcer. Pathogenesis: Habitat is the human stomach. Transmission is by ingestion. Organisms synthesize urease, which produces ammonia that damages gastric mucosa. Ammonia also neutralizes acid pH in stomach, which allows the organism to live in gastric mucosa. Clinically: It causes gastritis and peptic ulcer. Risk factor for gastric carcinoma. Laboratory Diagnosis: 1. The organism can be seen on Gram-stained smears of biopsy specimens of the gastric mucosa. 2. It can be cultured on Skirrow's agar. 3. It is urease-positive. 4. The "urea breath" test. In this test, radio-labeled urea is ingested. If the organism is present, it produces urease that cleaves the ingested urea, radiolabeled CO2 is evolved, and the radioactivity is detected in the breath. 5. Detection of Helicobacter antigen in the stool by ELISA 6. The presence of IgG antibodies in the patient's serum. Treatment: Tripple therapy as: Amoxicillin, metronidazole, and bismuth (PeptoBismol). Pseudomonas aeruginosa Aerobic gram-negative rods. Non–lactose-fermenting. Produce Pyocyanin (blue-green) exo-pigment. Oxidase-positive, which distinguishes it from members of the Enterobacteriaceae family. It is very resistant to antibiotics and can grow in improperly used disinfectants. Pathogenesis: 1. Habitat is environmental water sources, e.g., in hospital respirators and humidifiers. Also inhabits the skin, upper respiratory tract, and colon of about 10% of people. Transmission is via water aerosols, aspiration, and fecal contamination. 2. Endotoxin is responsible for fever and shock associated with sepsis. 3. Produces exotoxin A, which acts like diphtheria toxin (inactivates EF-2). 4. Pili and capsule are virulence factors that mediate attachment and inhibit phagocytosis, respectively. 5. Glycocalyx-producing strains predominate in chronic infections in cystic fibrosis patients. Severe burns and neutropenia are important predisposing factors. Clinically: Wound infection, UTI, pneumonia, and sepsis. One of the most important causes of nosocomial infections, especially in burn patients and those with cystic fibrosis. Causes endocarditis in intravenous drug users. Laboratory Diagnosis (1)Gram-stained smear: gram negative bacilli (2) Culture on nutrient agar gives bluish green exopigment and Non–lactose-fermenting colonies on MacConkey's agar. (3)Oxidase-positive colonies. - 74 -
Treatment: Antibiotics must be chosen on the basis of antibiotic sensitivities because resistance is common. Anti-pseudomonal penicillin as piperacillin, aminoglycoside as amikacine, ciprofloxacin and 3rd generation cephalosporin. Prevention: Disinfection of water-related equipment in the hospital, hand washing, and prompt removal of urinary and intravenous catheters. Bacteroides fragilis Anaerobic, gram-negative rods. Pathogenesis: Habitat is the human colon as normal flora, where it is the predominant anaerobe. Transmission occurs by spread from the colon to the blood or peritoneum. Lipopolysaccharide in cell wall is less potent than typical endotoxin. Its capsule is antiphagocytic. Predisposing factors to infection include bowel surgery and penetrating abdominal wounds. Clinically: Sepsis, peritonitis, and abdominal abscess. Laboratory Diagnosis 1. Specimen: pus or aspirated exudates, usually foul smell and it is better to take specimen as aspirate in a needle with expulsion of air then closure of its nozzle to decrease its exposure to air. 2. Gram-stained smear plus anaerobic culture. 3. Culture is done on gentamycin blood agar with anaerobic incubation for 24 hrs. 4. Identification based on biochemical reactions and gas chromatography. Serologic tests not useful. Treatment: Metronidazole, clindamycin, and cefoxitin are all effective. Abscesses should be surgically drained. Prevention: In bowel surgery, peri-operative cefoxitin can reduce the frequency of postoperative infections. Prevotella melaninogenica Anaerobic gram-negative rod resembling B. fragilis. Member of normal flora found primarily above the diaphragm (e.g., mouth) in contrast to B. fragilis, which is found below (e.g., colon). Often involved in brain and lung abscesses. Formerly called Bacteroides melaninogenicus. Gram-Negative Rods Related to the Respiratory Tract Haemophilus influenzae 1)Small gram-negative (coccobacillary) rods. 2) Requires factors X (hemin) and V (NAD) for growth. 3) Six capsular polysaccharide types, type b causes 95% of invasive disease. Type b capsule is polyribitol phosphate. Pathogenesis: Habitat is the upper respiratory tract. Transmission is via respiratory droplets. - Polysaccharide capsule is the most important determinant of virulence. - Unencapsulated ("untypeable") strains cause mucosal infections but not invasive infections. - IgA protease is produced. Most cases of meningitis occur in Clinically: Sinusitis, otitis media, and pneumonia are common. Epiglottitis is uncommon, but H. influenzae is the most important cause. H. influenzae used to be a leading cause of meningitis especially in children younger than 2 years of age. - 75 -
Laboratory Diagnosis 1. Gram-stained smear plus culture on chocolate agar. 2. Growth requires both factors X and V. 3. Determine serotype by using antiserum in various tests, e.g., latex agglutination. Capsular antigen can be detected in serum or cerebrospinal fluid. Treatment: Ceftriaxone is the treatment of choice for meningitis. Approximately 25% of strains produce β-lactamase. Prevention: Vaccine containing the type b capsular polysaccharide conjugated to diphtheria toxoid or other protein is given between 2 and 18 months of age. Rifampin can prevent meningitis in close contacts. Bordetella pertussis Small gram-negative small rods. It is the cause of Whooping cough (pertussis). Virulence and Pathogenesis: - Habitat is the human respiratory tract. Transmission is via respiratory droplets. Pertussis toxin stimulates adenylate cyclase. Toxin has two components: subunit A, which has the ADP-ribosylating activity, and subunit B, which binds the toxin to cell surface receptors. Pertussis toxin causes lymphocytosis in the blood. Tracheal cytotoxin damages ciliated epithelium of respiratory tract. - Endotoxin in the cell wall. - Clinically: Whooping cough is an acute tracheobronchitis that begins with mild upper respiratory tract symptoms followed by a severe paroxysmal cough, which lasts from 1 to 4 weeks. It is characterized by a series of hacking coughs, accompanied by production of copious amounts of mucus that end with an inspiratory "whoop". - -A pronounced leukocytosis with up to 70% lymphocytes is seen. - Although central nervous system anoxia and exhaustion can occur as a result of the severe coughing, death can occur due mainly to pneumonia. Laboratory Diagnosis - Gram-stained smear plus culture on Bordet-Gengou agar. - Identified slide agglutination with known antisera. - PCR tests, if available, are both sensitive and specific. Treatment: Erythromycin. Prevention: Pertussis vaccine contains the killed bacteria; Usually given to children in combination with diphtheria and tetanus toxoids (DTP). DTP vaccine is given to children in age of 2, 4, and 6 months then booster at 18 months and 4 years but it is contraindicated to give the pertussis vaccine after age of 4 years because it cause allergic encephalitis. The acellular vaccine containing pertussis toxoid and four other purified proteins is recommended rather than the killed vaccine. Usually given to children in combination with diphtheria and tetanus toxoids (DTaP). Legionella pneumophila Gram-negative rods, but stain poorly with standard Gram stain. It causes Legionnaires' disease (one of the"atypical" pneumonias) and Pontiac fever ( a mild, flu-like form of Legionella infection).Require increased iron and cysteine for growth in culture. - 76 -
Pathogenesis : Habitat is environmental water sources. Transmission is via aerosol from the water source. Person-to-person transmission does not occur. Predisposing factors include being older than 55 years of age, smoking, and having a high alcohol intake. Immuno-suppressed patients, e.g., renal transplant recipients, are highly susceptible. The organism replicates intracellularly, therefore cellmediated immunity is an important host defense. Smoking damages alveolar macrophages, which explains why it predisposes to pneumonia. Laboratory Diagnosis: -Microscopy with silver impregnation stain or fluorescent antibody. -Culture on charcoal yeast extract agar containing increased amounts of iron and cysteine .-Urinary antigen provides rapid diagnosis. -The main stay of diagnosis can be made serologically by detecting rise in antibody titer in patient's serum. Treatment: Azithromycin or erythromycin. Rifampin can be added in severe cases. Prevention: No vaccine or prophylactic drug is available. Gram-Negative Rods Related to Animal Sources (Zoonotic Organisms) Brucella Species 1. Brucellae are obligate parasites of animals and human, and are characteristically located intracellular. 2. Different species of Brucella are pathogenic to man, each is associated with particular animal species; Br. abortus(cattle), Br. melitensis(goats, sheep), Br. suis(swine), Br. canis (dogs). 3. Small gram-negative rods. It causes Brucellosis (undulant fever or Malta fever). Pathogenesis: Reservoir is domestic livestock. Transmission either by ingestion of contaminated milk products or through the skin by direct contact in an occupational setting such as an abattoir. They localize in the reticulo-endothelial system, namely, the lymph nodes, liver, spleen, and bone marrow. The mechanism of pathogenesis of these organisms is not well defined, except that endotoxin is involved. Brucella can survive and replicate intracellularly. Clinically: After an incubation period of 1 to 3 weeks, nonspecific symptoms such as fever, chills, fatigue, malaise, anorexia, and weight loss occur. The onset can be acute or gradual. The undulating (rising-and-falling) fever pattern that gives the disease its name occurs in a minority of patients. Enlarged lymph nodes, liver, and spleen are frequently found. Pancytopenia occurs. B. melitensis infections tend to be more severe and prolonged, whereas those caused by B. abortus are more selflimited. Osteomyelitis is the most frequent complication. Laboratory Diagnosis: Specimen: blood, bone marrow and lymph node biopsy. During the periods of fever: Blood culture---Repeated blood cultures-----5-10 ml blood from adults or 0.5-2 ml blood from children is inoculated in 50 ml (Blood culture bottle). Incubation is carried out aerobically for most Brucellae species, or in 10% CO2 for Br. abortus. - 77 -
Then subcultures on blood agar. Any growth appearing is identified by: 1)Morphology 2)Biochemical reactions 3)Agglutination with specific agglutinating sera. The blood cultures should be kept for 3-4 weeks before being discarded as negative. Serology: 1. Brucella agglutination test : Done using a wide range of dilutions of patients sera ( 1/ 20 – 1/ 5120 ), to avoid prozone phenomenon known to occur frequently with sera of brucellosis patients. The serum dilutions are tested against smooth and carefully standardized suspensions of Br. abortus and Br. melitensis. The tubes are incubated at 37Ċ for 24 hs before reading the test. A titer of 1/ 100 or more may be diagnostic. A rising titer is more indicative of infection. Zone phenomenon: is commonly met, where with low dilutions of the patient serum no agglutination occurs, whereas agglutination occurs at high dilution. Presence of blocking antibodies: these are IgA antibodies that interfere with agglutination by IgG and IgM and cause a serologic test to be negative in low serum dilutions (prozone phenomenon) although positive in higher dilutions. They are detected by adding antihuman globulin to the antigen- serum mixture ( coombs anti-globulin method). 2. ELISA: for detection of IgG, IgM or IgA. It has replaced coomb's test. 3. Polymerase chain reaction (PCR) for direct detection of organisms in clinical material. 4. Brucellin test: is a delayed type hypersensitivity test similar to tuberculin test. A protein brucella extract is injected intradermally, induration and erythema develop within 24 to 72 hs in chronic patients. Treatment:Tetracycline plus rifampin. Prevention: Pasteurize milk; vaccinate cattle. No human vaccine is available. Francisella tularensis Small gram-negative rods. The causative agent of Tularemia. Pathogenesis: Reservoir is many species of wild animals, especially rabbits, deer, and rodents. Transmission is by ticks , aerosols, contact, and ingestion. Organisms localize in reticuloendothelial cells. Clinically: vary from sudden onset of an influenza like syndrome to prolonged onset of a low-grade fever and adenopathy. Approximately 75% of cases are the "ulceroglandular" type, in which the site of entry ulcerates and the regional lymph nodes are swollen and painful. Other, less frequent forms include glandular, oculoglandular, typhoidal, gastrointestinal, and pulmonary. Laboratory Diagnosis: Culture is rarely done because special media are required and there is a high risk of infection of laboratory personnel. Diagnosis is usually made by serologic tests that detect antibodies in patient's serum. Treatment:Streptomycin. Prevention: Live, attenuated vaccine for persons in high-risk occupations. Protect against tick bites.
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Pasteurella multocida Small gram-negative rods. Pathogenesis: Reservoir is the mouth of many animals, especially cats and dogs. Transmission is by animal bites. Clinically: A rapidly spreading cellulitis at the site of an animal Laboratory Diagnosis: Gram-stained smear and culture on blood agar of a wound swab. Treatment: Penicillin G. Yersinia pestis Small gram-negative rods with bipolar ("safety pin") staining. One of the most virulent organisms, i.e., very low ID50. Causative of Bubonic and pneumonic plague. Pathogenesis:Reservoir is wild rodents, e.g., rats and squirrels. Transmission is by flea bite. - Virulence factors include (1)endotoxin (2) an exotoxin (3) two antigens (V and W) allow organism to grow within cells. (4)anenvelope (capsular) antigen that protects against phagocytosis. Clinically: The organisms inoculated at the time of the bite spread to the regional lymph nodes, which become swollen and tender. These swollen lymph nodes are the buboes that have led to the name bubonic plague. The organisms can reach high concentrations in the blood (bacteremia) and disseminate to form abscesses in many organs. The endotoxin-related symptoms, including disseminated intravascular coagulation and cutaneous hemorrhages, probably were the genesis of the term black death. - Respiratory droplet transmission of the organism from patients with pneumonic plague can occur. Laboratory Diagnosis: -Specimen: aspiration from bubo - Stained smear. Other stains, e.g., Wayson's stain show typical "safety-pin" appearance more clearly. - Cultures are hazardous and should be done only in specially equipped laboratories. Organism is identified by immunofluorescence. - Diagnosis can be made by serologic tests that detect antibody in patient's serum. Treatment: Streptomycin either alone or in combination with tetracycline. Strict quarantine for 72 hours. Prevention: - Control rodent population and avoid contact with dead rodents. - Killed vaccine is available for persons in high-risk occupations. - Close contacts should be given tetracycline. Mycobacteria The major pathogens are 1) Mycobacterium tuberculosis, the cause of tuberculosis 2) Mycobacterium leprae, the cause of leprosy 3)Atypical mycobacteria, such as Mycobacterium avium-intracellulare complex and Mycobacterium kansasii, can cause tuberculosis-like disease but are less frequent pathogens. Rapidly growing mycobacteria, such as Mycobacterium chelonei, occasionally cause human disease in immunocompromised patients or those in whom prosthetic devices have been implanted.
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Mycobacterium tuberculosis Aerobic, acid-fast rods. High lipid content of cell wall, which prevents dyes used in Gram stain from staining organism. Lipids include mycolic acids and wax D. Grows very slowly (it has a doubling time of 18 hours), which requires that drugs be present for long periods (months). Produces catalase, which is required to activate isoniazid to the active drug. Pathogenesis: Primary tuberculosis: occur after 1st exposure to the bacilli. It occurs in lung, skin, tonsils or intestine. Fate: (1) Complete resolution in case of high immunity. (2)Progression in case of decreased immunity and high dose of the infection. - Spread of the organism within the body occurs by two mechanisms: 1. A tubercle can erode into a bronchus, empty its caseous contents, and spread the organism to other parts of the lungs, to the gastrointestinal tract if swallowed, and to other persons if expectorated. 2. It can disseminate via the bloodstream to many internal organs. Dissemination can occur at an early stage if cell-mediated immunity fails to contain the initial infection or at a late stage if a person becomes immunocompromised. Dormant or latent tuberculosis: Deaths of most of the bacilli but few bacilli remain inactive and dormant surrounded by fibrous tissue with no clinical effect. Secondary tuberculosis: It occurs in any part of the body due to either secondary exposure to the TB bacilli or reactivation of dormant primary focus. Military Tuberculosis: wide spread blood stream dissemination. Immunity: 1. After recovery from the primary infection, resistance to the organism is mediated by cellular immunity. 2. Circulating antibodies also form, but they play no role in resistance 3. Granulomas and caseation mediated by cellular immunity, i.e., macrophages and CD4-positive T cells (delayed hypersensitivity). Cord factor (trehalose mycolate) correlates with virulence. No exotoxins or endotoxin. Immunosuppression increases risk of reactivation and dissemination. Pulmonary tuberculosis: It is caused by 1) M tuberculosis 2)M bovis Reservoirs are humans for M. tuberculosis and cows for M bovis. Transmission is via respiratory droplets produced by coughing. Lesions are dependent on the presence of the organism and the host response. There are two types of lesions: 1. Exudative lesions, which consist of an acute inflammatory response and occur chiefly in the lungs at the initial site of infection 2. Granulomatous lesions, which consist of a central area of giant cells containing tubercle bacilli surrounded by a zone of epithelioid cells. These giant cells, called Langhans' giant cells, are an important pathologic finding in tuberculous lesions. A tubercle is a granuloma surrounded by fibrous tissue that has undergone central caseation necrosis. Tubercles heal by fibrosis and calcification. The primary lesion of tuberculosis usually occurs in the lungs. The parenchymal lesion and the draining lymph nodes together are called a Ghon complex. - 80 -
Primary lesions usually occur in the lower lobes, whereas reactivation lesions usually occur in the apices. Reactivation lesions also occur in other well-oxygenated sites such as the kidneys, brain, and bone. Reactivation is seen primarily in immunocompromised or debilitated patients. Extra- pulmonary TB: can occur in any part of the body askidney, bones, meninges…..etc. Clinically:-Non specific symptoms as: Fever, fatigue, night sweats, and weight loss are common. -Pulmonary tuberculosis causes cough and hemoptysis. -Scrofula is mycobacterial cervical adenitis that presents as swollen non-tender lymph nodes, usually unilaterally. Both M. tuberculosis and Mycobacterium scrofulaceum cause scrofula. -Gastrointestinal tuberculosis is characterized by abdominal pain and diarrhea accompanied by more generalized symptoms of fever and weight loss. -Renal tuberculosis, dysuria, hematuria, and flank pain occur. "Sterile pyuria" is a characteristic finding. The urine contains white blood cells, but cultures for the common urinary tract bacterial pathogens show no growth. However, mycobacterial AFB of M tuberculosis cultures are often positive. -Miliary tuberculosis is characterized by multiple disseminated lesions that resemble millet seeds. Tuberculous meningitis and tuberculous osteomyelitis, especially vertebral osteomyelitis (Pott's disease), are important disseminated forms. Laboratory Diagnosis: Specimen:According to lesion: - Pulmonary TB: 3 morning sputum specimens on 3 subsequent days. - Urinary TB: morning urinary specimen – TB meningitis: CSF Microscopy: Acid-fast rods seen with Ziehl-Neelsen (hot) stain or Kinyoun (cold) stain seen by ordinary microscope. -auramine stain: which can be visualized by fluorescence microscopy for rapid screening. -Decontamination and concentration of contaminated specimens like sputum and stools: digestion of the specimen by treatment with N-acetyl-L-cysteine (NALC) to release tubercle bacilli, decontamination by 4% NaOH to kill contaminants, lastly centrifugation to concentrate the specimen. Culture: on solid Löwenstein-Jensen medium-----slow-growing (3–6 weeks) colony. -On liquid medium Middle brook 7H9- BACTEC medium, radioactive metabolites are present and growth can be detected by the production of radioactive carbon dioxide in about 2 weeks. A liquid medium is preferred for isolation because the organism grows more rapidly and reliably than it does on agar. Biochemically: M. tuberculosis produces produce niacin and catalase-positive. Nucleic acid amplification tests as PCR can be used to detect the presence of M. tuberculosis directly in clinical specimens such as sputum. Tests are available that detect either the ribosomal RNA or the DNA of the organism. Serologic tests for antibody in patient's serum not useful. - 81 -
Laboratory diagnosis of latent T.B 1. Skin Test: a delayed hypersensitivity skin test, which becomes positive 3-8 weeks after infection or BCG vaccination. 0.1 ml of PPD (purified protein derivative of tubercle bacilli) containing 5TU (tuberculin units) is injected intra-dermally in the volar aspect of the forearm. Reading 48 hours after inoculation. A) Positive test: if induration ≥10 mm. Induration is caused by a delayed hypersensitivity response. Positive skin test indicates that the person has been infected but not necessarily that the person has the disease tuberculosis. B) Anegative tuberculin test excludes infection in suspected persons. C)False negative reactions: patients with corticosteroid therapy, malnutrition, Hodgkin’s disease, measles and AIDS. D)False positive reactions are mainly due to infection with non-tuberculous Mycobacteria. 2. Interferon-gamma release assay (IGRA) (Quantiferon-TB): In this assay, blood cells from the patient are exposed to antigens from M. tuberculosis and the amount of interferon-gamma released from the cells is measured. Treatment *Long-term therapy (6–9 months) with three drugs, isoniazid (INH), rifampin, and pyrazinamide. A fourth drug, ethambutol, is used in severe cases (e.g., meningitis), in immunocompromised patients (e.g., those with AIDS), and where the chance of isoniazid-resistant organisms is high. *The necessity for prolonged therapy is attributed to (1) the intracellular location of the organism, (2) caseous material, which blocks penetration by the drug, (3) the slow growth of the organism, and (4) metabolically inactive "persisters" within the lesion. *Second line drugs: quinolones, aminoglycosides, ethionamide, capreomycin and cycloserine are used if resistance occurred to first line drugs: rifampicin , INH, pyrazinamide, ethambutole, and streptomycin. *Most patients become noninfectious within 2 weeks of adequate therapy. *Directly observed therapy (DOT): health care workers observe the patient taking the medication to overcome the problem of noncompliance is in which. Treatment of latent (asymptomatic) infections consists of isoniazid taken for 6 to 9 months. *Multidrug-resistant (MDR) strains: is defined as tuberculosis that is resistant to at least isoniazid and rifampicin the two most powerful first-line treatment anti-TB drugs. Isolates that are multiply resistant to any other combination of anti-TB drugs but not to INH and RMP are not classed as MDR-TB.have emerged and require other drug combinations. Prevention: Eradication of T.B. from infected cattle and pasteurization of milk to reduce infection with M. bovis. Improvement of socio-economic states (e.g. poverty, and malnutrition) Treatment of activeand latent T.B. BCGvaccine containing live, attenuated Mycobacterium bovis organisms may prevent or limit extent of disease but does not prevent infection with M. tuberculosis. Indications:(1)In Egypt, the vaccine is given in the first year of life, as a single intradermal injection in the deltoid region. (2)Tuberculin negative - 82 -
