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INTRODUCTION TO MEDICAL MICROBIOLOGY Medical microbiology is the study of the background material on various pathogenic organisms, and delineates the epidemiology, transmission, clinical manivestations, diagnosis, and treatment of diseases caused by these organisms. The lecture is divided into bacteriology, Mycology, Virology, Parasitology, and oral microbiology—the five sections that comprise a two course in medical microbiology. The bacteriology section familiarize students with basic concepts relating to bacterial architecture, pertinent phenotype characteristics, and general structurefunction relationships as they pertain to bacterial pathogenesis. This lecture, together with the lecture on host-parasite relationships, provides the necessary foundation for subsequent lectures designed to convey to the student simplifiede, clear explanations of mechanisms at play during the activation and destruction of microorganisms, bacterial genetics, bacterial metabolism, and ımmunology. A comprehensive survey of significant bacterial diseases is given, including their etiologic agents and mechanisms of pathogenesis. The first two lectures of the virology section deal with basic concepts while the subsequent three lectures deal with individual virus families and their medical significanse and importance. In the lecture on parasitology, each of the major parasitic diseases of humans is covered. Important features of the organisms as well as the epidemiology, transmission, clinical manifestations, and treatment of the diseases are included. The lecture on mycology presents succinct summary of the facts about medically important fungi. The first lecture of the oral microbiology section deal with development of the oral microflora while subsequent two lectures deal with the dental caries and the periodontal diseases. The Microbial World The development of suitable microscope and the discovery of microorganisms were first accomplished by Antony van Leeuwenhoek (1632-1723) of Delft, Holland, the “father of Protozoology and Bacteriology.” Leeuwnhoek was nonscientific and not university-educated, but he emerged rather suddenly as the inventor and producer of microscopes, and as a microscopist in 1673. Almost 100 years later the Danish biologist Otto Müller extended van Leeuwenhoek’s studies and organized bacteria

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into genera and species. This was the beginning of the taxonomic classification of microbes. In 1840, the German pathologist Friedrich Henle proposed criteria for proving that microorganisms were responsible for causing human disease (the “germ theory” of disease). Koch and Pasteur confirmed this theory in the 1870s and 1880s with a series of elegant experiments proving that microorganisms were responsible for causing anthrax, rabies, plague, cholera, and tuberculois. Other brilliant scientists went on to prove that a diverse collection of microbes was responsible for causing human disease. The era of chemotherapy was begun in 1910 when the German chemist Paul Ehrlich discovered the first antibacterial agent, a compound effective against the spirochete that causes syphilis. This was followed by Alexander Fleming’s discovery of penicillin in 1928, Gerhard Domagk’s discovery of sulfanamide in 1935, and Selman Waksman’s discovery of streptomycin in 1943. In 1946, the American microbiologist John Enders was the first to cultivate viruses in cell cultures, leading the way to the largescale production of virus cultures for vaccine development. Thousands of scientists have followed thses pioneers, each building on the foundation established by his or her predecessors, and each adding an observation that expanded our understanding of microbes and their role in disease. Microorganisms Microorganisms can be found in every ecosystem and in close association with every type of multicellular of organism. They populated the healthy human body by the billions as benign passengers (normal flora) and even as participants in bodily functions, for example, bacteria play a role in the degradation of intestinal contents. Microorganisms also serve as useful tools in many areas of current technology. However, in this lecture, we primarily consider the role of microorganisms: viruses, bacteria, fungi, and helminths in the initiation and spread of human diseases. Viruses Viruses are the smallest infectious particles, ranging in diameter from 18 to almost 300 nm (particles less than 200 nm cannot be seen with a light microscope). More than 40 genera of viruses have been implicated in human disease, and certainly, more will be discovered each year. Viruses consist of either DNA or RNA (but not both) and protein required for replication and pathogenesis. These components are then enclosed in a protein coat with or without a lipid membrane coat. These organisms are true parasites, requiring host cells for replication. Bacteria Bacteria are relatively simple in structure. They are procaryotic organisms__a simple unicellular organism with no nuclear membrane, mitochondria, Golgi bodies,

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or endoplasmic reticlum that reproduces by asexual division. Although the cell wall encircling bacteria is itself complex, there are two basic forms: a gram-positive cell wall with a thick peptidoglycan layer and a gram-negative cell wall with a thin peptidoglycan layer and an overlaying outer membrane. Some bacteria lack this cell wall structure and compensate by surviving only inside host cells or in a hypertonic inviroment. The human body is initiated by thousands of different bacterial species__some living transiently, others in a permanent parasitic relationship. Likewise, the enviroment that surrounds us, including the air we breathe, water we dring, and food we eat, is inhabitated by bacteria, many of which are relatively avirulent and some of which are capable of producing life-threatening disease. Fungi In contrast to bacteria, the cellular structure of fungi is more complex. These are eukaryotic organisms that contain a well-defined nucleus, mitochondria, Golgy bodies, and endoplasmic reticulum. Fungi can exist either in unicellular form (yeasts) that can replicate asexually or in multycellular (molds) that can replicate asexually and sexually. Parasites Parasites are the most complex microbes. Although all parasites are classified as eukaryotic, some are unicellular and others are multicellular. They range in size from tiny protozoa as small as 1 to 2 µm in diameter ( the size of many bacteria) to arthropods and tapeworms that can measure up to 10 m in length. Indeed, considering the size of some these parasites, it is hard to imagine how these organisms came to be classified as microbes. Their life cycles are equally complex, with some parasites establishing a permanet relationship with humans and others going through a series of developmental stages in a progression of animal hosts. MEDICAL BACTERIOLOGY Medical bacteriology is the study of the relatively small number of frank pathogenic and potentially pathogenic bacteria and the interacting bacterial and host factors that cause and influence infectious processes. Differences Between Eukaryotes and Prokaryotes With the exception of the viruses, microorganisms are classified as eucaryotic protists, which consist of the protozoa, fungi, and algae other than blue-green, and procaryotes, which consist of the bacteria and blue-green algae. Eucaryotic cells are structurally more complex than procaryotic cells, containing a true nucleus with a distinct nuclear membrane, endoplasmic reticulum, mitochndria, lysosomes, cell membrane, undergo typical chromosomal organization during cell division and may

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(fungi and algae) or may not (protozoa trophozoites) contain cell wall. On the other hand, the eucaryotic cells can be either unicellular or multicellular. Procaryotic cells are smaller and generally less complex than eucaryotic cells. They are unicellular, have neither a well-defined nucleus nor nuclear membrane, undergo amitotic division, and (with one exception) contain a cell wall of unique chemical composition. Other components like; endoplasmic reticulum, mitochondria, and lysosomes are not found. Bacterial Morphology and Structure Bacteria range in size from 0.1 to 20 µm in length and from 0.2 to 2 µm in diameter. They clearly visible under the light microscope at 900-1000 x utilizing the oil immersion abjective. Bacteria are found in four forms (Fig 1).
1. Coccus (spherical). Cocci may occur in clusters (Staphylococcus aureus), pairs

(Streptococcus pneumoniae), or in chains (Streptococcus pyogenes). 2. Bacilli (rod-shaped). Bacilli may be long with square-cut ends (B.anthracis) or short with rounded ends (Haemophilus influenzae); if the organisms are so short as to appear as cocci, they are referred to as coccobacillary. 3. Vibrio (curved). Curved bacteria have a single turn (Vibrio cholerae). 4. Spirillum, spirochete (spiral). Spiral organisms have a series of turns or twists. Cocci:

Fig. 1 Bacterial Morphology

COCCUS
A: Diplococcus B: Streptococcus C: Staphylococcus J: Tetrate

BACILLUS
D: Bacillus anthracis H: Vibrio(curved) E: Haemophilus influenzae I: Spiral F: Fusiform G : Flamentous

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Fig. 2 Bacterial cell structure

Bacterial Ultrastructure (Fig. 2) A. Cell Envelope Cytoplasmic Membrane The cytoplasmic membrane is a thin, ductile, and elastic membrane that encloses the cytoplasm and is composed mostly of proteins and lipoproteins embedded in a phospholipid bilayer. This membrane functions as a significant osmotic barrier to low molecular weight substances, and as a semipermeable membrane controlling the exchange of substances between the cell and surrounding medium. Several enzimatically mediated biosynthetic processes, as well as electron transport and oxidative phosphorylation, take place within the membrane. Mesosomes are intracellular membrane structures, formed by an invagination of the cytoplasmic membrane. They are more frequently seen in Gram-positive than Gram-negative bacteria. Mesosomes present at the septum of Gram-positive bacteria are involved in chromosomal separation; at other site they may be associated with cellular metabolism. Cell Wall The cell wall is a constituent of all bacteria, except members of the genus Mycoplasma. This structure confers rigidity and shape to the bacterial cell and acts as a barrier to low molecular weight substances. Inasmuch as mammalian cells lack cell walls, antimicrobial agents that inhibit cell wall synthesis (e.g., penicillin) will have sellective activity gainst bacteria and little or no host toxicity. Bacterial rigidity and shape are attriutable to layer, which lies closest to the cytoplasmic membrane and is referred to as peptidoglycan (syn: murein, mucopeptide).

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According to the structural and biochemical aspects of their cell wall, there are two groups of bacteria (Fig. 3):

Fig. 3 Bacterial cell wall structure

1. Gram Pozitive Bacteria: The walls of gram-pozitive (G+ ) organisms are thick, compact, and almost exclusively peptidoglycan. All gram-pozitive bacteria contain lipoteichoic acid (LTA) convalently linked to the cytoplasmic membrane, and as major surface antigens, may acts as a virulence factors. Several gram-positive bacteria have surface proteins that are linked to either the cytoplasmic membrane or peptidoglycan, and may acts as virulence factors or serve as the basis for a clasification scheme. 2. Gram Negative Bacteria: The cell wall of gram-negative bacteria are thinner, less compact, and more complex in their chemical composition. They are composed of a distinct outer membrane bilayer and a distinct, thin peptidoglycan layer separated from the cytoplasmic membrane by a periplasmic space. The outer membrane is composed essentially of lipoproteins, phospholipids, porins, and lipopolysaccharides. Lipoproteins are the most abundant proteins of gram negative bacteria; the lipid end is insrted into the outer membrane while the protein end is linked to peptidoglycan. Phospholipids from outer membrane matrix and contribute to outer membrane stabilization. Porins are outer membrane trimers that form channels that permit small molecular weight solutes to diffuse across the outer membrane; because porins are penetrated slowly by large molecules, they may contribute to the relatively high resistence of gram negative organisms to certain antimicrobial agents. Lipopolysaccharide (LPS) (syn: endotoxin) consist of glycolipid complex, referred to as lipid A, linked to a polysaccharide. LPS is toxic for humans, producing hypotension, fever, shock, intravascular coagulation, and/or tissue necrosis. The lipid

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A is responsible for the toxicity of the molecule, the core polysaccharide components constitute antigenic region ( O antigen ) to many gram negative organisms, and consist of major surface antigens with diverse epitopes that confer specificity upon the organisms. Protoplast refers to a shapeless gram positive bacterium whose cell wall hase been removed in a osmotically stabilized medium by lysosome hydrolysis or by inhibition of cell wall synthesis with an antimicrobial agent such as penicillin. Similar treatment of a gram negative bacterium results in a nearly cell wall-less organisms that has only retained some or all of its outer membrane components and is referred to as a spheroplast. Actively growing and multiplying protoplast and spheroplast are generally called L forms. Capsules Capsules are well difined mucoid structures, usually polysaccaride in nature, which closely surround the cell wall of some bacteria (Fig. 4). These capsules may be antiphagocytic, vaccinogenic, and/or identifiable in rapid diagnostic tests utilizing specific antiserum. Also, the dextran and levan capsules of Streptococcus mutans are the means by which the bacteria attach and stick to the tooth enamel.

Fig. 4 Bacterial capsule

Bacterial Slime Extracellular slime layers are produced by some bacteria. They are more loosely bound to the cell surface than capsules and are water soluble. The slime is composed of complex polysaccharides. It is a virulence factor, e.g. facilitating the attachment of Staphylococcus epidermidis onto artificial surfaces, such as intravascular cannulae and protect the organism from antibiotics and host defences. The capsule and slime layers are also called the glycocalyx Flagella Flagella are composed entirely of a single protein subunit called flagellin, which differs in primary structure among different bacterial speciese, and are responsible for bacterial motility that may inhance bacterial invasion. The surface of flagella is made up of protein antigens with diverse epitopes useful in the identification and classification of organisms. They can be single (monotrichous) or multiple (peritrichous). Spirochetes contain similar motility apparatus, protein in nature, which leis in the periplasmic space between the cytoplasmic membrane and the outer membrane.

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Pili Pili are short, hairlike, rigid, surface appendages originating in the cytoplasmic membrane. These appendages are found predominantly in gram negative organisms and are composed of protein subunits called pillin. There are two types of pili, referred to as sex (F) pili and common pili (fimbriae). 1. Sex pili. Sex pili, of which only 1-4 are found at random bacterial sites, mediate the conjugation of donor and recipient cells and may participate in DNA transfer. 2. Common pili. In contrast, as many as 200 common pili may be evenly distributed over the surface of an organism and may act as virulence factors by mediating adherence to host cell surface, e.g. Neisseria gonorrhoeae produce fimbriae that bind to specific receptors of cervical epithelial cells. B. Intracellular Structure The Cytoplasm The cytoplasm is the complex mixture of substances enclosed by the envelope. It consist of an amorphous equeous fluid in which are dissolved or suspended a myriad of metabolites, enzymes, ions, ribosomes, plasmids, and storage granules. Embedded in this fluid is a fibrous mass, called the nuclear material. Nuclear Material The bacterial material consists of a single circular molecule of double-stranded DNA, about 1 mm long when unfolded. It is tightly packed within the bacterium and is not surrounded by a nuclear membrane as in mammalian cells. Smaller extrachromosomal DNA molecules, called plasmids, that can replicate independently, may also be present. Plasmids are most commonly found in gram-negative bacteria, and although not usually essential for cellular survival, they often provide a selective advantage: many confer resistance to one or more antibiotics. Bacterial Ribosomes The cytoplasm has many ribosomes which contain RNA and proteins, and are involved in protein synthesis. These ribosomes are 70S monomers composed of 30S and 50S subunits in contrast to the 80S monomer mammalian ribosomes that are made up of 40S and 60S subunits.

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Cytoplasmic Granules are located in the bacterial cytoplasm. These granules represent stored food reserves consisting of protein, polysaccharide, and/or lipid. The size of theses granules can increase in a favourable enviroment and decrease when conditions are adverse, e.g. Corynebacterium diphtheriae may contain high energy phosphate reserves in granules termed volutin granules. Spores Some gram-positive, but never gram-negative, bacteria such as members of the genera Bacillus and Clostridium (soil bacteria) are spore formers. Under harsh enviromental conditions, such as the loss of a nutritional requirement, these bacteria can covert from vegetative state to a dormant state, or spore. The location of the spore within a cell is a characteristic of the bacteria and can assist in identification of the bacterium. The spore is a dehydrated, multishelled structure that protects and allows the bacteria to exist in “suspended animation”. They are highly resistant to adverse enviromental conditions and may survive desication, disinfectants or boiling water for several hours. Depletion of specific nutrients from the growth medium triggers a cascade of genetic events leading to the production of a spore. Spore formation begins with the invagination of the parent cell membrane, producing a double membrane that encapsulates and isolates a copy of the bacterial DNA and cytoplasmic contents in what will become the core of the spore. Then the core are surrounded by it is plasmic membrane, the peptidoglycan and the membrane of the septum. This wraps the DNA in the two layers of membrane and peptidoglycan that would normally divide the cell. This is surrounded by the cortex, which is made up of a thin inner layer of tightly crosslinked peptidoglycan surrounding a membrane (which used to be the cytoplasmic membrane) and a loose outer peptidoglycan layer. The cortex is surrounded by the tough, keratin-like protein coat which protects the spore (Fig. 5). The processes requires 6 to 8 hours for completion. The germination or transformation of spores into the vegetative state is stimulated by disruption of the outer coat by mechanical stress, pH, heat, or another stresser and requires water and a triggering nutrient. The process take about 90 minutes.

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Fig. 5 Spore formation

BACTERIAL PHYSIOLOGY Bacterial Growth Most bacteria will grow on artificial culture media. However, some bacteria, e.g. Mycobacterium leprae (leprosy) and Treponema pallidum (syphilis), cannot yet be grown in vitro; other bacteria, e.g. chlamydiae and rickettsiae, only replicate within host cells and are grown in tissue-culture. Under suitable conditions (nutrients, temperature and atmosphere) a bacterial cell will increase in size and then divide into two identical cells (binary fission). These two cells are able to grow and divide at the same rates as the parent cell, providing conditions remain stabe (Fig. 6). The time required for the number of bacteria in a culture to double is called the generation time; e.g. Escherichia coli has a generation time of about 30 min under optimal conditions.

Fig. 6 Binary fission

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Metabolism and the Conversion of Energy All cells require a constant supply of energy to survive. This energy, typically in the form of adenosine triphosphate (ATP), is drived from the controlled breakdown of various organic substrates (carbohydrates, lipids, and proteins). This process of substrate breakdown and conversion into usable energy is known as catabolism. The energy produced may then be used in the synthesis of cellular constituents (cell walls, proteins, fatty acids, and nucleic acids). The metabolic process generally begins with hydrolysis of large macromolecules in the external cellular enviroment by specific enzymes or exoenzymes. The small subunit molecules produced (monosaccharides, short peptides, and fatty acids) are transported across the cell membranes into the cytoplasm by active or passive transport mechanisms specific for the metabolite. The metabolites are converted by one or more pathways to one common universal intermediate, pyruvic acid. From pyruvic acid the carbon may be channeled towards energy production or the synthesis of a new carbohydrates, amino acids, lipids, and nucleic acids. Requirements for Bacterial Growth Most bacteria of medical importance require carbon, nitrogen, water, inorganic salts and a source of energy for growth. They have various gaseous, temperature and pH requirements, and can utilize a range of carbon, nitrogen and energy sources. Some bacteria also require special growth factors, including amino acids and vitamins. Growth requirements are important in selecting the various culture media required in diagnostic microbiology and in understanding the test for identifying bacteria 1. Carbon and Nitrogen Sources Bacteria are classified into two groups according to the type of compounds they can utilize as a carbon source. Autotrophs utilize organic carbon from carbon dioxide and nitrogen from ammonia, nitrites and nitrates; they are of minor medical importance. Heterotrophs require organic compounds as their major source of carbon and energy; they include most bacteria of medical importance. Heterotrophs may have very complex or very simple requirements for organic molecules. Bacteria must also have access to the major elements sulfur, phosphorus, potassium, calcium, magnesium, and iron, plus some minor elements. Most bacteria require varying numbers of growth factors, which are organic compounds required by the cell in order to grow, but which the organism cannot itself synthsize (for example, vitamins).

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2. Atmospheric Conditions Carbon dioxide (CO2). Bacteria require CO2 for growth; adequate amounts are present in air or are produced during metabolism by the organisms themselves. A few bacteria require additional CO2 for growth, e.g. Neisseria meningitidis. Oxygen. Bacteria may be classified into four groups according to their oxygen requirements: -obligate (strict) aerobe grow only in the presence of oxygen, e.g. Pseudomonas aeruginosa; -microaerophilic bacteria grow better in low oxygen concentrations, e.g. Campylobacter jejuni; -obligate (strict) anaerobe grow only in the absence of free oxygen, e.g. Bacteroides spp.; -facultative anaerobes grow in the presence or absence of oxygen, e.g. E.coli. Temperature. Bacteria can be classified according to the optimal temperature for growth: -psychrophiles: low temperatures (optima <20 ºC; some below 0 ºC); -mesophiles: 20-45 ºC; - thermophiles: >45 ºC. Nearly all pathogenic bacteria grow best at 37 ºC. The optimum temperature for growth is occationally higher, for example, for C.jejuni, it is 42 ºC. The ability of some bacteria to grow at low temperatures (0-4 ºC) is important in food microbiology; Listeria monocytogenes, a cause of food poisoning, will grow slowly at 4 ºC and has resulted in outbreaks of food poisoning associated with cook-chill products. pH. Most pathogenic bacteria grow best in slightly alkaline pH (pH 7.2-7.6). A few exceptions exist; Lactobacillus acidophilus, present in the vagina of postpubescent females, prefers an acid medium (pH 4.0). It produces lactic acid which keeps the vaginal secretions acid, thus preventing many pathogenic bacteria from establishing infection. Vibrio cholerae, the cause of cholera, prefers an alkaline enviroment (pH 8.5). 3. Energy production A distinctive feature of bacterial metabolism is the variety of mechanisms used to generate energy from these carbon sources. According to the biochemical mechanisms used, bacterial metabolism can be categorized into three types: Aerobic respiration is the metabolic precess in which molecular oxygen serves as the final electron acceptor of the electron transport chain. In this precess oxygen is

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reduced to water. Respiration is the energy-generating mode used by all aerobic bacteria Anaerobic respiration is the metabolic process in which inorganic compounds other than molecular oxygen serves as the final electron acceptors. Anaerobic respiration can be used as an alternative to aerobic respiration in some species (facultative organisms), but is obligatory in other species (some obligate anaerobes). Other obligate anaerobes use fermentation as their main mode of energy metabolism. This is particularly true among the anaerobic bacteria of medical importance. Fermentation is an alternative anaerobic process exhibited by some species. It is the metabolic process by which an organic metabolic intermediate derived from fermentable substrate serves as the final electron acceptor. Stages of Bacterial Growth Cycle When bacteria are added (inoculated) into a liquid growth medium, subsequent multiplication can be followed by determining the total number of viable organisms (viable counts) at various time intervals. The growth curve produced normally has four phases (Fig. 7): Lag phase of growth occurs while the cell adapts to its new surroundings. During this time, the metabolic activity of the organism is increased in preparation for bacterial division. An increase in bacterial RNA and protein synthesis is noted, as is an increase in the size of the bacterial particles. Exponential or log phase of growth, the next step in bacterial proliferation, represents the rapid cell growth and division. During this stage bacteria are especially susceptable to agents such as antibiotics, probably because the physiologic effeciency provides more frequent opportunities for interaction with the antibiotic and the bacterial envelope barriers are more easily traversed. Stationary growth phase of growth occurs when some essential nutrient becomes limiting in the bacterial inviroment, or alternatively, a toxic end product of cell metabolism accumulates that is deleterious for the growth process. Cell growth then slows and usually stops within a single cell generation time. If new or alternative nutrients are not encountered, cellular energy supplies are eventually depleted and the cells enter a death phase.

Fig. 7 Bacterial growth cycle

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Growth on Solid Media Liquid media can be solidified with agar which is extracted from algae. A temperature of 100 ºC is used to melt agar, which then remains liquid until the temperature falls to approximately 45 ºC, when it produces a transparent gel. Solid media are normally set in Petri dishes (agar plates). Most bacteria grow on solid media to produce colonies. Each colony comprises thousands of bacterial cells which emanated from a single cell. The morphology of the colony assists in bacterial identification. GROWTH ON LABORATORY MEDIA To culture bacteria in vitro, the microbiologist has to take into account the physiological requirements. Various types of liquid and solid madia have been developed for the diagnostic laboratory. Simple media. Many bacteria will grow in simple media, e.g. nutrient broth/nutrient agar which contains “peptone” (polypeptides and amino acids from the enzymatic digestion of meat) and “meat extract” (water-soluble components of meat containing mineral salts and vitamins). Enriched media. These contain additional nutrients for the isolation of fastidious bacteria which require special conditions, e.g. blood agar; chocolate agar (heated to lyse erythrocytes and release additional nutrients). Selective media. These are designed to facilitate growth of some bacteria, whilst suppressing the growth of others and include: MacConkey agar, which contains bile salts and allows the growth of bile-tolerant bacteria only; alkaline peptone broth, which allows selective growth of vibrios which prefer alkaline conditions; and antibiotics, which are frequently added to media to allow only certain bacteria to grow whilst suppressing or killing others. Indicator media. These are designed to aid the detection and recognition of particular pathogens. They are often based on sugar fermentation reactions which result in production of acid and the subsequent colour change of pH indicator, e.g. MacConkey agar contains lactose and a pH indicator (neutral red); lactose-fermenting bacteria (e.g. E.coli) produce acid and form pink colonies, whilst non-lactose fermenting bacteria (e.g. salmonella) do not produce acid and form pale yellow colonies. This property facilitates the recognition of possible salmonella colonies amongst normal bowel flora.

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BACTERIAL GENETICS The Bacterial Genome The bacterial genome is the total collection of genes (2000 to 4000 in number) carried by a bacterium both on it is chromosome and on it is extrachromosomal genetic elements, if any. The bacterial chromosome differs in several ways from the human chromosome: Bacterial Chromosome (such as E.coli) • Have single, double stranded circular Human Chromosome
23 chromosome, double stranded linear molecule of DNA 3.2 X 109 990 mm two distinct copies of each chromosom (they are therefor diploid)

molecule of DNA • 5 million base pairs • 1.3 mm in length • Have only one copy of chromosome (they are therefor haploid), so alteration of a gene (mutation) have a more obvious effect on the cell • Structure of the chromosome is maintained by by histones polyamines such as spermine and spermidine ________________________________________________________________________________

Bacteria may also contain extrachromosomal genetic elements such as plasmids or bacteriophages (bacterial viruses). These elements are independent of the bacterial chromosome and in most cases can be transmitted from one cell to another. Plasmids are small genetic elements that replicate independently of the bacterial chromosome. Most plasmids are circular, double stranded DNA molecules varying from 1500 to 400,000 base pairs. Like the bacterial chromosomal DNA, they can autonomously replicate and as such are referred to as replicons. Some plasmids, such as the E.coli F plasmid, are episomes, which means that they can integrate into the host chromosome. Plasmids carry genetic information, which may not be essential but can provide a selective advantage to the bacteria. For example: Antibiotic resistance, production of toxins and metabolizing some substrates. Bacteriophages (phages) are viruses that replicate inside of the bacterial cells. These extrachromosomal genetic elements can survive outside of a host cell because the nucleic acid genome (which may be DNA or RNA) is protected by a protein coat (Fig. 8). The typical replicative cycle begins with attachment of the phage to receptors on the cell surface, followed by injection of the nucleic acid into the bacterial cell, leaving all or most of the protein outside the cell (this is incontrast to viral infection of vertebrates cells, where the entire virus is taken up by the cell, and it

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is nucleic acid released intracellularly). The phage nucleic acid takes over the cell’s biosynthetic machinery to replicate it is own genetic material, and to synthesize phage-specific enzymes and proteins of the phage coat. When sufficient coat proteins and new phage have accumulated, these components self-asemble into mature phage particles with the DNA encapsulated by the phage coat. Release of the new phage particles is accomplished by a phage-specific enzyme (a lysozyme) that disolves the bacterial cell wall.

Fig. 8 Bacteriophage

Phage are classified as virulent or temperate depending on the nature of their relationship to the host bacterium (Fig. 9). A. Virulent phage. Infection of a bacterium with averulent phage inevitably results in the death of the cell by lysis, with release of newly replicated phage particles. Under optimal conditions, a bacterial cell infected with only one phage particle can produce hundreds of progeny phage in twenty minutes. Generally, phage that attack one bacterial species do not attack other bacterial species. B. Temperate phage. A bacterium infected with temperate phage can have the same fate as a bacterium infected with a virulent phage. However, an alternative outcome is also possible, namely, after entering the cell, the phage DNA, rather than replicating autonomously, can fuse or integrate with the chromosome of the host cell (prophage). Lysogenic bacteria are bacteria that carry a prophage; the phenomenon is termed lysogeny.

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Fig. 9 Life cycle of virulent and temparate phages

Replication of Bacterial DNA Replication of the bacteria genome is triggered by a cascade of of events linked to the growth rate of the cell. Replication of DNA is initiated at a specific sequence in the chromosome called OriC. The replication requires an enzyme (helicase) to unwind the DNA at the origin to expose the DNA, an enzyme (primase) to synthesize primers to start the process, the enzyme or enzymes (DNA dependent DNA polymerases) that copy the DNA but only in the 5′ to 3′ direction, and other enzymes. New DNA is synthesized semiconservatively, using both strands of the parental DNA as templates. New DNA synthesis occurs at growing forks and proceeds bidirectionally. One strand (the leading strand) is copied continuously in the 5′ to 3′ direction, whereas the other strand (the lagging strand) must be synthesized as many pieces of DNA using RNA primers (Okazaki’s fragments). The lagging strand DNA must be extended in the 5′ to 3′ direction as it is template becomes available. Then the pieces are ligated together by the enzyme DNA ligase. Protein Synthesis The synthesis of protein is primarily controlled by DNA. The design of each proteins is trasmitted from DNA to mRNA, which instructs the cellular machinery to assemble a particular protein. Proteins serve structural and enzymatic purposes and thereby determine the structure and metabolic function of any organism. The total genetic potential of DNA constitute the genotype of the bacterium; what actually becomes manifest discernible to the observer is the phenotype. The bacterial genome consist of 3,000 to 6,000 genes-distinct DNA sequences that specifiy the sequence of amino acids in polypeptide chain. Each gene determines a particular kind of amino acid assembly. The chain of nucleotides that constitute a

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gene is composed of groups of bases. Each set of three bases, known as a codon, specifies a particular amino acid. Since reading of the massage can begin at any base, three different transcriptions would be possible. GENETIC VARIATION The ability of bacteria to alter genetic information is fundamental to their survival in a changing enviroment. Such variation in the genome can occur in two ways, mutation and recombination (genetic transfer). Mutation During replication of DNA, copying errors called mutations may occur leading to changes in the sequence of nucleic acids. Many mutations occur spontaneously in nature (e.g., polymerase mistakes); however, mutations can also be induced by physical or chemical agents. Among the physical agents used to induce mutations in bacteria are heat, ultraviolet, and x-rays. Chemicals that are mutagens are nucleotidebase analogues, acridine derivatives, and DNA-reactive chemicals. Mutations can be classified according to the kind of chemical change that occurs in the DNA: 1. Base substitutions: A single base change can result in a transition, in which one purine (A or G) is replaced by another purine or in which a pyrimidine (C or T) is replaced by another pyrimidine. A transversion, in which for example, a purine is replaced by a pyrimidine and vice versa, may also result. 2. Additions and deletions: A single base pair, or a contiguous string of base pairs, can be added to or deleted from the DNA. More major changes which lead to significant alterations in the organism, are often detrimental and the mutant organism may not survive. However, under certain circumstances, such alteration can result in a mutant cell with a significant advantage, allowing it to autgrow other daughter cells; for example, antibiotic resistance mutants may be selected out when that particular antibiotic is present in the enviroment. Reconbination (Genetic transfer) Gene exchange between bacteria is possible by three mechanisms: transformation, transduction and conjugation. Transfer of genetic information among bacteria occurs most often between members of the same species; however, it does occur between totally unrelated organisms. Transformation Transformation is the process by which bacteria take up fragments of naked DNA from the surroundings and incorporate them into their genome. This process of transformation was first described in Streptococcus pneumoniae. How DNA gets

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across the bacterial membrane is not well understood. It is clear, however, that some bacteria have special receptors for DNA on their membranes, which facilitate the internalization of DNA. Transformation appears to be major source of genetic exchange for a number of bacteria including N.gonorrhoeae and S.pneumoniae. It is widely exploited by scientists and genetic engineers to introduce recombinant DNA forms.

Conjugation Conjugation refers to the process by which bacteria transfer genes from one cell to another by means of cell-to-cell contact. Specifically, the process requires the presence of the donor cell of hair-like projrctions called sex pili that make contact with specific receptor sites on the surface of the recipient cell. A plasmid isolated from E.coli called the F (fertility) factor has been intensively studiet and serves to illustrate the process of conjugation. It is related to plasmids moved from cell to cell by conjugation. Only bacteria that carry the F vactor (denoted as F+) produce sex pili, which can attach specifically to a bacterium that does not carry the F factor (denoted as F-). In a process that is not well understood, the pili bring the cells close together, a bridge is formed between the two cells, and the F factor DNA is copied and transferred from the F+ to the F- cell, converting it to an F+ cell (Fig. 10). The transfer of DNA is also directed by a set of genes carried on the F plasmid. First, the plasmid is cleaved on the strand at a specific site. Next, an end of DNA produced bu this cleavage enters the recipient cell and continues to move across until the entire strand is transferred, at which the points ends are joinned to form a circular molecule. This single-strand molecules is then converted to double-stranded DNA by the action of DNA polymerase. During this process, the complementary strand of DNA remains within the donor cell and is also converted to duplex DNA. In this way, the plasmid is transferred to a new cell while the donor cell maintains its copy.

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Fig. 10 F plasmid and conjugation

Occasionally, the F factor will integrate itself into the bacterial chromosome by homologous recombination (Fig. 11). When this happens, part and sometimes all of the bacterial chromosome can be transferred to a recipient cell by way of the pathway discussed above. Such cells are denoted Hfr (high frequency of recombination) to indicate that they often transfer chromosomal genes to recipient cells. Conjugation is a particularly devise for transferring plasmids (and for Hfr strains, chromosomal genes as well) from one bacterium to another (Fig. 12). Plasmids are used extensively in recombinant DNA technologies. Foreign DNA (e.g., from humans) can be inserted into bacterial plasmids where it can be easily reproduced and manipulated.

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Fig. 11 F, Hfr, and F-prime

Fig. 12 Transfer of Hfr from one bacterium to another

Transduction Transduction refers to transfer of genes from one cell to another via a phage vector without cell-to-cell contact. There are two ways in which this occur: Generalized transduction (Fig. 13). A random fragment of bacterial DNA, is accidentally packaged in the phage protein coat in place of the phage DNA. When this rare phage particle infects a cell it injects the bacterial DNA fragment into the cell. If this fragment becomes integrated into the recipient chromosome by recombination, the recipient cell will be stably transduced.

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Fig. 13 Generalized transduction

Specialized transduction Upon infection, temprate viruses can reproduce and lyse the host cell as described above. These viruses also have an alternative pathway available to them: They can insert DNA into the host chromosome, causing the bacterium no harm. Such a virus might remain dormant in the bacterium for many generations. However, in response to certain enviromental signals, the dormant virus can induced to lytically, killing the host cell and releasing hundreds of progeny phage. The temprate phage, in gaining its independence from the bacterial chromosome, may carry with it small pieces of donor bacterial DNA, which, when delivered to the next host cell, add a new attribute to the new host’s capabilities.

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GENETIC ENGINEERING Genetic engineering, also known as recombinant DNA technology, uses the techniques and tools developed by the bacterial geneticists to purify, amplify, modify, and express specific gene sequences. The basic tools of genetic engineering are (1) cloning vectors, which can be used to deliver the DNA sequences into receptive bacteria and amplify the desired sequence; (2) restriction enzyme, which are used to cleave DNA reproducibly at defined sequences; and (3) DNA ligase, the enzyme that links the fragment to the cloning vector. Cloning vectors must allow foreign DNA to be inserted into them but still must be able to replicate normally in the bacterial host. Many types of vectors such as plasmids, bacteriophages and cosmids are currently used. Genetic engineering has been used to isolate and express the genes for useful proteins in bacteria, yeasts, or even insect cells such insulin, interferone, growth hormones, and interleukin. Large amounts of pure immunogen for a vaccine can be prepared without the need to work with the intact disease organisms. For example, the hepatitis B surface antigen is produced by the yeast Saccharomyces cerevisiae. Recombinant DNA technology has also become essential to laboratory diagnosis, forensic science, agriculture, and many other disciplines. MICROBIAL FLORA IN HUMANS Important Microbial Definitions Pathogen: An organism which can invade the body and cause disease Infection: A disease caused by pathogen. Pathogenicity: The ability to cause disease. Virulence: The pathogen’s power to cause severe disease. Epidemiology: The study of the distribution and determinants of diseases in populations. Opportunistic microorganism: Is an organism that is unable to cause disease in healthy, immunocompetent individuals, but can infect people whose specific or nonspecific defenses have been impaired. Microbial Flora Medical microbiology is the study of the interactions between humans and microorganisms such as bacteria, viruses, fungi, and parasites. Although are primary interest is in diseases caused by these interactions, it must also be appreciated that microorganisms play a critical role in human survival. The normal commensal population of microbes participates in the metabolism of food products, provides essential growth factors, protects against infections with highly virulent microorganisms, and stimulates the immune response. In the absence of these organisms, life as we know it would be impossible.

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The microbial flora in and on the human body is in a continual state of flux determined by a variety of factors, such as age, diet, hormonal state, health, and personal hygiene. Whereas the human fetus lives in a protected, sterile enviroment, the newborn is exposed to microbes from the mother and enviroment. The infant’s skin is colonized first, followed by the oropharynx, gastrointestinal tract, and other mucosal surfaces. Throughout the life of an individual, this microbial population continues to change. Changes in health can drastically disrupt the delicate balance that is maintained among the heterogeneous organisms coexisting within as. For example, hospitalization can lead to the replacement of normally avirulent organisms in the oropharynx with gram-negative bacilli (e.g., Klebsiella, Pseudomonas) that can invade the lungs and cause pneumonia. Exposure of an individual to an organism can lead to one of three outcomes. The organism can (1) transiently colonize the person, (2) permanently colonize the person, or (3) produce disease. An understanding of medical microbiology requires knowledge not only of the different classes of microbes but also of their propensity for causing disease. A few infections are caused by strict pathogens (organisms always associated with human disease). Some examples of strict pathogens and the diseases they cause include Mycobacterium tuberculosis, Neiseria gonorrhoeae, plasmodium spp., and rabies virus. Most infections are caused by opportunistic pathogens (e.g., Staphylococcus aureus, Escherichia coli, Candida albicans). Respiratory Tract and Head Mouth, Oropharynx, and Nasopharynx The mouth is colonized with numerous organisms, with 10 to 100 anaerobes for every aerobic bacterium. The most common anaerobic bacteria are Peptostreptococcus, Veillonella, Actinomyces, and Fusobacterium ssp.; the most common aerobic bacteria are Streptococcus, Haemophilus, and Neisseria spp. The relative proportion of these organisms varies at different anatomic sites; for example, the microbial flora on the surface of a tooth is quite different from the flora in saliva or in the subgingival spaces. Most of the common organisms in the upper respiratory tract are relatively avirulent and are rarely associated with disease unless they are introduced into normally sterile sites (e.g., sinuses, middle ear, brain). Ear The most common organism colonizing the outer ear is coagulase-negative Staphylococcus. Other organisms colonizing the skin have been isolated from this site, as well as potential pathogens such as S.pneumonia, Pseudomonas aeruginosa, and the Enterobacteriaceae.

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Eye The surface of the eye is colonized with coagulase-negative staphylococci as well as rare numbers of organisms found in the nasopharynx (e.g., Haemophilus spp, Neisseria spp, and viridans streptococci). Lower respiratory tract The larynx, trachea, bronchioles, and lower airways are generally sterile, although transient colonization with secretions of the upper respiratory tract may occur after aspiration. Acute disease of the lower airway is usually caused by more virulent bacteria present in the mouth (e.g., S.pneumonia, S.aureus, H.influenzae, members of the family Enterobacteriaceae such as Klebsiella). Gastrointestinal Tract Esophagus Oropharyngeal bacteria and yeast, as well as the bacteria that colonize the stomach, can be isolated from the esophagus. Stomach Because the stomach contains hydrochloric acid and pepsinogen, the only organisms present are small numbers of acid-tolerant bacteria such as the lactic acidproducing bacteria (Lactobacillus and Streptococcus spp.) and Helicobacter pylori (is a cause of gastritis and ulcerative disease). Small intestine In contrast with the anterior portion of the digestive tract, the small intestine is colonized with many different bacteria, fungi, and parasites. Most of these organisms are anaerobes, such as Peptostreptococcus, Porphyromonas, and Prevotella. Common causes of gastroenteritis (e.g., Salmonella and Campylobacter spp.) can be present in small numbers as asymptomatic residents; however, their detection in the clinical laboratory generally indicates disease. Large intestine More microbes are present in the large intestine than anywhere else in the human body. It is estimated that more than 1011 bacteria per gram of feces can be found, with anaerobic bacteria in excess by more than 1000-fold. Various yeasts and nonpathogenic parasites can also establish residence in the large intestine. The most common bacteria include Bifidobacterium, Eubacterium, Bacteroides, Enterococcus, and the Enterobacteriaceae. E.coli is present in virtually all humans from birth until death. Although this organism represents less than 1% of the intestinal population, it is the most common aerobic organism responsible for intra-abdominal infections. Antibiotic treatment can rapidly alter the population, causing the proliferation of antibiotic-resistant organisms such as enterococci, Pseudomonas, and fungi.

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Genitourinary System In general, the anterior urethra and vagina are the only anatomic areas of the genitourinary system permanently colonized with microbes. Although the urinary bladder can be transiently colonized with bacteria migrating upstream from the urethra, these should be cleared rapidly by the bactericidal activity of the uroepithelial cells and the flushing action of voided urine. Anterior urethra The commensal population of the urethra consists of a variety of organisms, lactobacilli, and coagulase-negative staphylococci the most numerous. These organisms are relatively avirulent and are rarely associated with human disease. Vagina The microbial population of the vagina is more diverse and is dramatically influenced by hormonal factors. Newborn girls are colonized with lactobacilli at birth, and these bacteria predominate for aproximately 6 weeks. After that time, the levels of maternal estrogen have declined, and the vaginal flora changes to include staphylococci, streptococci, and Enterobacteriaseae. When estrogen production is initiated at puberty, the microbial flora again changes. Lactobacilli reemerge as the predominant organisms, and may other organisms are also isolated, including staphylococci, group B streptococci, Enterococcus, Gardnerella, Mycoplasma, Ureaplasma, and a variety of anaerobic bacteria. The lactobacilli metabolize the glycogen of the vaginal epithelium to produce lactic acid, resulting in a low pH that limits some potential pathogens. Skin Although many organisms come to contact with the skin surface, this relatively hostile enviroment does not support the survival of most organisms. Gram-positive bacteria (e.g., coagulase-negative Staphylococcus, corynebacteria, and propionibacteria) are the most common organisms found on the skin surface. Fungi Candida is also found on skin surfaces, particularly in moist sites. Gram-negative bacteria do not permanently colonize the skin surface because the skin is too dry. STERILIZATION, DISINFECTION, AND ANTISEPSIS Medical microbiology involves the study of the pathogenesis and chemotherapy of infectious diseases, as well as the examination of how diseases can be prevented. An important aspect of the control of infections is an understanding of the principles of sterilization, disinfection, and antisepsis.

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Sterilization refers to the destruction or elimination of all microorganisms by physical means, including heat (moist and dry heat), radiation, and filtration. Disinfection refers to the destruction of pathogenic or potentially pathogenic microorganisms by chemical means. The term usually applies to the treatment of inanimate objects but the term “skin disinfectant” has been used in medical practice. Antisepsis refers to the destruction or prevention of growth of pathogenic or potentially pathogenic microorganisms by chemical means. The term usually refers to the external application of a chemical to tissues. Bacteriostatic agent refers to chemical or biological agent that prevents the growth and multiplication of but does not destroy pathogenic or potentially pathogenic microorganisms. The term refers to those agents applied to inanimate objects or to external or internal tissues. Bactericidal agent refers to chemical or biological agent that destroys pathogenic and potentially pathogenic microorganisms. This term also refers to those agents applied to inanimate objects or to external or internal tissues. STERILIZATION Dry and Moist Heat Moist and Dry heat are the most common sterilizing methods used in hospitals and are indicated for most materials except those that are heat-sensitive or consist of toxic or volatile chemicals. Dry heat Dry heat destroys microorganisms by oxidation. There are three types of dry heat sterilization: flaming to red heat, incineration and hot-air ovens. Incineration is considered the best method to dispose of infected carcasses and organic wastes; sterilization of inoclating loops by passage through a bunsen burner is another effective form of incineration. Flaming to red heat. Metal instruments, such as dental reflection mirrors, can be sterilized by direct heating. In an emergency, scalpels can be sterilized by dipping the blade in methylated spirit and burning off the spirit. Hot-air ovens produce dry heat by gas or electricity in insulated double walled metal containers. The temperature and time required for effective sterilization is 171ºC for 1 hour, 160ºC for 2 hour or 121ºC for 16 hour. The process is useful on heat stable materials and on substances that steam cannot penetrate such as oil. The effectiveness is monitored with spore tests using Bacillus subtilis, which is relatively resistant to killing by dry air (in contrast with Bacillus stearothermophilus).

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Moist Heat Sterilization by moist heat is more rapid and efficient than dry heat; the presence of water causes protein denaturation resulting in disruption of cell membranes, and improves heat penetration. There are three types of moist heat sterilization: the autoclave, pasteurization, and boiling water. Autoclaving: Autoclaves work on the same principle as the domestic pressure cooker. Steam at atmospheric pressure can attain a temperature on only 100ºC. However, if steam is enclosed and put under pressure as in an autoclave or pressure cooker, the temperature at 1 pounds of pressure is increased to 121ºC. Under these conditions all known pathogens and essentially all known life form including bacterial spors and vegetative cells, viruses, and fungi are killed. The rate of killing organisms during the autoclave process is rapid but is influenced by the temperature and duration of autoclaving, size of the autoclave, flow rate of the steam, density and size of the load, placement of the load in the chamber. Care must be used to avoid creating air pockets, which inhibit penetration of steam into the load. The effectivenes of sterilization can be tested by chemical or biological methods: Chemical method: Browne’s sterilizer control tubes contain a chemical which changes colour when exposed to various temperatures. Biological method: An ampule including commercial preparations of Bacillus stearotermophilus spores is placed in the center of the load, is removed at the end of the autoclave process, and is incubated at 37ºC. If the sterilization process is successful, the organisms fail to sporulate and do not grow. Pasteurization is used primarily in the dairy industry. Pasteurization is the process whereby milk or milk products are exposed to 63 to 65ºC of temperature for 30 min. or exposed to 71 to 72ºC of temperature for 3 to 5 sec. and suddenly reduced under 10ºC. This way is effected for pathogenic nonspor-forming bacteria. Boiling water, even for as long as 30 min, is not recommended for sterilization, because it does not kill bacterial spors or certain viruses such as hepatitis B. Ultraviolet Light Ultraviolet (UV) light is a form of radiant energy in which relatively short wavelengths are caused inhibition of bacterial DNA synthesis. Mercury vapor lamps, which serve as a UV source, are sometimes used in enclosed areas such as operating rooms in an effort to control airborn infections; however, the ability of some potential pathogens to survive under these conditions render this procedure questionable. UV light can cause serious corneal damage and this is therefore never used during ophthalmological procedures.

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X- and Gamma Rays Sterilization by X- and gamma rays is used for delicate supplies such as syringes and sutures, as well as increasingly for food. Radiation kills bacteria and other life forms by damaging most cell constituents, including DNA, usually by free radical formation. Filtration Filtration is a mechanical method for eliminating bacteria from biological fluids and from the air. Filters for sterilizing are most commonly made from cellulose and are available with varying pore size. Apore size of 0.22 µm is small enough to remove bacteria. This filter would not ordinarily retain viruses, mycoplasma, and bacterial products (Fig. 13). DISINFECTION Microbes are also destroyed by disinfection procedures, although more resilient organisms can survive. The effectivenes of these procedures is influenced by the nature of the item to be disinfected, number and resilience of the contaminating organism or organisms, amount of organic material present (which can inactivate the disinfectant), type and concentration of disinfectant, and duration and temperature of exposure. Disinfectants are subdivided into: High-level disinfectants are used for items involved with invasive procedures that cannot withstand sterilization procedures (e.g., certain types of endoscopes, surgical instruments with plastic or other components that cannot be autoclaved). Disinfection of these and other items is most effective if treatment is preceded by cleaning the surface to remove organic matter. Examples of high-level disinfectants include treatment with moist heat (75ºC to 100ºC for 30 min) and use of liquids such as glutaraldehyde (2%), hydrogen peroxide (3 to 25%), peracetic acid (0.2%), chlorine dioxide, and other chlorine compounds (concentration is variable). Intermediate-level disinfectants such as alcohols (70%), iodophor compounds (30 to 50 ppm of free iodine/L), and phenolic compounds (0.4 to 5.0%) are used to clean surfaces or instruments in which contamination bacterial spores and other highly resilient organisms is unlikely. These have been referred to as semi-critical instruments and devices and include flexible fibrotic endoscopes, laryngoscopes, vaginal specula, and other items Low-level disinfectants like Quaternary ammonium compounds (0.4 to 1.6%) are used to treat non critical instruments and devices such as blood pressure cuffs, electrocardiogram electrodes, and stethoscopes. Although these items come into contact with patients, they do not penetrate through mucosal surfaces or into sterile tissues.

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ANTISEPSIS Antiseptic agents are used to reduce the number of microbes on skin surfaces. These compounds are selected for their safety and efficacy. Alcohols (70 to 90%) have excellent activity against all groups of organisms except spores and are nontoxic, although they tend to dry the skin surfaces because they remove lipids. They also do not have residual activity and are inactivated by organic matter. Thus, the surface of the skin should be cleaned before alcohol is applied. Iodophors ( 1 to 2% available iodine) are also excellent skin antiseptic agents, having a range of activity similar to that of alcohols. They are slightly more toxic to the skin than is alcohol, have limited residual activity, and are inactivated by organic matter. Iodophors and iodine preparations are frequently used with alcohols for disinfecting the skin surface. Chlorhexidine (0.5 to 4%) has broad antimicrobial activity, although it kills organisms at a much slower rate than does alcohol. Its activity persists, although organic material and high pH levels decrease its effectiveness. The activity of parachlorometaxylenol is limited primarily to gram-positive bacteria. Because it is nontoxic and has residual activity, it has been used in handwashing products. Triclosan is active against bacteria but not against other organisms. It is a common antiseptic agent in deodorant soaps and some toothpaster. Chemicals Avariety of chemicals are used for disinfection. They are evaluated as to whether they kill vegetative bacteria, mycobacteria, Pseudomonas, fungi, or spores. Surface-Active Agents These agents injure the bacterial cell by damaging the cytoplasmic membrane and altering cell permeability a. Cationic detergents are quaternary ammonium compounds that dissociate to yield positively charged ions that disrupt cytoplasmic membrane phospholipids. Examples of quaternary ammonium compounds include benzalkonium chloride and cetyipyridinium chloride. These compounds are bacteriostatic at low concentrations and bactericidal at high concentrations. However, organisms such as Pseudomonas, Mycobacterium, and the fungus Trichophyton, among others, are resistant to these compounds. Indeed, some Pseudomonas strains can grow rereadily in quaternary ammonium solutions. Many viruses and all bacterial spores are also resistant.

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b. Anionic detergents are soap and fatty acids that dissociated to yield negatively charged ions that disrupt cytoplasmic membrane lipoproteins.Their inability to kill spores or several gram-negative organisms associated with nosocomial (hospital acquired) diseases renders them of little value as disinfection. Phenoles Are compounds that bind to and denature proteins within the bacterial cytoplasmic membrane resulting in membrane damage, leakage of cell contents, and lysis the of the organism. These compounds are bactericidal for both gram negative and gram positive bacteria, including the spores formers. Phenolic compounds, such as AmphylR in concentrations of 2-5%, are excellent disinfectants for washing down surfaces such as operating room floor and laboratory benches. Alcohols Denature cell proteins and disorganize cytoplasmic membrane lipids resulting in a loss of membrane permeability. Alcohols are rapidly bactericidal against vegetative bacteria, mycobacteria, some fungi, and lipid-containing viruses. Unfortunately, alcohols have no activity against bacterial spores and have poor activity against some fungi and non-lipid-containing viruses. Activity is greater in the presence of water. Thus, 70% alcohol is more active than is 95% alcohol. Halogens Halogens such as compounds containing iodine or chlorine, are used extensively as disinfectants. Iodine compounds are the most effective halogens available for disinfection. Iodine is a highly reactive element that precipitate proteins and oxidizes enzymes. It is microbicidal against vertually all organisms, including spore-forming bacteria and mycobacteria. Elemental iodine can be dissolved in aqueous potassium iodide or alcohol, or it can be complexed with a carrier. The latter is reffered to as an iodophor. Povidone iodine is used most commonly, is relatively stable and nontoxic to tissues and metal sufaces, but is expensive compared with other iodine solutions. Chlorine compounds are are also used extensively as disinfectants. Three forms of chlorine may be present in water: elemental chlorine (Cl2), which is very oxidizing agent; hypochlorous acid (HOCl); and hypochlorite ion (OCl2). Chlorine also combines with ammonia and other nitrogenous compounds to form chloramines or Nchloro compounds. These compounds demonstrate good germicidal activity, although spore-forming organisms are 10- to 1000-fold more resistant to chlorine than are vegetative bacteria. Oxidizing Agents Examoles of oxidants include ozone, peracetic acid, and hydrogen peroxide, with the last used most commonly. Hydrogen peroxide effectively kills most bacteria at a concentration of 3 to 6% and kills all organisms including spores, at higher

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concentrations(10 to 25%). It is used to disinfect plastic implants, contact lenses, and surgical prostheses. Heavy Metals Inactivate microbial enzyme systems resulting in interference with protein synthesis. Most heavy metals are too toxic for human use, but some have been employed prophylactically with seccess. Ethylene oxide Ethylene oxide is a colorless gas, soluble in water and comming organic solvents, that is used to sterilize heat sensitive items. It interferes with protein synthesis by alkylating protein and thus blocking free amino groups. It is an excellent bactericidal agent against gram-negative and garm-positive organisms, including spore formers, and is used in the disinfection of hear-lung machines, polyethylene tubing, lensed instruments, biologicals, and other materials that would otherwise be damaged by heat or disinfecting solutions. Sterilization by ethylene oxide can be acchomplished by using 450 to 700 mg of ethylene oxide per liter of chamber space at 55 to 60ºC for two hours. Care must be taken when using ethylene oxide due to its potential tissue toxicity. Although a 24 to 72 h. period of aeration is required after disinfection in order to get rid of residual fumes. Formaldehyde is a gas whose mechanism of action and bactericidal activity is identical to that of ethylene oxide. It is used to decontaminate rooms and fabrics, but causes damage to the cutting edges of instruments. Like ethylene oxide, it is irritating to the skin and leaves residual fumes which necessitate similar periods of aeration. Formalin is formaldehyde in aqueous form. It is used at a 10% concentration to preserve tissues for histopathological study. At appropriate concentration, formalin is valuble in the perparation of several vaccines by virtue of it is ability to convert toxins to nontoxic, and vaccinogenic toxoids. Glutaraldehyde It is an alkylating agent that binds sulfhydryl or amino groups. In 2% aqueous solution, it is an excellent bactericidal agent against gram-negative and gram-positive organisms, including spore formers. Glutaraldehyde is highly effective disinfectant for lensed and other surgical instruments.

