UNIT 5 TEXT PDF

accompany this text. KEY TERMS Acute rheumatic fever Anti-DNase B ASO titer Commensalistic Endotoxin Exotoxin Group A streptococcus (GAS) Helicobacter pylori Impetigo Indigenous microbiota Infectivity Lancefield groups Lateral flow immunochromatographic assay (LFA) MALDI-TOF Microbio...

accompany this text. KEY TERMS Acute rheumatic fever Anti-DNase B ASO titer Commensalistic Endotoxin Exotoxin Group A streptococcus (GAS) Helicobacter pylori Impetigo Indigenous microbiota Infectivity Lancefield groups Lateral flow immunochromatographic assay (LFA) MALDI-TOF Microbiome Mutualistic Mycoplasma pneumoniae Parasitic Pathogenicity Plasmids Poststreptococcal glomerulonephritis Proteome Proteomics Rickettsiae Rocky Mountain spotted fever (RMSF) Scarlet fever Streptococcus pyogenes Streptolysin O Streptozyme Symbiotic Typhus Urease Virulence Virulence factors The collection of microorganisms that exists on the body—bacteria, viruses, and single-celled prokaryotic organisms (e.g., yeast and fungi)—is referred to as the human microbiome. The bacteria comprising the human microbiome keep us healthy in many ways. They protect us against disease-causing bacteria, aid in digesting our food, produce certain vitamins, and stimulate both the innate and adaptive immune systems. The establishment of an organism that leads to host injury is referred to as an “infection.” When a microbe causes damage to host cells or altered physiology that results in clinical signs and symptoms of disease, the phrase “infectious disease” applies. Traditional means of determining the cause of a bacterial infection have relied largely on growing the organism in culture and using stains to view the organism under the microscope. These infections can also be identified by immunoassays that detect bacterial antigens or antibodies and by molecular or proteomic techniques that detect nucleic acid or proteins from the organisms, respectively. This chapter will begin with an introduction to the human–microbe relationship and factors that influence the interactions between bacteria and the immune system. The chapter will then discuss laboratory methods that are commonly used to detect bacterial infections in the context of selected pathogenic bacteria. Human-Microbe Relationships When an individual is born, a dynamic relationship begins between the human host and the bacteria in the environment. Very quickly, various bacteria establish themselves on the surfaces of an individual, including the gastrointestinal tract, creating a symbiotic relationship. The bacteria and the host “live together,” often maintaining a long-term interaction. Symbiotic bacteria that reside on and colonize these surfaces are referred to as the indigenous microbiota (previously known as “normal flora”). The bacterial populations that exist on the body are not homogenous; they vary in composition and numbers, depending on the area of the body The microbial populations outnumber human cells by 10 to 1. Collectively, they may account for 2 to 6 pounds of an individual’s body weight. Although it was previously thought that a relatively limited number of bacteria make up the human microbiome, we now know that the microbial community is actually very diverse. Furthermore, greater than 90% of the bacteria that comprise the human microbiome cannot be cultured in vitro, most likely because their growth depends on specific conditions or substances that have not been duplicated in the laboratory. Our relationship with our indigenous microbiota exists through co-evolution, co-adaptation, and co-dependency between the bacteria and the host. For a microorganism to survive, the organism needs to colonize the host and acquire nutrients. Importantly, it must not stimulate the host’s immune response (in the case of the indigenous microbiota) or it must avoid or circumvent the immune responses. Once established, it needs to be able to replicate and disseminate to a preferred site in the body for survival and eventually be transmitted to a new susceptible host. Three types of symbiotic relationships can exist between humans and bacteria. In commensalistic relationships, there is no apparent benefit or harm to either organism. The indigenous microbiota are often referred to as “commensals” or the “commensalistic bacteria,” describing bacteria that are recovered in culture that do not represent a pathogen. An example of a commensalistic organism is Staphylococcus epidermidis, which colonizes and inhabits the human skin. In a mutualistic relationship, both humans and the bacteria benefit. One example is the Lactobacillus species that colonizes the epithelial surfaces of the vaginal canal. The human host provides conditions that allow the bacteria to grow and multiply, such as appropriate temperature, atmosphere, and nutrients. In exchange, the bacteria produce lactic acid, which prevents colonization of bacteria and yeast that may cause disease. Although the vast majority of the interactions between the host and members of the microbiome are harmless, the encounter with specific organisms or viruses occasionally results in harm to the host. In this case, a parasitic relationship exists between the other organisms and the host.  As previously mentioned, the establishment of an organism that leads to host injury is referred to as an infection. Although there is harm to the host, not all infections are symptomatic. In many instances, the infection may be subclinical; in other words, there are no signs or symptoms. An example is infection with Chlamydia trachomatis, a sexually transmitted organism. Only about 10% of infected males and 5% to 30% of infected females will show symptoms when infected with C. trachomatis. The organism may then be transmitted to other individuals or may result in complications such as pelvic inflammatory disease in women. Several terms are used to describe the interaction between the infecting organisms and the host. The terms infectivity, pathogenicity, and virulence are often used interchangeably; however, each term has different meanings when discussing an organism’s ability to cause an infection. Infectivity refers to an organism’s ability to establish an infection. More specifically, infectivity describes the proportion of individuals exposed to a pathogen through horizontal transmission (i.e., person-to-person spread) who will become infected. Another term that is used with similar meaning is contagious. For example, the measles virus is