contacts of a case. (3)Tuberculin negative medical staff and health care officers. Atypical Mycobacteria *These mycobacteria are called atypical because they differ from M. tuberculosis in various ways. They are also called "Mycobacteria other than M. tuberculosis," or MOTTS. *The most important difference is that the atypicals are found in the environment, whereas M. tuberculosis is found only in humans. The atypicals are subdivided into slow growers and rapid growers based on whether they form colonies in more than or less than 7 days. The following are important slow growers: 1. Mycobacterium avium-intracellulare complex (MAC) causes tuberculosis-like disease, especially in immunocompromised patients, such as those with AIDS. It is highly antibiotic-resistant. 2. Mycobacterium kansasii also causes tuberculosis-like disease but is less antibiotic-resistant than MAC. 3. Mycobacterium marinum causes "swimming pool granuloma or fish tank granuloma," which is a skin lesion at the site of an abrasion acquired in a swimming pool or an aquarium. 4. Mycobacterium scrofulaceum causes scrofula, which manifests as swollen, nontender cervical lymph nodes (cervical adenitis). The important rapid grower is Mycobacterium fortuitum-chelonei complex, which causes infections of prosthetic joints and indwelling catheters. It also causes skin and soft tissue infections at the site of puncture wounds. The organisms are usually resistant to most antituberculosis drugs. Mycobacterium leprae Aerobic, acid-fast rods. Cannot be cultured in vitro. Optimal growth at less than body temperature, so lesions are on cooler parts of the body, such as skin, nose, and superficial nerves.It is the causative of Leprosy. Pathogenesis: 1) Humans are the reservoir. Also found in armadillos, but it is uncertain whether they are a source of infections for humans. 2) Most important mode of transmission is probably nasal secretions of patients with the lepromatous form. Patients with the lepromatous form are more likely to transmit than those with the tuberculoid form because they have much higher numbers of organisms. Prolonged exposure is usually necessary. 3)Lesions usually occur in the cooler parts of the body, e.g., skin and peripheral nerves. 4) In tuberculoid leprosy, destructive lesions are due to the cellmediated response to the organism. Damage to fingers is due to burns and other trauma, because nerve damage causes loss of sensation. 5)In lepromatous leprosy, the cell-mediated response to M. leprae is lost and large numbers of organisms appear in the lesions and blood. Clinically: A) Tuberculoid leprosy: hypopigmented macular or plaque-like skin lesions, thickened superficial nerves, and significant anesthesia of the skin lesions occur. B) Lepromatous leprosy: multiple nodular skin lesions occur, resulting in the typical leonine (lion-like) facies. After the onset of therapy, patients with - 83 -
lepromatous leprosy often develop erythema nodosum leprosum (ENL), which is interpreted as a sign that cell-mediated immunity is being restored. Laboratory Diagnosis: 1) Specimen is taken by scrapping or biopsy from nasal mucosa, earlobe and skin lesions. 2) Staining with modified Z-N technique (decolourization with 5% H2SO4), shows acid fast bacilli (Bacilli are abundant in lepromatous type and few in tuberculoid type). 3) Histopathological examination reveals the characteristic lesion. 4) No culture is available 5) PCR Treatment: Dapsone plus rifampin for the tuberculoid form. Clofazimine is added to that regimen for the lepromatous form or if the organism is resistant to dapsone. Treatment is for at least 2 years. Prevention: Dapsone for close family contacts. Actinomycetes Actinomyces israelii Anaerobic, gram-positive filamentous, branching rods.It causes actinomycosis (abscesses with draining sinus tracts). Pathogenesis: - Habitat is human mouth, especially anaerobic crevices around the teeth. Transmission into tissues occurs during dental disease or trauma. Organism also aspirated into lungs, causing thoracic actinomycosis. Retained intrauterine device (IUD) predisposes to pelvic actinomycosis. - Organism forms sinus tracts that open onto skin and contain "sulfur granules," which are mats of intertwined filaments of bacteria. Laboratory Diagnosis Gram-stained smear plus anaerobic culture on blood agar plate. - "Sulfur granules" visible in the pus. - No serologic tests. Treatment: Penicillin G and surgical drainage. Nocardia asteroides Aerobic, gram-positive filamentous, branching rods. Weakly acid-fast. It is the cause of nocardiosis (especially lung and brain abscesses). Pathogenesis: Habitat is the soil. Transmission is via airborne particles, which are inhaled into the lungs. Immunosuppression and cancer predispose to infection. Laboratory Diagnosis: -Gram-stained smear and modified Ziehl-Neelsen stain. Aerobic culture on blood agar plate. No serologic tests. Treatment: Sulfonamides. Mycoplasmas 1)Smallest free-living organisms. 2) Not seen on Gram-stained smear because they have no cell wall, so dyes are not retained. 3) It is the only bacteria with cholesterol in cell membrane. 3)Can be cultured in vitro. A)Mycoplasma pneumoniae is the cause of atypical pneumonia B)Mycoplasma hominis has been implicated as an infrequent cause of pelvic inflammatory disease. C) Ureaplasma urealyticum may cause non-gonococcal urethritis. Mycoplasma pneumoniae Pathogenesis: -Habitat is the human respiratory tract. Transmission is via - 84 -
respiratory droplets.-It produces hydrogen peroxide, which may damage the respiratory tract. - During M. pneumoniae infection, autoantibodies are produced against red cells (cold agglutinins) and brain, lung, and liver cells. These antibodies may be involved in some of the extrapulmonary manifestations of infection. It causes atypical pneumonia and bronchitis. Clinically: The onset of Mycoplasma pneumonia is gradual, usually beginning with a nonproductive cough, sore throat, or earache. Small amounts of whitish, nonbloody sputum are produced. - Constitutional symptoms of fever, headache, malaise, and myalgias are pronounced. -The disease resolves spontaneously in 10 to 14 days. -The extrapulmonary manifestations include Stevens-Johnson syndrome, erythema multiforme, Raynaud's phenomenon, cardiac arrhythmias, arthralgias, hemolytic anemia, and neurologic manifestations such as Guillain-Barré syndrome. Laboratory Diagnosis: 1)Gram stain not useful. 2) Can be cultured on special bacteriologic media but takes at least 10 days to grow. 3)Positive cold-agglutinin test is presumptive evidence. 4) Complement fixation test for antibodies to Mycoplasma pneumoniae is more specific. Treatment: Erythromycin or tetracycline. Spirochetes Treponema pallidum It is Spirochetes. Not seen on Gram-stained smear because organism is too thin. Not cultured in vitro. It causes Syphilis. Pathogenesis: Habitat is the human genital tract. T. pallidum is transmitted by intimate contact, Sexually, from pregnant women to their fetuses and rarely, by blood transfusions. Organism multiplies at site of inoculation and then spreads widely via the bloodstream. Many features of syphilis are attributed to blood vessel involvement causing vasculitis. Clinically: The organism often infects the endothelium of small blood vessels, causing endarteritis. This occurs during all stages of syphilis but is particularly important in the pathogenesis of the brain and cardiovascular lesions seen in tertiary syphilis. 1)Primary syphilis, the Spirochetes multiply at the site of inoculation and a local, non-tender ulcer (chancre) usually forms in 2 to 10 weeks. The ulcer heals spontaneously, but Spirochetes spread widely via the bloodstream (bacteremia) to many organs. 2) One to three months later, the lesions of secondary syphilis may occur. These often appear as a maculopapular rash, notably on the palms and soles, or as moist papules on skin and mucous membranes (mucous patches). Moist lesions on the genitals are called condylomata lata. These lesions are rich in Spirochetes and are highly infectious, but they also heal spontaneously. Patchy alopecia also occurs. Constitutional symptoms of secondary syphilis include low-grade fever, malaise, anorexia, weight loss, headache, myalgias, and generalized lymphadenopathy. There may be internal organ involvement (meningitis, nephritis, hepatitis, etc). These stages may be asymptomatic, and yet the disease may progress. - 85 -
About one-third of these early (primary and secondary) syphilis cases will "cure" themselves, without treatment. 3)Another third remain latent, i.e., no lesions appear, but positive serologic tests indicate continuing infection. In the remaining one-third of people, the disease progresses to the tertiary stage. 4)Tertiary syphilis may show granulomas (gumma), especially of skin and bones; central nervous system involvement (e.g., tabes, paresis); or cardiovascular lesions (e.g., aortitis, aneurysm of the ascending aorta). In tertiary lesions, treponemes are rarely seen. 5)T. pallidum also causes congenital syphilis. The organism is transmitted across the placenta, typically after the third month of pregnancy, and fetal infection can occur. Skin and bone lesions are common, as is hepatosplenomegaly. Unless the disease is treated promptly, stillbirth or multiple fetal abnormalities occur. Laboratory Diagnosis Microscopy: Spirochetes are demonstrated in the lesions of primary or secondary syphilis, such as chancres or condylomatalata, by darkfield microscopy or by direct fluorescent antibody (DFA) test. They are not seen on a T.pallidum (DFA) Gram-stained smear. Serology:T. pallidum induce specific antibodies and nonspecific antibodies (reagin) which can be detected by the flocculation of lipids (cardiolipin) extracted from normal mammalian tissues, e.g., beef heart. Nonspecific Serologic Tests: These tests involve the use of nontreponemal antigens. Extracts of normal mammalian tissues (e.g., cardiolipin from beef heart) react with non specific antibodies in serum samples from patients with syphilis. These antibodies, which are a mixture of IgG and IgM, are called "reagin" antibodies. Tests used: VDRL (Venereal Disease Research Laboratory) RPR (rapid plasma reagin) tests These tests are positive in primary and secondary syphilis. The titer of these nonspecific antibodies decreases with effective treatment, in contrast to the specific antibodies, which are positive for life. These tests are inexpensive and easy to perform and therefore are used as a method of screening the population for infection. False-positive reactions occur in infections such as leprosy, hepatitis B, and infectious mononucleosis and in various autoimmune diseases. Therefore, positive results have to be confirmed by specific tests. Results of nonspecific tests usually become negative after treatment and should be used to determine the response to treatment. Falsely negative: may occur due to the prozone phenomenon. Specific Serologic Tests -Fluorescent Treponema antibody (FTA) - Treponema Palladium Hem-Agglutination (TPHA) assays with specific treponemal antibodies in the patient's serum. - 86 -
-These antibodies arise within 2 to 3 weeks of infection and remain positive for life after effective treatment and cannot be used to determine the response to treatment or re-infection. -They are more expensive and more difficult to perform than the nonspecific tests and therefore are not used as screening procedures. Treatment: Penicillin is effective in the treatment of all stages of syphilis. In primary and secondary syphilis, benzathine penicillin G is used.If the patient is allergic to penicillin, doxycycline can be used but must be given for prolonged periods. Prevention: Benzathine penicillin given to contacts. Borrelia burgdorferi The causative agent ofLyme disease. It is a Spirochete. Gram stain not useful. Can be cultured in vitro, but not usually done. Pathogenesis -The main reservoir is the white-footed mouse. Transmitted by the bite of ticks. -Organism invades skin, causing a rash called erythema chronicum migrans. It then spreads via the bloodstream to involve primarily the heart, joints, and central nervous system. The rash can be accompanied by nonspecific "flulike" symptoms such as fever, chills, fatigue, and headache. Secondary skin lesions frequently occur. Arthralgias, but not arthritis, are another common finding in the early stage. In stage 2, which occurs weeks to months later, cardiac and neurologic involvement predominates. Myocarditis, accompanied by various forms of heart block, occurs. Acute (aseptic) meningitis and cranial neuropathies, such as facial nerve palsy (Bell's palsy), are prominent during this stage. Bilateral facial nerve palsy is highly suggestive of Lyme disease. Laboratory Diagnosis Diagnosis usually made serologically by ELISA, i.e., by detecting IgM antibody. Confirm positive serologic test with Western blot assay. Treatment: Doxycycline for early stages; penicillin G for late stages. Borrelia recurrentis & Borrelia hermsii Borrelia recurrentis, Borrelia hermsii, and several other borreliae cause relapsingfever. During infection, the antigens of these organisms undergo variation. As antibodies develop against one antigen, variants emerge and produce relapses of the illness. This can be repeated 3 to 10 times. B. recurrentis is transmitted from person to person by the human body louse. Humans are the only hosts. B. hermsii and many other Borrelia species are transmitted to humans by soft ticks. Rodents and other small animals are the main reservoirs. These species of Borrelia are passed transovarially in the ticks, a phenomenon that plays an important role in maintaining the organism in nature. Clinically: during infection, the arthropod bite introduces spirochetes which then multiply in many tissues, producing fever, chills, headaches, and multiple-organ dysfunction. Each attack is terminated as antibodies arise. - 87 -
Diagnosis is usually made by seeing the large spirochetes in stained smears of peripheral blood. They can be cultured in special media. Treatment: Tetracycline may be beneficial early in the illness and may prevent relapses. Avoidance of arthropod vectors is the best means of prevention. Leptospira interrogans Spirochetes that can be seen on darkfield microscopy but not light microscopy. Can be cultured in vitro. Leptospira interrogans is the cause of leptospirosis. Pathogenesis: Habitat is wild and domestic animals. Transmission is chiefly via dog, livestock, and rat urine which contaminates water and soil. Swimming in contaminated water or consuming contaminated food or drink can result in human infection. Clinically: Human infection results when Leptospirae are ingested or pass through mucous membranes or skin. They circulate in the blood and multiply in various organs, producing fever and dysfunction of the liver (jaundice), kidneys (uremia), lungs (hemorrhage), and central nervous system (aseptic meningitis). The illness is typically biphasic, with fever, chills, intense headache, and conjunctival suffusion (diffuse reddening of the conjunctivae) appearing early in the disease, followed by a short period of resolution of these symptoms as the organisms are cleared from the blood. The second, "immune," phase is most often characterized by the findings of aseptic meningitis and, in severe cases, liver damage (jaundice) and impaired kidney function. Laboratory Diagnosis Darkfield microscopy and culture in vitro are available but not usually done. Diagnosis usually made by ELISA confirmed by western blotting in patient's serum. Treatment:Penicillin G. There is no significant antibiotic resistance. Prevention:Doxycycline effective for short-term exposure. Vaccination of domestic livestock and pets. Rat control. Chlamydiae Chlamydiae are obligate intracellular bacteria. They lack the ability to produce sufficient energy to grow independently and therefore can grow only inside host cells. Their cell walls resemble those of gram-negative bacteria but lack muramic acid. Chlamydiae have a special replicative cycle. The cycle begins when the extracellular, metabolically inert, "sporelike" elementary body enters the cell and reorganizes into a larger, metabolically active reticulate body. The latter undergoes repeated binary fission to form daughter elementary bodies, which are released from the cell. Within cells, the site of replication appears as an inclusion body, which can be stained and visualized microscopically. Chlamydia trachomatis Pathogenesis:Habitat is the human genital tract and eyes. Transmission is by sexual contact and during passage of neonate through birth canal. Transmission in trachoma is chiefly by hand-to-eye contact. Clinically: Nongonococcal urethritis, cervicitis, inclusion conjunctivitis, lymphogranuloma venereum, and trachoma. Also pneumonia in infants. - 88 -
Laboratory Diagnosis 1-Cytoplasmic inclusions seen on Giemsa-stained or fluorescent antibodystained smear.Glycogen-filled cytoplasmic inclusions can be visualized with iodine. 2- Organism grows in cell culture and embryonated eggs, but these are not often used. 3-PCR-based assay and an ELISA using patient's urine are available. Treatment:A tetracycline (such as doxycycline) or a macrolide (such as azithromycin). Prevention:Erythromycin effective in infected mother to prevent neonatal disease. Chlamydia pneumoniae It causes atypical pneumonia. Pathogenesis: Habitat is human respiratory tract. Transmission is by respiratory aerosol. Laboratory Diagnosis Serologic tests for antibody in patient's serum. Treatment A tetracycline, such as doxycycline. Chlamydia psittaci Disease: Psittacosis(Atypical pneumonia). Pathogenesis: Habitat is birds, both psittacine and others. Transmission is via aerosol of dried bird feces. Laboratory Diagnosis: Diagnosis usually made by testing for antibodies in patient's serum. Cytoplasmic inclusion seen by Giemsa or fluorescent antibody staining. Organism can be isolated from sputum by cell culture, but this is rarely done. Treatment: Tetracycline. Rickettsiae Rickettsiae are obligate intracellular bacteria. They are the agents of typhus, spotted fevers, and Q fever. Formerly, Rickettsiae were thought to be viruses as they are obligate intracellular parasites, because they are unable to produce sufficient energy to replicate extracellularly. Therefore, rickettsiae must be grown in cell culture, embryonated eggs, or experimental animals. Rickettsiae are very short rods that are barely visible in the light microscope. But they are bacteria as, they have cell wall resembles that of gram-negative rods, but they stain poorly with the standard Gram stain. Rickettsiae divide by binary fission within the host cell and treated by antibiotics. Several rickettsiaepossess antigens that cross-react with antigens of the OX strains of Proteus vulgaris. The Weil-Felix test, which detects anti-rickettsial antibodies in a patient's serum by agglutination of the Proteus organisms, is based on this cross-reaction. Transmission -The most striking aspect of the life cycle of the rickettsiae is that they are - 89 -
maintained in nature in certain arthropods such as ticks, lice, fleas, and mites andare transmitted to humans by the bite of the arthropod. The rickettsiae circulate widely in the bloodstream (bacteremia), infecting primarily the endothelium of the blood vessel walls. -The exception to arthropod transmission is C. burnetii, the cause of Q fever, which is transmitted by aerosol and inhaled into the lungs. -Virtually all rickettsial diseases are zoonoses (i.e., they have an animal reservoir), with the prominent exception of epidemic typhus, which occurs only in humans. It occurs only in humans because the causative organism, R. prowazekii, is transmitted by the human body louse. Pathogenesis The typical lesion caused by the rickettsiae is a vasculitis, particularly in the endothelial lining of the vessel wall where the organism is found. Damage to the vessels of the skin results in the characteristic rash and in edema and hemorrhage caused by increased capillary permeability. Disease
Organism
Arthropod Mammalian Important in the Vector Reservoir United States
R. rickettsii
Ticks
Dogs, rodents Yes (especially in southeastern states such as North Carolina)
R. akari
Mites
Mice
No
Spotted fevers Rocky Mountain spotted fever Rickettsialpox Typhus group Epidemic
R. prowazekii Lice
Humans
No
Endemic
R. typhi
Fleas
Rodents
No
Scrub
R. Mites tsutsugamushi
Rodents
No
C. burnetii
Cattle, sheep, Yes goats
Others Q fever
None
Typhus. Epidemic typhus is transmitted from person to person by the human body louse, Pediculus. Endemic typhus flea-borne caused by Rickettsia typhi (murine typhus) Chigger-borne scrub typhus caused by R. tsutsugamushi, and several other quite rare forms. Clinically: -Typhus begins with the sudden onset of chills, fever, headache, and other influenza-like symptoms approximately 1 to 3 weeks after the louse bite occurs. -Between the fifth and ninth days after the onset of symptoms, a maculopapular rash begins on the trunk and spreads peripherally. - 90 -
-Signs of severe meningoencephalitis, including delirium and coma, begin with the rash and continue into the second and third weeks. -In untreated cases, death occurs from peripheral vascular collapse or from bacterial pneumonia. - Brill-Zinsser disease: is a recurrent form of epidemic typhus. The signs and symptoms are similar to those of epidemic typhus but are less severe, of shorter duration, and rarely fatal. Laboratory Diagnosis Laboratory diagnosis of rickettsial diseases is based on serologic analysis rather than isolation of the organism. The serologic tests: - The indirect immunofluorescence and ELISA tests are most often used. -The Weil-Felix test is of historic interest.The Weil-Felix test is based on the cross-reaction of an antigen present in many rickettsiae with the O antigen polysaccharide found in P. vulgaris OX-2, OX-19, and OX-K. The test measures the presence of anti-rickettsial antibodies in the patient's serum by their ability to agglutinate Proteus bacteria. Treatment: The treatment of choice for all rickettsial diseases is tetracycline, with chloramphenicol as the second choice. Prevention -Anti-arthropod measures. - Personal hygiene and "delousing" with DDT. A typhus vaccine containing formalin-killed R. prowazekii organisms is effective. Coxiella burnetii Pathogenesis: Obligate intracellular parasites. C. burnetii has a spore-like stage that is highly resistant to drying, which enhances its ability to cause infection. Habitat is domestic livestock. Transmission is by inhalation of aerosols of urine, feces, amniotic fluid, or placental tissue. Clinically: It causes Q fever. The main organ involved in Q fever is the lungs. It begins suddenly with fever, severe headache, cough, and other influenza-like symptoms. Hepatitis is frequent enough that the combination of pneumonia and hepatitis should suggest Q fever. Rarely, chronic Q fever characterized by lifethreatening endocarditis occurs. Laboratory Diagnosis Diagnosis usually made by serologic tests. Weil-Felix test is negative. Stain and culture rarely done. Treatment: Tetracycline. Prevention: Killed vaccine for persons in high-risk occupations. Minor Bacterial Pathogens Only the more important of the minor bacterial pathogens are summarized in this section. Bartonella henselae Gram-negative rod. Causes cat-scratch fever in immunocompetent individuals and bacillary angiomatosis in immunocompromised, especially AIDS, patients. Found as normal flora in the mouth of cats. Transmitted to humans by cat bite or scratch and from cat to cat by fleas. Ehrlichia chaffeensis - 91 -
Member of rickettsia family. Causes human monocytic ehrlichiosis. Transmitted from dog reservoir to humans by ticks, especially Dermacentor, the dog tick. Endemic in southern states, e.g., Arkansas. Forms morulae in cytoplasm of monocytes. (A morula is a "mulberry-shaped" inclusion body composed of many E. chaffeensis cells.) Fusobacterium nucleatum Anaerobic gram-negative rod with pointed ends. Member of the normal human flora in mouth, colon, and female genital tract. Causes brain, lung, abdominal, and pelvic abscesses, typically in combination with other anaerobes and facultative bacteria. Gardnerella vaginalis Facultative gram-variable rod. Involved in bacterial vaginosis, along with Mobiluncus species, which are anaerobic. See "clue cells," which are vaginal epithelial cells covered with G. vaginalis cells. Positive "whiff" test found in bacterial vaginosis. Haemophilus ducreyi Small gram-negative rod. Causes chancroid. Sexually transmitted disease with painful ulcer on genitals (in contrast to syphilis, which is painless). To grow in culture, it requires factor X (heme) but not factor V (in contrast to H. influenzae, which requires both). Moraxella catarrhalis Small coccobacillary gram-negative rod that resembles the cocci of the genus Neisseria. Causes otitis media and sinusitis primarily in children. Also causes bronchitis and pneumonia, primarily in older people with chronic obstructive pulmonary disease. It is found only in humans and is transmitted by respiratory aerosol. Yersinia enterocolitica Gram-negative rods. Causes enterocolitis similar to that caused by Shigella and Salmonella. Also causes mesenteric adenitis, which can mimic appendicitis. Found in domestic animals and transmitted to humans by fecal contamination of food. Burkholderia cepacia Gram-negative rod resembling P. aeruginosa .Important cause of chronic infections in patients with cystic fibrosis. Formerly called Pseudomonas cepacia. Stenotrophomonas maltophilia Gram-negative rod resembling P. aeruginosa. Important cause of chronic infections in patients with cystic fibrosis. Formerly called Pseudomonas maltophilia.