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ANTIMICROBIAL AGENTS Until the twentieth century, there were no special agents available to treat infectious diseases, other than folk remedies and herbal concoctions. Amazingly, the hundreds of antimicrobial agents available today were developed within the last 80 years. In 1924, Fleming first isolated penicillin from penicillium notatum. It was introduced to clinical medicine during World War II by Chain and Florey and abruptly changed the practice of medicine. Later, in the 1940s and 1950s, streptomycin and the tetracyclines were developed and were followed rapidly by additional aminoglycosides, cephalosporins, quinolones, and other antimicrobials. Criteria for a successful antimicrobial agent are listed below: 1. Displays targeted toxicity to the offending microbe only. 2. Produces no side effects in the host. 3. Displays a narrow range of activity and does not harm normal host flora. 4. Kills the targeted microorganism (bactericidal), rather than merely inhibiting its growth (bacteriostatic). 5. Fails to induce resistence. 6. Is soluble in body fluids and tissues. Obviously, this ideal has not yet been met. In most cases, the goal of antimicrobial therapy is to reduce the total numbers of infecting microbes to a level low enough that host defenses can destroy them. Definitions Antimicrobial Agent: A substances that either kills, inhibits growth of, or prevents damage due to an infectious microorganism. Antibacterial, antifungal, antiprotozoal, antihelminthic, and antiviral agents are subsets of antimicrobial agents. Antibiotic: A substance produced by a fungus or bacterium that kills or inhibits growth of other microorganisms. Chemotherapeutic Agent: A substance, synthetically derived, used to treat any disease, infectious or non-infectious (e.g., cancer or hypertension). .....cidal: Indicate that the action of the agent will kill the targeted microbe. Bactericidal, fungicidal, and virucidal are derived terms. .....static: Indicates that the action of the agent will inhibit the growth of the targeted microbe but will not kill it. Bacteriostatic, and fungistatic are derived terms. Pharmacokinetics: The study of the concentration and activities of chemotherapeutic agents in patients. Synergy: The antimicrobial activity of two antimicrobial agents used together is greater than the additive effect of both agents used alone. Antagonism: The antimicrobial activity of one antimicrobial agent is diminished by another agent being administered concurrently.

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COMMON ANTIBACTERIAL AGENTS GROUPED BY MECHANISM OF ACTIVITY Antibiotics That İnhibit Cell Wall Synthesis (ß-lactams, and Others). ß-lactam antibiotics include the penicillins and cephalosporins, both of which possess a beta-lactam ring. These agents are a very potend bactericidal agent that attaches to penicillin-binding proteins (PBPs) on the bacterial cell membrane. The PBPs are enzymes (e.g., transpeptidase, carboxipetidase, endopeptidase) that are involved in the synthesis of the cell wall and the maintenance of the cell’s structure integrity Penicillins Penicillin compounds are highly effective antibiotics with extremely low toxicity. The base compound is an organic acid with a ß-lactam ring obtained from culture of the mold Penicillium chrysogenum. Penicillin derivatives are achieved by adding or modifying side chains to the structure of the basic ß-lactam ring: 1. Penicillin G. Penicillin G is a narrow spectrum activity includes gram-positive cocci such as streptococci (including S.pneumoniae, S. pyogenes, S. agalactiae), non-penicillinase producing staphylococci, and certain gram-negative cocci such as Neisseriae spp.. Also it is effective against spirochetes and some anaerobic bacteria. On the other hands, penicillin G, is ineffective against bacterial spp. that produced enzyme of penicillinase (or beta lactamase). Penicillin G is incompletely absorbed because it is inactivated by gastric acid. Thus it is used mainly as parenteral drug for serious infections with penicillin-sensitive organisms. The toxicity of penicillin G is extremely low, hypersensitivity to penicillin is commonly present. 1% to 8% of the general population are allergic to the penicillins. The hypersensitivity reactions range from immediate anaphylactic reactions to late manifestations such as a skin rash. 2. Penicillinase-resistant penicillins ( methicillin, oxacillin, and nafcillin). Are used to treat infections caused by penicillinase-producing staphylococci, including bacteremia, cellulitis, and ostemyelitis. Compared with penicillin G, the penicillin-resistant penicillins are less effective against penicillin G-sensitive organisms. 3. Aminopenicillins (ampicillin and amoxicillin). Aminopenicillins, developed by introducing an alfa-amino group into the benzyl chain, had enhanced activity against some gram-negative bacteria such as

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Escherichia coli, Proteus mirabilis, Salmonella, Shigella and Haemophilus influenzae. Because aminopenicillins are very sensitive to the penicillinase (betalactamase), so finally, they have been combined with bata-lactamase inhibitors to treat infections caused by beta-lactamase producing bacteria. Clavulanic acid + Amoxicillin Sulbactam + Ampicillin 4. Extended-spectrum penicillins (carbenicillin, mezlocillin, and piperacillin). Are a wider spectrum of action than penicillin G, with special against Pseudomonas aeruginosa and some strains of Proteus. They are not penicillinase resistant and is available pareterally to treat systemic infections. Cephalosporins. The cephalosporins are beta-lactam antibiotics derived from 7aminocephalosporanic acid, which was originally isolated for a Cephalosporium mold. The antibiotics have the same mechanism of action as the penicillin but have a wider antibacterial spectrum, are resistant to many beta-lactamase, and have improved pharmacokinetic properties. The antimicrobial agents are divided into “generations” based on their structure and spectrum of activity: 1. First-generation cephalosporins (cefazolin, cephalothin) have good activity against gram positive cocci, and also effective against some gram negative bacteria including E.coli, the genera Klebsiella and Shigella. 2. Second-generation cephalosporins (e.g., cefoxitin, cefuroxime). The spectrum of the 2 nd cephalosporins is expanded to include more gram negative. They are may be less active than the first generation cephalosporins against gram-positive cocci. 3. Third-generation cephalosporins (e.g., cefoperazone, cefotaxime, ceftriaxone). Generally, are less active than the first generation cephalosporins against gram positive cocci, but they are more active than the second-generation cephalosporins against gram negative bacteria. 3 rd generation cephalosporins are also effective against anaerobes. Other Beta-lactam Antibiotics Several beta-lactam antibiotics have slightly different biochemical structures from the penicillins and cephalosporins but have similar potent antibacterial activity. Imipenem is a carbepenem with excellent in vitro and in vivo activity for aerobic and anaerobic gram-positive and gram-negative bacteria. Aztreonam, a monobactam, is a narrow-spectrum antibiotic with activity specific for gram-negative bacilli.

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Other Important Cell Wall Active Agents. Vancomycin is a glycopeptide antimicrobial that prevents the formation of peptidoglycane by binding to the cell wall peptide percursors. This antibiotic elaborated by Streptomyces orientalis, an actinomycete found in soil samples from India and Indonesia. Vancomycin is poorly absorbed when administered orally; however, this property has proved useful for the treatment of gastrointestinal disease caused by S.aureus or Clostridium difficile. The drug is administrated intravenously for treatment of serious systemic infections in patients infected with methicillinresistant staphylococci or with a history of allergic to the penicillins. Bacitracin, which was isolated from Bacillus licheniformis, is a mixture of polypeptides used topically (e.g., creams, ointments, sprays) for skin infections caused by gram positive bacteria (particularly those caused by Staphylococcus and group A Streptococcus). Antibiotics That Alter Cell Membrane Polymyxins. The polymyxins are basic peptides (derived from Bacillus polymyxa) that act as cationic detergents to cause lysis of the lipoprotein cell membrane. Although polymyxin B and colistin are active against gram-negative bacteria (including Pseudomonas), serious nephrotoxicity has limited their internal use. They are used chiefly to treat local infections such as external otitis, eye infections, and skin infections with sensitive organisms. Antibiotics That Inhibit Protein Synthesis Aminoglycosides are bactericidal antibiotics commonly used to treat serious infections caused by many gram-negative bacilli and some gram-positive organisms. Streptococci and anaerobes are resistant to aminoglycosides. In 1943, a strain of Streptomyces griseus was isolated that elaborated streptomycin. Further strains of Streptomyces species furnished neomycin, kanamycin, tobramycin, amikacin, and Micromonospora organisms produced gentamycin and netilmicin. They are inhibit protein synthesis and to act directly on the 30S subunit of the ribosome. Because aminoglycosides are poorly absorbed after oral administration, they are injected intramuscularly or intravenously. Tetracyclines. The tetracyclines are broad-spectrum, bacteriostatic antibiotics that inhibit protein synthesis in bacteria by blocking the binding of tRNA to the 30S ribosomal subunit. Tetracyclines (e.g., tetracycline, doxycycline, minocycline) are effective in the treatment of infections caused by Chlamydia, Mycobacteria, Rickettsia, and other selected aerobic and anaerobic gram-positive and gram-negative bacteria. They are absorbed rapidly from the gastrointestinal tract, and distribute widely in most fluids and tissues, localizing particularly in bones and teeth. Because the tetracyclines will

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cause permanent discoloration of teeth, these antibiotics should not be used in pregnant women or childrens less than 8 years of age. Chloramphenicol. Chloramphenicol, a broad-spectrum, bacteriostatic antibiotic, inhibits bacterial protein synthesis by acting primarily on the 50S ribosomal unit. It is active against large number of gram-positive and gram-negative organisms, rickettsiae and some chlamydia, but is considered the drug of choice only for treatment of typhoid fever. The reason for this is, in addition to interfering with bacterial protein synthesis, chloramphenicol disrupts protein synthesis in human bone marrow cells and can produce aplastic anemia. Macrolides The macrolides are a group of related antimicrobials. They have a common macrocyclic lactam ring. The members of this group include erythromycin, clarithromycin, azihromycin and spiramycin. All the agents have similar antimicrobial spectrum, including gram-positive organisms, Neisseria, Haemophilus and Bordetella, and some gram-negative anaerobes. They are also active against Mycoplasma sp., Rickettsia sp. And Toxoplasma gondii. These agents disrupts protein synthesis by binding to the 50S ribosomal subunit. Erythromycin, is used mainly to treat pulmonary infections caused by Mycoplasma, Legionella, and gram-positive organisms in patients allergic to penicillins. Clindamycin Also blocks protein synthesis by binding to the 50S ribosomal subunit. It is active against staphylococci and anaerobic gram-negative bacilli but generally inactive against aerobic gram-negative bacteria. Clindamycin can be administered orally or intravenously with good penetration into tissues such as bone. Although intravenous administration of clindamycin is associated with relatively few side effects, oral administration can be responsible for gastrointestinal disturbances ranging from mild diarrhea to life-threatining pseudomembranous colitis. Antibiotics That Inhibit Nucleic Acid Synthesis Rifampin Asemisynthetic derivative of rifamycin B produced by Streptomyces mediterranei, bactericidal for Mycobacterium tuberculosis and is very active against aerobic grampositive cocci. Because resistance can develope rapidly, rifampin is usually combined with one or more other effective antibiotics. The drug inhibits DNA-dependent RNA polymerase.

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Quinolones (Fluoroquinolones) A group of orally effective antibacterial agents, are chemically related to nalidixic acid. The number of agens in this group of drugs has risen exponentially such as, ciprpfloxacin, Enoxacin, ofloxacin, lomefloxacin and others. They are bactericidal against most gram-negative organisms and many gram-positive organisms. The mechanism of action of the quinılınes is unique and involves antagonism of the DNA gyrase; the enzyme is involved in DNA synthesis. Metronidazole. Metronidazole was originally introduced as an oral agent for treatment of Trichomonas vaginalis. It is also effective in treatment of amebiasis, giardiasis, and serious anaerobic bacterial infections (including Bacteroides fragilis) but has no significant activity against aerobic or facultatively anaerobic bacteria. The antimicrobial properties of metronidazole appear to be intermediated, which results in DNA breakage. The drug diffuses well to all tissues, including the central nervous system. Antibiotics With Antimetabolic Activity Sulfonamides Strictly speaking, the sulfonamides cannot be classified as antibiotics because they are not produced by living organisms. They may be termed antiinfective or antimicrobials. The introduction of many newer antibiotics limited the use of sulfanamides until the introduction of trimethoprim-sulfomethoxazole combination. This combination, the most common use of the sulfonamides. Sulfonamides inhibit the synthesis of folic acid in bacteria. Folic acid is required for DNA synthesis. The sulfanamides are similar in structure to para-aminobenzoic acid (PABA) and compete with PABA, then they inhibit the synthesis of folic acid that the most important in the synthesis of bacterial DNA. The sulfonamides are bacteriostatic against many gram-positive and som gram negative bacteria. The combination of sulfomethoxazole with trimethoprim (SMXTMP) shows synergistic activity against a widw variety of gram-positive bacteria and some gram-negative bacteria such as Enterobacteriaceae, Pseudomonas spp., Haemophilus influenzae, S.aureus, S.pyogenes, and S.pneumoniae. SMX-TMP is indicated in the treatment of selected urinary tract infections and selected respiratory and gastrointestinal infections. BACTERIAL RESISTANCE Bacterial resistance to antibiotics may be present on a nongenetic basis or may develop on a genetic basis during therapy. Nongenetic resistence is most frequently attributable to the absence of targets for the drug in the bacteria. If the bacteria have no receptors that bind the drug or lack the metabolic pathway necessary for drug

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activity, the bacteria are intrinsically resistant ( e.g., vancomycin or erythromycin ). Inadequate permeability of a compound may also account for the ineffectiveness of tetracycline against some gram-negative bacteria. Certain microorganisms can escape the consequences of drug action by: 1.synthysizing en enzyme that destroys the antibiotic (e.g., the beta-lactamase that cleaves the beta-lactam rings of penicillin and cephalosporin. 2.Changing in permeability of the outer membrane (e.g., gram-negative bacilli) that prevent some antibiotics from entering the periplasmic space through transmembrane channels, called porins, that usually provide access. 3.Alterating macromolecules to which the antibiotic binds. 4. Altering some metabolic activity to which the antibiotic effects. Genetic resistance may be chromosomal in origin or may be transmtted by extrachromosomal plasmids. Chromosomal resistance to several unrelated antibiotics can be transferred to susceptible organisms by cell-to-cell contact or conjugation. The bacteria contain extrachromosomal DNA or resistance plasmids, act like viruses without coats. These plasmids are found in a variety of gram negative bacilli. HOST-PARASITE RELATIONSHIPS ECOLOGY Microorganisms have developed characteristics that allow them to survive and grow in a wide variety of enviroments and are found in virtually every ecological niche. They play a central role in global ecology, providing both an important food source and facilitating the degradation of organic matter. Saprophytes: Microorganisms that live on inanimate organic material Parasites: Microorganisms that grow in or on a living host, obtaining nutrients from the host. Parasitism: One organism benefits at the expense of the other Commensalism: One organism benefits without causing harm to the other, Staphylococcus epidermidis on intact skin represents an example of a commensal. Symbiosis: Both organisms benefit from their association. Lactobacilli in the vagina produced acid end products that keep the vaginal pH at a relatively low level, which contributes to inhibition of growth of pathogenic organisms, such as Neisseria gonorrhoeae. Certain bacteria in human gastrointestinal tract, including E.coli strains and others, produced vitamin K that is absorbed by the host.

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NORMAL FLORA The human body is colonized on the skin and mucosal surfaces with a large number of microorganisms which form the body’s normal flora. These organisms, far from causing disease, often provide benefit to the host, by competing with potential pathogens for attachment sites, by producing antimicrobial substances toxic to pathogens and by competing for nutrients with pathogens. Reduction in normal flora (e.g. by antibiotic therapy) may result in overgrowth of potential pathogens. PATHOGENIC MICROORGANISMS Pathogen: Defined as any organism capable of invading the body and causing disease. Opportunist: A microorgganism is not capabale of causing disease in an immunologically and physically intact host, but when the normal host defenses are defected in some way, this organism can cause disease. INFECTION Infection describes the clinical semptoms that occur when a microorganism invades a host. Acute infection is a rapid onset of observable semptoms. When the semptoms are minor or imperceptible, infections are often termed subclinical. Latent infection results when pathogens persist in the body without evoking a clinical response; periodically, overt infections occur following a change in the patient’s immune state. Chronic or persistent infections occur when pathogens are not eradicated completely and continue to evoke a clinical response. Communicable disease: A infection which is capable of spreading from person to person. Highly communicable diseases such as cholera are said to be contagious, and tend to occur as localized epidemic in which disease frequency is higher than normal. When epidemic becomes woeld-wide it is called a pandemic. Bacteremia: The presence of bacteria in the blood Septicemia: Proliferation of microorganisms in the blood. MICROBIAL STRATEDIES Transmission Ability to survive outside the host is an important factor in transmission of organism. Upper respiratory tract viruses (e.g., rhinoviruses) survive poorly outsite the host and successful transmission relies on the production of large quantities of

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infectious particles. Other organisms can survive outsite the host, e.g. Mycobacterium tuberculosis and do not require large numbers of infectious particles for efficient transmission. Methods of Transmission of Infectious Diseases The first step of the infectious process is the entery of the microorganism into the host by one of several parts: 1. Vertical/Direct: From the mother to the offspring in utero, during delivery, in the mother’s colostrum or breast milk, or by way of latent viruses in the germ cells. Syphilis, Congenital rubella syndrome, AIDS, and viral leukemia are examples of such diseases. 2. Horizontal/Indırect: Horizontal spread occurs when a disease is transmitted to susceptible hosts through the enviroment. For example, transmission can occur by way of ; a. By breathing in aerosols generated by sneezing or couphing (e.g., influenza virus, and tuberculosis). b. By ingesting contaminated food or water, as occurs with cholera, staphylococcal food poisoningi typhoid, and hepatitis A infection. c. By directly from an infected patient to the another individual by the following means: - During sexual activity, veneral disease such as syphilis, HIV, gonorrhea, chlamydia, and numerous others may be transmitted - By direct contact, as often occurs with rhinovirus-associated rhinitis and others. 3. Arthropod Vectors. An infected individual or animal host cen provide an inoculum of the etiologic agent to an arthropod vector, which transmits it to susceptible hosts. For example, the etiologic agents of Lyme borreliosis, and Rocky Mountain spotted fever are transmitted by ticks. 4. Wounds. An infected or colonized animal host can inject the etiologic agent. For example, cat-scratch disease occur when a normal individual is scratched by the family cat, whose claws are contaminated with the organisms (normal oral flora in cats). Attachment Many organisms have specific factors that allow attachment to mucosal surfaces, e.g. the cell surface lipoteichoic acid of group A streptococci and pili of N.gonorrhoea.

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Invassion Invasive bacteria are those that enter host cells or penetrate mucosal surfaces, thereby spreading from the initial site of infection. Invassion is facilitated by several bacterial enzymes, the most notable of which are collagenase and hyaluronidase. These enzymes degrade components of the extracellular matrix, thereby providing the bacteria with easier access to host cell surfaces. Bacterial Toxins Some bacteria cause disease by producing toxins, of which there are two general types: the exotoxins and the endotoxins. a. Exotoxins: The exotoxins, which are proteins, are secreted by both grampositive and gram-negative bacteria. In many cases, the toxin gene is encoded on a plasmid (tetanus neurotoxin of Clostridium tetani and enterotoxin of E.coli) or a lysogenic phage (Corynebacterium diphtheriae and Clostridium botulinum). The exotoxin proteins generally have two polypeptide components. One component is responsible for binding the protein to the host cell, whereas the second component is responsible for the toxic effect. In many cases, the toxin is completely responsible for causing characteristic symptoms of the disease. For example, the performed entertoxin present in food mediates the food poisoning caused by S.aureus and Bacillus cereus.The symptoms caused by performed toxin occur much sooner than for other forms of gastroenteritis because the effect is like eating a food poison and the bacteria do need to grow for the symptoms to occur. Most exotoxins are rapidly inactivated by moderate heating (60ºC), notable exceptions being staphylococcal enterotoxin and E.coli heat stable toxin. In addition, traetment with dilute formaldehyde destroys the toxic activity of most exotoxins, but does not affect their antigenicity. Formaldehyde-inactivated toxins, called toxoids, are thus useful in preparing vaccines. b. Endotoxins: These are heat-stable, lipopolysaccharide (LPS) components of the outer membranes of gram-negative but not gram-positive bacteria. They are not secreted, but released into the host’s circulation following bacterial cell lysis. An LPS consist of polysaccharide O (somatic antigen) and lipid A which is responsible for the toxicity of the molecule. The main physiologic effects of LPS endotoxins are fever, shock, hypotension, and thrombosis, collectively referred to as septic shock. IMMUNITY The term immunity refers to all mechanisms used by the body as protection against enviromental agents that are foreign to the body. Theses agents may be microorganisms or their products, chemicals, drugs, pollen, or animal hair and dander

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In vertebrates, immunity against microorganisms and their products, or against other foreign substances that may invade the body, is divided into two major categories : innate or natural immunity and acquired immunity. INNATE IMMUNITY Innate immunity is conferred by all those elements with which an individual is born and which are always present and available at very short notice to protect the individual from challenges foreign invaders. These elements include : Physiological and Chemical Barriers 1. Reflex: a. The cough and gag reflex prevent particles from entering the lung. b. Sneezing works to expel explosively infectious agents from the nasal pasages. c. Swallowing moves potential pathogens into the stomach, where they are destroyed by stomach acidity. 2. Intact skin: a. Sebaceous gland secretions may be inhibitory. b. A dry surface is not conducive to growth of microbes. c. Continuously sloughing epithelial cells carry adherent microbes away. d. Sweat removes microorganisms through a flushing action and contains inhibitory substances, such as lysozyme. 3. Conjuctive: a. The flushing action of blinking and tears prevents colonization b. Lysozyme and other antibacterial substances in tears are nonspecific effectors. Lysozyme hydrolyzes structural polysaccharide in bacterial cell wall. 4. Mucus membrane epithelium: a. Mucus layers entrap microorganisms, which are then swept by ciliary action into the throat to be swallowed. b. Lysozyme in mucus secretion is antibacterial. c. Epithelial cells may not possess certain receptors necessary for adherence of infectious agents. d. Nasal turbinates provide a barrier to free access into the lungs by airborne particles. 5. Gastrointestinal tract: a. Saliva acts to flush away microbes that have tensiently settled on the mucosa. e. Lysozyme in saliva destroye bacteria. c. Stomach acidity and proteolytic enzymes destroy or degrade many infectious agents.

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d. Bile secreted by the gallbladder is inhibitory to the growth of many bacteria. e. Normal peristalsis forces organisms to move along the GI tract and to be excreted with fecal matter. f. The mucosal layer protects epithelial cells. 6. Urinary tract: a. Flushing of urine periodically prevents bacterial population buildup. b. Acidity of urine is inhibitory to some organisms. c. Prostatic secretions containing spermine and zinc, which inhibit growth of some bacteria, are introduced into the urine in males. 7. Female vagina: a. Vaginal epithelium is sloughed rapidly and carries with it transient microbe. b. The secretions of the vaginal tract are acidic and contain antimicrobial substances; they also promote flushing of microorganisms from the body. Phagocytosis and Intracellular Killing Phagocytosis is the process whereby phagocytic cells engulf particulate matter such as bacteria. The major classes of phagocytic cells are polymorphonuclear neutrophils (PMNs) and macrophages. Both are produced in the bone marrow, but they differ in several ways. Phagocytosis involves several stages (Fig. 15) : Chemotaxis. Phagocytes are attracted to the site of inflammation (chemotaxis) in response to a variety of soluble stimuli (chemotaxins), including bacterial products and inflammantory mediators produced by the host. Attachment. Bacteria attach to the phagocytic cell membrane by non-specific mechanisms dependent on the physiochemical properties of the bacterial cell surface. Most organisms, especially encapsulated ones, do not attach to the phagocytic cell unless they are coated with serum factors known as opsonins. The best-characterized opsinins are IgG and a component of C3. Engulfment. Bacteria are engulfed by the phagocytic cells in a manner similar to that of amoebae engulfing particles, with small pseudopodia developing around the bacterial cell which eventually coalesce and internalize the organism in a “phagosome”. Intracellular killing. There are two principal mechanisms whereby phagocytic cells kill internalized bacteria: 1. oxygen-dependent killing: toxic oxygen radicals (hydrogen peroxide, superoxide ions) produced by the phagocyte cell membrane are secreted into the phagosome;

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2. lysosomal killing: the phagosome fuses with lysosomes (granules in the cytoplasm) containing enzymes that degrade the engulfed organism.

Fig. 15 Phagocytosis

Soluble and Circulating Nonspecific Effectors 1. Complement: The classical pathway of the complement system, comprised of a series of at least 20 proteins and glycoproteins (labeled C1-C9), acts to lyse microbial membranes and release inflammation-enhancing substances. Inasmuch as it is triggered by an antigen-antibody complex, initiation of this pathway cannot be considered nonspecific, although the complement effector proteins are nonspecific. The complement cascade can also be activated by the “properdin” or “alternate” pathway at the C3 stage. 2. Interferone-α (IF- α) proteins are released by cells once they have been infected by a virus. The interferones protect neighboring cells from viral multiplication by inducing the production of intracellular antiviral proteins. Interferone-α also enhances killer Cell activity.

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3. Interferone-γ (IF-γ) activates macrophages to phagocytize and destroy intracellular parasites more effectively; IF-γ also interrupts viral replication and promotes T-cell differentiation. Inflammation Inflammation is one of the first responses of the immune system to infection or irritation. It is serves to establish a physical barrier against the spread of infection, and to promote healing of any damaged tissue following the clearance of pathogens. The inflammatory response is characterized by the following quintet: redness (rubor), heat (calor), swelling (tumor), pain (dolor) and possible dysfunction of the organs or tissues involved (functio laesa). The peptidoglycan layer in bacterial cell walls (teichoic acid and peptidoglycan fragments of gram positive bacteria) and lipopolysaccharide in gram negative bacterial cell walls can activate the alternative complement pathway (properdin) in the abscence of antibody and, with mannose-binding protein, can activate the classic complement pathway. Complement activates inflammatory responses (stimulate mast cell to release histamine which increase vascular permeability) and also directly kill gram-negative bacteria and, to a much lesser extent, gram positive bacteria (the peptidoglycan of gram positive bacteria shields them from lysis). Kinins and clotting factors induced by tissue damage are also involved in inflammation (e.g., factor XII, bradykinin, fibrinopeptides). These factors increase vascular permeability and are chemotactic for leukocytes. Inflammation has two main components cellular and exudative. - The cellular component involves the movement of white blood cells from blood vessels into the inflamed tissue. This cells consisting mainly of the polymorphonuclear leukocytes, which accumulate within 30 – 60 minutes, phagocytize intruder or damaged tissue. If the cause of the inflammatory response persists beyond this point, within 5 – 6 hours the area will be infiltrated by mononuclear cells, which include macrophages and lymphocytes. - The exudative component involves the movement of fluid, usually containing many important proteins such as fibrin and immunoglobulins (antibodies). ACQUIRED IMMUNITY Acquired immunity is an immunity acquired by contact with the invader and is specific to that invader only. It is more specialized than innate immunity and it came into play relatively late. The initial contact with the foreign agent (immunization) triggers a chain of events that leads to the activation of certain cells (lymphocytes) and the synthesis of proteins, some of which exhibit specific activity against the foreign agent. Acquired immunity is induced by immunization, which can be achieved in several ways:

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Active immunization refers to immunization of an individual by administration of an antigen. Passive immunization refers to immunization through the transfer of specific antibody from an immunized individual to a non-immunized individual. Adoptive transfer refers to the transfer of immunity by the transfer of immune cells. Characteristics of the Immune Response The acquired immune response has several generalized features that characterize it and serve to distinguish it from other physiologic systems such as circulation, respiration, reproduction. These features are: 1. Specificity: The ability to discriminate among different molecular antities presented to it and to respond only to those uniquely required rather than making a random, undifferentiated response. 2. Adaptiveness: The ability to respond to previously unseen molecules that may in fact never have existed before on earth. 3. Discrimination between “self” and “nonself” : A cardinal feature of the specificity of the immune respons is it is ability to recognize and respond to molecules that are foreign or “nonself” and avoid making a response to those molecules that are “self”. 4. Memory: A property shared with the nervous system is the ability to recall previous contact with a foreign molecule. IMMUNOGENS AND ANTIGENS Acuired immune responses arise as a result of exposure to foreign stimuli. The compound that evokes the response is referred to either as “antigen” or as “immunogen”. The distinction between these terms is functional. An immunogen is any agent capable of inducing an immune response. In contrast, an antigen is any agent capable of binding specifically to components of the immune response, such as lymphocytes and antibodies. The distinction between the terms is necessary because there are many compounds that are incapable of inducing an immune response, yet they are capable of binding with components of the immune system that have been induced specifically against them. Thus, all immunogens are antigens, but not all antigens need be immunogens. By themselves, these compounds are incapable of inducing an immune response, but they are coupled with much larger entities, such as proteins, the resultant conjugate induces an immune response that is directed against various part of the conjugate, including the low-molecular-weight compound (hapten).

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Requirements for Immunogenisity A substance must possess the following three characteristic to be immunogenic: 1. Foreignness Animals normally do not respond immunologically to “self”. Thus, for example, if a rabbit is injected with it is own serum albumin, it will not mount an immune response; it recognizes the albumin as self. In contrast, if rabbit serum albumin is injected into a guinea pig, the guinea pig recognizes the rabbit serum albumin as “foreign” and mounts an immune response against it. In general, compounds that are part of self are not immunogenic to the individual. However, there are exceptional cases in which an individual mounts an immune response against his or her own tissues. This condition is termed autoimmunity. 2. High Molecular Weight Immunogenic compounds must have a certain minimal molecular weight. In general, compounds that have a molecular weight of less than 1000 daltons are not immunogenic; those of molecular weigh between 1000 and 6000 daltons may or may not be immunogenic; and those molecular weight greater than 6000 daltons are generally immunogenic. 3. Chemical Complexity In general, an increase in the chemical complexity of a compound is accompained by an increase in it is immunogenecity. Antigenicity An immune response induced by an antigen generates antibodies or lymphocytes that react specifically with the antigen. The antigen-binding site of an antibody or a receptor on a lymphocyte has an unique structure that allows a complementary fit to some structural aspect of the specific antigen. The portion of the antigen that binds specifically with the binding site of an antibody or a receptor on a lymphocyte is termed an antigenic determinant or epitope. Major Classes of Antigens The following major chemical families may be antigenic: 1. Carbonhydrates (polysaccharides). Polysaccharides are poteintially, but not always, immunogenic. An immune response, consisting primarily of antibodies, can be induced against many kinds of polysaccharide molecules, such as components of microorganisms and of eukaryotic cells.

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2. Lipids. Lipids are rarely immunogenic, but an immune response to lipids may be induced if the lipids are conjugated to protein carriers. Thus, in a sense, lipids may be regarded as haptens. 3. Nucleic acids are poor immunogens by themselves, but they become immunogenic when they are conjugated to protein carriers. 4. Proteins. Virtually all proteins are immunogenic. Thus, the most common immune responses are those to proteins. In general, proteins are multideterminant antigens. Immunologic Adjuvants To enhance the immune response to a given immunogen, various additives or vehicles are often used. An adjuvant is a substances that, when mixed with an immunogens, enhance the immune response against the immunogen. A hapten will become immunogenic when conjugated convalently to a carrier; it will not become immunogenic if mixed with an adjuvant. Thus, an adjuvant enhance the immune response to immunogens but dose not confer immunogenicity to haptens. During artificial immunization (e.g., vaccines), and adjuvant is used to enhance the response to antigen. Adjuvants usually prolong the presence of antigen in tissue and activate or promote uptake of the immunogen by macrophages and lymphocytes. There are various widely used adjuvants. One such adjuvant is Freund’s complete adjuvant, which consists of a water-in-oil emulsion and killed Mycobacterium tuberculosis or Mycobacterium butyricum. ANTIBODY STRUCTURE One of the major functions of the immune system is the production of soluble proteins that circulate freely and exhibit properties that contribute specifically to immunity and protection against foreign material. These soluble proteins are the antibodies, which belong to the class of proteins called globulins because of their blobular structure. Today they are known collectively as immunoglobulins (Ig). Structure of Immunoglobulin (Fig. 16) All immunoglobulin molecules consist of a basic unit of four polypeptide chains (Fig. 8), two identical heavy (H) chains and two identical light (L) chains, held together by a number of disulfide bonds. Each polypeptide chain is composed of a number of domains comprised of 100 to 110 amino acids residues, each forming a loop as a result of intrachain disulfide bonds. The N-terminal domain of each chain contains the area designated as the variable or V region. The V region contains several highly variable or hypervariable regions, which together from the antigenbinding pocket that confers the property of antigen specificity on the immunoglobulin

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molecule. The COOH-terminal domains (CH1, hinge, CH2, CH3, CH4) have been collectively defined as the constant region, because the polypeptide backbone is generally invariant within a particular class of immunoglobulin. Proteolytic treatment with the enzyme papain split the immunoglobuli molecule into three fragments of about equal size (Fig. 8). Two of these fragments were found to retain the antibody’s ability to bind antigen specifically. These two fragments are referred to as Fab (fragment antigen binding) fragments. The third fragment is called Fc (fragment-crystallizable). It cannot bind the antigen, but it is responsible for the biological functions of the antibody molecule after antigen has been bound to the Fab part of the intact molecule.

Fig. 16 Structure of immunoglobulin

Classes of Immunoglobulins (Fig. 17) 1. Ig G In healthy adults, constitutes approximately 85% of the total serum immunoglobulins in adults. Ig G is approximately equally distributed between intravascular and extravascular serum pools. One important biological function of Ig G it is unique ability to cross the placenta, which affords protection for the fetus amd newborn. Human Ig G has been divided into four subclasses on the basis of unique antigenic determinants. Relative subclass percentages of the total Ig G in serum are: Ig G1, 60 to 70%; Ig G2, 14 to 29%; Ig G3, 4 to 8%; and Ig G4, 2 to 6%. Except for the Ig G3 subclass, which has a rapid turnover, with a half-life of 7 days, the half-life of Ig G is approximately 23 days, which is the longest half-life of all Ig isotypes. The persistence in the serum makes Ig G the most suitable for passive immunization by transfer of antibodies. Ig G is the most versatile class of antibody, capable of carrying out numerous biological functions that range from neutralization of toxin to activation of

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complement and opsonization. It is predominate in secondary immune response to most antigens. 2. Ig M Ig M is a pentametric immunoglobulin of about 900,000 MW that constitutes about 10% of serum immunoglobulins in healthy individuals. Ig M antibodies are clinically significant because they predominate in early immune response to most antigens. Along with Ig D, Ig M is a major immunoglobulin that is expressed on the surface of B cells. Ig M antibody is present in pentametric form; of all classes of immunoglobulin it is the best agglutinating and complement-activating antibody. 3. Ig A Ig A is the major immunoglobulin in external secretions such as saliva, mucus, sweat, gastric fluid, and tears. It is, moreover, the major immunoglobulin of colostrum and milk, and it may provide the neonate with a major source of intestinal protection against pathogens. In contrast, 10% of the circulating serum Ig A is polymeric, while 90% is monomeric. Together, they constitute approximately 15% of the total serum immunoglobulins. Two subclasses of Ig A have been identified (Ig A1 and Ig A2). Ig A, has a half-life of 5.5 days In terms of complement activation, Ig A poorly activates the classical pathway. In contrast, Ig A reportedly activates the alternative pathway of complement to provide some direct protective functions. Ig A, once bound to bacterial or parasitic surface antigen, may bind receptors on inflammatory cells, leading to their destruction by means of antibody-dependent cell-mediated cytotoxicity. Moreover, its binding to viral or microbial surface antigens may restrict the mobility of microorganisms and prevent their binding onto mucosal epithelium. 4. Ig E Ig E (190,000 MW) exists in serum in a monomeric form. It is also termed reaginic antibody, has a half-life in serum of 2 days, the shortest half-life of all classes of immunoglobulins. While Ig E constitutes only 0.004% of the total serum immunoglobulins, is of paramount importance in hypersensitivity reactions. It also appears to be of importance in protection against parasitic infections. The Fc portion of Ig E binds with high affinity to receptors on mast cells and, on contact with antigen, it triggers the degranulation of mast cells, resulting in the release of pharmacologically active substances that mediate the hypersensitivity reactions. 5. Ig D Ig D is a monomer of approximately 180,000 MW. While Ig D is normally present in serum in small amounts (0.2% of total serum immunoglobulins), it is predominantly found with Ig M on the surface of human B lymphocytes. Despite suggestions that Ig D may be involved in B-cell differentiation, its principal function ia as yet unknown. Serum Ig D has a half-life of 2.8 days.

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Fig. 17 Classes of immunoglobulins

Kinetics of the Antibody Response Following Immunization Primary Response. In the primary response, the first class of antibody detected is generally Ig M, which in some instances may be the only class of immunoglobulin that is made. If production of Ig G antibody ensues, its appearance is generally accompained by a rapid cessation of production of Ig M. Secondary Response. Although production of antibody after priming contact with antigen may cease entirely within a few weeks (Fig. 18), the immunized individual is left with a cellular memory of this contact. This memory becomes apparent when a response is triggered by a second injection of the same antigen. After the second injection, Ig G antibodies appearce at higher concentrations, and with greater persistence, than Ig M, which may be greatly reduced or disappear altogether. This may be also accompained by the appearance of Ig A and Ig E.

Fig. 18 Immune response curve

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LYMPHOID TISSUES AND ORGANS Hematopoietic bone marrow stem cells give rise to all blood cells, including the lymphoid progenitor cells. Lymphocyte maturation, differentiation, and proliferation take place in the lymphatic organs. Theses include the primary lymphoid tissues and organs (The thymus gland in which T lymphocytes develop, and the bone marrow, in which B lymphocytes develop), and the secondary lymphoid tissues and organs (of which the spleen and lymph nodes are the most important), where mature lymphocytes encounter and respond to foreign antigens (Fig 19).

Fig. 19 Lymphoid tissues and organs

A. Primary Lymphoid Tissues and Organs Lymphoid tissues and organs are defined as primary because they include the sites where T and B lymphocyte production and initial maturation occur, prior to an encounter with an antigen. 1. Thymus gland Progenitor cells from the bone marrow migrate to the primary lymphoid organ, the thymus gland, where they differentiate into T lymphocytes. The thymus gland is a bilobed structure, derived from the endoderm of the third and fourth pharyngeal

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pouches (Fig. 20). During fetal development, the size of the thymus increases. The growth continues until puberty. Thereafter, the thymus undergoes atrophy with aging. The thymus is a lymphoepithelial organ and consists of epithelial cells organized into cortical and medullary areas that are infiltrated with lymphoid cells (thymocytes). The cortex is densely populated with lymphocytes of various sizes, most of which are immature. T lymphocytes mature in the cortex and migrate to the medulla, which they then leave to enter the peripheral blood circulation, through which they are transported to the secondary lymphoid organs. Thymocytes differentiate in the thymus into subpopulations characterized by specific surface glycoproteins such as CD3, plus CD4 and CD8. Maturation of the T lymphocytes involves the commitment of a given determinant or epitope of a foreign antigen. This recognition is achieved by a specific receptor on the T cell, wich is acquired during differentiation in the thymus.

Fig. 20 Thymus glans

2. Bursa of Fabricius and the Bone marrow A primary lymphoid organ was first discovered in birds. In birds, B cells undergo maturation in the bursa of Fabricius. This organ, situated near the cloaca. Mammals do not have a bursa of Fabricius. In embryonic life, B cells differentiate from hematopoietic stem cells in the fetal liver. After birth and for the life of the individual this function moves to the bone marrow, a structure that is considered to be a primary lymphoid organ with functions equivalent to that of the avian bursa. B. Secondary Lymphoid Tissues and Organs The secondary lymphoid tissues include the spleen, lymph nodes, and mucosaassociated lymphoid tissues. Within these tissues, mature T and B cells congregate, waiting for the appearance of antigen.

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1. Spleen This organ is highly effective at removing foreign substances and damaged blood cell elements (for example, platelets and aged red blood cells) present in the circulation. The spleen thus serves as the “oil filter” of the circulation. It is also the site where many of antibodies directed against foreign substances are synthesized. The spleen is organized into red pulp and white pulp, the latter being primarily found surrounding small arterioles (Fig. 21). White pulp is very rich in lymphoid cells, approximately 50% of the lymphocytes consising of mature B cells located in the follicles, and 35% consisting of mature T cells located in the periarteriolar sheet.; 3040% are T lymphocytes. The red pulp is a storage site for blood cells and the site of turnover of aged plateletes and erythrocytes.

Fig. 21 Spleen

2. Lymph Nodes These structures are located in numerous regions throughout the body. They are like “mini oil filters” that clear material from the tissues that drain to the lymph nodes. A lymph node consists of the following three layers (Fig. 22): - The cortex, the outer layer that contains mainly B cells and macrophages arranged in clusters called follicles. - The paracortex, which contains dendritic cells that bring antigens from the tissues to be presented to the T cells to initiate immune response. - The medulla, which contains B and T cells and antibody-producing plasma cells.

Fig.22 Lymph Node

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2. Mucosa-associated, secondary lymphoid tissues In addition to the spleen and lymphnodes, there are several other sites where lymphocytes interact with antigen and differentiate in order to produce antibodies, or become activated T cells. These secondary lymphoid tissues defend mucosal surfaces and are located at sites where the body is most likely to encounter pathogens, for example, the tonsils and adenoids. The largest percentage of lymphocytes in the body are located along the mucosal surfaces of the intestine in Peyer’s patches. These sites are where B and T cells reside and respond to antigenic challenges. Proliferation of the lymphocytes in response to infectious challenge causes these tissues to swel. CELLS INVOLVED IN IMMUNE RESPONSE All of the cells involved in host defense and immunity (Fig. 21) are derived during hematopoiesis from stem cells in the bone marrow. It is generally accepted that the lymphoid, monocyte/myeloid, and megakaryotic lines of leukocytes diverge and differentiate from a common, pluripotential stem cell.

Fig. 23 Cells involved in immune system

Polymorphonuclear Leukocytes (Fig. 24) Plymorphonuclear leukocytes (neutrophils) constitute 50% to 70% of circulating white blood cells and are a primary phagocytic defence against bacterial infection. These short-lived cells circulate in the blood for 7 to 10 hours and then migrate into the tissue, where they live for 3 days longer. Neutrophils are 11 to 14 µm in diameter, lack mitochondria, have a granulated cytoplasm in which granules stain with both acidic and basic stains, and have a multilobed nucleus. Neutrophils leave the blood

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and concentrate at the site of infection in response to chemotactic factors. They ingest bacteria by phagocytosis and expose the bacteria to antibacterial substances and enzymes contained in primary (azurophilic) and secondary (specific) granules. Azurophilic granules are contains myeloperoxidase, ß-glucoronidase, elastase, and cathepsin G. Specific granules serve as reservoirs for lysozyme and lactoferrin. Eosinophils are heavely granulated cells (11 to 15 µm in diameter) with a bilobed nucleus that stain with the acidic dye eosin. They are also phagocytic, motile, and granulated. The granules contain acid phosphatase, peroxidase, and eosinophilic basic proteins. Eosinophils play a role in the defense against parasitic infections. The eosinophilic basic proteins are toxic to many parasites. Basophils, another type of granulocyte, are not phagocytic but release the contents of their granules during allerging responses (type 1 hypersensitivity).

Fig. 24 Polymorphonuclear leukocytes

Mononuclear Phagocyte System The mononuclear phagocyte system (previously called the reticuloendothelial system) consists of monocytes in the blood and cells derived from monocytes, such as macrophages, alveolar macrophages in the lungs, dendritic cells, Kupffer cells in the liver, intraglumerular mesangial cells in the kidney, histocytes in connective tissue, synovial A cells, and microglial cells in the brain. Monocytes are 10 to 18 µm in diameter with a single-lobed, kidney bean-shaped nucleus (Fig. 23). They represent 3 to 8% of peripheral blood leukocytes. Different cytokines or tissue enviroments promote monocytes to differentiate into the various macrophages and dendritic cells. These mature forms have different morphologies corresponding to their ultimate tissue location and function and may not express all of the macrophage activities.

Fig. 25 Monocytes

Macrophages (large eaters) are long-lived cells that are phagocytic, contain lysosomes and, unlike neutrophils, have mitochondria (Fig. 24). Macrophages have the following basic functions: (1) phagocytosis, (2) antigen presentation to T cells to initiate specifi immune response, and (3) secretion of cytokines to activate and promote innate and immune responses.

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Fig. 26 Macrophage

Macrophages express cell surface receptors for the Fc portion of IgG and for the C3b product of the complement cascade. These receptors facilitate the phagocytosis of antigen, bacteria, or viruses coated with these proteins. Macrophages also express the class II MHC antigens (major histocompatibility complex), which allows these cells to present antigen to CD4 helper T cells to initiate the immune response. Macrophages secret interleukin-1, interleukin-6, interleukin-12, and tumor necrosis factor in response to bacterial interaction, which stimulate immune and inflammatory responses, including fever. A T-cell-derived lymphkine, interfero-γ, activates macrophages and enhances their phagocytic, killing, and antigen-presenting capabilities. Dendritic cells of spleen and lymph nodes, interdigitating cells of the thymus, as well as Langerhans cells in the skin probably also belong to this lineage of cells. These cells are not very phagocytic but they are potent antigen-presenting cells. Lymphocytes The lymphocytes are 6 to 10 µm in diameter, smaller than leukocytes. The two major classes of lymphocytes, B cells and T cells, have a large nucleus and smaller, agranular cytoplasm. Although B and T cells are indistinguishable by morphologic features, they can be distinguished on the basis of function and surface markers. Lymphoid cells that are not B and T cells are large granular lymphocytes also known as natural killer (NK) cells. B cells: The primary function of B cells is to make antibody, but they also internalize antigen and present the antigen to T cells to initiate or enhance the immune response. B cells can be identified by by the presence on their cell surfaces of immunoglobulins (Ig M and Ig D), class II MHC molecules, and receptors for the C3b and C3d products of the complement cascade. The B cell name is derived from its site of differentiation in birds, the bursa of Fabricius and the bone marrow of mammals. B cell differentiation also takes place in the fetal liver and fetal spleen. Activation of B cell leads to proliferation and differentiation into a plsma cells which have small nuclei and large cytoplasm, are factories for antibody production. T cells: Acquired their name because they develop in the thymus. T cells have the following two major functions in response to foreign antigen:

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1. Control, suppress (when necessary) and activate immune and imflammatory responses by releasing cytokines. 2. Directly kill virally infected cells, foreign cells(e.g., tissue grafts), and tumors. T cells were initially distinguished from B cells on the basis of their ability to bind and surround themselves (forming rosettes) with sheep erythrocytes through the CD2 molecule. All T cells express an antigene-binding T-cell receptor (TCR), which resembles but differs from antibody, and CD2 and CD3-associated proteins on their cell surface. The CD3 complex is the signal transduction unit for the TCR. There are two important types of T cells: 1. Helper T cells (CD4 T cells), wich serve as helper cells for other lymphocytes (B cells or CD8 T cells), phagocytes, and natural killer cells. 2. Cytotoxic T cells (CD8 T cells), which specialize and identifying and killing tumor cells, and cells infected with viruses. According to antigenic determinants and function, T cells have been divided into subtypes of cells including: A. Inducer T cells may be functionally divided into: 1. T-helper (TH) cells. The helper T cells (CD4) activate and control immune and imflammatory responses by releasing cytokines. Helper T cells interact with peptide antigens presented on class II MHC molecules expressed on antigen-presenting cells (macrophages and B cells). The vocabulary of cytokines secreted by specific CD4 T cell in response to antigenic challenge further distinquishes the CD4 T cell as Th0, Th1, Th2, or Th3. The Th0 cells produce IL-2, IFN-γ, and IL-4. Th1 cells promote imflammatory responses, which are especially important for controlling intracellular (mycobacterial and viral) and fungal infections. Th2 cells promote antibody and memory responses. Th3 cells are involved in production of IgA. 2. T-suppressor cells (TS). These cells bear the CD8 molecule on their outer membrane and interact with MHC-class I molecule. They are able to suppress the immune response leadind to a downward modulation or shutoff in reactivity of other effector cells. B. Effector T Cells 1. T-cytotoxic cells (TC). Tc cells are important for eliminating virally infected cells, foreign tissue tissue transplants, tumor cells . They have CD8 molecule interact with cells that bear MHC-class I molecule on their outer membrane. 2. T-delayed-type hypersensitivity (TDTH). These cells (Th1 cells) releases cytokines that induce the migration and activation of monocytes and macrophages, leading to the so-called delayed-type hypersensitivity inflammatory reactions. Natural Killer Cells (NK): These cells are large, nonphagocytic, granular lymphocytes that destroy abnormal host cells, such as those that are virus-infected or neoplastic. They are do not display a unique antigen specificity, but when activated

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with antibodies they became effector cells against appropriate target cell antigens. Target cell killing is accomplished by NK cell release of compounds that have a variety of toxic effects. The cytotoxic actions of NK cells are stimulated bu interferons and interleukine-2. Cytotoxic T cells that participate in the adaptive immune response are also characterized as killer cells. They apear later than the NK cells, at a time when the adaptive response has been mounted. Cytotoxic T cells kill target cells using mechanisms identical to those used by NK cells. CYTOKINES Information exchange between cells is afforded both by the direct interaction of molecules such as glycoproteins on cellular membranes of various immune cells, and by chemical signals or messengers called cytokines. Cytokines are soluble, antigennonspecific proteins that bind to cell surface receptors on a variety of cells. Cytokines usually function locally; thus, in some instances, the cell that secretes the cytokine also responds to that cytokine (autocrine response). Cytokines are secreted by many different cell types, and affect not only the function of cells of the immune system, but also other systems in the body. The term cytokine includes the molecules known as inteleukins (IL), interferons (IFN), tumor necrosis factors (TNF), and colony stimulating factor (CSF). Cytokines are being used in the treatment of immune deficiency disorders, cancer, and autoimmune diseases. Teble 1. Sommary of selected cytokines
Cytokine Produced by Major functions _______________________________________________________________________________________ Interleukin-1 Monocytes, many other cell Produces fever, promotes proliferation (IL-1) types of Th cells (Th2). Interleukin-2 (IL-2) Interleukin-3 (IL-3) Interleukin-4 (IL-4) Interleukin-5 (IL-5) Interleukin-6 (IL-6) Interleukin-7 (IL-7) Interleukin-8 Th cells (Th0 and Th1) T-cell growth factor

Primarily T cells Th cells (Th2) and mast cells Th cells (Th2) T cells and many others Fibroblasts, endothelial and some T cells Many cell types

Growth factor for hematopoietic stem cells and mast cells Growth factor for B cells and Th2; inhibits Th1 cells Stimulates B-cell growth and Ig secretion T-cell activation and IL-2 production; Stimulates B-cell Ig production Growth factor for Per-T and Per-B cells Chemotactic factor for PMN

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(IL-8) Interleukin-9 (IL-9) Interferon-gamma Tumor necrosis Factor T cells T cells (Th1) Monocytes Mast cell activation Activates NK cells, macrophages, and Inhibits Th2 cells Involved in inflammatory responses; activate endothelial cells and other cells of immune and nonimmune S. Growth and differentiation of hema.cells

Colony stimulating factor T cells and monocytes

____________________________________________________________________ The Major Histocompatibility Complex (MHC) MHC proteins play a pivotal role in “presenting” antigens to T cells. In fact, T cells do no respond to foreign peptides unless the antigen peptides are properly presented. [Note: In humans, the MHC is called the human leukocyte antigen (HLA) complex] Unlike most proteins that have a fixed, defined structure, key amino acid sequences in the MHC proteins vary widely from person to person, and thus they act as “identity markers” on the surface of the cells. Interestingly, these proteins were first detected by their effect on transplant reaction (that is, tissue incompatibility). The function of MHC (major-histocompatibility complex) molecules is to bind selected peptides generated inside a cell during the processing protein antigens. The complex formed between an MHC molecule and peptide moves to the cell surface, where the MHC molecule is inserted into the membrane of the cell with peptide on the outside. The peptide-MHC complex on the cell surface can now be recognized by a T cell with an appropriate receptor. The MHC codes for two major categories of cell surface molecules: MHC class I molecules consist of a single polypeptide chain. MHC class II molecules consist of two polypeptide chains. The outer region of every MHC class I and class II molecule contains a deep groove that functions as the peptide-binding site. T-cell responses are MHC-restricted: T-cells receptors on CD4 helper T cells recognize and respond to antigen only when bound to an MHC class II molecule on the surface of antigen-presenting cell. Antigen specific receptors on CD8 cytotoxic T cells recognize antigen only in association with MHC class I molecules. Thus, CD4 T cells are restricted by MHC class II, and CD8 T cells by MHC class I molecules.

