Medical Microbiology: with STUDENT CONSULT Online Access, 6e [6 ed.] 0323054706, 9780323054706

Table of contents :
Cover
Table of Contents
Section 1 Introduction
1 Introduction to Medical Microbiology
Section 2 Basic Principles of Medical Microbiology
3 Bacterial Metabolism and Genetics
4 Viral Classification, Structure, and Replication
5 Fungal Classification, Structure, and Replication
6 Parasitic Classification, Structure, and Replication
7 Commensal and Pathogenic Microbial Flora in Humans
8 Sterilization, Disinfection, and Antisepsis
Section 3 Basic Concepts in the Immune Response
9 Elements of Host Protective Responses
10 Humoral Immune Responses
11 Cellular Immune Responses
12 Immune Responses to Infectious Agents
13 Antimicrobial Vaccines
Section 4 General Principles of Laboratory Diagnosis
14 Microscopic Principles and Applications
15 In Vitro Culture Principles and Applications
16 Molecular Diagnosis
17 Serologic Diagnosis
Section 5 Bacteriology
18 Mechanisms of Bacterial Pathogenesi
19 Laboratory Diagnosis of Bacterial Diseases
20 Antibacterial Agents
21 Staphylococcus and Related Gram-Positive Cocci
22 Streptococcus
23 Enterococcus and Other Gram-Positive Cocci
24 Bacillus
25 Listeria and Erysipelothrix
26 Corynebacterium and Other Gram-Positive Rods
27 Nocardia and Related Bacteria
28 Mycobacterium
29 Neisseria and Related Bacteria
30 Enterobacteriaceae
31 Vibrio and Aeromonas
32 Campylobacter and Helicobacter
33 Pseudomonas and Related Bacteria
34 Haemophilus and Related Bacteria
35 Bordetella
36 Francisella and Brucella
37 Legionella
38 Miscellaneous Gram-Negative Rods
39 Clostridium
40 Anaerobic, Non-Spore-Forming, Gram-Positive Bacteria
41 Anaerobic Gram-Negative Bacteria
42 Treponema, Borrelia, and Leptospira
43 Mycoplasma and Ureaplasma
44 Rickettsia and Orientia
45 Ehrlichia, Anaplasma, and Coxiella
46 Chlamydia and Chlamydophila
47 Role of Bacteria in Diseas
Section 6 Virology
48 Mechanisms of Viral Pathogenesis
49 Antiviral Agents
50 Laboratory Diagnosis of Viral Diseases
51 Papillomaviruses and Polyomaviruses
52 Adenoviruses
53 Human Herpesviruses
54 Poxviruses
55 Parvoviruses
56 Picornaviruses
57 Coronaviruses and Noroviruses
58 Paramyxoviruses
59 Orthomyxoviruses
60 Rhabdoviruses, Filoviruses, and Bornaviruses
61 Reoviruses
62 Togaviruses and Flaviviruses
63 Bunyaviridae and Arenaviridae
64 Retroviruses
65 Hepatitis Viruses
66 Unconventional Slow Viruses Prions
67 Role of Viruses in Disease
Section 7 Mycology
68 Pathogenesis of Fungal Disease
69 Laboratory Diagnosis of Fungal Diseases
70 Antifungal Agents
71 Superficial and Cutaneous Mycoses
72 Subcutaneous Mycoses
73 Systemic Mycoses Due to Dimorphic Fungi
74 Opportunistic Mycoses
75 Fungal and Fungal-Like Infections of Unusual or Uncertain Etiology
76 Mycotoxins and Mycotoxicoses
77 Role of Fungi in Disease
Section 8 Parasitology
78 Pathogenesis of Parasitic Diseases
79 Laboratory Diagnosis of Parasitic Disease
80 Antiparasitic Agents
81 Intestinal and Urogenital Protozoa
82 Blood and Tissue Protozoa
83 Nematodes
84 Trematodes
85 Cestodes
86 Arthropods
87 Role of Parasites in Disease

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Table of contents Section I Introduction Section II Basic Principles of Medical Microbiology Section III Basic Concepts in the Immune Response Section IV General Principles of Laboratory Diagnosis Section V Bacteriology Section VI Virology Section VII Mycology Section VIII Parasitology Section I 1 Introduction to Medical Microbiology.htm? Section II 2 Bacterial Classification, Structure, and Replication.htm? 3 Bacterial Metabolism and Genetics.htm? 4 Viral Classification, Structure, and Replication.htm? 5 Fungal Classification, Structure, and Replication.htm? 6 Parasitic Classification, Structure, and Replication.htm? 7 Commensal and Pathogenic Microbial Flora in Humans.htm? 8 Sterilization, Disinfection, and Antisepsis.htm? Section III 09 Elements of Host Protective Responses.htm? 10 Humoral Immune Responses.htm? 11 Cellular Immune Responses.htm? 12 Immune Responses to Infectious Agents.htm? 13 Antimicrobial Vaccines.htm? Section IV 14 Microscopic Principles and Applications.htm? 15 In Vitro Culture Principles and Applications.htm? 16 Molecular Diagnosis.htm? 17 Serologic Diagnosis.htm? Section V

18 Mechanisms of Bacterial Pathogenesis.htm? 19 Laboratory Diagnosis of Bacterial Diseases.htm? 20 Antibacterial Agents.htm? 21 Staphylococcus and Related Gram-Positive Cocci.htm? 22 Streptococcus.htm? 23 Enterococcus and Other Gram-Positive Cocci.htm? 24 Bacillus.htm? 25 Listeria and Erysipelothrix.htm? 26 Corynebacterium and Other Gram-Positive Rods.htm? 27 Nocardia and Related Bacteria.htm? 28 Mycobacterium.htm? 29 Neisseria and Related Bacteria.htm? 30 Enterobacteriaceae.htm? 31 Vibrio and Aeromonas.htm? 32 Campylobacter and Helicobacter.htm? 33 Pseudomonas and Related Bacteria.htm? 34 Haemophilus and Related Bacteria.htm? 35 Bordetella.htm? 36 Francisella and Brucella.htm? 37 Legionella.htm? 38 Miscellaneous Gram-Negative Rods.htm? 39 Clostridium.htm? 40 Anaerobic, Non-Spore-Forming, Gram-Positive Bacteria.htm? 41 Anaerobic Gram-Negative Bacteria.htm? 42 Treponema, Borrelia, and Leptospira.htm? 43 Mycoplasma and Ureaplasma.htm? 44 Rickettsia and Orientia.htm? 45 Ehrlichia, Anaplasma, and Coxiella.htm? 46 Chlamydia and Chlamydophila.htm? 47 Role of Bacteria in Disease.htm? Section VI 48 Mechanisms of Viral Pathogenesis.htm?

49 Antiviral Agents.htm? 50 Laboratory Diagnosis of Viral Diseases.htm? 51 Papillomaviruses and Polyomaviruses.htm? 52 Adenoviruses.htm? 53 Human Herpesviruses.htm? 54 Poxviruses.htm? 55 Parvoviruses.htm? 56 Picornaviruses.htm? 57 Coronaviruses and Noroviruses.htm? 58 Paramyxoviruses.htm? 59 Orthomyxoviruses.htm? 60 Rhabdoviruses, Filoviruses, and Bornaviruses.htm? 61 Reoviruses.htm? 62 Togaviruses and Flaviviruses.htm? 63 Bunyaviridae and Arenaviridae.htm? 64 Retroviruses.htm? 65 Hepatitis Viruses.htm? 66 Unconventional Slow Viruses Prions.htm? 67 Role of Viruses in Disease.htm? Section VII 68 Pathogenesis of Fungal Disease.htm? 69 Laboratory Diagnosis of Fungal Diseases.htm? 70 Antifungal Agents.htm? 71 Superficial and Cutaneous Mycoses.htm? 72 Subcutaneous Mycoses.htm? 73 Systemic Mycoses Due to Dimorphic Fungi.htm? 74 Opportunistic Mycoses.htm? 75 Fungal and Fungal-Like Infections of Unusual or Uncertain Etiology.htm? 76 Mycotoxins and Mycotoxicoses.htm? 77 Role of Fungi in Disease.htm? Section VIII 78 Pathogenesis of Parasitic Diseases.htm?

79 Laboratory Diagnosis of Parasitic Disease.htm? 80 Antiparasitic Agents.htm? 81 Intestinal and Urogenital Protozoa.htm? 82 Blood and Tissue Protozoa.htm? 83 Nematodes.htm? 84 Trematodes.htm? 85 Cestodes.htm? 86 Arthropods.htm? 87 Role of Parasites in Disease.htm? Share please!!!

Viruses Viruses are the smallest infectious particles, ranging in diameter from 18 to 600 nanometers (most viruses are less than 200 nm and cannot be seen with a light microscope). Viruses typically contain either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) but not both; however, some viral-like particles do not contain any detectable nucleic acids (e.g., prions; see Chapter 66), while the recently discovered Mimivirus contains both RNA and DNA. The viral nucleic acids and proteins required for replication and pathogenesis are enclosed in a protein coat with or without a lipid membrane coat. Viruses are true parasites, requiring host cells for replication. The cells they infect and the host response to the infection dictate the nature of the clinical manifestation. More than 2000 species of viruses have been described, with approximately 650 infecting humans and animals. Infection can lead either to rapid replication and destruction of the cell or to a long-term chronic relationship with possible integration of the viral genetic information into the host genome. The factors that determine which of these takes place are only partially understood. For example, infection with the human immunodeficiency virus, the etiologic agent of the acquired immunodeficiency syndrome (AIDS), can result in the latent infection of CD4 lymphocytes or the active replication and destruction of these immunologically important cells. Likewise, infection can spread to other susceptible cells, such as the microglial cells of the brain, resulting in the neurologic manifestations of AIDS. Thus the diseases caused by viruses can range from the common cold to gastroenteritis to fatal catastrophes such as rabies, Ebola, smallpox, or AIDS. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) ? 2009 Elsevier

Bacteria

Bacteria are relatively simple in structure. They are prokaryotic organisms-simple unicellular organisms with no nuclear membrane, mitochondria, Golgi bodies, or endoplasmic reticulum-that reproduce by asexual division. The bacterial cell wall is complex, consisting of one of 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 overlying outer membrane (additional information about this structure is presented in Chapter 2). Some bacteria lack this cell wall structure and compensate by surviving only inside host cells or in a hypertonic environment. The size (1 to 20 ?m or larger), shape (spheres, rods, spirals), and spacial arrangement (single cells, chains, clusters) of the cells are used for the preliminary classification of bacteria, and the phenotypic and genotypic properties of the bacteria form the basis for the definitive classification. The human body is inhabited by thousands of different bacterial species-some living transiently, others in a permanent parasitic relationship. Likewise, the environment that surrounds us, including the air we breathe, water we drink, and food we eat, is populated with bacteria, many of which are relatively avirulent and some of which are capable of producing life-threatening disease. Disease can result from the toxic effects of bacterial products (e.g., toxins) or when bacteria invade normally sterile body sites. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) ? 2009 Elsevier

Fungi

In contrast to bacteria, the cellular structure of fungi is more complex. These are eukaryotic organisms that contain a well-defined nucleus, mitochondria, Golgi bodies, and endoplasmic reticulum (see Chapter 5). Fungi can exist either in a unicellular form (yeast) that can replicate asexually or in a filamentous form (mold) that can replicate asexually and sexually. Most fungi exist as either yeasts or molds; however, some fungi can assume either morphology. These are known as dimorphic fungi and include such organisms as Histoplasma, Blastomyces, and Coccidioides. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) ? 2009 Elsevier

Parasites Parasites are the most complex microbes. Although all parasites are classified as eukaryotic, some are unicellular and others are multicellular (see Chapter 6). They range in size from tiny protozoa as small as 1 to 2 ?m in diameter (the size of many bacteria) to tapeworms that can measure up to 10 meters in length and arthropods (bugs). Indeed, considering the size of some of 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 permanent relationship with humans and others going through a series of developmental stages in a progression of animal hosts. One of the difficulties confronting students is not only an understanding of the spectrum of diseases caused by parasites, but also an appreciation of the epidemiology of these infections, which is vital for developing a differential diagnosis and an approach to the control and prevention of parasitic infections. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) ? 2009 Elsevier

Microbial Disease

One of the most important reasons for studying microbes is to understand the diseases they cause and the ways to control them. Unfortunately, the relationship between many organisms and their diseases is not simple. Specifically, most organisms do not cause a single, well-defined disease, although there are certainly ones that do (e.g., Treponema pallidum, syphilis; poliovirus, polio; Plasmodium species, malaria). Instead, it is more common for a particular organism to produce many manifestations of disease (e.g., Staphylococcus aureus-endocarditis, pneumonia, wound infections, food poisoning) or for many organisms to produce the same disease (e.g., meningitis caused by viruses, bacteria, fungi, and parasites). In addition, relatively few organisms can be classified as always pathogenic, although some do belong in this category (e.g., rabies virus, Bacillus anthracis, Sporothrix schenckii, Plasmodium species). Instead, most organisms are able to establish disease only under well-defined circumstances (e.g., the introduction of an organism with a potential for causing disease into a normally sterile site such as the brain, lungs, and peritoneal cavity). Some diseases arise when a person is exposed to organisms from external sources. These are known as exogenous infections, and examples include diseases caused by influenza virus, Clostridium tetani, Neisseria gonorrhoeae, Coccidioides immitis, and Entamoeba histolytica. Most human diseases, however, are produced by organisms in the person's own microbial flora that spread to inappropriate body sites where disease can ensue (endogenous infections). The interaction between an organism and the human host is complex. The interaction can result in transient colonization, a long-term symbiotic relationship, or disease. The virulence of the organism, the site of exposure, and the host's ability to respond to the organism determine the outcome of this interaction. Thus the manifestations of disease can range from mild symptoms to organ failure and death. The role of microbial virulence and the host's immunologic response is discussed in depth in subsequent chapters. page 4 page 5

The human body is remarkably adapted to controlling exposure to pathogenic microbes. Physical barriers prevent invasion by the microbe; innate responses recognize molecular patterns on the microbial components and activate local defenses and specific adapted immune responses that target the microbe for elimination. Unfortunately, the immune response is often too late or too slow. To improve the human body's ability to prevent infection, the immune system can be augmented either through the passive transfer of antibodies present in immune globulin preparations or through active immunization with components of the microbes (antigens). Infections can also be controlled with a variety of chemotherapeutic agents. Unfortunately, many microbes can alter their antigenic complexion (antigenic variation) or develop resistance to even the most potent antibiotics. Thus the battle for control between microbe and host continues, with neither side yet able to claim victory (although the microbes have demonstrated remarkable ingenuity). There clearly is no "magic bullet" that has eradicated infectious diseases. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) ? 2009 Elsevier

Diagnostic Microbiology

The clinical microbiology laboratory plays an important role in the diagnosis and control of infectious diseases. However, the ability of the laboratory to perform these functions is limited by the quality of the specimen collected from the patient, the means by which it is transported from the patient to the laboratory, and the techniques used to demonstrate the microbe in the sample. Because most diagnostic tests are based on the ability of the organism to grow, transport conditions must ensure the viability of the pathogen. In addition, the most sophisticated testing protocols are of little value if the collected specimen is not representative of the site of infection. This seems obvious, but many specimens sent to laboratories for analysis are contaminated during collection with the organisms that colonize the mucosal surfaces. It is virtually impossible to interpret the testing results with contaminated specimens, because most infections are caused by endogenous organisms. The laboratory is also able to determine the antimicrobial activity of selected chemotherapeutic agents, although the value of these tests is limited. The laboratory must test only organisms capable of producing disease and only medically relevant antimicrobials. To test all isolated organisms or an indiscriminate selection of drugs can yield misleading results with potentially dangerous consequences. Not only can a patient be treated inappropriately with unnecessary antibiotics, but also the true pathogenic organism may not be recognized among the plethora of organisms isolated and tested. Finally, the in vitro determination of an organism's susceptibility to a variety of antibiotics is only one aspect of a complex picture. The virulence of the organism, site of infection, and patient's ability to respond to the infection influence the host-parasite interaction and must also be considered when planning treatment. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) ? 2009 Elsevier

Summary

It is important to realize that our knowledge of the microbial world is evolving continually. Just as the early microbiologists built their discoveries on the foundations established by their predecessors, we and future generations will continue to discover new microbes, new diseases, and new therapies. The following chapters are intended as a foundation of knowledge that can be used to build your understanding of microbes and their diseases. page 5 page 6 Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) ? 2009 Elsevier

Bacterial Metabolism Metabolic Requirements Bacterial growth requires a source of energy and the raw materials to build the proteins, structures, and membranes that make up and power the cell. Bacteria must obtain or synthesize the amino acids, carbohydrates, and lipids used as building blocks of the cell. The minimum requirement for growth is a source of carbon and nitrogen, an energy source, water, and various ions. The essential elements include the components of proteins, lipids and nucleic acids (C, O, H, N, S, P), important ions (K, Na, Mg, Ca, Cl) and components of enzymes (Fe, Zn, Mn, Mo, Se, Co, Cu, Ni). Iron is so important that many bacteria secrete special proteins (siderophores) to concentrate iron from dilute solutions, and our bodies will sequester iron to reduce its availability as a means of protection. Oxygen (O2 gas), although essential for the human host, is actually a poison for many bacteria. Some organisms, such as Clostridium perfringens, which causes gas gangrene, cannot grow in the presence of oxygen. Such bacteria are referred to as obligate anaerobes. Other organisms, such as Mycobacterium tuberculosis, which causes tuberculosis, require the presence of molecular oxygen for metabolism and growth and are therefore referred to as obligate aerobes. Most bacteria, however, grow in either the presence or the absence of oxygen. These bacteria are referred to as facultative anaerobes. Aerobic bacteria produce superoxide dismutase and catalase enzymes which can detoxify hydrogen peroxide and superoxide radicals that are the toxic byproducts of aerobic metabolism.

Growth requirements and metabolic byproducts may be used as a convenient means of classifying different bacteria. Some bacteria, such as certain strains of Escherichia coli (a member of the intestinal flora), can synthesize all the amino acids, nucleotides, lipids, and carbohydrates necessary for growth and division, whereas the growth requirements of the causative agent of syphilis, Treponema pallidum, are so complex that a defined laboratory medium capable of supporting its growth has yet to be developed. Bacteria that can rely entirely on inorganic chemicals for their energy and source of carbon (CO2) are referred to as autotrophs (lithotrophs), whereas many bacteria and animal cells that require organic carbon sources are known as heterotrophs (organotrophs). Clinical microbiology laboratories distinguish bacteria by their ability to grow on specific carbon sources (e.g., lactose) and the end products of metabolism (e.g., ethanol, lactic acid, succinic acid).

Metabolism, Energy, and Biosynthesis All cells require a constant supply of energy to survive. This energy, typically in the form of adenosine triphosphate (ATP), is derived 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), a process known as anabolism. Together these two processes, which are interrelated and tightly integrated, are referred to as intermediary metabolism.

The metabolic process generally begins with hydrolysis of large macromolecules in the external cellular environment by specific enzymes (Figure 3-1). The smaller molecules that are produced (e.g., 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. These mechanisms may use specific carrier or membrane transport proteins to help concentrate metabolites from the medium. The metabolites are converted via one or more pathways to one common, universal intermediate, pyruvic acid. From pyruvic acid the carbons may be channeled toward energy production or the synthesis of new carbohydrates, amino acids, lipids, and nucleic acids. page 23 page 24

Figure 3-1 Catabolism of proteins, polysaccharides, and lipids produces glucose, pyruvate, or intermediates of the tricarboxylic acid (TCA) cycle and, ultimately, energy in the form of adenosine triphosphate (ATP) or the reduced form of nicotinamide-adenine dinucleotide (NADH).

Metabolism of Glucose For the sake of simplicity, this section presents an overview of the pathways by which glucose is metabolized to produce energy or other usable substrates. Instead of releasing all the molecule's energy as heat (as for burning), the bacteria break down the glucose in discrete steps to allow the energy to be captured in usable forms. Bacteria can produce energy from glucose by-in order of increasing efficiency-fermentation, anaerobic respiration (both of which occur in the absence of oxygen), or aerobic respiration. Aerobic respiration can completely convert the six carbons of glucose to CO2 and H2O plus energy, whereas two- and three-carbon compounds are the end products of fermentation. For a more complete discussion of metabolism, please refer to a textbook on biochemistry.

Embden-Meyerhof-Parnas Pathway Bacteria use three major metabolic pathways in the catabolism of glucose. Most common among these is the glycolytic, or Embden-Meyerhof-Parnas (EMP), pathway (Figure 3-2) for the conversion of glucose to pyruvate. These reactions, which occur under both aerobic and anaerobic conditions, begin with activation of glucose to form glucose-6-phosphate. This reaction, as well as the third reaction in the series, in which fructose-6-phosphate is converted to fructose-1,6-diphosphate, requires 1 mole of ATP per mole of glucose and represents an initial investment of cellular energy stores.

Figure 3-2 Embden-Meyerhof-Parnas (EMP) glycolytic pathway results in conversion of glucose to pyruvate. ADP, adenosine diphosphate; ATP, adenosine triphosphate; iPO4, inorganic phosphate; NAD, nicotinamide adenine dinucleotide; NADH, reduced form of NAD. page 24 page 25

Figure 3-3 Fermentation of pyruvate by different microorganisms results in different end products. The clinical laboratory uses these pathways and end products as a means of distinguishing different bacteria.

Energy is produced during glycolysis in two different forms, chemical and electrochemical. In the first, the high-energy phosphate group of one of the intermediates in the pathway is used under the direction of the appropriate enzyme (a kinase) to generate ATP from adenosine diphosphate (ADP). This type of reaction, termed substrate-level phosphorylation, occurs at two different points in the glycolytic pathway (i.e., conversion of 3-phosphoglycerol phosphate to 3-phosphoglycerate and 2-phosphoenolpyruvic acid to pyruvate). Four ATP molecules per molecule of glucose are produced in this manner, but two ATP molecules were used in the initial glycolytic conversion of glucose to two molecules of pyruvic acid, resulting in a net production of two molecules of ATP. The reduced form of nicotinamide-adenine dinucleotide (NADH) that is produced represents the second form of energy, which may then be converted to ATP by a series of oxidation reactions. In the absence of oxygen, substrate-level phosphorylation represents the primary means of energy production. The pyruvic acid produced from glycolysis is then converted to various end products, depending on the bacterial species, in a process known as fermentation. Many bacteria are identified on the basis of their fermentative end products (Figure 3-3). These organic molecules, rather than oxygen, are used as electron acceptors to recycle the NADH, which was produced during glycolysis, to NAD. In yeast, fermentative metabolism results in the conversion of pyruvate to ethanol plus carbon dioxide. Alcoholic fermentation is uncommon in bacteria, which most commonly use the one-step conversion of pyruvic acid to lactic acid. This process is responsible for making milk into yogurt and cabbage into sauerkraut. Other bacteria use more complex fermentative pathways, producing various acids, alcohols, and often gases (many of which have vile odors). These products lend flavors to various cheeses and wines and odors to wound and other infections.

Tricarboxylic Acid Cycle

Figure 3-4 Tricarboxylic acid cycle occurs in aerobic conditions and is an amphibolic cycle. Precursors for the synthesis of amino acids and nucleotides are also shown. CoA, coenzyme A; FADH2, flavin adenine dinucleotide; GTP, guanosine triphosphate.

In the presence of oxygen, the pyruvic acid produced from glycolysis and from the metabolism of other substrates may be completely oxidized (controlled burning) to water and CO2 using the tricarboxylic acid (TCA) cycle (Figure 3-4), which results in production of additional energy. The process begins with the oxidative decarboxylation (release of CO2) of pyruvate to the high-energy intermediate, acetyl coenzyme A (acetyl CoA); this reaction also produces two NADH molecules. The two remaining carbons derived from pyruvate then enter the TCA cycle in the form of acetyl CoA by condensation with oxaloacetate, with the formation of the six-carbon citrate molecule. In a stepwise series of oxidative reactions the citrate is converted back to oxaloacetate. The theoretical yield from each pyruvate is 2 moles of CO2, 3 moles of NADH, 1 mole of flavin adenine dinucleotide (FADH2), and 1 mole of guanosine triphosphate (GTP). The TCA cycle allows the organism to generate substantially more energy per mole of glucose than is possible from glycolysis alone. In addition to the GTP (an ATP equivalent) produced by substrate-level phosphorylation, the NADH and FADH2 yield ATP from the electron transport chain. In this chain the electrons carried by NADH (or FADH2) are passed in a stepwise fashion through a series of donor-acceptor pairs and ultimately to oxygen (aerobic respiration) or other terminal electron acceptor (nitrate, sulfate, carbon dioxide, ferric iron) (anaerobic respiration). page 25 page 26

Figure 3-5 Aerobic glucose metabolism. The theoretical maximum amount of ATP obtained from one glucose molecule is 38, but the actual yield depends on the organism and other conditions.

Anaerobic organisms are less efficient at energy production than aerobic organisms. Fermentation produces only 2 ATP molecules per glucose, whereas aerobic metabolism with electron transport and a complete TCA cycle can generate as much as 19 times more energy (38 ATP molecules) from the same starting material (and it is much less smelly) (Figure 3-5). Anaerobic respiration uses organic molecules as electron acceptors, which produces less ATP for each NADH.

In addition to the efficient generation of ATP from glucose (and other carbohydrates), the TCA cycle provides a means by which carbons derived from lipids (in the form of acetyl CoA) may be shunted toward either energy production or the generation of biosynthetic precursors. Similarly, the cycle includes several points at which deaminated amino acids may enter (see Figure 3-4). For example, deamination of glutamic acid yields α-ketoglutarate, whereas deamination of aspartic acid yields oxaloacetate, both of which are TCA cycle intermediates. The TCA cycle therefore serves the following functions: 1. It is the most efficient mechanism for the generation of ATP. 2. It serves as the final common pathway for the complete oxidation of amino acids, fatty acids, and carbohydrates. 3. It supplies key intermediates (i.e., α-ketoglutarate, pyruvate, oxaloacetate) for the ultimate synthesis of amino acids, lipids, purines, and pyrimidines. The last two functions make the TCA cycle a so-called amphibolic cycle (i.e., it may function in the anabolic and the catabolic functions of the cell).

Pentose Phosphate Pathway

The final pathway of glucose metabolism considered here is known as the pentose phosphate pathway, or the hexose monophosphate shunt. The function of this pathway is to provide nucleic acid precursors and reducing power in the form of nicotinamide-adenine dinucleotide phosphate (reduced form) (NADPH) for use in biosynthesis. In the first half of the pathway, glucose is converted to ribulose-5-phosphate, with consumption of 1 mole of ATP and generation of 2 moles of NADPH per mole of glucose. The ribulose-5-phosphate may then be converted to ribose-5-phosphate (a precursor in nucleotide biosynthesis) or alternatively to xylulose-5-phosphate. The remaining reactions in the pathway use enzymes known as transketolases and transaldolases to generate various sugars, which may function as biosynthetic precursors or may be shunted back to the glycolytic pathway for use in energy generation. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

The Bacterial Genes and Expression The bacterial genome is the total collection of genes carried by a bacterium, both on its chromosome and on its extrachromosomal genetic elements, if any. Genes are sequences of nucleotides that have a biologic function; examples are protein-structural genes (cistrons, which are coding genes), ribosomal ribonucleic acid (RNA) genes, and recognition and binding sites for other molecules (promoters and operators). Each genome contains many operons, which are made up of genes. Eukaryotes usually have two distinct copies of each chromosome (they are therefore diploid). Bacteria usually have only one copy of their chromosomes (they are therefore haploid). Because bacteria have only one chromosome, alteration of a gene (mutation) will have a more obvious effect on the cell. In addition, the structure of the bacterial chromosome is maintained by polyamines, such as spermine and spermidine, rather than by histones.

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.

Transcription The information carried in the genetic memory of the DNA is transcribed into a useful messenger RNA (mRNA) for subsequent translation into protein. RNA synthesis is performed by a DNA-dependent RNA polymerase. page 26 page 27

The process begins when sigma factor recognizes a particular sequence of nucleotides in the DNA (the promoter) and binds tightly to this site. Promoter sequences occur just before the start of the DNA that actually encodes a protein. Sigma factors bind to these promoters to provide a docking site for the RNA polymerase. Some bacteria encode several sigma factors to allow transcription of a group of genes under special conditions, such as heat shock, starvation, special nitrogen metabolism, or sporulation. Once the polymerase has bound to the appropriate site on the DNA, RNA synthesis proceeds with the sequential addition of ribonucleotides complementary to the sequence in the DNA. Once an entire gene or group of genes (operon) has been transcribed, the RNA polymerase dissociates from the DNA, a process mediated by signals within the DNA. The bacterial, DNA-dependent RNA polymerase is inhibited by rifampin, an antibiotic often used in the treatment of tuberculosis. The transfer RNA (tRNA), which is used in protein synthesis, and ribosomal RNA (rRNA), a component of the ribosomes, are also transcribed from the DNA.

Promoters and operators control the expression of a gene by influencing which sequences will be transcribed into messenger RNA (mRNA). Operons are groups of one or more structural genes expressed from a particular promoter and ending at a transcriptional terminator. Thus all the genes coding for the enzymes of a particular pathway can be coordinately regulated. Operons with many structural genes are polycistronic. The E. coli lac operon includes all the genes necessary for lactose metabolism, as well as the control mechanisms for turning off (in the presence of glucose) or turning on (in the presence of galactose or an inducer) these genes only when they are needed. The lac operon includes a repressor sequence, a promoter sequence, and structural genes for the β-galactosidase enzyme, a permease, and an acetylase (Figure 3-6). The lac operon is discussed later in this chapter.

Translation Translation is the process by which the language of the genetic code, in the form of mRNA, is converted (translated) into a sequence of amino acids, the protein product. Each amino acid word and the punctuation of the genetic code is written in a set of three nucleotides, known as a codon. There are 64 different codon combinations encoding the 20 amino acids, the 20 amino acids plus start and termination codons. Some of the amino acids are encoded by more than one triplet codon. This feature is known as the degeneracy of the genetic code and may function in protecting the cell from the effects of minor mutations in the DNA or mRNA. Each tRNA molecule contains a three-nucleotide sequence complementary to one of the codon sequences. This tRNA sequence is known as the anticodon; it allows base pairing and binds to the codon sequence on the mRNA. Attached to the opposite end of the tRNA is the amino acid that corresponds to the particular codon-anticodon pair.

The process of protein synthesis (Figure 3-7) begins with the binding of the 30S ribosomal subunit and a special initiator tRNA for formyl methionine (fmet) at the methionine codon (AUG) start codon to form the initiation complex. The 50S ribosomal subunit binds to the complex to initiate mRNA synthesis. The ribosome contains two tRNA binding sites, the A (aminoacyl) site and the P (peptidyl) site, each of which allows base pairing between the bound tRNA and the codon sequence in the mRNA. The tRNA corresponding to the second codon occupies the A site. The amino group of the amino acid attached to the A site forms a peptide bond with the carboxyl group of the amino acid in the P site in a reaction known as transpeptidation, and the empty tRNA in the P site (uncharged tRNA) is released from the ribosome. The ribosome then moves down the mRNA exactly three nucleotides, thereby transferring the tRNA with attached nascent peptide to the P site and bringing the next codon into the A site. The appropriate charged tRNA is brought into the A site, and the process is then repeated. Translation continues until the new codon in the A site is one of the three termination codons, for which there is no corresponding tRNA. At that point the new protein is released to the cytoplasm and the translation complex may be disassembled, or the ribosome shuffles to the next start codon and initiates a new protein. The ability to shuffle along the mRNA to start a new protein is a characteristic of the 70S bacterial but not of the 80S eukaryotic ribosome. This has implications for the synthesis of proteins for some viruses. The process of protein synthesis by the 70S ribosome represents an important target of antimicrobial action. The aminoglycosides (e.g., streptomycin and gentamicin) and the tetracyclines act by binding to the small ribosomal subunit and inhibiting normal ribosomal function. Similarly the macrolide (e.g., erythromycin) and lincosamide (e.g., clindamycin) groups of antibiotics act by binding to the large ribosomal subunit.

Control of Gene Expression

Bacteria have developed mechanisms to adapt quickly and efficiently to changes and triggers from the environment. This allows them to coordinate and regulate the expression of genes for multicomponent structures or the enzymes of one or more metabolic pathways. For example, temperature change could signify entry into the human host and indicate the need for a global change in metabolism and up-regulation of genes important for parasitism or virulence. Many bacterial genes are controlled at multiple levels and by multiple methods. A coordinated change in the expression of many genes, as would be required for sporulation, occurs through use of a different sigma factor for the RNA polymerase. This would change the specificity of the RNA polymerase and allow mRNA synthesis for the necessary genes while ignoring unnecessary genes. Bacteria might produce more than six different sigma factors to provide global regulation in response to stress, shock, starvation, or to coordinate production of complicated structures such as flagella. page 27 page 28

Figure 3-6 A, The lactose operon is transcribed as a polycistronic messenger RNA (mRNA) from the promoter (P) and translated into three proteins: β-galactosidase (Z), permease (Y), and acetylase (A). The lac I gene encodes the repressor protein. B, The lactose operon is not transcribed in the absence of an allolactose inducer, because the repressor competes with the RNA polymerase at the operator site (O). C, The repressor, complexed with the inducer, does not recognize the operator because of a conformation change in the repressor. The lac operon is thus transcribed at a low level. D, Escherichia coli is grown in a poor medium in the presence of lactose as the carbon source. Both the inducer and the CAP-cAMP complex are bound to the promoter, which is fully "turned on," and a high level of lac mRNA is transcribed and translated. E, Growth of E. coli in a poor medium without lactose results in the binding of the CAP-cAMP complex to the promoter region and binding of the active repressor to the operator sequence, because no inducer is available. The result will be that the lac operon will not be transcribed. ATP, adenosine triphosphate; CAP, catabolite gene-activator protein; cAMP, cyclic adenosine monophosphate.

Coordination of a large number of processes on a global level can also be mediated by small molecular activators, such as cyclic adenosine monophosphate (cAMP). Increased cAMP levels indicate low glucose levels and the need to utilize alternative metabolic pathways. Similarly, the increased concentration of specific small molecules produced by individual bacteria is used to turn on virulence genes when a sufficient number of bacteria are present. This process is called quorum sensing. The trigger for biofilm production by Pseudomonas spp. is triggered by a critical concentration of N-acyl homoserine lactone (AHL) produced when sufficient numbers of bacteria (a quorum) are present. Activation of toxin production and more virulent behavior by S. aureus accompanies the increase in concentration of a cyclic peptide. To coordinate the expression of a more limited group of genes, such as for a specific metabolic process, the genes for the necessary enzymes would be organized into an operon. The operon would be under the control of a promoter or repressor DNA sequence that can activate or turn off the expression of a gene or a group of genes to coordinate production of the necessary enzymes and allow the bacteria to react to changes in concentrations of nutrients. The genes for some virulence mechanisms are organized into a pathogenicity island under the control of a single promoter to allow their expression under appropriate (to the bacteria) conditions. The many components of the Type III secretion devices of E. coli, Salmonella, or Yersinia are grouped together within a pathogenicity island. page 28 page 29

Transcription can also be regulated by the translation process. Unlike eukaryotes, the absence of a nuclear membrane in prokaryotes allows the ribosome to bind to the mRNA as it is being transcribed from the DNA. The position and speed of ribosomal movement along the mRNA can affect the presence of loops in the mRNA and the ability of the polymerase to transcribe new mRNA. This allows control of gene expression at both the transcriptional and translational levels.

Initiation of transcription may be under positive or negative control. Genes under negative control are expressed unless they are switched off by a repressor protein. This repressor protein prevents gene expression by binding to a specific DNA sequence called the operator, blocking the RNA polymerase from initiating transcription at the promoter sequence. Inversely, genes whose expression is under positive control are not transcribed unless an active regulator protein, called an apoinducer, is present. The apoinducer binds to a specific DNA sequence and assists the RNA polymerase in the initiation steps by an unknown mechanism. Operons can be inducible or repressible. Introduction of a substrate (inducer) into the growth medium may induce an operon to increase the expression of the enzymes necessary for its metabolism. An abundance of the end products (co-repressors) of a pathway may signal that a pathway should be shut down or repressed by reducing the synthesis of its enzymes.

Figure 3-7 Bacterial protein synthesis. 1, Binding of the 30S subunit to the messenger RNA (mRNA) with the formylmethionine transfer RNA (fmet-tRNA) at the AUG start codon allows assembly of the 70S ribosome. The fmet-tRNA binds to the peptidyl site (P). 2, The next tRNA binds to its codon at the A site and "accepts" the growing peptide chain. 3, 4, Before translocation to the peptidyl site. 5, The process is repeated until a stop codon and the protein are released.

The lactose (lac) operon responsible for the degradation of the sugar lactose is an inducible operon under positive and negative regulation (see Figure 3-6). Normally the bacteria use glucose and not lactose. In the absence of lactose the operon is repressed by the binding of the repressor protein to the operator sequence, thus impeding the RNA polymerase function. In the absence of glucose, however, the addition of lactose reverses this repression. Full expression of the lac operon also requires a protein-mediated, positive-control mechanism. In E. coli a protein called the catabolite gene-activator protein (CAP) forms a complex with cyclic adenosine monophosphate (cAMP), acquiring the ability to bind to a specific DNA sequence present in the promoter. When glucose decreases in the cell, cAMP increases to promote usage of other sugars for metabolism. The CAP-cAMP complex enhances binding of the RNA polymerase to the promoter, thus allowing an increase in the frequency of transcription initiation. The tryptophan operon (trp operon) contains the structural genes necessary for tryptophan biosynthesis and is under dual transcriptional control mechanisms (Figure 3-8). Although tryptophan is essential for protein synthesis, too much tryptophan in the cell can be toxic; therefore its synthesis must be regulated. At the DNA level the repressor protein is activated by an increased intracellular concentration of tryptophan to prevent transcription. At the protein synthesis level, rapid translation of a "test peptide" at the beginning of the mRNA in the presence of tryptophan promotes the formation of a double-stranded loop in the RNA, which terminates transcription. The same loop is formed if no protein synthesis is occurring, a situation in which tryptophan synthesis would similarly not be required. This regulates tryptophan synthesis at the mRNA level in a process termed attenuation, in which mRNA synthesis is prematurely terminated.

The expression of the components of virulence mechanisms are also coordinately regulated from an operon. Simple triggers, such as temperature, osmolarity, pH, nutrient availability, or the concentration of specific small molecules, such as oxygen or iron, can turn on or turn off the transcription of a single gene or a group of genes. Salmonella invasion genes within a pathogenicity island are turned on by high osmolarity and low oxygen, conditions present in the gastrointestinal tract. E. coli senses its exit from the gut of a host by a drop in temperature and inactivates its adherence genes. Low iron levels can activate expression of hemolysin in E. coli or diphtheria toxin from Corynebacterium diphtheriae, potentially to kill cells and provide iron. Quorum sensing for virulence factors of S. aureus and biofilm production by Pseudomonas spp. were discussed above.