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General Mycology Structure&Growth 1. The fungi are eukaryotes. 2. The fungal cell wall consists primarily of chitin (not peptidoglycan as in bacteria); thus, fungi are insensitive to antibiotics, such as penicillin, that inhibit peptidoglycan synthesis. 3. The fungal cell membrane contains ergosterol, in contrast to the human cell membrane, which contains cholesterol. The selective action of amphotericin B and azole drugs, such as fluconazole and ketoconazole, on fungi is based on this difference in membrane sterols. 4. Most fungi are obligate aerobes; some are facultative anaerobes; but none are obligate anaerobes. 5. All fungi require a preformed organic source of carbon—hence their frequent association with decaying matter. 6. The natural habitat of most fungi is, therefore, the environment. An important exception is Candida albicans, which is part of the normal human flora. Morphological Classification: Yeasts grow as single cells that reproduce by asexual budding. Molds grow as long filaments (hyphae) and form a mat (mycelium). Some hyphae form transverse walls (septate hyphae), whereas others do not (nonseptate hyphae). Nonseptate hyphae are multinucleated (coenocytic). Dimorphic fungi: They exist as molds in the environment at room temperature and as yeasts in human tissues at body temperature. Sexual classification: 1. Some fungi reproduce sexually by mating and forming sexual spores, e.g., zygospores, ascospores, and basidiospores. Zygospores are single large spores with thick walls Ascospores are formed in a sac called ascus Basidiospores are formed externally on the tip of a pedestal called a basidium. 2. The classification of these fungi is based on their sexual spores. Fungi that do not form sexual spores are termed "imperfect" and are classified as fungi imperfecti. 3. Most fungi of medical interest propagate asexually by forming conidia (asexual spores) from the sides or ends of specialized structures. The shape, color, and arrangement of conidia aid in the identification of fungi. Some important conidia are (1) arthrospores, which arise by fragmentation of the ends of hyphae and are the mode of transmission of Coccidioides immitis; (2) chlamydospores, which are rounded, thick-walled, and quite resistant (the terminal chlamydospores of Candida albicans aid in its identification) - 93 -
(3) blastospores, which are formed by the budding process by which yeasts reproduce asexually (some yeasts, e.g., Can. albicans, can form multiple buds that do not detach, thus producing sausagelike chains called pseudohyphae, which can be used for identification) (4) sporangiospores, which are formed within a sac (sporangium) on a stalk by molds such as Rhizopus and Mucor. Clinical classification of fungal diseases: 1- Superficial mycoses: these are fungal infections that are confined to the stratum corneum without tissue invasion e.g. Tinea versicolor. 2- Cutaneous mycoses: are fungal infections that involve the skin, nail or hair with tissue destruction and immunological reaction e.g. dermatophytes . 3- Subcutaneous mycoses: are infections of subcutaneous tissue without dissemination to distant sites as mycetoma. 4- Opportunistic mycoses :a) systemic candidiasisb) Cryptococcosis c) Aspergillosis d) Mucormycosis (zygomycosis) 5- Systemic (endemic) mycoses: are primary pulmonary lesions that may disseminate to any organ mainly in immune-compromised patients but can affect immune-competent personnel a)Histoplasmosis b) Coccidiodomycosis c)Blastomycosis d) Paracoccididomycosis 6- Allergies to fungal spores: as Aspergillus. Mainly type I hypersensitivity reactions or atopy manifesting as bronchial asthma, hay fever ... .etc. 7- Mycotoxicosis:these are diseases due to the consumption of food containing fungal toxins e.g.a) Mycotoxicosis that occurs after eating Amanita mushrooms. These fungi produce five toxins, two of which—amanitin and phalloidin—are among the most potent hepatotoxins. B) Aspergillus flavus produce aflatoxins in spoiled grains and peanuts. The aflatoxins are metabolized in the liver to epoxide, a potent carcinogen.Aflatoxin B1 induces a mutation in the p53 tumor suppressor gene, leading to a loss of p53 protein and a consequent loss of growth control in the hepatocyte. C) Ergotism, is caused by the mold Claviceps purpura, which infects grains and produces alkaloids (e.g., ergotamine and lysergic acid diethylamide [LSD]) that cause pronounced vascular and neurologic effects. Laboratory Diagnosis of fungal infections: Microscopic examination of a KOH preparation can reveal the presence of fungal structures. The purpose of the KOH is to dissolve the human cells, allowing visualization of the fungi. Other stains: Calcofluor white is a fluorescent dye that binds to fungal cell walls and is useful in the identification of fungi in tissue specimens. Methenaminesilver stain is also useful in the microscopic diagnosis of fungi in tissue. Culture: Sabouraud's dextrose agar(SDA) is often used to grow fungi because its low pH inhibits the growth of bacteria, allowing the slower-growing fungi to emerge. Inhibition of bacterial growth is due to the low pH of the - 94 -
medium and to the chloramphenicol and cycloheximide that are frequently added. The appearance of the mycelium and the nature of the asexual spores are frequently sufficient to identify the organism. DNA probes can be used to identify fungi growing in culture at a much earlier stage, i.e., when the colony size is much smaller. Serological Tests for the presence of antibodies in the patient's serum antibodies (a significant rise in the antibody titer must be observed to confirm a diagnosis). Tests for the presence of fungal antigens in CSF by the latex agglutination test as in Cryptococcus. PCR for the detection of DNA of the fungi. Antifungal Therapy The drugs used to treat bacterial diseases have no effect on fungal diseases. For example, penicillins and aminoglycosides inhibit the growth of many bacteria but do not affect the growth of fungi. This difference is explained by the presence of certain structures in bacteria, e.g., peptidoglycan and 70S ribosomes, that are absent in fungi. The most effective antifungal drugs, amphotericin B and the various azoles, exploit the presence of ergosterol in fungal cell membranes that is not found in bacterial or human cell membranes. Amphotericin B disrupts fungal cell membranes at the site of ergosterol and azole drugs inhibit the synthesis of ergosterol, which is an essential component of fungal membranes. Another antifungal drug, caspofungin (Cancidas), inhibits the synthesis of β-glucan, which is found in fungal cell walls but not in bacterial cell walls. Human cells do not have a cell wall. Mechanism of Action and Adverse Effects of Antifungal Drugs Name of Drug Amphotericin B (usedIV)
Mechanism of Action
Important Adverse Reactions
Binds to ergosterol and disrupts fungal cell membranes
Renal toxicity, fever, and chills; monitor kidney function; use test dose; liposomal preparation reduces toxicity
Azoles ( used oral or parentral)such Inhibits ergosterol synthesis as fluconazole, ketoconazole, itraconazole, voriconazole, posaconazole
Ketoconazole inhibits human cytochrome P450; this decreases synthesis of gonadal steroids resulting in gynecomastia
Echinocandins(used IV)such as caspofungin, mycafungin
Inhibits synthesis of Dglucan, a component of fungal cell wall
Well-tolerated
Inhibits DNA synthesis; FC converted to fluorouracil, which inhibits thymidine synthetase
Bone marrow toxicity
Disrupts mitotic spindle by binding to tubulin
Liver toxicity
Inhibits ergosterol
Well-tolerated on skin
Flucytosine (FC)
Griseofulvin (oral)
Azoles (topical) such as
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clotrimazole, miconazole
synthesis
Terbinafine (topical) Tolnaftate (topical)
Both inhibits ergosterol synthesis
Well-tolerated on skin
Nystatin(topical)
Binds to ergosterol and disrupts fungal cell membranes
Well-tolerated on skin
Opportunistic Mycosis Candida Candida is a gram positive, oval, budding yeast. The principal pathogen is Candida albicans. Other species are C.tropicalis, C.parapsilosis, C.glabrataandC.krusei. Some of these species can be resistant to certain antifungal drugs. Pathogenesis &Clinical findings: Candidais a member of the normal flora , it is an opportunistic pathogen i.e.only affects host that has been predisposed physically or physiologically by certain predisposing factors as: extremes of age, pregnancy, debilitated or immunosuppressed patients, Budding oval Candida yeasts diabetes mellitus, use of broad-spectrum antibiotics or corticosteroids or cytotoxic drugs Clinically: 1-Superficial candidiasis :e.g. oropharyngeal candidiasis (oral thrush), vulvovaginitis and cutaneous candidiasis. 2-Deep candidiasis : e.g. urinary tract infection, candidemia,& endocarditis Laboratory Identification: 1-Specimen: swabs and scrapings from surface lesions, sputum, exudates, urine and blood. 2- microscopic visualization: yeast cells appear as oval gram-positive budding yeasts with pseudohyphae. 3-Culture: Blood agar, Sabouraud dextrose agar(SDA) is used to inhibit bacterial growth. Some specialized (chromogenic) agars are available to allow for easy differentiation of the main Candida species. 4-Biochemical reaction: Germ tube formation: most strains of C. albicans produce true hyphae when incubated for 2–3 hours in human serum. Chlamydospores formation on cornmeal agar Sugar fermentation and assimilation Treatment: Topical antifungal drugs may be used for superficial infections. An echinocandin if patients are severely or critically ill Fluconazole, or voriconazole or amphotericin B if patients are clinically stable Cryptococcus neoformans Cryptococcusis opportunistic yeast, and most infections occur in - 96 -
immunocompromised patients, particularly those with HIV infection. It has a large capsule. It occurs widely in nature & abundantly in dried pigeon droppings or soil contaminated with birds' droppings. Pathogenesis and clinical findings: The fungus is usually inhaled and the lungs are the principal portal of entry. Meningitis (acute or chronic) is the principal clinical manifestation of cryptococcosis. Laboratory identification: 1- Specimens: cerebrospinal fluid (CSF), sputum or Cryptococcus India ink preparation blood. 2- Microscopic visualization: CSF mounted in India preparationpreparation ink shows budding yeasts appear surrounded by large gelatinous capsule. 3- Culture:Cryptococci can be cultured from CSF and blood cultures on Sabouraud Dextrose Agar (SDA) to give mucoid colonies of typical capsulated yeast cells. 4- Biochemical reaction:urease positive. They also give diagnostic brown pigments on bird seed (Niger seed) media. 5- Serological reaction: detection of cryptococcalcapsular antigen in CSF or serum using anticapsular antibodies (e.g. by latex agglutination). Treatment: A combination of either amphotericin B or fluconazole with flucytosine is usually given initially for 4–6 weeks. Aspergillus fumigatus is the most pathogenic Spp. Other spp as A. spores niger and A. flavus. These fungi are abundant in the environment (soil, dust, and decaying organic matter). vesicle A. The lungs are the principal habitat of aspergillosis. Airborne spores are inhalation of Aspergillus spores is the main route of infection. B. Diseases: 1) Aspegilloma: is a fungus ball in preformed Hypha cavities as in pulmonary TB or otomycosis caused by Aspergillus A.niger. 2) Allergic broncho-pulmonary aspergillosis: is due to hypersensitivity reaction to the inhalation of Aspergillus spores. often in patients with asthma or cystic fibrosis. High Ig E, eosiophils and the formation of mucus plugs which may contain Aspergillus mycelium 3) Invasive pulmonary aspergillosis: life-threatening infection in immunosuppressed patients. 4)Disseminated Aspergillosis in highly immunosuppressed patients. 5)Mycotoxicosis: It secrets Aflatoxin which is heatic carcinogen. Laboratory identification: Histological detection or culture definitive diagnosis can only be made by deep tissue biopsies or sterile body fluids. 1) Specimens: sputum. 2) Microscopic visualization: long branching hyphal strands may be seen in sputum on direct examination or after digestion with 10% KOH especially in allergic aspergillosis. In invasive aspergillosis microscopic results may be negative. Biopsy is needed for definitive diagnosis. 3)Culture: Aspergillus grows readily Sabouraud Dextrose Agar (SDA) (without addition of cycloheximide which inhibit Aspergillus). 4)Serological reaction: high levels of - 97 -
Aspergillus antibodies in serum. Aspergillus antibodies are usually elevated with aspergillomas. Mucormycosis Mucormycosis (zygomycosis, phycomycosis) is a disease caused by saprophytic molds (e.g., Mucor, Rhizopus, and Absidia) found widely in the environment. These organisms are transmitted by airborne asexual spores and invade tissues of patients with reduced host defenses. They proliferate in the walls of blood vessels, particularly of the paranasal sinuses, lungs, or gut, and cause infarction and necrosis of tissue distal to the blocked vessel. Patients with diabetic ketoacidosis, burns, or leukemia are particularly susceptible. Rhizopusoryzae, causes about 60% of cases of mucormycosis. In biopsy specimens, organisms are seen microscopically as nonseptate hyphae with broad, irregular walls and branches that form more or less at right angles. Cultures on SDA. Its microscopyshows spores contained within a sporangium. Treatment of the underlying disorder, plus administration of amphotericin B and surgical removal of necrotic infected tissue, has resulted in some remissions and cures. Pneumocystis Pneumocystis jeroveci is classified as a yeast on the basis of molecular analysis, but medically many still think of it as a protozoan or as an "unclassified" organism. It causes fatal pneumonia in HIV patients. Penicillium marneffei Penicillium marneffei is a dimorphic fungus that causes tuberculosis-like disease in AIDS patients. It grows as a mold that produces a rose-colored pigment at 25°C but at 37°C grows as a small yeast that resembles Histoplasma capsulatum. Bamboo rats are the only other known hosts. The diagnosis is made either by growing the organism in culture or by using fluorescent antibody staining of affected tissue. The treatment of choice consists of amphotericin B for 2 weeks followed by oral itraconazole for 10 weeks. Relapses can be prevented with prolonged administration of oral itraconazole. Cutaneous Infections: Malassezia furfur - It is a lipophilic yeast which constitutes a part of the normal skin flora. - It produces a common, mild superficial skin infection called pityriasisversicolor, which is a disfiguring skin condition characterized by the development of numerous brown scaly patches, and sometimes hypopigmentation of the skin. -Diagnosis is usually made by KOH preparations of skin scrapings. The lesions contain both budding yeast cells and hyphae(Spaghetti and meat balls). - Treatment is with topical antifungal creams or oral itraconazole. Dermatophytes Dermatophytes are a group of filamentous fungi that cause superficial infection of - 98 -
keratinized structures (skin, nails and hair). There are three genera of dermatophytes: 1)Trichophyton: Large, 2) Microsporum: Thick smooth, thin wall, septate, pencil-shaped
wall, spindle shape, multicellular.
3) Epidermophyton: Bifurcated hyphae with multiple, smooth, club shaped macroconidia (2-4 cells)
The natural reservoir of dermatophytes can be humans (anthropophilic), animals (zoophilic), or soil (geophilic). Common species causing human infection include E. floccosum, T. rubrum, T. mentagrophytes, T. tonsurans(anthropophilic) and M. canis(zoophilic). Pathogenesis and clinical findings Clinical infection is known as ring worm or tinea with active (extending) edge and healing centers. 1-Tinea pedis (Athelet's foot): between toes, there is itching, small vesicles, discharge, maceration and peeling2- Tinea corporis (ring worm): infection of non hairy skin of the body resulting in annular lesions.3-Tinea capitis (ring worm of scalp): infection of the skin of the scalp and hair follicles. There are circular bold patches with short hair stubs.4- Tinea ungium: infection of nail. 5-Favus: circular, cup-shaped crusts (scutula) grouped in patches like a piece of honeycomb. On healing, it leaves a shining bare patch devoid of hair. Laboratory Identification: 1- Tineacapitis lesions caused by Microsporum species can be detected by seeing fluorescence when the lesions are exposed to ultraviolet light from a Wood's lamp. 2- Specimens: skin scales, nail or hair after adding 10% KOH. 3- Microscopic visualization: branching hyphae and spores. 4- Culture: Dermatophytes grow readily on routine culture media e.g. SDA (with addition of cycloheximide and chloramphenicol). 5- Slide culture: to see the microscopic morphology of the micro- and macroconidia; the most reliable identification character. Treatment and prevention -Topically, clotrimazole, butenafine, miconazole, and terbinafine. -Oral: fluconazole, griseofulvin, terbinafine, and itraconizole. Subcutaneous Mycoses These are caused by fungi that grow in soil and on vegetation and are introduced into subcutaneous tissue through trauma. Sporotrichosis Sporothrixschenckii is a dimorphic fungus. The mold form lives on plants and the - 99 -
yeast form occurs in human tissue. When spores of the mold are introduced into the skin, typically by a thorn, it causes a local pustule or ulcer with nodules along the draining lymphatics. The lesions are typically painless. Sporotrichosis occurs most often in gardeners, especially those who prune roses, because they may be stuck by a rose thorn. In the clinical laboratory, round or cigar-shaped budding yeasts are seen in tissue specimens. In culture at room temperature, hyphae occurs bearing oval conidia in clusters at the tip of slender conidiophores (resembling a daisy). The drug of choice for skin lesions is itraconazole. It can be prevented by protecting skin when touching plants, moss, and wood. Chromomycosis This is a slowly progressive granulomatous infection that is caused by several soil fungi (Fonsecaea, Phialophora, Cladosporium, etc.) when introduced into the skin through trauma. These fungi are collectively called dematiaceous fungi, so named because their conidia or hyphae are dark-colored, either gray or black.Wartlike lesions with crusting abscesses extend along the lymphatics. The disease occurs mainly in the tropics and is found on bare feet and legs. In the clinical laboratory, dark brown, round fungal cells are seen in leukocytes or giant cells. The disease is treated with oral flucytosine or thiabendazole, plus local surgery. Mycetoma Mycotic mycetoma: Soil fungi (Petriellidium, Madurella) enter through wounds on the feet, hands, or back and cause abscesses, with pus discharged through sinuses. The pus contains compact colored granules. Actinomycotic mycetoma:Actinomycetes such as Nocardia can cause similar lesions. Sulfonamides may help the actinomycotic form. There is no effective drug against the fungal form; surgical excision is recommended. Dimorphic fungi Dimorphic fungi arecharacterized bymycelial (filamentous) growth at 22°C, but development of yeast forms at 35–37°C in the human body. There are a number of dimorphic fungi that can cause human infection including: Sporothrix shenckii (sporotrichosis) Histoplasma capsulatum (histoplasmosis) Coccidioides immitis (coccidioidomycosis) Histoplasmosis Histoplasma capsulatumis a dimorphic fungus principally found in North, Central and South America. In most humans, exposure to Histoplasmacauses asymptomatic infection, or only a mild, self-limiting chest infection. In some patients, however, a more chronic pulmonary infection occurs that resembles tuberculosis. Occasionally disseminated histoplasmosis develops. Treatment : amphotericin B or itraconazole. Coccidioidomycosis Coccidioides immitisis a fungus found only in soil in South Western USA and some parts of Central and South America. Infection occurs after inhalation of airborne spores. It is similar to histoplasmosis. - 100 -
General Virology Virus Size & Structure Viruses range in size from that of large proteins (~20 nm) to that of the smallest cells (~300 nm). Most viruses appear as spheres or rods in the electron microscope. Viruses contain either DNA or RNA, but not both. All viruses have a protein coat called a capsid that covers the genome. The capsid is composed of repeating subunits called capsomers. In some viruses, the capsid is covered with a lipoprotein envelope that becomes the outer surface. The structure composed of the nucleic acid genome and the capsid proteins is called the nucleocapsid. The repeating subunits of the capsid give the virus a symmetric appearance that is useful for classification purposes. Some viral nucleocapsids have spherical (icosahedral) symmetry, whereas others have helical symmetry. All human viruses that have a helical nucleocapsid are enveloped, i.e., there are no naked helical viruses that infect humans. Viruses that have an icosahedral nucleocapsid can be either enveloped or naked. Viral genome The genome of some viruses is DNA, whereas the genome of others is RNA. These DNA and RNA genomes can be either single-stranded or doublestranded. Some RNA viruses, such as influenza virus and rotavirus, have a segmented genome, i.e., the genome is in several pieces. All viruses have one copy of their genome (haploid) except retroviruses, which have two copies (diploid). Viral Proteins Viral surface proteins mediate attachment to host cell receptors. This interaction determines the host specificity and organ specificity(tropism) of the virus. The surface proteins are the targets of antibody, i.e., antibody bound to these surface proteins prevents the virus from attaching to the cell receptor. This "neutralizes" (inhibits) viral replication. Viruses also have internal proteins, some of which are DNA or RNA polymerases. The matrix protein mediates the interaction between the viral nucleocapsid proteins and the envelope proteins. Some viruses produce antigenic variants of their surface proteins that allow the viruses to evade our host defenses. Antibody against one antigenic variant (serotype) will not neutralize a different serotype. Some viruses have one serotype; others have multiple serotypes. Viral Envelope The viral envelope consists of a membrane that contains lipid derived from the - 101 -
host cell and proteins encoded by the virus. Typically, the envelope is acquired as the virus exits from the cell in a process called budding. Viruses with an envelope are less stable, i.e., they are more easily inactivated, than naked viruses (those without an envelope). In general, enveloped viruses are transmitted by direct contact via blood and body fluids, whereas naked viruses can survive longer in the environment and can be transmitted by indirect means such as the fecal–oral route. Atypical Virus-like Agents There are four exceptions to the typical virus as described above: 1. Defective viruses are composed of viral nucleic acid and proteins but cannot replicate without a "helper" virus, which provides the missing function. An example is Hepatitis D virus which cannot infect or replicate without the help of Hepatitis B virus. 2. Pseudovirions contain host cell DNA instead of viral DNA within the capsid. They are formed during infection with certain viruses when the host cell DNA is fragmented and pieces of it are incorporated within the capsid protein. Pseudovirions can infect cells, but they do not replicate. 3. Viroids consist solely of a single molecule of circular RNA without a protein coat or envelope. The RNA is quite small and apparently does not code for any protein. They cause several plant diseases but are not implicated in any human disease. 4. Prions Prions are infectious particles composed entirely of protein. They have no DNA or RNA. They cause diseases such as Creutzfeldt-Jakob disease and kuru in humans and mad cow disease and scrapie in animals. These diseases are called transmissible spongiform encephalopathies. The term spongiform refers to the sponge-like appearance of the brain seen in these diseases. The holes of the sponge are vacuoles resulting from dead neurons. Prion proteins are encoded by a cellular gene. When these proteins are in the normal, alpha-helix configuration, they are nonpathogenic, but when their configuration changes to a beta-pleated sheet, they aggregate into filaments, which disrupts neuronal function and results in the symptoms of disease. Prions are highly resistant to inactivation by ultraviolet light, heat, and other inactivating agents. As a result, they have been inadvertently transmitted by human growth hormone and neurosurgical instruments. Because they are normal human proteins, they do not elicit an inflammatory response or an antibody response in humans. Classification of viruses: -According to the envelope into, enveloped as influenza virus or non- enveloped as picornaviruses. -According to genome, DNA viruses as Herpes viruses or RNA viruses as Influenza viruses. Every type is further classified into whether it is single stranded (ss) or double stranded (ds) genome. RNA viruses are further classified into positive sense RNA or Negative sense RNA viruses. -According to symmetry into, icosahedral as herpesviruses or helical symmetry - 102 -
as influenza. Viral replication Virus replication is the intracellular phase of life cycle that starts by contact of a viron with the host cell depending completely on the cell ´s metabolic functions 1-Adsorbtion: Interaction between surface molecules on the target host cell and the receptor binding site on surface of the virus. 2-Penetration: depends on the nature of the virus (a)Enveloped Enter by fusion with plasma membrane or via endosomes at cell surface (b)Non- enveloped viruses: cross plasma membrane directly or through endosomes. 3-Uncoating: The physical separation of genome from its capsid to be free in the cell. It takes place in the cytoplasm and marks the beginning of eclipse phase. The eclipse phase is the periods in which no complete virus can be seen in the infected cell but the virus genome controls and directs the cell machinery to produce virus components. 4-Transcription: It is formation of virus mRNA and it depends on nucleic acid type: -ds DNA virus:mRNA is transcribed by DNA dependent RNA polymerase. -ss RNA (+sense): the ss RNA it self act as mRNA -ss RNA (-sense): mRNA is produced by RNA dependent RNA polymerase. -ss RNA of Retrovirus : complementary ss DNA is transcribed which is converted to ds DNA to be integrated into the cellular genome 5- Synthesis of viral proteins(Translation): ALL viruses use cellular ribosomes in this process to synthetise Early proteins (enzymes ) and Late proteins structural proteins. OVERVIEW OF VIRUS LIFE CYCLE Adsorption (cell Surface)
Penetration Uncoating (Cyto. or Nuc.) Eclipse Begins
Biosynthetic Period (Cyto. and/or Nuc.) Genome Replication Assembly (Cyto. or Nuc.)