IMMUNE RESPONSE

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The specific acquired immune response is manifest in two ways: Humoral immunity by production of immunoglobulins and cell-mediated immunity (Fig. 28). Humoral Immunity The triggering of the humoral response involves the cooperation of the T helper cells, B cells and macrophages. Th cells recognize the foreign antigen in combination with the MHC class II molecule and through a series of events act by triggering B cells. As a consequence of activation by antigen, Th cells secrete cytokines, soluble factors that effect B cells and activated them. When activated the B cells undergo several cell divisions to produce memory cells and plasma cells, the antibody producing cells. During the primary antibody response or the first encounter with an antigen, it takes several days for antibody to be detected in the serum. This lag time represents the time required for processing, selection, and transformation of enough plasma cells for production of antibody. During the secondary antibody response, antibody appears more quickly because of the presence of memory cells. Antibodies of different classes and types are prouced to different microbial antigens and to microbial products. These substances are generally important against extracellular organisms. The reaction between antigen and antibody serves to induce events such as: - In combination with complement, antibodies can lyse selected bacteria and viruses.This occurs more efficiently with gram-negative bacteria. - Antibodies in conjugation with complement neutralize some viruses, preventing their attachment to target cells. - Antibodies can also neutralize the effects of various bacterial toxins and enzymes. - Secretory Ig A antibodies prevent the adherence of bacteria and viruses to mucosal surface and neutralize viruses. - Antibodies also act as effective opsonins, thus enhancing phagocytosis Cellular Immune Responses (CIR) Cellular immune responses are mediated by monocyte-macrophage lineage cells, natural killer (NK) cells, and T cells. The natural killer cells provide provide early responses to infection, releasing cytokines and killing virally infected and tumor cells and antibody decorated cells. Cells of the monocyte-macrophage lineage bridge the gap between the innate and antigen-specific protective responses as phagocytic, killer, cytokine-producing, and antigen-presenting cells. The T cells plays a central role in activating and controlling immune and imflammatory responses through the release of cytokines. T cells are also very important for eliminating (killing) tumor cells and virally infected cells.

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The helper T cells (CD4) activate and control immune and imflammatory responses by releasing cytokines. Helper T cells interact with peptide antigens presented on class II MHC molecules expressed on antigen-presenting (macrophages and B cells) cells (Fig. 27).

Fig. 27 Antigen presentation to CD4 T cells

Cytotoxic and suppressor T cells (CD8) “patrol” the body for cells expressing virus or tumor-like abnormal proteins presented by the class I MHC molecules. Cellular immunity, as opposed to humoral immunity, encompasses a broad spectrum of immune phenomena mediated by cells of the immune system. The latter comprise various subsets of lymphocytes: T, B, and NK cells. Monocytes and granulocytes also participate in cellular immune reactions. They have the capability to mediate non-MHC-restricted cytotoxicity and to release a variety of enzymes and cytokines. In fact, CMI may be manifested in different ways depending on the antigen and the group of T cells involved. Manifestations of CMI include delayed hypersensitivity and cytotoxicity reactions.

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ANTIGEN-ANTIBODY INTERACTIONS Antibodies constitute the humoral arm of acquired immunity that provides protection against infectious organisms and their toxic products. Therefore, the interaction between antigen and antibody is of paramount importance. In addition, because of the exquisite specificity of the immune response, the interaction between antigen and antibody in vitro is widely used for diagnostic purposes, for the detection and identification of their antigen or antibody. The utilizzation of the in vitro reaction between antigen and serum antibodies is termed serology. A example of the use of serology for the identification and classification of antigens is the serotyping of various microorganisms by the use of specific antisera. The interaction of antigen with antibodies may result in a variety of consequence, including precipitation, agglutination and activation of complement. Primary Interactions Between Antigen and Antibody No covalent bonds are involved in the interaction between antibody and an epitope. Consequently, the binding forces are relatively weak. They consist mainly of van der Waals forces, electrostatic forces, and hydrophobic forces, all of which require a very clse proximity beween the interacting moieties. Thus the interaction requires a very close fit between an epitope and the antibody, a fit that is often compared to that between a lock and a key. Because of the low levels of energy involved in the interaction between antigen and antibody, antigen-antibody complex can be readily dissociated by low or high pH, by high salt concentrations, or by chaotropic ions,

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such as cyanates, which efficiently interfere with the hydrogen bounding of water molecules. Agglutination Reactions The interaction between a soluble antibody and an insoluble particulate antigen results in agglutination. The agglutination of an antigen as a result of cross-linking by antibodies is dependent on the correct proportion of antigen to antibody. Figure 26 depicts an example of an agglutination test for antibodies to the bacterium Brucella abortus present in the serum of an infected individual. At a high antibody levels, excess agglutination may not occur. This is referred to as a prozone. The term titer referse to the highest serum dilution at which agglutination still take place and beyond which, at higher dilution, no agglutination occurs. Zeta potential. The surfaces of certain particulate antigens may possess an electrical charge, as, for example, the net negative charge on the surface of red blood cells caused by the presence of sialic acid. When such charged particles are suspended in saline solution, an electrical potential termed the zeta potential is created between particles, preventing them from getting very close to each other. This introduces a difficulty in agglutinating charged particles by antibodies, in particular

Fig. 29 Dilution of antiserum

red blood cells by Ig G antibodies. The distance between the Fab arms of the Ig G molecule, even it is most extended form, is too short to allow effective bridging between two red blood cells across the zeta potential. Thus, although Ig G antibodies may be directed against antigens on the charged erythrocyte, agglutination may not occur because of the repulsion by the zeta potential. On the other hand, some of the Fab areas of Ig M pentamers are far enough apart and can bridge red blood cells separated by the zeta potential. This property of Ig M antibodies, together with their pentavalence, is a major reason for their effectiviness as agglutinating antibodies. The use of heterologous anti-immunoglobulin antibodies may bridg between antigenic particles that are bound nonagglutinating antibodies, leading to agglutination. This sequence of events is the basis for the coombs test. Latex Agglutination (LA). Latex polystyrene beads were first used to detect some antigenic factors in serum. Either antigen or Ig G antibody is nonspecifically

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absorbed to the surface of the latex polyeteren beads. Addition of the specific antibody or antigen visibly agglutinates the milky-white latex suspension. Although latex agglutination tests can be done in test tubes, they are usually performed on slides. LA is widely used for the test of pregnancy, which involves the detection of human chorionic gonadotropin (HCG) in the urine of pregnant women, and other factors. Hemagglutination (HA) Hemagglutination is a method that visualizes the antigen-antibody interaction by using red blood cells previously coated with the antigen under investigation. Using a variety of different methods, specific soluble antigens can be coupled to red blood cells. Then, when the patient’s serum is added, the antibody present binds the antigen on the cell surface, cross-links different cells together, and causes the cells to agglutinate. Coaggulations (CoA). Certain strains of Staphylococcus aureus, contain a cell surface known as protein A. Antibodies of the Ig G class adhere to protein A by their Fc portion, leaving the Fab ends free to complex homologous antigen. The presence of antigen results in the visible agglutination of the staphylococci. Precipitation Reactions Precipitation reactions occur on mixing, at the right proportions, of soluble multivalent antigen and at least divalent antibodies. The precipitation reaction may take place in aqueous media or in gels. Figure 30a depicts a qualitative precipitin reaction. When increasing concentrations of antigen are added to a series of tubes that contain a constant concentration of antibodies, variable amounts of precipitate form. If the amount of the precipitate is plotted against the amount of antigen added, a precipitin curve like the one shown in Figure 30b is obtained.

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Fig. 30 Precipitation reaction

There are three important areas under the curve shown in Figure 27c: (1) the zone of antibody excess, (2) the equivalence zone, and (3) the zone of antigen excess. In the equivalence zone, the proportion of antigen to antibody is optimal for maximal precipitation; in the zones of antibody excess or antigen excess, the proportions of the reactants do not lead to efficient cross-linking and formation of precipitate. Precipitation reactions between soluble antigens and antibodies can take place not only in solution but also in semisolid media such as agar gels. When soluble antigen and antibodies are placed in wells cut in the gel, the reactants diffuse in the gel and form gradients of concentration, with the highest concentrations closest to the wells. Somewhere between the two wells, the reacting antigen and antibodies will be present at proportions that are optimal for formation of a precipitate.

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Immunoassays Radioimmunoassay (RIA). RIA employs isotopically labeled molecules and permits measurments of extremely small amounts of antigen-antibody complexes. Solid-Phase Immunoassay Solid-phase immunoassay is one of the most widely used immunologic techniques. It is now automated and is widely used in clinical medicin for the detection of antigen or antibody. A good example is the use of solid-phase immunoassay for the detection of antibodies to the AIDS virus. For the detection of specific serum antibodies: Specific known antigen is bound to a solid support, such as a plastic tube, or polysteren surfaces. Then, the patient’s serum is layered over the antigen coated solid phase. A radioactive-labeled anti-immunoglobulin is then added to form antigenantibody-anti-immunoglobulin “sandwich”. After separation of the bound and free radioactive-tagget anti-immunoglobulin. The radiation can be detected by specific instrument If the test uses anti-immunoglobulins that are labeled with an enzyme that cen be detected by the appearence of a color on addition of proper substrate, the test is called an enzyme-linked immunosorbent assay (ELISA). Because of problems associated with disposal or radioactive waste and the cost of radiation measuring instruments, the ELISA is rapidly replacing SPRIA.

Fig. 31 Direct ELISA test

Immunofluorescence (IF) İmmunofluorescence is a method for localizing an antigen by the use of fluorescence-labeled antibodies. One fluorescent compound that is widely used in

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immunology is fluorescein isothiocyanate, which fluoresces with a visible greenish color when excited by ultraviolet light. There are two important and related procedures that employ fluorescent antibodies: direct immunofluorescence and indirect immunofluorescence (Fig. 29). Direct Immunofluorescence is primarily for detection of antigen and involves reacting the target microorganism with fluorescently labeled specific antibodies. Indirect Immunofluorescence involves first reacting the target with specific antibodies. This reaction is followed by subsequent reaction with fluorescently labeled anti-immunoglobulin. The indirect IF method is more widely used than the direct method, because a single fluorescent anti-immunoglobulin antibody can be used to localized antibody many different specificities.

Fig. 32 Indirect Immunofluorescence test

COMPLEMENT The complement system is an alarm and a weapon against infection, sepecially bacterial infection. This system is comprised of a number of soluble plasma proteins, some of which are proteolytic enzymes and others are regulatory and inflammatory proteins. Complement can be activated through three pathways: (a) the classical pathway that is initiated by antigen-antibody complexes, (b) the alternative pathway, in which complement components (C1-C9) become activated by the cell wall of some bacteria and yeasts, in combination with several serum factors such as B and D, and properdin or (c) by lecting binding to sugars on the bacterial cell surface. Biological Activity of Complement Components In addition to its function of producing cell lysis, the activation of complement yields fragments of complement components that possess various important biological activities are summarized below.

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Anaphylatoxins (C3a, C5a) is substance that induces the degranulation of mast cells and/or basophils, causing, among other things, release of histamine. Histamine has several important physiological functions (e.g., it increases capillary permeability and causes contraction of smooth muscle) that are associated with anaphylaxis and other allergic reactions. The anaphylaxis that are elaborated during the activation of complement are C3a and C5a. Chemotaxins (C5a) are substances that attract phagocytic cells and cause their migration to infected area. The chemotaxin produced during complement activation is C5a. Immune Adherence (C3b) is a phenomenon in which a particular antigen, coated with antibodies and in the presence of complement, adheres to various surface. The complement component C3b is responsible for immune adherence Opsonization (C3b) referse to the coating of particulate antigen by antibody and/or complement components that render the particle more attractive to phagocytic cells. The Complement Fixation Test The complement fixation test was used extensively in the past for the detection of hepatitis B surface antigen, and the Wassermann test for syphilis, but now less laborextensive, less expensive, and more sensitive analyses are used in clinical medicine. The complement fixation test is based on the competition, for complement, between various antigen-antibody complexes and the lytic system consisting of red blood cell specific antibodies and red blood cells (RBCs), which, together with complement, bring about the lysis of RBC (Fig. 33) The test consists of an indicator system composed of predetermined amounts of sheep red blood cells (SRBCs), rabbit antibodies to SRBC (also termed hemolysin), and complement ( C ), which is generally supplied as guinea pig serum. Preincubation of that fixed amount of complement with an antigen-antibody system (not related to SRBC and anti-SRBC) that would results in the attachment of complement to the antibody in this antigen-antibody complex. This attachment or fixation of complement results in the activation of the complement cascade and the consumption of complement components by the antigen-antibody complexes. Subsequent introduction of sensitized SRBC into the mixture does not result in lysis of the SRBC, because complement has been fixed or used by the first antigenantibody system.

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Fig. 33 Complement fixation test

IMMUNOPATHOGENESIS Hypersensitivity Response Under some circumstances, immunity, rather than providing providing protection, produces damaging and sometimes fatal results. Such deleterious reactions are known collectively as hypersensitivity or allergic reactions; antigen that commonly cause hypersensitivity or allergic reactions are referred to as allergens. Hypersensitivity reactions were divided into four classes: Type I: Anaphylactic reactions are mediated by Ig E antibodies, which bind through the Fc portion to receptors on mast cells and basophils. After reexposure to antigen, the Ig E antibodies trigger the mast cells and basophils to release pharmacologically active agents that are responsible for the characterisritic symptoms of anaphylaxis (Fig. 34). The combined pharmacologic effects of these mediators produce the immediate symptoms typical for this response: increased vascular permeability, constriction of smmoth muscles, and influx of eosinophils.

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Among the different types of allergic reactions caused in this manner are the folowing: Anaphylaxis. When a specific allergen (e.g., penicillin) is injected directly in to the circulation, it can react in widespread areas of the body with the basophils of the blood and mast cells located immediately outside the small blood vessels if these have sensitized by attachment of Ig E reagins. The histamine released into the circulation causes body-wide vasodilation as well as increased permeability of the capillaries with resultant marked loss of plasma from the circulation. Then much more severe consequences would ensue. This could include difficulty in breathing because of constriction of bronchiolar muscles, uterine cramps, or involuntary urination and defecation. In addition, widw-spread vascular permeability could produce a massive loss of fluid into tissue spaces (hives and edema) and a drastic fall in blood pressure (shock).

Fig. 34 Release of histamin from mast cell

Urticaria. Urticaria results from antigen entering specific skin areas (such as mosquito saliva injected during a mosquito bite) and causing localized anaphylactoid reactions. Histamine released locally to causes (1) vasodilation that induces an immediate red flare and (2) increased local permeability of the capillaries that leads to swelling of the skin in another a few minutes. The swellings are commonly called hives. Hay Fever. In hay fever, the allergen-Ig E reaction occurs in the nose. Histemine released in response to the reactions causes local vascular dilitation with resultant increased capillary pressure, as well as increased capillary permeability. Both of these effects cause rapid fluid leakage into the tissues of the nose, and the nasal linings become swollen and secretory. Asthma. Asthma often occurs in the allergic type of person. In these, the allergenIg E reaction occurs in the bronchioles of the lungs. Here, the most important product released from the mast cells seems to be the slow-reacting substances of anaphylaxis, which causes spasm of the bronchiolar smooth muscle. Consequently, the person has

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difficulty breathing untile the reactive products of the allergic reaction have been removed. It is widely recognized that many allergic reactions are due to food allergens. Various foods or their metabolic derivatives sensitize the gastrointestinal tract, leading to the production of Ig E antibodies. Subsequent exposure to these allergens may result in mast cell degranulation and the release of mediators that initiate urticaria or asthmatic disorders. Type II: Cytotoxic reaction iscaused by antibody binding to cell surface molecules and the subsequent activation of cytolytic responses by the classic complement cascade or by cellular mechanism. In the first pathway, antibody (usually Ig M, but also Ig G) activates the entire complement sequence and cause cell lysis. In the second pathway, antibody (usually Ig G) and C3b serves to engage receptors on phagocytic cells, causing destruction of the target cells. These reactions usually involve circulating blood cells, such as red cells, white cells, and platelets. Type III: İmmune complex reactions involve the formation of antigen-antibody complexes that can activate the complement cascade. Release of certain products of complement (C3a and C5a) causes a local increase in vessel permeability and permits the release of serum and chemotactic attraction of neutrophils. The neutrophils, in the process of ingesting the immune complexes, release degradative lysosomal enzymes that produce the tissue damage characteristic of these reaction. If the site of reaction is a vessel wall, the outcome is hemorrhage and necrosis; if the site is glomerular basement membrane, loss of integrity and release of protein and red cells into the urine results; and if the site is joint meniscus, destruction of synovial membranes and cartilage occurs. In all cases, however, the outcome depends on complement and granulocytes as mediators of tissue injury. Type IV: Cell-mediated immunity (CMI) reaction also called delayed-type hypersensitivity (DTH) or the tuberculin reaction is mediated by T cells (Th1) rather than by antibody. On activation, the T cells release cytokines lymphokines that cause accumulation and activation of macrophagesi which in turn, cause local damage. This type of reaction has a delayed onset and occurs 1-2 days after challenge with antigen. CMI is a crucial mode of immunologic reactivity for protection against intracellular parasites, such as viruses, many bacteria, and fungi. However, the nature of the reaction and its mediators also cause delayed-type hypersensitivity reactions. Autoimmune Response Autoimmunity is defined as the immune response to antigens of the host itself. Normally, a person is tolerized to self-antigens during the development of the immune system as a fetus and later in life by other mechanisms. However,

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deregulation of the immune response may be initiated by cross-reactivity microbial antigens (e.g., group A streptococcal infection), polyclonal activation of lymphocytes induced by tumors or infection (e.g., malaria, Epstein-Barr virus infection), or a genetic predisposition caused by lack of tolerization to specific antigens. Autoimmune reactions result from the presence of autoantibodies, activated T cells, and hypersensitivity reactions. People with certain MHC antigens are at higher risk for autoimmune responses. IMMUNODEFICIENCY Immunodeficiency may be result from genetic deficiencies, starvation, druginduced immunosuppression (e.g., steroid treatment, cancer chemotherapy, chemotherapeutic suppression of tissue graft rejection), cancer (especially of immune cells), or disease (e.g., AIDS) and may naturally occure in neonatous and pregnant women. Deficiencies in specific protective responses put a patient at high risk for serious disease due to the infectious agents that would be controlled by that response. 1. Hereditary deficiencies of complement components are associated with defects in activation of the classic complement pathway that lead to greater susceptibility to pyogenic (pus-producing) staphylococcal and streptococcal infections. 2. People with defective phagocytic function are more susceptible to bacteria infections but not to viral or protozoal infections. In children with Chronic granulomatous disease, the neutrophil granules are not capable to form superoxide anions. In patients with Vhediak-Hegashi syndrome, the neutrophil granules fuse when the cells are immature in the bone marrow. 3. People deficient in T-cell function are susceptible to opportunistic infections by (1) viruses especially enveloped and noncytolytic viruses and viruses that establish latent or recurrent infections, (2) intracellular bacteria, and (3) fungi. T-cell deficiencies can also prevent the maturation of B-cell antibody responses. T-cell deficiencies can rise from genetic disorders, infection (e.g., human immunodeficiency virus), cancer chemotherapy, or immunosuppressive therapy for tissue transplantation. 4. B-cell deficiencies may result in a complete lack of antibody production (hypogammaglobulinemia), or inability to produce specific subclasses of antibody. People deficient in antibody production are very susceptible to bacterial infection. IgA deficiency, whcih occurs in 1 to 700 white people, results in greater susceptibility to respiratory infections.

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Immunosuppression Immunosuppressive therapy is important for reducing excessive inflammatory or immune responses of macrophages and T cells or for preventing the rejection of tissue transplantats by T cells. Anti-inflammatory treatments primarily target the production of TNF and IL-1 by mononuclear cells. Corticosteroids prevent their production by macrophages and may be toxic to T cells. Immunosuppressive therapy for transplantation generally inhibits the action or causes the lysis of T cells. VACCINES The term vaccine is derived from vaccinia virus, a less virulent member of the poxvirus family that was used to immunize people against smallpox. Vaccines can be subdivided into two groups on the basis of whether they infect the person (live vaccines) or not (inactivated vaccines). Live Vaccines Live vaccines are prepared with organisms limited in their ability to cause disease (avirulent or attenuated). Live vaccines are specially useful for protection against infections caused by enveloped viruses, which require T-cell immune responses for resolution of the infection. Immunization with a live vaccine resembles the natural infection. Immunity is generally long-lived and, depending on the rout of administration, can mimic the normal immune response to the infecting agent. However, there are two problems with live vaccines, as follows: 1. The vaccine virus may still be dangerous for immunosuppressed people or pregnant women, who do not have the immunologic resources to resolve even a weakened virus infection. 2. The vaccine may revert to a virulent viral form. Live bacterial vaccines include the orally administered live, attenuated Salmonella typhi strain Ty2la vaccine for typhoid; The BCG for tuberculosis, which consists of an attenuated strain of Mycobacterium bovis; and an attenuated tularemia vaccine. Live virus vaccines include polio vaccine (Sabin); measles, mumps, rubella (administered together as the MMR) and now varicella zoster have been developed. Inactivated Vaccines Inactivated vaccines provide a large amount of antigen to produce a protective antibody response without the risk of infection by the agent. Inactivated vaccines can be produced through the chemical (e.g. formalin) or heat inactivation of bacteria, bacterial toxins, or viruses or through the purification of the components or subunits

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of the infectious agents. These vaccines are usually administered with an adjuvant which boosts their immunocenicity. Inactivated rather than live vaccines are used to confer protection against most bacteria and viruses that cannot be attenuated, may cause reccurrent infection, or have oncogenic potential. Inactivated vaccines are generally safe, except in people who have allergic reactions to vaccine components. For example, many vaccines are produced in eggs and so cannot be administered to people who are allergic to eggs. The immune response evoked by inactivated vaccines is predominantly humoral response and is more limited than that evoked by live vaccines. The disadvantages of inactivated vaccines in comparison with live vaccines are as following: 1. Immunity is not usually lifelong. 2. Immunity may be only humoral and not cell-mediated. 3. The vaccine dose not elicit a local IgA response. 4. Booster shots are required 5. Large doses must be used There are three major types of inactivated bacterial vaccines: toxoid, killed bacteria, and capsular or protein subunits of the bacteria. Inactivated viral vaccines are available for polio, hepatitis A, influenza, rabies, and other viruses. Subunit vaccine: Instaed of using whole organisms, vaccines can be composed of surface structures of bacteria and the viral attachment proteins. Vaccines against bacterial and viral pathogens are listed in the Tables respectively. and

Table . Bacterial Vaccines ___________________________________________________________________
Bacteria (Disease) Vaccine Component _______________________________________________________________________________ Corynebacterium diphtheriae (diphtheria) toxoid Clostridium tetani (tetanus) toxoid Bordetella pertussis (pertussis) killed cell or acellular Haemophilus influenzae B (Hib) capsule polysaccharide and capsule Polysaccharide-protein conjugate. Neisseria meningitidis A and C (meningitis) capsule polysaccharide Streptococcus pneumoniae capsule polysaccharide and capsule Polysaccharide-protein conjugate Vibro cholera (cholera) killed cell Salmonella typhi (typhoid) killed cell, polysaccharide Bacillus anthracis (anthrax) killed cell Yersinia pestis (plague) killed cell Francisella tularensis (tularemia) live attenuated Coxiella burnetii (Q fever) inactivated Mycobacterium tuberculosis (TB) live attenuated bacille Calmette-Guerin (Mycobacterium bovis) Borrelia burgdorferi (Lyme disease) subunit ________________________________________________________________________________

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Table . Viral vaccines ____________________________________________________________________
Virus Vaccine components ________________________________________________________________________________ Polio inactivated (IPV, Salk vaccine) attenuated (oral polio vaccine, Sabin v Measles attenuated Mumps attenuated Rubella attenuated Varicella-zoster attenuated Influenza inactivated Hepatitis B subunit Hepatitis A inactivated Adenovirus attenuated Yellow fever attenuated Rabies inactivated Smallpox live vaccinia virus ________________________________________________________________________________

Immunization Programs Infants are immunized with diphtheria, tetanus, pertussis, (DTP), and Hib inactivated vaccines as well as the inactivated or live oral polio vaccine. The inactivated hepatitis A vaccine can also be administered on this schdule or to adults at risk of infection. The hepatitis B vaccine is suggested for the first year of life as well as later in life. The live MMR and varicella-zoster vaccines are administered at 2 years of age, after the baby’s immune response has matured and maternal antibodies have dissipated. Booster immunizations of inactivated vaccines and the live measles vaccine are required later in life. Adults should be immunized with vaccines for S.pneumoniae, influenza, rabies, hepatitis B virus, and other diseases, depending on their jobs, the type of traveling they do, and other risk factors that may make them particularly susceptible to specific infectious agents.

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CLINICAL BACTERIOLOGY Bacterial Taxonomy and Nomenclature The classification of microorganisms is essential for the understanding of clinical microbiology. Bacteria are designated by a binominal system, with the genus name (capital letter) followed by the species name (without capital letter), for example, Escherichia coli and Staphylococcus aureus. Names are often abbreviated, for example, E.coli and S.aureus. Classification of Bacteria For descriptive purposes bacteria are often grouped by four main characteristics: the Gram reaction, shape, atmospheric requirements for respiration and the presence of spores (phenotypic classification). Analysis of the analytic characteristic of bacteria has also been used to classify bacteria at the genus, species, or subspecies level (analytic classification). The chromatographic pattern of cell wall mycolic acid is unique for many of individual species of mycobacteria and has been used for more than 25 years to identify the most commonly isolated species. The most precise method for classifying bacteria is by analysis of their genetic material (genotypic classification). Organisms were initially classified by the ratio of guanine to cytosine. STAPHYLOCOCCUS The name Staphylococcus is derived from the Greek term staphyl, meaning “a bunch of grapes.” This name refers to the fact that the cells of these gram-positive cocci grow in a pattern resembling a cluster of grapes; however, organisms in clinical material may also appear as single cells, pairs, or short chains. Most staphylococci are 0.5 to 1 µm in diameter and are nonmotile, aerobic or facultative anaerobic, and catalase-positive and grow in a medium containing 10% sodium chloride and at a temperature ranging from 18ºC to 40ºC. The organisms are present on the skin and mucouse membranes of humans and other mammals, and birds. Staphylococcus is an important pathogen in humans, causing a wide spectrum of life-threatening systemic diseases; infections of the skin, soft tissues, bones, and urinary tract; and apportunistin infections. The species most commonly associated with human diseases are S.aureus (the most virulent and bestknown member of the genus), S.epidermidis, S.saprophyticus, and S.haemolyticus. S.aureus colonies are golden as the result of the carotenoid pigments that form during their growth, hence the species name. It is also the only species found in humans that produces the enzyme coagulase; thus, all other species are commonly referred to as coagulase-negative staphylococci.

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Staphylococcus aureus S.aureus is ubiquitous, existing everywhere in nature. It constitutes part of the normal flora of the skin, nose, throat, gastrointestinal tract, and genital tract of 2550% of humans and animals. S.aureus is among the most resistance of the nonspore formers to adverse enviromental conditions and physical and chemical agents. The organism can survive for as long as 14 weeks in dried pus and is killed by 70% ethanol only after a 10-min contact period. Staphylococcus produce disease because of their ability to; spread in tissues and orm abscesses, produce extracellular enzymes or exotoxins and combat host defences. Cell Wall Virulence Factor 1. Capsule. A loose-fitting, polysaccharide layer (slime layer) is protect bacteria by inhibiting the chemotaxix and phagocytosis. It is also facilitate the adherence of bacteria to catheters and other synthetic material. 2. Teichoic acids are bound convalently to the peptidoglycan. It is mediate the attachment of staphylococci to mucosal surfaces through their specific binding to fibronectin. 3. Protein A is a cell wall component also covalently linked to peptidoglycan and its ability to bind to the Fc portion of Ig G and extracellular matrix glycoprotein. Protein A may contribute to adherence and possess antiphagocytic activity. Toxins 1. α, ß, δ, and γ toxins (produced by most strains of S.aureus) attack mammalian cell (including red blood cell) membranes, and are often referred to as hemolysins. α toxin exhibits dermonecrotic activity that contributes to tissue necrosis. 2. Toxic shock syndrome toxin-1 (TSST-1) is a protein produced by virtually all strains of S.aureus and responsible for the clinical manifestations of toxic shock syndrome. 3. Enterotoxin A, B, and D molecules are heat-stable proteins capable of withstanding boiling for 30 min and produced by 30% to 50% of all S.aureus strains. Synthesis is plasmid or chromosomally mediated. Enterotoxin A and D are responsible for staphylococcal food poisoning by inhibiting water absorption from the intestinal lumen and inducing diarrhea. Enterotoxin B damages the intestinal epithelium and produces colitis. 4. Exfolitative (epidermolytic) exotoxin is produced by some (5% to 10%) strains of S.aureus. Synthesis is plasmid or chromosomally mediated. By causing intraepidermal splitting of tissues and necrosis, it is responsible for the clinical manifestations seen in scalded skin syndrome (SSS).

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Enzymes 1. Lipases are lipid-hydrolyzing enzymes, which are allow the organisms to invade cutaneous and subcutaneous tissues by splitting fats and oils accumulating on the skin. All strains of S.aureus and more than 30% of the strains of coagulase-negative staphylococci produce several different lipases. 2. Leucocidin is an exotoksin that contributes to the survival of the organism, it destroys polymorphonuclear leukocytes. 3. Coagulase is an enzyme produced by the organism during it is growth. The role of coagulase in the pathogenesis is speculative, but coagulase may cause the formation of fibrin layer around a staphylococcal abscess, thus localizing the infection and protecting the organisms from phagocytosis. 4. Hyaluronidase is produced by over 90% of S.aureus strains. It is an enzyme that hydrolyzes the hyaluronic acid constituent of connective tissue ground substances and thus facilitates the spread of the organism through the tissues. 5. Staphylokinase (fibrinolysin) is produced by virtually all S.aureus strains. It dissolves fibrin clots and thus contributes to the spread of the organism from local sites. 6. Nuclease is the another enzyme for S.aureus. The role of this enzyme in the pathogenesis of infection is unknown. 7. Penicillinase. More than 90% of staphylococcal isolates were susceptible to penicillin in 1941, the year the antibiotic was first used clinically. Resistance to penicillin quickly developed, however, primarily because the organisms could produce penicillinase (ß-lactamase). 8. Catalase. All staphylococci produce catalase, which catalyzes the conversion of toxic hydrogen peroxide to water and oxygen. Hydrogen peroxide can accumulate during bacterial metabolism or after phagocytosis. Diseases of the S.aureus 1. Localized skin infections: Localized, pyogenic staphylococcal infections include impetigo, folliculitis, furuncles, and carbuncles. Impetigo, a superficial infection affecting mostly young children, occurs primarily on the face and limbs. Initially, a small macule (flattened red spot) is seen, and then a pus-filled vesicle (pustule) on an erythematous base develops. Folliculitis is a pyogenic infection in the hair follicles. The base of the follicle is raised and reddened, and thereis a small collection of pus beneath the epidermal surface. If this occurs at the base of the eyelid, it is called a stye. Furuncles (boils), an externsion of folliculitis, are large, painful, raised nodules with an underlying collection of dead and necrotic tissue. Carbuncles occur when furuncles coalesce and extend to the deeper subcutaneous tissue. Multiple sinus tracts are usually present.

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2. Scaled Skin Syndrome is a disease that occurs in infants and children 4 years of age or under. In this syndrome the organisms release exfolitative toxin, which is resposible for the extensive intraepidermal splitting and bullous necrosis of the tissue. 3. Toxic Shock Syndrome is a disease was initially described in children, although it is now recognized as primarily a disease in menstruating women. At present, 80% to 90% of patients with TSS are menstruating women. Highly absorbent tampons contribute to the initiation of the disease by providing a favarable enviroment for the growth of resident S.aureus. The disease is initiated with the localized growth of toxin-producing strains of S.aureus in the vagina or a wound, followed by release of the toxin into the blood stream. Clinical manifestations start abruptly and include fever, hypotension, and a diffuse macular erythematous rash. 4. Food Poisoning is the one of the most common food poisoning in the world. The organisms are usually introduced into food, such as processed meats, pastries, potato salad and ice cream. The contaminated foods is kept at room temperature, during which time the organisms multiply and release heat stable enterotoxin A or D. Following ingestion of the food, the onset of disease is abrupt and rapid with an incubation period of only 1-6 h. Staphylococcal food poisoning is characterized by severe vomiting, diarrhea, and abdominal pain. The absence of fever is an important observation in the differential diagnosis of staphylococcal food poisoning. 5. Colitis is a disease that is observed in patients whose normal bowel is altered by the oral administration of broad-spectrum antibiotics that selectively permit overgrowth by antibiotic resistant, enterotoxin B producing strains of S.aureus. Enterotoxin B dameges the intestinal epithelium and produces fever, diarrhea, and abdominal cramps. 6. Pneumoniae is a disease that occurs among immnosuppressed patients, the aged, infants less than one year of age, and frequently in children with measles and influenza. 7. Other diseases like osteomyelitis, septicemia, and septic arthritis. Laboratory Diagnosis Specimens obtained depend on the disease process and include lesion material, pus, sputum, blood, spinal fluid, and feces. İsolation and identification of S.aureus requires initial cultivation on blood agar and/or specific medium. Overnight incubation under aerobic conditions at 37ºC. The organism may be identified as a gram-positive, catalase positive coccus exhibiting coagulase.

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Treatment The antibiotics of choice are oxacillin (or other penicillinase-resistant penicillin) or vancomycin for oxacillin-resistant strains. The focus of infection (e.g., abscess) must be identified and drained. Treatment is symptomatic for patients with food poisoning Coagulase-Negative Staphylococci A. S. epidermidis is present in large numbers as part of the normal flora of the skin. As such it is frequently recovered from blood cultures, generally as a contaminant from skin. Despite its low virulence, it is a common cause of infection of implants such as heart vulves and catheters. Cell envelope factors that facilitate attachment to plastic surfaces act as virulence factors. Acquired drug resistance by S. epidermidis is even more frequent than by S. .aureus. B. S. saprophyticus. The organism is a frequent cause of cystitis in women, probably related to it is occurence as part of normal vaginal flora. It tends to be sensitive to most antibiotics, even penicillin G. S. saprophyticus can be distinguished from S. epidermidis and most other coagulase-negative staphylococci by its natural resistance to novobiocin. STREPTOCOCCUS The genus Streptococcus is a diverse collection of gram-positive cocci typically arranged in pairs or chains. Most species are facultative anaerobes, and some grow only in an atmosphere enhanced with carbon dioxide (capnophilic growth). There nutritional requirements are complex, necessitating the use of blood or serumenriched media for isolation. Carbohydrates are fermented, resulting in the production of lactic acid, and unlike Staphylococcus species, streptococci are catalase-negative. Streptococci can be classified by several schemes: 1. Hemolytic properties on blood agar. Alfa-hemolytic streptococci cause a chemical change in the hemoglobin of red cells in blood agar, resulting in the appearance of a green pigment that forms a ring around the colony. Beta-hemolytic streptococci cause gross lysis of red blood cells, resulting in a clear ring around the colony. Gamma-hemolytic is a term applied to streptococci that cause no color change or lysis of red blood cells. 2. Serologic (Lancefield) Groupings Many species of streptococci have in their cell walls a polysaccharide known as the C-carbohydrates, which is convalently linked to the cell wall peptidoglycan. The

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lancefield scheme classifies primarily ß-hemolytic streptococci into groups A through U on the basis of their C-carbohydrate. The clinically most important groups of ßhemolytic streptococci are types A and B. 3. Biochemical (physiologic) Properties GROUP A, ß-HEMOLYTIC STREPTOCOCCI Streptococcus pyogenes is the most virulent member of this group of gram-positve cocci. This bacteria is an important cause of a variety of suppurative and nonsuppurative diseases. Structure and Physiology Isolates of S. pyogenes are gram-positive, nonmotile cocci occuring in short or long chains, and occasionally singly and in pairs. Freshly isolated strains of group A streotococci are encapsulated, but the capsules are lost rapidly during the stationary phase of in vitro cultivation. Group A, like most streptococci, are less resistance to enviromental conditions than staphylococci, although they can survive on dry swabs for weeks. They are killed rapidly by physical and chemical agents. Structural features that are involved in the pathology or identification of the group A streptococci include: A. Antigenic structure Capsule. The outermost layer of the cell is the capsule, which is composed of hyaluronic acid, identical to that found in connective tissue. For this reason the capsule is nonimmunogenic (again in contrast with S. pneumoniae). C-carbonhydrates are cell wall polysaccharides whose antigenic diversity forms the basis for the classification of streptococci into 20 serogroups lettered from A to V. Lipoteicoic acid (LTA). The ability of group A streptococci to bind to epithelial cells in the mouth and on the skin is mediated by lipoteicoic acid exposed on the cell surface. M protein is a major antigen associated with virulent streptococci. In the abscence of M protein the strains are not infectious. The M protein also prevents interaction with complement. Protein F (fibronectin-binding protein) mediates attachment to fibronectin in the pharyngeal epithelium. B. Extracellular products Streptolysin S and O. Streptolysin S is an oxygen stable, nonimmunogenic cellbound hemolysin capable of lysing erythrocytes, as well as leukocytes and platelets,

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following direct cell contact. Streptolysin O is inactivated reversibly by oxygen. Unlike streptolysin S, antibodies are readily formed against streptolysin O and are useful for decumenting a recent infection (ASO test). In addition to its ability to lyse human erythrocytes, streptolysin O is also capable of killing leukocytes by lysis of their cytoplasmic granules with release of hydrolytic enzymes. Pyrogenic (erythrogenic) exotoxins are proteins responsible for the rash of scarlet fever. There are three antigenically distinct types designated A, B, and C, which are produced by more than 95% of group A streptococci strains. DNAse. Four immunologically distinct deoxyribonucleases (A through D) have been identified. These enzymes are not cytolytic but are capable of depolymerizing free DNA present in pus. This reduces the viscosity of the abscess material and facilitates spread of the organisms. Other enzymes such as hyaluronidase and streptokinase (fibrinolysin). Diseases of Group A Streptococci (Streptococcus pyogenes). Pharyngitis. Group A streptococcus is the major causes of bacterial pharyngitis, with group C and G occationally involved. This is primarily a disease of children between the age of 5 to 15 years, but infents and adults are also susceptible. The pathogen is spread by person-to-person contact via respiratory droplets. Pharyngitis generally develops 2 to 4 days after exposure to the pathogen, with an abrupt onset of sore throat, fever, malaise, and headache. The posterior pharynx can appear erythematous with an exudate, and cervical lymphadenopathy can be prominant. Scarlet fever is a complication of streptococcal pharyngitis seen when the infecting strain is lysogenized by a temperate bacteriophage that stimulates production of erythrogenic toxin. Within 1 to 2 days after initial clinical symptoms of pharyngitis, a diffuse erythematous rash will initially appear on the upper chest and then spread to the extremities. The area around the mouth is generally spared, as are the palms and soles. The tongue will initially be covered with a yellowish-white coating that will later be shed revealing a red, raw surface (“strawberry tongue”). Erysipelas. This disease can affect all age groups. İt is a disease of the skin and subcutaneous tissues usually occurring on the face or lower extremities and characterized by a fiery red, advancing erythema. Puerperal sepsis. This infection is initiated during, or following soon after, the delivery of a newborn. It can occur due to exogenous transmission (for example, by nasal droplets from an infected carrier, or from contaminated instruments), or endogenously, from the patient’s vaginal flora. This is a disease of the uterine endometrium in which patients suffer from a purulent vaginal discharge, and are systemically very ill. Acute rheumatic fever occurs most commonly among young children during the fall and winter, and can occur only when preceded by pharyngitis caused by any of group A streptococcal serotypes. The disease can occur in 0.1-3% of untreated patients from

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1 to 5 weeks after pharyngial onset. Although the mechanism of pathogenesis is not entirely understood, a plausible hypothesis is based upon the presence of crossreactive epitopes among M proteins and target tissues, including human cardiac tissue, and the binding of anti-M protein antibody to the tissue epitopes. The major clinical manifestations are carditis, polyarthritis, and subcutaneous nodules. Acute hemorrhagic glomerulonephritis occurs most commonly in children and can result when preceded by pharyngitis or skin disease caused by goup A streptococci. This disease can occur in less then 1 to 15% of untreated patients from 1 to 5 weeks after pharyngial or skin disease onset. Evidence supports the concept that renal damage is the result of immune complex diposition on the glomerular basement membrane. The major clinical manifestations are renal glomerular damage, hypertension, edema, proteinuria, and hematuria. Other disease such as impetigo, cellulitis, lymphangitis. Laboratory Diagnosis The specimens obtained depend on the disease process and include nose and throat swabs, lesion material, pus, sputum, blood for culture and immunoserology, urine, and spinal fluid. For isolation and identification of group A streptococci initial cultivation on blood agar or specialized selective agar are required. Overnight incubation anaerobically or under aerobic conditions in the presence of 10% carbon dioxide at 37ºC is optimal for isolation of the organism. The organism may be identified as beta-hemolytic, gram positive, catalese negative coccus, and is inhibited by bacitracin. Immunoserologic test. Both the anti-streptolysin O (ASO) and anti-Dnase B assays are useful in diagnosis. Treatment: Adequate drainage, debridement, and antibiotic therapy are essential for the treatment of localized, suppurative skin lesions. Penicillin is the drug of choice for acute diseases. Penicillin has no effect upon established rheumatic heart disease and acute hemorrhagic glomerulonephritis. Penicillin resistant strains have not been reported. Erythromycin is the drug of choice for penicillin allergic patients. GROUP B STREPTOCOCCİ Group B streptococci (S. agalactiae) are harbored in the female genital tract and male urethra of 15-25% of humans and animals, as well as in the pharynx and GI tract. The organism is transmitted from an infected mother to her infant in utero or at birth. Group B streptococci are encapsulated. These organisms far outnumber E.coli K1 as the leading cause of neonatal meningitis during the first 4 months of life. The antiphagocytic properties of the capsular polysaccharide allow the organisms to survivei multiply, invade epithelial cells, and induce an acute inflammatory response.

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In adults, the organisms may produce pneumoniae, septicemia, prosthetic joint disease, or puerperal sepsis originating from the female genital tract. Specimens for laboratory diagnosis depend on the disease process and include blood for culture, sputum, a cervical swab, and spinal fluid. These specimens are cultured on blood agar and incubated aerobically at 37ºC. Group B streptococci are beta-hemolytic, gram-positive, catalase negative cocci. They are only streptococci in which ability to hydrolyze hippurate and positive CAMP test. Early therapy with penicillin plus an aminoglycoside is essential for the prevention of progressive, fatal disease. Heavily colonized mothers can be treated with penicillin intrapartum to prevent subsequent colonization of their newborns. THE VIRIDANS GROUP OF STREPTOCOCCI The viridans group, often reffered to as “oral streptococci” do not contain C carbonhydrate but have been grouped on the basis of rRNA cataloging and nucleic acid hybridization studies. These organisms are normal inhabitants of the oral, respiratory, and gastrointestinal mucosa of humans and animals. They are opportunistic pathogens and have generally been thought to be of low virulence. Viridans streptococci are, however, the major etiologic agents of bacterial endocarditis. Patients who develop streptococcal endocarditis usually possess a previously damaged heart valve (from previous rheumatic fever and other cause). Gingival disease or dental manipulations, including dental prophylaxis, are often predisposing factors in the development of endocarditis. Viridans streptococci are able to adhere to epithelial and endothelial cells, and adherence is probably a key factor in their ability to cause disease. Streptococcus mutans has been definitively established as a major etiologic agent of dental caries in addition to being a cause of endocarditis. Extracellular sugars, called dextrans, serve as attachment mediators for tooth surfaces as well as heart valves. Specimens for laboratory diagnosis depend on the disease process and include blood for culture and urine. These specimens are cultured on blood agar and incubated aerobically at 37ºC. The species of the viridans group are alfa or gamma hemolytic, gram-positive, catalase negative cocci that are not inhibited by optochin. Although treatment with penicillin is effective, the occurence of penicillin resistant strains necessitate the use of penicillin plus an aminoglycoside. Streptococcus pneumoniae S. pneumoniae are gram-positive, nonmotile, encapsulated cocci. They are lancetshaped, and their tendency to occur in pairs accounts for their earlier designation as Diplococcus pneumoniae. S. pneumoniae is the most common cause of pneumonia and otitis media, and is an important cause of meningitis and bacteremia. The risk of disease is highest among young children, older adults, smokers, and persons with certain chronic illnesses. Like other streptpcocci, S. pneumoniae is fastidious and is

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routinely cultured on blood agar and releases an α-hemolysin. This bacteria is an obligate parasite of humans and harbored in the nasopharynxs of 25-70% of the population. Pneumococci are very sensitive to enviromental, physical, and chemical agents. They are killed rapidly by antiseptic agents. The polysaccharide capsule is the sole basis for classification and the only known virulence factor. Distinct epitopes enable the recognition of more than 85 serotypes of pneumococci, 23 of which are responsible for greater than 85% of pneumococcal disease. The capsule is inhibits phagocytosis and thus allows the organisms to establish themselves in host tissue, multiply, and produce disease. Disease of S. pneumoniae S. pneumoniae is the most common cause of lobar and lobular (broncho) pneumoniae. This organism also is the most common cause of meningitis among adults and a major cause of otitis media and sinusitis among children. Most infections are caused by endogenous spread from the colonized nasopharynx or oropharynx to distal site (e.g., lungs, sinuses, ears, blood, meninges). Colonization is highest in young children. Person to person spread through infectious droplets israre. Laboratory Diagnosis Specimens obtained depend on the disease process and include a nasopharyngeal swab, sputum (which may be rusty), blood for culture, spinal fluid, and pus. Gram stain of pus, sputum, and spinal fluid often shows gram-positive, lancet shaped, diplococci and numerous polymorphonuclear leukocytes. Primary isolation and identification are require initial cultivation on blood agar or in blood culture broth. Overnight incubation under aerobic conditions at 37ºC is optimal for isolation of the organism. The organism may be identified as an alfa hemolytic, gram-positive, catalase negative, coccus that is bile soluble and inhibited by optochin. Rapid identification of pneumococcus serotypes in spinal fluid can be accomplished by latex agglutination utilizing serotype-specific anticapsular antibody for the detection of capsular polysaccharide. Treatment. Althogh the penicillin is still the drug of choice, multiple-resistant strains are now appearing. Cephalosporins, Erythromycin, chloramphenicol, vancomycin are used for patients allergic to penicillin or for traetment of penicillinresistant strains. Common Pathogens of Typical Pneumonia Bacteria 1. Streptococcus pneumoniae 3. Staphylococcus aureus 2. Haemophilus influenzae

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Viruses 1. Influenza virus Types A and B 3. Respiratory syncytial virus

2. Parainfluenza viruses

ENTEROCOCCI Enterococci contain a C-carbohydrate that reacts with group D antisera. Therefore, in the past, they were considered group D streptococci. Today, DNA analysis and other properties have placed them in their own genus, Enterococcus. The clinically most important species are Enterococcus faecalis and Enterococcus faecium. Enterococci can be α-, ß-, or nonhemolytic. Enterococcus species are harbored in the GI tract of 25% of humans and animals, and do not cause disease in the normal host. Unlike streptococci, the enterococci are highly resistant to enviromental conditions and physical and chemical agents. There are no kown structural or extracellular virulance factors. Gastrointestinal or genitourinary tract manipulation can cause spread of the organisms to previously sterile sites with resultant biliary tract disease, urinary tract disease, septicemia, intraabdominal abscesses, and subacute bacterial endocarditis. Specimens for laboratory diagnosis depend on the disease process and include blood for culture and urine. These specimens are cultured on blood agar and incubated aerobically at 37ºC. Enterococci are alfa, beta, or gamma hemolytic, grampositive, catalase negative cocci that grow in 6.5% salt broth, grow in bile, and hydrolyze esculin. Enterococci are naturally resistant to ß-lactam antibiotics and aminoglycosides, but are sensitive to synergistic action of a combination of these classes. Newer antibiotic, such as the combination of quinupristin and dalfopristin are used to treat vancomycin-resistant infections. However, some enterococcal strains are resistant to all commercially available antibiotics. CORYNEBACTERIA Corynebacteria are small, slender, pleomorphic, garm-positive rods of distinctive morphology that tend to stain unevenly but do not form spores. They are nonmotile and noncapsulated. Gram stains of these bacteria reveal short chains (V or Y configurations) or clumps resembling Chinese “letters”. Metachromatic granules within the cells may be seen with special stain. Corynebacteria are aerobic or facultative anaerobic, nonmotile, and catalase-positive. Most species ferment carbohydrates, producing lactic acid. Although many species grow well on common laboratory media (e.g. blood agar), some species require supplementation with lipids for good growth. Corynebacteria are ubiquitous in plants and animals, and they normally colonize the skin, upper respiratory tract, gastrointestinal tract, and urogenital tract in humans.

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Although all species of corynebacteria can function as opportunistic pathogens, a few are more commonly associated with disease. The most famous of these is Corynebacterium diphtheriae, the etiologic agent of diphtheria. C. diphtheriae Diphtheria, caused by C. diphtheriae, is a life-threatening illness that is among the best studied of bacterial diseases. It is a disease that occurs mostly in infants and children, but may be seen in adults who fail to maintain protective levels of circulating antitoxin through toxoid boosters. Although, the prevalence of the disease is relatively low due to effective vaccination, diphtheria occurs with a high frequency in many areas of the world as a result of incomplete or no immunization. With the advent of effective antitoxin and antimicrobial therapy, the overall case fetality rate has been reduced from 40% to 5%. C. diphtheriae is considered the most important pathogen of this genus and is an obligate parasite of humans. It can survive for as long as 14 weeks in pseudomembraneous lesions and is readily destroyed by most physical and chemical agents. The active diphtheria exotoxin molecule is a heat-labile protein, which acts by inhibiting protein synthesis at the ribosome level. The mechanism of action of diphtheria exotoxin is well known. The “tox” gene that codes for the exotoxin is introduced into C. diphtheriae by a lysogenic bacteriophage. Diseases of C. diphtheriae The most common disease caused by C. diphtheriae is diphtheria, an acute communicable disease manifested by both local infection of the upper respiratory tract and the systemic effects of the toxin that are most notable on the heart and peripheral nerves. Spread person to person by exposure to respiratory droplets or skin contact. Respiratory diphtheria. Patients with diphtheria involving the respiratory tract will develop symptoms after a 2 to 6 day incubation period. Organisms will multiply locally, on epithelial cells in the pharynx or adjacent surfaces, and initially produce localized damage by exotoxin activity. The onset is sudden, with malaise, sore throat, exudative pharyngitis, and a low-grade temperature. The exudate evolves into a thick pseudomembrane, which is firmly adherent to the respiratory tissue and is difficult to dislodge without causing bleeding of the underlying tissue. As the patient recovers after the approximately 1-week cours of the disease, the membrane dislodges and is expectorated. Complications in patients with severe disease include breathing obstraction and myocarditis. Other forms of the disease caused by C. diphtheriae is cutaneous diphtheria. The disease is acquired by skin contact with other infected persons. The organism, which colonizes the skin surface, gains entry into the subcutaneous tissue through breaks in the skin. A papule will develope and evolve into a chronic nonhealing ulcer,

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sometimes covered with a greyish membrane. The ulcer may also be superinfected with S. aureus or group A Streptococcus Laboratory Diagnosis Specimens obtained depend on the disease process and include a nose, throat, nasopharyngeal, and wound swab. Specimens are cultivate on cysteine-tellurite agar and Loeffler’s coagulated serum. In addition, differential diagnosis necessitates primary cultivation on blood and chocolate agar. Identification of gray-black colonies on cystein-tellurite agar, the typical Chinese letter, beaded, barred, or palisading arrangment of pleomorphic rods with accentuated metachromatic granules by methylene blue staining of colonies on Loeffler’s coagulated serum, constitute presumptive evidence for C. diphtheriae. Definitive identification of the organism is based upon the demonstration of exotoxin pruduction by a virulence test in guinea pigs or by a modified in vitro “Elek” test utilizing specific antitoxin. Treatment Specific antitoxin must be administered immediately. Inasmuch as the antitoxin is generated in horses, a skin test to ensure the absence of hypersensitivity to horse protein is essential prior to its use. Although antibiotics have no effect upon the toxemic disease process, penicillin or erythromycin is effective in killing organism and thus preventing further toxin production. Prevention and Control 1. Active immunization with formalin-inactivated toxoid. Vaccination is highly effective. It is first administered early in infancy along with tetanus toxoid and killed Bordetella pertusis (DTP) and must be followed by periodic toxoid boosters throughout childhood and adulthood. 2. The schick test. This procedure has been used during outbreaks of diphtheria to determine which case contacts are susceptible to the disaese, and thus in need of immunization. A small amount of diphtheria toxin is injected intradermally into one forearm and a heated toxin control into the other. Redness and induration within 1 to 2 days at the test site only, or their appearance at the both sites with persistence at the test site but disappearance from the control site in 4-7 days, signifies susceptibility due to lack of sufficient antitoxin. In contrast, no reaction at both the test and control sites, or redness at both sites within 1 day, reaching a maximum in 2-3 days, and fading rapidly from both sites, signifies immunity due to the presence of sufficient neutralizing antitoxin.