extremely contagious and has a high degree of infectivity. Pathogenicity refers to the inherent capacity of an organism to cause disease. This is a qualitative trait of the organism determined by its genetic makeup. Some organisms, such as the HIV virus, are considered to be primary or true pathogens that are capable of causing harm to a majority of individuals who have intact immune systems. Although an organism may be pathogenic in nature, it may not always cause disease. The outcome of the host–pathogen interaction is determined by several factors, including the host’s immunologic status. Some microorganisms may only cause disease or infection in individuals who have compromised immune systems because of factors such as chemotherapy, radiation therapy, or various chronic diseases. These organisms are referred to as “opportunistic pathogens.” Virulence is a quantitative trait that refers to the extent of damage, or pathology, caused by the organism. For example, Yersinia pestis, the causative agent of bubonic and pneumonic plague, is considered to be extremely virulent and is likely to cause severe illness and death upon infection unless antibiotics are administered. If the bacterial strain is not capable of causing disease, the organism is said to be “avirulent.” Not all members of a bacterial species are necessarily capable of causing disease. For example, Escherichia coli resides as commensal bacteria in the gastrointestinal tract, but only some strains of E. coli are capable of causing diarrheal disease. The degree of damage is mediated by specific virulence factors. Virulence factors may increase an organism’s pathogenicity by contributing to the organism’s ability to establish itself on or in the host, invade or damage host tissue, or evade the host immune response. Bacterial Virulence Factors Bacterial properties or features that determine whether an organism is pathogenic and able to cause disease are referred to as “virulence factors.” Factors that increase a bacterium’s virulence may be classified as either structural components (e.g., endotoxin is a component of the cell walls of certain bacteria) or as extracellular substances produced by the bacteria, such as exotoxins. Various types of bacterial virulence factors are discussed in the sections that follow. For an organism to be pathogenic, it needs to possess genetic determinants that allow for the production of either the structural components or the extracellular products that contribute to its virulence. Genetic determinants located on the bacterial chromosome are generally responsible for the production of structural or surface molecules, which help the organism to attach to and colonize the host. The genetic information needed to produce the extracellular substances that are virulence factors is most frequently located on independent genetic elements called plasmids. Plasmids are self-replicating extrachromosomal DNA molecules that are located in the bacteria’s cytoplasm and contain a limited number of genes. In addition, plasmids are mobile genetic elements that can be transferred between bacteria through various mechanisms. Acquisition of exogenous DNA that codes for the production of virulence factors can convert an avirulent strain into a virulent strain. Structural Virulence Features Bacterial cells are classified as prokaryotic cells, whereas human cells are classified as eukaryotic cells. Although prokaryotic bacterial cells are relatively simple, they have a well-developed cell structure and contain several structural and genetic features that are not found in eukaryotic cells. One significant difference is that the bacterial chromosome is not enclosed inside of a membrane- bound nucleus but instead resides inside the bacterial cytoplasm. Also housed in the cytoplasm are internal cellular structures, such as ribosomes, mesosomes, and potentially plasmids, as well as other cytoplasmic inclusion bodies. Not present in bacterial cells are membrane-bound organelles such as the mitochondria found in eukaryotic cells (Fig. 20–1). FIGURE 20-1 Cross section of a bacterial cell. Other significant differences between eukaryotic and prokaryotic cells are features of the cell membrane and the presence of a cell wall in bacteria. Similar to the plasma membrane in eukaryotic cells, the bacterial plasma membrane plays a regulatory role in the transport of molecules in and out of the cell. The cell membrane in bacteria also contains various enzyme systems responsible for energy generation. In eukaryotic cells (except for plants and fungi) the plasma membrane is the outer surface of the cell. In contrast, bacteria have a cell wall as their outermost feature. The bacterial cell wall prevents osmotic lysis and confers shape and rigidity to the bacteria. Peptidoglycan is the primary component providing the shape and rigidity. The majority of antibiotics used to treat bacterial infections target the production of peptidoglycan in the cell wall. The bacterial cell wall structure has two different variations, which are classified as either gram-positive or gram-negative, depending on their staining characteristics. Both gram-positive and gram-negative cell walls have features that increase an organism’s virulence, as we will discuss later. One of the features found in the cell wall of gram-negative bacteria is the lipopolysaccharide (LPS) layer. There are three components that make up LPS—an outer core polysaccharide, an inner core polysaccharide, and lipid A. When released from a dead or dying cell, a portion of LPS called lipid A is exposed. Lipid A, also referred to as endotoxin, is a powerful stimulator of cytokine production that leads to a variety of systemic manifestations and potentially fatal endotoxic (gram-negative) shock (discussed later in this chapter). To be successful in establishing an infection, bacteria must first adhere to and colonize the host tissue. Furthermore, to persist in the host, they must be capable of evading the immune system. Structural features are generally involved in the adherence of bacteria or the evasion of the immune system. Whip-like structures called flagella can facilitate adherence as well as movement of the bacteria toward a host cell. Bacteria have surface structures or molecules that can bind specifically to complementary receptors on specific cells of host tissues. The most common surface structures involved in attachment are fimbriae, which are composed of protein. The fimbriae involved in