Replication of DNA page 29 page 30

Figure 3-8 Regulation of the tryptophan (trp) operon. A, The trp operon encodes the five enzymes necessary for tryptophan biosynthesis. This trp operon is under dual control. B, The conformation of the inactive repressor protein is changed after its binding by the co-repressor tryptophan. The resulting active repressor (R) binds to the operator (O), blocking any transcription of the trp mRNA by the RNA polymerase. C, The trp operon is also under the control of an attenuation-antitermination mechanism. Upstream of the structural genes are the promoter (P), the operator, and a leader (L), which can be transcribed into a short peptide containing two tryptophans (W), near its distal end. The leader mRNA possesses four repeats (1, 2, 3, and 4), which can pair differently according to the tryptophan availability, leading to an early termination of transcription of the trp operon or its full transcription. In the presence of a high concentration of tryptophan, regions 3 and 4 of the leader mRNA can pair, forming a terminator hairpin, and no transcription of the trp operon occurs. However, in the presence of little or no tryptophan the ribosomes stall in region 1 when translating the leader peptide because of the tandem of tryptophan codons. Then regions 2 and 3 can pair, forming the antiterminator hairpin and leading to transcription of the trp genes. Finally, the regions 1:2 and 3:4 of the free leader mRNA can pair, also leading to cessation of transcription before the first structural gene trpE. A, adenine; G, guanine; T, thymidine.

The bacterial chromosome is a storehouse of information by which the characteristics of the cell are defined and all cellular processes are carried out. It is therefore essential that this molecule be duplicated without errors. Replication of the bacterial genome is triggered by a cascade of events linked to the growth rate of the cell. Replication of bacterial DNA is initiated at a specific sequence in the chromosome called OriC. The replication process requires many enzymes, including an enzyme (helicase) to unwind the DNA at the origin to expose the DNA, an enzyme (primase) to synthesize primers to start the process, and the enzyme or enzymes (DNA-dependent DNA polymerases) that synthesize a copy of the DNA, but only if there is a primer sequence to add to and only in the 5' to 3' direction. page 30 page 31

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 fragments). The lagging-strand DNA must be extended in the 5' to 3' direction as its template becomes available. Then the pieces are ligated together by the enzyme DNA ligase (Figure 3-9). To maintain the high degree of accuracy required for replication, the DNA polymerases possess "proofreading" functions, which allow the enzyme to confirm that the appropriate nucleotide was inserted and to correct any errors that were made. During log-phase growth in rich medium, many initiations of chromosomal replication may occur before cell division. This process produces a series of nested bubbles of new daughter chromosomes, each with its pair of growth forks of new DNA synthesis. The polymerase moves down the DNA strand, incorporating the appropriate (complementary) nucleotide at each position. Replication is complete when the two replication forks meet 180 degrees from the origin. The process of DNA replication puts great torsional strain on the chromosomal circle of DNA; this strain is relieved by topoisomerases (e.g., gyrase), which supercoil the DNA. Topoisomerases are essential to the bacteria and are targets for the quinolone antibiotics.

Bacterial Growth Bacterial replication is a coordinated process in which two equivalent daughter cells are produced. For growth to occur, there must be sufficient metabolites to support the synthesis of the bacterial components and especially the nucleotides for DNA synthesis. A cascade of regulatory events (synthesis of key proteins and RNA), much like a countdown at the Kennedy Space Center, must occur on schedule to initiate a replication cycle. However, once it is initiated, DNA synthesis must run to completion, even if all nutrients have been removed from the medium.

Chromosome replication is initiated at the membrane, and each daughter chromosome is anchored to a different portion of membrane. Bacterial membrane, peptidoglycan synthesis, and cell division are linked together such that inhibition of peptidoglycan synthesis will also inhibit cell division. As the bacterial membrane grows, the daughter chromosomes are pulled apart. Commencement of chromosome replication also initiates the process of cell division, which can be visualized by the start of septum formation between the two daughter cells (Figure 3-10; see also Chapter 2). New initiation events may occur even before completion of chromosome replication and cell division. Depletion of metabolites (starvation) or a buildup of toxic byproducts (e.g., ethanol) triggers the production of chemical alarmones, which causes synthesis to stop, but degradative processes continue. DNA synthesis continues until all initiated chromosomes are completed, despite the detrimental effect on the cell. Ribosomes are cannibalized for deoxyribonucleotide precursors, peptidoglycan and proteins are degraded for metabolites, and the cell shrinks. Septum formation may be initiated, but cell division may not occur. Many cells die. Similar signals may initiate sporulation in species capable of this process (see Chapter 2).

Figure 3-9 Bacterial DNA replication. New DNA synthesis occurs at growing forks and proceeds bidirectionally. DNA synthesis progresses in the 5' to 3' direction continuously (leading strand) or in pieces (lagging strand). Assuming it takes 40 minutes to complete one round of replication, and assuming new initiation every 20 minutes, initiation of DNA synthesis precedes cell division. Multiple growing forks may be initiated in a cell before complete septum formation and cell division. The daughter cells are "born pregnant."

Figure 3-10 Bacterial cell division. Replication requires extension of the cell wall and replication of the chromosome and septum formation. Membrane attachment of the DNA pulls each daughter strand into a new cell. page 31 page 32

Figure 3-11 Phases of bacterial growth, starting with an inoculum of stationary-phase cells.

Population Dynamics When bacteria are added to a medium, they require time to adapt to the new environment before they begin dividing (Figure 3-11). This hiatus is known as the lag phase of growth. During the log or exponential phase, the bacteria will grow and divide with a doubling time characteristic of the strain and determined by the conditions. The number of bacteria will increase to 2 n, in which n is the number of generations (doublings). The culture eventually runs out of metabolites, or a toxic substance builds up in the medium; the bacteria then stop growing and enter the stationary phase. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Bacterial Genetics

Mutation, Repair, and Recombination Accurate replication of DNA is important to the survival of the bacteria, but mistakes and accidental damage to the DNA occurs. Despite efficient DNA repair systems, mutations and alterations to the DNA do occur. Most of these mutations have little effect on the bacteria or are detrimental, but some mutations may improve the chances of survival of the bacteria when challenged by the environment, the host, or therapy.

Mutations and Their Consequences A mutation is any change in the base sequence of the DNA. A single base change can result in a transition in which one purine is replaced by another purine, or in which a pyrimidine is replaced by another pyrimidine. A transversion, in which, for example, a purine is replaced by a pyrimidine and vice versa, may also result. A silent mutation is a change at the DNA level that does not result in any change of amino acid in the encoded protein. This type of mutation occurs because more than one codon may encode an amino acid. A missense mutation results in a different amino acid being inserted in the protein, but this may be a conservative mutation if the new amino acid has similar properties (e.g., valine replacing alanine). A nonsense mutation changes a codon encoding an amino acid to a stop codon (e.g., TAG [thymidine-adenine-guanine]), which will cause the ribosome to fall off the mRNA and end the protein prematurely. Conditional mutations, such as temperature-sensitive mutations, may result from a conservative mutation which changes the structure or function of an important protein at elevated temperatures.

More drastic changes can occur when numerous bases are involved. A small deletion or insertion that is not in multiples of three produces a frameshift mutation. This results in a change in the reading frame, usually leading to a useless peptide and premature truncation of the protein. Null mutations, which completely destroy gene function, arise when there is an extensive insertion, deletion, or gross rearrangement of the chromosome structure. Insertion of long sequences of DNA (many thousands of base pairs) by recombination, by transposition, or during genetic engineering can produce null mutations by separating the parts of a gene and inactivating the gene. Many mutations occur spontaneously in nature (e.g., by polymerase mistakes); however, physical or chemical agents can also induce mutations. Among the physical agents used to induce mutations in bacteria are heat, which results in deamination of nucleotides; ultraviolet light, which causes pyrimidine dimer formation; and ionizing radiation, such as x-rays, which produce very reactive hydroxyl radicals that may be responsible for opening a ring of a base or causing single- or double-stranded breaks in the DNA. Chemical mutagens can be grouped into three classes. Nucleotide-base analogues lead to mispairing and frequent DNA replication mistakes. For example, incorporation of 5-bromouracil into DNA instead of thymidine allows base pairing with guanine instead of adenine, changing a T-A base pair to a G-C base pair. Frameshift mutagens, such as polycyclic flat molecules like ethidium bromide or acridine derivatives, insert (or intercalate) between the bases as they stack with each other in the double helix. These intercalating agents increase the spacing of successive base pairs, destroying the regular sugar-phosphate backbone and decreasing the pitch of the helix. These changes cause the addition or deletion of a single base and lead to frequent mistakes during DNA replication. DNA-reactive chemicals act directly on the DNA to change the chemical structure of the base. These include nitrous acid (HNO2) and alkylating agents, including nitrosoguanidine and ethyl methane sulfonate, which are known to add methyl or ethyl groups to the rings of the DNA bases. The modified bases may pair abnormally or not at all. The damage may also cause the removal of the base from the DNA backbone.

Repair Mechanisms of DNA page 32 page 33

A number of repair mechanisms have evolved in bacterial cells to minimize damage to DNA. These repair mechanisms can be divided into the following five groups: 1. Direct DNA repair is the enzymatic removal of damage, such as pyrimidine dimers and alkylated bases. 2. Excision repair is the excision of a DNA segment containing the damage, followed by synthesis of a new DNA strand. Two types of excision-repair mechanisms, generalized and specialized, exist. 3. Recombinational or postreplication repair is the retrieval of missing information by genetic recombination when both DNA strands are damaged. 4. The SOS response is the induction of many genes (approximately 15) after DNA damage or interruption of DNA replication. 5. Error-prone repair is the last resort of a bacterial cell before it dies. It is used to fill in gaps with a random sequence when a DNA template is not available for directing an accurate repair.

Gene Exchange in Prokaryotic Cells Many bacteria, especially many pathogenic bacterial species, are promiscuous with their DNA. The exchange of DNA between cells allows the exchange of genes and characteristics between cells, thus producing new strains of bacteria. This exchange may be advantageous for the recipient, especially if the exchanged DNA encodes antibiotic resistance. The transferred DNA can be integrated into the recipient chromosome or stably maintained as an extrachromosomal element (plasmid) or a bacterial virus (bacteriophage) and passed on to daughter bacteria as an autonomously replicating unit.

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. However, Borrelia burgdorferi, the causative agent of Lyme disease, and the related Borrelia hermsii are unique among all eubacteria because they possess linear plasmids. Like the bacterial chromosomal DNA, plasmids 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, plasmids may encode the production of antibiotic resistance mechanisms, bacteriocins, toxins, virulence determinants, and other genes that may provide the bacteria with a unique growth advantage over other microbes or within the host (Figure 3-12). The number of copies of plasmid produced by a cell is determined by the particular plasmid. The copy number is the ratio of copies of the plasmid to the number of copies of the chromosome. This may be as few as one in the case of large plasmids or as many as 50 in smaller plasmids.

Figure 3-12 Plasmids. The pBR322 plasmid is one of the plasmids used for cloning DNA. This plasmid encodes resistance to ampicillin (Amp) and tetracycline (Tet) and an origin of replication (ori). The multiple cloning site in the pGEM plasmid provides different restriction enzyme cleavage sites for insertion of DNA within the β-galactosidase gene (lacZ). The insert is flanked by bacteriophage promoters to allow directional messenger RNA expression of the cloned sequence.

Large plasmids (20 to 120 kb), such as the fertility factor F found in E. coli or the resistance transfer factor (80 kb), can often mediate their own transfer from one cell to another by a process called conjugation (see the section on conjugation later in this chapter). These conjugative plasmids encode all the necessary factors for their transfer. Other plasmids can be transferred into a bacterial cell by means other than conjugation, such as transformation or transduction. These terms are discussed later in the chapter. page 33

page 34

Figure 3-13 Transposons. A, The insertion sequences code only for a transposase (tnp) and possess inverted repeats (15 to 40 base pairs) at each end. B, The composite transposons contain a central region coding for antibiotic resistances or toxins flanked by two insertion sequences (IS), which can be either directly repeated or reversed. C, Tn3, a member of the TnA transposon family. The central region encodes three genes-a transposase (tnpA), a resolvase (tnpR), and a β-lactamase-conferring resistance to ampicillin. A resolution site (Res site) is used during the replicative transposition process. This central region is flanked on both ends by direct repeats of 38 base pairs. D, Phage-associated transposon is exemplified by the bacteriophage mu.

Bacteriophages are bacterial viruses. These extrachromosomal genetic elements can survive outside of a host cell, because a protein coat protects the nucleic acid genome (which may be DNA or RNA). Bacteriophages infect bacterial cells and either replicate to large numbers and cause the cell to lyse (lytic infection) or in some cases integrate into the host genome without killing the host (the lysogenic state), such as the E. coli bacteriophage lambda. Some lysogenic bacteriophages carry toxin genes (e.g., corynephage beta carries the gene for the diphtheria toxin). Bacteriophage lambda remains lysogenic as long as a repressor protein is synthesized and prevents the phage from becoming unintegrated in order to replicate and exit the cell. This reaction can be triggered if the host cell DNA is damaged by radiation or by another means or if the cell can no longer make the repressor protein, a signal that the host cell is unhealthy and is no longer a good place for "freeloading." Transposons (jumping genes) are mobile genetic elements (Figure 3-13) that can transfer DNA within a cell, from one position to another in the genome, or between different molecules of DNA (e.g., plasmid to plasmid or plasmid to chromosome). Transposons are present in prokaryotes and eukaryotes. The simplest transposons are called insertion sequences and range in length from 150 to 1500 base pairs, with inverted repeats of 15 to 40 base pairs at their ends and the minimal genetic information necessary for their own transfer (i.e., the gene coding for the transposase). Complex transposons carry other genes, such as genes that provide resistance against antibiotics. Transposons sometimes insert into genes and inactivate those genes. If insertion and inactivation occur in a gene that encodes an essential protein, the cell dies.

Some pathogenic bacteria use a transposon-like mechanism to coordinate the expression of a system of virulence factors. The genes for the activity may be grouped together in a pathogenicity or virulence island, which is surrounded by transposon-like mobile elements, allowing them to move within the chromosome and to other bacteria. The entire genetic unit can be triggered by an environmental stimulus (e.g., pH, heat, contact with the host cell surface) as a way to coordinate the expression of a complex process. For example, the SPI-1 island of Salmonella encodes 25 genes that allow the bacteria to enter nonphagocytic cells.

Mechanisms of Genetic Transfer between Cells The exchange of genetic material between bacterial cells may occur by one of three mechanisms (Figure 3-14): (1) conjugation, which is the mating or quasisexual exchange of genetic information from one bacterium (the donor) to another bacterium (the recipient); (2) transformation, which results in acquisition of new genetic markers by the incorporation of exogenous or foreign DNA; or (3) transduction, which is the transfer of genetic information from one bacterium to another by a bacteriophage. Once inside a cell, a transposon can jump between different DNA molecules (e.g., plasmid to plasmid or plasmid to chromosome).

Transformation Transformation is the process by which bacteria take up fragments of naked DNA and incorporate them into their genomes. Transformation was the first mechanism of genetic transfer to be discovered in bacteria. In 1928, Griffith observed that pneumococcus virulence was related to the presence of a surrounding polysaccharide capsule and that extracts of encapsulated bacteria producing smooth colonies could transmit this trait to nonencapsulated bacteria, normally appearing with rough edges. Griffith's studies led to Avery, MacLeod, and McCarty's identification of DNA as the transforming principle some 15 years later.

Gram-positive and gram-negative bacteria can take up and stably maintain exogenous DNA. Certain species are naturally capable of taking up exogenous DNA (such species are then said to be competent), including Haemophilus influenzae, Streptococcus pneumoniae, Bacillus species, and Neisseria species. Competence develops toward the end of logarithmic growth, some time before a population enters the stationary phase. Most bacteria do not exhibit a natural ability for DNA uptake. Chemical methods or electroporation (the use of high-voltage pulses) can be used to introduce plasmid and other DNA into E. coli and other bacteria.

Conjugation page 34 page 35

Figure 3-14 Mechanisms of bacterial gene transfer. (From Rosenthal KS, Tan J: Rapid Reviews Microbiology and Immunology. St. Louis, Mosby, 2002.)

Conjugation occurs with most, if not all, eubacteria. Conjugation usually occurs between members of the same or related species, but has also been demonstrated to occur between prokaryotes and cells from plants, animals, and fungi. Conjugation occurs for E. coli, bacteroides, enterococci, streptococci, streptomycetes, and clostridia. Many of the large conjugative plasmids specify colicins or antibiotic resistance. Genetic transfer in E. coli was first reported by Lederberg and Tatum in 1946, when they observed sexlike exchange between two mutant strains of E. coli K12. Conjugation results in one-way transfer of DNA from a donor (or male) cell to a recipient (or female) cell through the sex pilus. Conjugative R (antibiotic resistance) for gram-positive bacteria, such as streptococci, streptomycetes, and clostridia, are brought together by the presence of an adhesin molecule on the surface of the donor cell instead of pili. The mating type (sex) of the cell depends on the presence (male) or absence (female) of a conjugative plasmid, such as the F plasmid of E. coli. The F plasmid is defined as conjugative because it carries all the genes necessary for its own transfer, including the ability to make sex pili and to initiate DNA synthesis at the transfer origin (OriT) of the plasmid. The F plasmid transfers itself, converting recipients into F+ male cells. If a fragment of chromosomal DNA has been incorporated into the plasmid, it is designated an F prime (F') plasmid. When it transfers into the recipient cell, it carries that fragment with it and converts it into an F' male. If the F plasmid sequence is integrated into the bacterial chromosome, the cell is designated an Hfr (high frequency recombination) cell. page 35 page 36

The DNA that is transferred by conjugation is not a double helix but a single-stranded molecule. Mobilization begins when a plasmid-encoded protein makes a single-stranded, site-specific cleavage at the OriT. The nick initiates rolling circle replication, and the displaced linear strand is directed to the recipient cell. The transferred single-stranded DNA is recircularized and its complementary strand synthesized. Integration of an F plasmid into the bacterial chromosome generates an Hfr cell. Conjugation results in transfer of a part of the plasmid sequence and some portion of the bacterial chromosomal DNA. Because of the fragile connection between the mating pairs, the transfer is usually aborted before being completed such that only the chromosomal sequences adjacent to the integrated F are transferred. Artificial interruption of a mating between an Hfr and an F- pair has been helpful in constructing a consistent map of the E. coli chromosomal DNA. In such maps the position of each gene is given in minutes (based on 100 minutes for complete transfer at 37° C), according to its time of entry into a recipient cell in relation to a fixed origin.

Transduction Genetic transfer by transduction is mediated by bacterial viruses (bacteriophages), which pick up fragments of DNA and package them into bacteriophage particles. The DNA is delivered to infected cells and becomes incorporated into the bacterial genomes. Transduction can be classified as specialized if the phages in question transfer particular genes (usually those adjacent to their integration sites in the genome) or generalized if the selection of the sequences is random because of accidental packaging of host DNA into the phage capsid.

Generalized transducing particles should contain primarily bacterial DNA and little or no phage DNA. For example, the P1 phage of E. coli encodes a nuclease that degrades the host E. coli chromosomal DNA. A small percentage of the resultant phage particles package the DNA fragments into their capsids. The encapsulated DNA, instead of phage DNA, is injected into a new host cell, where it can recombine with the homologous host DNA. Generalized transducing particles are valuable in the genetic mapping of bacterial chromosomes. The closer two genes are within the bacterial chromosome, the more likely it is that they will be co-transduced in the same fragment of DNA.

Recombination Incorporation of extrachromosomal (foreign) DNA into the chromosome occurs by recombination. There are two types of recombination: homologous and nonhomologous. Homologous (legitimate) recombination occurs between closely related DNA sequences and generally substitutes one sequence for another. The process requires a set of enzymes produced (in E. coli) by the rec genes. Nonhomologous (illegitimate) recombination occurs between dissimilar DNA sequences and generally produces insertions or deletions or both. This process usually requires specialized (sometimes site-specific) recombination enzymes, such as those produced by many transposons and lysogenic bacteriophages.

Generation of Vancomycin-Resistant Staphylococcus aureus by Multiple Genetic Manipulations

Until recently, vancomycin was the last-resort drug for S. aureus strains resistant to beta lactam (penicillin-related) antibiotics (e.g., methicillin resistant S. aureus [MRSA]). S. aureus acquired the vancomycin resistance gene during a mixed infection with Enterococcus faecalis (Figure 3-15). The gene for the vancomycin resistance gene was contained within a transposon (TN1546) on a multiresistance conjugative plasmid. The plasmid was probably transferred by conjugation between E. faecalis and S. aureus. Alternatively, after lysis of the E. faecalis, S. aureus acquired the DNA by transduction and became transformed by the new DNA. The transposon then jumped from the E. faecalis plasmid, recombined, and integrated into the S. aureus multiresistance plasmid, and the E. faecalis DNA was degraded. The resulting S. aureus plasmid encodes resistance to beta lactams, vancomycin, trimethoprim, and gentamycin/kanamycin/tobramycin antibiotics and to quaternary ammonium disinfectants and can transfer to other S. aureus strains by conjugation. (For more information, refer to Weigel in the bibliography at the end of the chapter.)

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 use of genetic engineering and "cloning" has revolutionized biology and medicine. The basic components of genetic engineering are: (1) cloning and expression vectors, which can be used to deliver the DNA sequences into receptive bacteria and amplify the desired sequence; (2) the DNA sequence to be amplified and expressed; (3) enzymes, such as restriction enzymes, which are used to cleave DNA reproducibly at defined sequences (Table 3-1) and DNA ligase, the enzyme that links the fragment to the cloning vector.

Cloning and expression vectors must allow foreign DNA to be inserted into them, but still must be able to replicate normally in a bacterial or eukaryotic host. Many types of vectors are currently used. Plasmid vectors, such as pUC, pBR322, and pGEM (Figure 3-16), are used for DNA fragments up to 20 kb. Bacteriophages, such as lambda, are used for larger fragments up to 25 kb. More recently, cosmid vectors have combined some of the advantages of plasmids and phages for fragments up to 45 kb. Most cloning vectors have been "engineered" to have a site for insertion of foreign DNA; a means of selection of the bacteria that have incorporated any plasmid (e.g., antibiotic resistance); and a means of distinguishing the bacteria that have incorporated those plasmids which contain inserted DNA. Expression vectors have DNA sequences to facilitate their replication in bacteria and eukaryotic cells and also the transcription of the gene into mRNA. page 36 page 37

Figure 3-15 Genetic mechanisms of evolution of methicillin and vancomycin resistant Staphylococcus aureus. Vancomycin resistant enterococcus (VRE) (in red) contains plasmids with multiple antibiotic resistance and virulence factors. During coinfection a methicillin resistant Staphylococcus aureus (MRSA) may have acquired the enterococcal resistance plasmid (e-plasmid) by transformation (after lysis of the enterococcal cell and release of its DNA) or more likely, by conjugation. A transposon in the e-plasmid containing the vancomycin resistance gene jumped out and inserted into the multiple antibiotic resistance plasmid of the MRSA. The new plasmid is readily spread to other S. aureus bacteria by conjugation.

Table 3-1. Common Restriction Enzymes Used in Molecular Biology Microorganism Acinetobacter calcoaceticus

Enzyme Recognition Site AccI

Bacillus amyloliquefaciens H

BamHI

Escherichia coli RY13

EcoRI

Haemophilus influenzae Rd

HindIII

H. influenzae serotype c, 1160

HincII

Providencia stuartii 164

PstI

Serratia marcescens

SmaI

Staphylococcus aureus 3A

Sau3AI

Xanthomonas malvacearum

XmaI

The DNA to be cloned can be obtained by purification of chromosomal DNA from cells, viruses, or other plasmids or by selective amplification of DNA sequences by a technique known as polymerase chain reaction (PCR). (PCR is explained further in Chapter 16.) Both the vector and the foreign DNA are cleaved with restriction enzymes (see Figure 3-16). Restriction enzymes recognize a specific palindromic sequence and make a staggered cut, which generates sticky ends, or a blunt cut, which generates blunt ends (see Table 3-1). Most cloning vectors have a sequence called the multiple cloning site that can be cleaved by many restriction enzymes. Ligation of the vector with the DNA fragments generates a molecule called recombinant DNA that is capable of replicating the inserted sequence. The total number of recombinant vectors obtained when cloning all the fragments that result from cleavage of chromosomal DNA is known as a genomic library, because there should be at least one representative of each gene in the library. An alternative approach to cloning the gene for a protein is to convert the mRNA for the protein into DNA using a retrovirus enzyme called reverse transcriptase (RNA-dependent DNA polymerase) to produce a complementary DNA (cDNA). A cDNA library represents the genes that are expressed as mRNA in a particular cell. page 37 page 38

Figure 3-16 Cloning of foreign DNA in vectors. The vector and the foreign DNA are first digested by a restriction enzyme. Insertion of foreign DNA into the lacZ gene inactivates the β-galactosidase gene, allowing subsequent selection. The vector is then ligated to the foreign DNA, using bacteriophage T4 DNA ligase. The recombinant vectors are transformed into competent Escherichia coli cells. The recombinant E. coli cells are plated onto agar containing antibiotic, an inducer of the lac operon, and a chromophoric substrate that turns blue in cells having plasmid but not insert; those cells with plasmid containing the insert remain white.

The recombinant DNA is then transformed into a bacterial host, usually E. coli, and the plasmid-containing bacteria are selected for antibiotic resistance (e.g., ampicillin resistance). The library can then be screened to find an E. coli clone possessing the desired DNA fragment. Various screening techniques can be used to identify the bacteria containing the appropriate recombinant DNA. The multiple cloning site used for inserting the foreign DNA is often part of the lacZ gene of the lac operon. Insertion of the foreign DNA into the lacZ gene inactivates the gene (acting almost like a transposon) and prevents the plasmid-directed synthesis of β-galactosidase in the recipient cell, which results in white bacterial colonies instead of blue colonies if β-galactosidase were able to cleave an appropriate chromophore. Genetic engineering has been used to isolate and express the genes for useful proteins such as insulin, interferon, growth hormones, and interleukin in bacteria, yeast, or even insect cells. Large amounts of pure immunogen for a vaccine can be prepared without the need to work with the intact disease organisms. The development of a vaccine against hepatitis B virus represents the first success of recombinant DNA vaccines approved for human use by the U.S. Food and Drug Administration. The hepatitis B surface antigen is produced by the yeast Saccharomyces cerevisiae. In the future it may be sufficient to inject plasmid DNA capable of expressing the desired immunogen (DNA vaccine) into an individual to let the host cells express the immunogen and generate the immune response. Recombinant DNA technology has also become essential to laboratory diagnosis, forensic science, agriculture, and many other disciplines.

Questions

1. How many moles of ATP are generated per mole of glucose in glycolysis, the TCA cycle, and electron transport? Which of these occur in anaerobic conditions and in aerobic conditions? Which is most efficient? 2. What products of anaerobic fermentation would be detrimental to host (human) tissue (e.g., C. perfringens)? 3. If the number of bacteria during log phase growth can be calculated by the following equation:

4. 5. 6. 7.

8.

in which Nt is the number of bacteria after time (t), t/d is the amount of time divided by the doubling time, and N0 is the initial number of bacteria, how many bacteria will be in the culture after 4 hours if the doubling time is 20 minutes and the initial bacterial inoculum contained 1000 bacteria? What are the principal properties of a plasmid? Give two mechanisms of regulation of bacterial gene expression. Use specific examples. What types of mutations affect DNA, and what agents are responsible for such mutations? Which mechanisms may be used by a bacterial cell for the exchange of genetic material? Briefly explain each mechanism. Discuss the applications of molecular biotechnology to medicine, including contributions and uses in diagnoses.

Bibliography Alberts B: Molecular Biology of the Cell, 4th ed. New York, Garland, 2002. Berg JM, Tymoczko JL, Stryer L: Biochemistry, 6th ed, New York, WH Freeman, 2006. Lewin B: Genes IX. Sudbury, Mass, Jones and Bartlett, 2007. Lodish H, et al: Molecular Cell Biology, 6th ed. New York, WH Freeman, 2007.

Nelson DL, Cox M: Lehninger Principles of Biochemistry, 4th ed. New York, Worth, 2004. Patel SS, Rosenthal KS: Microbial adaptation: Putting the best team on the field. Infect Dis Clin Pract 15:330-334, 2007. Watson JD, et al: Molecular Biology of the Gene, 4th ed. Menlo Park, Calif, Benjamin-Cummings, 1987. Weigel LM, et al: Genetic analysis of a high-level vancomycin-resistant isolate of Staphylococcus aureus. Science 302:1569-1571, 2003. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Classification Viruses range from the structurally simple and small parvoviruses and picornaviruses to the large and complex poxviruses and herpesviruses. Their names may describe viral characteristics, the diseases they are associated with, or even the tissue or geographic locale where they were first identified. Names such as picornavirus (pico, "small"; rna, "ribonucleic acid") or togavirus (toga, Greek for "mantle," referring to a membrane envelope surrounding the virus) describe the structure of the virus. The name retrovirus (retro, "reverse") refers to the virus-directed synthesis of DNA from an RNA template, whereas the poxviruses are named for the disease smallpox, caused by one of its members. The adenoviruses (adenoids) and the reoviruses (respiratory, enteric, orphan) are named for the body site from which they were first isolated. Reovirus was discovered before it was associated with a specific disease, and thus it was designated an "orphan" virus. Norwalk virus is named for Norwalk, Ohio; coxsackievirus is named for Coxsackie, New York; and many of the togaviruses, arenaviruses, and bunyaviruses are named after African places where they were first isolated. Viruses can be grouped by characteristics such as disease (e.g., hepatitis), target tissue, means of transmission (e.g., enteric, respiratory), or vector (e.g., arboviruses; arthropod-borne virus) (Box 4-3). The most consistent and current means of classification is by physical and biochemical characteristics, such as size, morphology (e.g., presence or absence of a membrane envelope), type of genome, and means of replication (Figures 4-2 and 4-3). DNA viruses associated with human disease are divided into seven families (Tables 4-1 and 4-2). The RNA viruses may be divided into at least 13 families (Tables 4-3 and 4-4). Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Virion Structure page 39 page 40

Box 4-1. Definition and Properties of a Virus Viruses are filterable agents. Viruses are obligate intracellular parasites. Viruses cannot make energy or proteins independently of a host cell. Viral genomes may be RNA or DNA but not both. Viruses have a naked capsid or an envelope morphology. Viral components are assembled and do not replicate by "division."

Box 4-2. Consequences of Viral Properties Viruses are not living. Viruses must be infectious to endure in nature. Viruses must be able to use host cell processes to produce their components (viral messenger RNA, protein, and identical copies of the genome). Viruses must encode any required processes not provided by the cell. Viral components must self-assemble.

Figure 4-1 Components of the basic virion.

Box 4-3. Means of Classification and Naming of Viruses *This is the current means of taxonomic classification of viruses. Structure: size, morphology, and nucleic acid (e.g., picornavirus [small RNA], togavirus) Biochemical characteristics: structure and mode of replication* Disease: encephalitis and hepatitis viruses, for example Means of transmission: arbovirus spread by insects, for example Host cell (host range): animal (human, mouse, bird), plant, bacteria Tissue or organ (tropism): adenovirus and enterovirus, for example

Figure 4-2 The DNA viruses and their morphology. The viral families are determined by the structure of the genome and the morphology of the virion.

Figure 4-3 The RNA viruses, their genome structure, and their morphology. The viral families are determined by the structure of the genome and the morphology of the virion. E, enveloped; N, naked capsid.

The units for measurement of virion size are nanometers (nm). The clinically important viruses range from 18 nm (parvoviruses) to 300 nm (poxviruses) (Figure 4-4). The latter are almost visible with a light microscope and are approximately one fourth the size of Staphylococcus bacteria. Larger virions can hold a larger genome that can encode more proteins, and they are generally more complex. The virion (the virus particle) consists of a nucleic acid genome packaged into a protein coat (capsid) or a membrane (envelope) (Figure 4-5). The virion may also contain certain essential or accessory enzymes or other proteins to facilitate initial replication in the cell. Capsid or nucleic acid-binding proteins may associate with the genome to form a nucleocapsid, which may be the same as the virion or surrounded by an envelope.

Table 4-1. Families of DNA Viruses and Some Important Members Family

Members*

POXVIRIDAE†

Smallpox virus, vaccinia virus, monkeypox, molluscum contagiosum

Herpesviridae

Herpes simplex virus types 1 and 2, varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, human herpesviruses 6, 7, and 8

Adenoviridae

Adenovirus

Papilloma viridae

Papilloma virus

Polyoma viridae JC virus, BK virus, SV40 Hepadnaviridae Hepatitis B virus Parvoviridae

Parvovirus B19, adeno-associated virus

*The italicized virus is the important, or prototype, virus for the family. † The size of type is indicative of the relative size of the virus. page 40 page 41

Table 4-2. Properties of Virions of Human DNA Viruses Family

Poxviridae

Herpesviridae

Genome* Viron Molecular Nature Shape Size (nm) DNA Mass × Polymerase† 106 Daltons 85-140 ds, Brick-shaped, 300 × +‡ linear enveloped 240 × 100 100-150

ds, linear

Icosahedral, enveloped

Capsid, + 100-110 Envelope, 120-200

Adenoviridae

20-25

ds, linear

Icosahedral

70-90

+

Hepadnaviridae 1.8

ds, Spherical, circular§ enveloped

42

+‡ [Verbar]

Polyoma and papilloma viridae

3-5

ds, Icosahedral circular

45-55

-

Parvoviridae

1.5-2.0

ss, linear

18-26

-

Icosahedral

*Genome invariably a single molecule. † Polymerase encoded by virus. ‡ Polymerase carried in the virion. §; Circular molecule is double-stranded for most of its length but contains a single-stranded region. [Verbar] Reverse transcriptase. ds, Double-stranded; ss, single-stranded.

Table 4-3. Families of RNA Viruses and Some Important Members Family† PARAMYXOVIRIDAE

Members* Parainfluenza virus, Sendai virus, measles virus, mumps virus, respiratory syncytial virus, metapneumovirus

ORTHOMYXOVIRIDAE Influenza virus types A, B, and C CORONAVIRIDAE

Coronavirus, SARS (severe acute respiratory syndrome)

Arenaviridae

Lassa fever virus, Tacaribe virus complex (Junin and Machupo viruses), lymphocytic choriomeningitis virus

Rhabdoviridae

Rabies virus, vesicular stomatitis virus

Filoviridae

Ebola virus, Marburg virus

Bunyaviridae

California encephalitis virus, LaCrosse virus, sandfly fever virus, hemorrhagic fever virus, Hanta virus

Retroviridae

Human T-cell leukemia virus types I and II, human immunodeficiency virus, animal oncoviruses

Reoviridae

Rotavirus, Colorado tick fever virus

Picornaviridae

Rhinoviruses, poliovirus, echoviruses, coxsackievirus, hepatitis A virus

Togaviridae

Rubella virus; western, eastern, and Venezuelan equine encephalitis virus; Ross River virus; Sindbis virus; Semliki Forest virus

Flaviviridae

Yellow fever virus, dengue virus, St. Louis encephalitis virus, West Nile virus, hepatitis C virus

Caliciviridae

Norwalk virus, calicivirus

Delta

Delta agent

†

The size of the type is indicative of the relative size of the virus. *The italicized virus is the important or prototype virus for the family. page 41 page 42

Table 4-4. Properties of Virions of Human RNA Viruses Genome* Family Molecular Nature Shape* Mass × 10 6 Daltons Paramyxoviridae 5-7 ss, - Spherical

Size (nm)

Virion Polymerase Envelope in Virion

150-300 +

+

Orthomyxoviridae 5-7

ss, -, seg

Spherical

80-120 +

+

Coronaviridae

6-7

ss, +

Spherical

80-130 3

+†

Arenaviridae

3-5

ss, -, seg

Spherical

50-300 +

+†

Rhabdoviridae

4-7

ss, -

Bullet-shaped 180 × 75

+

+

Filoviridae

4-7

ss, -

Filamentous

800 × 80

+

+

Bunyaviridae

4-7

ss, -

Spherical

90-100 +

+†

Retroviridae

2 3 (2-3)‡ ss, +

Spherical

80-110 +§

+

Reoviridae

11-15

ds, seg

Icosahedral

60-80

+

3

Picornaviridae

2.5

ss, +

Icosahedral

25-30

3

3

Togaviridae

4-5

ss, +

Icosahedral

60-70

3

+

Flaviviridae

4-7

ss, +

Spherical

40-50

3

+

Caliciviridae

2.6

ss, +

Icosahedral

35-40

3

3

*Some enveloped viruses are very pleomorphic (sometimes filamentous). † No matrix protein. ‡ Genome has two identical single-stranded RNA molecules. § Reverse transcriptase. ds, Double-stranded; seg, segmented; ss, single-stranded; + or -, polarity of single-stranded nucleic acid.

Figure 4-4 Relative sizes of viruses and bacteria. (Courtesy the Upjohn Company, Kalamazoo, Mich.)

The genome of the virus consists either of DNA or RNA. The DNA can be single or double stranded, linear or circular. The RNA can be either positive sense (+) (like messenger RNA [mRNA]) or negative sense (-) (analogous to a photographic negative), double stranded (+/-) or ambisense (containing + and - regions of RNA attached end to end). The RNA genome may also be segmented into pieces, with each piece encoding one or more genes. Just as there are many different types of computer memory devices, all of these forms of nucleic acid can maintain and transmit the genetic information of the virus. Similarly, the larger the genome, the more information (genes) it can carry and the larger the capsid or envelope structure required to contain the genome.

The outer layer of the virion is the capsid or envelope. These structures are the package, protection, and delivery vehicle during transmission of the virus from one host to another and for spread within the host to the target cell. The surface structures of the capsid and envelope mediate the interaction of the virus with the target cell through a viral attachment protein (VAP) or structure. Removal or disruption of the outer package inactivates the virus. Antibodies generated against the components of these structures prevent virus infection. The capsid is a rigid structure able to withstand harsh environmental conditions. Viruses with naked capsids are generally resistant to drying, acid, and detergents, including the acid and bile of the enteric tract. Many of these viruses are transmitted by the fecal-oral route and can endure transmission even in sewage. page 42 page 43

Figure 4-5 The structures of a naked capsid virus (top left) and enveloped viruses with an icosahedral (left) nucleocapsid or a helical (right) ribonucleocapsid. The helical ribonucleocapsid is formed by viral proteins associated with an RNA genome.

The envelope is a membrane composed of lipids, proteins, and glycoproteins. The membranous structure of the envelope can be maintained only in aqueous solutions. It is readily disrupted by drying, acidic conditions, detergents, and solvents such as ether, which results in inactivation of the virus. As a result, enveloped viruses must remain wet and are generally transmitted in fluids, respiratory droplets, blood, and tissue. Most cannot survive the harsh conditions of the gastrointestinal tract. The influence of virion structure on viral properties is summarized in Boxes 4-4 and 4-5.

Capsid Viruses The viral capsid is assembled from individual proteins associated into progressively larger units. All of the components of the capsid have chemical features that allow them to fit together and to assemble into a larger unit. Individual structural proteins associate into subunits, which associate into protomers, capsomeres (distinguishable in electron micrographs), and finally, a recognizable procapsid or capsid (Figure 4-6). A procapsid requires further processing to the final, transmissible capsid. For some viruses the capsid forms around the genome; for others the capsid forms as an empty shell (procapsid) to be filled by the genome.

Box 4-4. Virion Structure: Naked Capsid *Exceptions exist.

Component Protein Properties* Is environmentally stable to the following: Temperature Acid Proteases Detergents Drying Is released from cell by lysis Consequences* Can be spread easily (on fomites, from hand to hand, by dust, by small droplets) Can dry out and retain infectivity Can survive the adverse conditions of the gut Can be resistant to detergents and poor sewage treatment Antibody may be sufficient for immunoprotection

The simplest viral structures that can be built stepwise are symmetrical and include helical and icosahedral structures. Helical structures appear as rods, whereas the icosahedron is an approximation of a sphere assembled from symmetrical subunits (Figure 4-7). Nonsymmetrical capsids are complex forms and are associated with certain bacterial viruses (phages).

Box 4-5. Virion Structure: Envelope *Exceptions exist.