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Diphtheroides Diphtheroides are corynebacteria that occupy the skin, nose, throat, nasopharynx, urinary tract, and conjunctiva of normal individuals can, in rare instances, infect immunosuppressed hospitalized individuals, and produced septicemia with a high cases fatality rate. BACILLUS Species of the genus Bacillus are gram-positive, form endospores, and are either strictly or facultative aerobic. Most of the seventy or so species of Bacillus are found in soil and water, and are usually encountered in the medical laboratory as airborne contaminants. Bacillus anthracis, the organism responsible for anthrax, is the most important member of this genus. This species is considered one of the most feared agents of biological warfare. Bacillus anthracis Bacillus anthracis, has an illustrious place in medical history. It was the first bacterium shown to be the causative agent of an infectious disease by Koch in 1877. B.anthracis is a gram-positive, spore-forming, encapsulated, nonmotile rod with characteristic square cut ends. The organism occurs singly, in pairs, or in long chains with a “string of pearls” appearance. Spores are oval, centrally located and can survive in soil for years. The organism is facultative anaerobe that grows best aerobically on blood agar at 37ºC and is nonhemolytic. Colonies appear rough with an irregular edge that gives a “Medusa head” appearance. Pathogenesis The major factors responsible for the virulence of B.anthracis are: 1. A prominant polypeptide capsule made up exclusively of D-glutamic acid exhibits antiphagocytic activity but dose not stimulate protective antibody. 2. Three exotoxins: protective antigen (PA), lethal or toxic factor (LF), which is responsible for most of the toxicity, and edema factor (EF). Both edema toxin and lethal toxin as well as the capsule must be present for disease to occur. Diseases of B. anthracis Cutaneous anthrax is the result of infection with spores that gain access to humans through small abrasions or scratches in the skin while handling diseased animals or animal products, such as meats, hides, shaving brushes made from infected animals. Approximately 95% of anthrax infections are due to the inoculation of Bacillus spores through exposed skin surfaces. This disease characterized by the

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development of a painless papule at the site of inoculation that rapidly progresses (within 2-5 days) to a malignant pustule—a necrotic, black lesion sorrounded by a ring of vesicles containing dark, bluish-black, serosanguinous fluid and exotoxin. Mortality in patients with untreated cutaneous anthrax is 20%. Inhalation anthrax is initially seen as a viral respiratory ilness and then rapidly progresses to severe pulmonary disease. This disease is the result of transmission by droplet nuclei from a patient with respiratory disease or by the inhalation of spores from infected animals or animal products. Mortaly is high even in appropriately traeted patients, because the disease usually not suspected until the course is irreversible. GI anthrax is a very rare human disease with varied clinical presentations. Laboratory Diagnosis Specimens obtained depend on the disease process and include material from lesions, blood for culture, spinal fluid, and sputum. Gram stain of the specimen will reveal characteristic gram-positive rods with square cut ends and often in pairs or long chains; spores appear as an oval unstained “space” in the center of the organism. Primary isolation and identification procedures require initial cultivation on blood agar. Overnight incubation under aerobic conditions at 37ºC is optimal for isolation of the organism, which is identified as a nonhemolytic, gram-positive, nonmotile, spore-forming rod with square cut ends and occuring singly, in pairs, or in long chains. A direct immunofluorescence assay on the isolated organism or a virulence test in animals used for definitive identification. Traetment Ciprofloxacin is the drug of choice; penicillin, doxycycline, erythromycin or chloramphenicol can be used (if susceptible), but the bacteria are resistant to sulfonamides and extended-spectrum cephalosporins. The effectiveness of antimicrobial therapy among patients with inhalation anthrax is minimal. Prevention and Control 1. Carcasses of dead animals must be disposed of by incineration or deep underground burial. 2. Those at risk must wear masks and gloves and be clothed appropriately 3. Vaccination is an effective control measure in animal herds and has been used to protect humans in areas with endemic disease 4. The PA vaccin alone is effective for humans and should be used by those in high risk occupations.

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Bacillus cereus Bacillus cereus exists as a saprophyte in water and soil. The organism differs from B. anthracis in that it is motile, nonencapsulated, and beta-hemolytic. The organism may cause a self-limiting type of food poisoning, usually resulting from the imgestion of contaminated rice or meat dishes containing enterotoxin. LISTERIA The genus Listeria consists of seven species, with Listeria monocytogenes the only human pathogen. L.monocytogenes is a short, gram-positive, facultatively anaerobic bacillus. The short bacilli appear singly, in pairs, or in short chains and can be mistaken for S. pneumoniae or Enterococcus. The organism are motile at room temperature but not at 37ºC, and they exhibit a characteristic tumbling motion. Grows best aerobically or under microaerophilic conditions (3 to 5% oxygen) on blood agar at 37ºC. The organism produces a narrow zone of beta hemolysis. Although listeria are widely distributed in nature, human disease due to this organism is uncommon and is restricted to several well-defined populations: neonates, the elderly, pregnant women, and patients with defective cell-mediated immunity. Pathogenesis L. monocytogenes is a facultative, intracellular parasite, and has been used extensively to study phagocytosis and immune activation of macrophages. The organism attaches to and enters a variety of mammalian cells, apparently by normal phagocytosis, and ones internalized, escapes from the phagocytic vacuole by elaborating a membrane-damaging toxin called listeriolysin O and two different phospholipase C enzymes. The bacteria’s movement is mediated by a bacterial protein, ActA. Diseases of L.monocytogenes A. Neonatal diseases. Two forms of neonatal diseases have been described: Early-onset disease, acquired transplacentally in utero, and late-onset disease, acquired at or soon after birth. Early-onset disease, also called granulomatosis infantiseptica, is a devastating disease that has a high mortality rate unless treated promptly. It is characterized by the formation of dessiminated abscesses and granulomas in multiple organs. Late-onset disease occurs 2 to 3 weeks after birth in the form of meningitis or meningoencephalitis with septicemia.

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B. Adult listeriosis occurs mostly in immunocompromised hosts and pregnant females, the latter often resulting in abortion. The most common form in listeria infections is meningitis. Laboratory Diagnosis. Specimens (blood and spinal fluid) that obtained from patients are cultivate on blood agar and identified according to the charasteristic features discoused above. Treatment. The treatment of choice for severe disease is penicillin or ampicillin, alone or in combination with gentamicin. NEISSERIA The genus Neisseria consists of 10 species. Two species, Neisseria meningitidis (syn: the meningococcus) and Neisseria gonorrhoeae (syn: the gonococcus), are strictly human pathogens. The remaining species are commonly present on mucosal surfaces of the oropharynx and nasopharynx and occasionally colonize the anogenital mucosal membranes. Members of the genus are aerobic, gram-negative cocci typically arranged in pairs (diplococci) with adjacent sites flattenned together (resembling coffee beans). The bacteria are not motile and do not form endospores. All species are oxidase-positive, and most produce catalase. Acid is produced by oxidation of carbohydrates but not by fermentation. Neisseria gonorrhoeae N. gonorrhoeae is a fastidious organism, requiring complex media for growth and adversely affected by drying and fatty acids. Soluble starch is added to the media to neutralize the toxic effect of the fatty acids. It is highly susceptible to enviromental, physical, and chemical agents. The optimum growth temperature is 35ºC to 37ºC, with poor survival of the organism at cooler temperature. A humid atmosphere supplemented with CO2 is required or enhances growth of N. gonorrhoeae. Antigenic structure as it relates to virulence The outer surface is not covered with a true carbohydrate capsule, as is found in Neisseria meningitidis. Pili mediate attachment of the organism to epithelial and mucosal cell surfaces and are antiphagocytic. Three outer membrane proteins (OMPs) have been studied extensively: 1. Por proteins are porin proteins that form pores oe channels in the outer membrane. 2. Opa (opacity) proteins are a family of membrane proteins that mediate binding to epithelial cells.

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3. Rmp (reduction-modifiable) proteins. Prevents complement-mediated bactericidal antibody function and thus may contribute to dissemination of the disease. Another major antigen in the cell wall is lipooligosaccharide (LOS). This antigen is composed of lipid A and a core oligosaccharide, smilar to gram-negative lipopolysaccharide(LPS), and possesses endotoxin activity. Other important gonococcal proteins are an immunoglobulin (Ig) A1 protease. The enzyme inactivates local secretory Ig A and thus may play a role in facilitating the adherence of gonococci to mucosal surfaces. ß-lactamase, which degrades penicillin. Diseases of N. gonorrhoeae Gonorrhea is one of the most commonly reported sexually transmitted disease in the United States. A higher proportion of females than males are generally asymptomatic; these individuals act as the reservoir for maintaining and tranmitting gonococcal infections. More than one sexually transmitted disease may be acquired at the same time, for example gonorrhea in combination with syphilis, chlamydia, human immunodeficiency virus (HIV), hepatitis B virus. Patients with gonorrhea may therefore have to be treated for more than one pathogen. Genital infection in men is primarily restricted to the urethra. Purulent urethral discharge and dysuria developes after a 2-7 day incubation period. Although complications are rare, epididymitis, prostatitis, and periurethral abscesses can occur. The primary site of infection in women is the cervix, although gonococci can be isolated in the vagina, urethra and rectum. Vaginal discharge, dysuria, and abdominal pain are commonly reported in symptomatic patients. Other diseases associated with N. gonorrhoeae include purulent conjunctivitis particularly in newborns infected during vaginal delivery (ophthalmia), anorectal gonorrhea in homosexual males, and pharyngitis. Laboratory Diagnosis Specimens obtained depend on the disease process and include urethral, cervical, rectal, pharyngeal, and/or conjunctival exudates. The direct demonstration of gram-negative intracellular diplococci within PMNs is diagnostic only when observed in the urethral exudates of males with characteristic clinical manifestations. Gram stains of smears from female urethral and cervical exudates, from rectal, pharyngeal, and conjunctival exudates of male and females, are unreliable due to the potential presence of nonpathogens resembling gonococcal morphology, and presence of meningococcuses. All such specimens must be cultured and the isolated organism identified. These techniques require initial cultivation on Thayer-Martin medium. Incubation for 48 h at 35-37ºC under aerobic conditions in the presence of 3-10%

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carbon dioxide is optimal for isolation of the organism, which may be identified as a gram-negative, oxidase positive, diplococcus that ferments glucose, but not maltose, sucrose, or lactose. Treatment Ceftriaxone, cefixime, ciprofloxacin, or ofloxacin can be administered in uncomplicated cases. In vitro susceptibility should be determined in cases unresponsive to therapy, because antibiotic resistance is increasing. Penicillin should be avoided, because resistance is common. Doxycycline or azithromycin should be added for infections complicated by Chlamydia. Neisseria meningitidis N. meningitidis causes endemic or epidemic disease of worldwide prevalence. The most commonly recognized from of this disease is meningitis. N.meningitidis is an obligate parasite of humans, harbored in the nasopharynx, and transmitted by droplet nuclei from or direct intimate contact with a case or carrier. Factors contributing to sesceptibility include fatigue and exposure to inclement weather. Meningococci are destroyed rapidly in the enviroment and are highly susceptible to physical and chemical agents. This organism is a gram-negative, nonmotile, encapsulated, piliated diplococcus flattened on one site to give the appearance of a “kidney bean” or “coffee bean”. The antigenic diversity of the capsule forms the basis for classification of the meningococci into 13 serogroups. The great majority of meningicoccal disease is caused by serogroups A, B, C, W, and Y. The capsules are antiphagocytic and may facilitate meningeal invasion. Outer membrane proteins (OMPs) of the organism are also antigenically diverse, which enables serotyping meningococci within each serogroup. In addition, OMPs acts as porins The organisms are strict aerobes that grow best on chocolate agar at 35-37ºC in the presence of 3-10% carbon dioxide. Pathogenesis and Clinical Manifestations The basic pathogenesis process of primary meningococcal disease is initiated in the nasopharynx from a case or carrier. Adherence to the mucosal surface with resultant colonization is mediated by pili and possibly facilitated by secretory Ig A cleavage by Ig A1 protease. Phagocytosis is inhibited by the capsule and the organisms continue to multiply, producing nasopharyngitis. Most patients produce complementdependent bactericidal antibody, predominantly of the Ig M type, and opsonic antibody, both of which restrict the organisms to the mucosal surface of the nasopharynx and eventually cause their riddance.

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Disseminated meningococcal disease manifests most often as septicemia and meningitis. Onset may correlated with the absence of complement-dependent bactericidal antibody and opsonic antibody, the presence of serum Ig A antibody that blocks the initiation of immune lysis, or complement component deficiencies. The clinical manifestations of septicemia are the result of Lipooligosaccharide (LOS), which is an abundant component of the organism, is released upon multiplication and autolysis. LOS differs from LPS in that the former has shorter, nonrepeat, O-antigenic side chains and thus has a lower molecular weight. Meningitis is the most common complication of meningococcal septicemia. Clinical manifestations are fever, stiff neck, vomiting, severe headache, convulsions, bulging of the fontanelles, and progression to a coma within a few hours. Laboratory Diagnosis Specimens obtained depend on the disease process and mainly include nasopharynx swabs, blood culture, and cerebrospinal fluid. Gram stains of specimens may show gram-negative, intracellular and extracellular diplococci in association with PMNs. Primary isolations requires initial blood cultures and culture on either chocolate agar plates or, if a mixed flora is anticipated, Thyer-Martin medium, which is an enriched chocolate agar medium containing antibiotics to inhibit gram-positive organisms and gram-negative rods. Incubation for 48 h at 35-37ºC under aerobic conditions in the presence of 3-10% carbon dioxide is optimal for isolation of the organisms, which may be identified as gram-negative, oxidase-positive, diplococcus that ferments glucose and maltose, and agglutinates in the presence of serogroupspecific anticapsular antibody. Treatment: Early treatment with penicillin has reduced the case fatality rate in disseminated disease from 40-90% to 10-15%, but the antibiotic is ineffective in eradicating the carrier state. Chloramphenicol and ceftriaxone are effective in penicillin-allergic individuals. Polysaccharide vaccines conjugated with protein carriers offer protection for infants younger than 2 years. NOT: Common causes of bacterial meningitis in 2 to 18 years old : 1. Neisseria meningitidis 3. Literia monocytogenes 5. Streptococcus agalactiae 2. Streptococcus pneumoniae 4. Haemophilus influenzae

Escherichia coli is a major cause of meningitis in the newborn. Viral meningitis is often due to enteroviruses and sometimes herpes simplex virus.

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Moraxella Members of the genus Moraxella are nonmotile, gram-negative cocobacilli that are generally found in pairs. They are related to the neisseriae, and the two organisms can be confused on Gram stain. Moraxella are aerobic, oxidase-positive, fastidious organisms that do not ferment carbohydrates. The most important pathogen in the genus is Moraxella (formerly Branhamella) catarrhalis. This organism can cause infections of the respiratory system, middle ear, eye, CNS, and joints. These organisms can be cultured on blood or chocolate agar, and identified by a battery of biochemical tests. Acinetobacter Members of the genus Acinetobacter are nonmotile cocobacilli that are frequently confused with neisseriae in gram-stained samples. They are generally encapsulated, oxidase negative, obligately aerobic, and they do not ferment carbohydrates. Acinetobacter are widely distributed in nature and are capable of infecting virtually any body site, organ system, or tissue. ENTEROBACTERIACEAE The Enterobacteriaceae comprise a large and diverse family of gram-negative rods that are found worldwide in soil, water, and are part of the normal microbial flora of virtually all animals, including humans. Many members of the family are inhabitants of the human GI tract. Under normal circumstances, they do not cause disease in the GI tract, but they can cause disease in other organ systems. Other members of the family (e.g., Shigella, Salmonella, Yersinia pestis) are always associated with disease when isolated from human. Members of this family are moderate-sized (0.3-1.0 x 1.0-6.0 μm) gram-negative bacilli, either motile with peritrichous flagella or nonmotile, and do not form spores. All members grow aerobically and anaerobically (facultative anaerobes), with growth observed generally after 18 to 24 hours of incubation on a variety of nonselective and selective media such as Endo, Mac Conkey, SS agar. Enterobacteriaceae are usually characterized by three key properties: 1. Do not possess the cytochrome oxidase enzyme and are thus “oxidase negative”. 2. Possess enzymes that enable them to reduced nitrate to nitrite. 3. Are able to ferment glucose.

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Methods for Identification 1. Colony morphology. The red-colored colonies of lactose-fermenting organisms are readily differentiated on Mac Conkey or Endo agar from the colorless non-lactose fermenting colonies. 2. Biochemical reactions. a. Carbohydrates important for identifying Enterobacteriaceae include glucose, lactose, sucrose, and mannitol. b. Tests for the presence of enzymes, such as the “indol test” for tryptophanase, the urease test, and tests for the presence of dehydrogenase and dihydrolase enzymes. c. Methyl red, Voges Proskauer (MRVP) test for end products of glucose metabolism. d. The ability of an organism to produce hydrogen sulfide (H2S) gas and motility are also important differentiating characteristics. 3. Antigenic characterization. The serologic classification of Enterobacteriaceae is based on three major groups of antigens: a. Flagellar Antigens (H Antigens). The most external is the flagellar protein antigen. If the organism is not motile, it will not possess flagella or flagellar H antigens. b. Somatic Antigens (O Antigens). The heat-stable O polysaccharide is the major cell wall antigen and compose the outer region of lipopolysaccharides (LPSs) in the cell wall. c. Capsular Antigens (K Antigens). The capsular K antigens are either polysaccharides or proteins. The heat-labile K antigens may interfere with detection of the O antigens, necessitating removing the capsular antigen by boiling the suspension of organisms. The capsular antigen of Salmonella typhi is referred to as the Vi antigen. Pathogenesis Numerous virulence factors have been identified in the members of the family Enterobacteriaceae. Some are common to all genera, and others are unique to specific virulent strains. • Endotoxin is a virulence factor shared among all aerobic and some anaerobic gramnegative bacteria. • Capsule. Encapsulated Enterobacteriaceae are protected from phagocytosis by the hydrophilic capsular antigens, which repel the hydrophobic phagocytic cell surface. • Antigenic phase variation. Capsular and flagellar antigens can be alternately expressed or nor expressed, a feature that protects the bacteria from antibodymediated cell death.

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• Type III secretion systems. A variety of distinct bacteria (e.g., Yersinia, Salmonella, Shigella, Escherichia, Pseudomonas, Chlamydia) have a common effector system for delivering their virulence genes into targeted eukaryotic cells. This system, referred to as the type III secretion system, consists of approximately 20 proteins that facilitate secretion of bacterial virulence factor into host cells. • Resistance to serum killing • Antimicrobial resistance Escherichia coli The genus Escherichia consists of at least five species, with Escherichia coli the most frequently isolated. E.coli is present in the GI tract in large numbers and is the Enterobacteriaceae most frequently associated with bacterial sepsis, neonatal meningitis, infections of the urinary tract, and gastroenteritis in travelers to countries with poor hygiene. Diseases of the Organism Urinary Tract Infections. E. coli is responsible for more than 80% of all community-acquired urinary tract infections Neonatal Meningitis. E. coli, together with group B streptococci, are the most common causes of neonatal meningitis; 75% of these Escherichia strains possess the K1 capsular antigen. Septicemia. Typically, septicemia caused by gram-negative bacilli such as E.coli originates from infections in the urinary or gastrointestinal tract. Gastroenteritis. Strains of E. coli that cause gastroenteritis are subdivided into at least four groups: 1. Enterotoxigenic E.coli (ETEC) disease, most common in children in developing countries, is made more serious by the malnourished status of most patients. This disease is mediated by heat-labile toxin that is virtually identical to cholera toxin and a heat-stable toxin. Symptoms usually begin 24-72 h after ingestion of the organisms. Disease is characterized by watery diarrhea, cramping, and vomiting. 2. Enteroinvasive E. coli (EIEC) are able to invade and destroy the colonic epithelium, producing a disease characterized by fever and cramps, with blood and leukocytes in stool specimens (smilar to shigellosis). Disease has been associated with specific O serotypes of E.coli, which not produced enterotoxin. 3. Enteropathogenic E. coli (EPEC) are historically important agents of childhood diarrhea, particularly in impoverished countries.

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4. Enterohemorrhagic E. coli (EHEC) produce cytotoxin called verotoxin, which was so named because the toxin causes a cytopathic effect in the Vero cell line of tissue culture cells. Disease is most prevalent in the warm months of the year, with the greatest incidence in children under 5 years of age. Other infections are peritonitis and colesistitis Laboratory Diagnosis Specimens obtained depend on the disease process and include urine, bile salts, and rectal swab obtained during sigmoidoscopy. Specimens are inoculated onto blood agar and Mac Conkey agar. Colonies are usually detected after 24 h of incubation and are identified based on initial morphology and the species is verified by biochemical tests and serologing typing. E.coli are facultative anaerobes which ferment a wide range of sugars, including lactose, producing acid and gas. They are oxidase (-), Voges-Proskauer and citrate (-), but produce indol from tryptophane and are methyl red positive. They are actively motile due to the possession of flagella. Treatment. Treatment guided by in vitro susceptibility tests. Common Causes of Urinary Tract Infections 1. Escherichia coli 3. Klebsiella Salmonella Members of the genus Salmonella can cause a variety of diseases, including gastroenteritis and enteric (typhoid) fever. Salmonella classification has undergone numerous revisions; currently, all strains are grouped in a single species, Salmonella enterica. This species is further divided into over 1500 serotypes based on the cell wall (O), flagellar (H), and capsular (Vi). Originally thought to be different species, serotypes typhimurium and typhi are of particular clinical significance. Most strains of Salmonella are lactoz (-), and produce acid and gas during fermentation of glucose. They also produce H2S from sulfur-containing amino acids. Salmonella can colonize virtually all animals, including poultry, reptiles, livestock, redents, domestic animals, birds, and humans. Serotypes such as S.typhi and S.paratyphi are highly adapted to humans and do not cause disease in nonhuman hosts. Most infections results from the ingestion of contaminated food products and, in children, from direct fecal-oral spread. The most common sources of human infections are poultry, eggs, dairy products, and food prepared on contaminated work 2. Staphylococcus saprophyticus 4. Proteus and Enterobacter

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surfaces. It is now known that chicken eggs can become infected in the ovaries and carry the organism internally. Consumption of raw eggs is no longer recomended. S. typhi infections occur when food or water contaminated by infected food handlers is ingested. Also, person-to-person spread is common. Although exposure to Salmonella is frequent, a large inoculum (106-8 bacteria) is required for the development of symptomatic disease. Pathogenesis Salmonella invade epithelial cells of the small intestine. Disease may remain localized or become systemic, sometimes with disseminated foci. The organisms are facultative, intracellular parasites that survive in phagocytic cells. Development of clinical symptomes depends on : 1) infectious dose, 2) bacterial factors. Salmonella species are protected from stomach acids and the acid pH of the phagosome by an acid tolerance response (ATR) gene. Catalase and superoxide dismutase are other factors that protect the bacteria from intracellular killing. Clinical Diseases Enteritis Enteritis is the most common form of salmonellosis. Symptoms generally appear 6 to 48 h after consumption of the contaminated food or water, with the initial presentation of nausea, vomiting, and nonbloody diarrhea. Elevated temperature, abdominal cramps, myalgias, and headache are also common. Patients usually recover after 2-3 days and disease is limited to the GI tract in most cases. Septicemia All Salmonella species can cause bacteremia, although infections with S. cholerasuis, S. paratyphi, and S. typhi more commonly lead to a bacteremic phase. Enteric Fever S. typhi produce a febrile illness called typhoid fever. A mild form of this disease, referred to as paratyphoid fever, is produced by S. paratyphi A, Salmonella schottmuelleri (formerly S. paratyphi B), and Salmonella hirschfeldii (formerly S. paratyphi C). The disease is characterized by fever and, frequently abdominal symptoms. Nonspecific symptoms mau include chills, sweats, headache, anorexia, weakness, sore throat, cough, and myalgias. The incubation period various from 5 to 21 days. Patients who survive typhoid fever retain a high level of immunity to second infections. Laboratory Diagnosis In Patients with Enteritis Stool should be collected for diagnosis of gastroenteritis and inoculated onto selective media; colonies are usually detected after 24 h of incubation. Clinical

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laboratories identify the organisms to genus biochemically and may carry out limited serologic typing assays to place the organism into a group. Salmonella is a nonlactose fermenter, produced acid and gas (except S. typhi) from glucose, metabolizes citrate, and produces (except S. paratyphi A) hydrogen sulphide (H2S). Treatment: Unless patients are acutely ill with enteric fever, antibiotic treatment is not indicated. Ciprofloxacin, amoxicillin, or chloramphenicol is used to treat septicemia, although this is a last resort. In Patients with Enteric Fever During the acute and early stages of typhoid, blood, bone marrow and urine specimens are most likely to harbor the organism. (See Table below for timing of obtaining specimens for diagnosis of typhoid fever.)
Table. Collection of specimens for diagnosis of typhoid fever.

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Time Specimen First week Blood, bone marrow, bile Second and third week Blood, bone marrow, bile, stool, urine Anytime Abscess aspirate, tissue biopsy ________________________________________________________________________________

Specimens can be inoculated onto selected nonselective blood agar media; a selective medium (e.g., MacConkey agar and SS agar) is used for specimens normally contaminated with other organisms. After 24 h of incubation, the colonies are identified according to the biochemical features of the genus and serologic typing of serovars. Treatment. Chloramphenicol has been the drug of choice historically, but resistance of greater than 15% of isolates, the rare side effect of bone marrow suppression, and prolonged course required for effective treatment have all served to reduce its suitability. Ciprofloxacin and broad-spectrum cephalosporins are excellent alternatives to chloramphenicol. Prevention and Control Immunization is available against typhoid fever: 1. Killed-whole vaccine is given in two 0.5 ml doses, 4-6 weeks apart, and provides 65-75% protection for approximately 3 years. This vaccin causes a high rate of feverish reactions. 2. Vi-polysaccharide vaccin, given in a single 0.5 ml dose, provides protection equivalent to whole-cell vaccin, with fewer febrile site effects.

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3. Oral Ty21a live (attenuated) vaccine, given in three doses of one capsule each on alternative days. Shigella Shigella species cause shigellosis (bacillary dysentery)—a human intestinal disease that occurs most commonly among young children. Shigellae are nonmotile, unencapsulated, and lactose negative. Shigella species are carried primarily by humans and are not disseminated in nature; most infections are passed by the facaloral rout. Four species consisting of more than 45 O antigen-based serogroups have been described: S. dysenteriae, S. flexneri, S. boydii, and S. sonnei. S. sonnei responsible for most infections in developed countries, S. flexneri for infections in developing countries, and S. dysenteriae for the most sever infections. S.boydii is not commonly isolated. Shigellae can remain viable in food and water for as long as 6 months. Paradoxically, shigellae are very fragile in human feces collected for laboratory studies. These organisms die rapidly, usually within 30 min, in feces exposed to cold, drying conditions, or simply in feces placed in clean collection containers. Shigellae are readily killed by chlorination of water, heat, and chemical agents. Pathogenesis and Clinical manifestations The organisms survive stomach acidity, pass through the small intestine, and adhere to the mucosal epithelium of the terminal ileum and colon. Destruction of the superficial mucosal layer, and production of mucosal ulceration is occurs. An exotoxin (Shiga toxin) with enterotoxic and cytotoxic properties has been isolated from S. dysenteriae and its toxigenicity may play a secondary role in development of intestinal lesions. Shigellae cause classic bacillary dysentery, characterized by diarrhea with blood, mucos, and painful abdominal cramping. Abundant neutrophils, erythrocytes, and mucos are found in the stool. Infection is generally self-limited, although antibiotic treatment is recommended to reduce the risk of secondary spread to family members and other contacts. Laboratory Diagnosis Stool should be collected for diagnosis of dysenteria. Microbiologist will inspect the entire specimen and choose areas of blood or mucus to culture for optimal yield. Specimens are inoculated to selective media. Colonies are usually detected after 24 h of incubation. These colonies are identified based on initial morphology and verification of the species by biochemical tests and serologic typing. Shigella species are lactose (-), H2S (-), and are nonmotile.

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Treatment 1. Rehydration and restoration of electrolyte balance is essential. 2. Ampicillin, trimethoprim-sulfamethoxazole, and trimethoprim alone have been effective for treating cases and carriers. 3. For resistant strains, nalidixic acid or newer quinolones are alternative choices. Yersinia The genus Yersinia includes three species of medical importance: Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis. Yersinia pestis The etiologic agent of one of the devastating diseases in history was called Black Death (epidemic plague). During a 5-year period in the middle of the Fourteenth century, this disease claimed 25 million people—almost one fourth of the European. Y. pestis is a gram-negative, nonmotile, ellipsoidal rod that exhibits characteristic bipolar staining (safety pin appearance) with special polychromatic stains. The organism is a facultative intracellular parasite and is a facultative anaerobe that grows best aerobically on blood agar and MacConkey agar that contain bile salts at 28ºC and is nonhemolytic. Pathogenesis A common characteristic of the pathogenic Yersinia species is their ability to resist phgocytic killing. This property is mediated by the type III secretion system. Y. pestis has the following two additional plasmids that encode virulence genes: 1) fraction 1 (F1) gene, which codes for an antiphagocytic protein capsule, and 2) plasminogen activator (Pla) protease gene, which degrades complement components C3b and C5a, preventing opsonization and phagocytic migration, respectively. Pathogenesis and Clinical Manifestations of Plague Bubonic plague is initiated following the bite of an infected rat flea, usually on the lower extremitites. Within a few hours, the organisms are carried to the regional lymph nodes, usually in the groin, where rapid multiplication occurs. After 7 days of incubation period, chills, fever and an acute regional lymphadenitis referred to as a “bubo” are occured. In the absence of treatment patients will rapidly progress to bacteremia, and as many as 75% will die. Pneumonic plague is initiated by droplet nuclei from a patient with pulmonary disease. The organisms proceed directly to the lung parenchyma where they multiply

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rapidly. Death is rapid, occurring in less than 2 days in almost all patients, treated or untreated. Laboratory Diagnosis Specimens obtained depend on the disease process and include bubo aspirates, blood for smear and culture, sputum, and cerebrospinal fluid. These specimens are cultivate on blood agar and MacConkey’s agar. Overnight incubation under aerobic conditions at 28ºC is optimal for isolation of the organism. Definitive identification is made by direct immunofluorescence of the cultured organism, which is usually available only at regional Public Health Laboratories. Treatment Early treatment with streptomycin, tetracycline, or chloramphenicol is essential. Despite poor success in the treatment of patients with pulmonary, disseminated, and septicemic disease, prompt recognition and treatment of bubonic plague without respiratory complications has reduced the case fatality rate. Prevention and Control 1. Flea and rat control 2. An effective formalin-killed vaccin is available against bubonic, but not pneumonic plague and is recommended for those traveling to endemic areas, as well as for high-risk groups in the field and in the laboratory. Other Yersinia Species Y. enterocolitica and Y. pseudotuberculosis are both motile only when grown at 25ºC, do not exhibit bipolar staining, non-encapsulated, and are facultative anaerobe. In contrast to most pathogenic Enterobacteriaceae, these strains of Yersinia grow well at room temperature as well as at 37ºC. Most strains are lactose negative. Y. enterocolitica is a common cause of enterocolitis specially in colder areas of the world. The organism is harbored in the GI tract of wild and domestic animals and birds and may be found in water contaminated by animal and bird feces. Approximately most of all Y. enterocolitica infections are enterocolitis, as the name would imply. The gastroenteritis is characterized by diarrhea, fever and abdominal pain lasting for as long as 1 to 2 weeks. Y. tuberculosis can also produce an enteric disease with the same clinical features. Enteric infections with other Yersinia species are usually self-limited. If antibiotic therapy is indicated, most organisms are susceptible to broad-spectrum cephalosporins, aminoglycosides, chloramphenicol, and tetracyclines.

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OTHER ENTEROBACTERIACEAE Klebsiella Members of this genus are non-motile and have a prominent capsule that is responsible for the mucoid appearance of isolated colonies and enhanced virulence of the organism in vivo. The most commonly isolated member of this genus is Klebsiella pneumoniae, which as the name implies is associated with community acquired primary lobar pneumonia. Pneumonia caused by Klebsiella is frequently associated with necrotic destruction of alveolar spaces, with cavity formation and the production of blood-tinged sputum. Klebsiella are also associated with wound, and urinary tract infections. Proteus Proteus is non-encapsulated, motile by peritrichous flagella, gram-negative rods. Spontaneous swarming on the surface of solid media is a striking characteristic of most strains of P. vulgaris and P. mirabilis. Infections of the urinary tract caused by P.mirabilis are the most common diseases produced by this genus. Proteus strains produce large quantities of urease which splits urea into CO2 and NH3. This raises the urine pH and facilitates the formation of renal stones. The increased alkalinity of the urine is also toxic for the uroepithelium. In contrast with E. coli, the presence of pilli may actually decrease virulence of Proteus by enhancing phagocytosis of the bacilli. Enterobacter, Citrobacter, Serratia, Providencia Primary infections in immunocompetent patients are rarely caused by these organisms. They are more commonly associated with hospital-acquired infections in patients with a compromised immune system. Antibiotics therapy can be complicated, because resistance to multiple antibiotics is frequently seen. VIBRIO, AEROMONAS, and PLESIOMONAS Vibrio The genus Vibrio is composed of many species of curved bacilli, and 12 have been implicated in human infections. V. cholerae, V. parahemolyticus, and V. vulnificus are the most prominant. Vibrio cholerae Members of the genus Vibrio are short, curved, rod-shaped that differ from Enterobacteriaceae by their positive oxidase reaction and growth on alkaline but not

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acidic media. They are rapidly motile by means of a single polar flagellum. Vibrio species grows regularly on routine laboratory media (within temperature range from 18ºC to 37ºC), but can be enriched by incubation in alkaline broths, such as alkaline peptone water. Special selective media, such as thiosulfate-citrate-bile salts (TCBS) agar, are used for isolation of V. cholerae and relation vibrios from clinical specimens.. V. cholerae can grow in the absence of salts; most other species that are pathogenic in human require salt. Members of the genus are subdivided on the basis of their somatic O antigens, with more than 200 serogroups described to date. V. cholerae O1 and O139 are responsible for causing classic cholera, which can occur in epidemics or worldwide pandemics. V. cholerae O1 can be subdivided into the following two biotypes: El Tor and classical (cholerae) Vibrios can remain viable in food and water for up to 3 weeks. Shellfish harboring V. cholerae must be boiled for at least 10 min to kill the organisms. Chlorination of water and standard disinfectants are able to destroy the organism easily. Pathogenesis is the only virulence factor of V. cholerae that has been established definitively. Pathogenesis and Clinical Manifestations of Cholera Large numbers of organisms are ingested in contaminated food or water. Those that survive the gastric acidity attach, with the aid of flagella and pili, to the brush borders of epithelial cells in the small intestine. The vibrios multiply to large numbers rapidly on the mucosal surface and produce Cholera toxin (or chleragen), which binds to the ganglioside receptors on the host epithelial cells. The toxin A subunit enters into the mucosal cells, and effect a series of reactions that result in the hypersecretion of fluids and electrolytes into the intestinal lumen. After several hours to 3-days incubation, patients experience a sudden onset of severe watery diarrhea with vomiting and abdominal pain. The stool specimens are colorless and odorless, free of protein, and speckeled with mucus flecks (rice-water stool). Untreated, the death from sever dehydration causing hypovolemic shock may occur in hours to days, and the death rate may exceed fifty persent. Laboratory Diagnosis Stool is the only specimen used for diagnosis of cholera. Clear rice-water stool or stool containing mucus is highly suspicious. If transport is required, stool is place into Cary-Blair or alkaline peptone water. Stools should be inoculated into or onto standard laboratory media and TCBS selective agar. All colonies should be screened fo indophenol oxidase enzyme.

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Oxidase-positive isolates are then identified biochemically. Specific serologic typing is essential for definitive identification. Treatment 1.Fluid and electrolyte replacement is the primary treatment. 2.Tetracycline is the drug of choice. Prevention and Control 1. Emphasize proper hygiene practices, such as sewage disposal and treatment, using separate water sources for drinking and other activities. 2. Chlorine all water supplies. 3. Boil shellfish fo a minimum of 10 min before eating. Do not eat raw shellfish. 4. Vaccins are not recommended at this time because of their limited effectiveness. No countries currently require visitors to show evidence of cholera vaccination. Noncolera Vibrio Disease Like V. cholerae, the other pathogenic vibrios are free-living enviromental bacteria. Most species are saltwater inhabitants and require increased sodium chloride concentrations for growth. Gastroenteritis caused by V. parahaemolyticus can range from self-limiting diarrhea to a cholera like illness. Fatal septicemia caused by V. vulnificus, which is the most virulent of the noncholera vibrios Treatment Tetracycline, aminoglycosides, and chloramphenicol are considered effective antimicrobial agents. Aeromonas Aeromonas is a gram-negative, facultative anaerobic bacillus that morphologically resembles members of the Enterobacteriaceae. A total of 16 species of Aeromonas has been described, including 11 associated with human disease. The most important pathogens are A. Hydrophilla, A. Caviae, and A. veronii biovar sobria. The organisms are ubiquitous in fresh and brackish water. The two diseases associated with Aeromonas are gastroenteritis and wound infections.

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Plesiomonas The genus Plesiomonas consists of facultative anaerobic, gram-negative bacilli that are oxidase-positive, have multiple polar flagella, and are differentiated from Aeromonas by selected biochemical reactions. The organism is found in fresh water and estuarine waters and is acquired through contact with fresh water, the consumption of seafood, or exposure to amphibians or reptiles. The primary disease caused by P.shigelloides is a self-limited gastroenteritis with onset 48 hours after exposure to the organism. CAMPYLOBACTER AND HELICOBACTER Campylobacter Campylobacter are curved, spiral-shaped, or gullwing-shaped thin gram-negative bacilli whose natural habitat is the mammalian and avian GI tract. These organisms exhibit a rapid, darting motility mediated by one or two polar flagella. There is no demonstrable capsule. They microaerobes that grow best in 5-10% oxygen with added carbon dioxide. Special media, supplemented with blood or other nutrients and antibiotics to inhibit normal fecal flora, are used for isolation of campylobacters from feces. Because C. jejuni, the most common enteric pathogen, is able to grow at 42ºC, this temperature is used to inhibit other flora. Campylobacter species, specially C. jejuni and C. coli, caused gastroenteritis by colonizing the intestinal mucus layer and damaging host epithelial cell function, perhaps by means of an enterotoxin or a cytotoxin, but their exact pathogenic mechanism is unknown. C. fetus is associated with septicemia and is disseminated to multiple organ. Campylobacteriosis is usually acquired by ingestion of the organism on contaminated food or in contaminated water; domestic chickens and turkeys are often the source. For gastroenteritis, infection is self-limited and is managed by fluid and electrolyte replacement. Severe gastroenteritis and septicemia are treated with erythromycin (drug of choice), tetracyclines, and quinolones. Helicobacter Members of the genus Helicobacter are curved or spiral organisms. They have a rapid, corkscrew motility due to multiple polar flagella. Helicobacter pylori, the species of human significans, is microaerophilic, and produces urease. It causes acute gastritis, and duodenal and gastric ulcer. H. pylori is highly motile, and produces an abundance of urease. Helicobacters do not ferment or oxidize carbohydrates, although they can metabolize amino acids by

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fermentative pathways. Growth of H. pylori and other helicobacters requires a complex medium supplemented with blood, serum, carcoal, starch, or egg yolk, in microaerophilic conditions, and in a temperature range between 30ºC and 37ºC. Humans are the primary reservoir for H. pylori, and transmission is most likely by the fecal-oral route. Thus, it is expected that the risk of colonization will decreae with improved hygienic standards. Infections are common, particularly in people in a low socioeconomic class or in developing nations. Pathogenesis and Clinical Symptoms Multiple factors contribute to the gastric inflammation, alteration of gastric acid production, and tissue destruction that are characteristic of H. pylori disease. Initial colonization is facilitated by 1) blockage of acid production by a bacterial acid-inhibitory protein and 2) neutralization of gastric acids by the ammonia produced by bacterial urease activity. The actively motile helicobacters can then pass through the gastric mucus and adhere to the epithelial cells. Localized tissue damage is mediated by urease hypoproducts. H. pylori is protected from phagocytosis and intracellular killing by production of superoxide dismutase and catalase. The symptoms are apparently related to the inflamatory response. Patients develop pain, gas, dyspepsia, foul-smelling breath, and may experience nausea and vomiting. Further degradation of mucosal tissue leads to ulceration in the antrum of the stomach and duodenum. Laboratory Diagnosis A. Noninvasive Diagnostic Tests Serology. Infection with H. pylori stimulates a humoral immune reaction that persists as the result of continuous exposure to the bacteria. Because the antibody titers persist for many years, the test cannot be used to discriminate between past and current infection. The tests are useful, however, for documenting exposure to the bacteria. Breath Tests for Ureas. Breath tests involved administering radioactively labeled urea by mouth. If H. pylori are present in the patient’s stomach, the urease produced by the organism will split the urea to CO2 (radioactively labeled and exhaled) and NH3. B. Invasive Tests Invasive tests involved gastric biopsy specimens obtained by endoscopy. H. pylori can be detected by:

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1. A routine Gram stain with a modified counterstain using basic fuchsin insteat of safranin to enhance the faint staining of these thin bacteria. The presence of typical curved rods is confirmatory. 2. Biopsy tissue can be tested directly for the presence of urease. 3. Culturing the specimen for H. pylori is the third option. Ground tissue should be inoculated onto fresh chocolate agar or Skirrow campylobacter agar, and incubated under microaerophilic conditions (5-10% CO2) at 37ºC for up 1 week. Isolates of this organism are oxidase and catalase positive. Treatment Several treatment regimens have been effective but long term results have not been evaluated. Current therapies usually include: 1. Metronidazole + amoxicillin or Clarithromycin + amoxicillin 2. two antibiotics + bismuth subcitrate compound. Acid-reducing drugs greatly enhance the effect of antibiotic treatment. PSEUDOMONAS AND RELATED ORGANISMS Pseudomonas and related bacilli are a complex mixture of opportunistic pathogens of plants, animals, and humans. Despite the many genera, only a few are isolated commonly and are of interest to us here. Pseudomonas aeruginosa, Acinetobacter, and Moraxella catarrhalis. Pseudomonas Unlike the Enterobacteriaceae family, pseudomonads are free-living bacteria whose nutural habitat is soil and water. They are also found throughout the hospital enviroment in moist reservoirs, such as food, cut flowers, sinks, toilets, floor mops, respiratory therapy and dialysis equipment, and even disinfectant solutions. Most species are oxidase positive, which helps microbiologist to differentiate isolates from Enterobacteriaceae. These organisms rarely cause disease in immunocompetent hosts, but they are powerful opportunistic pathogens. The primary human pathogen in the genus is P. aeruginosa. Pseudomonas aeruginosa The pseudomonads are preferentially aerobic, although many of these organisms utilize proteins anaerobically. These bacteria oxidize but do not ferment carbonhydrates. Pseudomonads are important agents of nosocomial disease and disease in certain compromised hosts, including burn patients and those with cystic fibrosis.

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P. aeruginosa is resistant to many chemical disinfectants, including quaternary ammonium compounds. It can survive in moisture without nutrients for long periods of time. This organism is killed by boiling for 20 min and by autoclaving for a minimum of 15 min, as well as by chlorine-containing disinfectants. Many antibiotics, including penicillins, and cephalosporins, are ineffective against P. aeruginosa. P. aeruginosa is motile (it has polar flagella), encapsulated, and obligately aerobic (but it can use proteins to grow slowly anaerobically if necessary). Nutritional requirements are minimal, and the organism can grow on a wide variety of organic substrates. In fact, P. aeruginosa can even grow in laboratory water baths, hot tubes, wet IV tubing, and other water-containing vessels. This explains why the organism is responsible for so many nosocomial infections. P. aeruginosa produces diffusible green and blue pigments (pyoverdin and pyocyanin, respectively), and may also produce red and black pigments. Pathogenesis P. aeruginosa begins with attachment to and colonization of host tissue. Pili on the bacteria mediate adherence, and a glycocalyz capsule reduces the effectiveness of normal clearance mechanisms. Host tissue damage facilitates adherence and colonization. P. aeruginosa produces numerous toxins and extracellular products that promote local invasion and dissemination of the organism. These include: 1. Exotoxin A. Inhibitor of protein synthesis; produces tissue damages; immunosuppressive. 2. Exotoxin S. Inhibits protein synthesis; immunosuppressive 3. Proteases, including elastase, contribute to the invasiveness of the organism and to it is ability to destroy tissue. 4. Lipases and lecithinases also destroy tissue and blood cells, enhancing invasiveness and potentiating the inflammatory response. 5. Antibiotic resistance. Complicates antimicrobial therapy. Diseases of P. aeruginosa Bacteremia. Bacteremia caused by P.aeruginosa is clinically indistinguishable from other gram-negative infections, although the P.aeruginosa mortality rate is high. Endocarditis is most commonly observed in intravenous drug abusers. Respiratory tract disease. P.aeruginosa causes pneumonia in immunocompromised and hospitalized patients. External otitis and otitis media. Swimmers and divers often contract ear disease with this organism. The constant moisture and excoriated epithelium due to earplugs or cotton swabs used to dry out the external ear canals provide a suitable enviroment

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for P. aeruginosa colonization and growth. P. aeruginosa is the most common etiologic agant of middle ear disease. Burn infections. P. aeruginosa colonization of a burn wound, followed by localized vascular damage and tissue necrosis. Urinary tract disease. Pseudomonas is an important agent of UTI in hospitalized patients. Catheterized or otherwise compromised patients may develope Pseudomonas urinary tract disease. Laboratory Diagnosis Any infected tissue, blood urin, sputum, or other appropriate specimen may be collected for microbiological evaluation. Because pseudomonads have simple nutritional requirements, they grow easily on such common isolation media as blood agar or MacConkey agar. Aerobic incubation is required (unless nitrate is available), so growth in broth is generally confined to the broth-air interface. The colonial morphology (colony size, hemolytic activity, pigmentation, odor) combined with selected rapid biochemical tests (e.g., positive oxidase reaction) are used for the preliminary identification of isolates. P. aeruginosa grows rapidly and has flat colonies with a spreading border, green pigmentation (caused by production of blue pyocyanin and yellow fluorescein), and a characteristic sweet, grapelike odor. Treatment The antimicrobial therapy for Pseudomonas infections is frustrating, because 1) the bacteria are typically resistant to most antibiotics and the 2) infected patient with compromised host defenses cannot augment the antibiotic activity. Combined use of effective antibiotics (e.g., aminoglycoside and ß-lactam antibiotics) frequently required. Mono therapy is generally ineffective and can select for resistant strains. Acinetobacter Acinetobacters are recovered in nature and in the hospital. They survive on moist surfaces, including respiratory therapy equipment, and on dry surfaces such as the human skin (the latter feature is unusual for gram-negative bacilli). Acinetobacters are opportunistic pathogens that cause infections in the respiratory tract, urinary tract, and wounds; they also cause septicemia. Treatment of Acinetobacter infections is problematic because these organisms are often resistant to antibiotics. Specific therapy must be guided by in vitro susceptibility tests.

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Moraxella catarrhalis M. catarrhalis is a common cause of bronchitis and bronchopneumonia (in elderly patients), sinusitis, and otitis The latter two infections occur most commonly in previously healthy people. Most isolates produce ß-lactamase and are resistant to penicillin; however, these bacteria are uniformly susceptible to most other antibiotics. BORDETELLA, FRANCISELLA, AND BRUCELLA Bordetella Bordetella organisms are very small (0.2 to 1.0 µm), strictly aerobic, nonfermentative, gram-negative coccobacilli. Seven species are currently recognized, with three species responsible for human disease: Bordetella pertussis, the agent responsible for pertussis or whooping cough; Bordetella parapertussis, responsible for milder form of pertussis; and Bordetella bronchiseptica, responsible for respiratory disease in dogs, swine, laboratory animals, and occationally pertussis-like symptoms in humans. Bordetella pertussis B. pertussis organisms do not grow on common laboratory media; Bordet-Gengou medium, formulate with potatoes, glycerol, and added sheep blood, has been traditionally the medium of choice. These organisms are nonmotile and oxidize amino acids but do not ferment carbohydrates. The other Bordetella species are less fastidious and can grow on blood and Mac Conkey agars. Pathogenesis Infection with B. pertussis and the development of whooping cough require exposure to the organism, bacterial attachment to the ciliated epithelial cells of the respiratory tract, proliferation of the bacteria, and production of localized tissue damage and systemic toxicity. Attachment to cilliated epithelial cells is mediated primarily by two bacterial adhesins: filamentous hemaagglutinin and pertussis toxin. Two other adhesins have been identified in B. pertussis: pili and pertactin, the latter a protein on the surface of the bacteria. The intracellular survival of B. pertussis protects the bacteria from humoral antibodies and may permit persistent carriage. Pertussis toxin is a classic A-B toxin consisting of a toxic subunit (S1) and five binding subunits (S2 to S5). The S2 subunit binds to ciliated respiratory cells. The S3

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subunit binds to receptors on phagocytic cells. Two S4 subunits are present in each toxin molecule. B. pertussis produces several toxins that mediate the localized and systemic manifestations of disease. . S1 portion of pertussis toxin . hemolysin toxin . dermonecrotic toxin . tracheal toxin . lipoplysaccharide Clinical Syndromes of Pertussis of Whooping Couph B. pertussis is a human disease with no other recognized animal or enviromental reservoir. The disease is still endemic worldwide and affects more then 60 million people annually. Infection is initiated by inhalation of infectious aerosol droplets and attachment and proliferation of the bacteria on ciliated epithelial cells. After a 7-10 day incubation period the patient will experience the first of three stages. The catarrhal stage resembles a common cold, with serous rhinorrhea, sneezing, malaise, anorexia, and a low grade fever. Patients in the catarrhal stage pose the highest risk to their contacts After 1-2 weeks the paroxysmal stage begins, with classic whooping cough paroxysms. The paroxysms are characterized by a series of repetitive coughs fllowed by an inspiratory whoop.The paroxysms are frequently terminated with vomiting and exhaustion. After 2-4 weeks the disease inters the convalescent stage when the paroxysms diminish in number and severity, but secondary complications can occur. Laboratory Diagnosis Secretions collected on nasopharyngeal swabs consisting of calcium alginate or Dacron on wire handles are the best specimen. The swab must be inoculated immediately onto media at the patient’s bedside or transported in a moist, protective medium to the laboratory for immediate culture. Direct fluorescent antibody stains for the organism are the fastest diagnostic tool. Although method is only 60% sensitive, it is very specific. Specimens are inoculated onto special media, such as Regan-Lowe agar and freshly made Bordet-Gengou agar. Cultures are incubated in a humidified atmosphere without added carbon dioxide for a minimum of 10 days. Once the characteristic colonies (colonies are shiny and have a characteristic metallic or pearly sheen) appear, they can be identified using fluorescent antibody stain. B. pertussis is oxidase positive and microscopic examination should reveal very tiny gram-negative coccobacilli.