specific attachment to prokaryotic surface ligands are referred to as the common pili. Other pili used to exchange genetic information, called the F or sex pili, are fewer in number than common pili. The sex pili may also function in the attachment of bacteria to host cells. In addition to allowing the organism to attach to and colonize tissues, pili play a role in resisting phagocytosis by white blood cells (WBCs) and may undergo antigenic variation to evade the adaptive immune response. For example, the pili of Neisseria gonorrhoeae specifically adhere to human mucosal epithelial cells. N. gonorrhoeae can evade the immune response and resist phagocytosis by rearranging portions of its genome, thus changing the antigen makeup of the pili. Most strains of E. coli that colonize the gastrointestinal tract do not adhere to epithelial cells of the small intestine. The ability of certain strains of E. coli to cause gastrointestinal disease is associated with the expression of specific pili that allow for adherence to the epithelial cells of the small intestine. For example, enterotoxigenic E. coli (ETEC) strains have factor antigen I (CFA) pili that adhere to the cells in the small intestine; the organism then produces the toxins that cause diarrhea. Attachment may also occur via adhesions or surface molecules on the bacterial cell. Attachment caused by the presence of surface molecules is referred to as afimbrial (non–pili dependent) attachment. Host-cell receptors recognized by bacterial surface molecules include proteoglycans, laminins, collagens, elastin, and hyaluronan. Glycoproteins such as fibrinogen, fibronectin, and vitronectin are also potential receptors for bacteria. The ability of Streptococcus pyogenes pili to attach to host cells is caused by the production of F protein, which attaches to fibronectin. In addition to aiding in attachment, surface M protein, the major virulence factor of S. pyogenes, allows the organism to resist phagocytosis. A major structural feature that plays an important role in increasing an organism’s virulence is the presence of a capsule, which is usually polysaccharide in nature. Capsules contribute to the organism’s ability to resist innate and adaptive immune responses through a variety of means. They can block the attachment of antibodies, inhibit activation of complement, or act as a decoy when capsular material is released into the surrounding host environment. One of the most important features of a capsule is its role in blocking phagocytosis by WBCs. For example, S. pneumoniae’s primary determinant of virulence is a polysaccharide capsule that prevents the ingestion of pneumococci by alveolar macrophages. In addition, extracellular capsular antigens are released and bind to immunoglobulins, thus reducing the effective immune response against the organism itself. Strains of bacteria that do not possess a capsule are, in most cases, avirulent and not able to produce disease. Extracellular Virulence Factors Extracellular substances produced by bacteria also contribute to an organism’s virulence by breaking down primary or secondary defenses of the body, damaging the host tissues and cells, or facilitating the growth and spread of the organism. Substances that perform the latter function are called invasins. Several of the invasins include hyaluronidase, collagenase, phospholipases, lecithinases, coagulase, and various kinases. Endotoxin and Exotoxins Bacteria may also produce two types of toxins—endotoxin and exotoxins. Endotoxin (lipid A) is found in the LPS layer of the cell walls of all gram-negative bacteria. When the cell dies, the LPS layer is removed from the cell, releasing endotoxin in the host. The effects of endotoxin are complex. Endotoxin induces a variety of host responses, including the production of cytokines such as interleukin (IL)-1, IL-6, IL-8, tumor necrosis factor (TNF), and platelet-activating factor, which stimulate the production of prostaglandins and leukotrienes. This results in inflammation, increased heart rate, increased body temperature (fever), and a decrease in blood pressure. In addition, endotoxin activates the complement cascade, resulting in the formation of the anaphylatoxins C3a and C5a, which cause vasodilation and increase vascular permeability. The alternative coagulation cascade is also activated, producing coagulation, thrombosis, and acute disseminated intravascular coagulation (DIC), leading to hemorrhage and shock. A potential consequence of endotoxin release is septic shock, a life-threatening illness that is usually the result of a gram-negative bacteremia, or presence of bacteria in the blood. With septic shock, there is a large-scale release of inflammatory mediators that results in massive vasodilation and hypotension. Some refer to this as a “cytokine storm.” If the condition worsens, there is widespread organ damage, including renal failure, liver dysfunction, heart damage, and eventual death. Although immunogenic, endotoxin does not elicit a protective immune response, so no vaccine is available against this bacterial component. Unlike endotoxin, which has multiple effects on the body, exotoxins have a very specific and targeted activity. They are protein molecules that are released from living bacteria (mostly gram- positive bacteria) and are considered to be some of the most potent molecules known to harm living organisms. Exotoxins may be classified as neurotoxins, cytotoxins, or enterotoxins, according to their effect on cells. Exotoxins bind to specific receptors on host cells. Most exotoxins have several subunits that bind to the receptor (B subunits) and a subunit that is actually responsible for the specific activity of the toxin (A subunit). An example of a cytotoxin is the diphtheria toxin, which interferes with protein synthesis in epithelial cells. Neurotoxins include the tetanus toxin, which prevents the release of inhibitory transmitters from the presynaptic membrane of neuromuscular cells, leading to continuous excitement of muscle cells and spasms. Botulism toxin works in a reverse manner—the toxin prevents the release of acetylcholine (ACh), resulting in paralysis of the motor system. Examples of enterotoxins are toxins A and B produced by Clostridium difficile, which cause fluid secretion (diarrhea), mucosal injury, and inflammation (Fig. 20–2). Exotoxins are extremely immunogenic and induce the production of protective antibodies. Inactivated exotoxins called “toxoids” are used