Eclipse Ends
Release From Cell Infection of Other Cells
6- Synthssis of nucleic acid:Using a strand of parental nucleic acid as templete to produce progeny nucleic acids copies. 7-Assembly: of viral capsid with its genome to form mature virus. - 103 -
8-Release of virus from host cell: -Lytic viruses, usually non enveloped (eg. Polio): by lysis of the host cell. -Enveloped viruses(eg. Influenza): by budding from the cell surface All enveloped viruses acquire their envelope by budding through the external cell membrane as they exit the cell, except herpesviruses, which acquire their envelope by budding through the nuclear membrane. The matrix protein mediates the interaction of the nucleocapsid with the envelope. Lysogeny is the process by which viral DNA becomes integrated into host cell DNA, replication stops, and no progeny virus is made. Later, if DNA is damaged by, for example, UV light, viral DNA is excised from the host cell DNA and progeny viruses are made. The integrated viral DNA is called a prophage. Bacterial cells carrying a prophage can acquire new traits, such as the ability to produce exotoxins such as diphtheria toxin. Transduction is the process by which viruses carry genes from one cell to another. Lysogenic conversion is the term used to indicate that the cell has acquired a new trait as a result of the integrated prophage. Reassortment: is the exchange of segments of the genome RNA of influenza virus and it is important in the pathogenesis of the worldwide epidemics caused by this virus. Complementation occurs when one virus produces a protein that can be used by another virus. A medically important example is hepatitis D virus that uses the surface antigen of hepatitis B virus as its outer coat protein. Phenotypic mixing occurs when two different viruses infect the same cell and progeny viruses contain proteins of both parental viruses. Viral pathogenesis At the cellular level: Death of infected cells is probably caused by inhibition of cellular protein synthesis. Translation of viral mRNA into viral proteins preempts the ribosomes preventing synthesis of cellular proteins. Inclusion bodies are aggregates of virions in specific locations in the cell that are useful for laboratory diagnosis. Two important examples are Negri bodies in the cytoplasm of rabies virus-infected cells and owl's eye inclusions in the nucleus of cytomegalovirus-infected cells. Multinucleated giant cells form when cells are infected with certain viruses, notably herpesviruses and paramyxoviruses such as respiratory syncytial virus. Cytopathic effect (CPE) is a visual or functional change in infected cells typically associated with the death of cells. Malignant transformation occurs when cells are infected with oncogenic viruses. Transformed cells are capable of unrestrained growth. Some virus-infected cells appear visually and functionally normal, yet are producing large numbers of progeny viruses. The Infected Patient Viral infection in the person typically has four stages: incubation period, prodromal period, specific-illness period, and recovery period. The main portals of entry are the respiratory, gastrointestinal, and genital tracts, but through the skin, across the placenta, and via blood are important as well. - 104 -
Transmission from mother to offspring is called vertical transmission; all other modes of transmission, e.g., fecal–oral, respiratory aerosol, and insect bite, are horizontal transmission. Transmission can be from human to human or from animal to human. Most serious viral infections are systemic, i.e., the virus travels from the portal of entry via the blood to various organs. However, some are localized to the portal of entry, such as the common cold, which involves only the upper respiratory tract. Thesymptoms of viral diseases are usually caused by death of the infected cells and a consequent loss of function. For example, poliovirus kills neurons, resulting in paralysis. Immunopathogenesis is the process by which the symptoms of viral diseases are caused by the immune system rather than by the killing of cells directly by the virus. 1)Virus-infected cell are attacked and killed by cytotoxic T cells that recognize viral antigens on the cell surface. Damage to the liver caused by hepatitis viruses occurs by this mechanism. 2)The formation of virus–antibody complexes deposited in tissues can prouce symptoms as arthritis associated with parvovirus B19 or rubella virus infection. Virulence of viruses differs markedly from one virus to another and among different strains of the same virus. The genetic basis for these differences is not well understood. Strains with weakened (attenuated) virulence are often used in vaccines. Viruses can evade host defenses by producing multiple antigens, thereby avoiding inactivation by antibodies, and by reducing the synthesis of class I MHC proteins, thereby decreasing the ability of a cell to present viral antigens and blunting the ability of cytotoxic T cells to kill the virus-infected cells. Persistent Viral Infections Carrier state refers to people who produce virus for long periods of time and can serve as a source of infection for others. The carrier state that is frequently associated with hepatitis C virus infection is a medically important example. Latent infections are those infections that are not producing virus at the present time but can be reactivated at a subsequent time. The latent infections that are frequently associated with herpes simplex virus infection are a medically important example. Slow virus infections refer to those diseases with a long incubation period, often measured in years. Some, such as progressive multifocal leukoencephalopathy, are caused by viruses, whereas others, such as Creutzfeldt-Jakob disease, are caused by prions. The brain is often the main site of these diseases. Localized or Disseminated Infections Most viral infections are either localized to the portal of entry or spread systemically through the body. The best example of the localized infection is the common cold caused by rhinoviruses, which involves only the upper respiratory tract. Influenza is localized primarily to the upper and lower respiratory tracts. Respiratory viruses have a short incubation period because they replicate directly in the mucosa, but systemic infections like poliomyelitis - 105 -
and measles have a long incubation period because viremia and secondary sites of replication are required. One of the best-understood systemic viral infections is paralytic poliomyelitis. After poliovirus is ingested, it infects and multiplies within the cells of the small intestine and then spreads to the mesenteric lymph nodes, where it multiplies again. It then enters the bloodstream and is transmitted to certain internal organs, where it multiplies again. The virus reenters the bloodstream and is transmitted to the central nervous system, where damage to the anterior horn cells occurs, resulting in the characteristic muscle paralysis. It is during this obligatory viremia that circulating IgG antibodies induced by the polio vaccine can prevent the virus from infecting the central nervous system. Viral replication in the gastrointestinal tract results in the presence of poliovirus in the feces, thus perpetuating its transmission to others.
Diagnosis of viral infections: A. Microscopic Identification (1) Light microscopy can reveal characteristic inclusion bodies as negri bodies of rabies or multinucleated giant cells as the Tzanck smear, which shows herpesvirus-induced multinucleated giant cells in vesicular skin lesions. (2) UV microscopy is used for fluorescent antibody staining of the virus in infected cells. (3)Electron microscopy detects virus particles, as rota virus in stools or herpes simplex virus in skin vesicles. (4) ImmunoelectronMicroscopy: adding a homologous antibody to the specimen, aggregates of virus–antibody complexes are seen in the electron microscope. B. Viral isolation in cell culture: The Cell cultures are pieces of animal or human tissue to which trypsin is added to separate the cells. Cells are grown in a growth medium on glass or plastic tubes, bottles or plates with a flat side. A monolayer of cells is formed on the flat side into which the virus is inoculated. There are three types of cell cultures: 1. Primary cell lines prepared from organ fragments (e.g. monkey kidney) and they divide only several times. 2. Human diploid cell lines which are fibroblasts from human embryo tissue (e.g. lung tissue) with diploid chromosome number. They grow rapidly and can be subcultured up to 50 times. 3. Continuous (heteroploid) cell lines from tumor cells (e.gHeLa cell line from carcinoma of cervix). These can divide indefinitely. Virus growth in cell culture can be detected by: 1. Production of cytopathic effect (CPE) that can provide a presumptive identification. CPE is a change in the appearance of the virus-infected cells. This change can be in such features as size, shape, and the fusion of cells to form multinucleated giant cells (syncytia). CPE is usually a manifestation of virusinfected cells that are dying or dead. 2. Hemadsorption, i.e., attachment of erythrocytes to the surface of virusinfected cells as in influenza viruses. 3. Interference with the formation of a CPE by a second virus. For example, - 106 -
rubella virus, which does not cause a CPE, can be detected by interference with the formation of a CPE by certain enteroviruses such as echovirus or Coxsackie virus. 4. A decrease in acid production by infected, dying cells. This can be detected visually by a color change in the phenol red (a pH indicator) in the culture medium. The indicator remains red (alkaline) in the presence of virus-infected cells but turns yellow in the presence of metabolizing normal cells as a result of the acid produced. This technique can be used to detect certain enteroviruses. 5. A definitive identification of the virus grown in cell culture is made by using known antibody by serologic methods e.g. Complement fixation, hemagglutination inhibition, and neutralization of the CPE are the most frequently used tests. Other procedures such as fluorescent antibody, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy are also used in special instances. C. Serologic Procedures The presence of IgM can be used to diagnose current infection. The presence of fourfold or greater rising titer ofIgGbetween an acute and convalescent serum sample. D. Detection of Viral Antigens Viral antigens can be detected in the patient's blood or body fluids by various tests but most often by an ELISAasp24 antigen of human immunodeficiency virus and HBsAg of hepatitis B virus. E. Detection of Viral Nucleic Acids (1)genetic probe: Viral nucleic acids can be detected in the patient's blood or tissues with a piece of labeled complementary DNA or RNA (cDNA or cRNA). (2) polymerase chain reaction(PCR): If small amounts of viral nucleic acids are present in the patient, the polymerase chain reaction can be used to amplify the viral nucleic acids. AntiViral Drugs a) Using agents which prevent virus attachment, e.g: 1. Enfuvirtide, a "fusion inhibitor," which blocks entry of HIV into target CD4 cell. 2. Maraviroc, which inhibits binding of the gp120 envelope protein of HIV to the cell co-receptor CCR-5, are also useful b) Using agents which prevent virus uncoating, e.g: Pleconaril is a broad spectrum anti-picorna virus agent. It is orally bioavailable and reduces peak viral titres by more than 99%; symptoms are improved. In doing so it blocks attachment and uncoating of the viral particle Amantadine and rimantadine are active against influenza A viruses. They are believed to block cellular membrane ion channels. The target for both drugs is the matrix protein (M2). c) Using agents which block genome replication, e.g: Nucleoside analogues Nucleoside analogues are in fact pro-drugs, since they need to be phosphorylated - 107 -
before becoming effective. This is the key to their selectivity: Acyclovir is phosphorylated by HSV tk 200 times more efficiently than by cellular enzymes. The cell DNA polymerase is less sensitive to it than the viral DNA polymerase. Gancyclovir is 10 times more effective against CMV than acyclovir since it is specifically phosphorylated by a CMV-encoded kinase encoded by gene UL97 : c)Using agents which block virus assembly, maturation and release, e.g: Relenza taken as an aerosol and Tamiflu taken as a pill used to treat influenza. The latter is active against both A and B strains. Both function as neuraminidase inhibitors and prevent the release of budded viruses from the cell. Because they act late in the life cycle of the virus it is hoped that problems with resistance emergence will be minimised. Tamiflu is reported to be 90% effective as a prophylactic agent. Therapy of HIV Infection: Several distinct classes of drugs are now used to treat HIV infection: 3. Nucleoside-Analog Reverse Transcriptase Inhibitors (NRTI). These drugs inhibit viral RNA-dependent DNA polymerase (reverse transcriptase) and are incorporated into viral DNA (they are chain-terminating drugs). o Zidovudine (AZT = ZDV, Retrovir) first approved in 1987 o Didanosine (ddI, Videx) o Zalcitabine (ddC, Hivid) o Stavudine (d4T, Zerit) o Lamivudine (3TC, Epivir) 4. Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). In contrast to NRTIs, NNRTIs are not incorporated into viral DNA; they inhibit HIV replication directly by binding non-competitively to reverse transcriptase. o Nevirapine (Viramune) o Delavirdine (Rescriptor) 5. Protease Inhibitors. These drugs are specific for the HIV-1 protease and competitively inhibit the enzyme, preventing the maturation of virions capable of infecting other cells. o Saquinavir (Invirase) first approved in 1995 o Ritonavir (Norvir) o Indinavir (Crixivan) o Nelfinavir (Viracept) 6. Enfuvirtide, a "fusion inhibitor," which blocks entry of HIV into target CD4 cell. 7. Maraviroc, which inhibits binding of the gp120 envelope protein of HIV to the cell co-receptor CCR-5, are also useful.
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Hepatitis Viruses 1. Viruses that primarily attack the liver are called hepatitis viruses. There are several types of hepatitis viruses including types A, B, C, D, E, and possibly G. Types A, B, and C are the most common. 2. All hepatitis viruses can cause acute hepatitis. 3. Viral hepatitis types B and C can cause chronic hepatitis. 4. Symptoms of acute viral hepatitis include fatigue, flu-like symptoms, dark urine, light-colored stools, fever, and jaundice; however, acute viral hepatitis may occur with minimal symptoms that go unrecognized. Rarely, acute viral hepatitis causes fulminant hepatic failure. 5. The symptoms of chronic viral hepatitis often are mild and nonspecific, and the diagnosis of chronic hepatitis often is delayed. 6. Chronic viral hepatitis often requires treatment in order to prevent progressive liver damage, cirrhosis, liver failure, and liver cancer. Many patients infected with HAV, HBV, and HCV have few or no symptoms of illness. For those who do develop symptoms of viral hepatitis, the most common are flu- like symptoms including: - Loss of appetite - Nausea -Vomiting -Fever -Weakness -Tiredness Aching in the abdomen Less common symptoms include:-Dark urine -Light-colored stools -Fever -Jaundice 7. Beside hepatitis markers, All acute hepatitis Hepatitis viruses induce abnormalities in CBC including thrombocytopenia or leucopenia with relative lymphocytosis. Liver functions elevated AST and ALT 8. There are non hepatotropic viruses that can also cause liver inflammation including Herpes simplex, Cytomegalovirus, Epstein–Barr virus, or Yellow fever. Virolological Characteristics of Hepatitis viruses Hepatitis A
Hepatitis B
Hepatitis C
Hepatitis Delta
Hepatitis E
Circular RNA Similar to similar to plant Calicivirus viroid
Virus family
Picornavirus
Hepadnavirus
Flavivirus
Nucleic acid
RNA (+ sense)
DNA (partially double strand)
RNA (+ sense) RNA (- sense)
RNA (+ sense)
Disease caused
Infectious hepatitis
Serum hepatitis
Non-A, non-B hepatitis
Enteric non-A, non-B hepatitis
Size
~ 28nm
~40nm
30 - 60nm
~ 40nm
30 - 35 nm
Envelope
No
Yes
Yes
Yes
No
Hepatitis A Virus It causes Hepatitis A. Naked nucleocapsid virus with a single-stranded, positive-polarity RNA. It has a single serotype. Pathogenesis:Transmission: Fecal–oral route. Blood-borne transmission of HAV is uncommon because viremia is brief and of low titer. - 109 -
Epidemilogical characters of Hepatitis viruses Virus
Disease
Source
Transmission
A
Infectious hepatitis
Feces
Enteric Fecal-Oral
B
C
D
E
Serum hepatitis
Non-A, non-B hepatitis Post transfusion hepatitis
Delta agent
Enteric non-A, non-B hepatitis
Blood and Blood and Blood and body fluids body fluids body fluids Feces Sexual contact Sexual contact Sexual contact Parenteral Percutaneo-us Permucosal Vertical (mother to fetus)
Parenteral Percutaneous Permucosal Vertical (mother to fetus)
Parenteral Percutaneous Permucosal Vertical (mother to fetus)
Enteric Fecal-Oral
Sexual transmission
Yes (especially homosexual)
Chronic infection
No
Yes
Yes
Yes
No
Incubation period
15 - 20 days
45 - 160
14 - 180
15 - 64
16 - 60
Carcinogenesis No
Hepatocellular Hepatocellular Hepatocellular No carcinoma carcinoma carcinoma
Cirrhosis
Yes
Yes
Sometimes severe 1 -2% mortality
Superinfection with HBV - often Usually (80%) very severe asymptomatic with high Up to 4% mortality rate mortality Co-infection with HBV often severe
Usually mild except in pregnancy
Vaccine
Behavior Modification Blood screening
Safe water No vaccine
Severity of disease
Prevention
Chemotherapy
No
Usually mild. Very low mortality
Vaccine
Peginterferon/ Ribavirin
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Yes
Behavior Modification HBV vaccine
No
The virus replicates in the GI tract and then spreads to the liver during a brief viremic period. The virus is not cytopathic for the hepatocyte. Hepatocellular injury is caused by immune attack by cytotoxic T cells. Self limited within 3months with no chronicity. Laboratory Diagnosis The most useful test to diagnose acute infection is anti HAV IgM antibody. Other: Isolation of the virus from clinical specimens is not done or immune-electron microscopy of stools. Treatment: Supportive care should be given. Hepatitis A immune globulin can be administered early after infection (within two weeks) and gives some temporary immunity (up to five months). Prevention: Active immunization with a vaccine containing inactivated HAV is available. Two doses, an initial dose followed by a booster 6 to 12 months later, should be given. Passive immunization with immune serum globulin prior to infection or within 14 days after exposure can prevent or mitigate the disease. Observation of proper hygiene, e.g., sewage disposal and hand washing after bowel movements, is of prime importance. Hepatitis B Virus It causes hepatitis B and implicated as a cause of hepatocellular carcinoma. Characteristics: 1) Enveloped virus with incomplete circular double-stranded DNA; i.e., one strand has about one-third missing and the other strand is "nicked" (not covalently bonded). DNA polymerase is present in virion. 2) There are three important antigens: the surface antigen (HBsAg), the core antigen (HBcAg), and the e antigen (HBeAg). 3) Electron microscopy of a patient's serum reveals three different types of particles: a few 42-nm virions (Dane particles)and many 22-nm spheres and long filaments 22-nm wide, which are composed of surface antigen (HBsAg). In the patient's serum, long rods and spherical forms of HBsAg predominate. 4) HBV has four serotypes. 5) Three important antibodies appear during infection: Anti-HBsAb, anti-(HBcAb, and AntiHBeAb. Pathogenesis Transmitted by blood transfusion, needle brick injury, sexual intercourse , and by vertical from mother to fetus during birth or breast feeding . Hepato-cellular injury due to immune attack by cytotoxic (CD8) T cells. Antigen–antibody complexes may cause arthritis, rash, and glomerulonephritis. About 2% fulminant hepatitis and about 5% of HBV infections result in a chronic carrier state. Chronic hepatitis, cirrhosis, and hepatocellular carcinoma can occur. Hepatocellular carcinoma may be related to the integration of part of the viral DNA into hepatocyte DNA. Laboratory Diagnosis HBV has not been grown in cell culture. - 111 -
Three antigens: surface antigen (HBsAg), Core antigen (HBcAg) and HBe Ag and three antibodies: surface antibody (HBsAb), core antibody (HBcAb) and HBeAb. The most important laboratory test for the detection of early HBV infection is the immunoassay for surface antigen (HBsAg). HBsAg appears during the incubation period and is detectable in most patients during the Serology of HBV prodrome and acute disease. Detection of HbsAg for more than 6 months indicates a chronic carrier state. The presence of HBe Ag indicates a high viral replication and high infectivity. While Anti HBe Ab denotes low infectivity. An HBV-infected person who has neither detectable HBs antigen nor HBs antibody is said to be in the "window" phase. Diagnosis of this patient is made by detecting ant-HBc Ig M and high ALT (Surrogate marker). Anti HBs indicates immunity. It usually appear after disappearance of HBsAg. HBcAg is present in the hepatocyte only and not in blood. The HBVmarkers Panel – Interpretation Test
Results
Interpretation
HBsAg anti-HBc anti-HBs
Negative Negative Negative
The patient is susceptible to an HBV infection and has not been exposed previously to the virus The patient has not been vaccinated
HBsAg anti-HBc anti-HBs
Negative Positive Positive
The patient is immune to HBV as a result of having been infected previously (indicated by the presence of anti-HBc antibodies which would not occur if the patient had been vaccinated)
HBsAg anti-HBc anti-HBs
Negative Negative Positive
The patient is immune because of vaccination against HBV
HBsAg anti-HBc anti-HBc IgM anti-HBs
Positive Positive Positive Negative
The patient has an acute HBV infection. Any anti-HBs antibodies that have been made are complexed with the large amount of the antigen and are thus undetectable
HBsAg anti-HBc anti-HBcAg IgM anti-HBs
Positive Positive Negative Negative
The patient has a chronic HBV infection. The IgM anti-HBc has waned
HBsAg anti-HBc anti-HBs
Negative Positive Negative
The patient may be in the recovery phase of an acute HBV infection. This patient could be infected and thus a carrier of HBV. The inability to detect HBsAg may result from it being complexed with anti-HBs antibodies in the "window" phase Other possible interpretations are that the patient is distantly immune to HBV but - 112 -
the test was too insensitive to detect anti-HBs. There may also have been a false positive for anti-HBc and the patient is actually uninfected.