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Treatment Treatment with macrolide (i.e., erythromycin, azithromycin) is effective in eradicating organisms and reducing length of infectious stage. Treatment does not allevviate symptoms Prevention and Control Vaccination is the key to control; all susceptible populations should be immunized. Immunization of unvaccinated children older than 7 years of age or adults is not recommended at this time because of the decreased severity of disease among the age groups. Immunization with the current vaccine of whole killed cells of B. pertussis in a trivalent vaccin containing diphtheria and tetanus toxoids confers a high degree of protection. Francisella tularensis Francisella tularensis is the causative agent of tularemia (also called rabbit fever) in animals and humans. Tularemia was first isolated in 1912 from “plaguelike disease” among ground squirrels in Tulare County, CA. The organisms are harbored in the blood and tissues of wild and domestic animals, including rodents. In the United States, the chief reservoir hosts are wild rabbits and ground squirrels. Transmission is through the skin and/or conjunctiva from handling infected animals, through the skin from the bite of infected blood-sucking deer flies and wood ticks, through the GI tract from the ingestion of improperly cooked meat or contaminated water, and through the respiratory tract by aerosol inhalation. F. tularensis is a gram-negative, nonmotile, coccobacillary or pleomorphic rod. Virulent organisms appear to be encapsulated and are facultative intracellular parasites. Thses fastidious organisms are strict aerobes that grow best on bloodglocose-cysteine agar. Organisms entering through the unbroken or abraded skin, GI tract, or conjunctiva, establish residence locally and in the regional lymph nodes. They enter macrophages and mononuclear phagocytes with intracellular multiplication resulting in ulcer formation, and regional lymphadenopathy accompained by fever, nausea, vomiting, and/or abdominal pain. BRUCELLA Brucella is the causative agent of brucellosis (undulant fever, Malta fever). This disease is an acute or chronic recurrent disease transmissible from animals to humans. Although a wide range of animal reservior hosts exist for each of the four species capable of producing human disease, they exhibit significant animal-host specificity.

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The major reservoir hosts are cattle for Brucella abortus, goats and sheep for Brucella melitensis, swine for Brucella suis, and dogs (particularly beagles) for Brucella canis. Upon contact with the organisms, pregnant animals develop either an asymptomatic infection or uterine and mammary glad disease that culminates in abortion. Erythritol, an alcohol present in the placenta and fetal fluids of animals but not humans, srves both as a growth factor for virulent brucellae and enhances intracellular phagocytic growth, thereby accounting for the occurrence of fetal predilection and abortion only in animals. The animals shed the organisms in their milk for weeks or months during the carrier state and after recovery. Transmission to humans is from the ingestion of contaminated raw milk or dairy products in most parts of the world, and from the handling of infected animals by high risk individuals, such as veterinarians, butchers, meat packers, and farmers. Properties of the Organism The Brucella are gram-negative, nonmotile, encapsulated coccobacilli and are facultative intracellular parasites. They are strict aerobes, but B.abortus requires 510% carbon dioxide for growth. Optimal growth occurs on serum or blood enriched complex media. The role of structural or extracellular virulence factors has not been well defined, despite the presence of LPS and the identification of outer membrane proteins. Clinical Manifestations of Brucellosis Brucella gain entry through the broken or unbroken skin, by ingestion, or through the conjunctiva. After a variable incubation period of a few days to several weeks or months, the acute phase is ushered in and is characterized by an undulant fever, chills, sweating, and often times fatigue. Regional lymphadenopathy, hepatosplenomegaly, septicemia, osteomyelitis and an acute arthritis sometimes occur. Laboratory Diagnosis Specimens for laboratory diagnosis depend on the disease process and include lymph node and bone marrow aspirates and blood for culture and serology. Brucella are slow-growing, fastidious organisms on primary isolation. Primary isolation may be enhanced by using a biphasic blood culture system containing trypticase soy and/or Brucella agar enriched agar incubated aerobically under 5% carbon dioxide for minimum of 4 weeks or by using one of the newer automated blood culture systems. Organisms are identified as Brucella on the basis of biochemical reactions and are speciated on the basis of hydrogen sulfide production;

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growth in the presence of the dyes basic fuchsin and thionin; and agglutinin absorption assay. Treatment Prolonged treatment with combined doxycycline and rifampin or doxycycline and gentamicin, or with streptomycin, or gentamicin alone depending on the extent of the disease and the age of the patient, is essential due to the intracellular location of the organisms. PASTEURELLACEAE The family Pasteurellaceae consists of three genera: Haemophilus, Actinobacillus, and Pasteurella. Haemophilus the most common human pathogen. Members of the family are small, gram-negative bacilli, nonsporeforming, nonmotile, and aerobic or facultative anaerobes. HAEMOPHILUS Haemophilus are small, sometimes pleomorphic, gram-negative bacilli that are obligate parasites on the mucous membranes of humans and animal species. H.influenzae, an obligate human pathogen, is the Haemophilus species most commonly associated with disease. Other pathogenic Haemophilus include H.ducreyi, H.parainfluenzae, H.aphrophilus, H.haemolyticus. Microbial Physiology and Structure Most species of Haemophilus (from the Greek words for “blood-loving”) require supplementation of media with growth-stimulating factors, specifically X factor (hemin) and/or V factor (nicotinamide adenine dinucleotide, NAD). Although both factors are present in blood-enriched media, the blood must be gently heated to release the factors and destroy inhibitors of V factor. For this reason heated blood agar (i.e., chocolate agar) is used for the in vitro isolation of Haemophilus spp., which are differentiated by their requirements for X and V factors. X factor is a heme portion of hemoglobin; required for synthesis of essential enzymes. V factor is a co-enzyme. Present in erythrocytes but unavailable for some erythrocyte types, such as sheep RBCs, unless the cell membrane is lysed. Intact horse and rabbit RBCs provide V factor. The cell wall structure of Haemophilus is typical of other gram-negative bacilli. Lipopolysaccharide endotoxic activity is present, and the surface of the cell is covered with strain-and species-specific protein antigens. The surfaces of many but not all strains of H.influenzae are covered with polysaccharide capsule, with six

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antigenix serotypes (a through f). Serotype b is associated with invasive disease in greater than 95% of all Haemophilus infections. Other species of Haemophilus do not have capsules. Capsule is the primary virulence factor of the organism. No extracellular products are known to act as virulence factor. Pathogenesis and Clinical Manifestations of H.influenzae Infections H. influenzae, colonize the upper respiratory tract in virtually all individuals with in the first few months of life. These organisms can spread locally and cause disease in the ears (otitis media), sinuses (sinusitis), and lower respiratory tract, but disseminated disease is relatively uncommon. In contrast, encapsulated H. influenzae (particularly serogroup b) is infrequently present in the upper respiratory tract but is the most common cause of epiglottitis and pediatric meningitis. The organism is able to penetrate through the nasopharyngeal submucosa and enter the blood stream. In the absence of specific opsonic antibodies directed against the polysaccharide capsule, high-grade bacteremia can develop, with dissemination to the meninges or other distal foci. The clinical syndromes that accompany infections of H. influenzae are : Meningitis. H.influenzae type b is the most common cause of pediatric meningitis, which is indistinguishable clinically from that of the other acute bacterial maningitides. Clinical signs of the disease include; fever, vomiting, lethargy, nuchal rigidity. Epiglottitis is a life-threatening type of cellulitis that starts at the epiglottis and spreads into surrounding tissues. The child will have pharyngitis, fever and breathing difficulties, which can rapidly progress to complete obstruction of the airway and death. Arthritis. Infection of single large joints, secondary to bacteremic spread of H. influenzae type b, is the most common form of arthritis seen in children less than 2 years of age. Conjunctivitis and Brazilian Purpuric Fever. Epidemic, as well as endemic, conjunctivitis can be caused by H. influenzae biotype aegypticus. Aspecific strain of this organism has also been associated with Brazilian purpuric fever, a fulminant pediatric disease characterized by fever, vomiting and abdominal pain, and then rapid development of petechiae, purpura, shock and death. Otitis, Sinusitis, and Lower Respiratory Tract Disease. Laboratory Diagnosis Specimen Collection. In order to diagnose meningitis, cerebrospinal fluid (CSF) and blood cultures should be obtained. In pyogenic infections, purulent exudate is collected by aspiration or on swab. In nasocomial pneumonia, bronchoalveolar lavage is the specimen of choice for diagnosis

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In cases of epiglottitis, direct culture collection from the affected throat is not recommended, since the trauma may cause airway obstruction. Blood cultures are often positive. Microscopy. Gram stain is usually sensitive. Primary Isolation and Identification. Isolation of H. influenzae from clinical specimens is relatively easy if media supplemented with X and V factors are inoculated. Chocolate agar is used in most laboratories, the bacteria appear as 1 to 2 mm smooth opaque colonies after 24 hours of incubation. Growth on unheated blood agar plates can also be detected surrounding colonies of S. aureus [(satellite phenomenon) (süt anne etkisi)]. The staphylococci provide required growth factors by lysing the erythrocytes in the medium and secreting V factor. The size of the colonies of H. influenzae is much smaller than on chocolate agar because the V factor inhibitors are not inactivated. Identification of isolates is based on requirement for X and V factors, enzymatic and biochemical test results, hemolytic activity on rabbit blood. Treatment The current treatment recommendations for meningitis are either a combination of ampicillin and chloramphenicol (classic therapy) or cefotaxime (new therapy). Alternative therapies include cefuroxime, trimethoprim-sulfomethoxazole, and doxycycline. Prevention and Control H. influenzae type b polyribose-ribitol phosphate (PRP) capsular conjugate vaccines are available for children age 2 months and older. Haemophilus ducreyi H. ducreyi the etiologic agent for chancroid, or soft chancre, was first described by Ducrey. This sexually transmitted disease is distinguished from syphilis by the presence of a painful, nonindurated genital ulcer with well-defined margins. The disease has worldwide distribution, and is generally present in poor socioeconomic areas. Laboratory diagnosis is difficult because the organism sometimes fails to grow in vitro and, when it does grow, incubation for 3 or more days is required. In general, diagnosis is based on the clinical presentation of the patient and a history of chancroid in the community. Treatment with ceftriaxone, erythromycin, ciprofloxacin, amoxicillin+clavulanic acid has been effective.

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Pasteurella Pasteurella are small facultatively anaerobic, fermentative coccobacilli commonly found as commensals in the oropharynx of healthy animals. Most human infections result from animal contact (e.g., animal bites, scratches, shared food). P. multocida and, less commonly, P. canis are human pathogens. The following three general forms of disease are reported: 1. A localized cellulitis and lymphadenitis that occur after an animal bite or scratch. 2. Chronic respiratory disease 3. Systemic infection in immunocompromised patients P. multocida grow well on blood and chocolate agars but poorly on MacConkey agar and other media typically selective for gram-negative bacilli. It is susceptible to a variety of antibiotics. Penicillin is the antibiotic of choice. Actinobacillus Actinobacillus species are small, facultatively anaerobic, gram-negative bacilli that grow slowely (generally requiring 2 to 3 days of incubation). Actinobacillus actinomycetemcomitans is the most important human pathogen, and the other species are rarely encountered. Members of the genus Actinobacillus colonize the oropharynx of humans and animals and are responsible for periodontitis, endocarditis, bite wound infections, and opportunistic infections. A. actinomycetemcomitans is a relatively uncommon cause of subacute bacterial endocarditis. In patients in whom this disease occurs, however, typically there is preexisting valvular heart disease and evidence of oral disease (e.g., periodontitis, oral abscess, poor oral hygiene). The organism spreads from the oropharynx through the blood stream and adheres to the damaged heart vulve. An interesting characteristic of Actinobacillus noted in vitro is that the bacteria are sticky, adhering to the surfaces of blood culture bottles and agar plates in much the same way as they adhere to damaged heart vulves. Serious infections with Actinobacillus species are treated with ampicillin, either alone or in combination with aminoglycoside. Strains resistant to ampicillin can be treated with cephalosporins or fluroquinolones. MISCELLANEOUS GRAM-NEGATIVE BACILLI Legionella In the summer of 1976, public attention was focused on an outbreak of severe pneumonia that caused many deaths in members of the American Legion convention in Philadelphia. After months intensive investigations, a previously onknown gram-

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negative bacillus was isolated. Subsequent studies found this organism, named Legionella pneumophilia, to be cause of multiple epidemic and sporadic infections. Legionella genus consists 39 species and more than 60 serogroups. L. pneumophilia is the cause of 85% of all infections; serotypes 1 and 6 are most commonly isolated. Members of the genus Legionella are slender, pleomorphic, motile, gram-negative bacilli. The organisms are nutritionally fastidious; their growth is inhanced with iron salts and depends on the supplementation of media with L-cysteine. Growth of these bacteria on supplemented media but not on conventional blood agar media has been used as the basis for the preliminary identification of clinical isolates. The organisms are nonfermentative and derive energy from the metabolism of amino acids. Pathogenesis Respiratory tract disease caused by Legionella species develops in susceptible people who inhale infectious aerosols. Lagionella are facultative intracellular parasites that can multiply in alveolar macrophages and monocytes. The bacteria are not killed in the phagocytic cells through exposure to toxic superoxide and hydrogen peroxide, because phagolysosome fusion is inhibited. The bacilli proliferate in their intracellular vacuole and produce proteolytic enzymes, phosphatase, lipase, and nuclease, which eventually kill the host cell when the vacuole is lysed. Epidemiology Sporadic and epidemic legionellosis has a worldwide distribution. The bacteria commonly present in natural bodies of water, such as lakes and streams, as well as in air conditioning cooling towers and in water systems (e.g., showers, hot tubs). The organism can survive in moist enviroments for a long time, at relatively high temperatures, and in the presence of disinfectants such as chlorine. One reason for this is that the bacteria can parasitize amebae in the water and replicate in this protected enviroment (like their replication in human macrophages). Clinical Diseases Pontiac Fever. L. pneumophilia was responsible for causing a self-limited, febrile illness in people working in the Pontiac. The disease was characterized by fever, chills, myalgia, malaise, and headache but not clinical evidence of pneumonia. Legionnaires’ Disease (legionellosis) is characteristically more severe and causes considerable morbidity, leading to death unless therapy is initiated promptly. After an incubation period of 2 to 10 days, systemic signs appear abruptly (e.g., fever, and chills, a dry nonproductive cough, headache). Multiorgan disease involving the gastrointestinal tract, central nervous system, liver, and kidneys is common.

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Laboratory Diagnosis Microscopy. Legionellae in clinical specimens stain poorly with Gram stain. Nonspecific staining methods, such as those using Dieterle’s silver or Gimenez’s stain, can be used to visualize the organisms but are of little value if the specimens are contaminated with normal oral bacteria. Culture. The medium most commonly used for the isolation of legionellae is buffered charcoal-yeast extract (BCYE) agar, although other supplemented media have also been used. Legionellae grow in air or 3% to 5% carbon dioxide at 37ºC after 3 to 5 days. Serology. Legionellosis is commonly diagnosed with the use of the indirect fluorescent antibody test to measure a serologic response to infection. Treatment, Control, and Prevention Severe disease treated with azithromycin or levofloxacin; less severe disease can be treated with erythromycin or tetracycline. For enviromental sources associated with disease, treat with hyperchlorination, superheating, or copper-silver ionization. Eikenella These organisms colonize the human oropharynx and, in the setting of preexisting heart disease, can cause subacute bacterial endocarditis. E. corrodens is a moderate-sized, non-motile, non-spore-forming, facultatively anaerobic gram-negative bacilli. The organism is fastidious, requires 5% to 10% carbon dioxide to grow. Small colonies are observed after 48ºC hours of incubation on blood or chocolate agar. Pitting in agar is a usful differential characteristic, but fewer than half of all isolates exhibit pitting. The organism also produces a characteristic bleachlike odor. E. corrodens is most commonly isolate in the settings of a human bite wound or fistfight injury and in the periodontal pockets. Capnocytophaga Members of the genus Capnocytophaga are filamentous gram-negative bacilli capable of aerobic and anaerobic growth in the presence of carbon dioxide. These organisms colonize the human oropharynx and are associated with periodontitis, bacteremia, and, rarely, endocarditis.

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ANAEROBIC GRAM-POSITIVE COCCI and NON-SPOREFORMİNG BACILLI Anaerobic Gram-positive Cocci Peptostreptococcus These gram-positive cocci normally colonize the oral cavity, gastrointestinal tract, genitourinary tract, and skin. They produce infections when they spread from these sites to normally sterile sites. For example, sinusitis, pleuropulmonary infections, intra-abdominal infections, endometritis, pelvic abscesses and other infections. Members of the genus Peptostreptococcus are usually susceptible to penicillin, metronidazole, imipenem, and chloramphenicol. Anaerobic, Non-Spore-Forming, Gram-Positive Bacilli Actinomyces Actinomyces organisms are facultatively anaerobic or strictly anaerobic, grampositive bacilli. They are not acid-fast (in contrast to the morphologically similar Nocardia species), they grow slowly in culture, and they tend to produce chronic, slowly developing infections. They typically form delicate filamentous forms or hyphae, smilar to those of fungi, when detected in clinical specimens or isolated in culture. Numerous species have been described; Actinomyces israelii, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces odontolyticus, and Actinomyces viscosus are responsible for most human infections. Only, A. meyeri is a strict anaerobe. The other species grow best in anaerobic conditions but can grow aerobically. Pathogenesis and Clinical Diseases Actinomycosis Actinomycosis is a chronic, suppurative, granulomatous disease caused most commonly by Actinomyces israelii. The organisms are normal inhabitants of the oral cavity predominating in and around the teeth and gun margins. Most cases of actinomycosis are the cervicofacial type. The disease is the result of endogenous activation and is initiated by trauma, such as tooth extraction or pyogenic bacterial disease of the oral cavity. Human-human transmission does not occur. From the mouth, the organisms may spread to the face and neck to produce cervicofacial disease, be aspirated into the lung with resultant thoracic disease, or be swallowed to produce abdominal disease. Multiple draining abscesses occur.

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Laboratory Diagnosis The laboratory diagnosis is made by demonstrating “sulfur granules” in pus, sputum, or tissue biopsy material and by primary isolation and identification of the organism by strict anaerobic culture and biochemical reactions. Sulfur granules are lobulated bodies composed of delicate tangled masses of grampositive filaments, the ends of which are club shaped. Because the organisms are concentrated in sulfer granules and are spares in involved tissues, a large amount of tissue or pus should be collected. If sulfur granules are detected in a sinus tract or in tissue, the granule should be crushed between two class slides, stained, and examined microscopically. Thin, gram-positive, branching bacilli can be seen along the periphery of the granules. Actinomyces are fastidious and grow slowly under anaerobic conditions; it can take 2 weeks or more for the organisms to be isolated. Colonies appear white and have a domed surface that can become irregular after incubation for a week or more, resembling the top of a molar. The individual species of actinomyces can be differentiated by biochemical tests; however, this process can be time-consuming. Treatment Treatment of actinomycosis involves the combination of surgical debridement of the involved tissues and the prolonged administration of antibiotics. Actinomyces are uniformly susceptible to penicillin as well as to erythromycin and clindamycin. Propionibacterium Propionibacterium is a genus of anaerobic or microaerophilic rods of diphtheroidlike morphology. They are common inhabitants of normal skin, and, in rare instances, have been reported as causes of endocarditis. P.acnes, often a strict anaerobe, has been implicated as a contributing cause of acne. Lactobacillus Various species of Lactobacillus are part of the commensal flora of human mucous membranes. These organisms are usually long and sleneder with squared ends, and frequently occur in chains. They produce quantities of lactic acid during fermentation, and have been thought to assist in maintaining the acid pH of normal mucous epithelia. On the other hand, acid production by oral lactobacilli may play a role in the production of dental caries.

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CLOSTRIDIUM The genus Clostridium consists of gram-positive, spore-forming, anaerobic rods. Pathogens of the genus produce disease by virtue of their ability to elicit powerful exotoxins responsible for the clinical manifestations and, additionally, by their invasiveness in clostridial myonecrosis and anaerobic cellulitis. Clostridial spores are widely distributed in nature, particularly the soil. The organisms are also present in the GI tract of animals and humans. Properties of the Organisms Resistance to enviromental, physical, and chemical agents. Clostridial spores, like those of the members of the genus Bacillus, survive for years in the soil and are highly resistance to physical and chemical agents. The organisms are gram-positive, spore-forming, motile or nonmotile rods. A capsule, which is not associated with virulence, is produced by only one of the species capable of causing disease, Clostridium perfringens. All pathogens in the genus, with wxception of Clostridium tetani, produced oval, subterminal spores. C. tetani produces round, terminal spores that give the organism a characteristic “drumstic” or “tennis racket” appearance. The organisms are strict anaerobes that multiply only in the absence of oxygen or in an enviroment of low oxidation-reduction potential. Clostridia grow best at 37ºC on blood agar under anaerobic conditions. Several species are beta-hemolytic. On solid media, most pathogens produce colonies thet appear as a compact center with irregular edges surrounded by a loose meshwork of filaments spread delicately over the surface like a “spring of maidenhair fren”; some pathogens like C. perfringens produce colonies with entire edges. Those pathogenic species that ferment carbonhydrates with the production of acid and gas are referred to as saccharolytic. This fermentative characteristic is an important virulence factor operative in the pathogenesis of clostridial myonecrosis. Pathogenesis and Clinical Diseases TETANUS Tetanus, the etiologic agent of which is C.tetani, is a disease preventable by vaccination, yet thousands cases are reported each year in the more countries throughout the world as a result of incomplete or no vaccination. Transmission appears to be exclusively by the exogenus route. Spores are introduced from soil-contaminated objects or surgical instruments into major injuries, such as deep wounds and compound fractures. Spores may also be introduced as a result of minor injuries produced, for example, by contaminated rose thorns and slivers. Cases occur mostly among farmers and gardners, drug addicts who use

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contaminated needles, surgical patients treated with contaminated dressings, and newborns infected through a contaminated umbilicus at birth. Although the vegetative cells of C. tetani die rapidly when exposed to oxygen, spore formation allows the organism to survive in the most adverse conditions. C. tetani produces two toxins: Tetanospasmin or neurotoxin, one of the most powerful exotoxins known, is a heat-labile protein produced by vegetative cells and released during autolysis. It is transported from an infected locus by retrograde neuronal flow to the central nervous system (CNS). The toxin acts by blocking presynaptic inhibition that results in excitation of the cerntral nervous system. Tetanolysin is a heat-stable hemolysin of unknown significance. The incubation period for tetanus is variable, ranging from 2 to 50 days. The length of the incubation period is directly related to the distance of the primary wound infection from the central nervous system. The earliest sign is stiffiness of the jaw due to spasmic contraction masseter muscles (trismus). Later signs include sustained contraction of the facial muscles (risus sardonicus) with extension of the spasmic contractions to the back (opisthotonus), neck, and respiratory muscles. As the disease progresses, intermittent convulsive seizures are brought on by the slightest stimulus. Death is due to respiratory complications, usually pneumonia or asphyxiation. Laboratory Diagnosis of Tetanus Diagnosis must be prompt and thus presumptive based upon history of injury and clinical manifestations. Suspicion should be aroused if stiffiness of the jaw is present. Primary isolation and identification. If a wound is present, initial anaerobic cultivation is required on blood agar and in liquid media such as thioglycollate and cooked meat broth, in which the oxidation-reduction potential is low. Definitive identification of the organism is based upon the demonstration of a gram-positive rod with a round and terminal spore that gives the organism a “drumstick” appearance and by it is production of tetanospasmin as determined by a mouse virulence assay. The wound must also be cultured aerobically for secondary pyogenic invaders such as staphylococci and streptococci. Treatment If a wound is present, debridement is essential. Human tetanus immune globulin must be administered as soon as possible after onset to prevent additional toxin from fixing to nerve cells.Although antibiotics have no effect upon the toxemic disease process, penicillin or broad spectrum antibiotics are effective in inhibiting the growth of C. tetani and secondary pyogenic invaders

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Prevention and Control Active immunization is effective in the prevention of tetanus. Tetanus toxoid, which is formalin-inactivated toxin, is administered to normal infants and children together with diphtheria toxoid and killed Bordetella pertussis (diphtheria, tetanus, pertusis; DTP vaccine) at 2-4, and 6 months with booster doses at 15 months and 4-6 years. MYONECROSIS (GAS GANGRENE), ANAEROBIC CELLULITIS, SEPTICEMIA, and ORGAN ABSCESSES These disease are caused by several species of clostridia, but the most common etiologic agent is C. perfringens, which is also capable of producing food poisoning. Although there are five types of C. perfringens based upon exotoxin production, the great majority of human disease is caused by the alpha toxin producing type A. Clostridial myonecrosis and anaerobic cellulitis are the result of transmission by means of massive injury with soil contaminated objects or by contaminated surgical instruments. Food poisoning is the result of transmission by the ingestion of improperly cooked food contaminated with C. perfringens vegetative cells and left at room temperature. The pathogenic potential of C. perfringens is attributed primarily to the 12 toxins and enzymes. Some of these are listed below: Lecithinase C (alpha toxin) causing cell membrane disruption and lysis, tissue necrosis, and edema. Beta, epsilon, iota, and theta toxins are associated with increased capillary permeability Collagenase (kappa toxin) digests and liquefies the collagen in connective tissue. Hyaluronidase (mu toxin) hydrolyzes the hyaluronic acid of connective tissue ground substance. DNase depolymerizes DNA, resulting in tissue liquefaction. Enterotoxin is a heat-labile protein responsible for diarrheal food poisoning by inhibiting fluid absorption from the gut. Myonecrosis (Gas Gangrane) This life-threatening disease illustrate the full virulence potential of clostridia. The onset of disease, characterized by intense pain, generally begins within 1 week after clostridia are introduced into tissue by trauma or surgery. The progression from the time of onset through extensive muscle necrosis, shock, renal failure, and death is rapid, frequently occuring in less than 2 days. Macroscopic examination of muscle reveals devitalized necrotic tissue, with the presence of gas due to the metabolic activity of the rapidly dividing bacteria (hence, the name gas gangrene).

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Anaerobic Cellulitis is a disease whereby the organisms spread through fascial planes between muscle and do not invade muscle, causing sappuration and gas formation. Gas is prominent and tissue necrosis extensive, and the prognosis is better than for myonecrosis. Food Poisoning. Food poisoning is the result of ingesting improperly cooked meat, fish, or poultry containing greater than or equal to 108 C.perfringens vegetative cells. As the organisms sporulate, enterotoxin is produced in the GI tract. After an incubation period of 6-18 h., diarrhea occurs as a result of the inhibition of fluid absorption in the gut by the enterotoxin. Fever and vomiting are absent and the disease is self-limiting. Laboratory Diagnosis As for tetanus, the diagnosis of clostridial myonecrosis and anaerobic cellulitis must be immediate and thus presumptive based upon history of injury and clinical manifestations. The potential necessity for amputation points to the importance of a rapid and accurate diagnosis. Wounds must be cultured anaerobically for clostridia to confirm the diagnosis and aerobically for the presence of secondary pyogenic invaders, such as staphylococci and streptococci, that may facilitate extended growth of the organisms by further damaging tissue. Direct Gram stain of the leading edge of a necrotic process or exudate will usually reveal the typical boxcar-shaped C. perfringens; leukocytes are often absent or sparse due to destruction by clostridial toxins. Specimens from patients with the above clinical syndromes require initial anaerobic cultivation on bllod agar and in liquid media, such as thioglycolate and cooked meat broth. Identification of the organism is based upon the demonstration of a gram-positive, boxcar-shaped rod with no visible spores, and production of a “double zone” of hemolysis on blood agar, “stormy fermentation” in iron milk medium, and lecithinase on egg yolk medium. Diagnosis of food poisoning is based upon history and clinical manifestations. Treatment For patients with clostridial myonecrosis and anaerobic cellulitis, prompt and extensive debridement of the wound is essential. Penicillin or broad spectrum antibiotics are effective against C. perfringens and secondary pyogenic invaders. ANTIBIOTIC-ASSOCIATED GSTROINTESTINAL DISEASES Clostridium difficile is part of the normal intestinal flora in a small number of healthy people and hospitalized patients. The disease develops in people taking antibiotics because the agents alter the normal enteric flora, either permitting the overgrowth of these relatively resistant organisms or making the patient more

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susceptible to the exogenous acquisition of C. difficile. Vegetative cells multiply in the GI tract and release toxins A and B, which act in concert to cause negrosis of the mucosal surface of the colon. The host responds with a fibrinous exudate which, together with the area of necrosis, results in the formation of a pseudomembrane. Colitis without a pseudomembrane with resultant diarrhea often occurs. BOTULISM The two types of botulism, food-borne and infant, are caused by Clostridium botulinum and occur sporadically. In food-borne botulism, spores gain access to foods such as vegetables and some fruits grown in close association with the soil and maintained at a slightly alkaline or neutral pH during the canning process. The suitable pH, anaerobiasis, and faulty canning process, provide the favorable conditions necessary for spore germination and multiplying vegetative cells that produce neurotoxin intracellularly and release it into the food during cell autolysis. Transmission then occurs when individuals taste and ingest improperly prepared and improperly cooked canned foods containing preformed neurotoxin. In infant botulism, infants aged 2 days to 6 months ingest food containing spores that germinate in the GI tract. Vegetative cells then produce neurotoxin intracellularly and release it into the gut. The foods responsible for transmission are unknown, but contaminated honey has been suspected. Seven antigenically distinct botulinum toxins (A to G) have been described; human disease is associated with types A, B, E, and F. Only one toxin is produced by most individual isolates. Like tetanus toxin, C. botulinum toxin have neurotoxic activity. It is the most powerful exotoxin known, is a protein that is inactivated in food by boiling for 20 min. The toxin acts by suppressing or blocking the presyneptic release of acetylcholin responsible for muscle tone. Clinical Manifestations In the food-borne type of botulism, pre-formed neurotoxin is ingested with the food. The toxin is absorbed by the mucosa of the stomach and upper GI tract, take up by the blood stream, and selectively absorbed by peripheral neurons at the myoneural junctions, where it cause paralysis mostly of the pharyngeal, ocular, and respiratory muscles. After an incubation period of 12-46 h, the most common clinical manifestations are diplopia (double vision), dysphagia ( difficulty in swallowing), and dysphonia (thickness of speech). Death occurs in 12-24 h due to respiratory paralysis. In the infant type of botulism, following ingestion of spore-contaminated food, germination occurs in the GI tract. Vegetative cells multiply and produce neurotoxin intracellularly, which is released into the gut during autolysis. After an incubation

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period of 8-22 days, ptosis (droopy eyelids), dyspgagia, and fever result. The infants appears “floppy” due to loss of neck and limb muscle strength. Laboratory Diagnosis The rapidity with which death can occur with the food-borne type of botulism necessitates an immediate diagnosis based upon history (if possible) and clinical manifestations. For epidemiological purposes, serum specimens and food are analyzed for neurotoxin by mouse virulence assays. The infant type is usually diagnosed on clinical grounds, but may be confirmed by demonstrating the neurotoxin in the stool. Treatment Food-borne botulism. Immediate IV use of equine polyvalent antitoxin against serotypes A, B, and E. Infant botulism requires only supportive therapy. MYCOBACTERIUM The genus Mycobacterium consists of nonmotile, non-spore-forming, aerobic bacilli The organisms are slender, straight, or slightly bent and may appear “beaded” or “granular” after staining. The cell wall is rich in lipids, making the surface hydrophobic and the mycobacteria resistant to many disinfectants as well as to common laboratory stains (e.g., Gram stain and others). Once stained, the bacilli also cannot be easily decolorized with acid solutions. Hence they are termed “acid-fastbacilli.” Because the mycobacterial cell wall is complex and this group of organisms is fastidious, most mycobacteria grow slowely, divinding every 12 to 24 hours. Isolation of the slow-growing organisms (e.g., Mycobacterium tuberculosis, Mycobacterium avium-intracellulare and Mycobacterium kansasii ) can require 3 to 8 weeks of incubation whereas the other mycobacteria require incubation for 3 days or more. Mycobacterium leprae, the etiologic agent of leprosy, cannot be grown in cellfree cultures. Despite the abundance of mycobacterial species (more than 70 species), the following few species or groups cause most human infections: M. tuberculosis, M. leprae, M. avium-intracellulare, M. kansasii, M. fortuitum, M. chelonae, and M. abscessus. Physiology and Structure These organisms are the most resistant of the nonspore-forming bacteria and are resistant to drying, remain viable in dried sputum for as long as 8 months, and show a high degree of resistance to detergents, antibiotics and disinfecting agents. Mycolic

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acids in the cell wall of the organism contribute to enviromental survival. The resistance of these organisms to acids and alkalis allow the latter to be used in sputum concentration techniques employed in the laboratory diagnosis of tuberculosis. The basic structure of the cell wall is typical of gram-positive bacteria: an inner cytoplasmic membrane overlaid with a thick peptidoglycan layer and no outer membrane. The peptidoglycan sekeleton is covalently linked with arabinogalactan. This layer is overlaid with polypeptides and a hydrophobic layer of highly antigenic mycolic acids consisting of free lipids, glycolipids, and peptidoglycolipids. These lipids constitute 60% of the dry weight of the cell wall. The peptide chains in the outer layer constitute 15% of the cell weight and are biologically important antigens, stimulating the patient’s cellular immune response to infection (Fig. 35).

Fig. 35 M. tuberculosis cell wall

Mycobacterium are strict aerobes tha grow best on potato-egg (e.g., LowensteinJensen) and serum agar base (e.g., Middlebrook 7H10) media, both of which contain malachite green dye to inhibit the growth of other bacteria. Optimal growth occurs on or in these media at 37ºC in the presence of 5-10 carbon dioxide. The organisms are relatively slow growers. On solid media, colonies appear as dry, wrinkled, and cream colored (nonpigmented) after 10 days to 8 weeks. The production of niacin by M.tuberculosis is an important biochemical reaction in the laboratory identification of the organism, inasmuch as nonpathogenic and other pathogenic or potentially pathogenic mycobacteria do not produce niacin. Mycobacterium tuberculosis Pathogenesis and Immunology Tuberculosis is the classic human mycobacterial disease. The infection is acquired through the inhalation of aerosolized infectious particles, which then trave to the

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terminal airways. At these sites, the bacteria penetrate into unactivated alveolar macrophages. The phagocytized bacilli inhibit acidification of the phagosome and subsequent phagosome-lysosome fusion, and proceed to replicate freely (it is unclear whether the bacilli replicate in the phagosome or in the cytoplasm). The infected phagocytic cells are eventually destroyed, after which there are further cycles of phagocytosis by macrophages, mycobacterial replication, and cell lysis. Although phagocytosis is initiated by alveolar macrophages, circulating macrophages and lymphocytes are attracted to the infectious focus by the bacilli, cellular debris, and host chemotactic factors (e.g., complement component C5a). The histologic characteristic of this focus is formation of multinucleated giant cells of fused macrophages, also called Langhans’ cells. Infected macrophages can also spread during the initial phase of disease to the local lymph nodes as well as into blood stream and other tissues (e.g., bone marrow, spleen, kidneys, central nervous system). Three to four weeks after exposure to the organisms, DTH and CMI develop. The great majority of individuals develop a relatively high degree of CMI by a mechanism in which alveolar macrophages, activated by the lymphokine MAF (macrophage activating factor) released from T lymphocytes, phagocytize and digest the organisms. An adequate CMI response results in healing of most infected sites by fibrosis and calcification, although calcified areas may still harbor tubercle bacilli for years or for life. The calcified primary site lesions, including those in lymph nodes, are visible on X-ray and referred to as the Ghon complex. Epidemiology Although tuberculosis can be established in primates and laboratory animals such as guinea pigs, humans are the only natural reservoir. Patients with active pulmonary tuberculosis shed large numbers of organisms by coughing, thus creating aerosole droplet nuclei. Because of resistance to dessication, the organisms can remain viable in the enviroment for a long time. The principal mode of contagion is person-toperson transmission by inhalation of the aerosol, and repeated or prolonged contact is usually required for transmission of infection. Populations at greatest risk for disease are immunocompromised patients (particularly those with HIV infection), drug or alcohol abusers, homeless, and individuals exposed to diseased patients. Clinical Signs of Tuberculosis Although tuberculosis can involve any organ, most infections in immunocompetent patients are restricted to the lungs. The disease is insidious in onset. Patients typically have nonspecific complaints of malaise, weight loss, cough, and night sweats. Sputum may be scant or bloody and purulent. The clinical diagnosis is supported by (1) radiographic evidence of

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pulmonary disease, (2) positive skin test reactivity, and (3) the laboratory detection of mycobacteria either with microscopy or in cultures. Extrapulmonary tuberculosis resulting from hematogenous spread of the bacilli during the initial phase of multiplication can also be seen. The most common sites of infection include lymph nodes, pleura, and the genitourinary tract. With disseminated or miliary tuberculosis there is frequently no evidence of pulmonary disease. Laboratory Diagnosis Tuberculin Skin Test : Reactivity to intradermal injection of mycobacterial antigens can differentiate between infected and noninfected individuals. The tuberculin test is a measure of DTH as determined by the intradermal injection of 0.1 ml of intermediate strength purified protein derivative (PPD), which is a tuberculoprotein derived by fractionation of a broth culture filtrate of M.tuberculosis. Results are read 48-72 h after injection. Induration of greater than or equal to 10 mm is considered positive for exposure to M. tuberculosis. A positive PPD reaction usually developes within 3 to 4 weeks after exposure. Some infected patients may have less than 10 mm (5-9 mm) induration, but this level of reactivity generally represents exposure to other mycobacteria. Less than 5 mm induration is represents negative reaction. Specimens. The specimens obtained depend on the disease process and include sputum (the most common), gastric washing, urine, and cerebrospinal fluid. Microscopy. Detection of acid-fast bacilli in clinical specimens is extremely valuable for rapid laboratory confirmation of mycobacterial disease. The clinical specimen is stained with either carbolfuchsin (Ziehl-Neelsen) or fluorochrome dyes, decolorized with acid-alcohol solution, and then counterstained. The specimens are then examined with either a light microscope or, if fluorochrome dyes are used, a fluorescent microscope. The fluorochrome stain is more sensitive because the specimen can be rapidly scanned for fluorescence with low magnification and then confirmed using higher magnification. Primary Isolation. Isolation of mycobacteria from clinical specimens is complicated by the fact that most isolates grow slowly and can be obscured by the rapidly growing bacteria normally present in clinical specimens. Thus specimens such as sputum are initially treated with a decontaminating reagent (e.g., 2% sodium hydroxide). Because mycobacteria are tolerant to brief alkali treatment, this process kills the rapidly growing bacteria and permits the selective isolation of mycobacteria. Traditionally, specimens are inoculated onto both Lowenstain-Jansen and Middlebrook 7H10. Incubation under aerobic conditions at 37ºC is required for optimal growth, which may require as long as 8 weeks. This detection time has recently been shortened by the use of specially formulated broth cultures where the metabolism of 14C-labeled palmitic acid is measured in an ion chamber system (BACTEC).

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Identification. M. tuberculosis and M. bovis appear as acid-fast bacilli producing dry, wrinkled, cream-colored colonies. M. uberculosis is niacin positive, which distinguishes it from other members of the genus. Treatment Most mycobacteria are resistant to antibiotics used to treat other bacterial infections. Effective therapy for infection with M. tuberculosis requires use of at least two antimycobacterial (combined therapy) agents in order to avoid the selection of resistant organisms during treatment. Regimens recommended for treatment include isoniazid and rifampin for 9 months, with pyrazinamide and ethambutol or streptomycin added for drug resistant strains. Prevention and Control Vaccination with attenuated M. bovis (Bacillus of Calmette and Guerin or BCG) Is commonly used in contries where tuberculosis is endemic and responsible for significant morbidity and mortality. This vaccination significantly reduces the incidence of tuberculosis. One problem with BCG immunization is that all patients develop positive skin test reactivity; thus skin testing cannot be used to detect previous exposure to M. tuberculosis. For this reason BCG immunization are not not used in the United States and other contries where the incidence of tuberculosis is low. Mycobacterium avium and M. intracellulare (M. avium-intracellulare Complex) These species are generally considered together because differentiation by physiologic parameters is difficult and their diseases in humans are identical. The organisms are isolated in soil and water, as well as infected poultry, swine, and other animals. Although human exposure is common, significant human disease is rare except in immunocompromised patients. Patients with AIDS are at particular risk for disseminated disease with these mycobacteria, although the reasons for this are not entirely clear. Person-to-person spread does not occur. Mycobacterium leprae M.leprae, the etiologic agent of leprosy (Hansen’s disease), is an obligate parasite of humans and nine-banded armadillos and is most probably transmitted following intimate and prolonged contact with lepromatous patients who shed large numbers of organisms in their nasal secretions and ulcerative lesions.

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The organism is an acid-fast, nonmotile, nonencapsulated, obligate intracellular rod that has never been cultured in or on artificial media or in tissue culture. Mice and armadillos are susceptible to experimental disease. Pathogenesis and Immunity Leprosy manifests as tuberculoi leprosy or lepromatous leprosy, with intermediate forms also recognized. Patients with tuberculoid leprosy have a strong cellular immune reaction but a week humoral antibody response. Infected tissues typically have many lymphocytes and granulomas but relatively few bacilli. As in M. tuberculosis infections in immunocompetent patients, the bacteria produce cytokines that mediate macrophage activation, phagocytosis, and bacillary clearance. Patients with lepromatous leprosy, however, have a strong antibody response but a specific defect in the cellular response to M. leprae antigens. Thus, an abundance of bacilli are typically observed in dermal macrophages and the Schwann cells of the peripheral nerves. As would be expected, this is the most infectious form of leprosy. Clinical Manifestations Following introduction of M. leprae into the host, the organisms multiply slowly inducing a chronic granulomatous response characterized by an influx of mononuclear cells. After an incubation period of 3-5 years or longer, the disease manifests as the lepromatous or tuberculoid type with three intermediate stages. In lepromatous leprosy, the lesions occur as large maculae in cooler body tissues such as skin (especially nose and outer ears), testicles, and superficial nerve endings. The course of lepromatous leprosy is slow but progressive. Many macules, papules or nodules; extensive tissue destruction (e.g., nasal cartilage, bones, ears); lack of nerve enlargement occurs. Epidemiology Person-to-person spread by direct contact or inhalation of infectious aerosoles. People in close contact with patients who have lepromatous disease are at greatest risk. Laboratory Diagnosis: History and clinical manifestations are useful adjuncts but not definitive in the diagnosis of leprosy. Specimens depend on the disease process and include skin lesion biopsies (including the ear lobes) and nasal secretions. Definitive identification is based upon the demonstration of typical acid-fast bacilli within phagocytic foam cells; numerous packets of organisms or “globi” are observed in lepromatous leprosy, but are difficult or impossible to observe in the tuberculoid type.

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Treatment Although at one time the sulfone drug dapsone was the antimicrobial of choice in the treatment of leprosy, the development of a high degree resistance has prompted the recommendation of combined dapsone, rifampin, and clofazimine for patients with lepromatous leprosy and dapsone and rifampin for thoes with the tuberculoid type. NOCARDIA SYSTEMIC NOCARDIOSIS Systemic nocardiosis is a chronic granulomatous disease caused most commonly by Nocardia asteroides. The organisms are found in soil and water. The disease is the result of exogenous transmission and is usually initiated by inhalation. Most cases are opportunistic, occuring in immunosuppressed patients. Human-human transmission is possible but rare. The organisms are gram-positive, acid-fast, branching, filamentous, strict aerobic rods. Laboratory isolates are slow-growing, pigmented, rough-surfaced colonies that often smell “musty”. Following inhalation, a chronic lobar pneumonia develops with hematogenous dissemination most often to the CNS resulting in brain abscesses. The laboratory diagnosis is made by demonstrating gram-positive, acid-fast, branching filamentous rods in sputum, cerebrospinal fluid, or tissue biopsy material, and by primary isolation and identification of the organism by strict aerobic culture and biochemical reactions. The sulfanilamides, when administered early, can reduce significantly the 80% case fatality rate that occurs in the untreated disease. ANAEROBIC GRAM-NEGATIVE BACILLI The most important gram-negative anaerobes that colonize the human oral cavity (particularly the gingival sulcus), female genital tract, and gastrointestinal tract are the bacilli in the gener Bacteroides, Fusobacterium, Porphyromonas, and Prevotella. Anaerobes are the predominant bacteria at each of these sites, outnumbering aerobic bacteria by 10-to 1000-fold. Despite the abundance and diversity of these bacteria, most infections are caused by relatively a few species. Among these pathogens, the most important is Bacteroides fragilis, the prototypical endogenous anaerobic pathogen. Because these organisms are unable to survive in the presence of atmospheric oxygen, they cannot be passed easily from patient to patient. Thus, these organisms cause disease almost exclusively in their original hosts. A few species of anaerobes, notably some spore-forming species, clostridia, are aerotolerant. The anaerobic gram-negative bacilli group, are not aerotolerant but are

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able to survive some period of oxygen exposure in the original specimen and on an agar plate once colonies have matured. Physiology and Structure As indicated by their name, anaerobic bacteria are susceptible to oxygen. Exposure to air and drying will kill them rapidly. Most chemical disinfectants, heat, and sunlight are effective in killing these fragile bacteria. At one time, the genus Bacteroides consisted of almost 50 species, but many of these species have now been transferred to new genera. The genus Bacteroides now consists of the anaerobes previously categorized into the B. fragilis group and some closely related species. The microscopic morphology of anaerobic gram-negative bacilli encompasses the following: B. fragilis group. Theses organisms are highly pleomorphic, gram-negative bacilli that are often show vacuoles, swellings, and long filaments on the initial Gram stain. Fusobacteria. These bacteria are characterized by diverse morphologies. Fusobacterium nucleatum, one of the most common isolates, displays long, thin, fusiform gram-negative rods with pointed ends. It appearance is quite pathognomonic for the genus. Other fusobacteria, such as Fusobacterium mortiferum and Fusobacterium necrophorum may show bizarre, swollen and misshapen forms. Many species of fusobacteria are nondescript. Species of pigmented bacilli can be very small, asaccharolytic bacilli were classified as Porphyromonas, and saccharolytic, bile-sensitive bacilli were transferred into the genus Prevotella. Most gram-negative anaerobes respond weakly to Gram stain, so stained specimens must be carefully examined. The anaerobic gram-negative bacteria must be cultivated in an anaerobic atmosphere. Large laboratories perform anaerobic studies in plastic, self-contained anaerobic chambers that contain an atmospher of 5% hydrogen, 10% carbon dioxide, and 85% nitrogen. Materials are manipulated in a chamber with rubber gloves that are attached and inserted through ports for use by the technologist. Oxygen is removed by catalyzing the reaction of 2H2 + O2 → H2O. Specialized incubation systems, including plastic pouches and gas-impermeable jars, are used in clinical laboratories that do not use chambers. Because several important genera require the growth factors hemin and menadione (vitamin K1), they are usually added to all anaerobic media. Primary media usually contain blood. Laked blood, frozen and thawedi with it is damaged red cell membranes enhances the formation of fluorescence and pigments that are characteristic of several genera of pigmented anaerobes.

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In colonial morphology a few species of this group bacteria have very distinctive colonies that aid in their identification on the culture. The B. fragilis group yield large, shiny, opaque colonies on blood agar. Most species of Porphyromonas and some species of Prevotella, a gram negative bacilli, are pigmented. They use the heme component of RBCs in the medium to generate porphyrin compounds, which become components of various dark pigments. Colonies containing porphyrins will fluoresce under UV light before the pigments have developed. Colonies of some strains of F. nucleatum show typical bread-crumb appearance. colonies are shiny white and opaque. Anaerobes are identified by morphology, enzymatic and biochemical tests, the production of specific end products of glucose metabolism as detected using gasliquid chromatography (GLC) of spent culture medium, and by cell wall fatty acid composition. Definitive identification of many species of anaerobes can only be performed in specialized laboratories. However, rapid 2-day presumptive identification is within the capabilities of any clinical microbiology laboratory. Pathogenesis Each of the anaerobic species have adherence factors that allow them to multiply on mucous membrane surfaces and many of them produce proteolytic and lipolytic enzymes. Lipopolysaccharide. The gram-negative anaerobes contain LPS in their cell walls. The LPS of fusobacteria appears similar to that of aerobic gram-negative bacteria, but the LPS of members of the B. fragilis group is distinctly different. Both Fusobacterium and Bacteroides LPS contributes to abscess formation. The polysaccharide capsule of B. fragilis has been extensively studied as the principal virulence factor of these strains. The capsule is antiphagocytic and promotes abscess formation. Other genera, such as Fusobacterium and Prevotella, also include encapsulated species. Fimbriae. Several species of anaerobic gram-negative bacilli have been shown to posess pili or fimbriae, which mediate attachment to epithelial cells. Hemagglutinin. B. fragilis possesses a surface hemagglutinin, which may aid in invasion of host tissues. Extracellular products associated with pathogenicity are: Superoxide dismutase. The superoxide dismutase produced by B. fragilis and other anaerobes contributes to their survival in the presence of oxygen and may be a virulence factor that allows the organisms to persist in an infected site until the oxygen tension is lowered enough to permit multiplication. Phospholipases. Production of enzymes, such as phospholipases, by Prevotella melaninogenica nd Prevotella intermedia, two pigmenting strains found in the oral cavity, contributes to the ability of these organisms to colonize the gingival crevices.

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Collagenase. Porphyromonas gingivalis produces collagenase, which aids in the induction of necrosis in infected tissues. Proteases. Several anaerobes produce proteases that may cleave immunoglobulins and thus inhibit opsonization. Some fusobacteria produce lipases. Additional enzymes produced by several nonspore-forming anaerobes include hyaluronidase and fibrinolysin. Clinical Manifestations of Nonspore-Forming Anaerobic Infection Abscess formation. Abscesses ( specially in brain, lung, liver, perirectal, pelvic, and diaphragm) are the predominant feature of many diseases due to the nonsporeforming anaerobes. Other infections are: Periodontal infections, chronic sinusitis, necrotizing pneumonia, empyema, cellulitis (frequently with gas), endometritis, post abortal infection, and infected vascular gangrene. Laboratory Diagnosis A. The specimens obtained depend on the disease process and include: 1. Tissue biopsy material is the best specimen. If it is large enough, it can be transported in a sterile container in which anaerobes will survive for several hours. If it is small, it should be placed into a transport vial with a protected anaerobic enviroment. 2. Aspiration of abscess contents is the next best specimen. Specimens should be obtained from intact, disinfected skin with a needle and syringe. The material should be inoculated into an anaerobic transport vial for maitenance during transport to the laboratory. 3. Sterile body fluids are collected transcutaneous aspiration and transport in an aerobic transport vial. 4. Periodontal and subgingival exudates can be collected on sterile paper points that are placed immediately into a reduced transport fluid. 5. Swabs from areas contain normal flora are not acceptable because of the high degree of contamination by normal flora. B. For primary isolation and identification: 1. Direct Gram stains should always be performed; typical organism morphology may be present and provide a very early clue to the etiologic agents. 2. Specimens are inoculated onto special media supplemented with hemin and menadione. Standart media include anaerobic blood agar; a laked blood agar to enhance pigment formation; a selective agar containing antibiotics to suppress facultative gram-negative bacilli; Bacteroides-bile-esculin agar, a selective and

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differential plate for rapid identification of the B.fragilis group; and a broth enrichment backup culture. 3. All cultures are incubated anaerobically for 2-14 days before they are examined. 4. Isolates are identified based on colony morphology, Gram stain morphology, susceptibility to antibiotics, constitutive enzymes, biochemical reactions, and by the end products of glucose metabolism. A few rapid tests can identify most isolates to genus within 2 days of observing isolated colonies. Gas-liquid chromatographic analysis of volatile and nonvolatile fatty acids produced by metabolic processes of anaerobes is the definitive identification method. The cumbersome nature of the methods and the expense of the apparatus precludes the use of thses tests in most clinical laboratories. Treatment Surgical drainage and debridement are key factors in successful treatment of anaerobic diseases. Only a few types of diseases, including liver and brain abscesses, can be treated medically with antibiotics alone. Antimicrobials that have activity within abscesses are most effective for anaerobic diseases. Ampicillin-sulbactam and amoxicillin-clavulanic acid are effective against betalactamase producing anaerobes. Metronidazole has good penetration into abscesses and is effective for most gramnegative anaerobes. Imipenem is quite active against most anaerobes, although some limited resistance has been reported. Cefoxitin is still useful in limited situations, although resistant strains, especially among the B.fragilis group, are becomming more common. Clindamycin and chloramphenicol are effective and may be useful in some circumstances, but in general they are less desirable. SPIROCHETES TREPONEMA, BORRELIA, AND LEPTOSPIRA Spirochetes are unicellular, slender, helical-shaped, flexible gram-negative organisms actively motile by virtue of periplasmic flagella (syn: endoflagella, axial filaments) that lie in the periplasmic space between the cytoplasmic and outer membranes. The three genera of the order Spirochetales that contain human pathogens are Treponema, Borrelia, and Leptospira.