for some vaccines. For example, the toxin of Corynebacterium diphtheria is used in the vaccine to prevent diphtheria. Some exotoxins may act as “superantigens.” Unlike other antigens, these superantigens are not processed by antigen-presenting cells (APCs). Instead, they bind directly to class II major histocompatibility complexes (MHC II) on APCs outside of the normal antigen-binding groove. The MHC II receptor binds to the T-cell receptor (TCR) with the whole antigen (toxin) attached. Normally, only 0.0001% of T cells are activated in an immune response. However, a superantigen induces activation of up to 20% of T cells, resulting in a massive release of cytokines. The systemic events brought on by the release of large amounts of cytokines leads to what is referred to as “toxic shock syndrome.” Examples include the TSST-1 toxin produced by Staphylococcus aureus and the superantigens produced by S. pyogenes, the cause of streptococcal toxic shock syndrome (STSS). Clinical Correlations Capsular Antigens and Vaccine Development Most capsules evoke a strong humoral immune response and are used for the development of vaccines. For example, the vaccines for Haemophilus influenzae type b, S. pneumoniae, and certain types of Neisseria meningitidis consist of antigens derived from bacterial capsules (see Chapter 25). FIGURE 20-2 Gram stain of Clostridium difficile (a gram-positive rod). The clear areas in the cell represent a spore. Spores are resistant to disinfectants and contribute to the spread of the organism from person to person. (Courtesy of James Vossler.) Immune Defenses Against Bacterial Infections and Mechanisms of Evasion Immune Defense Mechanisms Although bacteria may possess various features that increase their virulence, they must be able to circumvent or overcome the host’s defense mechanisms. As we discussed in previous chapters, both innate and adaptive responses may occur after an encounter with foreign antigens. The first line of defense against potential pathogens involves intact skin and mucosal surfaces that serve as structural barriers. In addition, the epithelial surface may have enzymes and nonspecific antimicrobial defense peptides (ADPs), or defensins, and proteins that have antimicrobial activity. One example of an enzyme with specific antimicrobial activity is lysozyme, which is found in many secretions, including tears and saliva. Lysozyme destroys the peptidoglycan found in the cell wall of bacteria, especially gram-positive bacteria. Other enzymes include ribonucleases, which destroy RNA and have antimicrobial and antiviral activities. The body excretes a wide variety of ADPs and proteins that play a role in the innate defenses of the body. Some of the ADPs and proteins are only secreted by specific tissues or cells. One group of soluble peptides is the defensin peptides. Defensins are produced constitutively by the cells in the body. The three main classes of defensins are alpha, beta, and theta. Alpha defensins are produced by neutrophils, certain macrophage populations, and Paneth cells of the small intestine. This class of defensins is believed to disrupt the microbial membrane. Beta defensins are produced by neutrophils as well as epithelial cells lining the various organs, including the bronchial tree and genitourinary system. They are believed to increase resistance of epithelial cells to colonization. Theta defensins are not found in humans. Many antimicrobial proteins contribute to the innate immune response. For example, complement proteins can promote chemotaxis. Interleukins are involved in the regulation of immune responses and inflammatory reactions. Prostaglandins are involved in the dilation and constriction of blood vessels and modulation of inflammation. Leukotrienes are involved in inflammation and fever. Acute-phase reactants also play important roles. For example, C-reactive protein (CRP) activates the complement system and promotes phagocytosis by macrophages. Haptoglobin binds free plasma hemoglobin, which deprives the bacteria of iron. Ceruloplasmin is a glycoprotein with bactericidal activities. Bacteria, fungi, and viruses possess pathogen-associated molecular patterns (PAMPs), which are structural patterns consisting of carbohydrates, nucleic acids, or bacterial peptides. PAMPs are recognized by pattern recognition receptors (PRRs) expressed on the cells of the innate immune system. Engagement of the PRR with the appropriate PAMP triggers the release of immune mediators such as cytokines and chemokines, boosts production of various defensins and proteins, and initiates phagocytosis. The phagocytic process is enhanced by the activation of the alternative complement cascade, which is triggered by microbial cell walls or other products of microbial metabolism. Adaptive immune responses include the production of antibodies directed against bacterial antigens or extracellular products produced by bacteria such as exotoxins. Antibody formation is the main defense against extracellular bacteria. The binding of antibodies to invading bacteria is referred to as opsonization (see Chapter 5). Cell-mediated immunity (CMI), the other branch of the adaptive immune response, is helpful in attacking intracellular bacteria, such as Mycobacterium tuberculosis, Legionella pneumophila, Listeria monocytogenes, and Rickettsia species. Through the mechanism of delayed-type hypersensitivity, CD4 T cells produce cytokines, which activate macrophages to release enzymes that destroy the bacteria (see Chapter 14). The recruitment of inflammatory cells results in the formation of granulomas that surround the bacteria-infected cells to help prevent the spread of infection. Cytotoxic T cells are also recruited to the site of infection and mount an antigen- specific attack on the infected cells. Bacterial Evasion Mechanisms Bacteria have developed several ways to inhibit the immune system or make it more difficult for immune responses to occur. Three main mechanisms used by bacteria involve avoiding antibody, blocking phagocytosis, and inactivating the complement cascade (Fig. 20–3). Bacteria can evade antibodies by altering their bacterial antigens, a process called antigenic variation. They can also coat themselves with host proteins such as fibrin or immunoglobulin molecules (S. aureus) or fibronectin (Treponema pallidum) or hide their surface molecules through antigenic disguise (the hyaluronic acid capsule of S. pyogenes). Bacteria can also evade the specific immune