Components Membrane Lipids Proteins Glycoproteins Properties* Is environmentally labile-is disrupted by the following: Acid Detergents Drying Heat Modifies cell membrane during replication Is released by budding and cell lysis Consequences* Must stay wet Cannot survive the gastrointestinal tract Spreads in large droplets, secretions, organ transplants, and blood transfusions Does not need to kill the cell to spread May need antibody and cell-mediated immune response for protection and control Elicits hypersensitivity and inflammation to cause immunopathogenesis

page 43 page 44

Figure 4-6 Capsid assembly of the icosahedral capsid of a picornavirus. Individual proteins associate into subunits, which associate into protomers, capsomeres, and an empty procapsid. Inclusion of the (+) RNA genome triggers its conversion to the final capsid form.

Figure 4-7 Cryoelectron microscopy and computer-generated three-dimensional image reconstructions of several icosahedral capsids. These images show the symmetry of capsids and the individual capsomeres. During assembly, the genome may fill the capsid through the holes in the herpesvirus and papovavirus capsomeres. 1, Equine herpesvirus nucleocapsid; 2, simian rotavirus; 3, reovirus type 1 (Lang) virion; 4, intermediate subviral particle (reovirus); 5, core (inner capsid) particle (reovirus); 6, human papillomavirus type 19; 7, mouse polyomavirus; 8, cauliflower mosaic virus. Bar = 50 nm. (Courtesy Dr. Tim Baker, Purdue University, West Lafayette, Ind.)

The classic example of a virus with helical symmetry is the tobacco mosaic plant virus. Its capsomeres self-assemble on the RNA genome into rods that extend the length of the genome. The capsomeres cover and protect the RNA. Helical nucleocapsids are observed within the envelope of most negative-strand RNA viruses (see Figure 58-1).

Simple icosahedrons are used by small viruses such as the picornaviruses and parvoviruses. The icosahedron is made of 12 capsomeres, each with fivefold symmetry (pentamer or penton). For the picornaviruses, every pentamer is made up of five protomers, each of which is composed of three subunits of four separate proteins (see Figure 4-6). X-ray crystallography and image analysis of cryoelectron microscopy have defined the structure of the picornavirus capsid to the molecular level. These studies have depicted a canyon-like cleft, which is a "docking site" to bind to the receptor on the surface of the target cell (see Figure 56-2). page 44 page 45

Larger capsid virions are constructed by inserting structurally distinct capsomeres between the pentons at the vertices. These capsomeres have six nearest neighbors (hexons). This extends the icosahedron and is called an icosadeltahedron, and its size is determined by the number of hexons inserted along the edges and within the surfaces between the pentons. A soccer ball is an icosadeltahedron. For example, the herpesvirus nucleocapsid has 12 pentons and 150 hexons. The herpesvirus nucleocapsid is also surrounded by an envelope. The adenovirus capsid is composed of 252 capsomeres, with 12 pentons and 240 hexons. A long fiber is attached to each penton of adenovirus to serve as the viral attachment protein (VAP) to bind to target cells, and it also contains the type-specific antigen (see Figure 52-1). The reoviruses have an icosahedral double capsid with fiberlike proteins partially extended from each vertex. The outer capsid protects the virus and promotes its uptake across the gastrointestinal tract and into target cells, whereas the inner capsid contains enzymes for the synthesis of RNA (see Figures 4-7 and 61-2).

Enveloped Viruses

The virion envelope is composed of lipids, proteins, and glycoproteins (see Figure 4-5 and Box 4-5). It has a membrane structure similar to cellular membranes. Cellular proteins are rarely found in the viral envelope, even though the envelope is obtained from cellular membranes. Most enveloped viruses are round or pleomorphic. (See Figures 4-2 and 4-3 for the complete listing of enveloped viruses.) Two exceptions are the poxvirus, which has a complex internal and a bricklike external structure, and the rhabdovirus, which is bullet shaped.

Figure 4-8 Diagram of the hemagglutinin glycoprotein trimer of influenza A virus, a representative spike protein. The region for attachment to the cellular receptor is exposed on the spike protein's surface. Under mild acidic conditions the hemagglutinin changes conformation to expose a hydrophobic sequence at the "fusion region." CHO, N-linked carbohydrate attachment sites. (Modified from Schlesinger MJ, Schlesinger S: Domains of virus glycoproteins. Adv Virus Res 33:1-44, 1987.)

Most viral glycoproteins have asparagine-linked (N-linked) carbohydrate and extend through the envelope and away from the surface of the virion. For many viruses, these can be observed as spikes (Figure 4-8). Most glycoproteins act as VAPs, capable of binding to structures on target cells. VAPs that also bind to erythrocytes are termed hemagglutinins (HAs). Some glycoproteins have other functions, such as the neuraminidase of orthomyxoviruses (influenza) and the Fc receptor and the C3b receptor associated with herpes simplex virus glycoproteins, or the fusion glycoproteins of paramyxoviruses. Glycoproteins, especially the VAP, are also major antigens that elicit protective immunity. The envelope of the togaviruses surrounds an icosahedral nucleocapsid containing a positive-strand RNA genome. The envelope contains spikes consisting of two or three glycoprotein subunits anchored to the virion's icosahedral capsid. This causes the envelope to adhere tightly and conform (shrink-wrap) to an icosahedral structure discernible by cryoelectron microscopy. All of the negative-strand RNA viruses are enveloped. Components of the viral RNA-dependent RNA polymerase associate with the (-) RNA genome of the orthomyxoviruses, paramyxoviruses, and rhabdoviruses to form helical nucleocapsids (see Figure 4-5). These enzymes are required to initiate virus replication, and their association with the genome ensures their delivery into the cell. Matrix proteins lining the inside of the envelope facilitate the assembly of the ribonucleocapsid into the virion. Influenza A (orthomyxovirus) is an example of a (-) RNA virus with a segmented genome. Its envelope is lined with matrix proteins and has two glycoproteins: the hemagglutinin, which is the VAP, and a neuraminidase (NA) (see Figure 59-1). Bunyaviruses do not have matrix proteins. The herpesvirus envelope is a baglike structure that encloses the icosadeltahedral nucleocapsid (see Figure 59-1). Depending on the specific herpesvirus, the envelope may contain as many as 11 glycoproteins. The interstitial space between the nucleocapsid and the envelope is called the tegument, and it contains enzymes, other proteins, and even mRNA that facilitate the viral infection.

The poxviruses are enveloped viruses with large, complex, bricklike shapes (see Figure 54-1). The envelope encloses a dumbbell-shaped, DNA-containing nucleoid structure; lateral bodies; fibrils; and many enzymes and proteins, including the enzymes and transcriptional factors required for mRNA synthesis. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Viral Replication The major steps in viral replication are the same for all viruses (Figure 4-9 and Box 4-6). The cell acts as a factory, providing the substrates, energy, and machinery necessary for the synthesis of viral proteins and replication of the genome. Processes not provided by the cell must be encoded in the genome of the virus. The manner in which each virus accomplishes these steps and overcomes the cell's biochemical limitations is determined by the structure of the genome and of the virion (whether it is enveloped or has a naked capsid). This is illustrated in the examples in Figures 4-12 to 4-14. page 45 page 46

Figure 4-9 A general scheme of viral replication. Enveloped viruses have alternative means of entry (3) assembly, and exit from the cell (8' and 9'). The antiviral drugs for susceptible steps in viral replication are listed in magenta.

Box 4-6. Steps in Viral Replication

1. 2. 3. 4. 5.

Recognition of the target cell Attachment Penetration Uncoating Macromolecular synthesis a. Early messenger RNA (mRNA) and nonstructural protein synthesis: genes for enzymes and nucleic acid-binding proteins b. Replication of genome c. Late mRNA and structural protein synthesis d. Post-translational modification of protein 6. Assembly of virus 7. Budding of enveloped viruses 8. Release of virus

Figure 4-10 A, Single-cycle growth curve of a virus that is released on cell lysis. The different stages are defined by the presence or absence of visible viral components (eclipse period), infectious virus in the media (latent period), or macromolecular synthesis (early/late phases). B, Growth curve and burst size of representative viruses. (A modified from Davis BD, et al: Microbiology, 4th ed. Philadelphia, Lippincott, 1990; B modified from White DO, Fenner F: Medical Virology, 3rd ed. New York, Academic, 1986.)

A single round of the viral replication cycle can be separated into several phases. During the early phase of infection, the virus must recognize an appropriate target cell, attach to the cell, penetrate the plasma membrane and be taken up by the cell, release (uncoat) its genome into the cytoplasm, and if necessary, deliver the genome to the nucleus. The late phase begins with the start of genome replication and viral macromolecular synthesis and proceeds through viral assembly and release. Uncoating of the genome from the capsid or envelope during the early phase abolishes its infectivity and identifiable structure, thus initiating the eclipse period. The eclipse period, like a solar eclipse, ends with the appearance of new virions after virus assembly. The latent period, during which extracellular infectious virus is not detected, includes the eclipse period and ends with the release of new viruses (Figure 4-10). Each infected cell may produce as many as 100,000 particles; however, only 1% to 10% of these particles may be infectious. The noninfectious particles (defective particles) result from mutations and errors in the manufacture and assembly of the virion. The yield of infectious virus per cell, or burst size, and the time required for a single cycle of virus reproduction are determined by the properties of the virus and the target cell.

Recognition of and Attachment to the Target Cell page 46 page 47

Table 4-5. Examples of Viral Attachment Proteins

Virus Family Picornaviridae

Virus Rhinovirus

VAP VP1-VP2-VP3 complex

Adenoviridae

Adenovirus

Fiber protein

Reoviridae

Reovirus

σ-1

Rotavirus

VP7

Togaviridae

Semliki Forest virus

E1-E2-E3 complex gp

Rhabdoviridae

Rabies virus

G protein gp

Orthomyxoviridae Influenza A virus

HA gp

Paramyxoviridae Measles virus

HA gp

Herpesviridae

Epstein-Barr virus

gp350 and gp220

Retroviridae

Murine leukemia virus

gp70

Human immunodeficiency gp120 virus

gp, glycoprotein; HA, hemagglutinin; VAP, viral attachment protein.

The binding of the VAPs or structures on the surface of the virion capsid (Table 4-5) to receptors on the cell (Table 4-6) initially determines which cells can be infected by a virus. The receptors for the virus on the cell may be proteins or carbohydrates on glycoproteins or glycolipids. Viruses that bind to receptors expressed on specific cell types may be restricted to certain species (host range) (e.g., human, mouse) or specific cell types. The susceptible target cell defines the tissue tropism (e.g., neurotropic, lymphotropic). Epstein-Barr virus, a herpesvirus, has a very limited host range and tropism because it binds to the C3d receptor (CR2) expressed on human B cells. The B19 parvovirus binds to globoside (blood group P antigen) expressed on erythroid precursor cells.

Table 4-6. Examples of Viral Receptors

Virus Epstein-Barr virus

Target Cell B cell

Receptor* C3d complement receptor CR2 (CD21)

Human immunodeficiency virus

Helper T cell CD4 molecule and chemokine coreceptor

Rhinovirus

Epithelial cells

ICAM-1 (immunoglobulin superfamily protein)

Poliovirus

Epithelial cells

Immunoglobulin superfamily protein

Herpes simplex virus Many cells

Herpesvirus entry mediator (HVEA), nectin-1

Rabies virus

Neuron

Acetylcholine receptor, NCAM (neural cell adhesion molecule)

Influenza A virus

Epithelial cells

Sialic acid

B19 parvovirus

Erythroid precursors

Erythrocyte P antigen (globoside)

*Other receptors for these viruses may also exist. ICAM-1, Intercellular adhesion molecule.

The viral attachment structure for a capsid virus may be part of the capsid or a protein that extends from the capsid. A canyon on the surface of picornaviruses, such as the rhinovirus 14, serves as a "keyhole" for the insertion of a portion of the intercellular adhesion molecule (ICAM-1) from the cell surface. The fibers of the adenoviruses and the σ-1 proteins of the reoviruses at the vertices of the capsid interact with receptors expressed on specific target cells.

VAPs are specific glycoproteins of enveloped viruses. The HA of influenza A virus binds to sialic acid expressed on many different cells and has a broad host range and tissue tropism. Similarly, the α-togaviruses and the flaviviruses are able to bind to receptors expressed on cells of many animal species, including arthropods, reptiles, amphibians, birds, and mammals. This allows them to infect animals, mosquitoes, and other insects and to be spread by them.

Penetration Many interactions between the VAPs and the cellular receptors initiate the internalization of the virus into the cell. The mechanism of internalization depends on the virion structure and cell type. Most nonenveloped viruses enter the cell by receptor-mediated endocytosis or by viropexis. Endocytosis is a normal process used by the cell for the uptake of receptor-bound molecules such as hormones, low-density lipoproteins, and transferrin. Picornaviruses and papovaviruses may enter by viropexis. Hydrophobic structures of capsid proteins may be exposed after viral binding to the cells, and these structures help the virus or the viral genome slip through (direct penetration) the membrane. page 47 page 48

Enveloped viruses fuse their membranes with cellular membranes to deliver the nucleocapsid or genome directly into the cytoplasm. The optimum pH for fusion determines whether penetration occurs at the cell surface at neutral pH or whether the virus must be internalized by endocytosis, and fusion occurs in an endosome at acidic pH. The fusion activity may be provided by the VAP or another protein. The HA of influenza A (see Figure 4-8) binds to sialic acid receptors on the target cell. Under the mild acidic conditions of the endosome, the HA undergoes a dramatic conformational change to expose hydrophobic portions capable of promoting membrane fusion. Paramyxoviruses have a fusion protein that is active at neutral pH to promote virus-cell fusion. Paramyxoviruses can also promote cell-cell fusion to form multinucleated giant cells (syncytia). Some herpesviruses and retroviruses fuse with cells at a neutral pH and induce syncytia after replication.

Uncoating Once internalized, the nucleocapsid must be delivered to the site of replication within the cell and the capsid or envelope removed. The genome of DNA viruses, except for poxviruses, must be delivered to the nucleus, whereas most RNA viruses remain in the cytoplasm. The uncoating process may be initiated by attachment to the receptor or promoted by the acidic environment or proteases found in an endosome or lysosome. Picornavirus capsids are weakened by the release of the VP4 capsid protein to allow uncoating. VP4 is released by insertion of the receptor into the keyhole-like canyon attachment site of the capsid. Enveloped viruses are uncoated on fusion with cell membranes. Fusion of the herpesvirus envelope with the plasma membrane releases its nucleocapsid, which then "docks" with the nuclear membrane to deliver its DNA genome directly to the site of replication. The release of the influenza nucleocapsid from its matrix and envelope is facilitated by the passage of protons from inside the endosome through the ion pore formed by the influenza M2 matrix protein to acidify the virion.

The reovirus and poxvirus are only partially uncoated on entry. The outer capsid of reovirus is removed, but the genome remains in an inner capsid which contains the polymerases necessary for RNA synthesis. The initial uncoating of the poxviruses exposes a subviral particle to the cytoplasm, allowing synthesis of mRNA by virion-contained enzymes. An uncoating enzyme can then be synthesized to release the DNA-containing core into the cytoplasm.

Macromolecular Synthesis Once inside the cell the genome must direct the synthesis of viral mRNA and protein and generate identical copies of itself. The genome is useless unless it can be transcribed into functional mRNAs capable of binding to ribosomes and being translated into proteins. The means by which each virus accomplishes these steps depends on the structure of the genome (Figure 4-11) and the site of replication.

Figure 4-11 Viral macromolecular synthesis steps: The mechanism of viral mRNA and protein synthesis and genome replication are determined by the structure of the genome. 1. Double-stranded DNA (DS DNA) uses host machinery in the nucleus (except poxviruses) to make mRNA, which is translated by host cell ribosomes into proteins. Replication of viral DNA occurs by semiconservative means, by rolling circle, linear, and in other ways. 2. Single-stranded DNA (SS DNA) is converted into DS DNA and replicates like DS DNA. 3. (+) RNA resembles an mRNA that binds to ribosomes to make a polyprotein that is cleaved into individual proteins. One of the viral proteins is an RNA polymerase that makes a (-) RNA template and then more (+) RNA genome progeny and mRNAs. 4. (-) RNA is transcribed into mRNAs and a full-length (+) RNA template by the RNA polymerase carried in the virion. The (+) RNA template is used to make (-) RNA genome progeny. 5. DS RNA acts like (-) RNA. The (-) strands are transcribed into mRNAs by an RNA polymerase in the capsid. (+) RNAs get encapsidated and (-) RNAs are made in the capsid. 6. Retroviruses are (+) RNA that are converted to complementary DNA (cDNA) by reverse transcriptase carried in the virion. cDNA integrates into the host chromosome, and the host makes mRNAs, proteins, and full-length RNA genome copies. page 48 page 49

The cell's machinery for transcription and mRNA processing is found in the nucleus. Most DNA viruses use the cell's DNA-dependent RNA polymerase II and other enzymes to make mRNA. For example, eukaryotic mRNAs acquire a 3' polyadenylated (poly A) tail and a 5' methylated cap (for binding to the ribosome) and are processed to remove introns before being exported to the cytoplasm. Viruses that replicate in the cytoplasm must provide these functions or an alternative. Although poxviruses are DNA viruses, they replicate in the cytoplasm and therefore must encode enzymes for all these functions. Most RNA viruses replicate and produce mRNA in the cytoplasm, except for orthomyxoviruses and retroviruses. RNA viruses must encode the necessary enzymes for transcription and replication, because the cell has no means of replicating RNA. The mRNAs for RNA viruses may or may not acquire a 5' cap or poly A tail.

The naked genome of DNA viruses (except poxviruses) and the positive-sense RNA viruses (except retroviruses) are sometimes referred to as infectious nucleic acids, because they are sufficient for initiating replication on injection into a cell. These genomes can interact directly with host machinery to promote mRNA or protein synthesis, or both. In general, mRNA for nonstructural proteins is transcribed first (see Figure 4-12). Early gene products (nonstructural proteins) are often DNA-binding proteins and enzymes, including virus-encoded polymerases. These proteins are catalytic, and only a few are required. Replication of the genome usually initiates the transition to transcription of late gene products. Late viral genes encode structural proteins. Many copies of these proteins are required to package the virus, but are generally not required before the genome is replicated. Newly replicated genomes also provide new templates for more late gene mRNA synthesis. Different DNA and RNA viruses control the time and amount of viral gene and protein synthesis in different ways.

DNA Viruses

Figure 4-12 Replication of herpes simplex virus, a complex enveloped DNA virus. The virus binds to specific receptors and fuses with the plasma membrane. The nucleocapsid then delivers the DNA genome to the nucleus. Transcription and translation occur in three phases: immediate early, early, and late. Immediate early proteins promote the takeover of the cell; early proteins consist of enzymes, including the DNA-dependent DNA polymerase; and the late proteins are structural proteins, including the viral capsid and glycoproteins. The genome is replicated before transcription of the late genes. Capsid proteins migrate into the nucleus, assemble into icosadeltahedral capsids, and are filled with the DNA genome. The capsids filled with genomes bud through the nuclear and endoplasmic reticulum membranes into the cytoplasm, acquire tegument proteins, and then acquire their envelope as they bud through the viral glycoprotein modified membranes of the trans-Golgi network. The virus is released by exocytosis or cell lysis. page 49 page 50

Box 4-7. Properties of DNA Viruses

DNA is not transient or labile. Many DNA viruses establish persistent infections (e.g., latent, immortalizing). DNA genomes reside in the nucleus (except for poxviruses). Viral DNA resembles host DNA for transcription and replication. Viral genes must interact with host transcriptional machinery (except for poxviruses). Viral gene transcription is temporally regulated. Early genes encode DNA-binding proteins and enzymes. Late genes encode structural and other proteins. DNA polymerases require a primer to replicate the viral genome. The larger DNA viruses encode means to promote efficient replication of their genome. Parvovirus: requires cells undergoing DNA synthesis to replicate. Papovavirus: stimulates cell growth and DNA synthesis. Hepadnavirus: stimulates cell growth and encodes its own polymerase. Adenovirus: stimulates cellular DNA synthesis and encodes its own polymerase. Herpesvirus: stimulates cell growth, encodes its own polymerase and enzymes to provide deoxyribonucleotides for DNA synthesis, establishes latent infection in host. Poxvirus: encodes its own polymerase and enzymes to provide deoxyribonucleotides for DNA synthesis, replication machinery, and transcription machinery in the cytoplasm.

Replication of the DNA genome requires a DNA-dependent DNA polymerase, other enzymes, and deoxyribonucleotide triphosphates, especially thymidine (Box 4-7). Transcription of the DNA virus genome (except for poxviruses) occurs in the nucleus, using host cell polymerases and other enzymes for viral mRNA synthesis. Transcription of the viral genes is regulated by the interaction of specific DNA-binding proteins with promoter and enhancer elements in the viral genome. The viral promoter and enhancer elements are similar in sequence to those of the host cell to allow binding of the cell's transcriptional activation factors and DNA-dependent RNA polymerase. Cells from some tissues do not express the DNA-binding proteins necessary for activating the transcription of viral genes, and replication of the virus in that cell is thus prevented or limited. Different DNA viruses control the duration, timing, and quantity of viral gene and protein synthesis in different ways. The more complex viruses encode their own transcriptional activators, which enhance or regulate the expression of viral genes. For example, the herpes simplex virus encodes many proteins that regulate the kinetics of viral gene expression, including the VMW 65 (α-TIF protein, VP16). VMW 65 is carried in the virion, binds to the host cell transcription-activating complex (Oct-1), and enhances its ability to stimulate transcription of the immediate early genes of the virus. Genes may be transcribed from either DNA strand of the genome and in opposite directions. For example, the early and late genes of the SV40 papovavirus are on opposite, non-overlapping DNA strands. Viral genes may have introns requiring post-transcriptional processing of the mRNA by the cell's nuclear machinery (splicing). The late genes of papovaviruses and adenoviruses are initially transcribed as a large RNA from a single promoter and then processed to produce several different mRNAs after removal of different intervening sequences (introns).

Replication of viral DNA follows the same biochemical rules as for cellular DNA. Replication is initiated at a unique DNA sequence of the genome called the origin (ori). This is a site recognized by cellular or viral nuclear factors and the DNA-dependent DNA polymerase. Viral DNA synthesis is semiconservative, and viral and cellular DNA polymerases require a primer to initiate synthesis of the DNA chain. The parvoviruses have DNA sequences that are inverted and repeated to allow the DNA to fold back and hybridize with itself to provide a primer. Replication of the adenovirus genome is primed by deoxycytidine monophosphate attached to a terminal protein. A cellular enzyme (primase) synthesizes an RNA primer to start the replication of the papovavirus genome, whereas the herpesviruses encode a primase. Replication of the genome of the simple 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. Viral polymerases are usually faster but less precise than host cell polymerases, causing a higher mutation rate in viruses and providing a target for nucleotide analogues as antiviral drugs. Hepadnavirus replication is unique in that a circular, positive-strand RNA intermediate is first synthesized by the cell's DNA-dependent RNA polymerase. Viral proteins surround the RNA, an RNA-dependent DNA polymerase (reverse transcriptase) in this virion core makes a negative-strand DNA, and then the RNA is degraded. Positive-strand DNA synthesis is initiated but stops when the genome and core are enveloped, yielding a partially double-stranded circular DNA genome. page 50 page 51

Major limitations for replication of a DNA virus include availability of the DNA polymerase and deoxyribonucleotide substrates. Most cells in the resting phase of growth are not undergoing DNA synthesis, because the necessary enzymes are not present, and deoxythymidine pools are limited. The smaller the DNA virus, the more dependent the virus is on the host cell to provide these functions (see Box 4-7). The parvoviruses are the smallest DNA viruses and replicate only in growing cells, such as erythroid precursor cells or fetal tissue. Speeding up the growth of the cell can enhance viral DNA and mRNA synthesis. The T antigen of SV40, the E6 and E7 of papillomavirus, and the E1a and E1b proteins of adenovirus bind to and prevent the function of growth-inhibitory proteins (p53 and the retinoblastoma gene product), resulting in cell growth, which also promotes virus replication. The larger DNA viruses may encode a DNA polymerase and other proteins to facilitate DNA synthesis and are more independent. Herpes simplex virus encodes a DNA polymerase and scavenging enzymes, such as deoxyribonuclease, ribonucleotide reductase, and thymidine kinase, to generate the necessary deoxyribonucleotide substrates for replication of its genome.

RNA Viruses Replication and transcription of RNA viruses are similar processes, because the viral genomes are usually either an mRNA (positive-strand RNA) (see Figure 4-13) or a template for mRNA (negative-strand RNA) (Box 4-8; see Figure 4-14). During replication and transcription, a double-stranded RNA replicative intermediate, a structure not normally found in uninfected cells, is formed. The RNA virus genome must code for RNA-dependent RNA polymerases (replicases and transcriptases), because the cell has no means of replicating RNA. Because RNA is degraded relatively quickly, the RNA-dependent RNA polymerase must be provided or synthesized soon after uncoating to generate more viral RNA, or the infection will be aborted. Most viral RNA polymerases work at a fast pace but are also error prone, causing mutations. Replication of the genome provides new templates for production of more mRNA, which amplifies and accelerates virus replication.

Figure 4-13 Replication of picornaviruses: a simple (+) RNA virus. 1, Interaction of the picornaviruses with receptors on the cell surface defines the target cell and weakens the capsid. 2, The genome is injected through the virion and across the cell membrane. 2', The virion is endocytosed, and then the genome is released. 3, Alternatively, the genome is used as mRNA for protein synthesis. One large polyprotein is translated from the virion genome. 4, Then the polyprotein is proteolytically cleaved into individual proteins, including an RNA-dependent RNA polymerase. 5, The polymerase makes a (-) strand template from the genome and replicates the genome. A protein (VPg) is covalently attached to the 5' end of the viral genome. 6, The structural proteins associate into the capsid structure, the genome is inserted, and the virions are released on cell lysis.

Box 4-8. Properties of RNA Viruses

RNA is labile and transient. Most RNA viruses replicate in the cytoplasm. Cells cannot replicate RNA. RNA viruses must encode an RNA-dependent RNA polymerase. The genome structure determines the mechanism of transcription and replication. RNA viruses are prone to mutation. The genome structure and polarity determine how viral messenger RNA (mRNA) is generated and proteins are processed. RNA viruses, except (+) RNA genome, must carry polymerases. All (-) RNA viruses are enveloped. Picornaviruses, togaviruses, flaviviruses, caliciviruses, and coronaviruses (+) RNA genome resembles mRNA and is translated into a polyprotein, which is proteolyzed. A (-) RNA template is used for replication. Togaviruses, coronaviruses, and noroviruses have early and late genes. Orthomyxoviruses, paramyxoviruses, rhabdoviruses, filoviruses, and bunyaviruses (-) RNA genome is a template for individual mRNAs, but full-length (+) RNA template is required for replication. Orthomyxoviruses replicate and transcribe in nucleus, and each segment of the genome encodes one mRNA and template. Reoviruses (+/-) Segmented RNA genome is a template for mRNA. (+) RNA may also be encapsulated to generate the (+/-) RNA and then more mRNA. Retroviruses (+) Retrovirus RNA genome is converted into DNA, which is integrated into the host chromatin and transcribed as a cellular gene.

The positive-strand RNA viral genomes of the picornaviruses, caliciviruses, coronaviruses, flaviviruses, and togaviruses act as mRNA, bind to ribosomes, and direct protein synthesis. The naked positive-strand RNA viral genome is sufficient to initiate infection by itself. After the virus-encoded, RNA-dependent RNA polymerase is produced, a negative-strand RNA template is synthesized. The template can then be used to generate more mRNA and to replicate the genome. For the togaviruses and caliciviruses, the negative-sense RNA template is also used to produce a smaller RNA for the structural proteins (late genes). The mRNAs for these viruses are not capped at the 5' end, but the genome encodes a short poly A sequence. Transcription and replication of coronaviruses share many of these aspects but are more complex. page 51 page 52

Figure 4-14 Replication of rhabdoviruses: a simple enveloped (-) RNA virus. 1, Rhabdoviruses bind to the cell surface and are (2) endocytosed. The envelope fuses with the endosome vesicle membrane to deliver the nucleocapsid to the cytoplasm. The virion must carry a polymerase, which (3) produces five individual messenger RNAs (mRNAs) and a full-length (+) RNA template. 4, Proteins are translated from the mRNAs, including one glycoprotein (G) which is co-translationally glycosylated in the endoplasmic reticulum (ER), processed in the Golgi apparatus, and delivered to the cell membrane. 5, The genome is replicated from the (+) RNA template, and N, L, and NS proteins associate with the genome to form the nucleocapsid. 6, The matrix protein associates with the G protein-modified membrane, which is followed by assembly of the nucleocapsid. 7, The virus buds from the cell in a bullet-shaped virion.

The negative-strand RNA virus genomes of the rhabdoviruses, orthomyxoviruses, paramyxoviruses, filoviruses, and bunyaviruses are the templates for production of mRNA. The negative-strand RNA genome is not infectious by itself, and a polymerase must be carried into the cell with the genome (associated with the genome as part of the nucleocapsid) to make individual mRNA for the different viral proteins. As a result, a full-length positive-strand RNA must also be produced by the viral polymerase to act as a template to generate more copies of the genome. The (-) RNA genome is like the negatives from a roll of 35-mm film: Each frame encodes a photo/mRNA, but a full-length positive is required for replicating the roll. Except for influenza viruses, transcription and replication of negative-strand RNA viruses occur in the cytoplasm. The influenza transcriptase requires a primer to produce mRNA. It uses the 5' ends of cellular mRNA in the nucleus as primers for its polymerase and in the process steals the 5' cap from the cellular mRNA. The influenza genome is also replicated in the nucleus.

The reoviruses have a segmented, double-stranded RNA genome and undergo a more complex means of replication and transcription. The reovirus RNA polymerase is part of the inner capsid core; mRNA units are transcribed from each of the 10 or more segments of the genome while they are still in the core. The negative strands of the genome segments are used as templates for mRNA in a manner similar to that of the negative-strand RNA viruses. Reovirus-encoded enzymes contained in the inner capsid core add the 5' cap to viral mRNA. The mRNA does not have poly A. The mRNAs are released into the cytoplasm, where they direct protein synthesis or are sequestered into new cores. The positive-strand RNA in the new cores acts as a template for negative-strand RNA, and the core polymerase produces the progeny double-stranded RNA. The arenaviruses have an ambisense circular genome with (+) sequences adjacent to (-) sequences. The early genes of the virus are transcribed from the negative-sense portion of the genome, and the late genes of the virus are transcribed from the full-length replicative intermediate. Although the retroviruses have a positive-strand RNA genome, the virus provides no means for replication of the RNA in the cytoplasm. Instead, the retroviruses carry two copies of the genome, two transfer RNA (tRNA) molecules, and an RNA-dependent DNA polymerase (reverse transcriptase) in the virion. The tRNA is used as a primer for synthesis of a circular complementary DNA copy (cDNA) of the genome. The cDNA is synthesized in the cytoplasm, travels to the nucleus, and is then integrated into the host chromatin. The viral genome becomes a cellular gene. Promoters at the end of the integrated viral genome enhance the transcription of the viral DNA sequences by the cell. Full-length RNA transcripts are used as new genomes, and individual mRNAs are generated by differential splicing of this RNA.

The most unusual mode of replication is reserved for the deltavirus. The deltavirus resembles a viroid. The genome is a circular, rod-shaped, single-stranded RNA, which is extensively hybridized to itself. As the exception, the deltavirus RNA genome is replicated by the host cell DNA-dependent RNA polymerase II in the nucleus. A portion of the genome forms an RNA structure called a ribozyme, which cleaves the RNA circle to produce an mRNA.

Viral Protein Synthesis All viruses depend on the host cell ribosomes, tRNA, and mechanisms for post-translational modification to produce their proteins. The binding of mRNA to the ribosome is mediated by a 5' cap structure of methylated guanosine or a special RNA loop structure (internal ribosome entry sequence [IRES]), which binds within the ribosome to initiate protein synthesis. The cap structure, if used, is attached to mRNA in different ways by different viruses. The IRES structure was discovered first in the picornavirus genome and then in selected cellular mRNAs. Most but not all viral mRNA have a polyadenosine (polyA) tail, like eukaryotic mRNAs. page 52 page 53

Unlike bacterial ribosomes, which can bind to a polycistronic mRNA and translate several gene sequences into separate proteins, the eukaryotic ribosome binds to mRNA and can make only one continuous protein, and then it falls off the mRNA. Each virus deals with this limitation differently, depending on the structure of the genome. For example, the entire genome of a positive-strand RNA virus is read by the ribosome and translated into one giant polyprotein. The polyprotein is subsequently cleaved by cellular and viral proteases into functional proteins. DNA viruses, retroviruses, and most negative-strand RNA viruses transcribe separate mRNA for smaller polyproteins or individual proteins. The orthomyxovirus and reovirus genomes are segmented, and most of the segments code for single proteins for this reason.

Viruses use different tactics to promote preferential translation of their viral mRNA instead of cellular mRNA. In many cases, the concentration of viral mRNA in the cell is so large that it occupies most of the ribosomes, preventing translation of cellular mRNA. Adenovirus infection blocks the egress of cellular mRNA from the nucleus. Herpes simplex virus and other viruses inhibit cellular macromolecular synthesis and induce degradation of the cell's DNA and mRNA. To promote selective translation of its mRNA, poliovirus uses a virus-encoded protease to inactivate the 200,000-Da cap-binding protein of the ribosome to prevent binding and translation of 5' capped cellular mRNA. Togaviruses and many other viruses increase the permeability of the cell's membrane; thus the ribosomal affinity for most cellular mRNA is decreased. All these actions also contribute to the cytopathology of the virus infection. The pathogenic consequences of these actions are discussed further in Chapter 48. Some viral proteins require post-translational modifications, such as phosphorylation, glycosylation, acylation, or sulfation. Protein phosphorylation is accomplished by cellular or viral protein kinases and is a means of modulating, activating, or inactivating proteins. Several herpesviruses and other viruses encode their own protein kinase. Viral glycoproteins are synthesized on membrane-bound ribosomes and have the amino acid sequences to allow insertion into the rough endoplasmic reticulum and N-linked glycosylation. The high-mannose precursor form of the glycoproteins progresses from the endoplasmic reticulum through the vesicular transport system of the cell and is processed through the Golgi apparatus. The sialic acid-containing mature glycoprotein is expressed on the plasma membrane of the cell unless the glycoprotein expresses protein sequences for retention in an intracellular organelle. The presence of the glycoproteins determines where the virion will assemble. Other modifications, such as O-glycosylation, acylation, and sulfation of the proteins, can also occur during progression through the Golgi apparatus.

Assembly

Virion assembly is analogous to a three-dimensional interlocking puzzle that puts itself together in the box. The virion is built from small, easily manufactured parts that enclose the genome in a functional package. Each part of the virion has recognition structures that allow the virus to form the appropriate protein-protein, protein-nucleic acid, and (for enveloped viruses) protein-membrane interactions needed to assemble into the final structure. The assembly process begins when the necessary pieces are synthesized and the concentration of structural proteins in the cell is sufficient to drive the process thermodynamically, much like a crystallization reaction. The assembly process may be facilitated by scaffolding proteins or other proteins that are activated or release energy on proteolysis. For example, cleavage of the VP0 protein of poliovirus releases the VP4 peptide, which solidifies the capsid. The site and mechanism of virion assembly in the cell depend on where genome replication occurs and whether the final structure is a naked capsid or an enveloped virus. Assembly of the DNA viruses, other than poxviruses, occurs in the nucleus and requires transport of the virion proteins into the nucleus. RNA virus and poxvirus assembly occurs in the cytoplasm. Capsid viruses may be assembled as empty structures (procapsids) to be filled with the genome (e.g., picornaviruses), or they may be assembled around the genome. Nucleocapsids of the retroviruses, togaviruses, and the negative-strand RNA viruses assemble around the genome and are subsequently enclosed in an envelope. The helical nucleocapsid of negative-strand RNA viruses includes the RNA-dependent RNA polymerase necessary for mRNA synthesis in the target cell.

For enveloped viruses, newly synthesized and processed viral glycoproteins are delivered to cellular membranes by vesicular transport. Acquisition of an envelope occurs after association of the nucleocapsid with the viral glycoprotein-containing regions of host cell membranes in a process called budding. Matrix proteins for negative-strand RNA viruses line and promote the adhesion of nucleocapsids with the glycoprotein-modified membrane. As more interactions occur, the membrane surrounds the nucleocapsid, and the virus buds from the membrane. The type of genome and the protein sequence of the glycoproteins determine the site of budding. Most RNA viruses bud from the plasma membrane, and the virus is released from the cell at the same time. The flaviviruses, coronaviruses, and bunyaviruses acquire their envelope by budding into the endoplasmic reticulum and Golgi membranes and may remain cell-associated in these organelles. The herpes simplex virus nucleocapsid assembles in the nucleus and buds into and then out of the endoplasmic reticulum. The nucleocapsid is dumped into the cytoplasm, viral proteins associate with the capsid, and then the envelope is acquired by budding into a trans-Golgi network membrane decorated with the 10 viral glycoproteins. The virion is transported to the cell surface and released by exocytosis, on cell lysis, or transmitted through cell-cell bridges. page 53 page 54

Viruses use different tricks to ensure that all the parts of the virus are assembled into complete virions. The RNA polymerase required for infection by negative-strand RNA viruses is carried on the genome as a helical nucleocapsid. The human immunodeficiency virus and other retrovirus genomes are packaged in a procapsid consisting of a polyprotein containing the protease, polymerase, integrase, and structural proteins. This procapsid binds to viral glycoprotein-modified membranes, and the virion buds from the membrane. The virus-encoded protease is activated within the virion and cleaves the polyprotein to produce the final infectious nucleocapsid and the required proteins within the envelope. Assembly of viruses with segmented genomes, such as influenza or reovirus, requires accumulation of at least one copy of each gene segment. This can be accomplished if the segments assemble together like capsid subunits or randomly package more segments per virion than necessary. This will statistically generate a small but acceptable percentage of functional viruses. Errors are made by the viral polymerase and during viral assembly. Empty virions and virions containing defective genomes are produced. As a result, the particle to infectious virus ratio, also called particle to plaque-forming unit ratio, is high, usually greater than 10, and during rapid viral replication can even be 104. Defective viruses can occupy the machinery required for normal virus replication to prevent (interfere with) virus production (defective interfering particles).

Release

Viruses can be released from cells after lysis of the cell, by exocytosis, or by budding from the plasma membrane. Naked capsid viruses are generally released after lysis of the cell. Release of most enveloped viruses occurs after budding from the plasma membrane without killing the cell. Lysis and plasma membrane budding are efficient means of release. Viruses that bud or acquire their membrane in the cytoplasm (e.g., flaviviruses, poxviruses) remain cell-associated and are released by exocytosis or cell lysis. Viruses that bind to sialic acid receptors (e.g., orthomyxoviruses, certain paramyxoviruses) may also have a neuraminidase. The neuraminidase removes potential sialic acid receptors on the glycoproteins of the virion and the host cell to prevent clumping and facilitate release.