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TREPONEMA The treponemal species responsible for human disease are Treponema pallidum and T. carateum. T. pallidum is subdivided into subspecies pallidum, the etiologic agent of syphilis, subspecies pertenue, the agent responsible for yaws, and subspecies endemicum, the etiologic agent of endemic syphilis. Although DNA homology between Treponema pallidum subsp. pallidum and Treponema pallidum subsp. pertenue is almost 100%, subspecies nomenclature is based on differences in their clinical manifestations and geographic locations. The variant status of Treponema pallidum subsp. endemicum has warranted subspecies classification for this organism. Treponema pallidum subsp. pallidum T. pallidum subsp. pallidum is an obligate parasite of humans and does not occur in nature or in animals. The organism is a thin, coiled spirochete (0.1 x 5-15 µm) actively motile, exhibiting a shimmering motion in serious exudate and a directional, corkscrewlike motion in fibrinous exudate. The spirochetes cannot be seen with light microscopy in specimens stained with Gram or Giemsa stains, but can be best recognized and studied by dark field microscopy. None of pathogenic treponemes has been cultured in/on artificial media. Limited growth has been achieved in cultured rabbit epithelial cells but replication is slow and can be maitained only for a few generations. Treponema pallidum is extremely labile, unable to survive exposure to drying or disinfectants. Thus inanimate objects such as toilet seats cannot contribute to the spread of syphilis. Direct person-to-person contacts is required for transmission. Diseases of Treponema pallidum subsp. pallidum Syphilis. This occurs worldwide and is the third most common sexually transmitted disease in the developed world. The organism enters the body via skin or mucous membrane abrasions. Localized multiplication occurs, resulting in inflammatory cell infiltration, followed by endarteritis. The infection is divided into three phases: 1. Primary Stage. This stage of the disease occurs after an incubation period ranging from 10 to 90 days, but usually 3 weeks. Primary syphilis is characterized by the appearance of painless, indurated, well-circumscribed ulcer, known as chancre, accompained by a regional lymphadenopathy that is usually bilateral. At this time, the chancre and lymph nodes contain treponemes and the disease is communicable. The primary lesion heal in 1-6 weeks, presumably due to CMI. During this local healing process, organisms within the deeper tissues

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survive and undergo slow but continuous multiplication, setting up foci of infection. 2. Secondary Stage. This stage of the disease is initiated when organisms gain access to the circulation from these infected foci, produce a septicemia. It occurs simultaneously with or up to 6 months after healing of the primary lesion and is the end result of the confrontation. Secondary syphilis is characterized by fever, headach, generalized lymphadenopathy, a generalized rash with lesions characteristically on the palms and soles, mucous patches in the oral cavity. Late syphilis. One third of patients with secondary syphilis develop a latent infection, which may occur within 3 to 20 years after the initial infection. The typical granulomatous lesions of late syphilis are known as gummata. These lesions may involve the skin, mucous membrane, soft tissues, bone, eyes, central nervous system, and cardiovascular system. Congenital Syphilis is the result of transplacental transmission from an infected mother to the fetus, usually not until 18 weeks after gestation. In utero infections can lead to significant fetal disease, with death, multiorgan malformations, or latent infections. Most infected infants are born without clinical evidence of disease but then develop rhinitis followed by a widespread maculopapular rash. Late second tooth development, long bones destruction and cardiovascular syphilis are common in untreated infants who survive the initial course of disease. Laboratory Diagnosis As T. pallidum cannot be grown in vitro on standard laboratory media, diagnosis is based on microscopy and serology; Microscopy. Primary, secondary, and congenital syphilis can be diagnosed by dark field examination of fresh material from skin lesions. Serology. Diagnosis of syphilis in most patients is based on serological tests. Two types of tests are employed : 1. Non-specific antibody tests: These rapid tests determine the presence of IgM and IgG antibodies (also called reagin antibodies), which develop against lipids released from damaged cells during the early stages disease and also present on the cell surface of treponemes. The antigen used in these assays, cardiolipin, is extracted from beef heart; cardiolipin binds antibodies to T.pallidum lipids. The test commonly used are the veneral disease research laboratory (VDRL) test and the rapid plasma reagent (RPR) test. Both assays are based on the agglutination of cardiolipin antigen by the patient’s serum on a glass slide. As the assays are non-specific, fals positive results are common, and occur in other conditions (e.g., leprosy, tuberculosis, viral infections and others); results need confirmation by specific serological tests.

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2. Specific antibody tests: These tests are based on T. pallidum antigens and are used to confirm non-specific screening tests. Commonly used tests include the fluorescent treponemal antibody (FTA) test and the T. pallidum microhemagglutination assay (TP-MHA), in which erythrocytes coated with T. pallidum antigen are agglutinated by serum from patients with antibodies to T. pallidum. Specific tests to detect IgM antibodies are used to diagnosed congenital infections. Treatment Penicillin is the drug of choice in the treatment of all stages of syphilis and congenital syphilis. Penicillin resistant strains of T. pallidum have not been reported. Tetracycline, erythromycin, or chloramphenicol is administered if the patient is allergic to penicillin. Treponema pallidum subsp. pertenue T. pertenue is the causative agent of yaws. This is primarily a skin disease; destructive lesions (granuloma) of the skin, lymph nodes and bone can occur. Yaws is found primarily in South America and Central Africa. Spread is by direct contact with infected lesions. Diagnostic procedures and treatment are as for syphilis. Treponema carateum Pinta is caused by T. carateum and is found primarily in Central and South America. It occurs in all age groups. After a 1-3 week incubation period, small papules develop on the skin. These can enlarge, last for several years and result in hypopigmented lesions. Spread is via direct contact or insect vectors. Diagnosis and treatment is as for syphilis. BORRELIA Borrelia are associated with two important human infections, relapsing fever (primarily Borrelia recurrentis) and Lyme disease (Borrelia burgdorferi). Members of the genus Borrelia are identical appearing as loosely coiled, thin, flexible, actively motile spirochetes 3-30 µm in length and 0.2-0.5 µm in diameter. These organisms are gram-negative and stain well with Giemsa and Wright stains, and can be easily seen in smears of peripheral blood collected from patients with relapsing fever. Borrelia are microaerophilic and have complex nutritional requirements, making recovery in culture difficult. The few species that have been successfully cultured

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have generation times of 18 hours or longer. Because culture is generally unsuccessful, diagnosis of disease caused by borreliae is by microscopy (relapsing fever) or serology (Lyme disease). Diseases of the Borrelia Members of the genus Borrelia are responsible for two important human diseases: relapsing fever and Lyme disease. 1. Relapsing fever is a febrile illness characterized by recurrent episodes of fever and septicemia, separated by afebrile periods. Two forms of the disease are recognized: a. Borrelia recurrentis is the etiologic agent of epidemic or louse-borne relapsing fever is spread person-to-person by the human body louse (Pediculus humanus). The louse cannot infect the host during a blood meal or transmit borreliae to another louse by transovarian passage, in asmuch as the organisms are located only in the central ganglion and lymph nodes of the vector. Thus, humans can only become infected by crushing infected lice on their skin and creating an abrasion for entrance of the organisms. b. Endemic or tick-borne relapsing fever is caused by many species of borreliae and is spread from rodents (e.g., ground squirrels, chipmunks, rats, and opossums) via infected ticks. Ticks become infected with borreliae by feeding upon infected reservior hosts, after which the organisms gain access to their coxal and salivary glands and gonads. Borreliae are introduced into the human host from contaminated tick saliva, coxal fluid, and feces during a blood meal. The organism can be transmitted transovarialy from tick to tick, accounts the endemic nature of the disease. In general, the pathogenesis of epidemic and endemic relapsing fever are similar, although the clinical manifestations of the former may at times be somewhat more sever. After entry into the host from the louse or tick vector, the organisms enter the circulation, where they multiply to produce a septicemia. after an incubation period of 2-14 days, a sudden onset of fever of 38.9 – 40.6ºC is occurs, accompanied by chills and headache that last for 3 – 7 days. At this time borreliae antibody appears and causes destruction and elimination of the organisms. Surviving organisms within the tissues alter their antigenic structure by means of genetic mechanisms, and multiply in the presence of the antibody to the initial infecting strain, thereby resulting in another febrile period and a repeat of the pathogenesis and clinical manifestations. The number of relapses is usually 3 – 4. 2. Lyme disease, the etiologic agent of which is B. burgdorferi, is a worldwide tickborne disease. The primary reservoir hosts of B. burgdorferi are rodents and birds. Transmission occurs from infected Ixodes deer ticks. After an incubation period of from 3 – 30 days one or more skin lesions referred to as erythema migrancs (EM)

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develops at the site of tick bite. Chronic neurologic, cardiac, and rheumatic manifestations are also occurs after skin lesion. Laboratory Diagnosis Microscopy. Because of their relatively large size, the borreliae responsible for relapsing fever can be seen during the febrile period by preparing a Giemsa stain of the blood. In contrast, the microscopic examination of blood or tissues collected from patients with Lyme disease connot be recommended becaus B. burgdorferi is rarely seen in clinical specimens. Culture. The cultures are rarely performed in most clinical laboratoreis because the media are not readily available and replication is slow. The in vitro isolation of B. burgdorferi has had limited success. Serology. Because the borreliae responsible for relapsing fever undergo antigenic phase variation, serlogic tests are not useful. In contrast, serologic testing is an important confirmatory test for patients who clinically diagnosed as have Lyme disease. Treatment Early treatment of borreliae diseases with doxycycline, tetracycline, erythromycin, or chloramphenicol is effective. LEPTOSPIRA The genus Leptospira consist of two species: Leptospira interrogans (subdivided further 218 serotypes) and Leptospira biflexa. Leptospira are thin, coiled bacilli (0.1 x 6 – 12 µm) with a hook at one or both ends, obligatively aerobic and motile by means of two periplasmic flagella. They are best recognized by dark field microscopy and are grow best in serum enriched, semisolid media. However, the generation time is from 6 – 16 hours and incubation for up to 2 weeks may be required. L. interrogans is pathogenic for many wild and domestic animals, as well as humans. There are over 180 serotypes of L. interrogans but only a few are associated with human disease, namely icterhaemorrhagiae, canicola and pomona. L. biflexa is a free-living saprophyte found in moist enviromental sites and is not associated with disease. Leptospirosis, caused by L. interrogans, is a worldwide, acute, febrile disease that may manifest as a multiplicity of different clinical entities. The source of most of the reported cases are pet dogs and domestic livestock, which are harbored leptospirae in their renal tubules. Leptospirae enter through the abraded skin, oral or nasal

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mucosa, or conjunctiva following contact with urine-contaminated water by drinking, immersion of hands, or swimming. The pathogenesis of leptospirosis begins with the entrance of the leptospirae directly into the circulation from the portal of entry; the organisms multiply and produce a septicemia. The first phase of the disease manifests as a fever, chills, headage, GI disturbance, and conjunctival redness. In 2 – 7 days, the clinical manifestations diminish and the patient becomes afebrile. After 1 – 3 day afebrile period, a recurrence of fever, which persists for only 1 – 2 days. During either phase of the disease, jaundice, rash, hepatosplenomegaly, petechial hemarrhage, meningitis, and/or renal damage may occur. Laboratory Diagnosis Blood, cerebrospinal fluid, and urine may be cultured in specialized media. Positive blood and cerebrospinal fluid cultures should be obtained during the first phase febrile period. Urine cultures are most often positive during the second phase of the disease. Immunologic blood tests rather than culture methods are the assays used most frequently to diagnose leptospirosis. Treatment Penicillin and tetracycline are effective if administered within the first 2 – 4 days after initial onset of clinical manifestations. Later administration has no effect upon the course of the disease. RICKETTSIACEAE FAMILY The family Rickettsiaceae consist of aerobic, gram-negative small (0.3 x 1-2 µm) bacilli that, with one exception (Rochalimaea), are obligate intracellular parasites; they grow only in yolk sac of embryonated eggs, tissue cultures and in animal models. Four genera (Rickettsia, Coxiella, Rochalimaea, and Ehrlichia) are associated with human disease. The pathogenic species are maintained in animal reservoirs and transmitted by arthropod vectors (ticks, mites, lice, fleas). Humans are usually accidental hosts. Properties of the Organisms The cell wal structure of reckettsia, with a peptidoglycan layer and lipopolysaccharide, is typical of gram-negative bacilli. The bacteria do not have flagella or attachment proteins but are surrounded by a loosely adherent slime layer. All reckettsia are seen best with the Giemsa or Gimenes stains but react poorly with

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the Gram stain. With the exception of Rochalimaea, all rickettsia are strict intracellular parasites. The intracellular location of the rickettsia vary: Rickettsia species are generally found free in the cytoplasim, whereas Coxiella and Ehrlichia multiply in cytoplasmic vacuoles. Rickettsia rickettsii, the bacterium responsible for Rocky Mountain spotted fever, can also grown to high concentrations in the nucleus. Rochalimaea multiplies readly on the surface of eukaryotic cells but rapidly dies following phagocytosis. The rickettsia enter eukaryotic cells by phagocytosis. After phagocytosis occurs, rickettsia must be released into the cytoplasm or the organism fails to survive. A multiplication by binary fission then proceeds slowly compared with other bacteria (generation time, 6 – 10 hours) untile destruction of the host cell ocuurs, freeing the rickettsia to infect new cells. In contrast with the other rickettsia, multiplication of Coxiella and Ehrlichia proceeds within phagolysosomes rather than in the cytoplasm, and host cell lysis is low. Once released from the host cell, most rickettsia are unstable and die quickly. The exception is Coxiella, which is highly resistant to desiccation and remains viable in the enviroment for months to years. This characteristic is extremely important in the epidemiology of Coxiella infections. Rickettsial Infections Rickettsial infection should be suspected if fever, headache, myalgias, rash, and history of exposure to arthropods. Epidemic Typhus. R. prowazekii is the etiologic agent of epidemic typhus, also called louse-borne typhus, and the principal vector is the human body louse, Pediculus humanus. In contrast most other rickettsial diseases, humans are the primary reservoir. Epidemic typhus is associated with crowded, unsanitary conditions that favor the spread of body lice. The disease are characterized by headache, chills, fever, and a rash that spreads from the trunk to the extremities. Endemic Typhus. Endemic or murine typhus is caused by R. typhi. Rodents are the primary reservoir, and the rat flea is the principal vector. The associated rash usually remains on the trunk. Scrub Typhus. R. tsutsugamushi is the etiologic agent for scrub typhus, a disease transmitted to humans by mites. The reservoirs for this rickettsia are wild rodents. Disease is present in Eastern Asia, Australia, and Japan and other Western Pacific islands. Rocky Mountain Spotted Fever. R. rickettsii is responsible for Rocky Mountain spotted fever, which is the most common rickettsial disease in the United States. The disease is transmitted from wild rodents and dogs by infected ticks. Trench Fever. Rochalimaea (Bartonella) quintana is the etiologic agent of a rare febrile disease, trench fever. Patients are infected by human body lice and exhibit fever, rash, splenomegaly, and myalgias.

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Q Fever usually presents as an acute febrile illness with headache and frequently hepatitis. Although tick-borne Q fever is known, most cases are acquired by aerosols. The etiologic agent, Coxiella burnetii, is shed in the feces and genital secretions of infected domestic animals, such as sheep, cattle, and goats. Because of it is resistance to harsh enviromental conditions, the organism remains viable in aerosols and causes disease when inhaled by humans. Ehrlichiosis. Canine ehrlichiosis is transmitted by the dog tick and is characterized by an acute phase of pancytopenia and a chronic phase with fever, anemia, and occasional bleeding from the capillaries. Laboratory Diagnosis Microscopy. Rickettsial intracellular inclusions is best seen in specimes stained with Giemsa or Gimenes stains. The bacteria stain poorly with Gram stain. Rickettsia can also be detected in the prevascular endothelial cells by direct staining with fluorescein-labeled antibodies. Culture. Although rickettsia can be cultured in embryonated eggs or in cell cultures, the organisms are highly infectious and considered dangerous to handle except in the most experienced laboratories. For that reason the diagnosis for most infections is made by serologic testing. Serology. The Weil-Felix test detects cross-reacting antibodies to Rickettsiae which agglutinate certain strains of Proteus (OX-19 and OX-2). The test is nonspecific and has now been superseded by more specific serological tests based on purified rickettsial antigens (immunofluorescence assays and ELISA). Treatment and Prevention Treatment is with tetracycline and chloramphenicol. Infection can be prevented by avoidance of the various vectors. A vaccine to R. prowakzekii is available. CHLAMYDIACEAE FAMILY Members of the family Chlamydiaceae are obligate intracellular bacteria that were once regarded as viruses. Chlamydia possess inner and outer membranes similar to those of gram-negative bacteria, contain both DNA and RNA, possess prokaryotic ribosomes, synthesize their own proteins, nucleic acids, and lipids, and are susceptible to numerous antibiotics. Unlike other gram-negative bacteria, however, chlamydiae lack a peptidoglycan layer between the inner and outer membranes and undergo a unique growth cycle. The genus Chlamydia is divided into three species: C. trachomatis, C. pneumoniae and C. psittaci. Classification is based on the basis of antigens, the susceptibility to sulfonamides, the morphology of the elementary bodies, the formation of inclusions containing glycogen, and the natural host range.

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Morphology. All chlamydiae share a common group antigen but may be distinguished by species-specific antigens. They multiply in the cytoplasm of host cells following a well-defined developmental cycle. Chlamydiae have two distinct morphological forms: 1. The elementary body (EB): an infectious extracellular particle (300 – 400 nm); 2. The reticulate body (RB): an intracellular non-infectious particle (800 – 1000 nm). Life cycle: · Ebs bind to specific host cell receptors and enter by endocytosis. Target cells include conjunctival, urethral, rectal and endocervical epithelial cells. · Intracellular Ebs remain within phagosomes and replicate. Lysosomal fusion with the phagosome-containing Ebs is inhibited, probably by chlamydial cell wall components. · After approximately 8 h, Ebs become metabolically active and form RBs, which synthesize DNA, RNA and protein utilizing host cell adenosine triphosphate (ATP) as energy source. · RBs undergo multiple division and the phagosome becomes an “inclusion body” (visible by light microscope), which is contain as may as 1000 RBs. Glycogencontaining inclusion bodies, such as those formed only by C.trachomatis, stain orange-brown with iodine. · After approximately 24 h, the RBs recognize into smaller Ebs and, after a further 24 – 48 h, the host cell lyses and infective Ebs are released. C.trachomatis To understand C. trachomatis infections it is important to recognize the species has been subdivided into 2 biovars: trachoma and LGV (lymphogranuloma venereum). The human biovars have been further divided into 19 serotypes based on antigenic differences among the strains. The LGV biovar consists of 4 serovars (L1, L2, L2a, L3); the remaining 15 serovars (A, B, Ba, C, D, Da, E, F, G, Ga, H, I, Ia, J, and K) are in the trachoma biovar. Infections of C.trachomatis C. trachomatis is responsible for a wide range of clinical diseases: Trachoma. Trachoma, which is characterized by the pebbled appearance of the infected conjunctiva, is a chronic keratoconjunctivitis caused by serotypes A, B, Ba, and C. This disease is seen initially as a follicular conjunctivitis with a diffuse inflammation involving the entire conjunctiva. Progression of the disease leads to conjunctival scarring, producing inturned eyelids. Subsequently, inturned eyelashes

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cause constant abrasion of the cornea, pannus formation (invasion of vessels into the cornea), and loss of vision. Trachoma is transmitted eye-to-eye by droplet, hands, contaminated clothing, and by eye-seeking flies, which transmit ocular discharges to eyes of other person. Infections occurs predominantly in children. Adult inclusion conjunctivitis. An acute follicular conjunctivitis caused by serotypes A, B, Ba, D to K associated with genital infections. The infection is characterized by mucopurulent discharge, keratitis, corneal infiltrates, and occationally some corneal vascularization. Most cases occur in adults between the ages of 18 and 30, with probable genital infection before eye involvement. Infected secretions from the genital tract spread the infection through sexual activity, including anal-genital sex, and by direct contact. Adults can transfer the organism to their eyes after touching their genital tracts. Infants usually acquire the disease during passage through the infected birth canal of the mother. Neonatal conjunctivitis. Inclusion conjunctivitis in the newborn is acquired by passage through an infected maternal birth canal and associated with serotypes D— K. After an incubation period of 5—12 days, swelling of the lids, hyperemia, and copious purulent discharge appear. Without therapy or with tropical therapy only, infants are at risk for development of C. trachomatis pneumunia. Urogenital infections are associated with serotypes D—K. The most common cause of sexually transmitted disease in the developed world. Most genital tract infections in women are asymptomatic but can nevertheless spread to cause symptomatic disease, including cervicitis, endometritis, urethritis, salpingitis, and perihepatitis. Symptomatic infections produce mucopurulent discharge and hypertrophic ectopy and generally yield greater numbers of organisms on culture than do asymptomatic infections. Unlike genital infections in women, the majority of genital infections in men caused by C. trachomatis are symptomatic. Approximately 35% to 50% of cases of nongonococcal urethritis are caused by C. trachomatis. Postgonococcal urethritis results from coinfection with both N. gonorrhoeae and C. trachomatis, and symptomatic illness develops after successful treatment of the gonorrhea because of the longer incubation period of chlamydiae. Epididymitis may occur in association with urethritis due to chlamydiae or coliform bacteria. Lymphogranuloma venereum (LGV) is a chronic sexually transmitted disease caused by C. trachomatis serotypes L1, L2, and L3. The disease is highly prevalent in tropical climates (Africa, Asia, and South America). After an incubation period of 1—4 weeks, a prima lesion (ulcer) may appear at the site of infection (e.g., penis, urethra, glans, scrotum, vaginal wall, cervix, vulva). The second stage of infection is marked by inflammation and swelling of the lymph nodes draining the site of initial infection. Most commonly, the inguinal nodes are involved, producing painful, fluctuant “buboes” that gradually enlarge.

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Laboratory Diagnosis Culture: Swabs from the affected site collected in a special transport medium are inoculated into tissue culture. Growth of C. trachomatis is recognized by Giemsa staining to show intracellular bodies or by immunofluorescence staining with specific antisera. Direct antigen detection can be made by immunofluorescence or enzyme-linked immunosorbent assay (ELISA) methods. Both techniques utilize labelled specific antibodies to C. trachomatis. Serology: Antibodies to C. trachomatis may be detected by complement fixation or micro-immunofluorescence tests. · Assays based on the polymerase chain reaction and ligase chain reaction are also now available. Treatment: Since asymptomatic genital infection is common, all partners of infected patients should be treated. Tetracycline or doxycycline, followed by erythromycin and sulfasoxazole are still the therapies of choice. C. psittaci C. psittaci is the cause of psittacosis (parrot fever), which can be transmitted to humans by inhalation of aerosols containing viable organisms. The disease was first described in parrots, thus the name psittacosis (Psittakos, Greek for parrot). The natural reservior of C. psittaci is birds; virtually any species of bird can become infected and thus serve as a source of human infection. The organism is present in the blood, tissues, feces, and feathers of infected birds. The disease is flu-like illness and pneumonia. Laboratory diagnosis. By serology, e.g. complement fixation test. Culture of C. psittaci is also possible but is only performed in laboratories with high level containment facilities. Treatment. Tetracycline is the drug of choice for treating C. psittaci infections. C. pneumoniae Originally designated TWAR (Taiwan-associated respiratory disease) agent, but now called C. pneumoniae and is an established cause of atypical pneumonia. Infection is spread via direct human contact, with no apparent animal reservior. It is associated with respiratory infections, including pharyngitis, sinusitis and pneumonia (which can be srvere, particularly in the elderly). Diagnosis is made by serology and traetment includes either tetracycline or erythromycin.

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MYCOPLASMA AND UREAPLASMA The family Mycoplasmataceae consists of two genera, Mycoplasma, with 69 species and Uraeplasma with two species. Only three species have identified as human pathogens: Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma uraelyticum. Mycoplasma and Urealyticum are the smallest free living bacteria. They are pleomorphic, with an average diameter of 0.2 to 0.8 µm; filamentous forms also exist. Many of these bacteria are able to pass through 0.45 µm filters that are used to remove bacteria from solutions. In addition, mycoplasmas do not have a cell wall; the cytoplasmic contents are enclosed only by a plasma membrane, thus renders the organisms resistant to penicillins, cephalosporins, and other antibiotics that interfere with the integrity of the cell wall. For these reasons the mycoplasmas were thought originally to be viruses. However, the organisms devided by binary fission (typical of all bacteria) and are gram negative. Most mycoplasmas are facultatively anaerobic (M. pneumoniae is a strict aerobe), grow on artificial media, and require exogenous sterols supplied by the addition of animal serum to the growth medium. The mycoplasmas grow slowly with a generation time from 1 to 6 hours, and form small colonies that have a “fried egg” appearance. Colonies of Ureaplasma are extremely small, 10 to 50 µm in diameter. The three human pathogens can be differentiated by their ability to metabolize glucose (M. pneumoniae), arginine (M. hominis), or urea (U. urealyticum). Because these organisms do not have a cell wall, the major antigenic determinants are membrane proteins and glycolipids. Associated Infections · M. pneumoniae: pharyngitis; primary atypical pneumonia · Other mycoplasmas, including M. hominis and U. urealyticum, are found in human genital tracts. These organisms cause postpartum fever and may contribute to infertility. M. hominis can cause postsurgical wound infections. Laboratory Diagnosis Culture: mycoplasmas ans ureaplasmas can be grown on enriched media; penicillin is often added to inhibit other organisms. Although M. pneumoniae can be isolated from sputum following incubation for up to 3 weeks, diagnosis is normaly by serology. M. hominis grows after approximately 4 days, producing colonies with a fried egg appearance. U. urealyticum requires urea for growth and forms small colonies. Serology: M. pneumoniae infections can be diagnosed by serological tests for Ig G (fourfold rise in titres is indicative for current infection) or Ig M. Treatment. This is with macrolides (e.g. erythromycin) or tetracycline.

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VIROLOGY Conceptually, viruses are genetic elements packaged in protein coats. This packaged genetic element can penetrate a susceptible cell and replicate within the cell. The clinically important viruses range from 20 or 30 nm (picornavirus) to 300 nm (pox viruses). Their small size allowed them to pass through filters designed to retain bacteria. Virus Structure The electron microscope shows that viruses censist of a protein coat (capsid) and a core of nucleic acid. The larger viruses have an envelope composed of lipid, proteins, and glycoproteins (Fig. 35). This lipid envelope can be disrupted by detergents and solvents such as ether, which will then inactivate the virus. Some viruses also contain enzymes required for replication.

Fig. 36 Virus structure

Capsid A protein coat enclosing the genome and consisting of capsomeres (capsid subunit). The number of capsomeres differs in each virus family, and they usually appear in one of two geometric arrangements, cubical (icosahedral), or helical structures. Helical symmetry appear as rods or cylinder and are observed within the envelope of most negative-strand RNA viruses. Icosahedral symmetry occurs in both DNA and RNA viruses, the capsid forming an approximately spherical assembled from symmetrical subunits.

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The capsid is necessary for morphogenesis of the virus. It is also determine host specificity, protects the viral genome, enhances the efficiency of infection, and induces the formation of neutralizing antibody. Genome All true viruses have a nucleic acid that is either DNA or RNA. The genome may consist of double-stranded or single-stranded DNA or RNA and contains all necessary information for virus replication in susceptible cells. Nucleocapsid. The capsid-covered genome is referred to as the nucleocapsid. Envelope Lipid bilayer membrane surrounding the nucleocapsid of some viruses (enveloped viruses). This viral envelope is derived from the membrane of the host cell except that membrane proteins of the host are largely replaced by viral proteins such as: 1. Glycoproteins (peplomers) extend from the surface of the virion and for many viruses can be observed as spikes. The peplomers play a key role in viral attachment to a susceptible cell and in the induction of neutralizing antibodies against the enveloped virus. 2. Matrix proteins found between the nucleocapsid and envelope. These proteins strength the virion and facilitate their assembly. Virion A virus particle with all it is morphologic components is known as a virion VIRUS CULTIVATION Viruses are obligate, intracellular parasites and thus can only replicate in living cells. Viruses utilize the host cell metabolism to assist in the synthesis of viral proteins and progeny virions; the host cell range of viruses may be narrow or wide. For diagnostic purposes, most viruses are grown in cell cultures, either secondary or continuous cell lines; the use of embryonic eggs and laboratory animals for virus culture is reserved for specialized investigations. Cell culture lines are often derived from monkey kidney, human fetal lung, human amnion, and human cancer cells. Virus replication in cell cultures may be detected by : 1. Cytopathic effect (CPE): some viruses can be recognized by their effect on cell architecture, e.g. changes in cell morphology, cell lysis, the presence of inclusion bodies, or the formation of multinucleated cells (syncytia formation). Inclusion bodies are histologic changes in cells caused by the presence of viral components or changes in cell structure. 2. Haemadsorption: viruse expressing haemagglutinins on the cell surface may be recognized by absorption of red blood cells to infected cells.

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3. Immunofluorescence: the appearance of virus-coded proteins on the surface, in the nucleus or cytoplasm of infected cells may be detected by immunofluorescence techniques using virus protein-specific antibody. VIRAL INFECTION OF HOST CELLS Viras replication in host cells involves the following steps (Fig. 36). 1. Attachment (adsorption) occurs of the viral nucleocapsid (naked viruses) or of virus envelope components (enveloped viruses) to cell has specific viral receptors. 2. After attachment, the nucleocapsid enters the cell by endocytosis or by the fusion of the cell membrane with viral envelope. This fusion is usually mediated by a viral envelope protein. 3. Uncoating follows, which involves the proteolytic removal of viral protein coat and liberation of nucleic acid. The disassembly is achieved by unidentified host enzymes. 4. Synthesis of viral proteins. In order to direct the host cell ribosome to produce viral proteins (genome, capsid), virus specific mRNA must be produced. The mechanisms for virus specific mRNA production depend on the viral genome type. a) RNA or DNA b) Single-stranded or double-stranded c) Positive sense (base sequence configured as required for translation = mRNA) or negative sense (base sequence requires transcription). 5. Morphogenesis and maturation occur with assembly of components (nucleic acid, proteins) to form viral particles. 6. Release of the virus is by bursting of infected cells (lysis) or by budding through plasma membrane (host cell does not necessarily lose viability, therefore it can shed viral particles for extended periods). With some viruses, e.g. hepatitis B, the host cell remains viable and continues to release virus particles or subviral antigens at a slow rate. These persistent infections acts as a continuing source of new infectious viruses. During latent infections the virus does not undergo replication; the viral nucleic acid may remain in host-cell cytoplasm (e.g. herpes virus) or become incorporated into the host genome (e.g. human immunodeficiency virus HIV). A trigger is required to recommence viral nucleic acid replication, transcription and translation. Variations in the Synthetic Phase of Viral proteins The key viral product in the synthetic phase is the viral mRNA. The pathways leading from viral genomes to multiple copies of viral mRNA can be best be described under the following three categories: 1. RNA Viruses Other Than Retroviruses. It is important to remember that the host cell does not have an RNA dependent RNA polymerase; therefore, this polymerase

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Fig. 37 Virus replication

must be encoded in the viral genome. The initial step in the synthetic phase depends on whether the viral genome is single or double stranded; and, if single stranded, whether it is positive or negative sense. Viruses with single-stranded positive sense RNA (e.g., Picornaviridae) acts as mRNA, bind to ribpsomes, and direct protein synthesis. Then RNA dependent RNA polymerase is produced. Using the viral genome as template, this polymerase synthesizes a complimentary strand of RNA. Together, the original viral genome (positive sense) and the newly synthesized complimentary strand (ngative sense) form a double-stranded RNA known as the replicative form. Using this replicative form as the template, the viral polymerase synthesizes from the negative sense strand many copies of positive sense RNA. These positive sense RNAs serve as viral mRNA as well as viral genomes for progeny virions. Viruses with single-stranded negative sense RNA (e.g., Orthomyxoviridae) cannot be translated. It must be transcribed into positive sense RNA. This initial step is performed by an RNA dependent RNA polymerase found in the capsid. Once positive sense RNA are formed, the synthetic phase proceeds as described for Picornaviridae. Viruses with double-stranded RNA (e.g., Reoviridae). The capsid also contains an RNA dependent RNA polymerase that transcribes the negative sense of the uncoated viral genome into positive sense RNA 2. Retroviridae. The genome of retroviruses consists of a dimer of positive sense single-stranded RNA. Unlike other RNA viruses, its genome codes for a RNA

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dependent DNA polymerase (reverse transcriptase) instead of the RNA dependent RNA polymerase. The reverse transcriptase transcribes viral RNA into viral DNA some of which may be integrated into host cell DNA. The integrated viral DNA is known as provirus and the nonintegrated as episomal DNA. The host’s DNA dependent RNA polymerase then transcribes the episomal and proviral DNA into viral mRNA. 3. DNA Viruses. Most DNA viruses can take advantage of the cell’s DNA-dependent RNA polymerase and enzymes used to make mRNA. Poxviruses, which replicate in the cytoplasm, must provide all these functions. Replication of the symple DNA viruses (e.g., parvoviruses, papovaviruses) uses the host DNA-dependent DNA polymerases, whereas the larger, more complex viruses (e.g., adenoviruses, herpesviruses, poxviruses) encode their own polymerases. VIRUS GENETICS Viruses, like prokaryotic and eukaryotic cells, undergo variations during replication. Variations of viruses may be grouped according to the following three categories: mutation, genetic interaction, and nongenetic interaction. Nongenetic interaction is unique to viruses; it does not occur with prokaryotic or eukaryotic cells. Mutation. One type of variation is mutation characterized by a change in the structure of the viral genome during one replication cycle of a single virion. Mutations are commonly due to base substitution, deletions, or rearrangements. Because of the lack of proofreading mechanism during replication, RNA viruses generally have a much higher frequency of mutation than DNA viruses. Genetic interaction. When a single cell is infected by two strains of viruses of the same species, genetic recombination may occur. Both strains of virus contribute a part of their genomes to the recombinant. Nongenetic interaction. When a single cell is infected by two strains of viruses that need not be the same species, nongenetic interaction may occur. In nongenetic interaction, the genomes of progeny virions are derived entirely from the parentral virions of medical importance include complementation, interference, and phenotypic mixing. 1. Complementation. The defect in one virus is complemented by the other virus. For example, the Delta hepatitis virus is defective because it lacks the gene for the surface protein. When the same cell is infected by the hepatitis B virus, the surface protein (coded for by the hepatitis B virus) is formed in abundance and is used by both viruses for virion formation

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2. Interference. Both viruses compete for limiting amount of viral or cell protein essential for virus replication. Consequently, the formation of progeny virions from one or both parentral viruses is partially or completely inhibited. 3. Phenotyping Mixing. The genome of a virus may be wrapped in proteins supplied partly or completed by the other virus. VIRAL DISEASE Basic Steps in Viral Disease Viral disease in the body progresses through defined steps, just like viral replication in the cell. The early steps are as follows: 1. Acquisition (entry into the body) 2. Initiation of infection at a primary site. 3. An incubation period, when the virus is amplified and may spread to a secondary site The incubation period may proceed without symptoms (asymptomatic) or may produce nonspecific early symptoms, teremed the prodrome. The symptoms of the disease are caused by tissue damage and systemic effects caused by the virus and possibly the immune system. These symptoms may continue through the convalescence, while the body repairs the damage. Infection of the Target Tissue The virus gains entry into the body through breaks in the skin or through the mucoepithelial membranes that line the orifices of the body (eyes, respiratory tract, mouth, genitalia, and gastrointestinal tract). The skin is an otherwise excellent barrier to infection, and the orifices are protected by tears, mucus, ciliated epithelium, stomach acid, bile, and immunoglobulin A. Inhalation is probably the most common route of viral Infection On entry into the body, the virus replicates in cells that express viral receptors and that have the appropriate biosynthetic machinery. Many viruses initiate infection in the oral mucosa or upper respiratory tract. Symptoms may accompany viral replication at the primary site. The virus may replicate and remain at the primary site, may disseminate to other tissues via the blood stream or the mononuclear phagocyte and lymphatic system, or may disseminate through neurons. The blood stream and the lymphatic system are the predominant means of viral transfer in the body. The transfer of virus in the blood is termed viremia. The virus may be free in the plasma or may be cell-associated in lymphocytes or macrophages. Viruses teken up by phagocytic macrophages may be inactivated, may replicate, or may be delivered to other tissues by way of the mononuclear phagocyte system.

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Viruses can gain access to the central nervous system or brain (1) from the blood stream, (2) from infected meninges or cerebrospinal fluid, or (3) by means of the migration of infected macrophages or the infection of peripheral and sensory neurons. Host Defenses Against Viral Infection The skin is best barrier to infection, but openings in the skin, whether natural orifies (e.g., mouth, eyes, nose, ears, anus) or due to trauma such as abrasion or puncture, provide pathogens with access to the body. After the virus penetrates the natural barriers, it activates the antigen-nonspecific (innate) immune defenses (e.g., fever, interferon, macrophages, natural killer cells), which attempt to limit and control local viral replication and spread. Antigen-specific immune responses (e.g., antibodies, helper T cells) are the last to be activated. Interferon and cytotoxic T-cell responses may have involved primarily as antiviral defence mechanisms. The ultimate goal of the host response is to eliminate the virus and the cells harboring or replicating the virus. The immune response is the best and in most cases the only means of controlling a viral infection. Both humoral and cellular immune response are important for antiviral immunity. ANTIVIRALS Antivirals are substances capable of intercepting the virus replication cycle. There are literally hundreds of antivirals; but only a few have been approved by the FDA for the prophylaxis or treatment of specific virus infections in humans. The antivirals are grouped according to their modes of antiviral actions. 1. Substances interfering with virus attachment. Immune globulins contain neutralizing antibodies that prevent the attachment of specific viruses to sesceptible cells. In clinical practice, immune globulins are generally given before exposure or during incubation to modify the infection. 2. Interference with penetration or uncoating. Amantadine and rimantadine interfere with the penetration and/or uncoating of many enveloped viruses, but is effective only against influenza A infections in humans. 3. Interference with the synthetic phase. Interferons, Nucleoside analogs (acyclovir and azidovudine), ribavirin, and foscarnet. VIRUS CLASSIFICATION Viruses can be classified according to: 1. Disease or organ system involved 2. Nucleic acid type/virion structure 3. Replication strategy

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DETECTION OF VIRUS INFECTIONS 1. Direct (detection of virus particles, viral antigen, viral lesions or nucleic acid) by: microscopy; electron microscopy; particle agglutination; immunofluorescence; serology; PCR and RT-PCR. 2. Indirect (detection of virus-specific host response) by serology (complement fixation test, haemagglutination inhibition test ELISA etc). MAJOR VIRUS GROUPS DNA VIRUSES THE HERPESVIRUS FAMILY The herpesvirus family is one of the most important family of viruses in medical practice. These viruses are ubiquitous, infect virtually every person, induce a wide variety of diseases, respond to antiviral therapy, and enter into latency after recovery from primary infections. There are eight species of human pathogens in this family of viruses: herpes simplex 1 (HSV 1), herpes simplex 2 (HSV 2), varicella-zoster (VZV), Epstein-Barr (EBV), cytomegalo virus (CMV), human herpes 6 and 7 (HHV 6 and HHV 7), and the recently discovered human herpes 8 (HHV 8) associated with Kaposi’s sarcoma. Family Characteristics. The herpes viruses are large (120-220 nm in diameter), enveloped viruses that contain double-stranded DNA. The DNA core is is surrounded by icosahedral nucleocapsid containing 162 capsomeres. This is enclosed by a glycoprotein-containing envelope. The space between the envelope and the capsid, called the tegument, contains viral proteins and enzymes that help initiate replication. As enveloped viruses, the herpesviruses are sensitive to acid, solvents, detergents, and drying. Its replication strategy is similar to all enveloped DNA viruses. It codes for its own DNA polymerase. There is no DNA homology among herpesvirus species except for the 50% homology between herpes simplex virus types 1 and 2. Pathogenesis and Clinical Syndromes Diseases of Herpes simplex viruses (HSV) types 1 and 2. Primary targets of HSV are the skin and mucosa. The virus multiplies at the implantation site, damages the tissue, and forms a vesiculopapule. Lesions are usually not painful unless irritated. On moist areas, herpetic lesions usually ulcerate forming shallow ulcers. HSV is transmitted by direct contact via vesicle fluid, saliva, and

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gingival secretions. Common sites for herpetic lesions are the skin around the oral and genital orifices, the oral cavity, the cornea, the vagina and cervix, the anal canal, and the urethra. Oral herpes can be caused by HSV-1 or HSV-2. Primary herpetic gingivostomatitis in toddlers and children is almost always caused by HSV-1, whereas young adults may be infected with HSV-1 or HSV-2. The lesions begin as clear vesicles that may be widely distributed throughout the mouth, involving the palate, pharynx, gingivae, buccal mucosa, and tongue. People may experience recurrent mucocutaneous HSV infection (cold sores, fever blisters) even though they never had clinically apparent primary infection. The lesions usually occur at the corners of the mouth or next to the lips. Symptoms of the recurrent episode are less severe, more localized, and of shorter duration than those of a primary episode. Herpetic keratitis is almost always limited to one eye. It can cause recurrent disease, leading to permanent scarring, corneal damage, and blindness. Herpetic whitlow is an infection of the finger. The virus establishes infection through cuts or abrasions in the skin. Herpetic whitlow often occurs in nurses or physicians who attend patients with HSV infections. Eczema herpeticum is acquired by children with active eczema. Genital herpes is usually caused by HSV-2 but can also be caused by HSV-1 (responsible for 10 % of genital infections). Most primary genital infections are asymptomatic. Herpes encephalitis is an acute febrile disease that is usually caused by HSV-1. This disease occurs at all ages and at any time of the year. HSV meningitis is most often a complication of genital HSV-2 infection, and symptoms resolve on their own. HSV infection in the neonate is a devastating and usually fatal disease caused most orten by HSV-2. It may be acquired in utero but more commonly is contracted either during passage of the infant through the genital canal or postnatally from family members or hospital personnel. Treatment: The drug of choice is acyclovir. It is a potent inhibitor against HSV 1 and HSV 2 and is virtually nontoxic to humans. No vaccine or preventable drug is available. Diseases of varicella-zoster virus (VZV). Transmission of varicella is usually via respiratory droplets, which results in initial infection of the respiratory mucosa, followed by spread to the regional lymph nodes. From there, virus enters the blood stream, undergoes a second round of multiplication in cells of the liver and spleen, and is disseminated throughout the body by infected mononuclear leukocytes. Varicella (chickenpox) Infections are highly contagious. Following infection of a normal, healthy child, the first symptoms include fever, malaise, headache, and

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abdominal pain. Next is appearance of the virus-containing vesicles (pox) characteristic of the disease. Chickenpox is a mild-self-limiting disease among children. It can be severe and even fatal in adults, neonates, and the immunodeficient. Zoster (shingles). Due to the disseminated nature of the primary infection, latency is established in the dorsal root ganglion. Reactivation results in shingles (herpes zoster). Viral destruction of sensory ganglia leads to the pain associated with acute zoster. The likelihood of reactivation increases with age and with depressed cellular immune competence. A live attenuated varicella vaccin is recommended as one of the routine childhood vaccines. Acyclovir is used for treatment of VZV. Diseases of epstein-barr virus (EBV) Most transmission of EBV occurs by intimate contact with saliva that contains virus. EBV replicates in mucosal epithelium. The virus then spreads to the lymph nodes, where it infects B cells. EBV next travels via the blood to other organs, particularly targeting liver and spleen. The B cell infection is an abortive one, leading to B cell proliferation, accompanied by nonspecific increases in total IgM, IgG, and IgA. Infectious mononucleosis (IM). The “atypical lymphocytosis” characteristic of IM is caused by the active cytotoxic T cell response to the EBV antigens expressed by infected B cells. The typical IM syndrom appears after an incubation period of four to seven weeks, and includes fever, malaise, pharyngitis, lymphadenopathy (swollen glands), and, often, hepatosplenomegaly. Treatment is by acyclovir. No vaccine or preventable drug is available. Diseases of cytomegalovirus (CMV) Human CMV is transmitted by infected individuals through their tears, urine, saliva, semen or vaginal secretions, and braest milk. CMV can also cross the placenta. Virus is transmitted orally and sexually, in blood transfusions, in tissue transplants, in utero, at birth, and by nursing. CMV replicates initially in epithelial cells or the respiratory and gastrointestinal tracts, followed by viremia and infection of all organs of the body. CMV infectious mononucleosis. Whereas most CMV infections occur in childhood, primary infection as an adult may result in a mononucleosis syndrome that is clinically identical to that caused by EBV. Congenital infection. CMV is the most prevalent viral cause of congenital disease. Approximately 10 % of affected newborns show clinical evidence of disease, such as microcephaly, intracerebral calcification, hepatosplenomegaly, and rash (cytomegalic inclusion disease). Hearing loss and mental retardation are common consequences of congenital CMV infection.

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Disseminated infection occurs in the immunocompromised patients, leading to pneumonia, hepatitis and retinitis. Treatment of the infections is by Ganciclovir. No vaccine or preventable drug is available. Human Herpesviruses 6 and 7 HHV-6 was first isolate from the blood of patients with AIDS and grown in T-cell cultures. In 1988, HHV-6 was serologically associated with a common disease of children, exanthema subitum, commonly known as roseola. HHV-7 was isolated in a similar manner from the T-cells of a patient with AIDS who was also infected with HHV-6. However, HHV-7 remains an orphan virus with no disease association Human Herpesvirus 8 HHV-8 DNA sequences were discovered in biopsy specimens of Kaposi’s sarcoma, primary effusion lymphoma (a rare type of B-cell lymphoma) through the use of PCR analysis. Kaposi’s sarcoma is one of the characteristic opportunistic diseases associated with AIDS. THE POXVIRUS FAMILY Poxviruses are a large, complex group of viruses that cause disease in humans and other animals. Many of the basic characteristics of the poxviruses are uniquely different from those of other viruses. Some virologists have questiond the validity of classifying the poxviruses as viruses. Poxvirus are the largest viruses, measuring 230-300 nm in size, and are ovoid to brick-shaped. They have a capsid that is referred to as complex because it hase neither helical nor icosahedral symmetry. An outer membrane and envelope enclose the core and core membrane, which are flanked by two lateral bodies of unknown function. The viral genome is a double-stranded DNA. Three species are of medical interest: smallpox, vaccinia, and molluscum contagiosum viruses. Smallpox has been eradicated through a worldwide effort of vaccination case finding and quarantine. Vacciniavirus is the smallpox vaccin virus. Smallpox vaccination is not an innocuous procedure and is no longer recomended for universal usage. The vaccinvirus is now used extensively as vectors for selected genes (e.g., HbsAg, HIV gp 120, ect.). Molluscum contagiosum virus causes a benign self-limiting skin lesion characterized by umbilicated pearlike nodules.

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THE ADENOVIRUS FAMILY Adenoviruses are nonenveloped double-stranded DNA viruses, have icosahedral capsid symmetry, and measure about 80 nm in diameter. Virus encodes proteins to promote messenger RNA and DNA synthesis, including its own DNA polymerase. At least 47 serotypes are infectious for humans. Adenovirus resist drying, detergents, gastrointestinal tract secretions and even mild chlorine treatment. They can therefore be spread by the fecal-oral route, by fingers, by fomites (including towels and medical instruments), and in poorly chlorinated swimming pools. Primary targets are the epithelium of the respiratory and/or intestinal tract. Diseases caused by adenoviruses include acute respiratory infection, conjunctivitis (pink eye), hemorhagic cystitis and gastroenteritis. Virologic diagnosis is unnecessary in clinical practice and specific antiviral treatment is not available. THE PAPOVAVIRUS FAMILY Several members of the papovavirus family are common infectious agents of humans. The main interest in this family of viruses is their oncogenic potential. The medically important viruses belong to the genus Papillomavirus. There are many species or types of papillomaviruses; at least 70 types are infectious for humans. The papovaviruses are small, nonenveloped icosahedral capsid viruses with double-stranded and circular DNA genomes. Virus is acquired by close contact and infects epithelial cells of the skin or mucous membranes. Virus multiplication results in cell proliferation, and cell proliferation results in circumscribed tumor (wart). Disease of Papovaviruses Warts A warts is a benign, self-limited proliferation of skin that progresses with time. The appearance of the wart (domeshaped, flat, or plantar) depends on the HPV type and the infected site. Benign Head and Neck Tumors Single oral papillomas are the most benign epithelial tumors of the oral cavity Laryngeal papilloma Conjunctival papilloma

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Treatment and control Warts spontaneously regress, but the regression may take many months to years. Warts are removed because of pain and discomfort, for cosmetic reasons, and to prevent to spread to other parts of the body or to other people. At present, the best way to prevent transmission of warts is to avoid coming in direct contact with infected tissue. RNA VIRUSES THE ORTHOMYXOVIRUS FAMILY Orthomyxoviruses are the etiologic agent of influenza, which is one of the last few major epidemic diseases that inflict human. Family Characteristics. The genome of orthomyxovirus is single-stranded negative sense and segmented RNA. The virion of the virus is enveloped, has helical nucleocapsid, and contains RNA dependend RNA polymerase. The envelope contains two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), and is internally lined by the matrix (M1) and membrane (M2) proteins. The HA, has several functions: it is the viral attachment protein, binding to sialic acid on epithelial cell surface receptors; it promotes fusion of the envelope to the cell membrane; it hemagglutinates (binds and aggregates) human, chicken, and guinea pig red blood cells; and it elicits the protective antibody response. The NA, is facilitates the release of virus from infected cells Types and subtypes of the family. Influenza viruses are classified as types A, B, and C depending on their inner proteins, mainly the M proteins. Thus, all type A viruses share common internal antigens that are distinct from those shared by all type B viruses. Only the type A viruses are broken down into subtypes. The classification into subtypes depends on antigens associated with the outer viral proteins, H and N. Taking into consideration animal as well as human influenza viruses, 14 H and 9 N subtypes have been described. However, among human influenza viruses, only three H (H1, H2, and H3) and two N (N1 and N2) subtypes are found. Human influenza viruses are therefore designated, for example, as subtype H1N1, H2N2, H3N2, etc. Antigenic Variations. Influenzaviruses have a propensity of undergoing antigenic variations. Immunity acquired through infection by one virus is frequently insufficient to prevent infection by a variant. This antigenic variation is the main reason why it is difficult to prevent influenza through immunization and why recurrences of influenza in the same person are common. Key points to understand about antigenic variation are:

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1. Major antigenic change (antigenic shift) is due to the replasement of one H (or N) gene by another. After the shift, H of the virion is totally different immunologically from those before the shift. This process occurs only with the influenza A. Such changes are often associated with the occurence of pandemics. Antigenic shifts occur infrequently, taking place on average 10 years. 2. Minor antigenic change (antigenic drift) is due to mutation of the H (or N) gene of types A and B viruses. This process occurs every 2 to 3 years, causing local outbreaks of influenza A and B infection. Immunity acquired through infection with parentral virus is less effective against the mutant virus. Epidemiology Strains of influenza A virus are classified by the following four characteristics: 1. Type (A, B, and C) 2. Place of original isolation 3. Date of original isolation 4. Antigen (H and N) For example, a current strain of influenza virus might be designated A/Bangkok/1/79 (H3N2), meaning that it is an influenza A virus that was first isolated in Bangkok in January 1979 and contains H3 and N2 antigens. Strains of influenza B are designated by (1) type, (2) geography, and (3) date of isolation, but without specific mention of H and N antigens because influenza B does not undergo antigenic shift and pandemics like influenza A. New influenza A strains are generated through mutation and reassortment. The genetic diversity of influenza A is fostered by its segmented genomic structure and ability to infect and replicate in humans and many animal species (zoonose), including birds and pigs. Hybrid viruses are created by co-infection of a cell with different strains of influenza A virus, allowing the genomic segments to randomly associated into new virions. An exchange of the HA glycoproteins may generate an new virus that can infect an immunologically naive human population. In 1997, an A/Hong Kong (H5N1) strain was isolated from at least 18 humans and caused 6 deaths. The virus resembled a chicken virus, A/Chicken/Hong Kong (H5N1), leading to the destruction of all 1.6 million chickens in Hong Kong to destroy the potential source of the virus. Influenza infection is spread readily via small airborne droplets expelled during talking, breathing, and coughing. Children, immunosuppressed people (including pregnant women), the elderly, and people with heart and lung ailments (including smokers) are at highest risk for more serious disease, pneumonia, or other complications.