response through downregulation of MHC molecules and production of proteases that degrade immunoglobulin A (IgA). N. gonorrhoeae, H. influenzae, and Streptococcus sanguinis are all examples of bacteria that can cleave IgA. Connections Toll-Like Receptors (TLRs) Recall from Chapter 2 that one of the main groups of PRRs are the Toll-like receptors (TLRs). TLRs are expressed on key cells of the innate immune system, such as macrophages and dendritic cells. They recognize molecules that are commonly found in microbial pathogens but not on host cells. Once TLRs have bound to their ligands, cell-signaling pathways are triggered that result in the production of cytokines that enhance the inflammatory response, resulting in more efficient pathogen destruction. FIGURE 20-3 The strategies bacteria use to evade host defenses. (A) Inhibiting chemotaxis. (B) Blocking adherence of phagocytes to the bacterial cells. (C) Blocking digestion. (D) Inhibiting complement C3b binding. (E) Cleaving immunoglobulin A (IgA). Most of the evasion mechanisms target the process of phagocytosis. Bacteria can mount a defense at several stages in the phagocytic process, including chemotaxis, adhesion, and digestion. Some pathogens such as N. gonorrhoeae, for example, inhibit the release of chemotactic factors that would bring phagocytic cells to the area. The cell walls of S. pyogenes produce an M protein that interferes with adhesion to the phagocytic cell. Additionally, the presence of a polysaccharide capsule found in such organisms as N. meningitidis, S. pneumoniae, Y. pestis, and H. influenzae inhibits the binding of neutrophils and macrophages needed to initiate phagocytosis. Microorganisms use several different mechanisms to resist digestion. Some bacteria can block fusion of lysosomal granules with phagosomes after being engulfed by the phagocyte. Salmonella species are able to do this, as can M. tuberculosis and Mycobacterium leprae. In M. tuberculosis and M. leprae infections, each bacillus is contained in a membrane-enclosed fluid compartment called a pristiophorus vacuole (PV), which does not fuse with the lysosomes because of the complexity of the acid-fast cell walls.  An additional mechanism of resisting digestion involves the production of extracellular products after the bacteria are phagocytized. The primary effect is the release of lysosomal contents into the cytoplasm of the phagocytic cells, subsequently killing the WBC. Examples include leukocidin, produced by S. aureus; listeriolysin O, produced by L. monocytogenes; and streptolysin, produced by S. pyogenes. The last major defense some bacteria use is to block the action of complement. Organisms mentioned previously that produce a capsule do not bind the complement component C3b, which is important in enhancing phagocytosis. Such organisms cannot easily be phagocytized unless coated by other opsonins (e.g., IgG). Additionally, some organisms express molecules that disrupt one or more of the complement pathways. Protein H, produced by S. pyogenes, binds to C1 but does not allow the complement cascade to proceed further. Another example is Streptococcus agalactiae, also known as Group B streptococcus or GBS. GBS has a capsule that is rich in sialic acid (a common component of host-cell glycoproteins), causing degradation of C3b and making the organism resistant to complement-mediated phagocytosis. Laboratory Detection and Diagnosis of Bacterial Infections Five general ways can be used to detect the causative agent of a bacterial infection: (1) culture or growth of the causative agent, (2) microscopy, (3) detection of bacterial antigens in the clinical sample, (4) molecular detection of bacterial DNA or RNA, and (5) serology to detect antibodies produced in response to the infection. Bacterial Culture Traditional means of determining the cause of a bacterial infection rely largely on growing the organism in culture. Various broth and solid media may be used to recover the organism. Some media may contain substances that enhance the growth of certain organisms and are referred to as enriched media. Selective media contain substances or antibiotics that suppress the growth of commensalistic bacteria and support the growth of other bacteria. Differential media contain substrates that allow for the differentiation of bacteria based on their ability to use the substrate. For example, MacConkey agar selects for gram-negative bacteria and differentiates between lactose- and non–lactose-fermenting bacteria. Some organisms, such as Bordetella pertussis, the causative agent of whooping cough, have very specific growth requirements for which specialized media must be used. Although culture is the primary laboratory means of diagnosing bacterial infections, the culturing of bacterial pathogens has limitations. There are several bacterial pathogens for which clinically useful culture systems are not available. For other organisms, recovery in culture may take too long to be clinically useful. For example, although Mycoplasma pneumoniae, a leading cause of community acquired pneumonia, can be cultured, culturing is a challenge. The organism is extremely fastidious (difficult to grow) and may take weeks to recover in the laboratory. Other organisms present a danger to the laboratory technologist if they are not grown and handled using the most rigorous safety precautions (e.g., Y. pestis). Microscopic Visualization Visualization of the causative agents using microscopic techniques is most often done using differential or fluorescent stains. One example is the Gram stain for differentiating gram-positive bacteria, which stain purple with crystal violet (Fig. 20–4), from gram-negative bacteria, which stain pink with safranin. Other examples are the acid-fast stain for the detection of M. tuberculosis (Fig. 20–5), and the Giemsa stain for the detection of the causative agents of malaria. Direct fluorescent antibody assay, or DFA, involves the use of antibody conjugated with a fluorescent label to detect specific bacteria in a sample. Currently, many DFAs are being replaced by molecular tests because they lack sensitivity or because the reagents are not widely available. Although the various staining methods are not difficult to perform, a trained microscopist is necessary for proper interpretation. Another limitation of microscopy is that not all organisms may be visualized through microscopic means. FIGURE 20-4 (A) Gram stain of Staphylococci showing gram-positive cocci