Treatment: Alpha interferon and lamivudine, a reverse transcriptase inhibitor, can reduce the inflammation associated with chronic hepatitis B but does not cure the carrier state. Prevention: Pre-exposure prophylaxis: -by Active Immunization with recombinant HBV vaccine containing HBs Ag made by recombinant technology. Given in three doses 0, 1 month and 6 months. -It is given to high risk persons as medical and paramedical personnel. -Given compulsory to infants at 0,2 and 6 months of age Post-exposure prophylaxis: Passive–Active immunization: both the vaccine and hepatitis B immunoglobulin (HBIG) are given (at separate sites) as soon as possible in case of exposure to HBV infection as needle brick or baby of HbsAg positive mothers or after sexual exposure. Hepatitis C Virus It causes Hepatitis C and can lead to hepatocellular carcinoma. HCV is the most prevalent blood-borne pathogen in Egypt. Characteristics: HCV is a member of the flavivirus family. Enveloped virus with one piece of single-stranded, positive-polarity RNA. HCV has at least six genotypes and multiple subgenotypes based on differences in the genes that encode one of its two envelope glycoproteins. Genotype 4 is the most prevalent in Egypt. Pathogenesis Transmission: Most transmission is via blood. Sexual transmission and transmission from mother to child probably occurs as well. Hepatocellular injury probably caused by cytotoxic T cells. HCV replication itself does not kill cells, i.e., does not cause a cytopathic effect. More than 50% of infections result in the chronic carrier state. The chronic carrier state predisposes to chronic hepatitis and to hepatocellular carcinoma. Laboratory Diagnosis HCV infection is diagnosed by detecting antibodies to HCV in an ELISA. The test does not distinguish between IgM and IgG and does not distinguish between an acute, chronic, or resolved infection. Because false-positive results can occur in the ELISA, a RIBA (recombinant immuneSerology of HCV blot assay) should be performed as a confirmatory test. RT- PCR test detects the presence of viral RNA in the serum should be performed to - 113 -
determine whether active disease exists. HCV Core Ag testing recently introduced. Treatment: -Alpha interferon plus ribavirin mitigates chronic hepatitis but does not eradicate the carrier state. -Recent oral drugs sofosbuvir (Sovaldi): NS5B polymerase inhibitor that results in suppression of HCV replication and interrupts HCV life cycle. It is indicated for treatment of HCV infection genotypes 1, 2, 3, and 4 as part of a combination antiviral regimen. Prevention: -Post-transfusion hepatitis can be prevented by detection of antibodies in donated blood. -There is no vaccine, and hyperimmune globulins are not available. Hepatitis D Virus It causes Hepatitis D (hepatitis delta). Characteristics: Defective virus that uses hepatitis B surface antigen as its protein coat. HDV can replicate only in cells already infected with HBV, i.e., HBV is a helper virus for HDV. Genome is one piece of single-stranded, negative-polarity, circular RNA. Pathogenesis: -Transmitted by blood, sexually, and from mother to child. -As HBV, Chronic hepatitis and chronic carrier state occur. Laboratory Diagnosis Serologic testing (ELISA) detects either delta antigen or antibody to delta antigen. Treatment: Alpha interferon mitigates symptoms but does not eradicate the carrier state. Prevention: -Prevention of HBV infection by using the HBV vaccine and the HBV hyperimmune globulins will prevent HDV infection also. Hepatitis E Virus Causes outbreaks of hepatitis, primarily in developing countries. Similar to hepatitis A virus in the following ways: transmitted by fecal–oral route, no chronic carrier state, no cirrhosis, and no hepatocellular carcinoma. High mortality in pregnant females Acute hepatitis E virus (HEV) infection is diagnosed in immunocompetent individuals based on detection of anti-HEV immunoglobulin M (IgM). The anti-HEV IgM usually starts rising 4 weeks after infection and remains detectable for 2 months after the onset of illness. No antiviral therapy and no vaccine. Hepatitis F virus Hepatitis F virus (HFV) is a hypothetical virus linked to hepatitis. Several hepatitis F virus candidates emerged in the 1990s; none of these reports have been substantiated. GB virus C The GB virus C is another potential viral cause of hepatitis that is probably spread by blood and sexual contact. It was initially identified as Hepatitis G virus. There is very little evidence that this virus causes hepatitis, as it does not appear to replicate primarily in the liver. It is now classified as GB virus C
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DNA Enveloped Viruses Herpes viruses: enveloped double stranded DNA, icosahedral viruses 120-200 nm with glycoprotein spikes Human herpes viruses Viruses of humans Common names Human Herpes Virus,1 Herpes simplex virus Type 1 Human Herpes Virus,2 Herpes simplex virus Type 2 Human Herpes Virus,3 Varicella Zoster Virus (VZV) Human Herpes Virus,4 Epstein Barr virus (EBV) Human Herpes Virus,5 Cytomegalovirus (CMV) Human herpes virus 6 Exanthum subitum or roseola infantum Human herpes virus 7 Exanthum subitum Human herpes virus 8 Kaposi's sarcoma-associate herpes virus Herpes Simplex Virus Type 1 Characteristics: Enveloped virus with icosahedral nucleocapsid and linear doublestranded DNA. One serotype; cross-reaction with HSV-2 occurs. No herpes group–specific antigen. Pathogenesis: HSV-1: Transmission by saliva or direct contact with virus from the vesicle. HSV-2: Sexual contact in adults and during passage through the birth canal in neonates. HSV-1: Initial vesicular lesions occur in the mouth or on the face. The virus then travels up the axon and becomes latent in sensory (trigeminal) ganglia. Recurrences occur in skin innervated by affected sensory nerve and are induced by fever, sunlight, stress, etc. Dissemination to internal organs occurs in patients with depressed cell-mediated immunity with life-threatening consequences. HSV-1 encephalitis often affects the temporal lobe. HSV-2: Initial vesicular lesions occur on genitals. The virus then travels up the axon and becomes latent in sensory (lumbar or sacral) ganglion cells. Recurrences are less severe than the primary infection. HSV-2 infections in neonate can be life-threatening because neonates have reduced cell-mediated immunity. Asymptomatic shedding of HSV-2 in the female genital tract is an important contributing factor to neonatal infections. Clinically: HSV-1: Herpes labialis (fever blisters or cold sores), keratitis, encephalitis. HSV-2: Herpes genitalis, aseptic meningitis, and neonatal infection. Laboratory Diagnosis Virus causes cytopathic effect (CPE) in cell culture. It is identified by antibody neutralization or fluorescent antibody test. Tzanck smear of cells from the base of the vesicle reveals multinucleated giant cells with intranuclear inclusions. These giant cells are not specific for HSV-1; they are seen in the vesicular lesions caused by HSV-2 and varicella-zoster virus as well. A rise in antibody titer can be used to diagnose a primary infection but not recurrences. HSV encephalitis can be diagnosed using a PCR assay to detect HSV-1 DNA in spinal fluid. Treatment Acyclovir for encephalitis and disseminated disease. Acyclovir has no effect on the latent state of the virus. Trifluorothymidine for keratitis. Primary infections and localized - 115 -
recurrences are self-limited. Prevention HSV-1: Recurrences can be prevented by avoiding the specific inciting agent such as intense sunlight. Acyclovir can reduce recurrences. No vaccine is available. HSV-2: Recurrences can be reduced by the long-term use of oral acyclovir. Neonatal infection can be prevented by delivering the child by cesarean section if the mother has visible vesicular lesions in the birth canal. There is no vaccine. Varicella-Zoster Virus It causes varicella (chickenpox) in children and zoster (shingles) in adults. Pathogenesis: Transmission: Varicella is transmitted primarily by respiratory droplets. Zoster is not transmitted; it is caused by a reactivation of latent virus. Initial infection is in the oropharynx. It spreads via the blood to the internal organs such as the liver and then to the skin. After the acute episode of varicella, the virus remains latent in the spinal sensory ganglia and can reactivate to cause zoster years later, especially in older and immunocompromised individuals. Clinically: Varicella: A papulovesicular rash appears in croups on the trunk and spreads to the head and extremities. Fever and malaise. Shingles (Zoster): occurs in immunocompromised adults. It results from reactivation of latent VZV later in life at times of reduced cell-mediated immunity or local trauma. The virus affects the sensory nerves and ganglia leading to severe pain in the area supplied by these nerves, then crops of vesicles appear over the skin supplied by the affected nerves. The eruptions are unilateral, affecting the trunk and neck. Laboratory Diagnosis: 1. Virus causes CPE in cell culture and can be identified by fluorescent antibody test. Multinucleated giant cells seen in smears from the base of the vesicle. Intra-nuclear inclusions seen in infected cells. 2. A fourfold or greater rise in antibody titer in convalescent-phase serum is diagnostic. 3. PCR Treatment: No antiviral therapy is indicated for varicella or zoster in the immunocompetent patient. In the immunocompromised patient, acyclovir can treat and prevent dissemination of HSV-1. Prevention: Both the varicella vaccine and the zoster vaccine contain live, attenuated varicella-zoster virus. Immunocompromised patients exposed to the virus should receive passive immunization with varicella-zoster immune globulin (VZIG) and acyclovir to prevent disseminated disease. Epstein-Barr Virus Pathogenesis: -Transmission: Virus found in human oropharynx and B lymphocytes. It is transmitted primarily by saliva. Infection begins in the pharyngeal epithelium, spreads to the cervical lymph nodes, then travels via the blood to the liver and spleen. EBV establishes latency in B lymphocytes. -Infectious mononucleosis (kissing disease) fever, lymphadenopathy, enlarged spleen and liverand sore throat. -It is oncogenic virus: can cause Burkitt's lymphoma and nasopharyngeal carcinoma. Laboratory Diagnosis CBC: Lymphocytosis, including atypical lymphocytes (cytotoxic T cells). - 116 -
Pauall Bunnel test (Monospot test):Heterophil antibody agglutinates sheep or horse red blood cells. A significant rise in EBV-specific antibody to viral capsid antigen is diagnostic. Treatment & Prevention: Symptomatic & There is no vaccine. Cytomegalovirus Transmission: Virus is found in many human body fluids, including blood, saliva, semen, cervical mucus, breast milk, and urine. It is transmitted via contact with these fluids, across the placenta, or by organ transplantation. Pathogenesis: Initial infection usually in the oropharynx. In fetal infections, the virus spreads to many organs, e.g., central nervous system and kidneys. In adults, lymphocytes are frequently involved. A latent state occurs in leukocytes. Diseases: It causes: 1) cytomegalic inclusion body disease in infants: jaundice, with hepatosplenomegaly, peumonitis and CNS damage. 2)Infectious Mononucleosis like disease in adults. 3) Pneumonia and hepatitis in immune-compromised patients. Retinitis and enteritis, especially in AIDS patients. 4) Disseminated infection in immune-compromised patients can result from either a primary infection or reactivation of a latent infection. Laboratory Diagnosis Cell culture: It causes CPE and can be identified by fluorescent antibody test. "Owl's eye" nuclear inclusions are seenin urinary sediment. CMV IgM or A fourfold or greater rise in antibody titer in convalescent-phase serum is diagnostic. Treatment: Ganciclovir is beneficial in treating pneumonia and retinitis. Prevention:No vaccine is available. Ganciclovir suppresses retinitis. Do not transfuse CMV antibody-positive blood into newborns or antibody-negative immunocompromised patients. Human Herpesvirus 6: Roseola or exanthema subitum, a common exanthema disease of children. Human Herpesvirus 8:Causes Kaposi's sarcoma, especially in AIDS patients. Transmitted sexually. Diagnosis made by pathologic examination of lesion biopsy. No specific antiviral treatment and no vaccine. Pox viruses *Poxviruses are the largest viruses. Enveloped virus with linear double-stranded DNA. *It includes: -Vaiola causing small pox -Vaccinia: used in vaccine of small pox - Molluscum Contagiosum Virus causing wart like lesion. 1. Smallpox Virus: Disease: Smallpox. The disease smallpox has been eradicated by use of the vaccine. The last known case was in 1977 in Somalia. Causes of successful eradication of smallpox disease:1- The virus has Single stable serotype 2- No animal reservoir, human is the only host 3- The disease is easily diagnosed clinically so all the exposed can be immunized 4- has no carrier state, no subclinical state, no latency 5- develop solid immunity and antibody response is prompt 6- vaccine : is effective Pathogenesis: Transmission by respiratory droplets or direct contact with the virus from - 117 -
skin lesions.The virus infects the mucosal cells of the upper respiratory tract, then spreads to the local lymph nodes and by viremia to the liver and spleen and later the skin. Skin lesions progress in the following order: macule, papule, vesicle, pustule, crust which heal leaving a scar. Mortality is high. Laboratory Diagnosis: Virus identified by CPE in cell culture or "pocks" on chorioallantoic membrane. Electron microscopy reveals typical particles; cytoplasmic inclusions seen in light microscopy. Viral antigens in the vesicle fluid can be detected by precipitin tests. A fourfold or greater rise in antibody titer in the convalescent-phase serum is diagnostic. Treatment:None. Prevention: Vaccine contains live, attenuated vaccinia virus. Vaccine is no longer used except by the military, because the disease has been eradicated. 2. Molluscum Contagiosum Virus: Causes molluscumcontagiosum.pinkish, papular skin lesions with an umbilicated center. Lesions usually on the face, especially around the eyes. Transmitted by direct contact and sexually. Diagnosis made clinically; laboratory is not involved. There is no established antiviral therapy and no vaccine. Cidofovir may be useful in the treatment of the extensive lesions that occur in immunocompromised patients. DNA Non-enveloped Viruses Adenovirus Nonenveloped virus with icosahedral nucleocapsid and linear double-stranded DNA. There are 41 serotypes, some associated with specific diseases. Pathogenesis: Transmission by respiratory droplet primarily; iatrogenic transmission in eye disease; fecal–oral transmission with enteric strains.Virus preferentially infects epithelium of respiratory tract and eyes. After acute infection, persistent, low-grade virus production without symptoms can occur in the pharynx. Diseases:Conjunctivitis,upper and lower tract respiratory disease, especially pharyngitis and pneumonia. Enteric strains cause diarrhea. Some strains cause sarcomas in certain animals but not humans. Laboratory Diagnosis: Virus causes CPE in cell culture and can be identified by fluorescent antibody or complement fixation test. Antibody titer rise in convalescent-phase serum is diagnostic. Treatment:Symptomatic. Prevention:Live vaccine against types 3, 4, and 7 is used in the military to prevent pneumonia. Polyomaviruses:Nonenveloped virus with icosahedral nucleocapsid. They include: - Human papilloma virus -JC virus---- the cause of progressive multifocal leukoenchephalopathy. – BK virus causes cystitis. Human Papillomavirus Diseases: Papillomas (warts); planter warts, condylomata acuminata (genital warts); associated with carcinoma of the cervix and penis especially types16 & 18. Characteristics: Nonenveloped virus with icosahedral nucleocapsid and circular double-stranded DNA. There are at least 100 types, which are determined by DNA sequence not by antigenicity. Many types infect the epithelium and cause papillomas at specific body sites. - 118 -
Pathogenesis:Transmission byDirect contact of skin or genital lesions, sexually or vertical from mother to fetus. Two early viral genes, E6 and E7, encode proteins that inhibit the activity of proteins encoded by tumor suppressor genes, e.g., the p53 gene and the retinoblastoma gene, respectively. Human papilloma virus causes infections at cutaneous ( e. g., feet, hands, vocal cords, mouth) and mucosal sites that vary from benign warts to malignant squamous cell carcinoma. Most cervical, vulvar and penile cancers are associated with types 16 and 18. Laboratory Diagnosis:Diagnosis is made clinically and Pathologically by finding koilocytes in the lesions. DNA hybridization tests are available. Virus isolation and serologic tests are not done. Treatment:Treatment varies according to the site of lesions: liquid nitrogen is used for skin lesions, podophyllin for genital lesions, and salicylic acid for plantar lesions. Alpha interferon is also available. Prevention:A vaccine containing the capsid proteins of four HPV types (6, 11, 16 and 18) is available. Parvovirus B19 Nonenveloped virus with icosahedral symmetry and single-stranded DNA genome. There is one serotype. Pathogenesis: Transmission by respiratory droplets and transplacental. Fifth disease (erythema infectiosum, slapped cheek syndrome): bright red rash mainly in cheeks and flu-like symptoms. Aplastic anaemia (especially in patients with sickle cell anaemia) Fetal infections, cause severe anemia, leading to congestive heart failure and edema (hydrops fetalis). Arthritis due to immune complexes deposition Laboratory Diagnosis :ELISA to detect antibodies and PCR to detect DNA Prevention:There is no vaccine. RNA Enveloped Viruses Influenza Virus Pathogenesis: It is Orthomyxovirus. Enveloped virus with a helical nucleocapsid and segmented, single-stranded RNA of negative polarity. RNA polymerase is present in the virion. The two major antigens are the hemagglutinin (HA) and the Influenza virus neuraminidase (NA) on separate surface spikes. Antigenic shift in these proteins as a result of re-assortment (exchange) of RNA segments between different strains of the virus and accounts for the epidemics of influenza caused by influenza A virus. Influenza A viruses of animals are the source of the new RNA segments. Antigenic drift due to mutations also contributes. The virus has many serotypes because of these antigenic shifts and drifts. - 119 -
The antigenicity of the internal nucleocapsid protein determines whether the virus is an A, B, or C influenza virus. Transmission by respiratory droplets.Infection is limited primarily to the epithelium of the respiratory tract. Influenza A virus is the main cause of worldwide epidemics (pandemics). A pandemic caused by a swine-origin strain of H1N1 influenza A virus began in 2009. Uncomplicated influenza: Fever (38 - 40 °C), myalgias, headache, ocular symptoms, photophobia, tears, ache, dry cough, nasal discharge H1N1 strain, the 2009 "swine flu", also gives rise to gastro-intestinal symptoms (e.g. vomiting, diarrhea) Pulmonary complications, sequelae: Croup (acute laryngotracheobronchitis) in young children - symptoms include cough (like a barking seal), difficulty breathing, stridor (crowing sound during inspiration) Primary influenza virus pneumonia Secondary bacterial infection: This often involves Streptococcus pneumoniae, Staphylococcus aureus, Hemophilus influenzae H1N1 and H3N2 Swine Flu Swine flu, as its name suggests, is a type A influenza of pigs and does not normally infect humans. However, swine flu variants do sometimes spread to humans and in 2009, a new H1N1 swine flu started to circulate. This virus is unusual because it possesses a combination of genes that have not previously been observed in animal or human populations. In June 2010, WHO declared the pandemic over; however, the H1N1 "swine" flu continues to circulate around the globe along with the seasonal flu. It will likely continue to do so. In fact, H1N1 is one of the seasonal flu strains in the seasonal flu vaccine. Swine Influenza A H3N2 was first found in the United States in pigs in 2010 but in recent years a number of human infections have occurred in people with close contact to pigs. Although the virus can spread directly from human to human, there has been little spread. H5N1 Avian Flu There is concern about a recent outbreak of avian influenza due to a strain of H5N1 influenza A virus . This bird virus seems to be able to infect humans. The case fatality rate is high (~60%) in humans. Fortunately, as yet the virus does not readily spread from one human to another. However, there is concern that it might mutate, or undergo reassortment with a human influenza virus, and acquire the ability to spread rapidly from human to human while still being as virulent. Laboratory Diagnosis 1. A rapid ELISA test to detect influenza viral antigen in respiratory secretions is often used. 2. Virus grows in cell culture and embryonated eggs and can be detected by hemadsorption or hemagglutination. 3. A fourfold or greater antibody titer rise in convalescent-phase serum is diagnostic. 4. RT- PCR to detect the viral RNA. Treatment: The neuraminidase inhibitor, oseltamivir (Tamiflu), is the drug of - 120 -
choice. Zanamivir, another neuraminidase inhibitor, is also available. Amantadine and rimantadine are no longer used due to widespread resistance. Prevention:-Vaccines available: (1) A killed (subunit) vaccine containing purified HA and NA. (2) a vaccine containing a live, temperature-sensitive mutant of influenza virus. The virus in the live vaccine replicates in cool nasal passages where it induces secretory IgA, but not in warm lower respiratory tract. Both vaccines contain the strains of influenza A and B virus currently causing disease. (3)The killed vaccine :is not a good immunogen and must be given annually. The vaccine against "standard" influenza contains two A strains (H1N1 and H3N2) and one B strain. The vaccine against "swine" influenza contains only the novel H1N1 strain of swine-origin. -Oseltamivir (Tamiflu) can be used for prophylaxis in unimmunized people who have been exposed. Paramyxoviruses Paramyxoviruses: Enveloped virus with a helical nucleocapsid and one piece of single-stranded, negative-polarity RNA. RNA polymerase is present in virion. It includes:-Measles virus - Mumps virus -Parainfluenza virus -Respiratory Syncytial virus Measles Virus It has a single serotype. It causes Measles and a rare late complication Subacute sclerosing panencephalitis. Pathogenesis: Transmission by respiratory droplets.Initial site of infection is the upper respiratory tract. Virus spreads to local lymph nodes and then via the blood to other organs, including the skin. The maculopapular rash is due to cell-mediated immune attack by cytotoxic T cells on virus-infected vascular endothelial cells in the skin. Clinically, maculopapular rash, fever, Koplik’s spots on buccal mucosa. Complications: - encephalitis - giant cell pneumonia – Subacutes clerosing panencephalitis Laboratory Diagnosis:-The virus can be isolated in cell culture. -Serologic tests to detect measles Ig M. Treatment:symptomatic Prevention:Vaccine contains live, attenuated virus given subcutaneously to children at 15 months of age. Usually given in combination with mumps and rubella vaccines MMR. Mumps Virus It has a single serotype. it causes mumps Pathogenesis: Transmission by respiratory droplets. The initial site of infection is the upper respiratory tract. The virus spreads to local lymph nodes and then via the bloodstream to other organs, especially the parotid glands, testes, ovaries, meninges, and pancreas. -Clinically, a prodromal stage of fever, malaise, and anorexia is followed by tender swelling of the parotid glands, either unilateral or bilateral. There is a characteristic increase in parotid pain when drinking citrus juices. The disease is typically benign and resolves spontaneously within 1 week. -Complication: -orchitis (Sterility due to bilateral orchitis is a rare) -Aseptic - 121 -
meningitis -pancreatitis Laboratory Diagnosis: diagnosis is usually clinical. The virus can be isolated in cell culture from saliva, spinal fluid, or urine and detected by hemadsorption. Diagnosis can also be made serologically to detect rising antibody titer. Antibody to S antigen appears early and is short-lived, it indicates current infection. If antibody to V antigen is found, the patient has had mumps in the past. Treatment: symptomatic. Prevention:Vaccine contains live, attenuated virus. Usually given in combination with measles and rubella vaccines MMR. Given subcutaneously to children at 15 months of age. The vaccine is effective and long-lasting (at least 10 years) and causes few side effects. Parainfluenza Virus There are four serotypes. Pathogenesis: -Transmitted by respiratory droplets. -Infection and death of respiratory epithelium without systemic spread of the virus. Clinically: Bronchiolitis in infants, croup in young children, and the common cold in adults. Laboratory Diagnosis Isolation of the virus in cell culture is detected by hemadsorption. Immunofluorescence is used for identification. A fourfold or greater rise in antibody titer can also be used for diagnosis. Treatment: Symtomatic. No vaccine or drug is available. Respiratory Syncytial Virus Unlike other paramyxoviruses, it has only a fusion protein in its surface spikes. It has no hemagglutinin. It has a single serotype. Multinucleated giant cells caused by the viral fusion protein are a hallmark. Pathogenesis: Transmission by respiratory droplets. Infection involves primarily the lower respiratory tract in infants without systemic spread. Immune response probably contributes to pathogenesis. Diseases:Most important cause of bronchiolitis and pneumonia in infants. Also causes otitis media in older children. Laboratory Diagnosis 1. Enzyme immunoassay (rapid antigen test) detects RSV antigens in respiratory secretions. 2. Isolation in cell culture. Multinucleated giant cells visible. 3. Immunofluorescence is used for identification. 4. Serology is not useful for diagnosis in infants. 5. RT-PCR Treatment:Aerosolized ribavirin for very sick infants. Prevention:Passive immunization with palivizumab (monoclonal antibody) or immune globulins in infants who have been exposed is effective. Handwashing and the use of gloves may prevent nosocomial outbreaks in the newborn nursery.