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Influenza Type A. The primary target of the influenza virus type A is the respiratory tract. The virus is deposited on the respiratory tract through inhalation of droplets that contain the virus or through the direct contact with respiratory secretion positive for the virus. After an incubation period of 1 to 4 days, the “flu syndrome” begins with a brief prodrome of malaise and headache lasting a few hours. The prodrome is followed by the additional abrupt onset of fever, severe myalgia, and usually a nonproductive cough. The illness persists for approximately 3 days, and unless a complication occurs, recovery is complete within 7 to 10 days. Influenza in young children resembles other severe respiratory tract infections, causing bronchiolitis, croup, otitis media, and rarely febrile convulsions. Complications of influenza include bacterial pneumonia and myositis Early diagnosis and prompt treatment of secondary bacterial pneumonia are important in the management of influenza. Annual administration of influenza vaccin (inactivated) provides about 80% protection. Amantadine, if given within 1 or 2 days after onset, is effective in shortening the course and reducing the severity. Influenza Type B. Pathogenesis, diagnosis, treatment, epidemiology, and prevention of type B virus infections are similar to type A virus infections except : (1) amantadine is ineffective against type B virus and (2) genes that code for the H and N antigens of the type B virus are monomorphic. Therefore, type B virus does not undergo antigenic shift and does not cause pandemic. Influenza Type C. Type C virus infections are uncommon and have little medical importance. Laboratory Diagnosis The occurrence of the characteristic symptoms of influenza in a person during a community outbreak of the infection is often sufficient to the diagnosis. Laboratory methods can distinguish influenza from other respiratory viruses and identify its type and strain. Influenza viruses are obtained from respiratory secretions. 1. Cell culture in primary monkey kidney. A presence of virus and limited cytopathologic effects may be noted within as few as 2 days. Before the cytopathojogic effects develop, the addition of guinea pig erythrocytes may reveal hemadsorption (the adherence of these erythrocytes to HA-expressing infected cells). The addition of influenza virus-containing media to erythrocytes promotes the formation of a gel-like aggregate due to hemagglutination. Hemadsorption and hemagglutination are not specific to influenza viruses.

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2. Immunologic tests. Specific identification of the influenza virus requires immunologic tests, such as immunofluorescence or inhibition of hemadsorption and hemagglutination with specific antibody. 3. Serologic studies. ELISA or IF tests can be used to detect viral antigen in respiratory secretions or cell cultures and are more sensitive assays. Serologic studies do not aid in the diagnosis of an influenza infection and are generally used for epidemiologic purposes. Treatment and Prevention 1. Amantadine and rimantadine are first-generation antiviral agents effective against influenza A. Both drugs stop viral uncoating step by inhibition of the viral M2 membrane protein. These agents reduce both the duration and the severity of flu symptoms, but only if given early in infection (during the first 24 to 48 hours). 2. Zanamivir and oseltamivir are second-generation antiviral agents effective against influenza A and B. They inhibit viral neuraminidase, which is present in both influenza A and B viruses. [ neuraminidase—an enzyme essential for viral replication and the release of the virus from infected cells]. These drugs are effective only if given early in infection. 3. Vaccine. The airborne spread of influenza is almost impossible to limit. However, the best way to control the virau is through immunization. Killed (formalininactivated) influenza vaccine is available every year. Killed whole-virus vaccines are prepared from virus grown in embryonated eggs and then chemically inactivated. THE PARAMYXOVIRUS FAMILY Paramyxoviruses are enveloped, have helical nucleocapsid, and vary from 100 to 300 nm in diameter. The genomes of the virus family are single-stranded, negative sense, and nonsegmented RNA. This family of viruses includes the following human pathogens: measles, mumps, respiratory syncytial, and parainfluenza viruses. Measlesvirus is the cause of measles disease. Measles, which can be spread in respiratory secretions before and after the onset of characteristic symptoms, is one of the most contagious known. The virus multiplies at sites of implantation (upper respiratory). This multiplication is followed by a viremic phase during which the virus is widely disseminated throughout the body. The prodrome starts with high fever, cough, coryza, and conjunctivitis, in addition to photophobia. At this time the patient is most infectious. After 2 days of illness, the characteristic skin rash appear.

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One rare but conceptually important complication is subacute sclerosing panencephalitis (SSPE). In SSPE the measles virus persists for an unknown reason in the brain and acts as slow virus. Many months or years after clinical measles, the patient develops changes in personality, behavior, and memory. Blindness and spasticity follow. No specific virologic diagnosis is required. Clinical manifestations are sufficient for the diagnosis of measles. There is no specific antiviral agent against the measlesvirus. Preventive measures include immunization and the administration of immunoglobulins after exposure. Measles vaccine contains live attenuated measlesvirus. It is generally administered together with live attenuated mumps and rubellaviruses as the measles, mumps, and rubella (MMR) vaccine. Two doses of MMR vaccine given at least 1 month apart are recommended for all persons who are 15 months old. Mumpsvirus is the cause of acute benign viral parotitis; multiplies at implantation site (upper respiratory tract and reginal lymphnodes). Mumps, like measles, is a very communicable disease with only humans. The virus is spread by direct person-toperson contact and respiratory droplets. The symptomatic virus multiplication is followed by a viremic phase during which the virus is widely disseminated. Clinically important secondary targets include salivary glands, tests, meninges, and the brain. Usually, clinical illness is manifested as a parotitis that is almost always bilateral but may appear first in one parotid gland and then in the other. Orchitis (usually unilateral), oophoritis, pancreatitis, and meningoencephalitis may occur a few days after the onset of the virus infection but can occur in the absence of parotitis. Diagnosis is made by clinical manifestations and serology. Specific treatment is not available. Respiratory syncytial and parainfluenzaviruses produce superficial infections of the respiratory tract and are important pathogens associated with acute lower respiratory illness in infants and children. THE TOGAVIRUS FAMILY Togavirus is spherical and about 60 nm in diameter. It has an icosahedral capsid symmetry and an envelope. The genome of the virus is a single-stranded positive sense RNA. Medically important species of the family include the rubella, western equine encephalitis, and eastern equine encephalitis viruses. The pathogenesis of rubella is similar to measles except it is a mild disease. Primary infection during the first 12 weeks of pregnancy often leads to generalized fetal infection leading to congenital rubella syndrome (cataract, nerve defness, cardiac abnormalities, hepatosplenomegaly, purpura, and jaundice).

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THE RHABDOVIRUS FAMILY Rhabdoviruses are enveloped and have helical capsid symmetry. These viruses have a bullet-shaped morphology that is unique for this family. The genome is a single-stranded negatine sense RNA. The rabiesvirus is the only medically important species of rhabdoviruses. This is the cause of rabies: an acute lethal infection of the central nervous system. An epidemiologically important source of rabiesvirus is the sliva of the infected animals. Rabiesvirus has a wide animal reservoir (e.g., foxes, skunks, racoons, vampire bats). But, the principal reservoir for rabies in most of the world is the dog. Pathogenesis and Clinical manifestations. The rabiesvirus enters the human body through the bite of rabid animals. Multiplication takes place in muscle cells and the virus migrates along peripheral nerves to the CNS. Replication in the CNS causes nerve cell destruction and inclusion bodies (Negri bodies). Migration along peripheral nerves to the salivary glands also occurs. Following a highly variable incubation period, the prodrome phase of rabies ensues. Symptoms such as fever, malaise, headache, pain or itching at the site of the bite, fatigue, and anorexia typically occur. After a prodrome of 2 to 10 days, neurologic symptoms specific for rabies appear. Hydrophobia, the most characteristic symptoms of rabies, occurs in 20% to 50% of patients. It is marked by violent, jerky contractions of the diaphragm triggered by the patient’s attempts to swallow water. Focal and generalized seizures, disorientation, and hallucinations also frequently ocuur during the neurologic phase. Following the neurologic phase, which lasts from 2 to 10 days, the comatose phase developes. The phase almost universally leads to death resulting from neurologic and pulmonary complications. Diagnosis. In the management of animal bites, it is crucial to know if the biting animal is rabid. The method of choice is to detect rabies antigens in the brain smear of the suspected animal by immunofluorescence. Detection of intracytoplasmic negri bodies in affected neurons also used. Negri bodies are found within infected neurons and are matrices of viral nucleocapcids. Treatment. Specific antiviral traetment for rabies is not available. Prevention and Contral. Preexposure: 1. Mandatory immunization of dogs and cats 2. Immunization of persons at high risk of animal bites (e.g., veterinarians, park rangers, animal handlers, etc.). Postexposure:

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1. The first protective measure of major importance is local treatment of the wound. Washing the wound with soap and water, detergent, or another substance that inactivates the virus should be done immediately. 2. Postexposure immunoprophylaxis consists of the local infiltration and parenteral administration of human rabies immunoglobulins and a coursr of rabies vaccin. Rabies vaccin for human use contains inactivated rabies virus. THE PICORNAVIRUS FAMILY Picornaviridae is one of the largest families of viruses and includes some of the most important human and animal viruses. They are smal (28-30 nm in diameter), icosahedral, non-enveloped and have non-segmented single-stranded positive sense RNA. The family has more than 230 members that are divided into five genera: Enterovirus, Rhinovirus, Heparnavirus, Cardiovirus and Aphthovirus. Enteroviruses At least 72 serotypes of human enteroviruses exist, including the polioviruses, coxsackiviruses, and echoviruses. Hepatitis A virus was included in this group, but has been reclassified as a hepatovirus in the Heparnavirus genus, and is discussed in Hepatitis viruses. The capsids of these viruses are very resistant to harsh enviromental conditions and the conditions in the gastrointestinal tract, a fact that facilitates their transmission by the fecal-oral route. However, even though they may initiate infection in the gastrointestinal tract, the enteroviruses rarely cause enteric disease. Enterovirus Infections Enterovirus infections occur primarily through the ingestion of contaminated food or drinking contaminated water. The primary target is the intestinal mucosa including the lymphoid tissue. Multiplication in the primary target may be followed by transient viremia and dissemination of virus to secondary targets. Important secondary targets are: 1. Poliovirus infection: Poliomyelitis is an acute illnes in which the poliovirus selectively destroys the lower motor neurons of the spinal cord and brainstem, resulting in flaccid, asymmetrical weakness or paralysis. The classic presentation of paralytic poliomelitis is flaccid paralysis, most often affecting the lower limbs. This is due to viral replication in, and destruction of, the lower motor neurons in the anterior horn of the spinal cord. Respiratory paralysis may also occur, following infection of the brain stem. Specific antiviral agents for the treatment of poliomyelitis are not available. Management, therefore, is supportive and

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symptomatic. Vaccination is the only effective method of preventing poliomyelitis. Two types of poliovirus vaccine exist, a formalin-inactivated product known as the inactivated, killed, or Salk vaccine and an attenuated one known as the live, oral, or Sabin vaccine. Both vaccines can induce a protective antibody response. 2. Coxsackievirus and echovirus infections: These give rise to a large variety of clinical syndromes including: . Aseptic meningitis. May be caused by either a coxsackivirus or an echovirus . Herpangina (severe sore throat with vesicular ulcerated lesions around the soft palate and uvula) is caused by several type of coxsacki A virus . Hand-food-and-mouth disease (vesicular lesions on the hands, feet, mouth, and toungue) is caused by coxsacki A virus. . Pleurisy is an acute illness in which patients have an sudden onset of fever and unilateral low thoracic, pleuritic chest pain that may be excruciating. Coxsacki B virus is the causative agent. . Pericarditis, and myocardial infections caused by coxsack B virus occur sporadically in older children and adults but are most threatening in newborns. There are no vaccines for coxsackiviruses or echoviruses. Transmission of these viruses can presumably be reduced by improvments in hygiene and living condisions. Rhinoviruses Rhinoviruses are the most important cause of the common cold and upper respiratory tract infections. More than 100 serotypes of rhinovirus have been identified. Pathogenesis and Immunology Unlike the enteroviruses, rhinoviruses are unable to replicate in the gastrointestinal tract. The rhinoviruses are labile to acidic pH. Also, they grow best at 33ºC, a feature that may partly account for their predilection for the cooler enviroment of the nasal mucosa. Infection can be initiated by as little as one infectious viral particle. The virus enters through the nose, mouth, or eyes and initiates infection of the upper respiratory tract, including the throat. Most viral replication occurs in the nose, and the onset and the sevirity of the symptoms correlate with the time of viral shedding and the quantity of virus shed. Infected cells release bradykinin and histamine, which cause a “runny nose”. Immunity to rhinoviruses is transient and is unlikely to prevent subsequent infection because of the numerous serotypes of the virus.

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Epidemiology Rhinoviruses cause at least half of all upper respiratory tract infections. Other agents that cause the symptoms of the common cold are enteroviruses, adenoviruses, and parainfluenza viruses. Rhinoviruses can be transmitted by two mechanisms, as aerosoles and on fomites (e.g., by hands or on contaminated inanimate objects). Hands appear to be the major vector, and direct person-to-person contact is the predominant mode of spread. These nonenveloped viruses are extremely stable and can survive on such objects for many hours. Clinical Syndromes Common cold symptoms caused by rhinoviruses cannot readily be distinguished from those caused by other viral respiratory pathogens. An upper respiratory tract infection usually begins with sneezing, which is soon followed by rhinorrhea (runny nose), nasal obstruction, mild sore throat, headache, and malaise. The illness peaks in 3 to 4 days, but the cough and nasal symptoms may persist for 7 to 10 days or longer. Fever and rigors sometimes accompany rhinovirus infections. Otitis media in children and sinusitis in adults are common complication. Laboratory Diagnosis The clinical syndrome of the common cold is usually so characteristic that laboratory diagnosis is unnecessary. Treatment and Prevention Treatment is symptomatic with bed rest in severe cases. Rhinovirus is not a good candidate for a vaccine program. The multiple serotypes, the requirement for secretory IgA production, and the apparent antigenic drift in rhinoviral antigens pose major problems for the development of vaccines. HEPATITIS VIRUSES Acute hepatitis is a common disease; most are due to virus infections. Viruses that are primarily hepatotropic are frequently referred to as hepatitis viruses. There at least six hepatitis viruses pathogenic for humans; they are hepatitis A, B, C, D, E and G viruses. Although the target organ for each of these viruses is the liver, they differ greatly in their structure, mode of replication, and the mode of tranmission and in the course of the disease they cause. Each of the hepatitis viruses infects and damages the liver, causing the classic icteric symptoms of jaundice and the release of liver enzymes. These viruses are readily spread because infected people are contagious before, or even without, showing symptoms.

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HEPATITIS A VIRUS (HAV) Hepatitis A virus causes infectious hepatitis and is spread by the fecal-oral rout and occurs mainly in children and young adults. This virus is the member of picornavirus family and renamed enterovirus 72. It has the structural characteristic of a picornavirus. There is only one serotype of HAV. Epidemiology. Approximately 40% of acute hepatitis cases result from HAV. Spraed is by person-to-person contact, usually via a fecal-oral rout, or by exposure to contaminated food or water. The virus is present in blood only transiently, but in feces high concentrations of virus are present for several weeks, especially in the 2 weeks before the onset of jaundice. Virus is not present in urine or other body fluids, and a chronic carrier state does not occur. HAV is distinguished by it is ability to survive many months in fresh and salt water. HAV is resistant to detergents, acid, and temperatures elevated as high as 60˚C, which promotes its spread into food, even after handwashing. Pathogenesis. HAV is acquired by ingestion, probably replicates in the oropharynx and epithelial lining of the intestines, initiates a transient viremia, and is targeted to the liver. The virus binds to receptors on parenchymal cells of the liver and replicates. Virus is produced in these cells and is released into the bile and from there into the stool. Virus is shed into the stool approximately 10 days before symptoms or antibody can be detected. There is no chronic disease or carrier state with HAV. Clinical Manifestations. The incubation period is 15 to 50 days. This is followed by a typical viral-type illness with malaise, lassitude, myalgia, arthralgia and variable fever. Features of hepatitis gradually replace the prodromal illness after 2-7 days. Jaundice then developes, first seen in the sclerae and later in the skin. The fever resolves as jaundice becomes established. At this stage virus excretion ceases and the patient is no longer infectious. Most patients feel better once the jaundice appears. After a few days the appetite returns and the jaundice begins to resolve. Diagnosis. This is made by serology (ELISA) testing for specific HAV IgM (acute infection) or IgG (immune status). Treatment. Symptomatic Prevention. Passive immunoprophylaxis against the HAV are available. A killed vaccin is available.

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HEPATITIS B VIRUS (HBV) Hepatitis B virus is an exceedingly important virus in medical practice. HBV infections are common and worldwide in distribution. Infections by the HBV may result in acute hepatitis, chronic hepatitis, cirrhosis, and hepatoma. Structure of The Vırus. HBV is the member of the hepadnavirus, a small (42 nm in diameter) family of DNA viruses. Hepatitis B is an enveloped virus with icosahedral capsid symmetry. The genome is a circular, double-stranded DNA, encodes a reverse transcriptase, and replicates through an RNA intermediate. The nucleocapsid of HBV also includes a DNA polymerase and a protein kinase surrounded by the core antigen (HbcAg) and the hepatitis HBe antigen. HBcAg is the major component of the nucleocapsid. HbeAg is a cleavage product of the core antigen found on infected cells or free in serum. The surface antigen (HbsAg) originally termed the Australia antigen, is found in the envelope. The HbsAg elicits protective immunity Replication of The Virus. On penetration into the cell, the nucleocapsid and genome are delivered to the nucleus. The partial strand of the genome is completed to form a complete double-strand DNA circule and then transcribed into individual mRNAs by the host DNA-dependent RNA polymerase. A full-sized mRNA transcript is also synthesized as a template for replication of the genome. As with the retroviruses, HBV uses a reverse transcriptase to synthesize a complementary DNA (cDNA) from the full-seized positive strand RNA. Unlike the retrovirus, the cDNA is the negative strand of the virion rather than an intermediate from the genome to be integrated. The RNA template and polymerase are encapsulated within the nucleocapsid, and negative –starnd DNA synthesis occurs. The RNA is degraded as the DNA is synthesized. The positive-strand DNA is then synthesized from the negative DNA template as long as substrates are available. Pathogenesis. HBV typically causes an acute hepatitis which is characterized by liver cell necrosis and periportal histiocytic infiltration. The necrosis is centrilobular and multifocal. Rarely, massive necrosis occurs associated with considerable disruption of the reticulin framework of the liver. In a proportion estimated at approximately 10% of patients, chronic hepatitis occurs, associated with chronic persistent infection. Liver carcinoma is an important complication of chronic hepatitis with an excess risk of 100-fold. Clinical Manifestations. The incubation period is long from 3 to 6 months. It is followed by a prodromal viral illness and then a period of afebrile jaundice very similar to that of hepatitis A. Clinical distinction of acute hepatitis A from hepatitis B is rarely possible but cholestasis, common in convalecent hepatitis A, is rare in hepatitis B.

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Chronic hepatitis B occurs in 5% to 10% of HBV infections, usually after mild or inapparent initial disease. In up to 10% of patients may cause cirrhosis and liver failure. Epidemiology. It is estimated that there are 400-500 chronic carriers of the hepatitis B virus worldwide. The incidence of acute disease and prevalence of carriage varies considerably from country to country. In some parts of South-east Asia, 10-20% of the population may be carriers, whereas most countries in Europe and North America have carriage rates below 2%. Where carriage rates are high, acute infection occurs mainly in infants and young children. The routes of spread of hepatitis B are percutaneous (e.g., needle sharing, acupuncture, ear piercing, tatooing) or by very close personal contact with exchange of secretions (e.g., sexual, childbirth). The virus can be transmitted by contaminated blood or blood products. Laboratory Diagnosis. The initial diagnosis of hepatitis can be made from the clinical symptoms and the presence of liver enzymes in the blood. However, identification of the specific viral agent requires quantitation of the HBsAg and evaluation of the antibody response to HBsAg and HBcAg. HBsAg, HBcAg, and HbeAg are secreted into the blood during virus replication. The HbsAg and HbcAg are particulate, whereas the HbeAg is hidden, within the virion. No test is readily available for HbcAg, but HbsAg and HbeAg can be detected in clinical laboratory assays. Detection of HbeAg is the best correlate to the presence of active infection. Antibodies are produced against HBcAg and HbsAg, but so much HbsAg is produced during the symptomatic phase of infection that the antibody is complexed and not detectable. Indication of recent acute infection, especially during the period when neither HbsAg nor anti-HBs can be detected, is best established by measurment of IgM anti-HBc. Table 2
Pattern 1 2 3 4 5 6

Interpretation of Serologic Markers of HBV Infection
HbsAg + + + Anti-HBs + + + Anti-HBc + + + + Interpretation Early acute type B hepatitis. Acute* or chronic type B hepatitis. Acute* or chronic type B hepatitis. Recovery from type B hepatitis. Immediate recovery phase from type B* or low-level carrier state. Long after HBV infection or immunization with HBsAg.

* Anti-HBc IgM should be present.

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Treatment. There is no specific treatment of acute hepatitis B. Cases are therefore managed symptomatically, as for hepatitis A. Interferon-α has been found effective in the treatment of some cases of chronic HBV hepatitis. Prevention and Control Immunization. Both active and passive immunization against hepatitis B is possible. Hepatitis B vaccines contain HbsAg either derived from human plasma, or produced from yeast cells using recombinant DNA technology. A course of three injections provides active protection which lasts for 3-5 years. As approximately 10% of recipients do not produce an antibody response, postvaccination screening should be done and a booster dose given if necessary. A specific immunoglobulin (HBIG) prepared from the plasma of selected donors provides passive protection against hepatitis B. It is normally used in combination with vaccine to provide passive/active immunity: 1. For infants born to mothers who are HbsAg positive. 2. Following percutaneous or mucosal exposure to infected body fluids. 3. For sexual contacts of acute cases. 1. 2. 3. 4. Other measures. Universal testing of donated blood products. Use of condoms. Use of clean needles and other skin-piercing equipment. Carriers of HbsAg may be restricted from certain occupations. The disease is notifiable. HEPATITIS C VIRUS (HCV) HCV is the main causetive agent of parenterally transmitted non-A, non-B hepatitis. It is a new genus of the flavivirus family; possesses a single, positive RNA strand. This virus was identified by molecular biologic means in 1989 by screening infected chimpanzee blood for a viral RNA. These studies led to the development of ELISA and other tests for detection of the virus, which still cannot be grown in tissue culture. Pathogenesis. HCV has similar pathogenesis to HBV; the acute hepatitis is followed by chronic hepatitis at a high incidence (30-50%) with the risk of late cirrhosis or hepatocellular carcinoma. Apparently, HCV persists for many years in the host after clinical recovery. Clinical Manifestations. The incubation period of blood-borne hepatitis C is usually 3 or 4 weeks. A typical illness with malaise followed by jaundice is usual. Many cases are subclinical; the likelihood of early liver failure is not yet quantified.

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Epidemiology. The disease is transmitted by exposure to blood and blood fluids, probably contaminated blood transfusions (HCV is less efficiently transmitted than HBV). However, spread through contaminated needles and syringes is also important. Sexual tranmission probably occurs, but much less readily than with hepatitis B. Vertical transmission from an infected mothers is uncommon. Laboratory Diagnosis. Diagnosis is usually established by exclusion of hepatitis A and B. The HCV antigens produced recently by recombinant DNA technology appear to be specific and sensitive in detecting anti-HCV. This is made by ELISA test. In chronic hepatitis C virus infection, detection of antibodies is not sufficient to make a diagnosis. As the virus cannot yet be cultured, the only methods to detect persisting virus are detection of hepatitis C virus RNA by dot-blot hybridization or by PCR. Treatment. There is no specific treatment. Prolonged administration of interferonα to chronic HCV patients has been found beneficial in about 50% of the patients. Prevention. General measures of the prevention of hepatitis B apply also to hepatitis C. There is no vaccine or specific immunoglobulin available. HEPATITIS D VIRUS (HDV) Hepatitis D, or delta hepatitis, is caused by a defective setellite virus which cannot invade cells in the absence of HbsAg. It therefore only effects patients during the antigen-positive stages of acute hepatitis B, or long-term HbsAg carriers. Simultaneous infection with hepatitis B and D is thought to cause more severe disease than hepatitis B alone. Structure of The Virus. Hepatitis D virus, the delta agent, is an enveloped RNA virus of similar size to hepatitis B virus. The HDV is require a helper virus for replication. The RNA genome of this virus is so small that it cannot encode all the functions necessary for it is replication and structure. The presence of hepatitis B virus is required to provide the envelope proteins (HbsAg), enabiling hepatitis D virus to spraed from cell to cell, and to express it is pathogenic potential. Pathogenesis. The coexistence of hepatitis D and B virus is associated with an accelerated progression to carcinoma. Delta antigen and IgM antibody are detectable in the blood during infection. Clinical Manifestations. The delta agent increases the severity of hepatitis B infections. İndividuals infected with delta agent are much more likely to develop fulminant hepatitis than with the other hepatitides.

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Chronic infection with delta agent can occur in those individuals with chronic HBV. Laboratory Diagnosis. The diagnosis of hepatitis D generally rests on the finding of anti-HDV and HBsAg in the serum by ELISA test. Epidemiology. Infection due to hepatitis D is always associated with a coexistent hepatitis B infection, sharing its routes of transmission. The distributions of the two disease are therefore similar. Prevention and Control. Control measures are the same as for hepatitis B, although no specific immunoglobulin or vaccine exists. Controling the spraed of hepatitis B prevents the occurrence of hepatitis D. HEPATITIS E VIRUS (HEV) There is still another type of non-A non-B hepatitis that occurs primarily in less developed countries and in epidemics. This type of infections is enterically transmitted and is caused by a nonenveloped RNA virus about 30 nm in diameter. It does not cross react serologically with hepatitis A, B, C, or D viruses. It has tentatively named hepatitis E virus and related to caliciviruses. Pathogenesis may be similar to hepatitis A. Diagnosis is based on epidemiology and exclusion of other hepatitis. In research laboratories, immunoelectron microscopic examination of feces is useful. The mortality rate associated with HEV disease is 1% to 2%, approximately 10 times that associated with HAV disease. HEV infection is especially serious in pregnant women (mortality rate of approximately 20%). Specific antiviral treatment is not available. Prevention depends on strategy for minimizing fecal-oral transmission of infectious agents. HEPATITIS G VIRUS (HGV) HGV resembles HCV in many ways. HGV is a flavivirus, is transmitted in blood, and has a predilection for chronic hepatitis disease. HGV is identified by detection of the genome RNA. HUMAN IMMUNODEFICIENCY VIRUSES AND ACQUIRED IMMUNODEFICIENCY SYNDROME Acquired immunodeficiency syndrome (AIDS) was firs recognized as a clinical entity in the early 1980s among homosexual men in USA, although the causal agent and routes of transmission were not identified for some years. The number of cases

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has risen exponentially, and it is now estimated that over 40 million cases have occurred worldwide. Two-thirds of AIDS cases have occurred in Africa. Structure of The Virus. The human immunodeficiency virus (HIV) is an enveloped RNA virus belonging to the lentivirus subfamily of Retrovirus family. Two species, HIV 1 and HIV 2, are medically important. Both HIV 1 and HIV 2 show a 50% nucleic acid homology. Their p24 cross-react in serologic tests. Both causes AIDS in humans but infections by HIV 2 tend to be less severe and spread less efficiently. The HIV 2 infections are found mostly in West Africa while HIV 1 are now worldwide in distribution. The HIV genome consists of three structural genes (gag, pol, and env) and several regulatory genes (tat, nef, etc.) flanked by regulatory sequences known as lateral terminal repeats (LTR) Gag gene product is cleaved by a virus specified protease into three core proteins, one of which is p24 antigen; pol gene product, into three enzymes (reveres transcriptase, protease, and endonuclease); and env gene product, into two envelope antigens (gp 120 and gp 41). Key virion components are: 1. The gp 120 protein is the attachment site for the virion, and it is antibodies against gp 120 that neutralize the virus. 2. The gp 41 protein causes the viral envelope to fuse with the cell membrane of the host; it is also a key antigen in the diagnosis of HIV infection. 3. The p24 protein is detectable in early infection . 4. Reverse transcriptase (RT) is the virion associated polymerase that is essential in initiating the synthetic phase of the HIV replication cycle. The HIV mutates at very high freguency during its replication. Presumably, the mutations occur during the reveres transcription of the viral genome to proviral DNA. Therefore, HIV isolates are very heterogenous genomically, serologically, and biologically. The coexistence of genomically distinct HIV in the same patient has been documented. Mutation may effect the env gene (coding for gp 120) and produces mutants that are poorly neutralized by existing neutralizing antibody. Virus isolates may differ in cell tropism. For examples, some isolates grow chiefly in CD4 T cells, others grow chiefly in macrophages, and still others grow in both CD4 T cells and macrophages. Virus isolates may also vary in their ability to productively infect cells. Replication 1. Adsorption to virus-specific receptor on CD4 cells; and receptor-mediated endocytosis. 2. Synthesis of double-stranded complementary DNA (cDNA) by viral reveres transcriptase.

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3. Integration of proviral DNA into cellular chromosome leading to a state of chronic infection. 4. Transcription of cDNA to yield positive-stranded RNA which acts either as massenger RNA for viral protein synthesis or as viral genomic RNA. 5. Intracytoplasmic morphogenesis. 6. Release of virus particles by budding. Pathogenesis and Immunity The principal cell receptor for HIV attachment is the CD4 molecul. As expected, cells rich in CD4 molecules, such as CD4 T-lymphocytes and subpopulation of macrophages, are the principal target cells of HIV. HIV-induced immunosuppression (AIDS) results from a reduction in the number of CD4 T cells, which decimates the helper and delayed-type hypersensitivity functions of the immune response. During vaginal or anal sexual intercourse, HIV infects Langerhans dendritic cells in the epithelium, and these can then travel to lymph nodes. Anal sex may be of greater risk than other routes of infection. On injection of virus into blood, the virus is likely to infect dendritic and other monocyte-macrophage lineage cells. The virus reaches the lymph node within 2 days of infection, and there the CD4 T cells are infected. Macrophages express lesser amounts of CD4 than T cells but can still be infected with HIV. The virus does not kill the cells but may alter their function. Monocytes and macrophages are probably the major reservoirs and means of distribution for HIV. After infection of these cells, the virus replicates but rapidly established latency. The virus may remain latent for long periods, but when activated in CD4 T cells, the virus kills the cell. Reductions in the numbers of CD4 T cells may result from HIVinduced cytolysis and cytotoxic T-cell immune cytolysis. HIV inducess several cytopathic effects that may kill the cell. These include accumulation of nonintegrated circular DNA copies of the genome, increased permeability of the plasma membrane, and syncytia formation (formation of multinucleated cells). Changes in cell function also occur in CD4 T cells latently infected with HIV. HIV infection can reduce the expression of CD4 antigens and production of interleukin (IL-2), reducing the ability of the helper cell to respond to antigen and activate other T cells. The immune response to HIV restricts viral infection but contributes to pathogenesis. Neutralizing antibodies are generated against gp120 and participate in antibody-coated virus is infectious, however, and is taken up by macrophages. CD8 T cells can kill infected cells by direct cytotoxic action and by producing suppressive factors that restrict viral replication. However, CD8 T cells require activation by CD4 T cells, CD8 T-cell number decreases with CD4 T-cell number, and their reduction correlates with disease progression to AIDS.

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HIV has several ways of escaping immune control. Most significant are the virus’s ability to undergo mutation, alter its antigenicity and escape antibody clearance, and the targeted killing of the CD4 T cell. Clinical Manifestations There are three phases to the course of infection with HIV: Seroconversion illness. Seroconversion usually occurs 6 to 8 weeks after infection, but incubation can range from 4 to 12 weeks. This is often accompanied by a feverish illness which is variable in its presentation and severity. Antibodies to HIV become detectable in the serum. Latent phase. The latent phase of the HIV infection may last from 18 months to 15 years or more, with an average of about 8 years. For much of this time the individual is well, not unduly susceptible to infections and recovers apparently normally from common and seasonal infections. The total CD4 T helper cell population slowly declines, CD4 helper function is increasingly impaired. Symptomatic HIV infection. A sudden increase in the loss of CD4 cells heralds the end of the latent phase. Clinical evidence of immunodeficiency includes generalized infections with: . Troublesome bacterial infections, most importantly pneumococcal (Mycobacteria) and Salmonella infections. . Viral infections, herpes zoster is common and cytomegalovirus disease. . Fungal infections. Oral and genital candidiasis. . Protozoal infections. Pneumocystis and toxoplasma And tumours. Kaposi’s sarcoma, lymphomas. Epidemiology The principal sources of HIV are the blood and genital secretions of HIV infected persons. Other body secretions or fluids may contain HIV in low concentrations and are probably unimportant in transmission. Three main modes of transmission have been recognized: 1. Sexual contact with an infected person. 2. Inoculation of blood or blood products contaminated by HIV (mainly through needle sharing among IV drug users. 3. Perinatal transmission from an infected women to her fetus or infant. No other mode of transmission has been firmly established.

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Laboratory Diagnosis The diagnosis of HIV infection is made by three ways: 1. Demonstration of virus or virus components HIV is very difficult to culture, and culture is not routinely perforemed. The finding of the p24 viral antigen, the reverse transcriptase enzyme, or viral RNA in blood samples indicates the presence of recent infection or late-stage disease. Amplification of viral RNA or DNA proviruses by the PCR technique is the most sensitive method for early detection of virus in blood or tissue specimens. 2. Serology ELISA or agglutination procedures are used for routine screening. The ELISA test, however, can yield false-positive results and will not detect a recent infection. More specific procedures, such as the Western blot analysis, are subsequently used to confirm seropositive results. The Western blot assay, on the other hand, determines the presence of antibody to the viral antigens (p24 or p31) and glycoproteins (gp41 and gp120). HIV antibody may develope slowly, taking 4 to 8 weeks in most patients; however it may take 6 months or more in as much as 5% of those infected. 3. Immunology The status of an HIV infection can be implied from an analysis of the T-cell subsets. The absolute number of CD4 lymphocytes and the ratio of CD4 to CD8 are abnormally low im HIV-infected people. The particular concentration of CD4 lymphocytes identifies the stage of AIDS. Treatment Drug administration can be classified as; 1. Nucleoside analoque reverse transcriptase inhibitors ( azidothymidine, dideoxycytidine, Lamivudine, and other analoques) 2. Non-nucleoside reverse transcriptase inhibitors (nevirapine and others) 3. Protease inhibitors (saquinavir, ritonavir, and others) Azidothymidine (AZT) and other analogue reverse transcriptase inhibitors active against HIV, can delay the progression of HIV infection, reduce the occurrence of opportunistic infections in AIDS and improve the patient’s clinical condition by abolishing malaise and weight loss. They are not sufficiently effective to eradicate HIV infection. Unfortunately, the high mutation rate of HIV promotes the development of resistance to these drugs. A cocktail of several antiviral drugs (e.g., AZT, Lamivudine, protease inhibitor, each with different mechanisms of action, has less potential to breed resistance and has become a recommended therapy.

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Prevention and control The most effective control measure is the use of condoms. Other preventive measures include screening of blood products, needle exchange schemes, and voluntary testing of those in high-risk categories. No vaccine against HIV is available despite several trials. The development of a vaccine against HIV is, however, fraught with several problems unique to the virus. Theses problems are: 1. The antigenicity of the virus changes readily through mutation 2. The virus can be spread through syncytia and remains latent, thereby hiding from antibody. 3. Testing of the vaccine would be a problem because HIV is a human disease and long-term follow-up is required to monitor the efficacy of the vaccine. VIROIDS AND PRIONS Viroids are small molecules of single-stranded RNA less than 0.5 kb in size and are capable of replicating in sesceptible cells. The RNA have regions of internal complementarity allows for self-anneal by internal base pairing, thereby yielding stable rolike. It is not clear if viroids code for viroid specific proteins. Since the host does not have RNA replicase and the viroid genome is too small to code for such an enzyme, how viroids multiply remains unresolved. Viroids are conceptually but not medically important since all viroids identified thus far are plant pathogens. Virusoids are similar to viroids except that the virusoids is encapsulated in an RNA virus and that the virusoid needs helper function from the RNA virus for replication. Prions is an acronym derived from “proteinaceous particles.” Infectivity of prions is detroyed by most procedures that modify proteins but not by most that modify nucleic acids. The more important prions are the infectious agent of scrapie (laboratory animal model) and Creutzfeldt-Jakob disease (humans). Prionassociated diseases are chronic degenerative diseases of the brain, known as spongioform encephalopathies. Prion infectivity is associated with or inseparable from a purified protein known as prion protein (PrP), which has a molecular weight of 27-30 kDa. The PrP is derived from the host glycoprotein. In contrast to glycoprotein from uninfected host, PrP is resistant to hydrolysis by proteinase K. Creutzfeldt-Jakob disease is rare (prevalence of 1 per million). The brain of a patient dying from this disease may contain 1010 LD50 per gram of tissue. Transmission has been associated with cornea or transplantation, brain surgery, administration of growth hormone (extract from human pituitary glands), and transplantation of dura matter.

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Destruction of prions requires autoclaving at 132˚C for 1h or soaking in 1 N sodium hydroxide also for 1h. ORAL MICROBIOTA AND ITS DISEASES Microbial Flora of The Oral Cavity Various areas of the body normally support a microbiota even in the state ordinarily called health. The oral cavity supports one of the most concentrated and varied of such microbial populations, with main foci on the dorsum of the tongue, about gingival sulcus, and in coronal dental plaque. It has been estimated that about 400 different microbial species are capable colonizing the dentate oral cavity and that any individual may harbor over 150 different species. In utero, however, the fetus is normally germ-free. During birth, a child is presumably inoculated with the normal flora of the mother’s genital tract, that is, some or all of the following: lactobacilli, corynebacterium, micrococci, coliforms, alfa-, beta-, and anaerobic streptococci, yeasts, protozoa, and possibly viruses. With the exception of the Streptococcus salivarius, which can be cultured with some degree of regularity, most of these organisms are found sporadically, and not in high numbers. The oral flora is exclusively of aerobic type during the period preceding dentition, and that, thereafter, anaerobic forms definitely characterizes the microbial phases of the oral cavity. The early period is dominated by facultative species, to which are added gradually the various obligate anaerobes, but numerically the facultative types generally dominated at all ages. The presence of teeth would provide regions for the growth of organisms adapted to the environmental conditions these structures provide; for example, Streptococcus mutans seems to colonize preferentially in plaque. STRUCTURE OF THE ORAL CAVITY The oral cavity may be broadly subdivided into three major components: the teeth, the supporting structures of the teeth (periodontium), and other intraoral structures, including the lips, tongue, floor of the mouth, buccal mucosa, palate, temporomandibular joint, fauces, and the tonsils. This lecture focuses on the teeth and periodontium. Teeth Humans experience two dentitions: deciduous and permanent (adult). Deciduous Dentition. The deciduous dentition is composed of 20 teeth: 10 teeth for each dental arch (maxillary arch and mandibular arch). Eruption of the deciduous

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teeth usually begins between 6 and 71/2 months of age with the lower central incisors. The second molars are the last deciduous teeth to erupt, occurring between 20 and 24 months of age. Along with eruption of the deciduous teeth is an ever-increasing complex of bacterial microflora. Permanent Dentition. The permanent or adult dentition consists of 32 teeth. The integrity of the teeth in each dental arch is contingent on the arrangement of the teeth, with each tooth in close contact with neighboring teeth. Malpositioning of the teeth permits greater bacterial colonization and growth because bacterial plaque-retentive areas develope. These plaque-retentive areas harbor and foster the growth and development of greater numbers and varieties of bacteria. The permanent teeth begin erupting between 6 and 7 years of age; the final teeth, third molars “wisdom teeth,” erupt between 17 and 21 years of age. Teeth can be subdivided into four basic substructures; enamel, dentin, cementum, and pulp (Fig. 37). Dental enamel is the hardest material in the body and consists nearly entirely of inorganic salts (97%). This material covers the crown of the tooth and is usually resistant to abrasive wear. Dental caries begins when the demineralization of enamel occurs as result of acid production through the plaque constituents’ metabolism of sugars. Dentin, which is approximately 65% to 70% inorganic salts, is the calcified tissue that forms the main structure of the tooth. Dentin is the second structure subjected to dental caries. Pain is generally not perceived by patients unless dentin has been destroyed by the caries process. The cementum is thin and covers the roots of the teeth. One function of cementum is to incorporate collagenous periodontal ligament fibers and thus mediate the connection between teeth and alveolar bone. The cementum may also be colonized by bacteria, and dental caries may result (root caries). Dental pulp occupies the central area of the crown of the tooth and the roots. It is soft tissue, primarily a collagen mass with a network of blood vascular and nervous systems, that remains from the formative organ dentin. If dental caries continues through the enamel, cementum, and dentin, the pulp may be invaded. Periodontium The periodontium consists of the investing and supporting tissues of the tooth (gingiva, periodontal ligament, cementum, and alveolar bone). It has been divided into two parts: The gingiva, whose main function is protection of the underlying tissues, and the attachment apparatus, composed of the periodontal ligament, cementum, and alveolar bone. It is divided anatomically into marginal, attached, and interdental areas.

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Fig. 38 Tooth structure

. The marginal, or unattached, gingiva is the terminal edge or border of the gingiva surrounding the teeth collarlike fashion. . The attached gingiva is continuous with the marginal gingiva. It is firm and tightly bound to the alveolar bone. . The interdental gingiva occupies the gingival embrasure, which is the interproximal space beneath the area of tooth contact. The cementum is considered a part of the periodontium because, with the bone, it serves as the support for the fibers of the periodontal ligament. Tooth-Associated Materials The tooth-associated materials collectively included food debris, acquired pellicle, materia alba, bacterial plaque, and calculus. Food Debris Food debris is retained in the mouth after mastication of food. This debris, unless wedged between the teeth or inside the periodontal-tooth interface, is usually removed by saliva or oral musculature action. Acquired Pellicle Acquired pellicle is a very thin (0.1 to 0.8 µm), primarily protein film that forms on surfaces of the teeth. This pellicle is derived from components of saliva and crevicular fluid as well as bacterial and host tissue cell products and debris. It is acquired very soon (within minutes) after the teeth are cleaned. Acquired pellicle is considered the first stage in the formation of dental plaque, since bacterial adherence followed by colonization quickly ensues.

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Materia Alba Materia alba refers to soft accumulations of bacteria and tissue cells that lack the organized structure of dental plaque and are easily displaced with a water spray. Bacterial Plaque Bacterial plaque is a mat of densely packed, colonized microorganisms, growing and tenaciously attached to the teeth. It is embedded in a matrix of pellicle adhering to the teeth. Total bacterial counts are estimated to be approximately 108 to 1011 bacteria Per gram of plaque. Bacterial plaque is not removed by rinsing or forceful spraying with fluids but is easily removed by mechanical means. Subpopulations of the dental plaque develop further with varying compositions and metabolisms. Factors enhancing the rate of bacterial colonization in the oral cavity include oral hygiene, diet, malocclusion, malposed teeth, oral immune factors, saliva flow, and roughness of teeth surfaces, among others. Without proper brushing of the teeth, selected, extensive colonies of bacteria form on the surfaces of the teeth within 1 to 3 days. The colonies are localized within the pits and fissures of the coronal (crown) portion of the tooth and are also found along the gingiva. Within a few days these bacterial colonies begin to coalesce and fuse to form a continuous “matlike” deposit. Within approximately 7 to 10 days dental plaque increases in thickness on the surfaces of the teeth and in the region between the teeth and gingiva (sulcus). Streptococci and gram-positive bacilli form the pridominant structures of early plaque development, which is located primarily supragingivally (at or above the gingiva). Bacterial plaque, initially supragingival, continues to proliferate and extends underneath the gingiva (subgingival). As bacteria continue to proliferate subgingivally, relatively low oxygen tension enviroments are created. In this econiche below the gingiva, facultative and eventually strict anaerobic bacteria are favored for growth and development. Ultrastructurally, the subgingival plaque consists primarily of bacilli and flamentous bacteria. DENTAL AND ORAL INFECTIONS The normal flora of the oral cavity is responsible for a large proportion of oral infections (e.g. caries, abscesses, candidiasis). Lesions in the mouth can also be associated with generalized infections (e.g. Koplik spots in measles).

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DENTAL CARIES Dental caries continues to be a major problem in dentistry and should receive significant in everyday practice, not only from the standpoint of restorative procedures but also in terms of preventive practices designed to reduce the problem. Pathogenesis. The role of bacteria in causing dental caries is supported by two theories: . The Proteolytic-Chelation Theory. This theory postulated that oral bacteria attack organic components of enamel and that the breakdown products have chelating ability and thus dissolve the tooth minerals. . The Chemicoparasitic or Acidogenic Theory. This theory has been the most popular over the years and is probably the one most widely accepted today. It is generally agreed that dental caries is caused by acid resulting from the action of microorganisms on carbonhydrates. It is characterized by a decalcification of the inorganic portion and is accompanied or followed by a disintegration of the organic substance of the tooth. A number of microorganisms can produce enough acid to decalcify tooth structure, particularly aciduric streptococci, lactobacilli, diphtheroids, yeasts, staphylococci, and certain strains of sarcinae. The most frequently isolated bacteria associated with dental caries is Streptococcus mutans. Their numbers increase with initiation of dental caries and decrease when caries are treated, exhibiting a strongn correlation with this disease process. Streptococcus mutans produces an enzyme dextran-sucrase, which converts the sucrose of food to dextrin, and dextrin combines with salivary proteins to create a sticky, colorless film (plaque) on tooth surfaces. Plaque provides the haven for the activities of Lactobacilli and these produce of lactic acid, which attacks the enamel by decalcifying it. Interestingly, S.mutans is not present in the oral cavity of infants at birth and can be detected only after the primary teeth begin to erupt. The most common source of transmission of the bacteria is from the mother to the child. Dental caries extends through the enamel and into the dentin where the carious lesion must be restored. Without appropriate treatment at this time, the carious lesion may furthere extend through the dentin and into the dental pulp region. Sensitivity to heat and cold or sensitivity to percussion are possible symptoms related to extension of the caries to the pulp. After bacterial involvment of the pulpal regions during caries pathogenesis, a periapical lesion (granuloma or abscess) may develop at the tip of the root. Root canal therapy (endodontic therapy) is even more critical at this time and includes mechanical removal and debridement of the pulpal contents of the tooth. When this is performed, the periapical lesion frequently resolves without the use of antibiotic therapy.

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Clinical Manifestations. The normally hard tooth structure is converted to a soft, leather-like consistency. Laboratory Diagnosis. Caries is detected by examination of the saliva for numbers of streptococci and lactobacilli; this ascertains which patients are “caries prone.” However, these salivary tests have very limited application in clinical practice. Cultures of the caries lesion are not performed, since the clinical diagnosis is fairly accurate. Treatment. Mechanical removal of the caries lesion is the treatment for most cases. Extension of the carious lesion into the pulp of the tooth results in endodontic involvement. Mechanical removal and debridement of the pulpal contents is necessary. Systemic antibiotic therapy is used for the management and control of periapical lesions resulting from pulpal involvement. PERIODONTAL DISEASES Periodontal infections cause the most common diseases of the periodontium and are among the most common of all diseases in humans. The periodontal diseases can be broadly subdivided into two main categories: gingivitis and periodontitis. GINGIVITIS Gingivitis is an inflamation involving only the gingival tissues next to the tooth. The primary etiologic agent of gingivitis is bacterial plaque. This forms the basis for a classification of gingivitis, with “plaque-associated” gingivitis being the most common form. The primary bacteria involved in gingivitis is hemolytic streptococci. Other modifying secondary etiologic factors permit classification of other forms of gingivitis. Acute necrotizing ulcerative gingivitis (ANUG), commonly known as “trench-mouth,” “Vincent’s infection,” has several secondary etiologic factors and constitutes another form of gingivitis. Two microorganisms, Borrelia vincentii and Fusobacterium nucleatum, are generaly believed to be responsible for the disease. Because many of these organisms are normal inhabitants of the oral cavity, smears are of little value in establishing a diagnosis. Unlike conventional periodontal disease, ANUG is most often observed in young adults in their early twenties. It is the only periodontal infection in which bacterial invasion of the gingival tissue has been observed. The clinical features of ANUG are frequently seen by the practicing physician as periodontal tissues with gingival craters covered by a grayish-white pseudomembrane. Other clinical manifestations of the disease include inflamed, painful, bleeding gingival tissue, poor appetite, fever as high as 40ºC, general malaise, and a fetid odor. Although once thought to be communicable, the disease connot be transmitted solely by the transfer of organisms from one individual to another. Rather, it is

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believed that a localized reduction in tissue resistance, often precipitated by stress, produces an enviroment in which the fusospirochetes proliferate. The diagnosis of ANUG is made on the basis of the patient’s age, history, and clinical appearance. ANUG is most commonly confused with herpetic gingivostomatitis, which usually occurs in a younger age group, is preceded by a viral prodrome, and is characterized by the appearance of vesicular lesions on the mucosa in addition to a gingivitis. Recovery occurred within 36 hours after penicillin therapy and the application hydrogen peroxide. PERIODONTITIS Periodontitis is defined as, “an inflammatory disease of the supporting tissues of the teeth caused by specific microorganisms or groups of specific microorganisms, resulting in progressive destruction of the periodontal ligament and alveolar bone with pocket formation, recession, or both.” The clinical feature that distinguishes periodontitis from gingivitis is the presence of clinically detectable attachment lose. This often is accompanied by periodontal pocket formation (Fig. 39) and changes in the density and height of subjacent alveolar bone.

Fig. 39 Periodontal pocket formation

The Periodontal Microbiota In the oral cavity, bacteria colonize and accumulate principally in three regions: on the dorsum of the tongue, on areas of the teeth not cleaned by mastications, and on the teeth and gingivae in the vicinity of the gingival crevice or sulcus area. Samples from the healthy gingival sulcus contain relatively few (10 3 to 106) cultivable organisms, predominantly consisting of gram-positive facultative species and members of the genera Streptococcus and Actinomyces (e.g., S.sanguis, S.mitis, A.naeslundii, and A.viscosus). Small proportions of gram-negative species are also found, most frequently Prevotella intermedia, Fusobacterium nucleatum, and Capnocytophaga, Neisseria, and Veillonella spp. Microscopic studies indicate that a few spirochetes and motile rods also may be found.

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Certain bacterial species have been proposed to be protective or beneficial to the host, including S. sanguis, Veillonella parvula, and C. ochracea. These species probably function in preventing the colonization or proliferation of pathogenic microorganisms. One example of a mechanism by which this may occur is the production of H2O2 by S. sanguis; H2O2 is known to be lethal to cells of Actinobacillus actinomycetemcomitans. Subgingival bacterial counts range up to more than 108 in deep periodontal pockets. There is general agreement that periodontitis is an infectious disease associated with only a few of the bacterial species found in dental plaque. With the development of periodontitis, there is a shift toward a subgingival flora containing a higher proportion of gram-negative rods and decreased proportions of gram-positive species. In an established periodontal lesion, low numbers of cocci and high numbers of motile rods and spirochetes are seen. Increased proportions of Porphyromonas gingivalis, Bacteroides forsythus, and species of Prevotella, Fusobacterium, Campylobacter, and Treponema have been detected. P. gingivalis was isolated in 79% of periodontitis patients. However, it is still unknown whether the presence of the gram-negative bacteria is secondary to altered nutritional and anaerobic conditions because of the inflammatory processes and pocket formation or is itself responsible for the periodontal destruction. To become associated with destructive periodontitis, the microorganisms must comply with several criteria. . The species should be found more frequently and in higher proportions in cases of infection compared to non-diseased sites (association). . Absence of the species should be accompanied by a parallel remission of disease (elimination). . Production of antibodies or cellular immune response is directed specifically at that species (host response). . Potentially damaging metabolites are produced or properties possessed by a species (virulence factors). . Periodontal disease progression conferred by the presence of a species at a given level is evaluated in a prospective study (risk assessment). On the basis of these criteria, several species have been related to the etiology of destructive periodontal diseases, of which A. actinomycetemcomitans (A.a) and P. gingivalis have the strongest association. A. actinomycetemcomitans is the most important microorganism in juvenile periodontitis, while P. gingivalis is considered to be associated with chronic (adult) periodontitis and refractory periodontitis. In low numbers, Prevotella intermedia, and Prevotella nigrescence have been found in periodontally healthy subjects, but they may also be associated with the development of periodontitis. Furthermore, B. forsythus is found more frequently in periodontal patients, and it is levels are related to probing depth and periodontal breakdown. Other bacteria species with periodontal destruction include Fusobacterium nucleatum, Campylobacter rectus, P. micros, Treponema denticola, and Treponema vincentii. Like P. intermedia, these species are probably opportunistic pathogens with

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relatively low pathogenic potential and have to colonize the subgingival area for longer periods of time at elevated levels to be able affect the periodontium. Types of Periodontitis The disease periodontitis can be subclassified into the following three major types based on clinical, radiographic, historical, and laboratory characteristics. Chronic Periodontitis Chronic periodontitis is the most common form of periodontitis. This disease is most prevalent in adults but can be observed in children; therefore the age of > 35 years previously designated for the classification of this disease has been discarded. Chronic periodontitis is associated with the accumulation of plaque and calculus and generally has a slow to moderate rate of disease progression, but periods of more rapid destruction may be observed. Increases in the rate of disease progression may be caused by the impact of local, systemic, or enviromental factors that may influence the normal host-bacterial interaction. Microscopic examination of plaque from sites with chronic periodontitis have consistently revealed elevated proportions of spirochetes. Cultivation of plaque microorganisms from sites of chronic periodontitis reveals high percentages of anaerobic (90%) gram-negative (75%) bacterial species. In chronic periodontitis, the bacteria most often cultivated at high levels include P. gingivalis, B. forsythus, P. intermedia, C. rectus, Eikenella corrodens, F. nucleatum, A. actinomycetemcomitans, Peptostreptococcus micros, and Treponema and Eubacterium spp. Aggressive Periodontitis Aggressive periodontitis differs from the chronic form primarily by the rapid rate of disease progression seen in an otherwise healthy individual, an absence of large accumulations of plaque and calculus, and a family history of aggressive disease suggestive of a genetic trait. This form of periodontitis was previously classified as early onset periodontitis. The disease may be localized or generalized and rapidly progressive periodontitis. Aggressive forms of periodontitis usually affect young individuals at or after puberty and may be observed during the second and third dicated of life (i.e., 10 to 30 years of age). The microbiota associated with localized aggressive periodontitis is predominantly composed of gram-negative, capnophilic, and anaerobic rods. A. actinomycetemcomitans is generally accepted as the primary etiologic agent in most, but not all, cases of localized aggressive periodontitis. Other organisms found in significant levels include P. gingivalis, E. corrodense, C. rectus, F. nucleatum, B. capillus, Eubacterium brachy and Capnocytophaga spp. and spirochetes.