in clusters. (B) Gram stain of E. coli showing gram-negative rods. (Courtesy of James Vossler.) FIGURE 20-5 Photomicrograph of Mycobacterium tuberculosis bacteria using acid-fast Ziehl-Neelsen stain; magnified 1000X. The acid-fast stains depend on the ability of mycobacteria to retain the dye when treated with mineral acid or an acid-alcohol solution. (Courtesy of the CDC/Dr. George P. Kubica, Public Health image Library.) Antigen Detection Antigen detection assays are available for a wide variety of bacteria, viruses, parasites, and fungi in clinical samples. Testing methodologies include latex agglutination (LA), enzyme-linked immunosorbent assay (ELISA), and lateral flow immunochromatographic assays (LFA) that detect the presence of an analyte (e.g., bacterial antigen) in a sample. The LA and LFA assays are advantageous because of the relative ease by which the tests can be performed, their low cost, and the rapid turnaround time. Many of the LA and LFA assays are classified as “CLIA waived” tests. Under the Clinical Laboratory Improvement Amendments of 1988 (CLIA), simple, low- risk tests can be “waived” (i.e., laboratories performing the testing are not subject to routine inspection) and may be performed in physicians’ offices and various other locations. Bacteria and viruses for which antigen detection is widely used include S. pyogenes (strep throat), L. pneumophila (Legionnaire disease), rotavirus (pediatric diarrheal disease), respiratory syncytial virus, and influenza A and B. Antigen detection assays are highly specific, and because of advances in technology, the sensitivities of the assays have improved dramatically. In many cases, antigen detection assays, particularly LFA, have replaced other methods used to detect infections with bacteria, viruses, and fungi. Molecular Detection Rapid developments in the field of molecular diagnostics have allowed for the increased availability and use of nucleic acid–based assays in the clinical laboratory to detect pathogenic microorganisms. Compact hybridization and gene amplification assays that are easy to perform have made their way into physicians’ office laboratories. The most widely known molecular technology is the polymerase chain reaction (PCR), in which specific genetic sequences are amplified and detected. The development of real-time PCR or quantitative PCR (qPCR) has allowed for results to be obtained in a few hours, as compared with several days for traditional PCR. For some infectious agents, such as N. gonorrhoeae and C. trachomatis, nucleic acid–based assays are widely available and are more sensitive than traditional culture methods, making nucleic acid detection the method of choice for detection of these organisms. Although nucleic acid–based assays are more sensitive than other methods, there are limitations associated with nucleic acid–based testing. At the time of this writing, there are relatively few U.S. Food and Drug Administration (FDA)-approved assays on the market for several infectious agents, and the cost of the instrumentation and disposables is prohibitive to many organizations. However, FDA-approved assays for the detection of agents causing infections are becoming available at a rapid rate. As more assays receive FDA approval and additional technological advances occur, the use of molecular-based assays for the detection of agents responsible for various infectious diseases will become even more widespread. Serological Diagnosis Serology has historically been used to detect and confirm infections from organisms that are difficult to grow or for which other laboratory methods of diagnosing the infection are not available. Serology may also be useful in determining the causative agent when the clinical symptoms are not specific enough to identify the cause of the infection. For certain organisms (e.g., Anaplasma, Ehrlichia, Chlamydophila pneumoniae, Chlamydophila psittaci, Coxiella burnetii, Leptospira, Ricksettia spp., and T. pallidum), serological testing remains useful and, in some cases, is the best means of detecting exposure to or infection with an organism. Detection of either immunoglobulin M (IgM) or immunoglobulin G (IgG) antibodies may indicate recent or previous exposure to an organism, and antibody titers may be used to assess reactivation or reexposure to an infectious agent (see Chapter 5). Connections  Antibody Response Curve A serological pattern of IgM+, IgG- generally indicates an early-stage, acute infection, whereas an IgM–, IgG+ pattern usually signifies a past exposure. The best indication of a current infection is a four-fold rise in antibody titer when comparing two serum samples collected from a patient during the beginning and later stages of the infection. This is a good time to review the typical antibody-response curve shown in Chapter 5. The primary disadvantage of using serology in diagnosis is that there is usually a delay between the start of the infection and the production of antibodies to the infecting microorganism. Although IgM antibodies may appear relatively early (7 to 10 days after exposure), some infections have a rapid course; the need to initiate therapy limits the detection of IgM antibodies as a diagnostic tool in those instances. In some cases, demonstration of a high IgG antibody titer in the initial stage of infection is diagnostic; however, the high titer may be caused by a past infection, and the patient’s symptoms may have an entirely different cause. When testing for the presence of IgG antibodies, it is ideal to collect serum samples during both the acute and convalescent phases of the illness so that a rising titer to the suspected pathogen can be observed. Another limitation of serology is that immunosuppressed patients may be unable to mount an antibody response. Proteomics The proteome is the total complement, or set, of proteins expressed by an organism or cell. Proteomics is the analysis and study of the proteins expressed by an organism’s genome. Proteomics can be used to detect biomarkers—biological molecules found in blood, other body fluids, or tissues that are a sign of a normal or abnormal process, or of a condition or disease. The best-established clinical applications of proteomics so far are in the identification of markers for the early diagnosis of cancers. Proteomics, which is still in its infancy, has the potential to be used to detect agents causing an infectious disease, monitor the effectiveness of treatment, or identify changes