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Rubella Virus Characteristics: Rubella virus is a member of the togavirus family. Enveloped virus with an icosahedral nucleocapsid and one piece of single-stranded positivepolarity RNA. It has a single serotype. Pathogenesis:-Transmission by Respiratory droplets and across the placenta from mother to fetus.-The initial site of infection is the nasopharynx, from which it spreads to local lymph nodes. It then disseminates to the skin via the bloodstream. The rash is attributed to both viral replication and immune injury. During maternal infection, the virus replicates in the placenta and then spreads to fetal tissue. If infection occurs during the first trimester, a high frequency of congenital malformations occurs. The virus has teratogenic properties infecting the fetus where it stops cells from developing or destroys them. Clinically: Rubella (German measles): After an incubation period of 14 to 21 days, a brief prodromal period with fever and malaise is followed by a maculopapular rash, which starts on the face and progresses downward to involve the extremities. Posterior auricular lymphadenopathy is characteristic. The rash typically lasts 3 days. -Congenital rubella syndrome is characterized by congenital malformations, especially affecting the cardiovascular and central nervous systems, and by prolonged virus excretion. -Some children infected in utero can continue to excrete rubella virus for months following birth, which is a significant public health hazard because the virus can be transmitted to pregnant women. Laboratory Diagnosis Virus growth in cell culture is detected by interference with plaque formation by Coxsackie virus; rubella virus does not cause CPE. To determine whether an adult woman is immune, a single serum specimen to detect IgG antibody by ELISA. To detect whether recent infection has occurred, either a single serum specimen for rubella IgM antibody or a set of acute-and convalescent-phase sera for IgG antibody can be used. If recent infection has occurred, an amniocentesis can reveal whether there is rubella virus in the amniotic fluid by PCR or culture, which indicates definite fetal infection. Treatment: symptomatic. Prevention: Vaccine contains live, attenuated virus. Usually given in combination with measles and mumps vaccine. The vaccine is effective and long-lasting (at least 10 years) and causes few side effects, except for transient arthralgias in some women. It is given subcutaneously to children at 15 months of age (usually in combination with measles and mumps vaccine) and to unimmunized young adult women if they are not pregnant and will use contraception for the next 3 months. There is no evidence that the vaccine virus causes malformations. Because it is a live vaccine, it should not be given to immunocompromised patients or to pregnant women.
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Coronavirus Enveloped virus with helical nucleocapsid and one piece of single-stranded, positive-polarity RNA. Six different currently known strains of coronaviruses infect humans. Corona viruses includes Severe acute respiratory syndrome corona virus (SARS-CoV) and Middle east respiratory syndrome corona virus (MERS-CoV) Pathogenesis:-Transmission by respiratory droplets. Coronaviruses primarily infect the upper respiratory and gastrointestinal tract of mammals and birds. nfection is typically limited to the mucosal cells of the respiratory tract. At least 50% of infections are asymptomatic. Immunity is brief and reinfection occurs. -It is an important cause of the common cold. -SARS-CoV which causes SARS, has a unique pathogenesis because it causes both upper and lower respiratory tract infections and can also cause gastroenteritis. -Middle East respiratory syndrome coronavirus (MERS-CoV): this virus has a strong tropism for non-ciliated bronchial epithelial cells. The camelor bats may be the reservoir in MERS corona-virus. MERS can range from asymptomatic disease to severe pneumonia leading to the acute respiratory distress syndrome. -Severe acute respiratory syndrome (SARS) and Middle east respiratory syndrome (MERS)are severe atypical pneumonia with high mortality. Laboratory Diagnosis: The diagnosis primarily a clinical one. ELISA to detect rising antibody titer. RT-PCR-based test are available. Direct Immuno-fluorescenceto detect SARS or MERS-CoV Treatment: Supportive. Prevention:No vaccine available. Rabies Virus It is the only medically important member of the rhabdovirus family. Bullet-shaped enveloped virus with a helical nucleocapsid and one piece of single-stranded, negative-polarity RNA. RNA polymerase is present in virion. The virus has a single serotype. Pathogenesis: Main reservoir is wild animals such as skunks, raccoons, and bats. Transmission to humans is usually by animal bite usually dog or cat, but the virus is also transmitted by aerosols of bat saliva. Viral receptor is the acetylcholine receptor. Replication of virus at the site of the bite, followed by axonal transport up the nerve to the central nervous system. After replicating in the brain, the virus migrates peripherally to the salivary glands, where it enters the saliva. When the animal is in the agitated state as a result of encephalitis, virus in the saliva can be transmitted via a bite. Within the central nervous system, encephalitis develops, with the death of neurons and demyelination. Infected neurons contain an eosinophilic cytoplasmic inclusion called a Negri bodies. Clinically, It causes Rabies disease. Rabies is encephalitis.After I P of 2 weeks to 16 weeks or longer, the patient exhibits a prodrome of nonspecific symptoms such as fever, anorexia, and changes in sensation at the bite site. Within a few days, confusion, lethargy, and increased salivation develop. Hydrophobia due to pain on - 124 -
swallowing water. The disease progresses to seizures, paralysis, coma and death. Laboratory Diagnosis In animals:-Tissue can be stained with fluorescent antibody or with various dyes to detect cytoplasmic inclusions called Negri bodies. -The virus can be grown in cell culture. In humans:(1) can be diagnosed by fluorescent antibody staining of a biopsy specimen, usually taken from the skin of the neck at the hairline. (2) Isolation of the virus from sources such as saliva, spinal fluid, and brain tissue. (3) Serology by a rise in titer of antibody to the virus. (4) Negri bodies can be demonstrated in corneal scrapings and in autopsy specimens of the brain. Treatment: No antiviral therapy is available. Prevention: Preexposure: prevention of rabies consists of the vaccine only. It is given to highrisk groups, such as veterinarians. Preexposure immunization consists of three doses given on days 0, 7, and 21 or 28. Booster doses are given as needed to maintain an antibody titer of 1:5. Postexposure immunization involves the use of both the vaccine and human rabies immune globulin (RIG, obtained from hyperimmunized persons) plus immediate cleaning of the wound. This is an example of passive–active immunization. Tetanus immunization should also be considered. -The decision to give postexposure immunization depends on a variety of factors, such as (1) the type of animal (all wild animal attacks demand immunization) (2) whether an attack by a domestic animal was provoked, whether the animal was immunized adequately, and whether the animal is available to be observed; and (3) whether rabies is endemic in the area. -If the animal has been captured, it should be observed for 10 days and euthanized if symptoms develop. The brain of the animal should be examined by immunofluorescence. -If the decision is to immunize, both Human Diploid Cell Vaccine (HDCV) and Rabies Immuno Globulin (RIG) are given. Five doses of HDCV are given (on days 0, 3, 7, 14, and 28.RIG are given at different sites to prevent neutralization of the virus in the vaccine by the antibody in the RIG. As much as possible of the RIG is given into the bite site, and the remainder is given intramuscularly.
1.
2. 3. 4.
RNA Nonenveloped Viruses Picornaviruses: Picornaviruses are small (20–30 nm) nonenveloped viruses composed of an icosahedral nucleocapsid and a single-stranded RNA genome. The genome RNA has positive polarity, i.e., on entering the cell, it functions as the viral mRNA. The picornavirus family includes two groups of medical importance: the enteroviruses and the rhinoviruses. Among the major enteroviruses are - poliovirus-Coxsackie viruses – echoviruses-hepatitis A virus. Enteroviruses infect primarily the enteric tract, whereas rhinoviruses are found in the nose and throat (rhino = nose). - 125 -
Poliovirus -Naked nucleocapsid with single-stranded, positive-polarity RNA. Genome RNA acts as mRNA and is translated into one large polypeptide, which is cleaved by virus–encoded protease to form functional viral proteins. There are three serotypes. -Pathogenesis:Transmission by Fecal–oral route. Humans are the natural reservoir. -The virus replicates in the pharynx and the GI tract. It can spread to the local lymph nodes and then through the bloodstream to the central nervous system. -Clinical forms of poliomyelitis: (1) unapparent, asymptomatic infection, (2) abortive poliomyelitis, (3) non-paralytic poliomyelitis, and (4) paralytic poliomyelitis. Asymptomatic infection is quite common. Roughly 1% of infections are clinically apparent. The incubation period is usually 10 to 14 days. Paralysis is the result of death of motor neurons, especially anterior horn cells in the spinal cord. Flaccid paralysis is the predominant finding, but brain stem involvement can lead to life-threatening respiratory paralysis. -A postpolio syndrome: occurs many years after the acute illness manifested as marked deterioration of the residual function of the affected muscles occurs many years after the acute phase. The cause of this deterioration is unknown. Laboratory Diagnosis: -Isolation of the virus from spinal fluid indicates infection of the central nervous system. Isolation of the virus from stools indicates infection but not necessarily disease. It can be found in the GI tract of asymptomatic carriers. The virus can be detected in cell culture by CPE and identified by neutralization with type-specific antiserum. -A significant rise in antibody titer in convalescent-phase serum is also diagnostic. Treatment: Symptomatic relief and respiratory support, if needed. Physiotherapy for the affected muscles is important. Prevention:Disease can be prevented by both the inactivated (Salk) vaccine and the live, attenuated (Sabin) vaccine; both induce humoral antibody that neutralizes the virus in the bloodstream. However, only the oral vaccine induces intestinal IgA, which interrupts the chain of transmission by preventing GI tract infection. It also can replicate in GIT secreted in stools, infect others giving them immunity (Herd Immunity). The difference between Salk and Sabin vaccines of poliomyelitis Attribute
Killed (Salk) Live (Sabin)
Prevents disease
Yes
Yes
Interrupts transmission
No
Yes
Induces humoralIgG
Yes
Yes
Induces intestinal IgA
No
Yes
Herd immunity
No
Yes
Interferes with replication of virulent virus in gut
No
Yes
Reverts to virulence
No
Yes (rarely)
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Coinfection with other enteroviruses may impair immunization
No
Yes
Can cause disease in the immunocompromised
No
Yes
Route of administration
Injection
Oral
Requires refrigeration
No
Yes
Duration of immunity
Shorter
Longer
Coxsackie Viruses Divided into two groups (Group A and B) viruses defined by their different pathogenicity in mice. There are multiple serotypes in each group. Pathogenesis Transmission byFecal–oral route. The initial site of infection is the oropharynx, but the main site is the GI tract. The virus spreads through the bloodstream to various organs. Coxsackie Viruses cause: Group A:- Herpangina characterized by fever, sore throat, and tender vesicles in the oropharynx.-Hand-foot-and-mouth disease is characterized by a vesicular rash on the hands and feet and ulcerations in the mouth, mainly in children. GroupB:aseptic meningitis, pleurodynia, myocarditis, pericarditis, Also Coxsackie virus B4 may cause juvenile diabetes. Laboratory Diagnosis -The virus can be detected by CPE in cell culture and identified by neutralization.-A significant rise in antibody titer in convalescent-phase serum is diagnostic. Treatment: symptomatic. Prevention:No vaccine is available. Rhinoviruses Pathogenesis: -It causes common cold. Transmission by aerosol droplets and hand-to-nose contact. -There are more than 100 serotypes, which explains why the common cold is so common. Rhinoviruses are destroyed by stomach acid and therefore do not replicate in the GI tract, in contrast to other picornaviruses such as poliovirus, Coxsackie virus, and echovirus, which are resistant to stomach acid. -Infection is limited to the mucosa of the upper respiratory tract and conjunctiva. The virus replicates best at the low temperatures of the nose and less well at 37°C, which explains its failure to infect the lower respiratory tract. Laboratory Diagnosis Laboratory tests are rarely used clinically. The virus can be recovered from nose or throat washings by growth in cell culture. Serologic tests are not useful. Treatment and Prevention:- symptomatic -No vaccine is available because there are too many serotypes. Echoviruses 1. The prefix ECHO is an acronym for enteric cytopathic human orphan. 2. More than 30 serotypes have been isolated. They are transmitted by the fecal–oral route and occur worldwide. - 127 -
3. Echoviruses cause a variety of diseases such as aseptic meningitis, upper respiratory tract infection, febrile illness with and without rash, infantile diarrhea, and hemorrhagic conjunctivitis. 4. Along with Coxsackie viruses, echoviruses are one of the leading causes of aseptic (viral) meningitis. The diagnosis is made by isolation of the virus in cell culture. Serologic tests are of little value, because there are a large number of serotypes and no common antigen. There is no vaccine available. Caliciviruses Caliciviruses are small, nonenveloped viruses with single-stranded RNA of positive polarity. It includes norovirus and hepatitis E virus.Norwalk virus is the main human pathogen in the calicivirus family. Norwalk Virus (Norovirus) It is the main virus in the family Caliciviruses Nonenveloped virus with icosahedral nucleocapsid and one piece of single-stranded, positive polarity RNA. Pathogenesis:Transmission by fecal–oral route.Infection is typically limited to the mucosal cells of the intestinal tract. Many infections are asymptomatic. Immunity is brief and reinfection occurs. It causes gastroenteritis (watery diarrhea) especially in adults characterized by sudden onset of vomiting and diarrhea accompanied by low-grade fever and abdominal cramping. Laboratory Diagnosis:The diagnosis primarily a clinical one. A PCR-based test is available but not often done. Treatment: symptomatic. Treat diarrhea with fluid and electrolytes. Prevention:No vaccine or drug available. Reoviruses REO is an acronym for respiratory enteric orphan; when the virus was discovered, it was isolated from the respiratory and enteric tracts and was not associated with any disease. Rotaviruses are the most important human pathogens in the reovirus family. Rotavirus It causes gastroenteritis (diarrhea), especially in young children.Naked doublelayered capsid with 11 segments of double-stranded RNA. Rotavirus is resistant to stomach acid and hence can reach the small intestine. There are at least six serotypes. Pathogenesis: It is transmitted by the fecal–oral route.Rotavirus infection is limited to the GI tract, especially the small intestine. Rotavirus replicates in the mucosal cells of the small intestine, resulting in the excess secretion of fluids and electrolytes into the bowel lumen leading to diarrhea. No inflammation occurs, and the diarrhea is non bloody. Laboratory Diagnosis 1. Detection of rotavirus in the stool by ELISA 2. Demonstration of rotavirus in the stool was done by immunoelectron microscopy in which antibody aggregated the virions, allowing them to be visualized in the electron microscope. 3. Isolation of the virus is not done from clinical specimens. Treatment:Symptomatic. Prevention:-There are two rotavirus vaccines. One is a live attenuated vaccine - 128 -
that contains the single most common rotavirus serotype (G1) and the other is a live reassortant vaccine that contains five rotavirus strains. -Hygienic measures such as proper sewage disposal and handwashing are helpful. Arboviruses All arboviruses are transmitted by arthropods (arthropod-borne) such as mosquitoes and ticks from the wild animal reservoir to humans.