Reinitiation of the Replication The virus released to the extracellular medium is usually responsible for initiating new infections; however, traversal of cell-cell bridges, virus-induced cell-cell fusion, or vertical transmission of the genome to daughter cells can also spread the infection. These allow the virus to escape antibody detection. Some herpesviruses, retroviruses, and paramyxoviruses can induce cell-cell fusion to merge the cells into multinucleated giant cells (syncytia), which become huge virus factories. The retroviruses and some DNA viruses can transmit their integrated copy of the genome vertically to daughter cells on cell division. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Viral Genetics

Mutations spontaneously and readily occur in viral genomes, creating new virus strains with properties differing from the parental, or wild-type, virus. These variants can be identified by their nucleotide sequences, antigenic differences (serotypes), or differences in functional or structural properties. Most mutations have no effect or are detrimental to the virus. Mutations in essential genes inactivate the virus, but mutations in other genes can produce antiviral drug resistance or alter the antigenicity or pathogenicity of the virus. Errors in copying the viral genome during virus replication produce many mutations. This is because of the poor fidelity of the viral polymerase and the rapid rate of genome replication. In addition, RNA viruses do not have a genetic error-checking mechanism. As a result, the rates of mutation for RNA viruses are usually greater than for DNA viruses. Mutations in essential genes are termed lethal mutations. These mutants are difficult to isolate, because the virus cannot replicate. A deletion mutant results from the loss or selective removal of a portion of the genome and the function that it encodes. Other mutations may produce plaque mutants, which differ from the wild type in the size or appearance of the infected cells; host range mutants, which differ in the tissue type or species of target cell that can be infected; or attenuated mutants, which are variants that cause less serious disease in animals or humans. Conditional mutants, such as temperature-sensitive (ts) or cold-sensitive mutants, have a mutation in a gene for an essential protein that allows virus production only at certain temperatures. Whereas ts mutants generally grow well or relatively better at 30° C to 35° C, the encoded protein is inactive at elevated temperatures of 38° C to 40° C, preventing virus production.

Figure 4-15 Genetic exchange between viral particles can give rise to new viral types, as illustrated. Representative viruses include the following: 1, intertypic recombination of herpes simplex virus type 1 (HSV1) and type 2 (HSV2); 2, reassortment of two strains of influenza virus; 3, rescue of a papovavirus defective in assembly by a complementary defective virus (transcapsidation); and 4, marker rescue of a lethal or conditional mutation. page 54 page 55

New virus strains can also arise by genetic interactions between viruses or between the virus and the cell (Figure 4-15). Intramolecular genetic exchange between viruses or the virus and the host is termed recombination. Recombination can occur readily between two related DNA viruses. For example, coinfection of a cell with the two closely related herpesviruses (herpes simplex virus types 1 and 2) yields intertypic recombinant strains. These new hybrid strains have genes from types 1 and 2. Integration of retroviruses into host cell chromatin is a form of recombination. Recombination of two related RNA viruses, Sindbis and eastern equine encephalitis virus, resulted in creation of another togavirus, western equine encephalitis virus.

Viruses with segmented genomes (e.g., influenza viruses and reoviruses) form hybrid strains on infection of one cell with more than one virus strain. This process, termed reassortment, is analogous to picking 10 marbles out of a box containing 10 black and 10 white marbles. Very different strains of influenza A virus are created on coinfection with a virus from different species (see Figure 59-5). In some cases, a defective viral strain can be rescued by the replication of another mutant, by the wild-type virus, or by a cell line bearing a replacement viral gene. Replication of the other virus or expression of the gene in the cell provides the missing function required by the mutant (complementation), allowing replication to occur. A disabled infectious single-cycle herpes simplex virus (DISC-HSV) vaccine lacks an essential gene and is grown in a cell line that expresses that gene product to "complement" the virus. The virus that is produced can infect the normal cells of the vaccinated individual, but the virions that are produced lack the function necessary for replication in that person's cells. Rescue of a lethal or conditional-lethal mutant with a defined genetic sequence, such as a restriction endonuclease DNA fragment, is called marker rescue. Marker rescue is used to map the genomes of viruses such as herpes simplex virus. Virus produced from cells infected with different virus strains may be phenotypically mixed and have the proteins of one strain but the genome of the other (transcapsidation). Pseudotypes are generated when transcapsidation occurs between different types of virus, but this is rare.

Individual virus strains or mutants are selected by their ability to use the host cell machinery and to withstand the conditions of the body and the environment. Cellular properties that can act as selection pressures include the growth rate of the cell and tissue-specific expression of certain proteins required by the virus (e.g., enzymes, glycoproteins, transcription factors). The conditions of the body, its elevated temperature, innate and immune defenses, and tissue structure are also selection pressures for viruses. The viruses that cannot endure these conditions or evade the host defenses are eliminated. A small selective advantage in a mutant virus can shortly lead to its becoming the predominant viral strain. The high mutation rate of the human immunodeficiency virus promotes a switch in target cell tropism from macrophage to T cell, the development of antiviral drug-resistant strains after treatment, and the generation of antigenic variants during a patient's course of infection. The growth of virus under benign laboratory conditions allows weaker strains to survive because of the absence of the selective pressures of the body. This process is used to select attenuated virus strains for use in vaccines. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Viral Vectors for Therapy

Genetically manipulated viruses can be excellent delivery systems for foreign genes. Viruses can provide gene replacement therapy, can be used as vaccines to promote immunity to other agents or tumors, and can act as targeted killers of tumors. The advantages of using viruses are that they can be readily amplified by replication in appropriate cells, and they target specific tissues and deliver the DNA or RNA into the cell. Viruses that are being developed as vectors include retroviruses, adenoviruses, herpes simplex virus, adeno-associated virus (parvovirus), poxviruses (e.g., vaccinia and canarypox) (see Figure 54-3), and even some togaviruses. The viral vectors are usually defective or attenuated viruses, in which the foreign DNA replaces a virulence or unessential gene. The foreign gene may be under the control of a viral promoter or even a tissue-specific promoter. Defective virus vectors are grown in cell lines that express the missing viral functions "complementing" the virus. The progeny can deliver their nucleic acid but not produce infectious virus. Retroviruses and adeno-associated viruses can integrate into cells and permanently deliver a gene into the cell's chromosome. Adenovirus and herpes simplex virus promote targeted delivery of the foreign gene to receptor-bearing cells. Genetically attenuated herpes simplex viruses are being developed to specifically kill the growing cells of glioblastomas while sparing the surrounding neurons. Vaccinia virus carrying a gene for the rabies glycoprotein is already being used successfully to immunize raccoons, foxes, and skunks in the wild. Some day, virus vectors may be routinely used to treat cystic fibrosis, Duchenne muscular dystrophy, lysosomal storage diseases, and immunologic disorders.

Questions page 55 page 56

A 1.

Are resistant to detergents

B Picornaviruses

2.

Are resistant to drying

Togaviruses

3.

Replication in the nucleus

Orthomyxoviruses

4.

Replication in the cytoplasm

Paramyxoviruses

5.

Can be released from the cell without cell lysis

Rhabdoviruses

6.

Provide a good target for antiviral drug action

Reoviruses

7.

Undergo reassortment on coinfection with two strains

Retroviruses

8.

Make DNA from an RNA template

Herpesviruses

9.

Use a (+) RNA template to replicate the genome

Papovaviruses

10. Genome translated into a polyprotein

Adenoviruses Poxviruses Hepadnaviruses

1. Describe the features of these viruses that are similar, and those that are different. a. Poliovirus and rhinovirus b. Poliovirus and rotavirus c. Poliovirus and western equine encephalitis virus d. Yellow fever virus and dengue virus e. Epstein-Barr virus and cytomegalovirus 2. Match the characteristics from column A with the appropriate viral families in column B, based on your knowledge of their physical and genome structure and their implications. 3. Based on structural considerations, which of the virus families listed in question 2 should be able to endure fecal-oral transmission? 4. List the essential enzymes encoded by the virus families listed in question 2. 5. A mutant defective in the herpes simplex virus type 1 DNA polymerase gene replicates in the presence of herpes simplex virus type 2. The progeny virus contains the herpes simplex virus type 1 genome, but is recognized by antibodies to herpes simplex virus type 2. Which genetic mechanisms may be occurring?

6. How are the early and late genes of the togaviruses, papovaviruses, and herpesviruses distinguished, and how is the time of their expression regulated? 7. What are the consequences (no effect, decreased efficiency, or inhibition of replication) of a deletion mutation in the following viral enzymes? a. Epstein-Barr virus polymerase b. Herpes simplex virus thymidine kinase c. Human immunodeficiency virus reverse transcriptase d. Influenza B virus neuraminidase e. Rabies virus (rhabdovirus) G protein

Bibliography Big Picture Book of Viruses online: Available at http://www.virology.net/Big_Virology/BVHomePage.html Cann AJ: Principles of Molecular Virology. San Diego, Academic, 2001. Cohen J, Powderly WG: Infectious Diseases, 2nd ed. St Louis, Mosby, 2004. Electron microscopic images of viruses, by Linda Stannard, University of Capetown, South Africa, online: Available at www.uct.ac.za/depts/mmi/stannard/linda.html. Flint SJ, et al: Principles of Virology: Molecular Biology, Pathogenesis and Control of Animal Viruses, 2nd ed. Washington, DC, ASM Press, 2003. Knipe DM, Howley PM: Fields Virology, 4th ed. New York, Lippincott Williams & Wilkins, 2001. Richman DD, Whitley RJ, Hayden FG: Clinical Virology. New York, Churchill Livingstone, 1997. Rosenthal KS: Viruses: Microbial spies and saboteurs. Infect Dis Clin Practice 14:97-106, 2006. Specter S, Hodinka RL, Young SA: Clinical Virology Manual, 3rd ed. Washington, DC, ASM Press, 2000. Strauss JM, Strauss EG: Viruses and Human Disease, 2nd ed. San Diego, Academic, 2007. Virology on the web: Available at http://www.virology.net/garryfavweb.html Viruses in cell culture: Available at www.uct.ac.za/depts/mmi/stannard/linda.html

The Importance of Fungi The fungi represent a ubiquitous and diverse group of organisms, the main purpose of which is to degrade organic matter. All fungi lead a heterotrophic existence as saprobes (organisms that live on dead or decaying matter), symbionts (organisms that live together and in which the association is of mutual advantage), commensals (organisms living in a close relationship in which one benefits from the relationship, and the other neither benefits nor is harmed), or as parasites (organisms that live on or within a host from which they derive benefits without making any useful contribution in return; in the case of pathogens, the relationship is harmful to the host). Fungi have emerged in the past two decades as major causes of human disease (Table 5-1), especially among those individuals who are immunocompromised or hospitalized with serious underlying diseases. Among these patient groups, fungi serve as opportunistic pathogens, causing considerable morbidity and mortality. The overall incidence of specific invasive mycoses continues to increase with time (Table 5-2), and the list of opportunistic fungal pathogens likewise increases each year. In short, there are no nonpathogenic fungi! This increase in fungal infections can be attributed to the ever-growing number of immunocompromised patients, including transplant patients, individuals with AIDS, patients with cancer and undergoing chemotherapy, and those individuals who are hospitalized with other serious underlying conditions and who undergo a variety of invasive procedures. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Fungal Taxonomy, Structure, and Replication

The fungi are classified in their own separate kingdom, Kingdom Fungi. They are eukaryotic organisms that are distinguished from other eukaryotes by a rigid cell wall composed of chitin and glucan and a cell membrane in which ergosterol is substituted for cholesterol as the major sterol component (Figure 5-1). page 57 page 58

Table 5-1. Incidence and Case-Fatality Ratios of Selected Invasive Fungal Infections Pathogen

Candida species

No. of Cases per Case-Fatality Ratio Million per Year (%) for First Episode Incidence 72.8 33.9

Cryptococcus neoformans

65.5

12.7

Coccidioides immitis

15.3

11.1

Aspergillus species

12.4

23.3

Histoplasma capsulatum

7.1

21.4

Agents of zygomycosis 1.7

30.0

Agents of hyalohyphomycosis

1.2

14.3

Agents of phaeohyphomycosis

1.0

0

Sporothrix schenckii

70%), the initial disease is asymptomatic but establishes chronic persistent disease. The predominant symptom is chronic fatigue. The chronic, persistent disease often progresses to chronic active hepatitis within 10 to 15 years and to cirrhosis (20% of chronic cases) and liver failure (20% of cirrhotic cases) after 20 years. HCV-induced liver damage may be exacerbated by alcohol, certain medications, and other hepatitis viruses to promote cirrhosis. HCV promotes the development of hepatocellular carcinoma after 30 years in up to 5% of chronically infected patients.

Clinical Case 65-1. Hepatitis C Virus

In a case reported by Morsica, et al (Scand J Infect Dis 33:116-120, 2001), a 35-year-old woman was admitted with malaise and jaundice. Elevated blood levels of bilirubin (71.8 µmol/L (normal value 95%) protein bound. They are distributed to all major organs, although concentrations in cerebrospinal fluid are low. All of the echinocandins are very well tolerated and have few drug-drug interactions. Among the three echinocandins, all have similar spectrum and potency against Candida and Aspergillus species. Caspofungin is approved for the treatment of invasive candidiasis, including candidemia, and for treatment of patients with invasive aspergillosis refractory to or intolerant of other approved antifungal therapies. Anidulafungin is approved for the treatment of esophageal candidiasis and candidemia, and micafungin is approved for treatment of esophageal candidiasis and candidemia, and for prevention of invasive candidiasis. page 707 page 708

Antimetabolites

Flucytosine (5-fluorocytosine, 5-FC) is the only available antifungal agent that functions as an antimetabolite. It is a fluorinated pyrimidine analogue that exerts antifungal activity by interfering with the synthesis of DNA, RNA, and proteins in the fungal cell (see Figure 70-1). Flucytosine enters the fungal cell via cytosine permease and is deaminated to 5-fluorouracil (5-FU) in the cytoplasm. The 5-FU is converted to 5-fluorouridylic acid, which then competes with uracil in the synthesis of RNA, with resultant RNA miscoding and inhibition of DNA and protein synthesis. The antifungal spectrum of flucytosine is limited to Candida spp., Cryptococcus neoformans, Rhodotorula spp., Saccharomyces cerevisiae, and selected dematiaceous molds (see Table 70-2). Although primary resistance to flucytosine is rare among isolates of Candida spp., resistance may develop among Candida and Cryptococcus neoformans during flucytosine monotherapy. Flucytosine is not active against Aspergillus spp., the Zygomycetes, or other hyaline molds. Flucytosine is water soluble and has excellent bioavailability when administered orally. High concentrations of flucytosine may be achieved in serum, cerebrospinal fluid, and other body fluids. Major toxicities are observed when flucytosine serum concentrations exceed 100 µg/ml and include bone marrow suppression, hepatotoxicity, and gastrointestinal intolerance. Monitoring of serum concentrations of flucytosine is important in avoiding toxicity. Flucytosine is not used as monotherapy, owing to the propensity for secondary resistance. Combinations of flucytosine with either amphotericin B or fluconazole have been shown to be efficacious in treating both cryptococcosis and candidiasis.

Allylamines The allylamine class of antifungal agents includes terbinafine, which has systemic activity, and naftifine, which is a topical agent (see Table 70-1). These agents inhibit the enzyme squalene epoxidase, which results in a decrease in ergosterol and an increase in squalene within the fungal cell membrane (see Figures 70-1 and 70-4).

Terbinafine is a lipophilic antifungal agent with a broad spectrum of activity that includes dermatophytes, Candida spp., Malassezia furfur, Cryptococcus neoformans, Trichosporon spp., Aspergillus spp., Sporothrix schenckii, and Penicillium marneffei (see Table 70-2). It is available in oral and topical formulations and achieves high concentrations in fatty tissues, skin, hair, and nails. Terbinafine is efficacious in the treatment of virtually all forms of dermatomycoses, including onychomycosis, and exhibits few side effects. It has shown clinical effectiveness in the treatment of sporotrichosis, aspergillosis, and chromoblastomycosis and has shown promise for the treatment of infections due to fluconazole-resistant Candida spp. when used in combination with fluconazole.

Griseofulvin Griseofulvin is an oral agent used in the treatment of infections due to the dermatophytes. It is thought to inhibit fungal growth by interaction with microtubules within the fungal cell, resulting in inhibition of mitosis. (see Table 70-1 and Figure 70-1). Griseofulvin is considered a second-line agent in the treatment of dermatophytoses. Newer agents, such as itraconazole and terbinafine, are more rapid acting and provide greater efficacy. Griseofulvin is also associated with a number of mild side effects, including nausea, diarrhea, headache, hepatotoxicity, rash, and neurologic side effects. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Topical Antifungal Agents

A wide variety of topical antifungal preparations is available for the treatment of superficial cutaneous and mucosal fungal infections (see Table 70-1). Topical preparations are available for most classes of antifungal agents, including polyenes (amphotericin B, nystatin, pimaricin), allylamines (naftifine and terbinafine), and numerous imidazoles and miscellaneous agents (see Table 70-1). Creams, lotions, ointments, powders, and sprays are available for use in the treatment of cutaneous infections and onychomycosis, whereas mucosal infections are best treated with suspensions, tablets, troches, or suppositories. Whether one uses topical or systemic therapy for treatment of cutaneous or mucosal fungal infections usually depends on the status of the host and the type and extent of infection. Whereas most cutaneous dermatophytic infections and oral or vaginal candidiasis will respond to topical therapy, the refractory nature of infections such as onychomycosis or tinea capitis ("ringworm" of the scalp) usually calls for long-term systemic therapy. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Investigational Antifungal Agents page 708 page 709

At the present time, there are several antifungal agents in various stages of clinical evaluation. These "investigational" agents include some with established modes of action, as well as some novel classes of antifungal agents, such as a liposomal formulation of nystatin, novel triazole agents (albaconazole, isavuconazole, and ravuconazole), echinocandins (aminocandin), an inhibitor of chitin synthesis (nikkomycin Z) and sordarin and azasordarin derivatives (see Table 70-1). The mechanisms of action and spectra of activity of liposomal nystatin, the novel triazoles, and echinocandins are essentially the same as that of the currently available members of each class (see Tables 70-1 and 70-2). To a varying degree, the newer agents in each class offer the potential for more favorable pharmacokinetic and pharmacodynamic proprieties, decreased toxicities or drug-drug interactions, or possible improved activity against certain pathogens that are refractory to presently available agents. In contrast, completely new agents such as the sordarins and azasordarins interact with a novel target, elongation factor 3, which is essential for fungal protein synthesis. Inhibition of chitin synthesis in the fungal cell wall by nikkomycin Z provides another novel approach that may be useful in concert with other inhibitors of cell wall or cell membrane synthesis. The development of agents with novel mechanisms of action is both necessary and promising for future advances in the area of antifungal therapy. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Combinations of Antifungal Agents in the Treatment of Mycoses

The high mortality of opportunistic fungal infections has spurred the development of new antifungal agents, including some with novel mechanisms of action (see Table 70-1). In addition to aggressive use of new antifungal agents such as voriconazole and caspofungin as monotherapy, the use of azole-, echinocandin-, and polyene-based combinations for treatment of the more difficult to treat mycoses, such as opportunistic mold infections, is the focus of intense interest and discussion. The rationale behind combination therapy is that by using combinations of antifungal agents, one may achieve a better clinical outcome than with monotherapy. The push towards the use of combination antifungal therapy is especially strong for those infections such as invasive aspergillosis, where the associated mortality is unacceptably high. In considering combination therapy, one seeks to achieve synergy and avoid antagonism. Synergy is achieved when the outcome obtained with the combination of agents is significantly better than that obtained with either drug alone. Conversely, antagonism is when the combination is less active or efficacious than either drug alone. In the case of antifungal therapy, there are several mechanisms that one may consider in developing an effective combination treatment strategy. (1) Inhibition of different stages of the same biochemical pathway. This is a classical approach for achieving synergy with antiinfective agents. An example of this approach to antifungal therapy would be the combination of terbinafine with an azole, where both agents attack the sterol pathway at different points (see Figure 70-4), resulting in inhibition of ergosterol synthesis and disruption of the fungal cell membrane. (2) Increased penetration of one agent into the cell by virtue of the permeabilizing action of another agent on the fungal cell wall or cell membrane. The combination of amphotericin B (cell membrane disruption) and flucytosine (inhibition of nucleic acid synthesis intracellularly) is a classic example of this interaction. (3) Inhibition of the transport of one agent out of the cell by another agent. Many fungi employ energy dependent efflux pumps to actively pump antifungal agents out of the cell, thereby avoiding the toxic effects of the antifungal. Inhibition of these pumps by agents such as reserpine has been shown to enhance the activity of the azole antifungal agents against Candida spp. (4) Simultaneous inhibition of

different fungal cell targets. Inhibition of fungal cell wall synthesis by an agent such as caspofungin, coupled with disruption of cell membrane function by amphotericin B or azoles, is an example of this type of combination. Although the potential value of combination antifungal therapy is appealing, there are several possible downsides to this strategy that must be considered. Antagonism among antifungal agents when used in combination is also a distinct possibility and may occur via several different mechanisms. (1) The action of one agent results in a decrease in the target of another agent. The action of azole antifungal agents depletes the cell membrane of ergosterol, which is the primary target for amphotericin B. (2) The action of one antifungal agent results in the modification of the target of another agent. The inhibition of ergosterol synthesis by azole antifungal agents results in the accumulation of methylated sterols, to which amphotericin B binds less well. (3) Blocking of the target site of one agent by another. Lipophilic agents, such as itraconazole, may adsorb to the fungal cell surface and inhibit the binding of amphotericin B to membrane sterols. Despite these possible positive and negative scenarios, the data supporting the achievement of synergy when various combinations are used clinically are limited. Likewise, antagonism may be demonstrated in the laboratory, but significant antagonism has not been observed clinically with antifungal combinations. By considering all of the laboratory and clinical data for antifungal combination therapy, one arrives at a very limited number of instances where combination therapy has been shown to be beneficial in the treatment of invasive mycoses (Table 70-3). The strongest data exists for the treatment of cryptococcosis, where the combination of amphotericin B and flucytosine has been shown to be beneficial in the treatment of cryptococcal meningitis. The data are less strong for the combination of flucytosine with fluconazole or amphotericin B with triazoles; however, these combinations appear to be beneficial in treating cryptococcosis as well.

Candidiasis is generally treated adequately with a single antifungal agent such as amphotericin B, caspofungin, or fluconazole; however, combination therapy may be useful in selected situations. The combination of amphotericin B and fluconazole has proven benefits in treating candidemia. Likewise, the combination of terbinafine plus an azole is promising in the treatment of refractory oropharyngeal candidiasis. Flucytosine in combination with either amphotericin B or triazoles has positive effects on survival and tissue burden of infection in animal models of candidiasis. Currently, combination therapy of candidiasis should be reserved for specific individual settings such as meningitis, endocarditis, hepatosplenic infection, and candidiasis that is recurrent or refractory to single agent therapy. page 709 page 710

Table 70-3. Summary of Potentially Useful Antifungal Combinations for Treatment of Common Mycoses Infection Candidiasis

Antifungal combination AmB + FCZ

Comments

AmB + FC

Clinical success in humans with peritonitis

Cryptococcosis AmB + FC

Good clinical success in humans with candidemia

Good clinical success in humans with cryptococcal meningitis

AmB + FCZ

Clinical success in humans with cryptococcal meningitis

FC + FCZ

Clinical success in humans with cryptococcal meningitis

Aspergillosis

AmB + FC

In vivo benefit (animal model); minimal human data

AmB + azoles

No benefit in animals

AmB + echinocandins

In vivo benefit (animal model); minimal human data

Triazoles + echinocandins

In vivo benefit (animal model); minimal human data

AmB, amphotericin B; FCZ, fluconazole; FC, flucytosine.

Although the clinical setting of invasive aspergillosis is where combination therapy is most attractive, the data to support its use are lacking. At the present time, there are no clinical trials published that evaluate the use of combination therapy in the treatment of invasive aspergillosis. Studies in vitro and in animals have produced variable results. Combinations of echinocandins with azoles or amphotericin B have yielded positive results. Likewise, amphotericin B plus rifampin appears synergistic. Studies with flucytosine or rifampin plus amphotericin B or azoles have been inconsistent. Despite the desperate need for better treatment options for invasive aspergillosis, there is little evidence that combination therapy will improve clinical outcome. Combination therapy should be used with caution until more clinical data is available. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Mechanisms of Resistance to Antifungal Agents

Given the prominent role of Candida spp. as etiologic agents of invasive mycoses, it is not surprising that most of our understanding of the mechanisms of resistance to antifungal agents comes from studies of Candida albicans and other species of Candida. Much less is known of resistance mechanisms in Aspergillus spp. and Cryptococcus neoformans, and almost no information on antifungal resistance mechanisms is available for other opportunistic fungal pathogens. In contrast to mechanisms of resistance to antibacterial agents, there is no evidence that fungi are capable of destroying or modifying antifungal agents as a means of achieving resistance. Likewise, antifungal resistance genes are not transmissible from cell to cell in the manner that occurs with many bacterial resistance genes. It is apparent, however, that multidrug efflux pumps, target alterations, and reduced access to drug targets are important mechanisms of resistance to antifungal agents, just as they are for antibacterial resistance (Table 70-4). In contrast to the rapid emergence and spread of high-level multidrug resistance that occurs in bacteria, antifungal resistance usually develops slowly and involves the emergence of intrinsically resistant species or a gradual, stepwise alteration of cellular structures or functions that results in resistance to an agent to which there has been prior exposure.

Polyenes Resistance to polyenes, and amphotericin B in particular, remains uncommon despite extensive use over more than 30 years. Decreased susceptibility to amphotericin B has been reported in isolates of Candida lusitaniae, Candida glabrata, Candida krusei, and Candida guilliermondii. Although primary resistance may be seen, most resistance to amphotericin B among Candida spp. is secondary to amphotericin B exposure during therapy. Aspergillus spp. are generally susceptible to amphotericin B; however, Aspergillus terreus is unique in that it appears to be resistant both in vitro and in vivo. Although secondary resistance to amphotericin B has been reported in Cryptococcus neoformans, it is quite rare. page 710

page 711

Table 70-4. Mechanisms Involved in the Development of Resistance to Antifungal Agents in Pathogenic Fungi Fungus Aspergillus fumigatus

Amphotericin B

Flucytosine

Candida albicans

Decrease in ergosterol Replacement of polyene-binding sterols Masking of ergosterol

Loss of permease activity Loss of cytosine deaminase activity Loss of uracil phosphoribosyltransferase activity

Candida glabrata Candida krusei

Itraconazole Altered target enzyme, 14α-demethylase Decreased azole accumulation

Fluconazole

Echinocandins

Overexpression or mutation of 14α-demethylase Overexpression of efflux pumps, CDR and MDR genes

Mutation in FKS1 gene

Alteration or Loss of permease activity decrease in ergosterol content

Overexpression of efflux pumps (CgCDR genes)

Mutation in FKS1 gene

Alteration or decrease in ergosterol content

Active efflux Reduced affinity for target enzyme, 14α-demethylase

Mutation in FKS1 gene

Candida lusitaniae

Alteration or decrease in ergosterol content Production of modified sterols

Cryptococcus Defects in sterol neoformans synthesis Decreased ergosterol Production of modified sterols

Alterations in target enzyme Overexpression of MDR efflux pump

The mechanism of amphotericin B resistance appears to be due to qualitative and quantitative alterations in the fungal cell. Amphotericin B-resistant mutants of Candida spp. and Cryptococcus neoformans have been shown to have a reduced ergosterol content, replacement of polyene-binding sterols (ergosterol) by ones that bind polyenes less well (fecosterol), or masking of ergosterol in the cell membranes so that binding with polyenes is hindered due to steric or thermodynamic factors. The molecular mechanism of amphotericin B resistance has not been determined; however, sterol analysis of resistant strains of Candida spp. and Cryptococcus neoformans suggest that they are defective in ERG2 or ERG3, genes encoding for the C-8 sterol isomerase and C-5 sterol desaturase enzymes, respectively.

Azoles The ubiquitous use of azoles, especially fluconazole, for the treatment and prevention of fungal infections has given rise to reports of emerging resistance to this class of antifungal agents. Fortunately, primary resistance to fluconazole is rare among most species of Candida causing bloodstream infection. Among the five most common species of Candida isolated from the blood of infected patients (Candida albicans, Candida glabrata, Candida parapsilosis, Candida tropicalis, and Candida krusei), only Candida krusei is considered intrinsically resistant to fluconazole. Among the remaining species, approximately 10% of Candida glabrata exhibit primary resistance to fluconazole, and less than 2% of Candida albicans, Candida parapsilosis, and Candida tropicalis are resistant to this agent. The new triazoles (voriconazole, posaconazole, and ravuconazole) are more potent than fluconazole against Candida spp., including activity against Candida krusei and some fluconazole-resistant strains of other Candida spp.; however, there is a strong positive correlation between the activity of fluconazole and that of the other triazoles, suggesting some degree of cross-resistance within the class. Primary resistance to fluconazole is also rare among clinical isolates of Cryptococcus neoformans. Secondary resistance has been described in isolates obtained from individuals with AIDS and relapsing cryptococcal meningitis. Only a small number of isolates of Aspergillus spp. have been shown to demonstrate resistance to itraconazole. In contrast to Candida, cross-resistance between itraconazole and the new triazoles is not complete among isolates of Aspergillus spp.; cross-resistance between itraconazole and posaconazole, but not voriconazole, has been reported. page 711 page 712

Azole resistance in Candida spp. can be due to the following mechanisms: a modification in the quantity or quality of the target enzymes, reduced access of the drug to the target, or some combination of these mechanisms. Thus point mutations in the gene (ERG11) encoding the target enzyme, lanosterol 14α-demethylase, leads to an altered target with decreased affinity for azoles. Overexpression of ERG11 results in overproduction of the target enzyme, creating the need for higher concentrations of the drug within the cell to inactivate all the target enzyme molecules. Up-regulation of genes encoding for multidrug efflux pumps results in active efflux of the azole antifungal agents out of the cell. Up-regulation of genes encoding the major facilitator type efflux pump (MDR) leads to fluconazole resistance, and up-regulation of genes encoding the ATP-binding cassette transporters (CDR) leads to resistance to multiple azoles. These mechanisms may act individually, sequentially, or simultaneously, resulting in strains of Candida that exhibit progressively higher levels of azole resistance. The mechanisms of azole resistance in Aspergillus spp. are poorly characterized, given the paucity of strains with documented resistance. It appears that both increased drug efflux and alterations in the 14α-demethylase target enzyme serve as mechanisms for resistance to itraconazole among isolates of Aspergillus spp. Similarly, secondary resistance to fluconazole among isolates of Cryptococcus neoformans has been associated with overexpression of MDR efflux pumps and alteration of the target enzyme. Cryptococcus neoformans has also been shown to have a CDR-type efflux pump.

Echinocandins

Caspofungin, anidulafungin, and micafungin all demonstrate potent fungicidal activity against Candida spp., including azole-resistant strains. Clinical isolates of Candida spp. with reduced susceptibility to the echinocandins are very rare. Efforts to produce caspofungin-resistant mutants of Candida albicans in the laboratory have shown that the frequency with which these mutants arise is very low (1 in 108 cells), suggesting a low potential for the emergence of resistance in the clinical setting. Clinical isolates of Aspergillus spp. with reduced susceptibility to echinocandins are non-existent at the present time, and efforts to produce resistance in the laboratory setting have been unsuccessful. The mechanism of resistance to caspofungin that has been characterized in laboratory-derived mutants of Candida albicans is one of an altered glucan synthesis enzyme complex that shows a decreased sensitivity to inhibition by caspofungin. These strains have point mutations in the FKS1 gene that encodes for an integral membrane protein (FKS1), which is the catalytic subunit of the glucan synthesis enzyme complex. The FKS1 mutation results in strains that are resistant to all of the echinocandins but retain susceptibility to polyene and azole antifungal agents. Although the FKS1 gene is essential in Aspergillus species as well, similar mutations have not been demonstrated thus far.

Flucytosine Primary resistance to flucytosine is uncommon among clinical isolates of Candida spp. and Cryptococcus neoformans. Secondary resistance, however, is well documented to occur among both Candida spp. and Cryptococcus neoformans during monotherapy with this agent.

Flucytosine resistance may develop due to decreased uptake of the drug (loss of permease activity) or by loss of enzymatic activity necessary to convert flucytosine to 5-FU (cytosine deaminase) and 5-fluorouridylic acid (FUMP pyrophosphorylase). Uracil phosphoribosyltransferase, another enzyme in the pyrimidine salvage pathway, is also important in the formation of FUMP (5-fluorouracilmonophosphate), and loss of its activity is sufficient to confer resistance to flucytosine.

Allylamines Although clinical failures can occur during treatment of fungal infections with terbinafine and naftifine, they have not been shown to be due to resistance to these agents. It has been shown that the CDR1 multidrug efflux pump can use terbinafine as a substrate, suggesting that efflux-mediated resistance to allylamines is a possibility.

Clinical Factors Contributing to Resistance Antifungal therapy may fail clinically, despite the fact that the drug employed is active against the infecting fungus. The complex interaction of the host, the drug, and the fungal pathogen may be influenced by a wide variety of factors, including the immune status of the host, the site and severity of the infection, presence of foreign body (e.g., catheter, vascular graft), the activity of the drug at the site of infection, the dose and duration of therapy, and patient compliance with the antifungal regimen. It must be recognized that the presence of neutrophils, use of immunomodulating drugs, concomitant infections (e.g., HIV), surgical procedures, age, and nutritional status of the host all may be more important in determining the outcome of the infection than the ability of the antifungal agent to inhibit or kill the infecting organism.

Antifungal Susceptibility Testing

In vitro susceptibility testing of antifungal agents is designed to determine the relative activity of one or more agents against the infecting pathogen in hopes of selecting the best option for treatment of the infection. Thus antifungal susceptibility tests are performed for the same reasons that tests with antibacterial agents are performed. Antifungal susceptibility tests will (1) provide a reliable estimate of the relative activity of two or more antifungal agents against the tested organism, (2) correlate with in vivo antifungal activity and predict the likely outcome of therapy, (3) provide a means with which to monitor the development of resistance among a normally susceptible population of organisms, and (4) predict the therapeutic potential of newly developed investigational agents. page 712 page 713

Standardized methods for performing antifungal susceptibility testing are reproducible, accurate, and available for use in clinical laboratories. Antifungal susceptibility testing is now increasingly and appropriately used as a routine adjunct to the treatment of fungal infections. Guidelines for the use of antifungal testing as a complement to other laboratory studies have been developed. Selective application of antifungal susceptibility testing, coupled with broader identification of fungi to the species level, is especially useful in difficult to manage fungal infections. One must keep in mind, however, that the in vitro susceptibility of an infecting organism to the antimicrobial agent is only one of several factors that may influence the likelihood that therapy for an infection will be successful. (See previous page.)

Questions

1. What is the mechanism of action of the echinocandin antifungal agents? Why is this an advantage for this class of agents? 2. Describe the mechanisms of resistance to the azoles that are known for Candida albicans. 3. Why is combination therapy with antifungal agents attractive? Give an example of a mechanism that would likely produce synergy.

Bibliography Arikan S, Rex JH: Antifungal agents. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Espinel-Ingroff A, Pfaller MA: Susceptibility test methods: Yeasts and filamentous fungi. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Ghannoum MA, Rice LB: Antifungal agents: Mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 12:501-517, 1999. Johnson MD, et al: Combination antifungal therapy. Antimicrob Agents Chemother 48:693-715, 2004. Rex JH, Pfaller MA: Has antifungal susceptibility come of age? Clin Infect Dis 35:982-989, 2002. White TC: Mechanisms of resistance to antifungal agents. In Murray PR, et al. (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. page 713 page 714 Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Superficial Mycoses Agents of superficial mycoses are fungi that colonize the keratinized outer layers of the skin, hair, and nails. Infections due to these organisms elicit little or no host immune response and are nondestructive and thus asymptomatic. They are usually of cosmetic concern only and are easy to diagnose and treat.

Pityriasis (Tinea) Versicolor Pityriasis versicolor is a common superficial fungal infection that is seen worldwide. In certain tropical environments, it may affect up to 60% of the population. It is caused by the lipophilic yeast Malassezia furfur.

Morphology When viewed in skin scrapings, M. furfur appears as clusters of spherical or oval, thick-walled yeastlike cells, 3 to 8 µm in diameter (Figure 71-1). The yeast cells may be mixed with short, infrequently branched hyphae that tend to orient end to end. The yeastlike cells represent phialoconidia and show polar bud formation with a "lip" or collarette around the point of bud initiation on the parent cell (Figure 71-2). In culture on standard media containing or overlaid with olive oil, M. furfur grows as cream-colored to tan yeastlike colonies composed of budding yeastlike cells; hyphae are infrequently produced.

Epidemiology Pityriasis versicolor is a disease of healthy persons that occurs worldwide, but it is most prevalent in tropical and subtropical regions. Young adults are most commonly affected. M. furfur is not found as a saprophyte in nature, and pityriasis versicolor has not been documented in animals. Human infection is thought to result from the direct or indirect transfer of infected keratinous material from one person to another.

Clinical Syndromes The lesions of pityriasis versicolor are small hypo- or hyperpigmented macules. The upper trunk, arms, chest, shoulders, face, and neck are most often involved, but any part of the body may be affected (Figure 71-3). The lesions are irregular, well-demarcated patches of discoloration that may be raised and covered by a fine scale. Because M. furfur tends to interfere with melanin production, lesions are hypopigmented in dark-skinned individuals. In light-skinned subjects, the lesions are pink to pale brown and become more obvious when they fail to tan after exposure to sunlight. Little or no host reaction occurs, and the lesions are asymptomatic, with the exception of mild pruritus in severe cases. Infection of the hair follicles, resulting in folliculitis, perifolliculitis, and dermal abscesses, is a rare complication of this disease.

Laboratory Diagnosis page 715 page 716

Figure 71-1 Pityriasis versicolor. PAS-stained skin scraping showing yeastlike cells and short, infrequently branched hyphae that are often oriented end to end (×100). (From Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997.)

The laboratory diagnosis of pityriasis versicolor is made by the direct visualization of the fungal elements on microscopic examination of epidermal scales in 10% KOH with or without calcofluor white. The organisms are usually numerous and may also be visualized with H&E or PAS stains (see Figure 71-1). The lesions will also fluoresce with a yellowish color upon exposure to a Wood lamp. Although not usually necessary for establishing the diagnosis, culture may be performed using synthetic mycologic media supplemented with olive oil as a source of lipid. Growth of yeastlike colonies appear following incubation at 30° C for 5 to 7 days. Micr oscopically, the colonies are comprised of budding yeastlike cells with occasional hyphae.

Treatment Although spontaneous cure has been reported, the disease is generally chronic and persistent. Treatment consists of the use of topical azoles or selenium sulfide shampoo. For more widespread infection, oral ketoconazole or itraconazole may be used.

Figure 71-2 Scanning electron micrograph of Malassezia furfur demonstrating the liplike collarette around the point of bud initiation on the parent cell.

Figure 71-3 Pityriasis versicolor. Multiple, pale brown, hyperpigmented patches on chest and shoulders. (From Chandler FW, Watts JC: Pathologic Diagnosis of Fungal Infections. Chicago, ASCP, 1987.)

Tinea Nigra Tinea nigra is a superficial phaeohyphomycosis caused by the black fungus Hortaea werneckii (formerly Exophiala werneckii).

Morphology Microscopically, H. werneckii appears as dematiaceous, frequently branched, septate hyphae, 1.5 to 3.0 µm wide. Arthroconidia and elongate budding cells are also present (Figure 71-4). H. werneckii also grows in culture on standard mycologic media at 25° C, where it is a black mold producing annelloconidia (conidia possessing annelids or rings), which often slide down the sides of the conidiophore.

Epidemiology

Figure 71-4 Tinea nigra. Dematiaceous hyphae of Hortaea werneckii (H&E, ×100). (From Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997.) page 716 page 717

Figure 71-5 Tinea nigra. Darkly pigmented macules with irregular edges present on the palm. (From Chandler FW, Watts JC: Pathologic Diagnosis of Fungal Infections. Chicago, ASCP, 1987.)