in a disease caused by a microorganism or virus. Advances in proteomic analysis are attributable largely to the introduction of mass spectrometry (MS) platforms capable of screening complex biological fluids for individual protein and peptide biomarkers. Proteomic-based approaches are being used to discover biomarkers that can be used in the detection and diagnosis of bacterial, viral, parasitic, or fungal infections. Specific protein biomarkers may also reveal the biological state of a particular organism (e.g., reproducing or dormant) or pathological changes within an infected host. For example, the definitive diagnosis and monitoring of chronic hepatitis B virus (HBV) infection currently relies on liver biopsy. Research involving proteomic analysis of serum samples shows that the expression of at least seven serum proteins changes significantly in chronic HBV patients and that expression patterns correlate with fibrosis stage and inflammatory grade. Such markers could provide a non-invasive and definitive way to assess prognosis and guide treatment. Proteomic analysis primarily relies on the use of MS platforms. Such platforms include matrix- associated laser desorption ionization time-of-flight MS (MALDI-TOF MS) and surface- enhanced laser desorption ionization time-of-flight MS (SELDI-TOF MS). Currently, the single most important application of MALDI-TOF is the identification of bacterial and fungal isolates growing from a culture. To perform MALDI-TOF MS, the sample is placed on a solid surface matrix. The matrix is then placed in the instrument, where pulses of laser light vaporize the specimen, generating high- molecular-weight ions that absorb energy from the proteins and carbohydrates in the sample in a process known as desorption. The ions are then accelerated by an electrical field and a vacuum. Because the ions have the same energy, yet a different mass, they reach the detector at different times. The smaller ions reach the detector first because of their greater velocity, whereas the larger ions take longer owing to their larger mass. Hence, the analyzer is called “TOF” because the mass is determined from the ions’ “time of flight” from being ionized and reaching the detector (Fig. 20–6). Identification of the microorganism in the sample is based on the generation of a characteristic “protein fingerprint” called a profile (Fig. 20–7). SELDI-TOF is a variant of MALDI-TOF. SELDI-TOF has a higher sensitivity to low- molecular-weight proteins than MALDI-TOF and has been shown to detect various biomarkers associated with certain cancers better than MALDI-TOF. Research has shown that MALDI-TOF and SELDI-TOF can be used to detect certain parasitic and fungal infections in patients. The use of MALDI-TOF and SELDI-TOF to detect biomarkers associated with bacterial, viral, fungal, and parasitic agents shows promise in the detection and diagnosis of infections cause by these agents. The remainder of this chapter reviews the infections caused by several bacteria and the clinical laboratory tests used to detect these infections. FIGURE 20-6 MALDI-TOF. The sample is ionized with a laser beam, breaking it into various-sized particles. The particles are accelerated into a tube with a magnetic field. Particles move through the tube via a vacuum. Smaller particles move faster through the tube (time of flight). The number of particles of each size is measured, creating a pattern unique to each organism and allowing for identification of the organism. (Courtesy of James Vossler.) FIGURE 20-7 An example of a profile or pattern generated with MALDI-TOF. The pattern is compared with a database of known profiles, allowing for microorganism identification. (Courtesy of James Vossler.) Group A Streptococci (Streptococcus pyogenes) Classification and Structure Streptococci are gram-positive cocci; these spherical, ovoid, or lancet-shaped organisms are often arranged in pairs or chains when observed on Gram stain (Fig. 20–8). The streptococci are initially differentiated by their effect on sheep red blood cells (RBCs) when grown in culture. Those that completely lyse or hemolyze the blood cells are classified as being β-hemolytic. If the organisms only partially hemolyze the cells, causing them to appear green, they are classified as being α-hemolytic. If the organisms exhibit no effect on the cells, they are referred to as being γ- hemolytic. The β-hemolytic streptococci are further classified according to a group-specific carbohydrate composition that divides these bacteria into 20 groups designated A through H and K through V. These are known as the Lancefield groups, based on the pioneering work of Dr. Rebecca Lancefield. A member of the β-hemolytic streptococci is S. pyogenes, a major cause of bacterial pharyngitis (a throat infection) and childhood impetigo (a skin infection). Because S. pyogenes has a Group A carbohydrate, the organism is also referred to as Group A streptococcus (GAS). Additional cell wall components, the M and T proteins, allow for further classification and differentiation (Fig. 20–9). The M protein is a filamentous molecule consisting of two alpha- helical chains twisted into a ropelike structure that extends out from the cell surface. Some strains possess a hyaluronic acid capsule outside the cell wall that contributes to the bacterium’s antiphagocytic properties. Serotyping and molecular techniques can be used to identify a particular strain of GAS. Serotyping involves identification of the M protein antigens by precipitation with type-specific anti-sera. More than 80 different serotypes have been identified by this method. However, serotyping has limitations, including limited availability of typing sera, new M protein types that do not react with the anti-sera, and difficulty in interpreting the results. Genotyping techniques involving PCR amplification of a portion of the emm gene, which codes for the M protein, followed by sequence analysis circumvents these problems. Pulsed-field gel electrophoresis (PFGE) has also been used for epidemiological studies. In PFGE, DNA from GAS is separated by using an alternating current to obtain a unique pattern or “fingerprint.” The patterns from multiple sources may be compared when there is a Group A streptococcal outbreak. FIGURE 20-8 Gram stain of Streptococcus pyogenes, also known as Group A streptococci, which are gram-positive cocci that grow in pairs and chains. (Courtesy of James Vossler.) FIGURE 20-9 Diagram of antigenic