Family
Genus
Classification of Major Arboviruses Viruses of Medical Interest in the Americas
Togavirus Alphavirus Eastern equine encephalitis virus, western equine encephalitis virus Flavivirus Flavivirus St. Louis encephalitis virus, yellow fever virus, dengue virus, West Nile virus, Japanese encephalitis Bunyavirus Bunyavirus California encephalitis virus, Rift Vally fever virus Reovirus
Orbivirus
Colorado tick fever virus
Yellow Fever Virus -Member of the flavivirus family. Causes yellow fever in the tropical areas of Africa and South America. "Jungle" yellow fever is transmitted from monkeys to humans by mosquitoes Haemogogus. "Urban" yellow fever is transmitted from human to human by the mosquito Aedes aegypti. Yellow fever is characterized by jaundice and fever. -It begins with the sudden onset of fever, headache, myalgias, and photophobia.It progress to involve the liver, kidneys, and heart. Prostration and shock occur, accompanied by upper gastrointestinal tract hemorrhage with hematemesis ("black vomit"). Diagnosis in the laboratory can be made either by isolating the virus or by detecting a rise in antibody titer. There is no antiviral therapy. There is a live, attenuated vaccine for humans. Dengue Virus Member of the flavivirus family. It has four serotypes. It causes denguefeverin tropical areas. Dengue is the most common insect-borne viral disease in the world. The pathogenesis:Transmitted by Aedes aegypti mosquitoes from one human to another. -Classic dengue (break- bone fever) begins suddenly with an influenza-like syndrome: fever, malaise, cough, headache and severe pains in muscles and joints (break- bone) occur. Enlarged lymph nodes, a maculopapular rash, and leucopenia are common. After a week or so, the symptoms regress. -Dengue hemorrhagic fever and Hemorrhagic shock syndrome: The initial picture is the same as classic dengue, but then shock and hemorrhage, especially into the gastrointestinal tract and skin, develop. Hemorrhagic shock syndrome is due to the production of large amounts of crossreacting antibody at the time of a second dengue infection. The patient recovers from classic dengue caused by one of the four serotypes, and antibody against that - 129 -
serotype is produced. When the patient is re-infected with another serotype of dengue virus, an anamnestic, heterotypic response occurs with production of large amounts ofantibodies. Immune complexes of virus and antibody are formed that activate complement, causing increased vascular permeability and thrombocytopenia. The antibodies increase the entry of virus into monocytes and macrophages with the consequent liberation of a large amount of cytokines. In either scenario, shock and hemorrhage result. The diagnosis: -isolation of the virus in cell culture -serologic tests that demonstrate the presence of IgM antibody or a fourfold or greater rise in Ig G antibody titer in acute and convalescent sera. -No antiviral therapy or vaccine for dengue is available. Outbreaks are controlled by using insecticides - Personal protection includes using mosquito repellent and wearing clothing that covers the entire body Rift valley fever virus It is a Buyaviridae; spherical enveloped virus with segmented negative sense RNA virus. It is the causative agent of Rift valley fever maintained and transmitted in subSaharan Africa by Aedes mosquito with transoval transmission. Reservoirs are sheep, cattle and camels. Clinically: febrile illness with 10% retinitis and vasculitis that can lead to blindness. Fulminant disease with hepatitis and haemorrhagic fever or encephalitis can occur. Diagnosis:- cell culture to isolate the virus -ELISA to detect rising antibody titer -PCR -Ribavirin can be used in treatment besides supportive measures. Zika Virus An arbovirus the flavivirus genus (family Flaviviridea) Transmission by the Aedes aegypti mosquitoes bite, vertical transmission, from infected male to female. After an infected mosquito bite, the disease symptoms usually appear following an incubation period of 3-12 days with Fever, Non-purulent conjunctivitis, Headache, Myalgia, Arthralgia, Asthenia, Maculopapular rash, Oedema of the lower limbs. Complications: 1) Guillian Barre’syndrome 2) eningoencephalitis 3)Thrombocytopenia purpura 4) Conginital transmission with microcephaly Lab Diagnosis: Nucleic Acid detection of Zika viral RNA using real time Reverse Transcriptase Polymerase Chain Reaction performed on an “acute sample” collected during the first five (5) days of illness Serological detection of anti-Zika IgM and IgG antibodies using EnzymeLinked Immunosorbent Assay (ELISA) and immunofluorescence assays may be performed on blood samples collected 6 days after the onset of symptoms Eastern and western Equine Encephalitis Virus Member of the togavirus family. Causes encephalitis but western equine is less severe than estern equine encephalitis Transmitted to humans (and horses) by mosquitoes from small wild birds, such as sparrows. Humans and horses are "deadend" hosts because viremia is low. There is no antiviral therapy and no vaccine for humans. - 130 -
HUMAN CANCER VIRUSES (Oncogenic viruses) Tumor viruses are viruses that can produce tumors when they infect approprite hosts. The viruses strongly associated with human cancers are I- DNA tumor viruses 1-Human papilloma virus(papovavirdae): Genital tumors Squamous cell carcinoma Oropharyngeal carcinoma. 2-Epstein -Barr viruses (Herpesviridae): Burkitt's lymphoma Nasopharyngeal carcinoma B cell lymphoma 3- Human herpes virus -8(Herpesviridae):kaposi sarcoma 4-Hepatitis -B virus(Hepadnaviridae): Hepatocellular carcinoma II- RNA tumor viruses 1-Retro viruses (human T- lymphotroic virus-I) (Retroviradae): Adult T-cell leukaemia & Lymphoma 2- HCV (Flaviviridea): Hepatocellular carcinoma. Mechanisms of cancer production Two ideas address the fundamental question of the source of the genes for malignancy provirus and oncogene. 1- Oncogenes; cellular oncogenesare genes that are stimulatory for growth and which cause cancer when hyperactive through encoding protein that potentially can transform human cells. proto-oncogenes: Normal versions of the transforming genes that are present in normal cells. viraloncogens : A gene transmitted to a cell by a virus. That gene encodes for protein that potentially can transform normal cells into malignant cell. Evidence for the provirus mode consists of finding copies of viral DNA integrated into cell DNA only in cells that have been infected with the tumor virus. The corresponding uninfected cells have no copies of the viral DNA. 2-Tumor supressor genes: Tumour suppressor genes e.g. retinoblastoma susceptibility gene and p53 gene; are genes normally present in the body. Their products are anti-opncogenes i.e. suppress the effect of oncogenes inhibiting the growth of cancer. Suppression or mutation of these Tumor Suppressor Genes leads to carcinogenesis. So, carcinogenesis by tumour viruses occurs through: a) Introduction of new transforming genes ( i.e viral oncogenes) (direct-acting) b) Activation or over expression of pre-existing cellular genes(proto-oncogenes) which are normally suppressed.(indirect –acting). Inactivation of tumour suppressor genes. More than half of human cancers have a mutated p53 gene in the DNA of malignant cells. All tumor viruses have the ability to integrate their own genome into that of the host cells.(except HCV) - 131 -
DNA tumor viruses have two life styles: 1) In permissive cells, all parts of viral genome are expressed and this leads to viral replication ,cell lysis and cell death. Non permissive cells, viral DNA is usually but not always integrated into the cell chromosomes at random sites.Only part of the viral genome is expressed and viral structural proteins are not made so replication is aborted. The oncogens carried by DNA tumor viruses are true viral genes. They are necessary in lytic infections because they participate in the control of viral and cellular DNA transcription(i.e DNA tumor viruses encode viral oncoproteins that are important to viral replication but also affect cellular growth control pathways ;direct acting tumor viruses ) RNA tumor viruses They are of two general types with respect to tumor induction: 1. Direct transforming (Acute transforming); the highly oncogenic viruses that carry an oncogen of cellular origin that have no role in viral replication. 2. Indirect or (slowly transforming); weakly oncogenic viruses that do not contain an oncogen and induce leukaemias after long incubation period by indirect mechanisms (by altering the expression of a pre-exisiting cellular genes). Human T-Cell Lymphotropic Virus (HTLV-1 and HTLV-2) Characteristics: HTLV is a member of the retrovirus family. It causes malignant transformation of CD4-positive T cells. Transmission:HTLV is transmitted primarily by blood transfusion, sexually, and by breast feeding. Diseases: - HTLV induces malignant transformation of CD4-positive T lymphocytes by activating IL-2 synthesis.Only a small fraction of infected individuals contract leukemia, indicating that HTLV infection alone is insufficient to cause cancer. - It also causes HTLV-associated myelopathy (tropical spastic paraparesis), which is a demyelinating disease of the brain and spinal cord. Laboratory Diagnosis Detect anti-HTLV antibodies in the patient's serum using the ELISA test. Western blot assay is used to confirm a positive ELISA result. PCR assay can detect the presence of HTLV RNA or DNA within infected cells. Treatment and Prevention: No specific antiviral treatment for HTLV infection and no antiviral drug will cure latent infections by HTLV. No vaccine against HTLV. Preventive measures include discarding donated blood if anti-HTLV antibodies are present, using condoms to prevent sexual transmission, and encouraging women with HTLV antibodies to refrain from breast feeding. Human Immunodeficiency Virus Disease: Acquired immunodeficiency syndrome (AIDS). Characteristics: belonges to Lentiviruses. Enveloped virus with two copies (diploid) of a single-stranded, positive-polarity RNA genome. Contains reverse transcriptase enzyme which makes a DNA copy of the genome, which integrates into host cell DNA. Antigenicity of the gp120 protein changes rapidly; therefore, there are many serotypes. There are three main structural genes
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Gene
Proteins Encoded by Gene
Function of Proteins
gag
p24, p7
Nucleocapsid
p17
Matrix
Reverse transcriptase
Transcribes RNA genome into DNA
Protease
Cleaves precursor polypeptides
Integrase
Integrates viral DNA into host cell DNA
Gp120
Attachment to CD4 protein
Gp41
Fusion with host cell
pol
env
The natural host range of HIV is limited to humans, although certain primates can be infected in the laboratory. There is evidence that chimpanzees living in West Africa were the source of HIV-1. Human immunodeficiency virus type 2 (HIV-2) was isolated and restricted to AIDS patients in West Africa. The proteins of HIV-2 are only about 40% identical to those of the original HIV isolates. Transmission: blood contact, sexually. Also transplacental and perinatal transmission and through breast feeding. Pathogenesis: Target cell is CD4 cell. Two receptors are required for HIV to enter cells; One receptor is CD4 protein found primarily on helper T cells. The other receptor for HIV is a chemokine receptor such as CCR5. HIV infects and kills helper T cells, which predisposes to opportunistic infections. It infects macrophages which transmit it through out the body. Other cells bearing CD4 proteins on the surface, e.g., astrocytes, are infected also. Cytotoxic T cells are the main host defense against HIV The NEF protein is an important virulence factor. It reduces class I MHC protein synthesis, thereby reducing the ability of cytotoxic T cells to kill HIV-infected cells. Clinically, after exposure to infection flu like symptoms occur that resolve. Then the patient enters in a latentphase in which no clinical manifestation is present but the virus remains and replicates in lymphoid tissues. The duration of latent period is different in different patients and may extend to 10 years or more. AIDS stage: the number of CD4 cells gradually decrease and the immune response fails and the patient enter in the AIDs stage when the number of CD4 is below 200cell/µl. The patient is now exposed to opportunistic infections as: Bacterial as: Salmonellosis and Atypical mycobacteria Viral as Herpes virus. Protozoal infections - 133 -
Malignancy as Kaposi sarcoma Neurologic manifestations. Laboratory Diagnosis 1. Screening test: detecting antibody with ELISA; most will have seroconverted by 4 weeks. 2. Western blot as confirmatory test. 3. Determine the "viral load," i.e., the amount of HIV RNA in the plasma, using PCRbased assays. A high viral load predicts a more rapid progression to AIDS than a low viral load. 4. CD4 number by flow cytometry for following treatment. Treatment 1. Nucleoside analogues, such as zidovudine (AZT), lamivudine (3TC, inhibit HIV replication by inhibiting reverse transcriptase. 2. Nonnucleoside inhibitors of reverse transcriptase as nevirapine and efavirenz. 3. Protease inhibitors, e.g., indinavir and ritonavir prevent cleavage of precursor polypeptides. 4. Highly active antiretroviral therapy (HAART) consists of two nucleoside inhibitors and one protease inhibitor. Clinical improvement occurs, but the virus persists. 5. Enfuvirtide, a "fusion inhibitor," which blocks entry of HIV into target CD4 cell. 6. Maraviroc, which inhibits binding of the gp120 envelope protein of HIV to the cell coreceptor CCR-5, are also useful. 7. Treatment of the opportunistic infection depends on the organism. Prevention 1. Screening of blood prior to transfusion for the presence of antibody. "Safe sex," including the use of condoms. 2. AZT with or without a protease inhibitor should be given to HIV-infected mothers and their newborns. 3. Zidovudine (AZT), lamivudine (3TC), and a protease inhibitor should be given after a needle-stick injury. There is no vaccine. Slow Viruses & Prions Slow virus infections : The term "Slow virus infections" does not refer to the growth rate of the virus but because these diseases have a prolonged incubation period (which can be months or years), and a protracted, progressive clinical course. Slow virus diseases may be caused by conventional viruses or by the unconventional prions. Slow virus diseases caused by conventional viruses 1. Progressive multifocal leukoencephalopathy (PML) A progressive, fatal, demyelinating disease with memory loss, loss of coordination, mentation problems, vision problems, etc. The disease is caused by polyomavirus family, usually JC virus. PML develops in up to 5% of patients with AIDS. PML may be due to reactivation of a JC virus latent infection, probably in the kidney. There is also abundant virus in brain. 2. Subacute sclerosing panencephalitis (SSPE) A rare complication of measles virus infection and develops approximately 1 - 134 -
to 10 years after the initial infection. It is progressive and fatal and is characterized by mental and motor deterioration. SSPE is associated with defective forms of the virus in the brain and so it is difficult to isolate infectious virus from patients 3. Progressive rubella panencephalitis (PRP) PRP is a very rare consequence of rubella virus infection and also results in mental and motor deterioration. The initial infection is usually congenital or soon after birth and the onset of PRP occurs at 8 to 19 years of age. The course of the disease may extend over many years. 4. Human immunodeficiency virus and AIDS : Long latent period up to 7-10 and may be more between exposure to infection and appearance of AIDS. During that period, active replication of HIV occurs in lymphoid organs and destruction of immune cells (CD4). Unconventional viruses or agents: Prions: infectious proteinacious material. These agents: cause diseases that are confined to the CNS have a prolonged incubation period show a slow, progressive, fatal course of disease show a spongiform encephalopathy characteristically result in vacuolation of neurons can cause formation of fibrillar aggregates, which contain PrP and have amyloid-like characteristics Diseases in animals: Scrapie in sheep and goat. Mad Cow Disease (Bovine spongiform encephalopathy: A disease of cows with neurological manifestation due to meal containing meat and bone of goats diseased with scrapie which may transmit the prion to cows. In Humans: Kuru Creutzfeldt-Jakob disease (CJD) New variant CID :occurs in a yonger age than CJD. Gerstmann-Sträussler-Scheinker syndrome : a hereditary form of CJD Fatal Familial Insomnia Prions are composed of protein only. They have no detectable nucleic acid and are highly resistant to UV light, formaldehyde, and heat. They are encoded by a cellular gene. The pathogenic form increases in amount by inducing conformational change in normal form. Normal conformation is alpha helix; abnormal is beta-pleated sheet. In GSS syndrome, a mutation occurs that enhances the probability of the conformational change to the beta-pleated sheet form. Pathogenesis: Transmission: CJD has been transmitted by pituitary extracts, brain electrodes, and corneal transplants but In most cases of CJD, mode of transmission is unknown. Kuru was transmitted by ingestion or inoculation of human brain tissue. Variant CJD probably is transmitted by ingestion of cow brain tissue in undercooked food. -Aggregation of prion filaments within neurons occurs, vacuoles within neurons cause spongi-form changes in brain, no inflammation or immune response occurs. - 135 -
DIAGNOSIS During life, a probable diagnosis is based on the clinical picture. A positive diagnosis can sometimes be made due to the presence of PrP SC in peripheral lymphoid tissue; for example, tonsil biopsies have been used. The final diagnosis is usually made from post-mortem brain biopsy showing spongiform changes. TREATMENT: No treatment; fatal disease. Minor Viral Pathogens Viral Hemorrhagic Fevers: All types of VHF are characterized by fever and bleeding disorders and all can progress to high fever, shock and death in many cases. Some of the VHF agents cause relatively mild illnesses, such as the Scandinavian nephropathia epidemica (a Hantavirus), while others, such as Ebola virus, can cause severe, life-threatening disease. Clinically; (1)Severe multisystem syndrome (2)Damage to overall vascular system (3)Symptoms often accompanied by hemorrhage asIncludes conjunctivitis, petechia, echymosis Caustive viruses: The family Arenaviridae: as Lassa fever (Lassa virus), Argentine (Junin virus), Bolivian (Machupo virus), hemorrhagic fevers. They are enveloped helical viruses with negative sense ssRNA genome. The family Bunyaviridae: as the members of the Hantavirus genus that cause hemorrhagic fever with renal syndrome (HFRS), The Rift Valley fever (RVF) virus. The family Filoviridae includeEbola virus and Marburg virus. The family Flaviviridae: as dengue and yellow fever Pathogenesis:In most VHFs, it is likely that several mechanisms contribute to symptoms, including liver damage, disseminated intravascular coagulation (DIC), and bone marrow dysfunction. In DIC, small blood clots form in blood vessels throughout the body, removing platelets necessary for clotting from the bloodstream and reducing clotting ability. DIC is thought to cause bleeding in Rift Valley, Marburg, and Ebola fevers. Diagnosis Definitive diagnosis is usually made at a reference laboratory with advanced biocontainment capabilities. 1) Serology 2)PCR 3)immunohistochimestry 4)Viral isolation 5)Electron microscopy 6) CBC 7) the prothrombin (PT) and activated partial thromboplastin times (PTT). Management • Supportive treatment − Ribavirin : Not approved by FDA. Effective in some individuals with Arenaviridae and Bunyaviridae only. − Convalescent-phase plasma in Argentine HF, Bolivian HF and Ebola • Strict isolation of affected patients is required • Report to health authorities Prevention: With the exception of Yellow fever vaccine neither vaccines nor experimental vaccines are readily available. Prophylactic (preventive) ribavirin may be effective - 136 -
for some bunyavirus and arenavirus infections. VHF isolation guidelines dictate that all VHF patients (with the exception of dengue patients) should be cared for using strict contact precautions, including hand hygiene, double gloves, gowns, shoe and leg coverings, and faceshield or goggles. Lassa, Ebola, and Marburg viruses may be particularly prone to nosocomial (hospital-based) spread. Airborne precautions should be utilized including, at a minimum, a fit-tested, HEPA filter-equipped respirator (such as an N-95 mask), a battery-powered, airpurifying respirator, or a positive pressure supplied air respirator to be worn by personnel coming within 1,8 meter (six feet) of a VHF patient. Multiple patients should be cohorted (sequestered) to a separate building or a ward with an isolated air-handling system. Environmental decontamination is typically accomplished with hypochlorite (e.g. bleach) or phenolic disinfectants. Ebola Virus Member of the Filovirus family. 5 Spp;Zaire( Most virulent), Bundibugyo,Sudan, Tai Forest, Reston elola viruses. All affect human except reston which affect non human primates. Causes Ebola hemorrhagic fever, which has a very high mortality rate. Animal reservoir is fruit bats from which it is transmitted to animals as monkeys. Transmission to humans in Africa occurs through bush meat. Human-to-human transmission, especially in hospital setting, is by blood and other body fluids. Clinically: Acute onset; typically 8–10 days after exposure(range 2–21 days). 1)Initial: Fever, chills, myalgias, malaise, anorexia 2) After 5 days: GI symptoms, such as nausea, vomiting, watery diarrhea, abdominal pain 3)Other: Headache, conjunctivitis, hiccups, rash, chest pain, shortness of breath, confusion, seizures 4)Hemorrhagic symptoms in 18% of cases Hypovolemic shock and multi-organ failure, Hemorrhagic disease, Death may occur.Non-fatal cases typically improve 6–11 days after symptoms onset Diagnosis is usually 1) clinical one 2) serologic tests 3) electron microscope, see long "thread-like" viruses. 4) RT-PCR. 5) Culturing the virus is very dangerous and should be done only in special laboratories. Treatment:No approved Ebola-specific prophylaxis or treatment -Therapeutics in development with limited human clinical trial data (1) Convalescent serum (2) Therapeutic medications as A)Zmapp – chimeric human-mouse monoclonal antibodies B)Tekmira – lipid nanoparticle small interfering RNA C)Brincidofovir – oral nucleotide analogue with antiviral activity Vaccines – in clinical trials 1)Chimpanzee-derived adenovirus with an Ebola virus gene inserted 2)Attenuated vesicular stomatitis virus with an Ebola virus gene inserted Prevention : as other hemorrhagic fevers. Hantavirus (Sin Nombe Virus) Member of the bunyavirus family. Causes hantavirus pulmonary syndrome. Sin Nombre virus (SNV) is a robovirus, i.e., it is rodent-borne. Deer mice are the reservoir, and the virus is acquired by inhalation of dried urine and feces. Diagnosis made by detecting viral RNA in lung tissue or by serologic tests. No antiviral therapy and no vaccine. - 137 -
APPLIED MICROBIOLOGY NORMAL FLORA (1)Normal flora is the term used to describe the various bacteria and fungi that are permanent residents of certain body sites, especially the skin, oropharynx, colon, and vagina. - The viruses and parasites, which are the two other major groups of microorganisms, are usually not considered members of the normal flora, although they can be present in asymptomatic individuals. The normal flora organisms are often referred to as commensals. (2)Commensals are organisms that derive benefit from another host but do not damage that host. Resident flora: normally present in the site and reestablish itself after their removal by antiseptics. Transient: potentially pathogenic organisms acquired from the environment and don't reestablish itself after its removal by hand washing or antiseptic use. (3)CSF, Blood, urine, serous cavities is sterile sites with no normal flora. Medically Important Members of the Normal Flora Location Important Organisms Less Important Organisms Skin Staphylococcus Staphylococcus aureus, Corynebacterium epidermidis (diphtheroids), various streptococci, Pseudomonas aeruginosa, anaerobes (e.g., Propionibacterium), yeasts (e.g., Candida albicans) Nose Staphylococcus aureus S. epidermidis, Corynebacterium (diphtheroids), various streptococci Mouth Viridans streptococci Various streptococci, Eikenellacorrodens Dental Streptococcus mutans Prevotellaintermedia, Porphyromonasgingivalis plaque Gingival Various anaerobes, e.g., crevices Bacteroides, Fusobacterium, streptococci, Actinomyces Throat Viridans streptococci Various streptococci (including Streptococcus pyogenes and Streptococcus pneumoniae), Neisseria species, Haemophilusinfluenzae, S. epidermidis Colon Bacteroidesfragilis, Bifidobacterium, Eubacterium, Fusobacterium, Escherichia coli Lactobacillus, various aerobic gram-negative rods, Enterococcus faecalis and other streptococci, Clostridium Vagina Lactobacillus, E. coli, Various streptococci, various gram-negative rods. B. group B streptococci fragilis, Corynebacterium (diphtheroids), C. albicans Urethra S. epidermidis, Corynebacterium (diphtheroids), various streptococci
Importance of the normal flora: 1.
They can cause disease, especially in immunocompromised and debilitated individuals.
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2.
3.
4.
Although these organisms are nonpathogens in their usual anatomic location, they can be pathogens in other parts of the body e.g. Ecoliis normal flora in colon but the main pathogen in UTI. They constitute a protective host defense mechanism. The nonpathogenic resident bacteria occupy attachment sites on the skin and mucosa that can interfere with colonization by pathogenic bacteria. The ability of members of the normal flora to limit the growth of pathogens is called colonization resistance. If the normal flora is suppressed, pathogens may grow and cause disease. For example, antibiotics can reduce the normal colonic flora that allows Clostridium difficile, which is resistant to the antibiotics, to overgrow and cause pseudomembranous colitis. They may serve a nutritional function. The intestinal bacteria produce several B vitamins and vitamin K.
ANAEROBIC INFECTIONS Anerobic organisms: 1. Spore-forming gram positive bacilli: a) Clostridiumtetanib)Clostridiumperfringinsc)Clostridium botulinumd)Clostridiumdifficile 2. Non spore forming bacilli: a. Gram negative bacilli:Bacteroides, Fusobacterium, Prevotella, Porphyromonas. b. Gram-positive bacilli:Lactobacillus, Propionibacterium, Actinomyces, Eubacterium, Arachina. c. Gram-positive cocci:PeptostreptococciandPeptococci. d. Gram-negative cocci: Veillonella, Acidominococcus. e. Spirochaetes: Treponema Pathogenesis: Predisposing factors: 1)presence of foreign body 2) Mixed infection with facultative anaerobe which consume oxygen 3)cutting of blood supply 4)Al lthese factors increase the oxidation reduction potential favouring the anaerobic growth and infection. Sites: a)Oral cavity: periodontitis, sinusitis, otitis media, mastoiditis and vincent angina. B) Respiratory: sinusitis, otitis media, mastoiditis necrotizing pneumonia, lung abscess and empyema. c) CNS: Brain abscess d)Abominal: appendicitis, peritonitis. e) Pelvic or genital: salpingitis, vulvovaginitis, pelvic and intrauterine abscesses. It is characterized by 1) A foul smelling discharge due to short chain fatty acids 2) presence of necrotic tissues and gas in tissues 3) are often polymicrobial(the anaerobic bacteria are found in mixed infections with other anaerobes, facultative anaerobes and aerobes) Diagnosis: : 1)Specimen aspirated pus with closure of nozzle of syringe 2) foul smell of discharge 3) direct microscopy of a gram stained film (The presence of organism in smear and its absence in aerobic culture point to anerobic infection) 4) culture on gentamycin blood agar with anaerobicincubation 4) Identification by Gram-stain, biochemical reactions, gas-liquid chromatography PYREXIA OF UNDETERMINED ORIGIN(PUO) PUO: fever > 38.3 on several occasions lasting for 3 weeks without definitive diagnosis despite one week investigations in a hospital. - 139 -
Causes: 1) infections 2)neoplasms as lymphoma 3)connective tissue disorders as Systemic lupus erythmatosus. Infectious causes of PUO: Specific infection:A-Viral infections:-CMV- EBV- HIV - HSV B- Bacterial infection:tuberculosis - enteric fever – brucellosis- relapsing fever – endemic and epidemic typhus, and Q fever - Lyme disease C-Fungal; Candidiasis, zygomycosis, histoplasmosis,blastomyc.osis. D-Protozoa: malaria, filaria 2. Nonspecific infection:-Pyelonephritis– meningitis – pneumonia - subacute bacterial endocarditis (SBE) -osteomyelitis - biliaryinfection- prostatitisabdominal or pelvic abscess - dental abscess - peritonsillar abscess. URINARY TRACT INFECTIONS (UTI) 1) Bacterial :1)-Escherichia coli: the commonest urinary pathogen causing 6090 % of urinary infections 2)Klebsiella 3) Proteus: associated with renal stones 4)-Pseudomonas: associated with hospital acquired infections 5)S. aureus(upper UTI) 6) S. epidermidis(catheter) 7) S. saprophyticus 8) Enterococci (Strept. faecalis) 9)Mycobacterium tuberculosis 2) Fungal : Candidaalbicans 3) Protozoal: T. vaginalis,S. haematobium Diagnosis: A ‘mid stream’ urine sample under aseptic precaution is obtained.The urine sample can be obtained by catheterization or suprapubic aspiration. For Mycobacterium tuberculosis: three early morning urine samples on three consecutive days Significant bacteriurea: Bacterial count > 105CFU/mlis suggestive of UTI. But counts as low as 102 - 103 CFU/mlis considered significant in the presence of symptoms. PHARYNGITIS Bacterial:1)Streptococcus pyogenes 2)Corynebacterium diphtheria 3) vicent angina caused by Borrelia vincenti 4)Mycoplasma 5)Chlamydia. Viral: CMV, EPV, adenovirus, rhinovirus and influenza. Fungal: Candidaalbicans
SINUSITIS Acute
1. 2. 3. 4.
Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis. Others: Staphylococcus aureus andother Streptococci species, anaerobic bacteria and, less commonly, Gram negative bacteria. 5. Viruses: influenza, parainfluenza, rhinoviruses and adenoviruses 6. Fungal as Mucor, Rhizopus and Aspergillus spp.These infections are typically seen in patients with diabetes or other immune deficiencies Chronic: 1) A combination of anaerobic and aerobic bacteria: Staphylococcus aureus (including methicilin resistant S.aureus ), coagulase-negative Staphylococci, and Gram negative enteric organisms. 2) Fungi - 140 -
ACUTE BRONCHITIS Viruses (the main cause): as rhinovirus, coronavirus, adenovirus, influenza Less common: Mycoplasma pneumoniae, Chlamydiapneumonia, Bordetellapertusis. CHRONIC BRONCHITIS Acute exacerbation of COPD and chronic bronchitis is caused by 1)H influenza 2)S pneumonia 3) M catarrhlis 4) viral infections as influenza, adenovirus, coronavirus. PNEUMONIA Community acquired: Bacteria: 1)pneumococci 2)Mycoplasma pneumonia 3) Chlamydia pneumonia 4) Chlamydia psittaci 5)legionella pneumophilia 6) M. tuberculosis 7) Klebsiella pneumonia, Staphaureus. 8) Coxiellaburnetii Viruses: influenza, parainfluuenza, respiratory syncytial virus (RSV), SARS, MERS-CoV, mumps, measles. Fungi: Pneumocystis jiroveci. Atypical pneumonia: A case in which causative organism cannot beisolates by routine culture or clinical picture is not the same as pneumococcal pneumonia. Bacteria-Mycoplasma pneumonia - Chlamydia pneumonia - Chlamydia psittaci legionella pneumophilia -Coxiellaburnetii Viruses: as influenza, mumps, measles, parainfluuenza, respiratory syncytial virus (RSV). Adenovirus, coronavirus as (SARS and MERS-CoV). Fungi: Pneumocystis jiroveci. Hospital acquired pneumonia: pneumonia developing more than 48 hours after admission. -Enterobacteria as E coli, Klebsiella, Enterobacter. –Pseudomonas aeruginosa. -Staph aureus. –MRSA Ventillator associated pneumonia:-P aeruginosa –S. aureu –Enterobacteria, AcinitobacterSpp, haemophilus Spp. INFECTIVE ENDOCARDITIS 1. 80% of native valve endocarditis is due to Streptococcus viridians or staphylococci. 2. S epidermidis in early (2 months) prothetic valve endocarditis. 3. Enterococci in case of manipulation of GIT or genitourinary tract. 4. Others: E coli, Klebsiella, Pseudomonas , Listeria …etc. 5. HACEK organisms 6. Culture negative endocarditis (5%):Coxiella burnetii, Chlamydia, Mycoplasma, Bartonella, Brucella. Lab Diagnosis by Blood culture: three sets obtained in the 1st 24 h and incubated for 3 weeks. INFECTIVE DIARRHEA 1) Bacteria: Entero-toxin mediated:1) S.aureus 2)V.cholera 3) Entrotoxigenic E coli 4)Bacillus cereus 5)Clostridium perfringens 4) Enterohaemorrhgic E coli
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Invasive –inflammatory: RBCS and pus cells is present in stool analysis. 1) Salmonella eg S typhimurium 2) Shigella 3)Cambylobacterjejuni 4) Enteropathogenic Ecoli 5) Enteroinvasive coli Both: 1) V. parahaemoliticus 2)Yersinia enterocolitica Pseudo-membranouscolitisorantibiotic associated diarrhea: Clostridium difficile 2) Viruses: 1) rota virus 2) norovirus(Norwalk virus) 3) Protozoa: Giardia lamblia, Entamoeba, Cryptosporidia, Microsporidia. Causes of Food Poisoning: Staphylococcus aureus – most common cause, no fever-short incubation time due to preformed enterotoxins. Salmonella typhimurum and enteritidis, fever and long incubation period due to multiplication of bacilli and invasion of gut wall. Clostridium perfringins – caused by cooked meat products that have not been properly stored Bacillus cereus – “fried rice” food poisoning Clostridium botulinum – neurological symptoms only, no GI symptoms. Vibrio parahemolyticus Organism Stap. Aureus
Incubation period 1-4hrs
Salmonella
12-48 hrs
Cl. perfringins
6-18 hrs
Cl. botulinum B. cereus
12-24 hrs 2-6 hrs
V.parahemolyticus 6-36 hrs
C/P
Pathogenesis
Food type
Outcome
Vomiting, diarrhea
Enterotoxin
Recovery in 1-2 days
Vomiting, diarrhea, fever Diarrhea
Proliferation in gut
Carbohydrate, milk, milk products Meat and eggs
Enterotoxin
Meat
Neurological Vomiting, diarrhea Vomiting, diarrhea, fever
Neurotoxin Enterotoxin
Canned food Reheated rice
Hemolysis
Sea food
Recovery after treatment Recovery in 1-2 days Fatal Recovery in few days Recovery in few days
SEXUALLY TRANSMITTED DISEASES A. Bacterial -Chancroid (Haemophilus ducreyi)-Chlamydia (Chlamydia trachomatis) -Gonorrhea (Neisseria gonorrhoeae) - Syphilis (Treponema pallidum) -Non gonococcal urethritis (Ureaplasma urealyticum). B. Viral -Genital herpes (HSV) -Genital warts (condyloma acuminata) (HPV) -Human immunodeficiency virus (HIV) –Molluscum contagiosum (MCV) C. Protozoal Trichomoniasis (Trichomonas vaginalis) D. Parasitic -Pubic lice (Pediculosis pubis) -Scabies (itch mites)
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INFECTIVE CAUSES OF VAGINAL DISCHARGE 1) Bacterial vaginosis 2)candidiasis 3) Trichomonas vaginalis Bacterial vaginosis: is non specific vaginitis due to change of the vaginal ecology withabsence of inflammation. Reduction of the normal lactobacilli and increase in other organisms especially Gardnerella vaginalis and Bacteroides Spp. These anaerobic organisms produce proteolytic enzymes which degrade vaginal peptides into offensive smelling products. Diagnosis: The amsel criteria; three of these four criteria are diagnostic: 1)watery grey offensive vaginal discharge. 2)Vaginal pH>4.5 3)positive amine test: add one drop of 10% KOH to vaginal discharge gives fishy odour 4)presence of clue cells: epithelial cells studded withadherentcoccobacilli on saline mount(the single best predictor of bacterial vaginosis. CONJUNCTIVITIS
A. Bacteria: 1) Hemophilus aegyptius 2)Strep. pneumoniae 3)Moraxella lacunata 4)Gonococci 5) Staphylococcus aureus 6)Pseudomonas aeruginosa 7)Diphtheria bacilli 8)Chlamydia trachomatis
B. Viruses: Adenoviruses - Echoviruses -Herpes simplex virus (HSV) MENINGITIS Causative organisms: Bacteria: most important are: 1) N. meningitides (2)Haemophilus influenza (3)Streptococcus pneumonia In the neonatal period: 1) Group B streptococci 2)Listeria monocytogenes 3) E coli Others: M tuberculosis, Pseudomonas aeruginosa, Klebsiella and staphylococci. Viruses (aseptic meningitis): -Enteroviruses (coxsackieviruses and echoviruses) are the main viral causes Mumps -Herpes simplex virus 1 &2 -cytomegalovirus(CMV) - Epstein Barr virus (EBV) -HIV - lymphocytic choriomeningitis virus Fungi: Cyyptococcus neoformans, Histoplasma capsulatum Protozoa: Naegleria fowleri –Acanthamoeba spp Lab. diagnosis of meningitis 1. CSF is obtained by lumbar puncture under complete aseptic conditions. 2. Physical examination: In pyogenic meningitis, CSF is turbid and under tension. In tuberculous meningitis, CSF coagulates on standing. In viral meningitis, CSF is clear. 3. Chemical examination: In pyogenic meningitis, protein level increases while glucose level decreases. In aseptic (viral) meningitis, protein level moderately increases while glucose level is within normal. 4. Cellexamination:
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In pyogenic meningitis the number of cells may be 20.000/cmmor higher and mainly polymorphs. In tuberculous or viral meningitis, the number of cells does not exceed 1000/cmm and are mainly lymphocytes. 5. Bacteriologic examination : CSF is centrifuged and the deposit examined by : Microscopic examination of direct smear stained by Gram or ZiehlNeelsen. Culture: CSF deposit is cultured on blood agar and chocolate agar. If tuberculous meningitis is suspected, culture is made on Lowenstein-Jensen medium. Detection of antigens in CSF by immunoelectrophoresis and latex agglutination. Blood culture should be done. Molecular techniques: PCR and DNA hybridization for viral or bacterial nucleic acids. ORGANISMS TRANSMITTED BY BLOOD TRANSFUSION: 1)HIV 2) Hepatitis viruses: B, C, D, and G 3) CMV, EBV, HTLV : are transmitted in blood cells not in plasma. 4)Treponemapallidum is transmitted in fresh blood. 5) Malaria MILK BORNE DISEASES A. From infected animals: 1)Tuberculosis 2)Brucellosis 3)Q-fever 4)Staphylococcal food poisoning from udder abscesses . B. Milk contaminated by milk handlers: 1)Salmonella infections 2)Cholera 3)Staphylococcal food poisoning . 4)Hepatitis A and E andenteroviruses. WATER-BORNE DISEASES 1)Salmonella infections : enteric fever and gastroenteritis 2)Cholera 3)Shigellosis 4)Poliomyelitis 5)Hepatitis-A, hepatitis E 6)Weil's disease (leptospirosis) . Indicators of faecal pollution of water are : - E. coli -Strept. Faecalis - ClostridiumPerferingens DISEASES TRANSMITTED FROM MOTHER TO FOETUS OR NEWBORNS (vertical transmission) A)Prenatal transmission (transplacentally): Viruses Bacteria 4. Congenital syphilis (T. Prenatal transmission pallidum) (transplacentally): 1. CMV Protozoa 2. Congenital rubella syndrome 5. Toxoplasmosis (Toxoplasma (Rubella virus) "Protozoa) 3. Parvovirus B19
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B)At time of birth (transmitted during delivery through the birth canal): Viruses Bacteria 1) HBV 1) Neisseria gonorrheae 2) HCV 2) Chlamydia trachomatis 3) HSV2 3) Group b streptococci 4) HIV 4) E.coli 5) HPV 5) Listeria monocytogenes C) Transmitted By breast feeding: 1)CMV 2)Human T cell leukemia Virus 3)HIV 4)Ebola INFECTIOUS DISEASES WITH BACTERAEMIA Bacteraemia is a feature of some diseases. It can be diagnosed by blood culture. 1) Enteric fever 2) Puerperal sepsis 3) Brucellosis 4)Endocarditis 5)Relapsing fever 6) Meningitis 7)Ttyphus 8)Osteomylitis 9)Pneumonia FEVER WITH JAUNDICE "HEPATITIS" 1) Hepatitis viruses: HAV, HBV, HCV, HDV, HEV or HGV 2)Yellow fever 3)CMV infections 4)Infectious mononucleosis (EBV) 5)Weil's disease (L. icterohaemorrhagiae) 6)Q fever 7)Other causes, e. g., liver abscesses, hydatid cyst, tumours THE VALUE OF DIRECT SMEAR IN DIAGNOSIS Proper examination of direct smears stained by different stains is very useful in several situations: 1.Detection of organisms in direct smear may be sufficient for diagnosis in Bacterial meningitis. Leprosy (nasal scrapings by modified Z.N. stain). Vincent angina (pus cells, spirochaetes and fusiform bacilli). M tuberculosis Syphilis (smears from chancer show spirochaetes). Relapsing fever during the fever (Borrelia in blood films). Acute male gonorrhoea (gram negative diplococci in pus cells). Secondary case of cholera during an epidemic (motile curved in stools). 2.The information given by direct smear may be urgently neededto start treatment before culture results appear as in: Meningitis (CSF deposit may show the causative organism). Tuberculosis (acid fast bacilli in sputum by Z.N.). Diphtheria like organisms are reported to help clinicians start treatment. ORGANISMS CAUSING TOXAEMIA (TOXIN IN BLOOD) C. diphtheria, CL. tetani, Cl. botulinum, Cl. perfringens, Cl. difficile , Stph. aureusTSST, and Strepococcus pyogenes toxin. ZOONOSIS A zoonosis is any infectious disease that can be transmitted (in some instances, by a vector) from animals to humans or from humans to animals (the latter is sometimes called reverse zoonosis or anthroponosis).
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Main organisms casing Zoonosis Bacteria Plague Anthrax Brucellosis Borrelia (Lyme disease and others) Bovine tuberculosis Escherichia coli O157:H7 Psittacosis Typhus Q-Fever Francisellatularensis Pasturellamultucida
Viruses Hantavirus Lassa fever Yellow fever Ebola Equine encephalitis H1N1 flu Dengue fever Rabies Rift Valley fever Hemorrhagic fever West Nile virus "mad cow disease"
Antibiotic Resistant Bacteria • Naturally resistant bacteria: 1. Enterococcus spp 2. Pseudomonas Spp 3. Acinitobacter Spp • Aquired resistance: 1. Methicillin Resistant Staphaureus(MRSA) 2. Penicillin Resistant peumococci Treatment by vancomycin or teicoplanin 3. Vancomycin intermediate Staphaureus (VISA) 4. Vancomycin Resistant Staphaureus (VRSA ) 5. Vancomycin Resistant Enterococci (VRE) Treatment of VISA, VRSA or VRE by Linezolid, Streptogramins, Daptomycin, Tigcycline 6. Extented spectrum Beta lactamase (ESBL) producing Enterobacteriaceae: usually Ecoli and klebsiella spp; it is resistant to all beta lactam antibiotics and can be treated by Carbapenems, colistin, fluroquinolones
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HOSPITAL ACQUIRED INFECTIONS (NOSOCOMIAL INFECTION) -An infection occurring in a patient in a hospital or other health care facility in whom the infection was not present or incubating at the time of admission. This includes infections acquired in the hospital but appearing after discharge, and also occupational infections among staff of the facility -Infections are considered Nosocomial if they first appear 48 hours or more after hospital admission or within 30 days after discharge. -"Healthcare associated infections" (HCAI) applies to infections acquired where persons receive healthcare (example, hospital care, home care, ambulance care…etc). In developed countries, the prevalence is 5-10% of admissions to acute-care hospitals. In developing countries, the prevalence is usually higher than 15%. Common clinical types: 1) Urinary tract infection (UTI): Of particular interest is catheter-associated urinary tract infections (CAUTI) Commonest organisms: E. coli, Klebsiella, Pseudomonas and Proteus. 2) Surgical site infection (SSI): The infection may be superficial (skin) or more serious involving tissues under the skin, organs or implanted materials. SSI may be of theater-origin or ward-origin Commonest organisms: Staphylococcus aureus (particularly, MRSA), coagulase negative Staphylococci, vancomycin resistant Enterococci (VRE), Candida. 3) Respiratory tract infection: Of particular interest is ventilator-associated pneumonia (VAP), which is a lung infection that develops in a person who is on a ventilator. Commonest organisms: S. aureus(MRSA), Gram negative bacilli. 4) Blood stream infections: Bacteraemia or speticaemia. Commonest organisms: S. aureus, Enterococci, Bacteroids,Gram negative bacilli, and Candida 5) Gastrointestinal tract infections: Commonest organisms: Clostridium difficile (causing pseudomembranous colitis). Factors increasing the risk of infections: 1) Susceptible host: a) Extremes of age b) Lower host resistance due to chronic diseases, immunosuppressive therapy C) Instrumentation; catheters, endoscopes…etc 2) Microorganisms: - Microorganisms present in health care units are highly pathogenic and drug resistant (such as MRSA, MRSE, VRE). 3) Sources of infections: a-Endogenous: from the normal flora of the patient himself due to lower host resistance. b-Exogenous: transmitted to patients by various modes: - 147 -
Exogenous cross-infection: Mainly through hands of healthcare workers, visitors, patients. Exogenous environmental infections: several types of micro-organisms survive well in the hospital environment (hospital flora): On linen, equipment and supplies. Invasive procedures: urinary catheters, I.V. lines, inhalation therapy, surgery. Mode of transmission: 1- Contact: - Direct: by hands of other patients, healthcare workers..etc - Indirect: by contaminated objects used in common for patients. 2- Air- borne, and droplet infection 3- Vector- borne: mosquitoes, flies…etc INFECTION CONTROL Each hospital should implement an infection control program. The infection control committee will be responsible for this implementation. This committee includes representative staff from key departments, the hospital director and the infection control team (the microbiologist and the infection control nurses). Goals of the infection control program: • Improves the quality and reduces the cost of patient care. • Prevent disease transmission by breaking one or more of the links in the chain of transmission. • Basic infection control measures based on reducing the risk of transmission of pathogens from a known or unknown source. INFECTION CONTROL COMMITTEE 1) It is a multidisciplinary committee responsible for monitoring IC program policy implementation and recommendation for corrective actions. 2) It includes representatives from different concerned hospital departments and management, and they should meet monthly. 3) It establishes standards for patient care, reviews and assesses IC reports and identifies areas of intervention. INFECTION CONTROL TEAM 1) The optimal structure varies with hospitals types, needs and resources but ideally ICT should compose of ICD & ICNs with a separate office. According to WHO one ICN for 250 beds. 2) Hospital can appoint public health specialist or epidemiologist or infectious disease specialist, microbiologist to work as Infection Control Director (ICD). 3) Infection control nurse (ICN), having experience in infection control issues. Objectives of the infection control program: 1- Minimize and if possible eliminates the possibility of Nosocomial infection. 2- Promote rational use of antimicrobials. 3- Implementation of sound infection control polices and procedures. Infection control polices and procedures. a- Hand washing b- Decontamination policies (cleaning, disinfection and sterilization)c- Proper housekeeping measuresd- Isolation precautions. - 148 -
e) Early recognition and reporting of HAIs. F)Infection control precautions. g) Personal Protective Equipment PPE: Like gloves, gowns, masks/respirators, eye protection. H)Patient accommodation. I)waste disposal. F)Occupational health management. Investigation of outbreaks: An outbreak is the occurrence of a larger than usual number of cases of a specific infection acquired in an institution or certain population. Examples of outbreaks include: MRSA outbreak, food poisoning…etc Identifying the cause of outbreaks involves the following steps: 1. Identifying the source/reservoir of infection. 2. Isolation of incriminated organisms, and identification to species levels. 3. Typing of similar species to recognize identical strains in patients and sources of infection. The typing methods include: a- Non- molecular typing (bio-typing, antibiotic resistance pattern, phage typing, sero- typing). b- Molecular typing (plasmid profile analysis, restriction fragment length polymorphism(RFLP), PCR-RFLP, pulsed-field gel electrophoresis (PFGE), ribotyping. Surveillance of nososcomial infections: Surveillance is defined as close and continuous observation of an individual or group.Its role in the hospital is the collection of accurate, reliable and timely data on infections and notifiable diseases. Active surveillance (Prevalence and incidence studies). Targeted surveillance (site, unit, priority-oriented target). Requirements For Surveillance: 1)Trained investigators. 2)Standardized methodology. 3)Risk- adjusted rates for comparisons. The important objectives of surveillance are: 1- To determine base-line infection rates. 2- To detect potential epidemics. 2- To assess the effectiveness of infection control measure. 3- To identify areas of concerns in terms of infection control. INFECTION CONTROL PRECAUTIONS Standard Precautions: Should be applied for ALL patients 1)Hand hygiene2)Respiratory hygiene/cough etiquette 3)Use of personal protective equipment (PPE). 4)Prevention of needle sticks/sharps injuries 5)Cleaning and disinfection of the environment and equipment. Transmission-based Precautions
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A)Contact precautions:Use for protection against infections which spread by contact In addition to Standard Precautions: – Use non-sterile, clean, disposable gloves, gown, apron (only if gown is not impermeable) – Use disposable or dedicated reusable equipment (which must be cleaned and disinfected before use on other patients) - 149 -
– Limit patient contact with non-infected persons – Place patient in a single room or cohort with similar patients B)Droplet precautions:Use for protection against respiratory pathogens transmitted by large droplets • In addition to Standard Precautions: – Use a surgical/medical mask – Maintain a distance ≥ 1 meter between infectious patient and others. – Place patient in a single room or cohort with similar patients. – Limit patient movement. C)Airborne precautions:Use for protection against inhalation of tiny infectious droplet nuclei. Use airborne precautions during performing of any aerosolgenerating procedures associated with risk pathogen transmission like bone cutting, dental procedures. • In addition to Standard Precautions: – Use particulate respirator /N 95 mask – Place the patient in adequately ventilated room (≥ 12 air changes /h) – Limit patient movement TYPES OF HAND HYGIENE • Routine Hand wash with plain soap &water is the mechanical removal of soil and transient bacteria (for 40-60 sec). • Aseptic hand wash is removal & destruction of transient flora using antimicrobial soap & water (for 40-60 sec). • When hands are visibly soiled do wash hands with soap and water. • Alcohol hand rub 2cc gel is use (for 15-20 sec). • Use alcohol-based hand rub when hands are not visibly soiled. • Surgical hand scrub: removal / destruction of transient flora and reduction of resident flora using anti-microbial soap with effective rubbing (for least 35 min). • • • • •
RESPIRATORY HYGIENE AND COUGH ETIQUETTE Part of standard precautions. Education of health care workers, patients and visitors. Source control measures ( cover mouth to prevent dissemination of infectious droplets) Proform Hand hygiene Spatial separation (> 1 meter) of persons with acute febrile respiratory symptoms. Personal Protective Equipments o Gloves – protect hands o Gowns/aprons – protect skin and/or clothing o Masks and respirators– protect mouth/nose Respirators /N95 mask – protect respiratory tract from airborne infectious agents o Goggles – protect eyes o Face shields – protect face, mouth, nose, and eyes
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References Abla M. El-Mishad: Manual of Medical Microbiology & Immunology volume I 7th edition, 2008, El-Ahram Press Egypt. Brooks, G.F., Carroll, K.C., Butel, J.S., Morse, S.A. and Mietzner, T.A. (2013:) Medical Microbiology. Jawetz, Melnick and Adelbergs, 26th Edition, McGraw-Hill Companies. Richard A. Harvey R A, Cornelissen C N (2013): Lippincott Illustrated Reviews: Microbiology (Lippincott Illustrated Reviews Series) 3rd Edition, LIPPINCOTT WILLIAMS & WILKINS. Levinson W (2014): Review of Medical Microbiology and Immunology, 13th edition, McGraw-Hill Companies.
The Author: Dr Ayman A. Allam Current Job: Professor of Microbiology and Immunology, Faculty of Medicine, Zagazig University, Egypt Qualifications:
MBBCH with grade very good with honor in Faculty of Medicine, Zagazig University.11,1990. Master degree of Microbiology and Immunology with estimate excellent in Faculty of Medicine, Zagazig University, Session 4/1997. Title of thesis was (Infection control in Zagazig University Surgery Hospital). MD of Microbiology and Immunology in Faculty of Medicine, Zagazig University, Session 11/2001. Title of thesis was (Study of HLA Typing Type II (DRBI) by PCR in Egyptian Rheumatoid Arthritis Patients). Diploma of Internal Medicine with estimate very good, Faculty of Medicine, Al-Azhar University, Session 11/2003.
Fields of Interest (Career Wise):
Infectious diseases Microbiology Allergy and immunology Antimicrobial resistance
Membership of Societies and Associates:
Egyptian Society of Medical Microbiology and Immunology The Egyptian society of Allergy and Clinical Immunology
Registration: In Egyptian Medical Syndicate as 1. Consultant Microbiology 2. Consultant Allergy and Immunology 3. Internal medicine specialist
In Saudi Commission for Health Specialties as 1. Consultant Microbiology and Immunology