Tinea nigra is a tropical or subtropical condition. It is likely contracted by traumatic inoculation of the fungus into the superficial layers of the epidermis. It is most prevalent in Africa, Asia, and Central and South America. Children and young adults are most often affected, with a higher incidence in females.

Clinical Syndromes Tinea nigra appears as a solitary, irregular, pigmented (brown to black) macule, usually on the palms or soles (Figure 71-5). There is no scaling or invasion of hair follicles, and the infection is not contagious. Owing to its superficial location, there is little or no discomfort or host reaction. Because the lesion grossly may resemble a malignant melanoma, biopsy or local excision may be considered. Such invasive procedures may be avoided by a simple microscopic examination of skin scrapings of the affected area.

Laboratory Diagnosis Tinea nigra is easily diagnosed by microscopic examination of skin scrapings placed in 10% to 20% KOH. The pigmented hyphae and yeast forms are confined to the outer layers of the stratum corneum and are easily detected on H&E stained (see Box 69-1) sections (see Figure 71-4). Once fungal elements are detected, skin scrapings should be placed on mycologic media with antibiotics. A dematiaceous yeastlike colony should appear within 3 weeks, becoming velvety with age. Microscopic examination reveals two-celled, cylindrical, yeastlike cells and, depending upon the age of the colony, toruloid hyphae.

Treatment The infection responds well to topical therapy, including Whitfield ointment, azole creams, and terbinafine.

White Piedra

White piedra is a superficial infection of hair caused by yeast-like fungi of the genus Trichosporon: T. inkin, T. asahii, T. beigelii, or T. mucoides.

Morphology

Figure 71-6 These images are not available online due to electronic permissions.

Microscopic examination reveals hyphal elements, arthroconidia (rectangular cells resulting from the fragmentation of hyphal cells), and blastoconidia (budding yeast cells) (Figure 71-6).

Epidemiology This condition occurs in tropical and subtropical regions and is related to poor hygiene.

Clinical Syndromes White piedra affects the hairs of the groin and axillae. The fungus surrounds the hair shaft and forms a white to brown swelling along the hair strand. The swellings are soft and pasty and may be easily removed by running a section of the hair between the thumb and forefinger. The infection does not damage the hair shaft.

Laboratory Diagnosis When microscopic examination reveals hyphal elements, arthroconidia, and/or budding yeast cells, infected hair should be placed on mycologic media without cycloheximide (cycloheximide will inhibit Trichosporon spp.). Trichosporon spp. will form cream-colored, dry, wrinkled colonies within 48 to 72 hours upon incubation at room temperature. The various species of Trichosporon can be identified in the same manner as other yeast isolates. Sugar assimilations, KNO3 assimilation (negative), urease production (positive) and morphology on cornmeal agar (both arthroconidia and blastoconidia are present) should be determined.

Treatment Treatment may be accomplished by the use of topical azoles; however, improved hygiene and shaving of the infected hair are also effective and usually negate the necessity of medical treatment.

Black Piedra Another condition affecting the hair, primarily the scalp, is black piedra. The causative agent of black piedra is Piedraia hortae. page 717 page 718

Morphology The organism grows as pigmented (brown to reddish-black) mold. As the culture ages, asci-containing, spindle-shaped ascospores are formed within specialized structures. These structures (asci and ascospores) are also produced within the rock-hard hyphal mass that surrounds the hair shaft.

Epidemiology Black piedra is uncommon and has been reported from tropical areas in Latin America and Central Africa. It is thought to be a condition of poor hygiene.

Clinical Syndromes Black piedra presents as small, dark nodules that surround the hair shafts. It is asymptomatic and generally involves the scalp. The hyphal mass is held together by a cement-like substance and contains asci and ascospores, the sexual phase of the fungus.

Laboratory Diagnosis

Examination of the nodule reveals branched, pigmented, hyphae held together by a cement-like substance. P. hortae can be cultured on routine mycologic media. Very slow growth may be observed at 25° C and may begin as a yeast-like colony, later becoming velvety as hyphae develop. Asci may be observed microscopically, usually ranging from 4 to 30 µm and containing up to 8 ascospores.

Treatment Treatment of black piedra is easily accomplished by a haircut and proper, regular washings. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Cutaneous Mycoses The cutaneous mycoses include infections caused by dermatophytic fungi (dermatophytosis) and nondermatophytic fungi (dermatomycosis) (Table 71-1). Owing to the overwhelming importance of dermatophytes as etiologic agents of cutaneous mycoses, the majority of this section will deal with those fungi. The nondermatophytic fungi will be discussed regarding their role in onychomycosis. The superficial and cutaneous infections caused by Candida spp. will be discussed in Chapter 74.

Dermatophytoses (Clinical Cases 71-1 and 71-2)

The term dermatophytosis refers to a complex of diseases caused by any of several species of taxonomically related filamentous fungi in the genera Trichophyton, Epidermophyton, and Microsporum (Tables 71-1 through 71-3). These fungi are known collectively as the dermatophytes, and all possess the ability to cause disease in humans and/or animals. All have in common the ability to invade the skin, hair, or nails. In each case, these fungi are keratinophilic and keratinolytic and so are able to break down the keratin surfaces of these structures. In the case of skin infections, the dermatophytes invade only the upper, outermost layer of the epidermis, the stratum corneum. Penetration below the granular layer of the epidermis is rare. Likewise with hair and nails, being part of the skin, only the keratinized layers are invaded. The various forms of dermatophytosis are referred to as "tineas" or ringworm. Clinically the tineas are classified according to the anatomic site or structure affected: (1) tinea capitis of the scalp, eyebrows and eyelashes; (2) tinea barbae of the beard; (3) tinea corporis of the smooth or glabrous skin; (4) tinea cruris of the groin; (5) tinea pedis of the foot; (6) tinea unguium of the nails (also known as onychomycosis). The clinical signs and symptoms of dermatophytosis vary according to the etiologic agents, the host reaction, and the site of infection.

Morphology Each genus of dermatophytic mold is characterized by a specific pattern of growth in culture and by the production of macroconidia and microconidia (see Table 71-2). Further identification to species level requires consideration of colony morphology, spore production, and nutritional requirements in vitro.

Microscopically, the genus Microsporum is identified by observation of its macroconidia, whereas microconidia are the characteristic structures of the genus Trichophyton (see Table 71-2). Epidermophyton floccosum does not produce microconidia, but its smooth-walled macroconidia borne in clusters of two or three are quite distinctive (Figure 71-7). Microsporum canis produces characteristic large, multicellular (5 to 8 cells per conidium), thick- and rough-walled macroconidia (Figure 71-8). Trichophyton rubrum produces microconidia that are teardrop or peg shaped and borne along the sides of hyphae (Figure 71-9), whereas T. mentagrophytes produces both single, cigar-shaped macroconidia and grapelike clusters of spherical microconidia (Figure 71-10). T. tonsurans produces variably sized and shaped microconidia, with relatively large spherical conidia often located right alongside small, parallel-walled conidia and other microconidia of various sizes and shapes (Figure 71-11). page 718 page 719

Table 71-1. Common and Uncommon Agents of Superficial and Cutaneous Dermatomycoses and Dermatophytoses Fungus

Type of Infection TP TCO TCR TCA TBA TVR O TN BP WP

Dermatophytic Trichophyton rubrum

X X

X

T. mentagrophytes

X X

X

X X

X X

T. tonsurans

X

X

T. verrucosum

X

X

T. equinum

X

T. violaceum

X

T. schoenleinii

X

X

T. megnini Epidermophyton floccosum

X X

Microsporum canis

X X

X X

M. audouinii

X

Nondermatophytic Scopulariopsis brevicaulis Scytalidium spp.

X X

X

Malassezia spp. Candida albicans

X X

X

X

Aspergillus terreus

X

Acremonium spp.

X

Fusarium spp.

X

Trichosporon spp.

X

Piedraia hortae Hortaea werneckii

X X

TP, tinea pedis; TCO, tinea corporis; TCR, tinea cruris; TCA, tinea capitis; TBA, tinea barbae; TVR, tinea versicolor; O, onychomycosis; TN, tinea nigra; BP, black piedra; WP, white piedra; X, etiologic agents of.

In skin biopsies, all of the dermatophytes are morphologically similar and appear as hyaline septate hyphae, chains of arthroconidia, or dissociated chains of arthroconidia that invade the stratum corneum, hair follicles, and hairs. When the hair is infected, the pattern of fungal invasion can be either ectothrix, endothrix, or favic depending on the dermatophytic species (Figure 71-12). Septate hyphae may be seen within the hair shaft in all three patterns. In the ectothrix pattern, arthroconidia are formed on the outside of the hair (Figure 71-13; see Figure 71-12); in the endothrix pattern, arthroconidia are formed inside the hair (see Figure 71-12); and in the favic pattern, hyphae, arthroconidia, and empty spaces resembling air bubbles ("honeycomb" pattern) are formed inside the hair (see Figure 71-12). The dermatophytes can usually be seen on H&E stain; however, they are best visualized with special stains for fungi, such as GMS and PAS (see Figure 71-13 and Chapter 69).

Ecology and Epidemiology page 719 page 720

Clinical Case 71-1. Dermatophytosis in an Immunocompromised Host Squeo, et al. (J Am Acad Dermatol 39:379, 1998) describe a case of a 55-year-old renal transplant recipient with onychomycosis and chronic tinea pedis who presented with tender nodules on his left medial heel. He then developed papules and nodules on his right foot and calf. A skin biopsy demonstrated periodic acid-Schiff (PAS) positive, thick-walled, round cells, 2 to 6 microns in diameter in the dermis. Skin biopsy culture grew Trichophyton rubrum. T. rubrum has been described as an invasive pathogen in immunocompromised hosts. The clinical presentation, histopathology, and early fungal culture growth suggested Blastomyces dermatitidis in the differential diagnosis before the final identification of T. rubrum.

Dermatophytes can be classified into three different categories based on their natural habitat (see Table 71-3): (1) geophilic, (2) zoophilic, and (3) anthropophilic. The geophilic dermatophytes live in the soil and are occasional pathogens of both animals and humans. Zoophilic dermatophytes normally parasitize the hair and skin of animals but can be transmitted to humans. Anthropophilic dermatophytes generally infect humans and may be transmitted directly or indirectly from person to person. This classification is quite useful prognostically and emphasizes the importance of identifying the etiologic agent of dermatophytoses. Species of dermatophytes that are considered anthropophilic tend to cause chronic, relatively noninflammatory infections that are difficult to cure. In contrast, the zoophilic and geophilic dermatophytes tend to elicit a profound host reaction, causing lesions that are highly inflammatory and respond well to therapy. In some instances, these infections may heal spontaneously.

Table 71-2. Characteristic In Vitro and In Vivo Features of Dermatophytes Genus

In vitro In vivo hair Macroconidia Microconidia Invasion Fluorescencea

Epidermophyton Smooth Absent walled, borne in clusters of two or three

NA

NA

Microsporum

Numerous, Rare large, thick and roughwalledb

Ectothrix

+/-c

Trichophyton

Rare, smooth, thin-walled

Numerous, Endothrixe +/-f spherical, teardrop orpegshapedd

a

Fluorescence with a Wood lamp. Except M. audouinii. c M. gypseum not fluorescent. d Except T. schoenleinii. e T. verrucosum, ectothrix; T. schoenleinii, favic. f T. schoenleinii is fluorescent. NA, not applicable. b

Clinical Case 71-2. Tinea Capitis in an Adult Woman Martin and Elewski (J Am Acad Dermatol 49:S177, 2003) describe an 87-year-old woman with a 2-year history of a pruritic, painful, scaling scalp eruption and hair loss. Her previous treatment for this condition included numerous courses of systemic antibiotics and prednisone without success. Of interest in her social history was that she had recently acquired several stray cats that she kept inside her home. On physical exam, there were numerous pustules throughout the scalp, with diffuse erythema, crusting, and scale extending to the neck. There was extremely sparse scalp hair and prominent posterior cervical lymphadenopathy. She had no nail pitting. A Wood light examination of the scalp produced negative findings. A skin biopsy specimen and fungal, bacterial, and viral cultures were obtained. Bacterial culture grew rare Enterococcus species, whereas viral cultures showed no growth. The scalp biopsy specimen revealed an endothrix dermatophyte infection. Fungal culture grew Trichophyton tonsurans. The patient was treated with griseofulvin and Selsun shampoo. When seen at a 2-week follow-up visit, the patient demonstrated new hair growth and a resolution of her pustular eruption. With the brisk clinical response and culture growth of T. tonsurans, treatment with griseofulvin was continued for 8 weeks. The scalp hair grew back normally without permanent alopecia. Adults with alopecia require an evaluation for tinea capitis, including fungal cultures.

The dermatophytes are worldwide in distribution (see Table 71-3), and infection may be acquired from the transfer of arthroconidia or hyphae, or keratinous material containing these elements, from an infected host to a susceptible, uninfected host. Dermatophytes may remain viable in desquamated skin scales or hair for long periods, and infection may be either by direct contact or indirect via fomites. Individuals of both sexes and all ages are susceptible to dermatophytosis; however, tinea capitis is more common in prepubescent children, and tinea cruris and tinea pedis are primarily diseases of adult males. Although dermatophytoses occur worldwide, especially in tropical and subtropical regions, individual dermatophyte species may vary in their geographic distribution and in their virulence for humans (see Table 71-3). For example, Trichophyton concentricum, the cause of tinea imbricata, is confined to the islands of the South Pacific and Asia, whereas T. tonsurans has replaced Microsporum audouinii as the principal agent of tinea capitis in the United States. Infections due to dermatophytes are generally endemic but may assume epidemic proportions in selected settings (e.g., tinea capitis in school children). On a worldwide scale, T. rubrum and T. mentagrophytes account for 80% to 90% of all dermatophytoses.

Clinical Syndromes Dermatophytoses manifest a wide range of clinical presentations, which may be affected by factors such as the species of dermatophytes, the inoculum size, the site of infection, and the immune status of the host. Any given disease manifestation may result from several different species of dermatophytes, as shown in Table 71-1. page 720 page 721

Table 71-3. Classification of Dermatophytes According to Ecologic Niche Ecologic Species Principal Geographic Prevalence Niche Hosts Distribution Anthropophilic Epidermophyton Worldwide Common floccosum Microsporum audouinii

Worldwide

Common

M. ferrugineum

Africa, Asia Endemic

Trichophyton concentricum

Asia, Pacific Islands

Endemic

T. megnini

Europe, Africa

Endemic

T. mentagrophytes var. interdigitale

Worldwide

Common

T. rubrum

Worldwide

Common

T. schoenleinii

Europe, Africa

Endemic

T. soudanese

Africa

Endemic

T. tonsurans

Worldwide

Common

T. violaceum

Europe, Common Africa, Asia

Zoophilic

Microsporum canis

Cat, dog, Worldwide horse

Common

M. gallinae

Fowl

Worldwide

Rare

M. nanum

Swine

Worldwide

Rare

M. persicolor

Vole

Europe, USA

Rare

Trichophyton equinum

Horse

Worldwide

Rare

Worldwide

Common

T. Rodent mentagrophytes var. mentagrophytes var. erinacei

Hedgehog Europe, New Zealand, Africa

Occasional

var. quinckeanum

Mouse

Worldwide

Rare

T. sinii

Monkey

India

Occasional

T. verrucosum

Cow

Worldwide

Common

Microsporum gypseum

Worldwide

Occasional

M. fulvum

Worldwide

Occasional

From Hiruma M, Yamaguchi H: Dermatophytes. In Anaissie EJ, McGinnis MR, Pfaller MA (eds): Clinical Mycology. New York, Churchill Livingstone, 2003. page 721 page 722

Figure 71-7 Epidermophyton floccosum. Lactophenol cotton blue showing smooth-walled macroconidia.

Figure 71-8 Microsporum canis. Lactophenol cotton blue showing rough-walled macroconidia (black arrow) and microconidia (red arrow).

Figure 71-9 Trichophyton rubrum. Lactophenol cotton blue showing multicelled macroconidia (black arrow) and teardrop- and peg-shaped microconidia (red arrow).

Figure 71-10 Trichophyton mentagrophytes. Lactophenol cotton blue showing cigar-shaped macroconidia (black arrow) and grapelike clusters of microconidia (red arrow).

The classic pattern of dermatophytosis is the "ringworm" pattern of a ring of inflammatory scaling with diminution of inflammation toward the center of the lesion. Tineas of hair-bearing areas often present as raised, circular or ring-shaped patches of alopecia with erythema and scaling (Figure 71-14) or as more diffusely scattered papules, pustules, vesicles, and kerions (severe inflammation involving the hair shaft) (Figure 71-15). Hairs infected with certain species, such as M. canis, M. audouinii, and T. schoenleinii, often fluoresce yellow-green when exposed to a Wood light (see Table 71-2). Infections of smooth skin commonly present as erythematous and scaling patches that expand in a centripetal pattern with central clearing. Dermatophytoses of the foot and hand may often become complicated by onychomycosis (Figure 71-16), in which the nail plate is invaded and destroyed by the fungus. Onychomycosis (tinea unguium) is caused by a variety of dermatophytes (see Table 71-1) and is estimated to affect approximately 3% of the population in most temperate countries. It is a disease seen mostly in adults, with toenails affected more commonly than fingernails. The infection is usually chronic, and the nails become thickened, discolored, raised, friable, and deformed (see Figure 71-16). T. rubrum is the most common etiologic agent in most countries. A rapidly progressive form of onychomycosis that originates from the proximal nailfold and involves the upper and underside of the nail is seen in AIDS patients.

Laboratory Diagnosis The laboratory diagnosis of dermatophytoses relies on the demonstration of fungal hyphae by direct microscopy of skin, hair, or nail samples and the isolation of organisms in culture. Specimens are mounted in a drop of 10% to 20% KOH on a glass slide and examined microscopically. Filamentous, hyaline hyphal elements characteristic of dermatophytes may be seen in skin scrapings, nail scrapings, and hairs. In examining specimens for fungal elements, calcofluor white has been used with excellent results.

Figure 71-11 Trichophyton tonsurans. Lactophenol cotton blue showing microconidia (black arrow). page 722 page 723

Figure 71-12 Schematic of A, Ectothrix hair infection. B, Endothrix hair infection. C, Favic hair infection.

Cultures are always useful and can be obtained by scraping the affected areas and placing the skin, hair, or nail clippings onto standard mycologic media such as Sabouraud agar, with and without antibiotics, or dermatophyte test medium. Colonies develop within 7 to 28 days. Their gross and microscopic appearance and nutritional requirements can be used in identification.

Treatment

Figure 71-13 Arthroconidia surrounding a hair shaft. Ectothrix hair infection caused by M. canis (GMS-H&E, ×160). (From Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997.)

Figure 71-14 Tinea capitis due to M. canis. (From Hay RJ: Cutaneous and subcutaneous mycoses. In Anaissie EJ, McGinnis MR, Pfaller MA [eds]: Clinical Mycology. New York, Churchill Livingstone, 2003.)

Figure 71-15 Tinea barbae caused by T. verrucosum. (From Chandler FW, Watts JC: Pathologic Diagnosis of Fungal Infections. Chicago, ASCP, 1987.) page 723 page 724

Figure 71-16 Onychomycosis caused by T. rubrum. (From Hay RJ: Cutaneous and subcutaneous mycoses. In Anaissie EJ, McGinnis MR, Pfaller MA [eds]: Clinical Mycology. New York, Churchill Livingstone, 2003.)

Dermatophytic infections that are localized and that do not affect hair or nails can usually be treated effectively with topical agents; all others require oral therapy. Topical agents include azoles (miconazole, clotrimazole, econazole, tioconazole, and itraconazole), terbinafine, and haloprogin. Whitfield ointment (benzoic and salicylic acids) is an optional agent for dermatophytosis, but responses are usually slower than those seen with agents with specific antifungal activity. Oral antifungal agents with systemic activity against dermatophytes include griseofulvin, itraconazole, fluconazole, and terbinafine. The azoles and terbinafine are more rapidly and broadly efficacious than griseofulvin, especially for the treatment of onychomycosis.

Onychomycosis Caused by Nondermatophytic Fungi A number of nondermatophytic molds, as well as Candida species, have been associated with nail infections (see Table 71-1). These organisms include Scopulariopsis brevicaulis, Scytalidium dimidiatum, S. hyalinum, and a variety of others, including Aspergillus, Fusarium, and Candida species. Among these organisms, S. brevicaulis and Scytalidium spp. are proven nail pathogens. The other fungi certainly may be the cause of nail pathology; however, the interpretation of nail cultures with these organisms should be done with caution, as they may simply represent saprophytic colonization of abnormal nail material. Criteria used to determine an etiologic role for these fungi include isolation on multiple occasions and the presence of abnormal hyphal or conidial structures on microscopic examination of nail material. Infections due to S. brevicaulis, S. dimidiatum, and S. hyalinum are notoriously difficult to treat, because they are not usually susceptible to any antifungals. Partial surgical removal of infected nails, coupled with oral itraconazole or terbinafine or intensive treatment with 5% amorolfine nail lacquer or Whitfield ointment, may be useful in achieving a clinical response.

Case Study and Questions

Darrell, a 24-year-old medical student, just loves his new bulldog puppy, Delbert. He recently purchased Delbert from a local "backyard" breeder. Darrell has taken to giving Delbert frequent "smooches" on his muzzle, which Delbert loves, because he knows a treat is soon to follow. After about 3 months of proud puppy ownership and "smooching," Darrell noticed that his mustache began itching, and his upper lip was beginning to swell. Over a 1-week period, his upper lip became swollen and inflamed, and small pustular areas became apparent among the sparse hairs of his moustache. Similar changes were also becoming apparent on Delbert's muzzle. This concerned Darrell, so he promptly took Delbert to the vet. The vet took one look at the pair, wrote a prescription for Delbert, and told Darrell that he should make a visit to the dermatologist. 1. What was the likely cause of Darrell/Delbert's affliction? Be specific. 2. How would you go about making a diagnosis? 3. How would you go about treating this infection? 4. Who gave what to whom?

Bibliography Chandler FW, Watts JC: Pathologic Diagnosis of Fungal Infections. Chicago, ASCP, 1987. Hay RJ: Cutaneous and subcutaneous mycoses. In Anaissie EJ, McGinnis MR, Pfaller MA (eds): Clinical Mycology. New York, Churchill Livingstone, 2003. Hiruma M, Yamaguchi H: Dermatophytes. In Anaissie EJ, McGinnis MR, Pfaller MA (eds): Clinical Mycology. New York, Churchill Livingstone, 2003. Summerbell RC, et al: Trichophyton, Microsporum, Epidermophyton, and other agents of superficial mycoses. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Lymphocutaneous Sporotrichosis (Clinical Case 72-1) Lymphocutaneous sporotrichosis is caused by Sporothrix schenckii, a dimorphic fungus that is ubiquitous in soil and decaying vegetation. Infection with this organism is chronic and is characterized by nodular and ulcerative lesions that develop along lymphatics that drain the primary site of inoculation (Figure 72-1). Dissemination to the other sites such as bones, eyes, lungs, and central nervous system is extremely rare ( Native American > Hispanic > Asian

Serum CF antibody titer > 1:32 Pregnancy

Late pregnancy and postpartum

Skin test

Negative

Depressed Malignancy, chemotherapy, steroid cell-mediated immunity treatment, HIV infection

From Mitchell TG: Systemic fungi. In Cohen J, Powderly WG (eds): Infectious Diseases, 2nd ed. St Louis, Mosby, 2004.

Primary disease usually resolves without therapy and confers a strong, specific immunity to reinfection, which is detected by the coccidioidin skin test. In patients symptomatic for 6 weeks or longer, the disease progresses to secondary coccidioidomycosis, which may include nodules, cavitary disease, or progressive pulmonary disease (5% of cases); single or multisystem dissemination follows in approximately 1% of this population. Extrapulmonary sites of infection include skin, soft tissues, bones, joints, and meninges. Persons in certain ethnic groups (e.g., Filipino, African American, Native American, and Hispanic) run the highest risk of dissemination, with meningeal involvement a common sequela (Table 73-3). In addition to ethnicity, males (9:1), women in the third trimester of pregnancy, individuals with a cellular immunodeficiency (including AIDS, organ transplantation recipients, and those treated with tumor necrosis factor [TNF] antagonists), and persons at the extremes of age are at high risk for disseminated disease (see Table 73-3). The mortality in disseminated disease exceeds 90% without treatment, and chronic infection is common.

Laboratory Diagnosis The diagnosis of coccidioidomycosis includes the use of histopathologic examination of tissue or other clinical material, isolation of the fungus in the culture, and serologic testing (see Table 73-2). Direct microscopic visualization of endosporulating spherules in sputum, exudates, or tissue is sufficient to establish the diagnosis (see Figure 73-8) and is preferred over culture because of the highly infectious nature of the mold when grown in culture. Clinical exudates should be examined directly in 10% to 20% KOH with calcofluor white, and tissue from biopsy can be stained with H&E or specific fungal stains such as GMS or PAS (see Figure 73-8).

Clinical specimens may be cultured on routine mycologic media at 25° C. Colonies of C. immitis develop within 3 to 5 days, and typical sporulation may be seen in 5 to 10 days. Owing to the highly infectious nature of the fungus, all plates or tubes should be sealed using gas-permeable tape (plates) or screw caps (tubes) and only examined within a suitable biosafety cabinet. The identification of C. immitis from culture may be accomplished by using the exoantigen immunodiffusion test or nucleic acid hybridization. Conversion of the mold into spherules in vitro is not usually attempted outside of a research setting. page 742 page 743

Several serologic procedures exist for initial screening, confirmation, or prognostic evaluation (see Table 73-2). For initial diagnosis, the combined use of the immunodiffusion (ID) test and the latex particle agglutination (LP) test detects approximately 93% of cases. The complement fixation (CF) and tube precipitin (TP) tests may also be used for diagnosis, as well as prognosis. Prognostic studies frequently employ serial CF titers; rising titers are a bad prognostic sign, and falling titers indicate improvement. A test to detect antigen in urine is commercially available, but it is unclear what role it will play in diagnosis.

Treatment Most individuals with primary coccidioidomycosis do not require specific antifungal therapy. For those with concurrent risk factors (see Table 73-3), such as organ transplant, HIV infection, high doses of corticosteroids, or when there is evidence of unusually severe infection, treatment is necessary. Primary coccidioidomycosis in the third trimester of pregnancy or during the immediate postpartum period requires treatment with amphotericin B.

Immunocompromised patients or others with diffuse pneumonia should be treated with amphotericin B followed by an azole (either fluconazole or itraconazole) as maintenance therapy. The total length of therapy should be at least 1 year. Immunocompromised patients should be maintained on an oral azole as secondary prophylaxis. Chronic cavitary pneumonia should be treated with an oral azole for at least 1 year. In cases where the response is suboptimal, the alternatives are to switch to another azole (e.g., from itraconazole to fluconazole), increase the dose of the azole in the case of fluconazole, or switch to amphotericin B. Surgical treatment is required in the event of rupture of a cavity into the pleural space, hemoptysis, or for localized refractory lesions. The treatment of nonmeningeal extrapulmonary disseminated infections is based on oral azole therapy with either fluconazole or itraconazole. In the case of vertebral involvement or inadequate clinical response, treatment with amphotericin B is recommended, along with appropriate surgical debridement and stabilization. Meningeal coccidioidomycosis is managed with the administration of fluconazole or itraconazole (secondary choice because of poor CNS penetration) indefinitely. Intrathecal administration of amphotericin B is recommended only in the event of failure of azole therapy, owing to its toxicity when administered by this route. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Histoplasmosis (Clinical Case 73-3) Clinical Case 73-3. Disseminated Histoplasmosis

Mariani and Morris (Infect Med 24(Suppl 8):17-19, 2007) describe a case of disseminated histoplasmosis in a patient with AIDS. The patient was a 42-year-old El Salvadoran woman who was admitted to the hospital for evaluation of progressive dermatosis involving the right nostril, cheek, and lip, despite antibiotic therapy. She was HIV-positive (CD4 lymphocyte count 21/microliter) and had lived in Miami for the past 18 years. The lesion first appeared on the right nostril 3 months before admission. The patient sought medical attention and was treated unsuccessfully with oral antibiotics. Over the following 2 months, the lesion increased in size, involving the right nares and malar region, and was accompanied by fever, malaise, and a 50-pound weight loss. A necrotic area developed on the superior aspect of the right nostril, extending to the upper lip. A presumptive diagnosis of leishmaniasis was entertained, based in part on the patient's country of origin and a possible exposure to a sandfly bite. Laboratory studies revealed anemia and lymphopenia. A chest x-ray was normal, and a CT scan of the head showed a soft-tissue mass in the right nasal cavity. Histopathologic evaluation of a skin biopsy showed chronic inflammation, with intracytoplasmic budding yeasts. Culture of the biopsy grew Histoplasma capsulatum, and results of a urine Histoplasma antigen test were positive. The patient was treated with amphotericin B followed by itraconazole with good results.

This case underscores the ability of H. capsulatum to remain clinically latent for many years, only to reactivate upon immunosuppression of the host. Cutaneous manifestations of histoplasmosis are usually a consequence of progression from primary (latent) to disseminated disease. Histoplasmosis is not endemic to South Florida but is endemic to much of Latin America, where the patient had lived prior to moving to Miami. A high index of suspicion and confirmation with skin biopsies, cultures, and testing for urinary antigen are crucial for timely and appropriate treatment of disseminated histoplasmosis.

Histoplasmosis is caused by two varieties of Histoplasma capsulatum: H. capsulatum var. capsulatum and H. capsulatum var. duboisii (see Table 73-1). H. capsulatum var. capsulatum causes pulmonary and disseminated infections in the eastern half of the United States and most of Latin America, whereas H. capsulatum var. duboisii causes predominately skin and bone lesions and is restricted to the tropical areas of Africa (see Figure 73-2).

Morphology page 743 page 744

Figure 73-9 Histoplasma capsulatum mold phase showing tuberculate macroconidia.

Both varieties of H. capsulatum are thermally dimorphic fungi existing as a hyaline mold in nature and in culture at 25° C and as an intracellular budding yeast in tissue and in culture at 37° C (Figures 73-9, 73-10, and 73-11; see Table 73-2). In culture, the mold forms of H. capsulatum var. capsulatum and var. duboisii are indistinguishable macroscopically and microscopically. The mold colonies grow slowly and develop as white or brown hyphal colonies after several days to a week. The mold form produces two types of conidia: (1) large (8 to 15 µm), thick-walled, spherical macroconidia with spikelike projections (tuberculate macroconidia) that arise from short conidiophores (see Figures 73-1 and 73-12) and (2) small, oval microconidia (2 to 4 µm) with smooth or slightly rough walls that are sessile or on short stalks (see Figures 73-1 and 73-12). The yeast cells are thin-walled, oval, and measure 2 to 4 µm (var. capsulatum) (see Figure 73-10) or thicker-walled and 8 to 15 µm (var. duboisii) (see Figure 73-11). The yeast cells of both varieties of H. capsulatum are intracellular in vivo and are uninucleated (see Figures 73-10 and 73-11).

Epidemiology

Histoplasmosis capsulati is localized to the broad regions of the Ohio and Mississippi River valleys in the United States and occurs throughout Mexico and Central and South America (see Figure 73-2 and Table 73-1). Histoplasmosis duboisii, or African histoplasmosis, is confined to the tropical areas of Africa, including Gabon, Uganda, and Kenya (see Figure 73-2 and Table 73-1).

Figure 73-10 Giemsa-stained preparation showing intracellular yeast forms of Histoplasma capsulatum var. capsulatum.

Figure 73-11 H&E-stained tissue section showing intracellular yeast forms of Histoplasma capsulatum var. duboisii. (From Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997.)

The natural habitat of the mycelial form of both varieties of H. capsulatum is soil with a high nitrogen content, such as that found in areas contaminated with bird or bat droppings. Outbreaks of histoplasmosis have been associated with exposure to bird roosts, caves, and decaying buildings or urban renewal projects involving excavation and demolition. Aerosolization of microconidia and hyphal fragments in the disturbed soil, with subsequent inhalation by exposed individuals, is considered to be the basis for these outbreaks (Figure 73-12). Although attack rates may reach 100% in certain of these exposures, most cases remain asymptomatic and are detected only by skin testing. Immunocompromised individuals and children are more prone to develop symptomatic disease with either variety of Histoplasma. Reactivation of the disease and dissemination is common among immunosuppressed individuals, especially those with AIDS.

Clinical Syndromes

Figure 73-12 Natural history of the mold (saprobic) and yeast (parasitic) cycle of Histoplasma capsulatum. page 744 page 745

The usual route of infection for both varieties of histoplasmosis is via inhalation of microconidia, which in turn germinate into yeasts within the lung and may remain localized or disseminate hematogenously or by the lymphatic system (see Figure 73-12). The microconidia are rapidly phagocytosed by pulmonary macrophages and neutrophils, and it is thought that conversion to the parasitic yeast form takes place intracellularly.

Histoplasmosis Capsulati

The clinical presentation of histoplasmosis caused by H. capsulatum var. capsulatum is dependent upon the intensity of exposure and immunologic status of the host. Asymptomatic infection occurs in 90% of individuals following a low-intensity exposure. In the event of an exposure to a heavy inoculum, however, most individuals exhibit some symptoms. The self-limited form of acute pulmonary histoplasmosis is marked by a flulike illness with fever, chills, headache, cough, myalgias, and chest pain. Radiographic evidence of hilar or mediastinal adenopathy and patchy pulmonary infiltrates may be seen. Most acute infections resolve with supportive care and do not require specific antifungal treatment. In rare instances, usually following very heavy exposure, acute respiratory distress syndrome may be seen. In approximately 10% of patients, inflammatory sequelae such as persistent lymphadenopathy with bronchial obstruction, arthritis, arthralgias, or pericarditis may be seen. Another rare complication of histoplasmosis is a condition known as mediastinal fibrosis, in which persistent host response to the organism may result in massive fibrosis and constriction of mediastinal structures, including the heart and great vessels. Progressive pulmonary histoplasmosis may follow acute infection in approximately 1 in 100,000 cases per year. Chronic pulmonary symptoms are associated with apical cavities and fibrosis and are more likely to occur in patients with prior underlying pulmonary disease. These lesions generally do not heal spontaneously, and persistence of the organism leads to progressive destruction and fibrosis secondary to the immune response to the organism. Disseminated histoplasmosis follows acute infection in 1 in 2000 adults and is much higher in children and immunocompromised adults. Disseminated disease may assume a chronic, subacute, or acute course. Chronic disseminated histoplasmosis is characterized by weight loss and fatigue, with or without fever. Oral ulcers and hepatosplenomegaly are common.

Subacute disseminated histoplasmosis is marked by fever, weight loss, and malaise. Oropharyngeal ulcers and hepatosplenomegaly are prominent. Bone marrow involvement may produce anemia, leukopenia, and thrombocytopenia. Other sites of involvement include the adrenals, cardiac valves, and the central nervous system. Untreated subacute disseminated histoplasmosis will result in death in 2 to 24 months. Acute disseminated histoplasmosis is a fulminant process that is most commonly seen in severely immunosuppressed individuals, including those with AIDS, organ transplant recipients, and those receiving steroids or other immunosuppressive chemotherapy. In addition, children younger than 1 year and adults with debilitating medical conditions are also at risk, given sufficient exposure to the fungus. In contrast to the other forms of histoplasmosis, acute disseminated disease may present with a septic shock-like picture, with fever, hypotension, pulmonary infiltrates, and acute respiratory distress. Oral and gastrointestinal ulcerations and bleeding, adrenal insufficiency, meningitis, and endocarditis may also be seen. If untreated, acute disseminated histoplasmosis is fatal within days to weeks.

Histoplasmosis Duboisii In contrast to classic histoplasmosis, pulmonary lesions are uncommon in African histoplasmosis. The localized form of histoplasmosis duboisii is a chronic disease characterized by regional lymphadenopathy, with lesions of skin and bone. Skin lesions are papular or nodular and eventually progress to abscesses, which then ulcerate. About one third of patients will exhibit osseous lesions characterized by osteolysis and involvement of contiguous joints. The cranium, sternum, ribs, vertebrae, and long bones are most frequently involved, often with overlying abscesses and draining sinuses. A more fulminant disseminated form of histoplasmosis duboisii may be seen in profoundly immunodeficient individuals. Hematogenous and lymphatic dissemination to bone marrow, liver, spleen, and other organs occurs and is marked by fever, lymphadenopathy, anemia, weight loss, and organomegaly. This form of the disease is uniformly fatal unless promptly diagnosed and treated.

Laboratory Diagnosis

Table 73-4. Laboratory Tests for Histoplasmosis Test Antigen

Sensitivity (% True Positives) in Disease States Disseminated Chronic Pulmonary Self-limited* 92 21 39

Culture

85

85

15

Histopathology 43

17

9

Serology

100

98

71

*Includes acute pulmonary histoplasmosis, rheumatologic syndrome, and pericarditis. From Wheat LJ: Endemic mycoses. In Cohen J, Powderly WG (eds): Infectious Diseases, 2nd ed. St Louis, Mosby, 2004. page 745 page 746

The diagnosis of histoplasmosis may be made by direct microscopy, culture of blood, bone marrow, or other clinical material, and by serology, including antigen detection in blood and urine (Table 73-4; see Table 73-2). The yeast phase of the organism can be detected in sputum, bronchoalveolar lavage fluid, peripheral blood films, bone marrow, and tissue stained with Giemsa, GMS, or PAS stains (see Figure 73-10). In tissue sections, cells of H. capsulatum var. capsulatum are yeastlike, hyaline, spherical to oval, 2 to 4 µm in diameter, uninucleate, and have single buds attached by a narrow base. The cells are usually intracellular and clustered together. The cells of H. capsulatum var. duboisii are also intracellular, yeastlike, and uninucleate but are much larger (8 to 15 µm) and have thick "doubly-contoured" walls. They are usually in macrophages and giant cells (see Figure 73-11).

Because of the high organism burden in patients with disseminated disease, cultures of respiratory specimens, blood, bone marrow, and tissue are of value. They are less useful in self-limited or localized disease (see Table 73-4). Growth of the mycelial form in culture is slow, and once isolated, the identification must be confirmed by conversion to the yeast phase or by use of exoantigen testing or nucleic acid hybridization. As with the other dimorphic pathogens, cultures of Histoplasma must be handled with care in a biosafety cabinet. Serologic diagnosis of histoplasmosis employs tests for both antigen and antibody detection (see Table 73-2). Antibody detection assays include a complement fixation (CF) assay and immunodiffusion (ID) test. These tests are usually used together to maximize sensitivity and specificity, but neither is useful in the acute setting; CF and ID are often negative in immunocompromised patients with disseminated infection. Detection of Histoplasma antigen in serum and urine by enzyme immunoassay has become very useful, particularly in diagnosing disseminated disease (see Tables 73-2 and 73-4). The sensitivity of antigen detection is greater in urine specimens than in blood and ranges from 21% in chronic pulmonary disease to 92% in disseminated disease. Serial measurements of antigen may be used to assess response to therapy and for establishing relapse of the disease.

Treatment Since most patients with histoplasmosis recover without therapy, the first decision must be whether specific antifungal therapy is necessary or not. Some immunocompetent patients with more severe infection may exhibit prolonged symptoms and may benefit from treatment with itraconazole. In cases of severe acute pulmonary histoplasmosis with hypoxemia and acute respiratory distress syndrome, amphotericin B should be administered acutely, followed by oral itraconazole to complete a 12-week course.