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Herpesviruses, includind EBV-1 and HCMV, also have been associated with localized aggressive periodontitis. Periodontitis as a Manifestation of Systemic Disease Several hematologic and genetic disorders have been associated with the development of periodontitis in affected individuals. INFECTIONS OF THE ORAL MUCOSA Oral Syphilis The primary lesion of acquired syphilis is the chancre, which is most common periorally on the lip or corners of the mouth and occurs approximately 3 weeks after contact with an infected lesion. Clinically, chancres usually present as painless ulcerations. Lymphadenopathy with tenderness is also noted. The chancre heals spontaneously in about a month. Swabs of the ulcer cannot be relied upon for diagnosis as non-pathogenic spiral treponemas may be present in the normal flora. Diagnosis is confirmed serologically. In secondary syphilis ulcers may develop on skin and mucous membranes, including the lips, tongue and palate. The tertiary lesion of acquired syphilis is the gumma. Unlike the other lesions of syphilis, the gumma is not seen in proximity to the initial infection and, in fact, is noted 2 to 10 years later. Clinically, the gumma is rare in the mouth but, when present, is seen as a deep penetrating ulcer, often involving the palate or tongue. Unlike the primary and secondary forms of syphilis, the gumma is not infectious. Acute Bacterial Sialoadenitis. Acute bacterial sialoadenitis is an acute infection of the parotid gland originally seen almost exclusively in debilitated, elderly, dehydrated, or postoperative patients. This disease is usually (80%) characterized by the sudden onset of unilateral firm eryhematous swelling and localized pain and tenderness at the angle of the mandible, which then spread to involve the parotid gland. The gland increas in size so that the overlaying skin becomes shiny and tense. A purulent discharge may be expressed from the parotid duct. Simultaneously, the patient experiences systemic signs of infection characterized by fever, leukocytosis, chills, and malaise. The organism most commonly isolated from the infected gland is penicillinresistant Staphylococcus aureus. Discharge from the parotid duct in a patient suspected of having acute bacterial parotitis should be immediately cultured and analyzed for antibiotic sensitivity. After a culture is obtained, the patient should be empirically started on an antistaphylococcal semisynthetic penicillin, because most infections are caused by

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penicillin-resistant staphylococci. The penicillin-allergic patient can be placed on clindamycin. Candidiasis Of the fungal infections that affect the mouth, candidiasis is the most common. Approximately 50% of the population has Candida albicans in the normal oral flora. Usually this organism is of no clinical significance. If changes ocuur in the oral enviroment, however, candidal organisms can proliferate and cause infection. Candidiasis is most frequent in patients at either age extreme; in newborns the infection is called thrush. It has been reported that fungal proliferation is seen frequently under the palate of maxillary prostheses. Any patient whose marrow status changes because of diseases such as aplastic anemia or drugs is at high risk to develop candidiasis. Similarly, patients reciving immunosuppressive therapy such as asteroids or debilitated individuals such as patients with diabetes may develop fungal infections of this type. Mucocutaneous candidiasis is one of the most frequent signs of symptomatic HIV infection. Prolonged use of antibiotics, particularly broadspectrum agents, may produce candidiasis. Clinically, candidiasis has a wide range of manifestations. Patients may be totally asymptomatic or complain of pain or burning or of having a coated feeling in the mouth. On examination, the clinician may note raised, curdy white areas that often appear to lump in heaps. The tongue may take on a coated white appearance. The borders of the white areas may be erythromatous. The white areas can usually be scraped off with firm pressure using a wooden tongue blade, leaving a raw bleeding surface. A smear of the material reveals the presence of hyphae when observed microscopically. Treatment is made by nistatin oral suspension. Candidiasis beneath dentures can be traeted with nistatin ointment applied to the prosthesis. Systemic medications for mucosal candidiasis include ketoconazole tablets. Dental Abscesses The grouping of dental abscesses includes acute alveolar, apical, dentoalveolar, periodontal and submucosa abscesses. They often result from pre-existing oral infection or trauma. These abscesses are often associated primarily with anaerobic bacteria and facultative streptococci. Actinomycosis Actinomyces israelii may cause infection of the mandible and associated tissue. Sinus formation may occur, with “sulphur granule” discharge which contains clumps of the organism. Diagnosis is based on history, clinical presentation, and culture of drainage. The infection is usually responsive to a prolonged antibiotic course;

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penicillin is the drug of choice. Surgical debridement of the infected region is often helpful. Herpetic Stomatitis Herpetic stomatitis is an acute gingivostomatitis caused by herpes simplex, usually type 1. A large number of vesicles cover the lips, gingiva and tongue. It is usually self-limiting, within 10-14 days. The virus may remain latent in trigeminal nerve ganglia and later reactivate from a dormant state to present secondary infection. The lesions produced by this reactivation are commonly referred to as cold sores or herpes labialis. The frequency of recurrence varies among patients, but 25% of patients affected bu recurrent herpes labialis have one or more episodes per mouth. Clinically, vesicular lesions commonly occurs at the corners of the mouth or beneath the nose. The lesions of secondary herpes infections are recurrent, completely disappearing and then returning. It has been proposed that their appearance is seasonal and related to cold, sunlight, and stress. Treatment is with acyclovir, particularly in the early stages. It should be remembered that the vesicle harbors live virus particles. Thus, the dentist should avoid contact with the patient with active herpes to avoid infection. Herpangina Herpangina, a viral disease that affects the mouth, is caused by a group A coxsackie virus. The disease, which is most common in young children under 4 years of age, demonstrates seasonal variation, being most frequent in the summer months. Clinically, the initial manifestations of the disease is the acute onset of fever, malaise, myalgia, runny nose, throat pain, and dysphagia. The predominant clinical lesions associated with herpangina occur in the mouth and are manifested as multiple, small, ovoid vesicular painful lesions occurring on the soft palate and oropharynx. The vesicles may rupture, leaving an ulcer. The acute symptoms of herpangina persist for about 3 days and the lesions heal without scarring in about a week. Treatment of herpangina is palliative and supportive. Bed rest, aspirin, and fluids are recommended. Palliative rinses, such as Xylocaine may be helpful to control discomfort. The diagnosis can usually be made based on the history and clinical appearance.

BASIC MYCOLOGY AND CLASSIFICATION OF FUNGI

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Characteristics Fungi are eukaryotic organisms; they are distinct from plants in not containing chlorophyll. Fungi are macroscopic (mushrooms) or microscopic (moulds and yeasts). Only a few species cause human disease. Microscopic fungi are non-motile; they may grow as single cells (yeasts) or filamentous structures (mycelia), some of which may be branched. Classification Fungi are classified according to their method of sexual reproduction. Those that do not reproduce sexually are called ‘fungi imperfecti’; those that reproduce sexually may be self-fertile or require strains of an opposite type to allow sexual fusion to occur. Four groups of fungi cause human disease. • Yeasts: have round or oval cells that multiply by budding, e.g. Cryptococcus neoformans. • Yeast-like fungi: grow predominantly as yeasts that can bud. They may also form chains of elongated filamentous cells called pseudohyphae, e.g. Candida albicans. • Filamentous fungi: grow as filaments (hyphae) and produce an intertwined network called a mycelium. They produce asexual spores (conidia), which may be single or multi-celled. Conidia are produced in long chains on an aerial hyphae (condiophore): e.g. Aspergillus, Trichophyton and Zygomycetes (including Mucor). • Dimorphic fungi: have two forms of growth: filamentous at 22 C (saprophytic phase) and yeast-like at 37 C (parasitic phase); examples include Blastomyces, Coccidioides, Histoplasma capsulatum. Fungal Infections Fungal infections can be divided into two groups, superficial and deep mycoses. Most deep mycoses are opportunist infections occurring in immunocompromised patients.

Superficial Mycoses

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Fungi
Candida albicans Dermatophytes: Epidermophyton Microsporum Trichophyton Malassezia furfur

Type of fungus
Yeast-like Filamentous

Principal infections
Oral thrush, vaginitis, cutaneous candidiasis Tinea (ringworm) of skin and hair Pityriasis versicolor

Epidemiology
Worldwide Worldwide

Worldwide, most common in the tropics _______________________________________________________________________________

Dimorphic

Laboratory diagnosis • Direct microscopy can be made of clinical material, including skin scrapings and sputum; the characteristic morphology facilitates identification. • Culture: fungi can be isolated on most routine media, but may require a prolonged incubation. Antibiotic-containing selective media (e.g. Sabouraud’s glucose agar), which inhibit bacterial growth, are often used. Incubation at both 37 C and 28 C facilitates the isolation of common filamentous fungi and yeasts. • Serology: serological tests are available for the diagnosis of some fungal infections (e.g. candida and aspergillus), but these lack spesificity and sensitivity. Cryptococcus neoformans C. neoformans is a capsulate yeast found worldwide. Its natural habitat is soil, particularly soil contaminated with bird droppings. • C. neoformans has a polysaccharide capsule which can be detected by mixing the organism in Indian ink. It can be observed in cerebrospinal fluid (CSF) by this method; other methods of detection include latex agglutination of CSF or serum. • Infection is acquired by inhalation of the fungus. Human infections are rare. • Primary infection occurs in the lungs and is usually asymptomatic. Acute pneumonia may occur with fungaemia and infection in various organs particularly the brain and meninges. Treatment is with amphotericin.

Deep Mycoses

201 Fungi Aspergillus fumigatus Candida albicans Type of fungus Filamentous Yeast-like Principal infections Lung infection, disseminated aspergillosis Lung infection, oesophagitis, endocarditis, candidaemia with disseminated candidiasis Meningitis Lung infection Lung infection Lung infection Lung infection Rhinocerebral infection, lung infection, disseminated infection (mucormycosis) Epidemiology Worldwide Worldwide

Cryptococcus neoformans Histoplasma capsulatum Coccidioides immitis Blastomyces dermatidis Paracoccidioides brasiliensis Rhizopus arrhizus and related fungi

True yeast Dimorphic Dimorphic Dimorphic Dimorphic Filamentous

Worldwide USA Central/ South America Africa, America South America Worldwide

Candida The genus Candida contains a number of species, including C. albicans (the most frequently isolated pathogen), C. parapsilosis and C. tropicalis. C. albicans is a commensal of the mouth and gastrointestinal tract. • Superficial candida infections are common and include vaginal and oral candidiasis (thrush), skin and nail infections. These infections may arise as a complication of antibiotic therapy which reduces temporarily the bacterial flora. • Invasive candida infections may involve the gastrointestinal tract (e.g. oesophagus), lungs and urinary tract. Candidaemia may result in abscesses in various organs (e.g. brain, liver). These infections occur primarily in immunocompromised patients.

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Candida can also colonize prosthetic materials, e.g. intravascular catheters and peritoneal dialysis cannulae, resulting in septicaemia and peritonitis, respectively. Candida is a rare cause of endocarditis.

Laboratory diagnosis. By direct microscopy of appropriate clinical material for oval Gram-positive cells, some of which may be budding or producing pseudomycelia; culture; and serology for candida antibodies in patients with deep-seated infections. Treatment. Topical with nystatin; parenteral therapy is with fluconazole or amphotericin B. Malassezia furfur M. furfur is part of the normal human flora. It produces thick-walled, budding cells and curved hyphae. • It causes pityriasis versicolor, a superficial scaly skin infection with depigmentation.

Diagnosis. By microscopy of skin scales showing yeast cells. Treatment. Topical or oral with azole antifungals. Aspergillus

• •

The genus Aspergillus contains a number of species, including A. fumigatus (the most frequent human pathogen) and A. niger. It is a common saprophyte worldwide, frequently found in soil and dust. Outbreaks of aspergillosis in immunocompromised patients have occurred because of construction work adjacent to hospitals which may result in the release of large numbers of spores. Infections are acquired by inhalation of spores (conidia), resulting in diffuse lung infection, or, occasionally, a large mycelial mass (aspergilloma). Infection can also spread to other sites, including the adjacent blood vessels and sinuses, or become disseminated to the liver, kidneys and brain. The fungus also causes chronic infections of the ear. Aspergillus infections are most frequent in immunosuppressed patients, e.g. patients with leukaemia, transplant patients and patients with the acquired immunodeficiency syndrome (AIDS). Aspergillus is also associated with allergic alveolitis which occurs in atopic patients with recurrent exposure to aspergillus spores; symptoms include fever, cough and bronchospasm.

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Laboratory diagnosis. By: direct examination of appropriate samples for branching hyphae; culture; serology (of limited value). Treatment. Amphotericin B or itraconazole. Dermatophytes The dermatophytes are a group of related filamentous fungi, also referred to as the ringworm fungi which infect skin and related structures. Three clinically important genera have been described.
1) Epidermophyton: E. floccosum is the only important species. It infects the skin

(tinea corporis), nails (tinea unguium), groin (tinea cruris) and feet (tinea pedis). 2) Microsporum: these infect hair and skin; M. audouini causes epidemic ringworm of the scalp (tinea capitis) in children. M. canis, which is a parasite of cats and dogs, occasionally causes ringworm in children. 3) Trichophyton: these infect skin, hair and nails. T. mentagrophytes is the most common cause of tinea pedis. T. rubrum causes severe recurrent skin and nail infections. Epidemiology. The natural habitat is man, animals or soil; human infection results from spread from any of these reservoirs. Dermatophyte infections are found worldwide with different species predominating in various climates. Laboratory diagnosis • Skin scrapings, hair or nail clippings from active lesions are examined microscopically in 30% potassium hydroxide on a glass slide; the presence of hyphae confirms the diagnosis. Occasionally, the dermatophyte species can be identified by typical morphology. • Samples can be cultured on Sabouraud’s medium at room temperature.Subsequent species identification is based on growth rate, colony appearance and microscopic morphology. • Infected hair may fluoresce under ultraviolet light (Wood’s light) and is characteristic of certain infections, e.g. M. canis. Treatment. Depends on the site and severity of infection. Options include topical imidazoles (e.g. clotrimazole, miconazole); oral griseofulvin, itraconazole or terbinafine. Mucormycosis

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The term ‘mucormycosis’(or ‘zygomycosis’) refers to infections caused by a variety of filamentous fungi belonging to the order Mucorales. Medically important species include Rhizopus arrhizus and Absidia corymbifera. Epidemiology. The Mucorales are found worldwide, in soil and decaying organic matter. Infections occur primarily in the immunocompromised. As with aspergillus, hospital outbreaks of infection have occurred in association with building work. Infection • Pulmonary: an often fatal infection of immunocompromised patients may result in disseminated infection (e.g. brain, liver, gastrointestinal tract). • Rhinocerebral: an infection of the nasal sinuses may spread rapidly to involve the face, orbit, and brain. It occurs particularly in uncontrolled diabetes mellitus and is often fatal if treatment is delayed. Laboratory diagnosis. Microscopy and culture of appropriate specimens are undertaken. Treatment. Amphotericin, plus debridement of necrotic tissue in rhinocerebral infection. Coccidioides immitis C. immitis is a dimorphic fungus found in soil in hot arid areas of south-west USA, Central and South America. Infection. This follows inhalation of arthrospores and is often subclinical. A mild, self-limiting pneumonia, often accompanied by a maculopapular rash, occurs in some patients. Severe progressive disseminated disease (e.g. meningitis, osteomyelitis) may occur, principally in immunosuppressed patients. Laboratory diagnosis. Direct microscopy and culture of appropriate specimens (culture should only be attempted in specialized centres because of the risk of infection); serology (e.g. latex agglutination) for IgM antibodies. Treatment. Amphotericin. Blastomyces dermatitidis B. dermatitidis is a dimorphic fungus found in soil in North and South America, and Africa. Man is probably infected by inhalation.

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Infections. Primary pulmonary infection may be complicated by haematogenous spread to involve the skin (granulomatous ulcers), bone and joints, brain and other organs. Laboratory diagnosis. Microscopy and culture of appropriate specimens are undertaken. Treatment. Amphotericin B or itraconazole. Histoplasma capsulatum H. capsulatum is a dimorphic fungus found worldwide, but infections occur most commonly in North, Central and South America. The natural habitat is soil, particularly in sites enriched with bat droppings (e.g. caves). Infections. Infection is acquired by inhalation of microconidia, which germinate in the lung to produce budding yeast cells. Pulmonary infection is normally selflimiting, but chronic pulmonary disease with cavitations (similar to tuberculosis) may occur in patients with underlying lung disease. Disseminated histoplasmosis (liver, bone, brain, skin) may occur, particularly in immunocompromised patients. Laboratory diagnosis. Microscopy of stained blood films or histological sections of tissue (oral yeast-forms seen within mononuclear phagocytes); culture; or serology (e.g. complement fixation test). Treatment. Amphotericin or itraconazole. Paracoccidioides brasiliensis A dimorphic fungus found in soil in Central and South America, P. brasiliensis causes pulmonary infection and mucocutaneous lesions including ulceration of the mucous membranes of the nasal and oral pharynx, which may progress to destruction of the palate and nasal septum. Disseminated infections occur with haematogenous spread to various sites, including the spleen, liver, bone and brain. Laboratory diagnosis. Direct microscopy of pus, sputum or tissue biopsy; culture; serology (e.g. complement fixation test). Treatment. Itraconazole, or amphotericin plus sulphadiazine.

PARASITOLOGY

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Although the term “parasite” may be applied broadly to all infectious agents, including bacteria, viruses, and fungi, it is traditionally reserved for parasitic protozoa, helminths (worms), and arthropods. The classification of parasites used in this chapter is simplified and uses common terminology. CLASSIFICATION Parasites are classified within the kingdom Animalia and are separated into two subkingdoms, Protozoa and Metazoa. The subkingdom Protozoa comprises unicellular animals in which all life functions occur in a single cell. The Metazoa are multicellular animals in which life functions occur in cellular structures organized as tissue and organ systems. Protozoa The subkingdom Protozoa comprises various groups follows: • Amebae (Sarcodina) move by the use of cytoplasmic protrusions called pseudopods. Reproduction is asexual. An example of this group is Entamoeba histolytica, the cause of amebic dysentery. • Flagellates (Mastigophora) use specialized whiplike structures called flagella for motility. Reproduction is asexual. Human pathogenic flagellates include Giardia lamblia (gastrointestinal tract), Tricomonas vaginalis (genital tract), Trypanosoma species (blood/tissue) and Leishmania species. • Apicomplexa organisms are often referred to as sporozoa or coccidia. These 0unicellular organisms have a system of organelles at their apical end that produce substances that help the organism penetrate host cells to become an intracellular parasite. The Apicomplexa include Cryptosporidium parvum, Isospora belli, Plasmodium species, Toxoplasma gondii and Pneumocystis carinii. They undergo asexual (schizogony) and sexual (gametogony) reproduction and have a variety of hosts, including humans. • Ciliates (Ciliophora) are represented by only a single species that is parasitic in humans, Balantidium coli. Ciliates move by the use of cilia that cover the organism’s surface.

Metazoa

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The subkingdom Metazoa, which includes all animals that are not Protozoa, has two groups of organisms of major importance in this text, the helminths (worms) and the arthropods (crabs, insects, ticks, etc.). • Helminths are multicelled worms, which are classified as flatworms and roundworms. Flatworms (Platyhelminthes) that parasitize humans are classified into two major groups; flukes and tapeworms. Flukes (Trematodes) are leaf-shaped, dorsoventrally flattened, and possess oral and ventral suckers for attachment. All species excepting the schistosomes are hermaphroditic. Tapeworms (Cestodes) are ribbon-shaped with an anterior attachment organ, the scolex, and a segmented body. Each segment contains both male and female sex organs. Roundworms (Nematodes) are cylindrical and possess a well-developed intestinal tract. The sexes are separate. • The arthropods have exoskeletons with jointed appendages. Arthropods of major medical importance are classified as insects or arachnids. Insects have a body that is divided into the head, thorax, and abdomen. Three pairs of legs originated from the thorax. An example of an insect parasite is the body louse. Many species of insects are not, by strict definition, parasites, but they serve as vectors for a wide variety of diseases. For example, mosquitoes transmit malaria, filariasis, and arboviruses. Arachnids have four pairs of legs in the adult stages. The body is divided into the cephalothorax and abdomen. This group contains mites, ticks, spiders, and scorpions. Mites and ticks appear unsegmented because the the cephalothorax is fused with the abdomen. DEFINITIONS Below are definitions of terms that are used in describing parasite life cycles and host-parasite interrelationships: Host: An organism that harbors or nourishes another organism. Definitive Host: A host that harbors the adult or sexual form of the parasite. Intermediate Host: A host that harbors larval or asexual stages of the parasite. Reservoir Host: A nonhuman host that can maintain the infection in nature in the absence of human hosts. Adult: The sexually mature stage of helminths and arthropods.

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Larva: Immature stages of helminths and arthropods Vector: An arthropod that is responsible for the transmission of parasite from host to host. If the arthropod is not essential to the life cycle, it is a mechanical vector; if it is essential, it is a biologic vector. Symbiosis: The relationship between two dissimilar organisms that are adapted to living together is called synbiosis and the associates are symbionts. The association may be beneficial or harmful to either of the associates. Symbiosis may be divided into categories: • Mutualism: A symbiotic association that is beneficial to both parties. • Commensalism: One of the associated organisms is benefits and the other is neither benefits or harms. • Parasitism: One organism (the parasite) benefits at the expense of the other (the host). Organisms that live on or in the skin of their hosts are ectoparasites. This relationship is an infestation. Most parasitic arthropods belong to this category. Parasites of the digestive tract, extraintestinal organs and tissues, and those that are intracellular within the host are referred to as endoparasites and produce infection, irrespective of their size. DIAGNOSTIC TECHNIQUES FOR PARASITIC INFECTIONS With few exceptions laboratory diagnosis is essential for a definitive diagnosis of parasitic diseases. There are some general techniques that used in clinical laboratories. Fecal Examination Fecal examination for ova and parasites is used for most intestinal parasites. In clinical jargon, this is often referred to as “stool for O and P” when an order is written to collect fecal specimens for parasitologic examination. Collection of fecal specimens. Collection should be made in a clean container without contamination by water or urine. The specimen must be free of oil, magnesia, aluminum salts, and barium. Preservation of fecal samples. For periods of several hours and sometimes longer good preservation is obtained by refrigeration at 3 to 10˚C. For reliable and lasting preservation, chemical fixation is required. • Formalin. Eggs and larvae of helminths found in stool specimens are satisfactorily fixed and preserved for later examination in 10% formalin heated to 65˚C (1 part of feces to 5-10 parts of 10% formalin).

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• MIF. The MIF (merthiolate-iodine-formaldehyde) solution can be used to preserve trophozoites and cysts of protozoa as well as eggs and larvae of helminths in fecal specimens. Direct smear saline suspension. Fecal specimens should first examined microscopically in direct smear preparations containing 1 to 2 mg of feces evenly suspended in 1 drop of physiologic saline solution under a coverglass. These films should be free of air bubbles and macroscopic debris. An unstained film of this type is useful for observing motile trophozoites of intestinal protozoa, helminth eggs, and Strongyloides larvae and this considerable diagnostic value for protozoan cysts. For studying the internal diagnostic characteristics of protozoan cysts, iodine stain is used. Permanent fecal film. To make a permanent, stained film of fresh feces for study of intestinal protozoa, a representative fleck of the material is smeared as evenly as possible in a thin film on a clean slide and immediately immersed in Schaudinn’s solution. Fecal concentration techniques attempt to concentrate ova of helminths and cysts of protozoa from relatively large amounts of stool. The method used by most laboratories is called the formalin-ethyl acetate sedimentation method. Ethyl acetate removes fatty substances and some debris from the stool specimen. Repeated decanting and resuspending the sediment following centrifugation provides a small amount of sediment that is rich in ova and cysts. Another technique, the zinc sulfate flotation method, is similar except that the final step suspends the sediment in a zinc sulfate solution of precise specific gravity. After concentration, the surface film of the solution, which contains the ova and cysts, is removed with a loop and examined. Antigen detection systems are new and promising supplements to the microscopic examination of stool. These systems rely on the use of antibodies to specific antigens excreted in the stool of infected individuals. Antigen in the stool binds to antibody. Enzyme technques that produce a visible colored product are used to detect the bound antibody. Blood Films Next to feces, blood provides the most common medium for recovery of various stages of animal parasites. From this source, diagnosis is routinely made of malaria, African trypanosomiasis, and others. Giemsa stain is preferred for most parasitic infections. Thin films are made by drawing out a drop of blood on a microscope slide so that the red cells from an even layer that is one cell thick. The film is then fixed and

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stained. This preparation allows optimal determination of the morphology of the parasites, especially malaria organisms. Thick films are made by stirring several drops of blood on a microscope slide into a single circular area. The blood is allowed to dry. The slide is placed in water that removes hemoglobin from the RBCs. The slide is then stained. This technique allows the concentration of organisms in a small area for easier detection. Serologic Tests Serologic tests, which detect antibody to the parasite, are becoming increasingly valuble as specific antigens are identified and used in sensitive tests, such as ELISA and in immunoblotting methods. PROTOZOA AMOEBAE The amebae are primitive, unicellular microorganisms, with a simple two stage life cycle: the actively motile feeding stage (trophozoite) and the quiescent, resistant, infective stage (cyst). Replication is accomplished by binary fission (splitting the trophozoite) or by the development of numerous trophozoites within the mature multinucleated cyst. Motility of amebae is accomplished by extension of a pseudopod (“false foot”). The amebic trophozoites remain actively motile as long as the enviroment is favorable. Cyst formation occurs under adverse conditions. Most amebae found in man are commensal organisms (Entamoebae coli, Entamoebae gingivalis, etc.). However, Entamoebae histolytica is an important human pathogen. Some free-living amebae (Naegleria fowleri, Acanthamoebae species) are present in worm, freshwater ponds or swimming pools and can be opportunistic human pathogens, causing meningoencephalitis or keratitis. Entamoebae histolytica (The Dysentery Amebae) E.histolytica has worldwide distribution, primarily in subtropical and tropical regions. Infected cases (symptomatic or asymptomatic carriers) act as a reservoir. Spread is usually via water or food contaminated with cysts. Morphology, Biology, and Life Cycle E.histolytica has four distinct stages in its life cycle: trophozoite, precyst, cyst, and metacyst. The stages commonly recognized in the feces are trophozoites and cysts; only trophozoites are present in the tissues.

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The trophozoite in its natural habitat in the large intestine and in extraintestinal foci generally varies from 12 to 30 μ in diameter. It is contain a single spherical nucleus that has a small central karyosome and peripheral chromatin, which is evenly distributed on the nuclear membrane in small granules. The cytoplasm is divided into endoplasm, the granular portion that often contains vacules (with ingested RBCs), and ectoplasm the portion that is clear. The pseudopod is a protrusion of cytoplasm. Usally clear ectoplasm is extended in a blunt or fingerlike projection followed by the flowing of endoplasm into the pseudopod. This process is the method of locomotion in amebae. The cyst is the resistant stage that is excreted in the stool. The mature cyst has a refractile cyst wall and contains four nuclei that resemble the nuclei of the trophozoites. Young cysts often contain deeply staining bundles of crystalline RNA that are called chromatoidal bodies. Cysts range in size from 10 to 20 μ in diameter. Pathogenesis Following ingestion the cysts pass through the stomach, where exposure to gastric acid stimulates release of the pathogenic trophozoite in the small intestine. The trophozoites divide and produce necrosis and ulceration in the large intestine. Invasion through the gut wall into the peritoneal cavity and blood stream spread to other organs (primarily the liver) may occur. The cysts are excreted in the stool. Trophozoites are found in the tissues in invasive disease. Cysts do not form in tissues. Clinical Manifestations Dysentery (acute amebic colitis) is associated with diarrhea containing blood and mucus. Liver abscesses and, less commonly, lung and brain abscesses are found. Laboratory Diagnosis Microscopy of freshly passed stools for the presence of trophozoites and cysts, or for trophozoites in abscesses. E.histolytica must be differentiated morphologically from other intestinal protozoa. There are other species of Entamoeba that resemble E.histolytica, such as the E.coli and E.gingivalis, which is found in human mouth. Treatment The treatment of amebiasis has two major components: the elimination of tissue invading organisms and the elimination of organisms from the lumen of the intestine. The drugs that are most commonly used for tissue invasion are metronidazole, emetine and others. Drugs that are used to treat the luminal organisms are iodoquinol and diloxanide furoate.

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FLAGELLATES Human pathogenic flagellates include Giardia lamblia (gastrointestinal tract), Trichomonas vaginalis (genital tract), Trypanosoma species (blood/tissues) and Leishmania species (blood/tissues). Giardia lamblia (Giardiasis) Giardia lamblia Stiles, 1915 has a cosmopolitan distribution and is common in both warm and temparate climates. Morphology and Life Cycle G.lamblia (G.intestinalis) has trophozoite and cystic stages. The trophozoite (9 to 21 μ in length) is delicate but very active. When seen from the ventral aspect, the trophozoite appears broadly rounded anteriorly and tapering to a point posteriorly; when viewed in profile, it is relatively thin and its anterior half is concave ventrally, forming an adhesive disc. It bears four pairs of flagella, all arising from a complex system of axonemes extending along the midline. Approximately in the center of the trophozoite there is a deeply staining, short, rod-shaped organelle that is believed to be the parabasal body. In the anterior portion of the body there are two ovoid nuclei, each with a central karyosome, one nucleus laying on each side of the midline. By means of the eight flagella Giardia is able to move very actively, and by applying its cup-shaped anterior ventral disc it becomes firmly attached to epithelial surfaces. Multiplication is by longitudinal binary fission. The oval cyst contains four nuclei, the median bodies, and numerous linear structures that are the intracytoplasmic components of the flagella. The primary habita of the trophozoites is the epithelial brush border of the upper two thirds of the small intestine, where myriad active organisms may be present. The stage commonly recovered in the feces is the cyst; trophozoites are seen in the stool only when it is diarrheal. Pathogenesis Infection results from the ingestion of cysts from feces. Contaminated water is frequent source and a few food borne outbreaks have been reported. Cysts are ingested and gastric acid stimulates the release of trophozoites in the small intestine, which then multiply by binary fission. Trophozoites attach to the intestinal villi by a sucking disc. İnflammation of the epithelium may occur, but systemic invasion is rare. Cyst formation occurs as the organisms move through colon. Clinical Manifestations

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Some infections may be asymptomatic. When symptoms occur, they may vary from severe acute diarrhea to chronic mild diarrhea associated with flatulence, abdominal discomfort. In some instances, significant intestinal malabsorption ocuurs. Laboratory Diagnosis Stool microscopy for cysts and trophozoites. If clinical suspicion is high and stool examination is negative, duodenal aspiration or biopsy may be helpful. Treatment. Metronidazole Trichomonas vaginalis (Trichomoniasis) This organism is found in the vagina and urethra in females and in the urethra, seminal vesicles, and prostate in males. Morphology and Structure Trophozoites average about 13 μ in the longest dimension and have a single nucleus located at the anterior end. There are four anterior flagella. An undulating membrane extends from the anterior pole to about halfway down the body. Beneath the undulating membrane there is a supporting structure called the costa. There is a rigid rod, the axostyle, which runs the length of the body and protrudes from the posterior end. T.vaginalis has no cyst form. Epidemiology This parasite has worldwide distribution with sexual intercourse as the primary mode of transmission. Occasionally, infections have been transmitted by fomites (toilet articles, clothing), although this is limited by the ability of the trophozoite form. Infants may be infected by passage through the mother’s infected birth canal. Clinical Manifestations Most infected women are asymptomatic or have a scant, watery vaginal discharge. Vaginitis may occur with more extensive inflammation and erosion of the epithelial lining, associated with itching, burning, and painful urination. Men are primarily asymptomatic carriers who serve as a reservoir for infections in women. Laboratory Diagnosis

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Motile organisms can be demonstrated in wet mounts of vaginal secretions in about 80% of infections. Culture of vaginal discharge is the most sensitive method of diagnosis, but this technique requires several days of incubation. Treatment Metronidazole is usually effective in a single dose. It is important to treat the patient’s sexual partners even if they are asymptomatic. Trichomonas tenax is normal habitat of the mouth, particularly in diseased gums, in tartar around the teeth, and in carious teeth. It is not pathogenic but its presence indicates poor oral hygiene. Leishmania Species The genus Leishmania is named in honor of William Leishman, who discovered the species (L.donovani) that causes kala-azar. In man and reservoir hosts (dogs, rodents) the organism in the amastigote form is a parasite of macrophage cells, in which it multiplies by binary fission, causing the death of the host cells. Leishmania species are obligate, intracellular parasites transmitted to mammalian hosts by sandflies. There are three main species that produce human disease: L.donovani, L.tropica, and L.braziliensis. These species are associated with various clinical syndromes. Morphology and Life Cycle When a sand fly bites an infected person or reservoir host, it sucks up parasitized macrophages or temporarily free parasites (amastigotes) in the blood or tissue juices. Soon after the amastigotes reach the midgut of the fly, they transform into the flagellated promastigote forms, which after rapid multiplication transform into infective promastigotes and migrate forward. From the foregut they are regurgiated or otherwise introduced into the skin of the next individual when the sand fly takes another blood meal. Two stages in the life cycle are known: the amastigote in man and reservoir mammals and the promastigote in the sand fly and in cultures. The amastigote is spherical or subspherical and measures 2 to 5 μm in its greatest dimension. It lives and reproduces by longitudinal binary fission in macrophages of skin, mucosa, lymph nodes, and reticuloendothelial system. In preparations stained with Giemsa’s or Wright’s stain, the cytoplasm is pale blue and the relatively large nucleus is red. In the cytoplasm, usually lying in the medium line of the cell, is a deep red rodlike structure called the kinetoplast; a delicate filament called the axoneme extends from near the kinetoplast to the cell membrane.

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The promastigote is pyriform or spindle-shaped, measuring roughly 15 to 25 μm by 1.5 to 3.5 μm, with a single flagellum as long as or longer than the cell. The nucleus near the middle of the cell, and the kinetoplast is at the base of the flagellum. Visceral Leishmaniasis Visceral leishmaniasis (kala-azar) is caused by Leishmania donovani. It can be asymptomatic. Fever, weight loss diarrhoea are present, with hepatosplenomegaly and renal involvement. Darkening of the skin, mainly the face and hands, malabsorption and anaemia may occur. Most infections resolve without therapy. Diagnosis is made by histological examination of smears of white cells; bone marrow or lymph node aspirates for amastigotes; serology. Sodium stibogluconate or pentamidine isethionate are the drugs of choice in treatment. Cutaneous Leishmaniasis Cutaneous leishmaniasis is caused by L.tropica and L.major complexes. Cutaneous disease with a papule at the bite site, followed by necrosis of epidermis and ulceration. Infection remain localized, although lesions may be multiple. Diagnosis is made by histological examination for amastigotes in smears or biopsy material; serology. Sodium stibogluconate is also used for treatment of this disease. Mucocutaneous Leishmaniasis This disease is usually caused by L.braziliensis. Infection mainly involves the mucous membranes of the upper respiratory tract (palate, nose) and related tissue, with ulceration, tissue destruction and resulting disfugurement. Histological examination of biopsies and serology are used for diagnosis of the disease. Treatment is made by sodium stibogluconate or amphotericin B. Trypanosoma Species Trypanosomes are haemoflagellates and live in the blood and tissue of human hosts. The life cycle involves two hosts: blood-sucking insects and mammals. It causes two diseases. • African trypanosomiasis (sleeping sickness) caused by T.brucei gambiense and T.brucei rhodesiense, and is transmitted by the tsetse fly. • American trypanosomiasis (Chagas’ disease) caused by T.cruzi, and is transmitted by the reduviid bug.

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Trypanosoma brucei gambiense (West African sleeping sickness) Morphology and Life Cycle The infective stage of the organism is the trypomastigote, present in the salivary glands of transmitting tsetse flies. Trypomastigote varies greatly in length from 17 to 30 μm. It has an undulating membrane that runs the length of the organism. In the long, slender forms, the undulating membrane terminates in a free flagellum at the anterior end. There is a prominent nucleus in the midbody region. The kinetoplast is found near the origin of the flagellum and undulating membrane at the posterior of the organism. The trypomastigotes enter the wound created by the fly bite and find their way into blood and lymph node, eventually invading the central nerves system. Reproduction of the trypomastigotes in blood, lymph, and spinal fluid is by binary fission. These trypomastigotes in blood are then infective for biting tsetse flies, where further reproduction occurs in the midgut. The organism then migrate to the salivary glands where an epimastigote form (having a free flagellum but only a partial undulating membrane) continues reproduction to the infective trypomastigote stage. Tsetse become infective 4 to 6 weeks after feeding on blood from a diseased patient. Clinical Manifestations A nodule or chancre can form at the site of the bite, which usually resolves spontaneously. The parasite enters the blood stream and results in lymphadenopathy, irregular fever and myalgia. CNS involvment (sleeping sickness) may occur with lethargy and encephalitis, leading to convulsions, coma and occasionally death. Laboratory Diagnosis Microscopy of thick and thin blood films, lymph node aspirates and cerebrospinal fluid for trypomastigotes; serology (e.g., ELISA). Treatment. Suramin; melarsoprol if CNS involvment. Trypanosoma brucei rhodesiense (East African sleeping sickness) The life cycle is similar to T.brucei gambiense, with trypomastigote and epimastigote stages. Transmission is by the tsetse fly. It is found in East Africa. The organism has a shorter incubation than T.brucei gambiense. The illness is more severe and progresses rapidly and may involve the heart, kidney and CNS. Mortality is greater than for the West African form. Diagnosis and treatment. As above.

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Trypanosoma cruzi (Chagas’ disease or American disease) T.cruzi is found in North, central and South America. The life cycle is similar to other trypanosomes, except when the infected bug bites humans, trypomastigotes are released simultaneously in the faeces; these enter the wound following scratching. Spread is via the lymphatic and blood systems and results in invasion of many organs, including liver, heart, muscle and brain. Within host cells, trypomastigotes transform into amastigote, which multiply by binary fission and form either further amastigotes or trypomastigotes; the latter are ingested by a feeding insect, multiply in the intestine and are then passed in the faeces. Chagas’ disease may be asymptomatic, acute or chronic. A painful nodule may form at the bite. Acute infection results in high fever, erythematous rash, oedema and myocarditis. Chronic disease may develop years after the initial infection. Organisms proliferate in various organs, including the brain, spleen, liver, heart and lymph nodes, resulting in lymphadenopathy, hepatosplenomegaly, myocarditis and cardiomegaly. Granulomas and cysts may form in the brain. Cardiac disease is the most common presentation, with congestive cardiac failure. Laboratory diagnosis is made by microscopy of biopsies of affected tissues; thick blood films for trypomastigotes in the acute phase; serological tests (e.g., ELISA). Treatment is by nifurtimox. COCCIDIA The coccidae include Cryptosporidium parvum, Isospora belli, Plasmodium species, Toxoplasma gondii and Pneumocystis carinii. They undergo asexual (schzogony) and sexual (gametogony) reproduction and have a variety of hosts, including humans. Cryptosporidium parvum These organisms are seen in the brush border of the intestinal epithelium of both small and large bowel. Parasites have been seen in the gallbladder, tonsillar epithelium, and bronchi in patients with AIDS. Morphology, Biology, and Life Cycle The form of the organism excreted in the stool is called an oocyst. It contains four sporozoites. Following ingestion, the sporozoites escape from the oocyst and penetrate the intestinal epithelium, where they undergo asexual multiplication. The organisms appear to be attached to the cell’s surface at the brush border, but are actually under the cell membrane external to the cytoplasm. Sexual stage develop and unite to form an oocyst that is shed in the feces. Some oocysts release sporozoites

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before they are excreted in the stool. These sporozoites enter intestinal epithelial cells and continue the cycle (internal autoinfection). Clinical Manifestations In immunocompetent individuals, the infection is associated with low-grade fever, nausea, occasional vomiting, and a self-limited watery diarrhea. The illness rarely lasts more than 2 weeks. In immunocompromised patients, the diarrhea is chronic, often severe, and may be fatal. It has a major problem in patients with AIDS. Laboratory Diagnosis Microscopy of faecal specimens by a modified acid-fast stain for the presence of oocysts. Treatment There is no effective treatment available. Immunocompetent patients do not require treatment and the infection resolves spontaneously. Isospora belli I.belli is a coccidian parasite which causes diarrhoea and malabsorption in immunocompromised patients. Morphology, Biology, and Life cycle I.belli has a life cycle similar to that of Cryptosporidium, but it is located deep within the cytoplasm of the intestinal epithelial cell. The oocysts excreted in the stool. Two spherical sporoblast are seen within the oocyst. The sporoblasts mature into sporocysts, each containing four sporozoites. Clinical Manifestations The acute infection is similar to cryptosporidiosis, but is often accompanied by eosinophilia. Patients with AIDS and other immunosuppressed individuals may develop chronic, debilitating diarrhea. Laboratory Diagnosis is dependent on finding oocysts in fecal concentrates or in modified acid-fast stains of stool.

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Treatment is usually not needed in the self-limited illness seen in immunocompetent individuals, but the combination of trimethroprim and sulfamethoxazole appears effective. Toxoplasma gondii T.gondii, is found in a large number of mammals including man, and in birds. It is an intracellular parasite that has the ability to infect any nucleated cell. Major target organs are the lymph nodes, the brain, skeletal muscle, and the retina. Morphology, Biology, and Life Cycle The definitive host for T.gondii is the domestic cat and other felids. The cat acquires the infection by eating infected rodents or birds that contain tissue cysts or by ingesting oocysts from the feces of other cats. In the cat, the parasite undergoes reproduction in the intestinal epithelium. Sexual stages in the intestine result in the production of oocysts that are excreted in the cat’s feces. The oocysts are infectious for a wide variety of birds and mammals including humans and other cats. When animals or humans ingest oocysts from cat feces, organisms escape from the oocyst and develop into trophozoites called tachyzoites. Tachyzoites are elongated, crescentshaped organisms. The posterior end of the organism is more rounded than the anterior end that contains the apical complex. There is a single nucleus. Tachyzoites actively penetrate host cells, multiply intracellularly, and cause the rupture and death of the host cell. While the tachyzoite is transiently free in the extracellular enviroment, it is susceptible to lysis by antibody and complement. Once it has entered a cell, it is protected from the lethal effects of humoral antibody. In macrophages, the tachyzoites survive by inhibiting the fusion of lysosomes with the phagosome containing the parasite. Eventually, some tachyzoites form cysts, more frequently in brain, skeletal muscle, and heart. The organisms that develop within the cysts are called bradyzoites. Humans become infected from: . ingestion of undercooked meat contaminated with trophozoites; . ingestion of infected oocytes from the cat feces; . transplacental transmission; . cardiac transplantation; the recipient receives a heart containing toxoplasma cysts. Clinical Manifestations The two major categories of infection are acquired and conginital. Most acquired infections are asymptomatic. When clinical evidence of infection is present, the most common presentation of acquired infection is lymphodenopathy,usually cervical, without associated symptoms. Some acquired infections may have systemic manifestations, such as fever, headache, myalgia, lymphadenopathy, rash, and

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splenomegaly. The complex of symptoms may mimic infectious mononucleosis or a “flu-like” illness. There are rare case reports of acquired toxoplasmosis causing hepatitis, encephalitis, myocarditis, and pneumonia. Congenital infections occur only when the mother acquires the infection immediately prior to or during pregnancy. Women who have antibody to toxoplasma prior to becoming pregnant are not at risk for having an infected fetus or infant. Although congenital toxoplasmosis may be asymptomatic, it can cause mild-tosevere disease. Infants with inapparent infection at birth may develop significant ocular and neurologic sequelae in later childhood. Infections acquired by the fetus early in the gestational period are more likely to result in serious complications recognized at birth. Laboratory Diagnosis . Serology (e.g. ELISA): by rising IgG antibodies in paired sera, or IgM antibodies to differentiate between active and previous infection. . Histology: by examination of appropriate biopsies for cysts. Treatment Toxoplasmosis is a self-limiting disease in most cases, and is often diagnosed relatively late in the acute stage. The risks of treatment must therefore be balanced against the likely benefit. Howevere, treatment is always justified in myocardial or central nervous system disease. The treatment of choice is a combination of sulphonamide and pyrimethamine. Pneumocystis carinii Historically, P.carinii has been placed with the sporozoites, but recent DNA studies suggest that it may be a fungus. It is causes pneumonia in immunocompromised individuals, such as AIDS patients, premature infants, malnourished children, patients undergoing immunosuppressive therapy, and in children with congenital immunodeficiencies. P.carinii is seen in the pulmonary alveoli as thick-walled cysts with intracystic bodies and as trophozoites. The illness may be abrupt or insidious in onset. Fever and difficulty breathing are common symptoms. Death from progressive pulmonary failure usually occurs in untreated patients. Diagnosis is made by direct examination of material, including bronchial biopsies, washings or aspirates, and open lung biopsies. Parasites are identified by histopathological stains or specific fluorescein-labelled antibodies. Serology may also be used.

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Treatment of Pneumocystis infection is usually with Co-trimoxazole or with pentamidine. Prophylaxis is used in AIDS patients and in HIV infected patients with low CD4 lymphocyte counts. Plasmodium spp. Plasmodium are coccidian or sporozoan parasites of erythrocytes. The four species that cause malaria disease in humans are P.vivax, P.falciparum, P.malariae, and P.ovale. Morphology, Biology, and Life Cycle All plasmodia share a common life cycle but with some important variations. Anopheles mosquito introduces infective stages (sporozoites) into the human host in its saliva during biting. The sporozoites liver parenchymal cells and multiply asexually untile the parasites rupture from the cell. The released parasites infect erythrocytes and begin a cycle of asexual reproduction (schizogony) within the RBCs. This asexual cycle in the RBCs has the following stages: 1. The ring stage is the earliest form seen in the red cell. It consists of a ring of cytoplasm containing a dot of nuclear material. The central area of the ring is a vacule containing hemoglobin. 2. The trophozoite is the ameboid growing form of the organism before there is any division of nuclear material. As the trophozoite develops, brownish or black granular material appears within the cytoplasm. This malaria pigment is the ironcontaining, end product of hemoglobin metabolized by the parasite. 3. The early schizont stage begins with the first nuclear division. The cytoplasm of the parasite has not yet undergo division. 4. The mature schizont (segmenter) has undergo complete division of the nuclear material and the cytoplasm to form individual organisms called merozoites. When the erythrocyte containing the mature schizont ruptures, the merozoites are released and penetrate other RBCs to continue the cycle. After several asexual cycles, some merozoites do not progress through asexual development, but remain compact organisms that enlarge and differentiate into either male (microgametocyte) or female (macrogametocyte) forms. These gametocytes circulate in the blood and are unable to develop further unless taken up by an anopheline mosquito during a blood meal. The sexual reproductive cycle occurs in the mosquito’s digestive system. Sporozoites form, migrate to salivary glands and are inoculated into a new host.

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Characteristics of the different species follow: P.vivax infects young RBCs. The parasite cause enlargement of the infected cell and produce stainable alterations of the red cell membrane called Schüffner’s dots. The trophozoites are very ameboid and the mature schizont contains 12-24 merozoites. P.falciparum is able to infect RBCs of any age. The ring stage is small and delicate and many have two chromatin (nuclear) dots. Gametocytes are cresent shaped. Only rings and gametocytes are seen in the peripheral blood. The more advanced stages of asexual development take place in venules where the infected red cells adhere to the vascular endothelium. P.malariae infects mature RBCs and these cells do not enlarge as the parasite develop. The trophozoites tend to extend from one side of the red cell to the other in a band. Mature schizonts contain 6-12 merozoites. P.ovale infects young RBCs causes them to enlarge and produces Schüffner’s dots. The infected cells are often ovoid and have ragged margins. The trophozoite remains relatively compact, and the mature schizont usually has 4-12 merozoites. Epidemiology In tropical and subtropical areas plasmodia are dependent on correct conditions for breeding of Anopheles mosquitos. P.falciparum is responsible for more than 80% cases in tropical areas. In endemic areas, repeated infections/exposure result in relative immunity and less severe disease. Victors to endemic areas are more severely affected. Transmission via contaminated blood transfusions or needle-sharing can ocuur rarely. Clinical Manifestations of Malaria . Malaria ia one of the important imported diseases. . The incubation period is variable (10-40 days, but may be prolonged). . Fever/sweet: symptoms are related to release of toxins when schizonts burst and therefore intervals between bouts of pyrexia are dependent on the erythrocyte cycle of the Plasmodium species. . Anaemia: this is due to erythrocyte haemolysis. It is most severe with P.falciparum malaria and may result in haemoglobinuria (blackwater fever). . Cerebral malaria: high levels of parasitaemia associated with P.falciparum may result in erythrocyte debris blocking capillaries in the brain; the resultant hypoxia causes confusion and eventually coma, with a high mortality.

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Laboratory Diagnosis Thick and thin blood films are taken. The optimal stain for malaria parasites is Giemsa; however, parasites should be easily recognized in films stained with Wright’s stain. The typical morphology of the parasite within erythrocytes allows the differentiation of Plasmodium species. Treatment Chloroquine is the treatment of choice for malaria due to P.vivax, P.ovale and P.malariae. Supplementing treatment with primaquine is important to destroy the liver hypnozoite stages of P.vivax and P.ovale. Chloroquine is also the drug of choice for P.falciparum malaria, but chloroquineresistance is now common in some areas of the world. Alternative drugs include quinine and the combination of pyrimethamine and sulphadoxine. Prevention This is important for travellers to endemic areas. Avoidance of mosquito bites is necessary. Prophylactic antimalarials may be taken. CILIATES The only cilliate species pathogenic to man is Balantidium coli, a common pathogen of pigs. The parasites are found in the colon. The trophozoites vary in size and can reach 150 µ in their largest dimension. Trophozoites are covered with cilia and have a funnellike cytostome at the anterior pole. The cytoplasm contains a very large, kidney-shaped macronucleus and an inconspicuous micronucleus. Cysts measure about 60 µ in diameter. B. coli is found worldwide, with pigs and cattle as important reservoirs. Infection is via the fecal-oral route, with occational out-breaks following contamination of water supplies. B.coli cysts are ingested and trophozoites are formed, which invade the mucosa of the large intestine and terminal ileum. The majority of infections are asymptomatic but the parasite has the ability to produce colonic ulcerations similar to those found in amebiasis. Diagnosis is made by finding trophozoites or cysts in the stool. Treatment with tetracycline or metronidazole is effective.

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HELMINTHS (WORMS) NEMATODES Nematodes, or roundworms, are cylindrical in shape and have a complete intestinal tract, with the mouth located at the anterior end and the anus at the posterior end. The sexes are separate. The female worm is larger than the male. The male has a sharply curved posterior end or an expansion of the posterior, called a bursa, which is used in clasping the female. The male also possesses one or two copulatory spicules that aid in the mating process. The testes and ovaries are cylindrical. The ovaries are continuous with the uteri. Usually, the vulva is located near the midbody of the female. Larvae develop within the egg, and in some species, hatch from the egg to continue development in the soil. The first stage larva is noninfectious. After additional development it becomes infectious. The common intestinal nematodes, with the exception of Enterobius vermicularis, are often referred to as soil-borne nematodes because infection is acquired by the ingestion of eggs from soil or by the active penetration of the skin by larvae dwelling in the soil. The intestinal nematodes do not have intermediate hosts, but they require a period of maturation outside the human host. There are no significant reservoir hosts for the intestinal nematodes. Ascaris lumbricoides Structure and Life cycle Adult worms are cylindrical; the female is up to 35 cm long, the male up to 30 cm long. Following ingestion of an infective egg, larval worms are released in the duodenum, which penetrate the intestinal wall to reach the blood stream, passing via liver and heart to the polmunary circulation. These larval worms pass into the alveoli, migrate via the bronchi, trachea and pharynx, are swallowed and return to the small intestine. Worms can be expelled orally if vomiting occurs. Male and female worms mature and mate in the intestine; up to 200 000 eggs Per day are produced by the female and passed in stools. Epidemiology. A.lumbricoides is found throughout the world, but particularly in areas of poor sanitation where faecal-oral transmission may occur. Eggs can survive for long periods (several years), making Ascaris the most common pathogenic helminth worldwide. Clinical Manifestations/Complications.

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