Chronic pulmonary histoplasmosis also warrants treatment, since it is known to progress if left untreated. Treatment with amphotericin B followed by itraconazole for 12 to 24 months is recommended. Disseminated histoplasmosis usually responds well to amphotericin B therapy. Once stabilized, the patient may be switched to oral itraconazole to be administered over 6 to 18 months. Patients with AIDS may require lifelong therapy with itraconazole. Histoplasmosis of the central nervous system is universally fatal if not treated. The therapy of choice is amphotericin B followed by fluconazole for 9 to 12 months. Patients with severe obstructive mediastinal histoplasmosis require amphotericin B therapy. Itraconazole may be used for outpatient therapy. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Paracoccidioidomycosis Paracoccidioidomycosis is a systemic fungal infection caused by the dimorphic pathogen Paracoccidioides brasiliensis. This infection is also known as South American blastomycosis and is the major dimorphic endemic fungal infection in Latin American countries. Primary paracoccidioidomycosis usually presents in young people as a self-limited pulmonary process. At this stage, it rarely displays a progressive acute or subacute course. Reactivation of a primary quiescent lesion may occur years later, resulting in chronic progressive pulmonary disease with or without involvement of other organs.

Morphology

The mold phase of P. brasiliensis grows slowly in vitro at 25° C. White colonies become apparent in 3 to 4 weeks, eventually taking on a velvety appearance. Glabrous, wrinkled, brownish colonies may also be seen. The mycelial form is nondescript and nondiagnostic: hyaline, septate, hyphae with intercalated chlamydoconidia. Specific identification requires conversion to the yeast form or by exoantigen testing. The characteristic yeast form is seen in tissue and in culture at 37° C. Variable-sized (3 to 30 µm or more in diameter), oval to round, yeastlike cells with double refractile walls and single or multiple buds (blastoconidia) are characteristic of this fungus (Figure 73-13). The blastoconidia are connected to the parent cell by a narrow isthmus, and six or more of various sizes may be produced from a single cell: the so-called "mariner's" or "pilot-wheel" morphology. The variability in size and number of blastoconidia and their connection to the parent cell are identifying features (see Figure 73-13). These features are best disclosed by the GMS stain but may also be seen in H&E-stained tissues or in KOH mounts of clinical material.

Epidemiology page 746 page 747

Figure 73-13 GMS-stained yeast form of Paracoccidioides brasiliensis showing multiple budding "pilot wheel" morphology. (From Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997.)

Paracoccidioidomycosis is endemic throughout Latin America but is more prevalent in South America than in Central America (see Figure 73-2). The highest incidence is seen in Brazil, followed by Colombia, Venezuela, Ecuador, and Argentina. All patients diagnosed outside of Latin America previously had lived in Latin America. The ecology of the endemic areas includes high humidity, rich vegetation, moderate temperatures, and acid soil. These conditions are found along rivers from the Amazon jungle to small indigenous forests in Uruguay. P. brasiliensis has been recovered from soil in these areas; however, its ecologic niche is not well established. The portal of entry is thought to be either by inhalation or traumatic inoculation (Figure 73-14), although even this is poorly understood. Natural infection has only been documented in armadillos.

Figure 73-14 Natural history of the mold (saprobic) and yeast (parasitic) cycle of Paracoccidioides brasiliensis.

Although infection occurs in children (peak incidence 10 to 19 years), overt disease is uncommon in both children and adolescents. In adults, disease is more common in men age 30 to 50 years. Most patients with clinically apparent disease live in rural areas and have close contact with the soil. There are no reports of epidemics or human-to-human transmission. Depression of cell-mediated immunity correlates with the acute progressive form of the disease.

Clinical Syndromes

Paracoccidioidomycosis may be subclinical or progressive with acute or chronic pulmonary forms or acute, subacute, or chronic disseminated forms of the disease. Most primary infections are self-limited; however, the organism may become dormant for long periods of time and reactivate to cause clinical disease concomitant with impaired host defenses. A subacute disseminated form is seen in younger patients and immunocompromised individuals with marked lymphadenopathy, organomegaly, bone marrow involvement, and osteoarticular manifestations mimicking osteomyelitis. Recurrent fungemia results in dissemination and frequent skin lesions. Pulmonary and mucosal lesions are not seen in this form of the disease. Adults most often present with a chronic pulmonary form of the disease marked by respiratory problems, often as the sole manifestation. The disease progresses slowly over months to years, with persistent cough, purulent sputum, chest pain, weight loss, dyspnea, and fever. Pulmonary lesions are nodular, infiltrative, fibrotic, and cavitary. Although 25% of patients exhibit only pulmonary manifestations of the disease, the infection can disseminate to extrapulmonary sites in the absence of diagnosis and treatment. Prominent extrapulmonary locations include skin and mucosa, lymph nodes, adrenal glands, liver, spleen, central nervous system, and bones. The mucosal lesions are painful and ulcerated and usually are confined to the mouth, lips, gums, and palate. More than 90% of these individuals are male.

Laboratory Diagnosis The diagnosis is established by the demonstration of the characteristic yeast forms on microscopic examination of sputum, bronchoalveolar lavage fluid, scrapings or biopsy of ulcers, pus draining from lymph nodes, cerebrospinal fluid, or tissue (see Table 73-2). The organism may be visualized by a variety of staining methods, including calcofluor fluorescence, H&E, GMS, PAS, or Papanicolaou stains (see Figure 73-13). The presence of multiple buds distinguishes P. brasiliensis from Cryptococcus neoformans and Blastomyces dermatitidis.

Isolation of the organism in culture requires confirmation by demonstration of thermal dimorphism or exoantigen testing (detection of exoantigen 1, 2, and 3). Cultures should be manipulated in a biosafety cabinet. Serologic testing using either ID or CF to demonstrate antibody may be helpful in suggesting the diagnosis and in evaluating response to therapy (see Table 73-2). page 747 page 748

Treatment Itraconazole is the treatment of choice for most forms of the disease and generally must be given for at least 6 months. More severe or refractory infections may require amphotericin B therapy followed by either itraconazole or sulfonamide therapy. Relapses are common with sulfonamide therapy, and both dose and duration require adjustment based on clinical and mycologic parameters. Fluconazole has some activity against this organism, although frequent relapses have limited its use for the treatment of this disease. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Penicilliosis Marneffei Penicilliosis marneffei is a disseminated mycosis caused by the dimorphic fungus Penicillium marneffei. This infection involves the mononuclear phagocytic system and occurs primarily in HIV-infected individuals in Thailand and Southern China (see Figure 73-2).

Morphology

P. marneffei is the only species of Penicillium that is a pathogenic dimorphic fungus. In its mold phase in culture at 25° C, it exhibits sporulating structures that are typical of the genus (see Figure 73-1). Identification is aided by the formation of a soluble red pigment that diffuses into the agar (see Table 73-3).

Figure 73-15 GMS-stained yeast forms of Penicillium marneffei, including forms with single, wide, transverse septa (center). (From Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997.)

At 37° C in culture and in tissue, P. marneffei grows as a yeastlike organism that divides by fission and exhibits a transverse septum (Figure 73-15). The yeast form is intracellular in vivo and in this way resembles H. capsulatum, although it is somewhat more pleomorphic and elongated and does not bud (see Table 73-2 and Figures 73-10 and 73-15).

Epidemiology

P. marneffei has emerged as a prominent mycotic pathogen among HIV-infected individuals in Southeast Asia (see Figure 73-2). Imported cases have been reported in Europe and the United States. Although infection has been seen in immunocompetent hosts, the vast majority of infections since 1987 have been in patients with AIDS or other immunocompromised hosts residing in, or who have visited, Southeast Asia or Southern China. Penicilliosis marneffei has become an early indicator of HIV infection in that part of the world. P. marneffei has been isolated from bamboo rats and occasionally from soil. Laboratory-acquired infection has been reported in an immunocompromised individual exposed to the mycelial form in culture.

Clinical Syndromes Penicilliosis marneffei is caused when a susceptible host inhales conidia of P. marneffei from the environment, and disseminated infection develops. The infection may mimic tuberculosis, leishmaniasis, and other AIDS-related opportunistic infections such as histoplasmosis and cryptococcosis. Patients present with fever, cough, pulmonary infiltrates, lymphadenopathy, organomegaly, anemia, leukopenia, and thrombocytopenia. Skin lesions reflect hematogenous dissemination and appear as molluscum contagiosum-like lesions on the face and trunk.

Laboratory Diagnosis P. marneffei is readily recovered from clinical specimens, including blood, bone marrow, bronchoalveolar lavage specimens, and tissue. In culture at 25° C to 30° C, isolation of a mold t hat exhibits typical Penicillium morphology and a diffusible red pigment is highly suggestive. Conversion to the yeast phase at 37° C is confirmatory. Microscopic detection of the elliptical fission yeasts inside phagocytes in buffy coat preparations or smears of bone marrow, ulcerative skin lesions, or lymph nodes is diagnostic (see Figure 73-15). Serologic tests are under development.

Treatment

Amphotericin B with or without flucytosine is the treatment of choice. Administration of amphotericin B for 2 weeks should be followed by itraconazole for another 10 weeks. AIDS patients may require lifelong treatment with itraconazole to prevent relapses of the infection. Fluconazole therapy has been associated with a high rate of failure and is not recommended. page 748 page 749

Case Study and Questions Jane and Joan were two avid "outdoorspersons" in their mid-30s. In the past 5 years, they had been spelunking in southern Missouri, backpacking in northern Wisconsin, and camping in Arizona. Most recently, they had been renovating an old farmhouse in rural Iowa, and in the process had to tear down an old chicken coop that was attached to the back of the house. About 1 week into the process, they both suffered from a flulike illness, and Jane developed a cough and shortness of breath. They went to the family practice clinic to get "checked out." At the clinic, Joan appeared fine, but Jane was noted to be quite short of breath and appeared ill. The doctor thought it would be a good idea to get a chest x-ray on Jane. Joan got one too, just in case. Jane's chest x-ray showed a diffuse bilateral pneumonia. Although Joan's x-ray did not show pneumonia, it was noted that she had a solitary nodule in the right upper lobe. 1. What dimorphic fungal pathogens were Jane and Joan exposed to? 2. What constitutes a dimorphic fungus? 3. Aside from dimorphism, what feature is common to all of the endemic mycoses? 4. Describe the life cycles of the six dimorphic endemic pathogens. 5. What do you think is the cause of Jane's pneumonia? How would you make the diagnosis? 6. How would you treat her pneumonia?

7. What do you think accounts for Joan's lung nodule? How would you make the diagnosis? How would you treat her?

Bibliography Brandt ME, Warnock DW: Histoplasma, Blastomyces, Coccidioides, and other dimorphic fungi causing systemic mycoses. In Murray PR, et al. (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Chu JH, et al: Hospitalization for endemic mycoses: A population-based national study. Clin Infect Dis 42:822, 2006. Connor DH, et al: Pathology of Infectious Diseases. Stamford, Conn, Appleton & Lange, 1997. Kauffman CA: Histoplasmosis: A clinical and laboratory update. Clin Microbiol Rev 20:115, 2007. Mitchell TG: Systemic fungi. In Cohen J, Powderly WG (eds): Infectious Diseases, 2nd ed. St Louis, Mosby, 2004. Perea S, Patterson TF: Endemic mycoses. In Anaissie EJ, Mc Ginnis MR, Pfaller MA (eds): Clinical Mycology, New York, Churchill Livingstone, 2003. Vanittanakom N, et al: Penicillium marneffei infection and recent advances in the epidemiology and molecular biology aspects. Clin Microbiol Rev 19:95, 2006. Wheat LJ: Endemic mycoses. In Cohen J, Powderly WG (eds): Infectious Diseases, 2nd ed. St Louis, Mosby, 2004. page 749 page 750 Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Candidiasis It is clear that the most important group of opportunistic fungal pathogens is the Candida species. Candida spp. are the fourth most common cause of nosocomial bloodstream infections (BSI), exceeding that of any individual gram-negative pathogen (Table 74-2, Clinical Case 74-1). Between 1980 and the present, the frequency of Candida BSI has risen steadily in hospitals of all sizes and in all age groups. (See Chapter 5, Table 5-2.) page 751 page 752

Table 74-1. Predisposing Factors for Opportunistic Mycoses Factor Antimicrobial agents (number and duration)

Possible Role in Infection Promote fungal colonization Provide intravascular access

Major Opportunistic Pathogens Candida spp., other yeastlike fungi

Adrenal corticosteroid Immunosuppression Cryptococcus neoformans, Aspergillus spp., Zygomycetes, other molds, Pneumocystis Chemotherapy

Immunosuppression Candida spp., Aspergillus spp., Pneumocystis

Hematologic/solid organ malignancy

Immunosuppression Candida spp., Aspergillus spp., Zygomycetes, other molds and yeastlike fungi, Pneumocystis

Previous colonization Translocation across Candida spp. mucosa Indwelling catheter (central venous, pressure transducer, Swan-Ganz)

Direct vascular access Contaminated product

Candida spp., other yeastlike fungi

Total parenteral nutrition

Direct vascular access Contamination of infusate

Candida spp., Malassezia spp., other yeastlike fungi

Neutropenia (WBC 95% cure rate) and tolerability. Unfortunately, preliminary data from India suggests an increasing relapse rate in patients treated with miltefosine, indicating that drug resistance could develop and that strategies must be developed to prevent it. Prevention of the various forms of leishmaniasis involves prompt treatment of human infections and control of reservoir hosts, along with insect vector control. Protection from sand flies by screening and insect repellents is also essential. The protection of forest and construction workers in endemic areas is most difficult, and disease in those places may be effectively controlled only by vaccination. Work to develop a vaccine is ongoing. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Trypanosomes

Trypanosoma, another hemoflagellate, causes two distinctly different forms of disease (Table 82-3). One is called African trypanosomiasis, or sleeping sickness, and is produced by Trypanosoma brucei gambiense and T. b. rhodesiense. It is transmitted by tsetse flies. The second infection is called American trypanosomiasis, or Chagas' disease, produced by T. cruzi. It is transmitted by true bugs (triatomids, reduviids, also called kissing bugs; Clinical Case 82-4).

Table 82-3. Trypanosoma Species Responsible for Human Diseases Parasite Trypanosoma brucei gambiense and T. b. rhodesiense

Vector Disease Tsetse fly African trypanosomiasis (sleeping sickness)

Trypanosoma cruzi

Reduviids American trypanosomiasis (Chagas' disease) page 848 page 849

Clinical Case 82-4. Trypanosomiasis

Herwaldt and colleagues (J Infect Dis 181:395-399, 2000) describe a case in which the mother of an 18-month-old boy in Tennessee found a triatomine bug in his crib, which she saved because it resembled a bug shown on a television program about insects that prey on mammals. An entomologist identified the bug as Triatoma sanguisuga, a vector of Chagas' disease. The bug was found to be engorged with blood and infected with Trypanosoma cruzi. The child had been intermittently febrile for the preceding 2 to 3 weeks but was otherwise healthy except for pharyngeal edema and multiple insect bites of unknown type on his legs. Whole-blood specimens obtained from the child were negative by buffy-coat examination and hemoculture but positive for T. cruzi by PCR and DNA hybridization, suggesting that he had low-level parasitemia. Specimens obtained after treatment with benznidazole were negative. He did not develop anti-T. cruzi antibody; 19 relatives and neighbors were also negative. Two of three raccoons trapped in the vicinity had positive hemocultures for T. cruzi. The child's case of T. cruzi infection-the fifth reported U.S. autochthonous case-would have been missed without his mother's attentiveness and the availability of sensitive molecular techniques. Given that infected triatomine bugs and mammalian hosts exist in the southern United States, it is not surprising that humans could become infected with T. cruzi. Furthermore, given the nonspecific clinical manifestations of the infection, it is likely that other cases have been overlooked.

Trypanosoma brucei gambiense Physiology and Structure

The life cycle of the African forms of trypanosomiasis is illustrated in Figure 82-12. The infective stage of the organism is the trypomastigote, which is present in the salivary glands of transmitting tsetse flies. The organism in this stage has a free flagellum and an undulating membrane running the full length of the body (Figure 82-13). The trypomastigotes enter the wound created by the fly bite and find their way into blood and lymph, eventually invading the CNS. Reproduction of the trypomastigotes in blood, lymph, and spinal fluid is by binary or longitudinal fission. These trypomastigotes in blood are then infective for biting tsetse flies, where further reproduction occurs in the midgut. The organisms then migrate to the salivary glands, where an epimastigote form (with a free flagellum but only a partial undulating membrane) continues reproduction to the infective trypomastigote stage. Tsetse flies become infective 4 to 6 weeks after feeding on blood from a diseased patient.

Epidemiology

Figure 82-12 Life cycle of T. brucei.

T. b. gambiense is limited to tropical West and Central Africa, correlating to the range of the tsetse fly vector. The tsetse flies transmitting T. b. gambiense prefer shaded stream banks for reproduction and proximity to human dwellings. Persons who work in such areas are at greatest risk of infection. An animal reservoir has not been proved, although several species of animals have been infected experimentally.

Clinical Syndromes

Figure 82-13 These images are not available online due to electronic permissions. page 849 page 850

The incubation period of Gambian sleeping sickness varies from a few days to weeks. T. b. gambiense produces chronic disease, often ending fatally, with CNS involvement after several years' duration. One of the earliest signs of disease is an occasional ulcer at the site of the fly bite. As reproduction of organisms continues, the lymph nodes are invaded, and fever, myalgia, arthralgia, and lymph node enlargement result. Swelling of the posterior cervical lymph nodes is characteristic of Gambian disease and is called Winterbottom sign. Patients in this acute phase often exhibit hyperactivity. Chronic disease progresses to CNS involvement, with lethargy, tremors, meningoencephalitis, mental retardation, and general deterioration. In the final stages of chronic disease, convulsions, hemiplegia, and incontinence occur, and the patient becomes difficult to arouse or respond, eventually progressing to a comatose state. Death is the result of CNS damage and other infections such as malaria or pneumonia.

Laboratory Diagnosis Organisms can be demonstrated in thick and thin blood films, in concentrated anticoagulated blood preparations, and in aspirations from lymph nodes and concentrated spinal fluid (see Figure 82-13). Methods for concentrating parasites in blood may be helpful. Approaches include centrifugation of heparinized samples and anion-exchange chromatography. Levels of parasitemia vary widely, and several attempts to visualize the organism over a number of days may be necessary. Preparations should be fixed and stained immediately to avoid disintegration of the trypomastigotes. Serologic tests are also useful diagnostic techniques. Immunofluorescence, ELISA, precipitin, and agglutination methods have been used. Most reagents are not available commercially. Referral laboratories have used PCR to detect infections and to differentiate species (T. b. gambiense vs. T. b. rhodesiense), but these methods are not routinely used in the field.

Treatment, Prevention, and Control Suramin is the drug of choice for treating the acute blood and lymphatic stages of the disease, with pentamidine as an alternative. Suramin and pentamidine do not cross the blood-brain barrier; therefore, melarsoprol is the drug of choice when central nervous system (CNS) involvement is suspected. Difluoromethylornithine (DFMO) is a cytostatic drug with activity against the acute and late (CNS) stages of the disease. The most effective control measures include an integrated approach to reduce the human reservoir of infection and the use of fly traps and insecticide; however, economic resources are limited, and effective programs have been difficult to sustain.

Trypanosoma brucei rhodesiense Physiology and Structure The life cycle of T. b. rhodesiense is similar to that of T. b. gambiense (see Figure 82-12), with both trypomastigote and epimastigote stages and transmission by tsetse flies.

Epidemiology The organism is found primarily in East Africa, especially the cattle-raising countries, where tsetse flies breed in the brush rather than along stream banks. T. b. rhodesiense also differs from T. b. gambiense in that domestic animal hosts (cattle and sheep) and wild game animals act as reservoir hosts. This transmission and vector cycle makes the organism more difficult to control than T. b. gambiense.

Clinical Syndromes The incubation period for T. b. rhodesiense is shorter than that for T. b. gambiense. Acute disease (fever, rigors, and myalgia) occurs more rapidly and progresses to a fulminating, rapidly fatal illness. Infected persons are usually dead within 9 to 12 months if untreated. This more virulent organism also develops in greater numbers in the blood. Lymphadenopathy is uncommon, and CNS invasion occurs early in the infection, with lethargy, anorexia, and mental disturbance. The chronic stages described for T. b. gambiense are not often seen, because in addition to rapid CNS disease, the organism produces kidney damage and myocarditis, leading to death.

Laboratory Diagnosis Examination of blood and spinal fluid is carried out as for T. b. gambiense. Serologic tests are available; however, the marked variability of the surface antigens of trypanosomes limits the diagnostic usefulness of this approach.

Treatment, Prevention, and Control

The same treatment protocol applies as for T. b. gambiense, with early treatment for the more rapid neurologic manifestations. Similar prevention and control measures are needed: tsetse fly control and use of protective clothing, screens, netting, and insect repellent. In addition, early treatment is essential to control transmission, detect infection, and determine treatment in domestic animals. Control of infection in game animals is difficult, but infection can be reduced if measures to control the tsetse fly population, specifically eradication of brush and grassland breeding sites, are applied.

Trypanosoma cruzi Physiology and Structure The life cycle of T. cruzi (Figure 82-14) differs from T. brucei with the development of an additional form called an amastigote (Figure 82-15). The amastigote is an intracellular form with no flagellum and no undulating membrane. It is smaller than the trypomastigote, is oval, and is found in tissues. The infective trypomastigote, which is present in the feces of a reduviid bug ("kissing bug"), enters the wound created by the biting, feeding bug. The bugs have been called kissing bugs because they frequently bite people around the mouth and in other facial sites. They are notorious for biting, feeding on blood and tissue juices, and then defecating into the wound. The organisms in the feces of the bug enter the wound; penetration is usually aided when the patient rubs or scratches the irritated site. page 850 page 851

Figure 82-14 Life cycle of T. cruzi.

Figure 82-15 Amastigote stage of T. cruzi in skeletal muscle. (From Ash LR, Orihel TC: Atlas of Human Parasitology, 2nd ed. Chicago, American Society of Clinical Pathologists, 1984.)

The trypomastigotes then migrate to other tissues (e.g., cardiac muscle, liver, brain), lose the flagellum and undulating membrane, and become the smaller, oval, intracellular amastigote form. These intracellular amastigotes multiply by binary fission and eventually destroy the host cells. Then they are liberated to enter new host tissue as intracellular amastigotes or to become trypomastigotes infective for feeding reduviid bugs. Ingested trypomastigotes develop into epimastigotes in the midgut of the insect and reproduce by longitudinal binary fission. The organisms migrate to the hindgut of the bug, develop into metacyclic trypomastigotes, and then leave the bug in the feces after biting, feeding, and defecating, initiating a new human infection.

Epidemiology T. cruzi occurs widely in both reduviid bugs and a broad spectrum of reservoir animals in North, Central, and South America. Human disease is found most often among children in South and Central America, where 16 to 18 million people are infected. There is a direct correlation between infected wild-animal reservoir hosts and the presence of infected bugs whose nests are found in human homes. Cases are rare in the United States, because the bugs prefer nesting in animal burrows, and because homes are not as open to nesting as those in South and Central America.

Clinical Syndromes

Chagas' disease may be asymptomatic, acute, or chronic. One of the earliest signs is development at the site of the bug bite of an erythematous and indurated area called a chagoma. This is often followed by a rash and edema around the eyes and face (Romaña's sign). The disease is most severe in children younger than 5 years of age and frequently is seen as an acute process with CNS involvement. Acute infection is also characterized by fever, chills, malaise, myalgia, and fatigue. Parasites may be present in the blood during the acute phase; however, they are sparse in patients older than 1 year of age. Death may ensue a few weeks after an acute attack, the patient may recover, or the patient may enter the chronic phase as organisms proliferate and enter the heart, liver, spleen, brain, and lymph nodes. Chronic Chagas' disease is characterized by hepatosplenomegaly, myocarditis, and enlargement of the esophagus and colon as a result of the destruction of nerve cells (e.g., Auerbach plexus) and other tissues that control the growth of these organs. Megacardia and electrocardiographic changes are commonly seen in chronic disease. Involvement of the CNS may produce granulomas in the brain, with cyst formation and a meningoencephalitis. Death from chronic Chagas' disease results from tissue destruction in the many areas invaded by the organisms, and sudden death results from complete heart block and brain damage.

Laboratory Diagnosis page 851 page 852

T. cruzi can be demonstrated in thick and thin blood films or concentrated anticoagulated blood early in the acute stage. As the infection progresses, the organisms leave the bloodstream and become difficult to find. Biopsy of lymph nodes, liver, spleen, or bone marrow may demonstrate the organisms in the amastigote stage. Culture of blood or inoculation into laboratory animals may be useful when the parasitemia is low. Serologic tests are also available. In endemic areas, xenodiagnosis is widely used. Gene amplification techniques, such as polymerase chain reaction, have been used to detect the organism in the bloodstream. These approaches are not widely available and have not been adapted for use in the field.

Treatment, Prevention, and Control Treatment of Chagas' disease is limited by the lack of reliable agents. The drug of choice is nifurtimox. Although it has some activity against the acute phase of disease, it has little activity against tissue amastigotes and has a number of side effects. Alternative agents include allopurinol and benznidazole. Education regarding the disease, its insect transmission, and the wild-animal reservoirs is critical. Bug control, eradication of nests, and construction of homes to prevent nesting of bugs are also essential. The use of dichlorodiphenyltrichloroethane (DDT) in bug-infested homes has demonstrated a drop in the transmission of malaria and Chagas' disease. Screening of blood by serologic means or excluding blood donors from endemic areas prevents some infections that would otherwise be associated with transfusion therapy. Development of a vaccine is possible because T. cruzi does not have the wide antigenic variation observed with the African trypanosomes.

Case Study and Questions

The patient, a 44-year-old heart transplant patient, complained to her primary physician about headache, nausea, and vomiting approximately 1 year after transplant. She had no skin lesions. A computed tomographic scan of the head demonstrated ring-enhancing lesions. A biopsy of the lesions was performed. All cultures (bacterial, fungal, viral) were negative. Special stains of the tissue revealed multiple cystlike structures of varying size. 1. What was the differential diagnosis of infectious agents in this patient? What was the most likely etiologic agent? 2. What other tests would have been done to confirm the diagnosis? 3. What aspects of the medical history might suggest a risk for infection with this agent? 4. What were the therapeutic options and the likelihood that therapy would be successful?

Bibliography Baird JK: Effectiveness of antimicrobial drugs. N Engl J Med 352: 1565-1577, 2005. Bruckner DA, Labarca JA: Leishmania and Trypanosoma. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Connor DH, et al: Pathology of Infectious Diseases, vol 2. Stamford, Conn, Appleton & Lange, 1997. Conway DJ: Molecular epidemiology of malaria. Clin Microbiol Rev 20:188-204, 2007. Croft SL, Sundar S, Fairlamb AH: Drug resistance in leishmaniasis. Clin Microbiol Rev 19:111-126, 2006. Hotez PJ, et al: Control of neglected tropical diseases. N Engl J Med 357:1018-1027, 2007. Jones JL, et al: Toxoplasma gondii infection in the United States, 1999-2004, decline from the prior decade. Am J Trop Med Hyg 77:405-410, 2007. Karp CL, Auwaerter PG: Coinfection with HIV and tropical infectious diseases. I. Protozoal pathogens. Clin Infect Dis 45:1214-1220, 2007.

Marciano-Cabral F, Cabral G: Acanthamoeba spp. as agents of disease in humans. Clin Microbiol Rev 16:273-307, 2003. Rogers WO: Plasmodium and Babesia. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Shiff C: Integrated approach to malaria control. Clin Microbiol Rev 15:278-293, 2002. Talisuna AO, Bloland P, D'Alessandro U: History, dynamics, and public health importance of malaria parasite resistance. Clin Microbiol Rev 17:235-254, 2004. Visvesvara GS: Pathogenic and opportunistic free-living amebae. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Zintl A, et al: Babesia divergens, a bovine blood parasite of veterinary and zoonotic importance. Clin Microbiol Rev 16:622-636, 2003. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Enterobius vermicularis Physiology and Structure E. vermicularis, the pinworm, is a small, white worm that is familiar to parents who find them in the perianal folds or vagina of an infected child. Infection is initiated by ingestion of embryonated eggs (Figure 83-1). Larvae hatch in the small intestine and migrate to the large intestine, where they mature into adults in 2 to 6 weeks. Fertilization of the female by the male produces the characteristic asymmetrical eggs. These eggs are laid in the perianal folds by the migrating female. As many as 20,000 eggs are deposited on the perianal skin. The eggs rapidly mature and are infectious within hours.

Epidemiology E. vermicularis occurs worldwide but is most common in the temperate regions, where person-to-person spread is greatest in crowded conditions such as in daycare centers, schools, and mental institutions. An estimated 500 million cases of pinworm infection are reported worldwide, and this is the most common helminthic infection in North America.

Infection occurs when the eggs are ingested and the larval worm is free to develop in the intestinal mucosa. These eggs may be transmitted from hand to mouth by children scratching the perianal folds in response to the irritation caused by the migrating, egg-laying female worms, or the eggs may find their way to clothing and play objects in daycare centers. They can also survive long periods in the dust that accumulates over doors, on windowsills, and under beds in the rooms of infected people. Egg-laden dust can be inhaled and swallowed to produce infestation. In addition, autoinfection ("retrofection") can occur, wherein eggs hatch in the perianal folds and the larval worms migrate into the rectum and large intestine. Infected individuals who handle food can also be a source of infection. No animal reservoir for Enterobius is known. Physicians should be aware of the related epidemiology of Dientamoeba fragilis; this organism correlates well with the presence of E. vermicularis, with D. fragilis transported in the pinworm eggshell.

Clinical Syndromes Many children and adults show no symptoms and serve only as carriers. Patients who are allergic to the secretions of the migrating worms experience severe pruritus, loss of sleep, and fatigue. The pruritus may cause repeated scratching of the irritated area and lead to secondary bacterial infection. Worms that migrate into the vagina may produce genitourinary problems and granulomas. page 853 page 854

Table 83-1. Nematodes of Medical Importance Parasite Enterobius vermicularis

Common Name Pinworm

Disease Enterobiasis

Ascaris lumbricoides Roundworm

Ascariasis

Toxocara canis

Visceral larva migrans

Dog ascaris

Toxocara cati

Cat ascaris

Visceral larva migrans

Baylisascaris procyonis

Raccoon ascaris

Neural larva migrans

Trichuris trichiura

Whipworm

Trichuriasis

Ancylostoma duodenale

Old World hookworm

Hookworm infection

Necator americanus

New World hookworm

Hookworm infection

Ancylostoma braziliense

Dog or cat hookworm

Cutaneous larva migrans

Strongyloides stercoralis

Threadworm

Strongyloidiasis

Trichinella spiralis

Trichinosis

Wuchereria bancrofti Bancroft filaria

Filariasis

Brugia malayi

Malayan filaria

Filariasis

Loa loa

African eye worm

Loiasis

Mansonella species

Filariasis

Onchocerca volvulus

Onchocerciasis

Dirofilaria immitis

Dog heartworm

Dirofilariasis

Dracunculus medinensis

Guinea worm

Dracunculosis

Worms attached to the bowel wall may produce inflammation and granuloma formation around the eggs. Although the adult worms may occasionally invade the appendix, there remains no proven relationship between pinworm invasion and appendicitis. Penetration through the bowel wall into the peritoneal cavity, liver, and lungs has been infrequently recorded.

Laboratory Diagnosis

Figure 83-1 Life cycle of E. vermicularis.

The diagnosis of enterobiasis is usually suggested by the clinical manifestations and confirmed by detection of the characteristic eggs on the anal mucosa. Occasionally, the adult worms are seen by laboratory personnel in stool specimens, but the method of choice for diagnosis involves use of an anal swab with a sticky surface that picks up the eggs (Figure 83-2) for microscopic examination. Sampling can be done with clear tape or commercially available swabs. The sample should be collected when the child arises and before bathing or defecation to pick up eggs laid by migrating worms during the night. Parents can collect the specimen and deliver it to the physician for immediate microscopic examination. Three swabbings, one per day for 3 consecutive days, may be required to detect the diagnostic eggs. The eggs are rarely seen in fecal specimens. Systemic signs of infection such as eosinophilia are rare.

Treatment, Prevention, and Control

Figure 83-2 E. vermicularis egg. The thin-walled eggs are 50 to 60 × 20 to 30 µm, ovoid, and flattened on one side (not because children sit on them, but this is an easy way to correlate the egg morphology with the epidemiology of the disease). page 854 page 855

The drug of choice is albendazole or mebendazole. Pyrantel pamoate and piperazine are effective, but reinfection is common. To avoid reintroduction of the organism and reinfection in the family environment, it is customary to treat the entire family simultaneously. Although cure rates are high, reinfection is common. Repeat treatment after 2 weeks may be useful in preventing reinfection. Personal hygiene, clipping of fingernails, thorough washing of bed clothes, and prompt treatment of infected individuals all contribute to control. When housecleaning is done in the home of an infected family, dusting under beds, on window sills, and over doors should be done with a damp mop to avoid inhalation of infectious eggs.

Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Ascaris lumbricoides Physiology and Structure A. lumbricoides are large (20 to 35 cm in length), pink worms that have a more complex life cycle than E. vermicularis (Figure 83-3) but are otherwise typical of an intestinal roundworm. The ingested infective egg releases a larval worm that penetrates the duodenal wall, enters the bloodstream, is carried to the liver and heart, and then enters the pulmonary circulation. The larvae break free in the alveoli of the lungs, where they grow and molt. In about 3 weeks, the larvae pass from the respiratory system to be coughed up, swallowed, and returned to the small intestine.

Figure 83-3 Life cycle of A. lumbricoides.

As the male and female worms mature in the small intestine (primarily jejunum), fertilization of the female by the male initiates egg production, which may amount to 200,000 eggs per day for as long as a year. Female worms can also produce unfertilized eggs in the absence of males. Eggs are found in the feces 60 to 75 days after the initial infection. Fertilized eggs become infectious after approximately 2 weeks in the soil.

Epidemiology A. lumbricoides is prevalent in areas where sanitation is poor and where human feces are used as fertilizer. Because food and water are contaminated with Ascaris eggs, this parasite, more than any other, affects the world's population. Although no animal reservoir is known for A. lumbricoides, an almost identical species from pigs, A. suum, can infect humans. This species is seen in swine growers and is associated with the use of pig manure for gardening. Ascaris eggs are quite hardy and can survive extreme temperatures and persist for several months in feces and sewage. Ascariasis is the most common helminthic infection worldwide, with an estimated 1 billion people infected.

Clinical Syndromes (Clinical Case 83-1) Infections caused by the ingestion of only a few eggs may produce no symptoms; however, even a single adult Ascaris worm may be dangerous, because it can migrate into the bile duct and liver and damage tissue. Furthermore, because the worm has a tough, flexible body, it can occasionally perforate the intestine, creating peritonitis with secondary bacterial infection. The adult worms do not attach to the intestinal mucosa but depend on constant motion to maintain their position within the bowel lumen.

After infection with many larvae, migration of worms to the lungs can produce pneumonitis resembling an asthmatic attack. Pulmonary involvement is related to the degree of hypersensitivity induced by previous infections and the intensity of the current exposure and may be accompanied by eosinophilia and oxygen desaturation. Also, a tangled bolus of mature worms in the intestine can result in obstruction, perforation, and occlusion of the appendix. As mentioned previously, migration into the bile duct, gallbladder, and liver can produce severe tissue damage. This migration can occur in response to fever, drugs other than those used to treat ascariasis, and some anesthetics. Patients with many larvae may also experience abdominal tenderness, fever, distention, and vomiting.

Laboratory Diagnosis page 855 page 856

Clinical Case 83-1. Hepatic Ascariasis Hurtado and colleagues (N Engl J Med 354:1295-1303, 2006) describe a case of a 36-year-old woman who presented with recurrent right-upper-quadrant (RUQ) abdominal pain. One year earlier, she also presented with RUQ abdominal pain, abnormal liver function tests, and positive serology for hepatitis C. An abdominal ultrasonographic examination showed biliary dilatation, and endoscopic retrograde cholangiopancreatography (ERCP) showed multiple stones in the common bile duct, the left hepatic duct, and the left intrahepatic duct. The majority of the stones were removed. Examination of the bile-duct aspirate was negative for ova and parasites. One month prior to the present admission, the patient experienced recurrent RUQ pain and jaundice. Repeat ERCP again showed multiple stones in the common and left main hepatic ducts; partial removal was accomplished.

One month later, the patient was admitted with severe epigastric pain and fever. The patient was born in Vietnam and had immigrated to the United States when she was in her early 20s. She had no history of recent travel. An abdominal CT scan with contrast showed abnormal perfusion of the left hepatic lobe and dilatation of the left biliary radicles with multiple filling defects. ERCP showed partial obstruction of the left main hepatic duct, a few small stones, and purulent bile. Magnetic resonance imaging (MRI) showed diffuse enhancement of the left lobe and left portal vein suggestive of inflammation. Cultures of blood grew Klebsiella pneumoniae, and examination of a stool sample revealed a few Strongyloides stercoralis rhabditiform larvae. Biliary stents were placed, and the patient was treated with levofloxacin. Two weeks later, the patient was admitted to the hospital, where a partial hepatectomy was performed for treatment of recurrent pyogenic cholangitis. Gross examination of the left hepatic lobe showed ectatic bile ducts containing bile-stained calculi. Microscopic examination of the calculous material revealed collections of parasite eggs and a degenerated and fragmented nematode. Klebsiella species were identified in cultures by the microbiology laboratory. The findings were consistent with recurrent pyogenic cholangiohepatitis with infection by Ascaris lumbricoides and Klebsiella species. In addition to antibiotics for the bacterial infection, the patient was treated with ivermectin for the Strongyloides infection and albendazole for the Ascaris organisms. The aberrant migration of A. lumbricoides into the pancreatobiliary tree with subsequent deposition of eggs, followed by death and degeneration of both worm and eggs, became a nidus for calculus formation and secondary bacterial infection. Although unusual in the United States, hepatic ascariasis is estimated to contribute to more than 35% of cases of biliary and pancreatic disease in the Indian subcontinent and parts of Southeast Asia.

Examination of the sediment of concentrated stool reveals the knobby-coated, bile-stained, fertilized and unfertilized eggs. Eggs are oval, 55 to 75 mm long, and 50 mm wide. The thick-walled outer shell can be partially removed (decorticated egg). Occasionally, adult worms pass with the feces, which can be quite dramatic because of their large size (20 to 35 cm long). Roentgenologists may also visualize the worms in the intestine, and cholangiograms often disclose their presence in the biliary tract of the liver. The pulmonary phase of the disease may be diagnosed by the finding of larvae and eosinophils in sputum.

Treatment, Prevention, and Control Treatment of symptomatic infection is highly effective. The drug of choice is albendazole or mebendazole; pyrantel pamoate and piperazine are alternatives. Patients with mixed parasitic infections (A. lumbricoides, other helminths, Giardia lamblia, and Entamoeba histolytica) in the stool should be treated for ascariasis first to avoid provoking worm migration and possible intestinal perforation. Education, improved sanitation, and avoidance of human feces as fertilizer are critical. A program of mass treatment in highly endemic areas has been suggested, but this may not be economically feasible. Furthermore, eggs can persist in contaminated soil for 3 years or more. Certainly, improved personal hygiene among people who handle food is an important aspect of control. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Toxocara and Baylisascaris Physiology and Structure

Toxocara canis, Toxocara cati, and Baylisascaris procyonis are ascarid worms that are naturally parasitic in the intestines of dogs, cats, and raccoons, respectively. These organisms may accidentally infect humans, producing disease states known as visceral larva migrans (VLM), neural larva migrans (NLM), and ocular larva migrans (OLM). When ingested by humans, the eggs of these worms can hatch into larval forms that cannot follow the normal developmental cycle as in the natural host. They can penetrate the human gut and reach the bloodstream and then migrate as larvae to various human tissues. The Toxocara species are the most common causes of VLM and OLM, whereas B. procyonis is increasingly recognized as a cause of fatal NLM. Although the Toxocara species do not develop beyond the migrating larval form, B. procyonis larvae continue to grow to a large size within the human host.

Epidemiology Wherever infected dogs and cats are present, the eggs are a threat to humans. Likewise, contact with raccoons or their feces presents a significant risk of infection with B. procyonis. This is especially true for children who are exposed more readily to contaminated soil and who tend to put objects in their mouths.

Clinical Syndromes (Clinical Case 83-2) page 856 page 857

Clinical Case 83-2. Baylisascariasis

Gavin and colleagues (Pediatr Infect Dis J 21:971-975, 2002) describe a case of a previously normal 2.5-year-old boy who was admitted to hospital with fever and recent onset of encephalopathy. Past history was significant for pica and geophagia, and he was receiving ferrous sulfate for iron-deficiency anemia. He was in good health until 8 days before admission, when a temperature of 38.5° C and mild cough developed. Three days before admission, he developed increasing lethargy and marked somnolence. He was irritable, confused, and ataxic. The family lived in suburban Chicago, and there were no sick contacts or pets at home. There was no travel history. On admission, he was febrile and lethargic but irritable and agitated when disturbed. Neck stiffness with generalized hypertonicity, hyperreflexia and bilateral extensor plantar responses were present. The white blood cell count (WBC) was elevated, and eosinophilia was present. CSF examination revealed an elevated protein and WBC with 32% eosinophils. Gram, acid-fast, and India ink stains and bacterial and cryptococcal antigen tests were all negative. Broad-spectrum antibacterial and antiviral therapy was begun empirically; however, the patient became comatose, with opisthotonus, decerebrate posturing, hypertonicity, and tremulousness. Cranial MRI demonstrated areas of increased signal involving both cerebellar hemispheres. Bacterial, fungal, mycobacterial, and viral cultures of blood and CSF were negative. Viral serologies were negative, as were tests for antibodies against Toxocara, cysticercosis, coccidioidomycosis, blastomycosis, and histoplasmosis. A detailed epidemiologic history revealed that 18 days before hospitalization, the family attended a picnic in a nearby suburb. Numerous raccoons were observed regularly in the vicinity, and the patient was observed playing with and eating dirt beneath the trees. CSF and serum antibodies against third-stage Baylisascaris procyonis were demonstrated by indirect immunofluorescent assay (IFA), with titers increasing from 1/4 to 1/1024 over a 2-week period. The patient was treated with albendazole and corticosteroids for 4 weeks but has remained severely affected with marked generalized spasticity and cortical

blindness. Subsequent examination of soil and debris from the child's play site revealed thousands of infective B. procyonis eggs. This case underscores the devastating effects of NLM. In many regions of North America, large populations of raccoons with high rates of endemic B. procyonis infection (e.g., 60%-80%) live in proximity to humans, which suggests that the risk of human infection is probably substantial.

The clinical manifestations of VLM, NLM, and OLM in humans are related to the migration of the larvae through tissues. The larvae may invade any tissue of the body, where they can induce bleeding, the formation of eosinophilic granulomas, and necrosis. Patients may be asymptomatic and have only eosinophilia, but they can also have serious disease directly related to the number and location of the lesions caused by the migrating larvae, as well as the degree to which the host is sensitized to the larval antigens. The organs most frequently involved are the lungs, heart, kidneys, liver, skeletal muscles, eyes, and central nervous system. NLM is a common sequela of infection with B. procyonis and is attributed to the extensive somatic larval migration of this species. Continued growth and migration within the CNS produces extensive mechanical tissue damage. Signs and symptoms due to the migrating larvae include cough, wheezing, fever, rash, anorexia, seizures, fatigue, and abdominal discomfort. On examination, patients may have hepatosplenomegaly and nodular pruritic skin lesions. Death may result from respiratory failure, cardiac arrhythmia, or brain damage. Ocular disease can also occur with the movement of larvae through the eye and may be mistaken for malignant retinoblastoma. Prompt diagnosis is required to avoid unnecessary enucleation.

Laboratory Diagnosis

The diagnosis of VLM, NLM, and OLM is based on clinical findings, the presence of eosinophilia, known exposure to dogs, cats, or raccoons and serologic confirmation. Enzyme-linked immunosorbent assays are available and appear to offer the best serologic marker for disease. The examination of feces from infected patients is not useful, because egg-laying adults are not present. However, examination of fecal material from infected pets often supports the diagnosis. Tissue examination for larvae may provide a definitive diagnosis but may be negative because of sampling error.

Treatment, Prevention, and Control Treatment is primarily symptomatic, since antiparasitic agents are not of proven benefit. Anthelmintic therapy with albendazole, mebendazole, diethylcarbamazine or thiabendazole is often used. Corticosteroid therapy may be lifesaving if the patient has serious pulmonary, myocardial, or central nervous system involvement, since a major component of the infection is an inflammatory response to the organism. To date, despite anthelmintic treatment of cases of B. procyonis NLM, there are no neurologically intact survivors. This zoonosis can be greatly reduced if pet owners conscientiously eradicate worms from their animals and clean up pet fecal material from yards and school playgrounds. Children's play areas and sandboxes should be carefully monitored. Raccoons should not be encouraged to visit homes or yards for food, and the keeping of raccoons as pets should be strongly discouraged. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Trichuris trichiura Physiology and Structure

Commonly called whipworm because it resembles the handle and lash of a whip, T. trichiura has a simple life cycle (Figure 83-4). Ingested eggs hatch into a larval worm in the small intestine and then migrate to the cecum, where they penetrate the mucosa and mature to adults. Some 3 months after the initial infection, the fertilized female worm starts laying eggs and may produce 3000 to 10,000 eggs per day. Female worms can live for as long as 8 years. Eggs passed into the soil mature and become infectious in 3 weeks. T. trichiura eggs are distinctive, with dark bile staining, a barrel shape, and the presence of polar plugs in the egg shell (Figure 83-5). page 857 page 858

Figure 83-4 Life cycle of T. trichiura.

Epidemiology

Like A. lumbricoides, T. trichiura has worldwide distribution, and its prevalence is directly correlated with poor sanitation and the use of human feces as fertilizer. No animal reservoir is recognized.

Clinical Syndromes

Figure 83-5 T. trichiura egg. The eggs are barrel shaped, measuring 50 × 24 µm, with a thick wall and two prominent plugs at the ends. Internally, an unsegmented ovum is present.

The clinical manifestations of trichuriasis are generally related to the intensity of the worm burden. Most infections are with small numbers of Trichuris organisms and are usually asymptomatic, although secondary bacterial infection may occur because the heads of the worms penetrate deep into the intestinal mucosa. Infections with many larvae may produce abdominal pain and distention, bloody diarrhea, weakness, and weight loss. Appendicitis may occur as worms fill the lumen, and prolapse of the rectum is seen in children because of the irritation and straining during defecation. Anemia and eosinophilia are also seen in severe infections.

Laboratory Diagnosis

Stool examination reveals the characteristic bile-stained eggs with polar plugs. Light infestations may be difficult to detect because of the paucity of eggs in the stool specimens.

Treatment, Prevention, and Control The drug of choice is albendazole or mebendazole. As with A. lumbricoides, prevention of T. trichiura depends on education, good personal hygiene, adequate sanitation, and avoidance of the use of human feces as fertilizer. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Hookworms Ancylostoma duodenale and Necator americanus Physiology and Structure

Figure 83-6 Life cycle of human hookworms. page 858 page 859

The two human hookworms are A. duodenale (Old World hookworm) and N. americanus (New World hookworm). Differing only in geographical distribution, structure of mouthparts, and relative size, these two species are discussed together as agents of hookworm infection. The human phase of the hookworm life cycle is initiated when a filariform (infective form) larva penetrates intact skin (Figure 83-6). The larva then enters the circulation; is carried to the lungs; and like A. lumbricoides, is coughed up, swallowed, and develops to adulthood in the small intestine. The adult N. americanus has a hooklike head, which accounts for the name commonly used. Adult worms lay as many as 10,000 to 20,000 eggs per day, which are released into the feces. Egg laying is initiated 4 to 8 weeks after the initial exposure and can persist for as long as 5 years. On contact with soil, the rhabditiform (noninfective) larvae are released from the eggs and within 2 weeks develop into filariform larvae. The filariform larvae can then penetrate exposed skin (e.g., bare feet) and initiate a new cycle of human infection. Both species have mouthparts designed for sucking blood from injured intestinal tissue. A. duodenale has chitinous teeth, and N. americanus has shearing chitinous plates.

Epidemiology Transmission of hookworm infection requires the deposition of egg-containing feces on shady, well-drained soil and is favored by warm, humid (tropical) conditions. Hookworm infections are reported worldwide in places where direct contact with contaminated soil can lead to human disease, but they occur primarily in warm subtropical and tropical regions and in southern parts of the United States. It is estimated that more than 900 million individuals worldwide are infected with hookworms, including 700,000 in the United States.

Clinical Syndromes

Skin-penetrating larvae may produce an allergic reaction and rash at sites of entry, and larvae migrating in the lungs can cause pneumonitis and eosinophilia. Adult worms produce the gastrointestinal symptoms of nausea, vomiting, and diarrhea. As blood is lost from feeding worms, a microcytic hypochromic anemia develops. Daily blood loss is estimated at 0.15 to 0.25 ml for each adult A. duodenale and 0.03 ml for each adult N. americanus. In severe, chronic infections, emaciation and mental and physical retardation may occur related to anemia from blood loss and nutritional deficiencies. Also, intestinal sites may be secondarily infected by bacteria when the worms migrate along the intestinal mucosa.

Laboratory Diagnosis Stool examination reveals the characteristic non-bile-stained segmented eggs shown in Figure 83-7. Larvae are not found in stool specimens unless the specimen was left at ambient temperature for a day or more. The eggs of A. duodenale and N. americanus cannot be distinguished. The larvae must be examined to identify these hookworms specifically, although this is clinically unnecessary.

Treatment, Prevention, and Control

Figure 83-7 Human hookworm egg. The eggs are 60 to 75 µm long and 35 to 40 µm wide, are thin shelled, and enclose a developing larva.

The drug of choice is albendazole or mebendazole; pyrantel pamoate is an alternative. In addition to eradication of the worms to stop blood loss, iron therapy is indicated to raise hemoglobin levels to normal. Blood transfusion may be necessary in severe cases of anemia. Education, improved sanitation, and controlled disposal of human feces are critical preventive measures. Wearing shoes in endemic areas helps reduce the prevalence of infection.

Ancylostoma braziliense Physiology and Structure

A. braziliense, a species of hookworm, is naturally parasitic in the intestines of dogs and cats and accidentally infects humans. It produces a disease properly called cutaneous larva migrans but also called ground itch and creeping eruption. The filariform larvae of this hookworm penetrate intact skin but can develop no further in humans. The larvae remain trapped in the skin of the wrong host for weeks or months, wandering through subcutaneous tissue and creating serpentine tunnels.

Epidemiology Similar to the situation with Ascaris worms, the threat of infection with A. braziliense is greatest among children coming into contact with soil or sandboxes contaminated with animal feces containing hookworm eggs. Infections are prevalent throughout the year on beaches in subtropical and tropical regions; in the summer, infection is reported as far north as the Canadian-U.S. border.

Clinical Syndromes The migrating larvae may provoke a severe erythematous and vesicular reaction. Pruritus and scratching of the irritated skin may lead to secondary bacterial infection. About half of patients develop transient pulmonary infiltrates with peripheral eosinophilia (Löffler's syndrome), presumably resulting from pulmonary migration of the larvae.

Laboratory Diagnosis Occasionally, larvae are recovered in skin biopsy or after freezing of the skin, but most diagnoses are based on the clinical appearance of the tunnels and a history of contact with dog and cat feces. The larvae are rarely found in sputum. page 859 page 860

Treatment, Prevention, and Control

The drug of choice is albendazole; ivermectin and thiabendazole are alternatives. Antihistamines may be helpful in controlling pruritus. This zoonosis, as with animal Ascaris infection, can be reduced by educating pet owners to treat their animals for worm infections and to pick up pet feces from yards, beaches, and sandboxes. In endemic areas, shoes or sandals should be worn to prevent infection. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Strongyloides stercoralis Physiology and Structure Although the morphology of these worms and the epidemiology of their infections are similar to the hookworm, the life cycle of S. stercoralis (Figure 83-8) differs in three aspects: (1) Eggs hatch into larvae in the intestine and before they are passed in feces, (2) larvae can mature into filariforms in the intestine and cause autoinfection, and (3) a free-living, nonparasitic cycle can be established outside the human host.

Figure 83-8 Life cycle of S. stercoralis.

In direct development, like the hookworm, a skin-penetrating S. stercoralis larva enters the circulation and follows the pulmonary course. It is coughed up and swallowed, and adults develop in the small intestine. Adult females burrow into the mucosa of the duodenum and reproduce parthenogenetically. Each female produces about a dozen eggs each day, which hatch within the mucosa and release rhabditiform larvae into the lumen of the bowel. The rhabditiform larvae are distinguished from the larvae of hookworms by their short buccal capsule and large genital primordium. The rhabditiform larvae are passed in the stool and may either continue the direct cycle by developing into infective filariform larvae or develop into free-living adult worms and initiate the indirect cycle.

In indirect development, the larvae in soil develop into free-living adults that produce eggs and larvae. Several generations of this nonparasitic existence may occur before new larvae become skin-penetrating parasites. Finally, in autoinfection, rhabditiform larvae in the intestine do not pass with feces but become filariform larvae. These penetrate the intestinal or perianal skin and follow the course through the circulation and pulmonary structures, are coughed up, and then are swallowed; at this point, they become adults, producing more larvae in the intestine. This cycle can persist for years and can lead to hyperinfection and massive or disseminated, often fatal infection.

Epidemiology Similar to hookworms in its requirements for warm temperatures and moisture, S. stercoralis demonstrates low prevalence but a somewhat broader geographical distribution, including parts of the northern United States and Canada. Sexual transmission also occurs. Animal reservoirs, such as domestic pets, are recognized.

Clinical Syndromes Individuals with strongyloidiasis frequently are afflicted with pneumonitis from migrating larvae similar to that seen in ascariasis and hookworm infection. The intestinal infection is usually asymptomatic. However, heavy worm loads may involve the biliary and pancreatic ducts, the entire small bowel, and the colon, causing inflammation and ulceration leading to epigastric pain and tenderness, vomiting, diarrhea (occasionally bloody), and malabsorption. Symptoms mimicking peptic ulcer disease coupled with peripheral eosinophilia should strongly suggest the diagnosis of strongyloidiasis. page 860 page 861

Clinical Case 83-3. Strongyloides Hyperinfection

Gorman and colleagues (Infect Med 23:480, 2006) describe a case of necrotizing myositis complicated by diffuse alveolar hemorrhage and sepsis following corticosteroid therapy. The patient was a 46-year-old Cambodian man with a history of Raynaud phenomenon. He presented to the rheumatology clinic with worsening symptoms of Raynaud syndrome and diffuse muscle aches. He was employed as a truck driver and had immigrated from Cambodia 30 years earlier. Pertinent laboratory studies included markedly elevated creatine kinase and aldolase levels. Pulmonary function studies showed decreased forced vital capacity, forced expiratory volume, and carbon monoxide diffusing capacity. A high resolution CT scan of the chest showed mild ground-glass changes in both lung bases and interlobular septate thickening. Muscle biopsy showed myocyte necrosis and random atrophy but no inflammatory cells. Bronchoscopy was unremarkable, and all cultures were negative. The patient was started on prednisone for presumed necrotizing myopathy secondary to undifferentiated connective tissue disease.

He was admitted to the hospital 1 month later with profound muscle weakness and dyspnea, which improved with the administration of methylprednisolone and intravenous immunoglobulin. Three weeks later, the patient was readmitted with fever, nausea, vomiting, abdominal pain, and diffuse joint pain. A CT scan of the abdomen suggested small bowel intussusception and colitis, but his symptoms improved without treatment. Another high resolution CT scan of the chest showed early honeycombing and worsening interstitial infiltrates. The patient was scheduled for a lung biopsy; however, while awaiting the biopsy, he suffered an abrupt and fulminant deterioration, with hemoptysis and hypoxemic respiratory failure that required intubation and mechanical ventilation. Chest x-ray showed new, diffuse, bilateral infiltrates. The patient developed an acute abdomen accompanied by purpura on the lower trunk. An abdominal CT showed pancolitis. Refractory septic shock caused by Escherichia coli bacteremia and lactic acidosis ensued. Bronchoscopy showed diffuse alveolar hemorrhage, and numerous larvae of Strongyloides stercoralis were demonstrated on staining of an aspirate of endotracheal secretions. Serology was positive for anti-Strongyloides antibodies. Despite treatment with ivermectin, albendazole, cefepime, vancomycin, vasopressors, steroids, and dialysis, the patient died. This case of Strongyloides hyperinfection syndrome emphasizes the importance of screening and treating persons at risk for latent S. stercoralis infection (endemic in tropical and subtropical areas) before the initiation of immunosuppressive therapy. Contact precautions should be taken in patients with hyperinfection syndrome because of the risk of infection to healthcare workers and visitors upon exposure to infectious larvae in the patient's stool and secretions.

Autoinfection may lead to chronic strongyloidiasis that can last for years, even in nonendemic areas. Although many of these chronic infections may be asymptomatic, as many as two thirds of patients have recurring episodic symptoms referable to the involved skin, lungs, and intestinal tract. Individuals with chronic strongyloidiasis are at risk of developing severe, life-threatening hyperinfection syndrome if the host-parasite balance is disturbed by any drug or illness that compromises the host's immune status (Clinical Case 83-3). Hyperinfection syndrome is seen most commonly in individuals immunocompromised by malignancies (especially hematologic malignancies), corticosteroid therapy, or both. Hyperinfection syndrome has also been observed in patients who have undergone solid organ transplantation and in malnourished people. Loss of cellular immune function may be associated with the conversion of rhabditiform larvae to filariform larvae, followed by dissemination of the larvae via the circulation to virtually any organ. Most commonly, extraintestinal infection involves the lung and includes bronchospasm, diffuse infiltrates, and occasionally cavitation. Widespread dissemination that involves the abdominal lymph nodes, liver, spleen, kidneys, pancreas, thyroid, heart, brain, and meninges is common. Intestinal symptoms of hyperinfection syndrome include profound diarrhea, malabsorption, and electrolyte abnormalities. Notably, hyperinfection syndrome is associated with a mortality rate of approximately 86%. Bacterial sepsis, meningitis, peritonitis, and endocarditis secondary to larval spread from the intestine are frequent and often fatal complications of hyperinfection syndrome.

Laboratory Diagnosis

The diagnosis of strongyloidiasis may be difficult because of the intermittent passage of low numbers of first-stage larvae in stool. Examination of concentrated stool sediment reveals the larval worms (Figure 83-9), but in contrast with hookworm infections, in S. stercoralis infections, eggs are generally not seen. Collecting samples from three stools, one per day for 3 days (as for G. lamblia), is recommended because S. stercoralis larvae may occur in "showers," with many present one day and few or none the next. Several authors favor the Baermann funnel gauze method of concentrating living S. stercoralis larvae from fecal specimens. This method uses a funnel with a stopcock and a gauze insert. The funnel is filled with lukewarm water to a level just covering the gauze, and a specimen of stool is placed on the gauze, partially in contact with the water. The larvae in the stool migrate through the gauze into the water and then sediment into the neck of the funnel where they may be detected by low-power microscopy. When absent from stool, larvae may be detected in duodenal aspirates or in sputum in the case of massive infection. Finally, culture of the larvae from stool using charcoal cultures or an agar plate method may be used, although these are not routine in most laboratories. Demonstration of anti-Strongyloides antibodies in blood may be useful as a screening test or as an adjunct for diagnosis.

Figure 83-9 S. stercoralis larvae. The larvae are 180 to 380 µm long and 14 to 24 µm wide. They are differentiated from hookworm larvae by the length of the buccal cavity and esophagus and by the structure of the genital primordium. page 861 page 862

Treatment, Prevention, and Control All infected patients should be treated to prevent autoinfection and potential dissemination (hyperinfection) of the parasite. The drug of choice is ivermectin, with albendazole or mebendazole as an alternative. Patients in endemic areas who are preparing to undergo immunosuppressive therapy should have at least three stool examinations to rule out S. stercoralis infection and thus avoid the risks of autoinfection. Strict infection-control measures should be enforced when clinicians care for patients with hyperinfection syndrome, because stool, saliva, vomitus, and body fluids may contain infectious filariform larvae. As with hookworm, control of Strongyloides species requires education, proper sanitation, and prompt treatment of existing infections.

Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Trichinella spiralis Physiology and Structure T. spiralis is the most important cause of human disease, but other species, such as T. pseudospiralis and T. britovi may also cause trichinosis. The adult form of this organism lives in the duodenal and jejunal mucosa of flesh-eating mammals worldwide. The infectious larval form is present in the striated muscles of carnivorous and omnivorous mammals. Among domestic animals, swine are most frequently involved. Figure 83-10 illustrates the simple, direct life cycle, which terminates in the musculature of humans, where the larvae eventually die and calcify.

Figure 83-10 Life cycle of T. spiralis.

Figure 83-11 These images are not available online due to electronic permissions.

The infection begins when meat that contains encysted larvae is digested. The larvae leave the meat in the small intestine and within 2 days develop into adult worms. A single fertilized female produces more than 1500 larvae in 1 to 3 months. These larvae move from the intestinal mucosa into the bloodstream and are carried in the circulation to various muscle sites throughout the body, where they coil in striated muscle fibers and become encysted (Figure 83-11). The muscles invaded most frequently include the extraocular muscles of the eye; the tongue; the deltoid, pectoral, and intercostal muscles; the diaphragm; and the gastrocnemius muscle. The encysted larvae remain viable for many years and are infectious if ingested by a new animal host. The muscle larvae of T. pseudospiralis do not induce the formation of a cyst and generate less inflammation than that of T. spiralis.

Epidemiology Trichinosis occurs worldwide in humans, and its greatest prevalence is associated with the consumption of pork products. In addition to its transmission from pigs, many carnivorous and omnivorous animals harbor the organism and are potential sources of human infection. Notably, polar bears and walruses in the Arctic account for outbreaks in human populations, especially with a strain of T. spiralis (T. natira) that is more resistant to freezing than the T. spiralis strains found in the continental United States and other temperate regions. It is estimated that more than 1.5 million Americans carry live Trichinella cysts in their musculature and that 150,000 to 300,000 acquire new infection annually.

Clinical Syndromes page 862

page 863

Trichinosis is one of the few tissue parasitic diseases still seen in the United States. As with other parasitic infections, most patients have minimal or no symptoms. The clinical presentation depends largely on the tissue burden of organisms and the location of the migrating larvae. Patients in whom no more than 10 larvae are deposited per gram of tissue are usually asymptomatic; those with at least 100 generally have significant disease; and those with 1000 to 5000 have a very serious course that occasionally ends in death. In mild infections with few migrating larvae, patients may experience only an influenza-like syndrome with slight fever and mild diarrhea. With more extensive larval migration, persistent fever, gastrointestinal distress, marked eosinophilia, muscle pain, and periorbital edema occur. "Splinter" hemorrhages beneath the nails, a common finding, are probably caused by vasculitis resulting from toxic secretions of the migrating larvae. In heavy infections, severe neurologic symptoms, including psychosis, meningoencephalitis, and cerebrovascular accident, may occur. Patients who survive the migration, muscle destruction, and encystment of larvae in moderate infections experience a decline in clinical symptoms in 5 or 6 weeks. Lethal trichinosis results when myocarditis, encephalitis, and pneumonitis combine; the patient dies 4 to 6 weeks after infection. Respiratory arrest often follows heavy invasion and muscle destruction in the diaphragm.

Laboratory Diagnosis The diagnosis is usually established with clinical observations, especially when an outbreak can be traced to consumption of improperly cooked pork or bear meat. The laboratory may confirm the diagnosis if the encysted larvae are detected in the implicated meat or in a muscle biopsy specimen from the patient. Marked eosinophilia is characteristically present in patients with trichinosis. Serologic procedures are also available for confirmation of the diagnosis. Significant antibody titers are usually absent before the third week of illness but then may persist for years.

Treatment, Prevention, and Control Treatment of trichinosis is primarily symptomatic, since there are no good antiparasitic agents for tissue larvae. Treatment of the adult worms in the intestine with mebendazole may halt the production of new larvae. Steroids, along with thiabendazole or mebendazole, are recommended for severe symptoms. In infections caused by T. pseudospiralis, albendazole may be effective. Education regarding disease transmission from pork and bear meat is essential, especially the recommendation that pork and bear meat be cooked until the interior is gray. Microwave cooking and smoking or drying meat do not kill all larvae. Laws regulating the feeding of garbage to pigs help control transmission, as may regulations controlling the foraging of bears in garbage pits and public parks. Freezing pork, as conducted in federally inspected meat packing plants, has reduced transmission. Quick freezing of pork at -40° C effectively destro ys the organisms, as does low-temperature storage at -15° C for 20 days or more.

Figure 83-12 Life cycle of W. bancrofti.

Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Wuchereria bancrofti and Brugia malayi Physiology and Structure Because of their many similarities, W. bancrofti and B. malayi are discussed together. Human infection is initiated by the introduction of infective larvae, present in the saliva of a biting mosquito, into a bite wound (Figure 83-12). Various species of Anopheles, Aedes, and Culex mosquitoes are vectors of Bancroft and Malayan filariasis. The larvae migrate from the location of the bite to the lymphatic system, primarily in the arms, legs, or groin, where larval growth to adulthood occurs. From 3 to 12 months after the initial infection, the adult male worm fertilizes the female, which in turn produces the sheathed larval microfilariae that find their way into the circulation. The presence of microfilariae in blood is diagnostic for human disease and is infective for feeding mosquitoes. In the mosquito, the larvae move through the stomach and thoracic muscles in developmental stages and finally migrate to the proboscis. There they become infective, third-stage larvae and are transmitted by the feeding mosquito. The adult form in humans can persist for as long as 10 years.

Epidemiology page 863 page 864

Infection with W. bancrofti occurs in tropical and subtropical areas and is endemic in central Africa, along the Mediterranean coast, and in many parts of Asia, including China, Korea, Japan, and the Philippines. It is also present in Haiti, Trinidad, Surinam, Panama, Costa Rica, and Brazil. No animal reservoir has been identified. B. malayi is found primarily in Malaysia, India, Thailand, Vietnam, and parts of China, Korea, Japan, and many Pacific islands. Animal reservoirs such as cats and monkeys are recognized.

Clinical Syndromes In some patients, there is no sign of disease, even though blood specimens may show the presence of many microfilariae. In other patients, early acute symptoms are fever, lymphangitis and lymphadenitis with chills, and recurrent febrile attacks. The acute presentation is thought to result from the inflammatory response to the presence of molting adolescent worms and dead or dying adults within the lymphatic vessels. As the infection progresses, the lymph nodes enlarge, possibly involving many parts of the body, including the extremities, the scrotum, and the testes, with occasional abscess formation. This results from the physical obstruction of lymph in the vessels caused by the presence of adult worms and host reactivity in the lymphatic system. This process may be complicated by recurrent bacterial infections, which contribute to the tissue damage. The thickening and hypertrophy of tissues infected with the worms may lead to the enlargement of tissues, especially the extremities, progressing to filarial elephantiasis. Filariasis of this type is thus a chronic, debilitating, and disfiguring disease requiring prompt diagnosis and treatment. Occasionally, ascites and pleural effusions secondary to rupture of the enlarged lymphatic vessels into the peritoneal or pleural cavity may be observed.

Laboratory Diagnosis

Eosinophilia is usually present during acute inflammatory episodes; however, demonstration of microfilariae in the blood is required for definitive diagnosis. As with malaria, microfilariae can be demonstrated in Giemsa-stained blood films in infections with W. bancrofti and B. malayi (Figures 83-13 and 83-14). Concentration of anticoagulated blood specimens and urine specimens are also valuable procedures. Buffy coat films concentrate the white blood cells and are useful for the detection of microfilariae. The presence of small numbers of microfilariae in blood can be detected by a membrane-filtration technique in which anticoagulated blood is mixed with saline and forced through a 5-µm membrane filter. After several washes with saline or distilled water, the filter is examined microscopically for living microfilariae, or it is dried, fixed, and stained as for a thin blood film. W. bancrofti and B. malayi have both nocturnal and subperiodic periodicity in the production of microfilariae. Nocturnal periodicity results in greater numbers of microfilariae in blood at night, whereas with the subperiodic form, microfilariae are present at all times, with a peak in the afternoon.

Figure 83-13 Giemsa stain of sheathed W. bancrofti microfilaria in blood smear; 245 to 295 µm long × 7 to 10 µm wide.

W. bancrofti, as well as B. malayi and Loa loa, demonstrate a sheath on their microfilariae. This can be the first step in identifying the specific types of filariasis. Further identification is based on study of head and tail structures (Figure 83-15). Clinically, an exact species identification is not critical, because treatment for all the filarial infections, except Onchocerca volvulus, is identical. Serologic testing is also available through reference laboratories so that a diagnosis can be reached. Detection of circulating filarial antigens is promising but is not widely available as a diagnostic test.

Treatment, Prevention, and Control

Figure 83-14 Giemsa stain of sheathed B. malayi microfilaria in blood smear; 180 to 230 µm long × 5 to 6 µm wide. page 864 page 865

Figure 83-15 Differentiation of microfilariae. Identification of microfilariae is based on the presence of a sheath covering the larvae, as well as the distribution of nuclei in the tail region. A, W. bancrofti. B, B. malayi. C, L. loa. D, O. volvulus. E, Mansonella perstans. F, Mansonella streptocerca. G, Mansonella ozzardi.

Treatment is of little benefit in most cases of chronic lymphatic filariasis. The drug of choice for treatment of W. bancrofti and B. malayi infections is diethylcarbamazine (DEC). Ivermectin and albendazole may also be used, often in combination with DEC. Supportive and surgical therapy for lymphatic obstruction may be of some cosmetic help. Education regarding filarial infections, mosquito control, use of protective clothing and insect repellents, and treatment of infections to prevent further transmission is essential. Control of B. malayi infections is more difficult because of the presence of disease in animal reservoirs. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Loa loa

Physiology and Structure The life cycle of L. loa is similar to that illustrated in Figure 83-12, except the vector is a biting fly called Chrysops, the mango fly. Approximately 6 months after infection, the production of microfilariae starts and can persist for 17 years or more. Adult worms can migrate through subcutaneous tissues, through muscle, and in front of the eyeball.

Epidemiology L. loa is confined to the equatorial rain forests of Africa and is endemic in tropical West Africa, the Congo basin, and parts of Nigeria. Monkeys in these areas serve as reservoir hosts in the life cycle, with mango flies as vectors.

Clinical Syndromes

Figure 83-16 Giemsa stain of sheathed L. loa microfilaria in blood smear; 230 to 250 µm long × 6 to 9 µm wide.

Symptoms usually do not appear until a year or so after the fly bite, because the worms are slow in reaching adulthood. One of the first signs of infection is the so-called fugitive or Calabar swellings. These swellings are transient and usually appear on the extremities, produced as the worms migrate through subcutaneous tissues, creating large, nodular areas that are painful and pruritic. Because eosinophilia (50% to 70%) is observed, Calabar swellings are believed to result from allergic reactions to the worms or their metabolic products. Adult L. loa worms can also migrate under the conjunctiva, producing irritation, painful congestion, edema of the eyelids, and impaired vision. The presence of a worm in the eye can obviously cause anxiety in the patient. The infection may be long lived and in some cases asymptomatic.

Laboratory Diagnosis The clinical observation of Calabar swellings or migration of worms in the eye, combined with eosinophilia, should alert the physician to consider infection with L. loa. The microfilariae can be found in the blood (Figure 83-16). In contrast to the other filariae, L. loa is primarily present during the daytime. Serologic testing can also be useful for confirming the diagnosis but is not readily available.

Treatment, Prevention, and Control

Diethylcarbamazine is effective against adults and microfilariae; however, destruction of the parasites may induce severe allergic reactions that require treatment with corticosteroids. Albendazole or ivermectin (not approved by FDA) has been shown to be effective in reducing microfilarial loads. Surgical removal of worms migrating across the eye or bridge of the nose can be accomplished by immobilizing the worm with instillation of a few drops of 10% cocaine. Education regarding the infection and its vector, especially for people entering the known endemic areas, is essential. Protection from fly bites by using screening, appropriate clothing, and insect repellents, along with treatment of cases, is also critical in reducing the incidence of infection. However, the presence of disease in animal reservoirs (e.g., monkeys) limits the feasibility of controlling this disease. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Onchocerca volvulus Physiology and Structure

Figure 83-17 These images are not available online due to electronic permissions.

Infection occurs after the introduction of O. volvulus larvae through the skin during the biting and feeding of the Simulium or blackfly vector (Figure 83-17). The larval worms migrate from the skin to subcutaneous tissue and develop into adult male and female worms. The adults become encased in fibrous subcutaneous nodules within which they may remain viable for as long as 15 years. The female worm, after fertilization by the male, begins producing as many as 2000 nonsheathed microfilariae each day. The microfilariae exit the capsule and migrate to the skin, the eyes, and other body tissues. These nonsheathed microfilariae appearing in skin tissue are infective for feeding blackflies.

Epidemiology O. volvulus is endemic in many parts of Africa, especially in the Congo basin and the Volta River basin. In the western hemisphere, it occurs in many Central and South American countries. Onchocerciasis affects more than 18 million people worldwide and causes blindness in approximately 5% of infected people. Several species of the blackfly genus Simulium serve as vectors but none so appropriately named as the principal vector, Simulium damnosum ("the damned blackfly"). These blackflies, or buffalo gnats, breed in fast-flowing streams, which makes control or eradication by insecticides almost impossible, because the chemicals are rapidly washed away from the eggs and larvae. There is a greater prevalence of infection in men than women in endemic areas because of their work in or near the streams where the blackflies breed. Studies in endemic areas in Africa have shown that 50% of men are totally blind before they reach 50 years of age. This accounts for the common term river blindness, which is applied to the disease onchocerciasis. This fear of blindness has created an additional problem in many parts of Africa, because whole villages leave the area near streams and farmland that could produce food. The migrating populations then find themselves in areas where they face starvation. page 866

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Clinical Syndromes (Clinical Case 83-4) Clinical onchocerciasis is characterized by infection involving the skin, subcutaneous tissue, lymph nodes, and eyes. The clinical manifestations of the infection are due to the acute and chronic inflammatory reaction to antigens released by the microfilariae as they migrate through the tissues. The incubation period from infectious larvae to adult worms is several months to a year. The initial signs of disease are fever, eosinophilia, and urticaria. As the worms mature, copulate, and produce microfilariae, subcutaneous nodules begin to appear on any part of the body. These nodules are most dangerous when they are present on the head and neck, because the microfilariae may migrate to the eyes and cause serious tissue damage, leading to blindness. The mechanisms for development of eye disease are thought to be a combination of both direct invasion by the microfilaria and antigen-antibody complex deposition within the ocular tissues. Patients progress from conjunctivitis with photophobia to punctate and sclerosing keratitis. Internal eye disease with anterior uveitis, chorioretinitis, and optic neuritis may also occur. Within the skin, the inflammatory process results in loss of elasticity and areas of depigmentation, thickening, and atrophy. A number of skin conditions, including pruritus, hyperkeratosis, and myxedematous thickening, are related to the presence of this parasite. A form of elephantiasis called hanging groin also occurs when the nodules are located near the genitalia.

Clinical Case 83-4. Onchocerciasis

Imtiaz and colleagues (Infect Med 22:187-189, 2005) describe the case of a 21-year-old man who immigrated from the Sudan to the United States 1 year prior to presenting with a maculopapular rash that was associated with severe pruritus. The rash and pruritus had been present for the past 3 to 4 years. In the past, the patient had undergone multiple treatments for this condition, including corticosteroids, without relief. The patient denied any systemic symptoms but did complain of blurred vision. On physical examination, his skin was somewhat thickened over different parts of the body, and he had scattered maculopapular lesions with increased pigmentation; some lesions had keloid nodules, as well as wrinkling. There was no lymphadenopathy. The remainder of his evaluation was unremarkable. Because of the presence of intense pruritus unresponsive to treatment, blurred vision, and the prevalence of onchocerciasis in his native country, skin snips were taken from the scapular area. Microfilariae of Onchocerca volvulus were revealed on microscopic examination. Ivermectin was prescribed, to which the patient's condition responded. Onchocerciasis, although not common in the United States, should be considered in immigrants and expatriates with suggestive symptoms if they came from areas in which the disease is endemic.

Figure 83-18 Giemsa stained unsheathed O. volvulus microfilaria; 300 to 315 µm long × 5 to 9 µm wide.

Laboratory Diagnosis The diagnosis of onchocerciasis is made by the demonstration of microfilariae in skin snip preparations from the infrascapular or gluteal region. A sample is obtained by raising the skin with a needle and shaving the epidermal layer with a razor. The specimen is incubated in saline for several hours and is then inspected with a dissecting microscope for the presence of nonsheathed microfilariae (Figure 83-18). In patients with ocular disease, the organism may also be seen in the anterior chamber with the aid of a slit lamp. Serologic methods using recombinant antigens have been useful as have assays using PCR to detect onchocercal DNA in skin snip specimens.

Treatment, Prevention, and Control

Figure 83-19 Cross-section of an adult female O. volvulus in an excised nodule showing numerous microfilariae. page 867 page 868

Surgical removal of the encapsulated nodule is often performed to eliminate the adult worms and stop production of microfilariae (Figure 83-19). In addition, treatment with ivermectin is recommended. A single oral dose of ivermectin (150 mg/kg) greatly reduces the number of microfilariae in the skin and eyes, thus diminishing the likelihood of developing a disabling onchocerciasis. In endemic areas, the dose of ivermectin can be repeated every 6 to 12 months to maintain suppression of dermal and ocular microfilariae. Suppression of dermal microfilariae reduces the transmission of this vectorborne disease, and thus mass chemotherapy may prove to be a successful strategy for the prevention of onchocerciasis. At present there is no firm evidence that O. volvulus is becoming resistant to ivermectin; however, whenever a single agent is used for disease control with varying doses over a long period of time, it is prudent to be on guard for the possibility of resistance developing.

Education regarding the disease and its transmission is essential. Protection from blackfly bites through the use of protective clothing, screening, and insect repellents, as well as prompt diagnosis and treatment of infections to prevent further transmission, are critical. Although control of blackfly breeding is difficult because insecticides wash away in the streams, some form of biologic control of this vector may reduce fly reproduction and disease transmission. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Dirofilaria immitis Several mosquito-transmitted filariae infect dogs, cats, raccoons, and bobcats in nature and occasionally are found in humans. D. immitis, the dog heartworm, is notorious for forming a lethal worm bolus in the dog's heart. This nematode may also infect humans, producing a nodule called a coin lesion in the lung. Only very rarely have these worms been found in human hearts. The coin lesion in the lung presents a problem for the radiologist and the surgeon because it resembles a malignancy requiring surgical removal. Unfortunately, no laboratory test can provide an accurate diagnosis of dirofilariasis. Peripheral eosinophilia is rare, and the radiographic features are insufficient to allow the clinician to distinguish pulmonary dirofilariasis from bronchogenic carcinoma. Serologic tests are not sufficiently sensitive or specific to preclude the surgical intervention. A definitive diagnosis is made when a thoracotomy specimen is examined microscopically, revealing the typical cross sections of the parasite. Transmission of the filarial infections can be controlled by mosquito control and the prophylactic use of the drug ivermectin in dogs. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Dracunculus medinensis The name D. medinensis means "little dragon of Medina." This is a very ancient worm infection thought by some scholars to be the "fiery serpent" noted by Moses with the Israelites at the Red Sea.

Figure 83-20 Life cycle of D. medinensis.

Physiology and Structure

D. medinensis is not a filarial worm but is a tissue-invading nematode of medical importance in many parts of the world. The worms have a very simple life cycle, depending on freshwater and a microcrustacean (copepod) of the genus Cyclops (Figure 83-20). When Cyclops species harboring larval D. medinensis are ingested in drinking water, the infection is initiated with liberation of the larvae in the stomach. These larvae penetrate the wall of the digestive tract and migrate to the retroperitoneal space, where they mature. These larvae are not microfilariae and do not appear in the blood or other tissues. Male and female worms mate in the retroperitoneum, and the fertilized female then migrates to the subcutaneous tissues, usually in the extremities. When the fertilized female worm becomes gravid, a vesicle is formed in the host tissue, which will ulcerate. When the ulcer is completely formed, the worm protrudes a loop of uterus through the ulcer. On contact with water, the larval worms are released. The larvae are then ingested by the Cyclops species in freshwater, where they are then infective for humans or animals drinking the water containing the Cyclops species.

Epidemiology D. medinensis occurs in many parts of Asia and equatorial Africa, infecting an estimated 10 million people. Reservoir hosts include dogs and many fur-bearing animals that come into contact with drinking water containing infective Cyclops species. page 868 page 869

Human infections usually result from ingestion of water from so-called "step wells" where people stand or bathe in the water, at which time the gravid female worm discharges larvae from lesions on the arms, legs, feet, and ankles to infect Cyclops species in the water. Ponds and standing water are occasionally the source of infection when humans use them for drinking water.

Clinical Syndromes

Symptoms of infection usually do not appear until the gravid female creates the vesicle and the ulcer in the skin for the liberation of larval worms. This occurs usually 1 year after initial exposure. At the site of the ulcer, there are erythema and pain, as well as an allergic reaction to the worm. There is also the possibility of abscess formation and secondary bacterial infection, leading to further tissue destruction and inflammatory reaction with intense pain and sloughing of skin. If the worm is broken in attempts to remove it, there may be toxic reactions, and if the worm dies and calcifies, there may be nodule formation and some allergic reaction. Once the gravid female worm has discharged all the larvae, it may retreat into deeper tissue, where it is gradually absorbed, or it may simply be expelled from the site.

Laboratory Diagnosis Diagnosis is established by observing the typical ulcer and by flooding the ulcer with water to recover the larval worms when they are discharged. Occasionally, x-ray examination reveals worms in various parts of the body.

Treatment, Prevention, and Control The ancient method of slowly wrapping the worm on a twig is still used in many endemic areas (Figure 83-21). Surgical removal is also a practical and reliable procedure for the patient. There is no evidence that any chemotherapeutic agent has a direct effect on D. medinensis, although various benzimidazoles may have an antiinflammatory effect and either eliminate the worm or make surgical removal easier. Treatment with mebendazole has been associated with aberrant migration of the worms, with the result that they were more likely to emerge at anatomic sites other than the lower limbs.

Education regarding the life cycle of the worm and avoidance of water contaminated with Cyclops species are critical. Protection of drinking water by prohibiting bathing and washing of clothing in wells is essential. Persons who live in or travel to endemic areas should boil water before drinking it. The treatment of water with chemicals and the use of fish that consume Cyclops species as food also help control transmission. Prompt diagnosis and treatment of cases also limit further transmission. These preventive measures have been incorporated into an ongoing global effort to eliminate dracunculiasis with dramatic success. The annual incidence of worldwide disease has been reduced by 98%, with complete eradication in 7 countries.

Figure 83-21 Removal of a D. medinensis adult from an exposed ulcer by winding the worm slowly around a stick. (From Binford CH, Conner DH: Pathology of Tropical and Extraordinary Diseases. Washington, DC, Armed Forces Institute of Pathology, 1976.)

Case Study and Questions

A 10-year-old boy was brought in by his father for evaluation of crampy abdominal pain, nausea, and mild diarrhea that had persisted for approximately 2 weeks. On the day before evaluation, the boy reported to his parents that he passed a large worm into the toilet during a bowel movement. He flushed the worm before the parents could see it. Physical examination was completely unremarkable. The boy had no fever, cough, or rash and did not complain of anal pruritus. His travel history was unremarkable. Examination of a stool specimen revealed the diagnosis. 1. Which intestinal parasites of humans are nematodes? 2. Which nematode was likely in this case? What organisms may be found in stool? 3. What was the most likely means of acquisition of this parasite? 4. Was this patient at risk of autoinfection? 5. Describe the life cycle of this parasite. 6. Can this parasite cause extraintestinal symptoms? What other organs may be invaded and what might stimulate extraintestinal invasion?

Bibliography Barry M: The tail end of guinea worm - Global eradication without a drug or a vaccine. N Engl J Med 356:2561-2564, 2007. Bruschi F, Murrell KD: New aspects of human trichinellosis: The impact of new Trichinella species. Postgrad Med J 78:15-22, 2002. Cairncross S, Muller R, Zagaria N: Dracunculiasis (Guinea worm disease) and the eradication initiative. Clin Microbiol Rev 15:223-246, 2002. Despommier D: Toxocariasis: Clinical aspects, epidemiology, medical ecology and molecular aspects. Clin Microbiol Rev 16:265-272, 2003. Garcia LS: Diagnostic Medical Parasitology, 4th ed. Washington, DC, ASM Press, 2001. page 869 page 870

Keiser PB, Nutman TB: Strongyloides stercoralis in the immunocompromised population. Clin Microbiol Rev 17:208-217, 2004. Gavin PJ, Kazacos KR, Shulman ST: Baylisascariasis. Clin Microbiol Rev 18:703-718, 2005. Hotez PJ, et al: Hookworm infection. N Engl J Med 351:799-807, 2004. Hotez PJ, et al: Control of neglected tropical diseases. N Engl J Med 357:1018-1027, 2007. McPherson T, Nutman TB: Filarial nematodes. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Procop GW, Neafie RC: Less common helminths. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Sheorey H, Biggs BA, Traynor P: Nematodes. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press, 2007. Strickland GT: Hunter's Tropical Medicine and Emerging Infectious Diseases. Philadelphia, WB Saunders, 2000. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Fasciolopsis buski A number of intestinal flukes are recognized, including F. buski, Heterophyes heterophyes, Metagonimus yokogawai, Echinostoma ilocanum, and Gastrodiscoides hominis. F. buski is the largest, most prevalent, and most important intestinal fluke. The other flukes are similar to F. buski in many respects (epidemiology, clinical syndromes, treatment) and are not discussed further. It is important only that physicians recognize the relationship among these different flukes.

Physiology and Structure This large intestinal fluke has a typical life cycle (Figure 84-1). Humans ingest the encysted larval stage (metacercaria) when they peel the husks from aquatic vegetation (e.g., water chestnuts) with the teeth. The metacercariae are scraped from the husk, swallowed, and develop into immature flukes in the duodenum. The fluke attaches to the mucosa of the small intestine with two muscular suckers, develops into an adult form, and undergoes self-fertilization. Egg production is initiated 3 months after the initial infection with the metacercariae. The operculated eggs pass in feces to water, where the operculum at the top of the eggshell pops open, liberating a free-swimming larval stage (miracidium). Glands at the pointed anterior end of the miracidium produce lytic substances that allow the penetration of the soft tissues of snails. In the snail tissue, the miracidium develops through a series of stages by asexual germ cell propagation. The final stage (cercaria) in the snail is a free-swimming form that, after release from the snail, encysts on the aquatic vegetation, becoming the metacercariae, or infective stage.

Epidemiology Because it depends on the distribution of its appropriate snail host, F. buski is found only in China, Vietnam, Thailand, parts of Indonesia, Malaysia, and India. Pigs, dogs, and rabbits serve as reservoir hosts in these endemic areas.

Clinical Syndromes The symptomatology of F. buski infection relates directly to the worm burden in the small intestine. Attachment of the flukes in the small intestine can produce inflammation, ulceration, and hemorrhage. Severe infections produce abdominal discomfort similar to that of a duodenal ulcer, as well as diarrhea. Stools may be profuse, a malabsorption syndrome similar to giardiasis is common, and intestinal obstruction can occur. Marked eosinophilia is also present. Although death can occur, it is rare. page 871 page 872

Table 84-1. Medically Important Trematodes Trematode Fasciolopsis buski

Common Intermediate Biologic Reservoir Name Host Vector Host Giant Snail Water plants Pigs, dogs, intestinal (e.g., water rabbits, fluke chestnuts) humans

Fasciola hepatica

Sheep Snail liver fluke

Water plants Sheep, (e.g., cattle, watercress) humans

Opisthorchis (Clonorchis) sinensis

Chinese Snail, liver fluke freshwater fish

Uncooked fish

Dogs, cats, humans

Uncooked crabs, crayfish

Pigs, monkeys, humans

Paragonimus Lung westermani fluke

Snail, freshwater crabs, crayfish

Schistosoma species

Blood fluke

Snail

None

Primates, rodents, domestic pets, livestock, humans

Laboratory Diagnosis Stool examination reveals the large, golden, bile-stained eggs with an operculum on the top (Figure 84-2). The measurements and appearance of F. buski eggs are similar to that of the liver fluke F. hepatica, and differentiation of the eggs of these species usually is not possible. Large (approximately 1.5 to 3.0 cm) adult flukes can rarely be found in feces or specimens collected at surgery.

Treatment, Prevention, and Control

Figure 84-1 Life cycle of Fasciolopsis buski (giant intestinal fluke).

The drug of choice is praziquantel, and the alternative is niclosamide. Education regarding the safe consumption of infective aquatic vegetation (particularly water chestnuts), proper sanitation, and control of human feces reduces the incidence of disease. In addition, the snail population may be eliminated with molluscacides. When infection occurs, treatment should be initiated promptly to minimize its spread. Control of the reservoir hosts also reduces transmission of the worm. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Fasciola hepatica A number of liver flukes are recognized, including F. hepatica, Opisthorchis sinensis, O. felineus, and Dicrocoelium dendriticum. Only F. hepatica and O. sinensis are discussed in this chapter, although the eggs of other flukes are occasionally detected in the feces of patients in other geographical areas.

Physiology and Structure Commonly called the sheep liver fluke, F. hepatica is a parasite of herbivores (particularly sheep and cattle) and humans. Its life cycle (Figure 84-3) is similar to that of F. buski, with human infection resulting from the ingestion of watercress that harbors the encysted metacercariae. The larval flukes then migrate through the duodenal wall and across the peritoneal cavity, penetrate the liver capsule, pass through the liver parenchyma, and enter the bile ducts to become adult worms. Approximately 3 to 4 months after the initial infection, the adult flukes start producing operculated eggs that are identical to those of F. buski, as seen in stool examination.

Epidemiology page 872

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Figure 84-2 Fasciolopsis buski egg, 130 to 150 µm long and 65 to 90 µm wide, with a thin operculum at one end.

Infections have been reported worldwide in sheep-raising areas, with the appropriate snail as an intermediate host. These areas include the former Soviet Union, Japan, Egypt, and many Latin American countries. Outbreaks are directly related to human consumption of contaminated watercress in areas where infected herbivores are present. Human infection is rare in the United States, but several well-documented cases have been reported in travelers from endemic areas.

Clinical Syndromes (Clinical Case 84-1)

Figure 84-3 Life cycle of Fasciola hepatica (sheep liver fluke).

Migration of the larval worm through the liver produces irritation of this tissue, tenderness, and hepatomegaly. Pain in the right upper quadrant, chills, fever, and marked eosinophilia are commonly observed. As the worms take up residence in the bile ducts, their mechanical irritation and toxic secretions produce hepatitis, hyperplasia of the epithelium, and biliary obstruction. Some worms penetrate eroded areas in the ducts and invade the liver to produce necrotic foci referred to as liver rot. In severe infections, secondary bacterial infection can occur, and portal cirrhosis is common.

Laboratory Diagnosis

Stool examination reveals operculated eggs indistinguishable from the eggs of F. buski. Exact identification is a therapeutic problem because treatment is not the same for both infections. Whereas F. buski responds favorably to praziquantel, F. hepatica does not. When exact identification is desired, examination of a sample of the patient's bile differentiates the species; if the eggs are present in bile, they are F. hepatica, not F. buski, which is limited to the small intestine. Eggs may appear in stool samples from people who have eaten infected sheep or cattle liver. The spurious nature of this finding can be confirmed by having the patient refrain from eating liver and then rechecking the stool.

Treatment, Prevention, and Control In contrast to F. buski, F. hepatica responds poorly to praziquantel. Treatment with bithionol or the benzimidazole compound triclabendazole has been effective. Preventive measures are similar to those for F. buski control; people who live in areas frequented by sheep and cattle should especially avoid ingestion of watercress and other uncooked aquatic vegetation.

Clinical Case 84-1. Fascioliasis

Echenique-Elizondo and colleagues (JOP 6:36-39, 2005) described a case of acute pancreatitis due to the liver fluke Fasciola hepatica. The patient was a 31-year-old female who was admitted to the hospital because of a sudden onset of nausea and upper abdominal pain. She was otherwise healthy and gave a negative history of drug abuse, alcohol ingestion, gallstone disease, abdominal trauma or surgery. On physical exam, she was markedly tender in the epigastric region and had hypoactive bowel sounds. Serum chemistries showed elevated pancreatic enzymes (amylase, lipase, pancreatic phospholipase A2, and elastase). Her white blood count was elevated, as were tests for alkaline phosphatase and bilirubin. Serum blood urea nitrogen, creatinine, LDH and calcium were normal. Abdominal ultrasonography and CT scan showed diffuse enlargement of the pancreas, and a cholangiogram demonstrated dilatation and numerous filling defects in the common bile duct. An endoscopic sphincterotomy was performed, with extraction of numerous large flukes that were identified as F. hepatica. The patient was treated with a single oral dose of triclabendazole (10 mg/kg). Follow-up demonstrated normal blood chemistries and no evidence of disease 2 years postprocedure.

Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Opisthorchis sinensis Physiology and Structure

O. sinensis, also referred to as Clonorchis sinensis in the older literature, is commonly called the Chinese liver fluke. (Figure 84-4) illustrates its life cycle, which involves two intermediate hosts. This trematode differs from other fluke cycles in that the eggs are eaten by the snail, and then reproduction begins in the soft tissues of the snail. O. sinensis also requires a second intermediate host, freshwater fish, where the cercariae encyst and develop into infective metacercariae. When uncooked freshwater fish harboring metacercariae are eaten, flukes develop first in the duodenum and then migrate to the bile ducts, where they become adults. The adult fluke undergoes self-fertilization and begins producing eggs. O. sinensis may survive in the biliary tract for as long as 50 years, producing approximately 2000 eggs per day. These eggs pass with feces and are once again eaten by snails, reinitiating the cycle.

Epidemiology O. sinensis is found in China, Japan, Korea, and Vietnam, where it is estimated to infect approximately 19 million people. It is one of the most frequent infections seen among Asian refugees, and it can be traced to the consumption of raw, pickled, smoked, or dried freshwater fish that harbor the viable metacercariae. Dogs, cats, and fish-eating mammals can also serve as reservoir hosts.

Figure 84-4 Life cycle of Opisthorchis sinensis (Chinese liver fluke).

Clinical Case 84-2. Cholangitis Due to Clonorchis (Opisthorchis) sinensis

Stunell, et al (Eur Radiol 16:2612-2614, 2006) describe a 34-year-old Asian woman who presented to a local emergency department with a 2-day history of right upper quadrant abdominal pain, fever, and rigors. She had emigrated from Asia to Ireland 18 months before and gave a history of intermittent upper abdominal pain occurring over a 3-year period. On examination, she appeared acutely ill and was clammy to the touch. She was febrile, tachycardic, and had mild scleral icterus. Her abdomen was tender, with guarding in the right upper quadrant. Routine hematologic and biochemical studies revealed a marked leukocytosis and obstructive liver function tests. Contrast-enhanced CT of the abdomen demonstrated evidence of multiple ovoid opacities within dilated intrahepatic bile ducts in the right lobe of the liver. The remainder of the liver parenchyma appeared normal. Upon stabilization of the patient, an endoscopic retrograde cholangiopancreatography (ERCP) was performed for biliary decompression. ERCP demonstrated intra- and extrahepatic bile duct dilatation, with multiple filling defects and strictures. A stool sample sent for analysis confirmed the presence of ova and adult flukes of Clonorchis (Opisthorchis) sinensis. The patient recovered with medical management (praziquantel) and had negative stool samples 30 days after treatment. This case, as well as Case 84-1, demonstrates the various complications of liver fluke infestation. Notably, praziquantel is the drug of choice for treating the Oriental liver fluke (Clonorchis sinensis), whereas triclabendazole is used to treat fascioliasis, thus emphasizing the importance of an epidemiologic history and identification of the fluke.

Clinical Syndromes (Clinical Case 84-2)

Infection in humans is usually mild and asymptomatic. Severe infections with many flukes in the bile ducts produces fever, diarrhea, epigastric pain, hepatomegaly, anorexia, and occasionally jaundice. Biliary obstruction may occur, and chronic infection can result in adenocarcinoma of the bile ducts. Invasion of the gallbladder may produce cholecystitis, cholelithiasis, and impaired liver function, as well as liver abscesses.

Laboratory Diagnosis The diagnosis is made by recovering the distinctive eggs from stool. The eggs measure 27 to 35 µm × 12 to 19 µm and are characterized by a distinct operculum with prominent shoulders and a tiny knob at the posterior (abopercular) pole (Figure 84-5). In mild infections, repeated examinations of stool or duodenal aspirates may be necessary. In acute symptomatic infection, there are usually eosinophilia and an elevation of serum alkaline phosphatase levels. Radiographic imaging procedures may detect abnormalities of the biliary tract. page 874 page 875

Figure 84-5 These images are not available online due to electronic permissions.

Treatment, Prevention, and Control The drug of choice is praziquantel. Prevention of infection is accomplished by not eating uncooked fish and by implementing proper sanitation policies, including the disposal of human, dog, and cat feces in adequately protected sites so that they cannot contaminate water supplies with the intermediate snail and fish hosts. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Paragonimus westermani Physiology and Structure P. westermani, commonly called the lung fluke, is one of several species of Paragonimus that infect humans and many other animals. Figure 84-6 shows a familiar fluke life cycle from egg to snail to infective metacercaria. The infective stage occurs in a second intermediate host: the muscles and gills of freshwater crabs and crayfish. In humans who ingest infected meat, the larval worm hatches in the stomach and follows an extensive migration through the intestinal wall to the abdominal cavity, then through the diaphragm, and finally to the pleural cavity. Adult worms reside in the lungs and produce eggs that are liberated from ruptured bronchioles and appear in sputum or, when swallowed, in feces.

Epidemiology

Figure 84-6 Life cycle of Paragonimus westermani (Oriental lung fluke).

Paragonimiasis occurs in many countries in Asia, Africa, India, and Latin America. It can be seen in refugees from Southeast Asia. Its prevalence is directly related to the consumption of uncooked freshwater crabs and crayfish. It is estimated that approximately 3 million people are infected with this lung fluke. As many as 1% of all Indochinese immigrants to the United States are infected with P. westermani. A wide variety of shore-feeding animals (e.g., wild boars, pigs, and monkeys) serve as reservoir hosts, and some human infections result from ingestion of meat containing migrating larval worms from these reservoir hosts. Human infections endemic to the United States are usually caused by a related species, P. kellicotti, which is found in crabs and crayfish in eastern and midwestern waters.

Clinical Syndromes (Clinical Case 84-3) The clinical manifestations of paragonimiasis may result from larvae migrating through tissues or from adults established in the lungs or other ectopic sites. The onset of disease coincides with larval migration and is associated with fever, chills, and high eosinophilia. The adult flukes in the lungs first produce an inflammatory reaction that results in fever, cough, and increased sputum. As the destruction of lung tissue progresses, cavitation occurs around the worms, sputum becomes blood tinged and dark with eggs (so-called rusty sputum), and patients experience severe chest pain. The resulting cavity may become secondarily infected with bacteria. Dyspnea, chronic bronchitis, bronchiectasis, and pleural effusion may be seen. Chronic infections lead to fibrosis in the lung tissue. The location of larvae, adults, and eggs in ectopic sites may produce severe clinical symptoms depending on the site involved. The migration of larval worms may result in invasion of the spinal cord and brain, producing severe neurologic disease (visual problems, motor weakness, and convulsive seizures) referred to as cerebral paragonimiasis. Migration and infection may also occur in subcutaneous sites, the abdominal cavity, and the liver. page 875

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Clinical Case 84-3. Paragonimiasis Singh, et al (Indian J Med Microbiol 23:131-134, 2005) describe a case of pleuropulmonary paragonimiasis mimicking pulmonary tuberculosis. The patient was a 21-year-old man who was admitted to the hospital for progressive dyspnea, with a 1-month history of headache, fever, cough with scant hemoptysis, fatigue, pleuritic pain, anorexia, and weight loss. He had a history of antituberculous therapy for 6 months without improvement clinically. Two months prior to admission, after ingesting three raw crabs, he had a 3-day episode of watery diarrhea. On hospital admission, the patient was cachectic and afebrile. There was bilateral dullness to percussion and absent breath sounds in the lower two thirds of the chest. He was found to be anemic and had clubbing without lymphadenopathy, cyanosis, of jaundice. A chest radiograph showed bilateral pleural effusions that were also confirmed by CT. Ultrasound-guided thoracentesis of the right lung yielded about 200 ml of yellowish fluid. The fluid was exudative and contained 2700 WBC per ml, 91% of which were eosinophils. Gram stain of the fluid was negative, as was culture for bacteria and fungi. Sputum smears revealed operculated yellowish eggs consistent with Paragonimus westermani infection. The patient was treated with a 3-day course of praziquantel and responded well. Notably, the right-sided plural effusion did not recur after the thoracentesis and praziquantel treatment. This case emphasizes the importance of making an etiologic diagnosis of a pleuropulmonary process in order to differentiate paragonimiasis from tuberculosis in regions where both are endemic infectious diseases.

Laboratory Diagnosis

Figure 84-7 These images are not available online due to electronic permissions.

Examination of sputum and feces reveals golden brown, operculated eggs (Figure 84-7). Pleural effusions, when present, should be examined for eggs. Chest x-ray films often show infiltrates, nodular cysts, and pleural effusion. Marked eosinophilia is common. Serologic procedures are available through reference laboratories and can be helpful, particularly in cases with extrapulmonary (e.g., central nervous system) involvement.

Treatment, Prevention, and Control The drug of choice is triclabendazole; praziquantel is an alternative. Education regarding the consumption of uncooked freshwater crabs and crayfish, as well as the flesh of animals found in endemic areas, is critical. Pickling and wine soaking of crabs and crayfish do not kill the infective metacercarial stage. Proper sanitation and control of the disposal of human feces are essential. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Schistosomes

Schistosomiasis is a major parasitic infection of tropical areas, with some 200 million infections worldwide. The three schistosomes most frequently associated with human disease are Schistosoma mansoni, S. japonicum, and S. haematobium. They collectively produce the disease called schistosomiasis, also known as bilharziasis or snail fever. As discussed earlier, the schistosomes differ from other flukes: they are male and female rather than hermaphroditic, and their eggs do not have an operculum. They also are obligate intravascular parasites and are not found in cavities, ducts, and other tissues. The infective forms are skin-penetrating cercariae liberated from snails, and these differ from other flukes in that they are not eaten on vegetation, in fish, or in crustaceans. Figure 84-8 illustrates the life cycle of the different schistosomes. Infection is initiated by ciliated, free-swimming cercaria in fresh water that penetrate intact skin, enter the circulation, and develop in the intrahepatic portal circulation (S. mansoni and S. japonicum) or in the vesical, prostatic, rectal, and uterine plexuses and veins (S. haematobium). The female has a long, slender, cylindrical body, whereas the shorter male, which appears cylindrical, is actually flat. The cylindrical appearance derives from folding the sides of the body to produce a groove, the gynecophoral canal, in which the female resides for fertilization. Both sexes have oral and ventral suckers and an incomplete digestive system, which is typical of a fluke. As the worms develop in the portal circulation, they elaborate a remarkable defense against host resistance. They coat themselves with substances that the host recognizes as itself; consequently, there is little host response directed against their presence in blood vessels. This protective mechanism accounts for chronic infections that may last 20 to 30 years or longer. page 876 page 877

Figure 84-8 Life cycle of schistosomes.

After developing in the portal vein, the male and female adult worms pair up and migrate to their final locations, where fertilization and egg production begin. S. mansoni and S. japonicum are found in mesenteric veins and produce intestinal schistosomiasis; S. haematobium occurs in veins around the urinary bladder and causes vesicular schistosomiasis. On reaching the submucosal venules of their respective locations, the worms initiate oviposition, which may continue at the rate of 300 to 3000 eggs daily for 4 to 35 years. Although the host inflammatory response to the adult worms is minimal, the eggs elicit an intense inflammatory reaction, with mononuclear and polymorphonuclear cellular infiltrates and the formation of microabscesses. In addition, the larvae inside the eggs produce enzymes that aid in tissue destruction and allow the eggs to pass through the mucosa and into the lumen of the bowel and bladder, where they are passed to the external environment in the feces and urine, respectively. The eggs hatch quickly on reaching fresh water to release motile miracidia. The miracidia then invade the appropriate snail host, where they develop into thousands of infectious cercariae. The free-swimming cercariae are released into the water, where they are immediately infectious for humans and other mammals. The infection is similar in all three species of human schistosomes, in that disease results primarily from the host's immune response to the eggs. The very earliest signs and symptoms are due to the penetration of the cercariae through the skin. Immediate and delayed hypersensitivity to parasite antigens result in an intensely pruritic papular skin rash. The onset of oviposition results in a symptom complex known as Katayama syndrome, which is marked by fever, chills, cough, urticaria, arthralgias, lymphadenopathy, splenomegaly, and abdominal pain. This syndrome is typically seen 1 to 2 months after primary exposure and may persist for 3 months or more. It is thought to result from the massive release of parasite antigens, with subsequent immune complex formation. Associated laboratory abnormalities include leukocytosis, eosinophilia, and polyclonal gammopathy.

The more chronic and significant phase of schistosomiasis is due to the presence of eggs in various tissues and the resulting formation of granulomas and fibrosis. The retained eggs induce extensive inflammation and scarring, the clinical significance of which is directly related to the location and number of eggs. Because of differences in some aspects of disease and epidemiology, these worms are discussed as separate species. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Schistosoma mansoni Physiology and Structure S. mansoni usually resides in the small branches of the inferior mesenteric vein near the lower colon. The species of Schistosoma can be differentiated by their characteristic egg morphology (Figures 84-9 to 84-11). The eggs of S. mansoni are oval, possess a sharp lateral spine, and measure 115 to 175 µm × 45 to 70 µm (see Figure 84-9).

Epidemiology

Figure 84-9 Schistosoma mansoni egg. These eggs are 115 to 175 µm long and 45 to 70 µm wide, contain a miracidium, and are enclosed in a thin shell with a prominent lateral spine. page 877 page 878

Figure 84-10 These images are not available online due to electronic permissions.

The geographical distribution of the various species of Schistosoma depends on the availability of a suitable snail host. S. mansoni is the most widespread of the schistosomes and is endemic in Africa, Saudi Arabia, and Madagascar. It has also become well established in the western hemisphere, particularly in Brazil, Suriname, Venezuela, parts of the West Indies, and Puerto Rico. Cases originating in these areas may present in the United States. In all of these areas, there are also reservoir hosts, specifically primates, marsupials, and rodents. Schistosomiasis may be considered a disease of economic progress; the development of massive land irrigation projects in desert and tropical areas has resulted in the dispersion of infected humans and snails to previously uninvolved areas.

Clinical Syndromes (Clinical Case 84-4)

Figure 84-11 Schistosoma haematobium egg. These eggs are similar in size to those of Schistosoma mansoni but can be differentiated by the presence of a terminal, rather than lateral, spine.

Clinical Case 84-4. Schistosomiasis

Ferrari (Medicine [Baltimore] 78:176-190, 1999) described a case of neuroschistosomiasis due to Schistosoma mansoni in an 18-year-old Brazilian man. The patient was admitted to the hospital because of the recent onset of paraplegia. He was in good health until 33 days prior to admission, when he noted the onset of progressive low back pain with radiation to the lower limbs. During this period, he was evaluated three times in another institution, where x-ray films of the lower thoracic, lumbar, and sacral spine were normal. He received antiinflammatory agents, with only transient relief in his symptoms. Four weeks after the pain began, the disease progressed acutely with sexual impotence, fecal and urinary retention, and paraparesis progressing to paraplegia. At this time, the pain disappeared, replaced by a marked impairment of sensation in the lower limbs. On admission to the hospital, he gave a history of exposure to schistosomal infection. Neurologic examination revealed flaccid paraplegia, marked sensory loss, and absence of superficial and deep reflexes at and below the level T11. The CSF contained 84 WBC/mm3 (98% lymphocytes, 2% eosinophils) and 1 red blood cell, 82 mg/dL total protein, and 61 mg/dL glucose. Myelography, CT-myelography, and magnetic resonance imaging (MRI) showed a slight widening of the conus. The diagnosis of neuroschistosomiasis was confirmed by the demonstration of viable and dead eggs of S. mansoni on rectal mucosal biopsy. The concentration of CSF IgG against soluble egg antigen of S. mansoni quantitated by ELISA was 1.53 µg/ml. He was treated with prednisone and praziquantel. Despite therapy, his condition remained unaltered at follow-up 7 months later. S. mansoni is the most frequently reported cause of schistosomal myeloradiculopathy (SMR) worldwide. SMR is among the most severe forms of schistosomiasis, and prognosis depends largely on early diagnosis and treatment.

As noted before, cercarial penetration of intact skin may be seen as dermatitis with allergic reactions, pruritus, and edema. Migrating worms in the lungs may produce cough; as they reach the liver, hepatitis may appear. Infections with S. mansoni may produce hepatic and intestinal abnormalities. As the flukes take up residence in the mesenteric vessels and egg laying begins, fever, malaise, abdominal pain, and tenderness of the liver may be observed. Deposition of eggs in the bowel mucosa results in inflammation and thickening of the bowel wall with associated abdominal pain, diarrhea, and blood in the stool. Eggs may be carried by the portal vein to the liver, where inflammation can lead to periportal fibrosis and eventually to portal hypertension and its associated manifestations. page 878 page 879

Chronic infection with S. mansoni produces a dramatic hepatosplenomegaly with large accumulations of ascitic fluid in the peritoneal cavity. On gross examination, the liver is studded with white granulomas (pseudotubercles). Although S. mansoni eggs are primarily deposited in the intestine, eggs may appear in the spinal cord, lungs, and other sites. A similar fibrotic process occurs at each site. Severe neurologic problems may follow when eggs are deposited in the spinal cord and brain. In fatal schistosomiasis caused by S. mansoni, fibrous tissue, reacting to the eggs in the liver, surrounds the portal vein in a thick, grossly visible layer ("clay pipestem fibrosis").

Laboratory Diagnosis

The diagnosis of schistosomiasis is usually established by the demonstration of characteristic eggs in feces. Stool examination reveals the large golden eggs with a sharp lateral spine (see Figure 84-9). Concentration techniques may be necessary in light infections. Using rectal biopsy, the clinician can see the egg tracks laid by the worms in rectal vessels. Quantitation of egg output in stool is useful in estimating the severity of infection and in following the response to therapy. Serologic tests are also available but are largely of epidemiologic interest only. The development of newer tests using stage-specific antigens may allow the distinction of active from inactive disease and thus have greater clinical application.

Treatment, Prevention, and Control The drug of choice is praziquantel, and the alternative is oxamniquine. Anthelmintic therapy may terminate oviposition but does not affect lesions caused by eggs already deposited in tissues. Schistosomal dermatitis and Katayama syndrome may be treated with the administration of antihistamines and corticosteroids. Education regarding the life cycles of these worms and molluscacide control of snails are essential. Improved sanitation and control of human fecal deposits are critical. Mass treatment may one day be practical, and the development of a vaccine may be forthcoming. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Schistosoma japonicum Physiology and Structure

S. japonicum resides in branches of the superior mesenteric vein around the small intestine and in the inferior mesenteric vessels. S. japonicum eggs (see Figure 84-10) are smaller, are almost spherical, and possess a tiny spine. These eggs are produced in greater numbers than those of S. mansoni and S. haematobium. Because of the size, shape, and numbers of these eggs, they are carried to more sites in the body (liver, lungs, brain), and infection with a few S. japonicum adults can be more severe than infections involving similar numbers of S. mansoni or S. haematobium.

Epidemiology This Oriental blood fluke is found only in China, Japan, the Philippines, and on the island of Sulawesi, Indonesia. Epidemiologic problems correlate directly with a broad range of reservoir hosts, many of which are domestic (cats, dogs, cattle, horses, and pigs).

Clinical Syndromes The initial stages of infection with S. japonicum are similar to those of S. mansoni, with dermatitis, allergic reactions, fever, and malaise, followed by abdominal discomfort and diarrhea. Katayama syndrome associated with the onset of oviposition is observed more commonly with S. japonicum than with S. mansoni. In chronic S. japonicum infection, hepatosplenic disease, portal hypertension, bleeding esophageal varices, and accumulation of ascitic fluid are commonly seen. Granulomas that appear as pseudotubercles in and on the liver are common, along with the clay pipestem fibrosis as described for S. mansoni. S. japonicum frequently involves cerebral structures when eggs reach the brain and granulomas develop around them. The neurologic manifestations include lethargy, speech impairment, visual defects, and seizures.

Laboratory Diagnosis Stool examination demonstrates the small, golden eggs with tiny spines; usually, rectal biopsy is similarly revealing. Serologic tests are available.

Treatment, Prevention, and Control The drug of choice is praziquantel. Prevention and control may be achieved by measures similar to those for S. mansoni, especially education of populations in endemic areas regarding proper water purification, sanitation, and control of human fecal deposits. Control of S. japonicum must also involve the broad range of reservoir hosts and consider the fact that people work in rice paddies and on irrigation projects where infected snails are present. Mass treatment may offer help, and a vaccine may be developed someday. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 21 September 2009) © 2009 Elsevier

Schistosoma haematobium Physiology and Structure After development in the liver, these blood flukes migrate to the vesical, prostatic, and uterine plexuses of the venous circulation, occasionally the portal bloodstream, and only rarely other venules. Large eggs with a sharp terminal spine (see Figure 84-11) are deposited in the wall of the bladder and occasionally in the uterine and prostatic tissues. Those deposited in the bladder wall can break free and are found in urine.

Epidemiology S. haematobium occurs throughout the Nile Valley and in many other parts of Africa, including islands off the eastern coast. It also appears in Asia Minor, Cyprus, southern Portugal, and India. Reservoir hosts include monkeys, baboons, and chimpanzees. page 879 page 880

Clinical Syndromes

Early stages of infection with S. haematobium are similar to those of infections involving S. mansoni and S. japonicum, with dermatitis, allergic reactions, fever, and malaise. Unlike the other two schistosomes, S. haematobium produces hematuria, dysuria, and urinary frequency as early symptoms. Associated with hematuria, bacteriuria is frequently a chronic condition. Egg deposition in the walls of the bladder may eventually result in scarring, with loss of bladder capacity and the development of obstructive uropathy. Patients with S. haematobium infections involving many flukes frequently demonstrate squamous cell carcinoma of the bladder. It is commonly stated that the leading cause of cancer of the bladder in Egypt and other parts of Africa is S. haematobium. The granulomas and pseudotubercles seen in the bladder may also be present in the lungs. Fibrosis of the pulmonary bed caused by egg deposition leads to dyspnea, cough, and hemoptysis.

Laboratory Diagnosis Examination of urine specimens reveals the large, terminally spined eggs. Occasionally, bladder biopsy is helpful in establishing the diagnosis. S. haematobium eggs may appear in stool if worms have migrated to mesenteric vessels. Serologic tests are also available.

Treatment, Prevention, and Control The drug of choice is praziquantel. At present, education, possible mass treatment, and development of a vaccine are the best approaches to the control of S. haematobium disease. The basic problems of irrigation projects (e.g., dam building), migratory human populations, and multiple reservoir hosts make prevention and control extremely difficult. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Cercarial Dermatitis

Several nonhuman schistosomes have cercariae that penetrate human skin, producing a severe dermatitis ("swimmer's itch"), but these schistosomes cannot develop into adult worms. The natural hosts are birds and other shore-feeding animals from freshwater lakes throughout the world and a few marine beaches. The intense pruritus and urticaria from this skin penetration may lead to secondary bacterial infection from scratching the sites of infection. Treatment consists of oral trimeprazine and topical applications of palliative agents. When indicated, sedatives may be given. Control is difficult because of bird migration and the transfer of live snails from lake to lake. Molluscacides such as copper sulfate have produced some reduction in the snail populations. Immediate drying of the skin when people leave such waters offers some protection.

Case Study and Questions A 45-year-old Egyptian man was referred for evaluation of hematuria and urinary frequency of 2 months' duration. This individual had lived in the Middle East for most of his life but for the past year lived in the United States. He denied previous renal or urologic problems. His physical examination was unremarkable. A midstream urine specimen was grossly bloody. 1. What was the differential diagnosis of hematuria in this patient? 2. What was the etiologic agent of this patient's urologic process? 3. What exposures might put an individual at risk for this infection? 4. What are the major complications of this infection? 5. How is this disease treated?

Bibliography Connor DH, et al: Pathology of Infectious Diseases, vol 2. Stamford, Conn, Appleton & Lange, 1997. Garcia LS: Diagnostic Medical Parasitology, 4th ed, Washington, DC, ASM Press, 2001.

Jones MK, McManus DP: Trematodes. In Murray PR, et al (eds): Manual of Clinical Microbiology, 9th ed. Washington, DC, ASM Press. 2007. Keiser J, Utzinger J: Emerging foodborne trematodiasis. Emerg Infect Dis 11:1507-1514, 2005. Markell EK, John DT, Krotoski WA: Markell and Voges' Medical Parasitology, 8th ed. Philadelphia, WB Saunders, 1999. Meltzer E, et al: Schistosomiasis among travelers: New aspects of an old disease. Emerg Infect Dis 12:1696-1700, 2006. Strickland GT: Hunter's Tropical Medicine and Emerging Infectious Diseases. Philadelphia, WB Saunders, 2000. Printed from STUDENT CONSULT: Medical Microbiology 6E (on 20 September 2009) © 2009 Elsevier

Taenia solium Physiology and Structure The larval stage, or cysticercus ("bladder worm"), of Taenia species consists of a scolex, which is invaginated into a fluid-filled bladder. Larval cysts develop in the tissues of the intermediate host, are 4 to 6 mm long × 7 to 11 mm wide and have a pearl-like appearance in the tissues. After a person ingests pork muscle containing a larval worm, attachment of the scolex with its four muscular suckers and crown of hooklets initiates infection in the small intestine (Figure 85-1). The worm then produces proglottids until a strobila of proglottids is developed, which may be several meters in length. The sexually mature proglottids contain eggs, and as these proglottids leave the host in feces, they can contaminate water and vegetation ingested by swine. The gravid proglottids have a similar length and width (1 cm × 1 cm) and contain few (