components of Streptococcus pyogenes. Virulence Factors GAS is one of the most common and ubiquitous pathogenic bacteria and causes a variety of infections. The M protein is the major virulence factor of GAS and has a net-negative charge at the amino-terminal end that helps to inhibit phagocytosis. In addition, the presence of the M protein limits deposition of C3 on the bacterial surface, thereby diminishing complement activation. The M protein, along with lipoteichoic acid and protein F, help GAS attach to host cells. Immunity to GAS appears to be associated with antibodies to the M protein. There are more than 100 serotypes of this protein, and immunity is serotype-specific. Therefore, infections with one strain will not provide protection against another strain. Additional virulence factors include various exotoxins that may be produced during the course of an infection. Pyrogenic exotoxins A, B, and C are responsible for the rash seen in scarlet fever and also appear to contribute to pathogenicity. Additional extracellular substances include the enzymes streptolysin O, deoxyribonuclease B (DNase B), hyaluronidase, nicotinamide adenine dinucleotide (NAD), and streptokinase. Antibodies produced against these substances are useful in the diagnosis of infection and for developing complications or sequelae associated with GAS infection (discussed later in the chapter). Clinical Manifestations of Group A Streptococcal Infection S. pyogenes can be responsible for infections ranging from pharyngitis (a throat infection) to life- threatening illnesses such as necrotizing fasciitis and streptococcal toxic shock syndrome. The two major sites of infections in humans are the upper respiratory tract and the skin, with pharyngitis (“strep throat”) and streptococcal pyoderma (a skin infection) being the most common clinical manifestations. Symptoms of pharyngitis include fever, chills, severe sore throat, headache, tonsillar exudates, petechial rash on the soft palate, and anterior cervical lymphadenopathy (Fig. 20–10). The most common skin infection is streptococcal pyoderma (also known as impetigo), characterized by vesicular lesions on the extremities that become pustular and crusted (Fig. 20–11). Such infections tend to occur in young children. Other complications include otitis media, erysipelas, cellulitis, puerperal sepsis, and sinusitis. Septic arthritis, acute bacterial endocarditis, and meningitis also can result from a pharyngeal infection. Humans are the primary reservoir for GAS, and transmission of GAS is from person to person. A small percentage of individuals develop scarlet fever. Although usually associated with pharyngeal infections, scarlet fever may occur with streptococcal infections at other sites. Symptoms include a fever of higher than 101°F, nausea, vomiting, headache, and abdominal pain. A distinct scarlet rash initially appears on the neck and chest and then spreads all over the body. Scarlet fever results from infection with a GAS that elaborates streptococcal pyrogenic exotoxins (erythrogenic toxins). Two of those toxins, streptococcal pyrogenic exotoxin A (SpeA) and streptococcal pyrogenic exotoxin C (SpeC), can act as superantigens that may induce toxic shock syndrome. Toxic shock syndrome is a life-threatening multisystem disease that often initiates as a skin or soft tissue infection and may proceed to shock and renal failure because of overproduction of cytokines. FIGURE 20-10 Pharyngitis, or sore throat, is characterized by swelling and reddening of the pharynx. Note the inflammation of the oropharynx and petechiae, or small red spots on the soft palate caused by Streptococcal pharyngitis. (Courtesy of the CDC/Henry F. Eichenwald, Public Health Image Library.) FIGURE 20-11 Impetigo is a dermatological streptococcal infection that is characterized by thick, golden-yellow discharge that dries, crusts, and sticks to the skin. It is also caused by the S. aureus bacteria. Necrotizing fasciitis may occur when a GAS skin infection invades the muscles in the extremities or the trunk. The onset is quite acute and is a medical emergency. Exotoxins produced by S. pyogenes cause a rapidly spreading infection deep in the fascia, resulting in ischemia, tissue necrosis, and septicemia if not treated promptly. The disease may be associated with predisposing conditions such as chronic illness in the elderly or varicella in children, but healthy persons can be affected as well. Reporting of necrotizing fasciitis and toxic shock syndrome is part of a surveillance program conducted by the Centers for Disease Control and Prevention. Although the incidence of this syndrome has declined in the United States, a significant number of cases are still reported each year. Group A Streptococcal Sequelae The reason GAS receives so much attention is the potential for the development of two serious sequelae, acute rheumatic fever and poststreptococcal glomerulonephritis (sequelae are conditions that are the consequence of a previous disease or injury). The sequelae result from the host response to infection. Serological testing plays a major role in the diagnosis of these two diseases because the organism itself may no longer be present by the time symptoms appear. Acute rheumatic fever develops as a sequela to pharyngitis or tonsillitis in 2% to 3% of infected individuals. It does not occur because of skin infection. The latency period is typically 1 to 3 weeks after onset of the sore throat. Characteristic features of acute rheumatic fever include fever, pain caused by inflammation in the joints, and inflammation of the heart. The disease is most likely caused by antibodies or cell-mediated immune responses originally produced against streptococcal antigens that cross-react with antigens present in human heart tissue. Chief among the antibodies thought to be involved are those directed toward the M proteins, which have at least three epitopes that resemble antigens in heart tissue, permitting cross- reactivity to occur. Titers of some antibodies may remain high for several years following infection. The second main complication following a streptococcal infection is acute glomerulonephritis, a condition characterized by damage to the glomeruli in the kidneys. This condition may follow infection of either the skin or the pharynx, whereas rheumatic fever follows only upper respiratory tract infections. Glomerulonephritis caused by a streptococcal infection is most common in children between the ages of 2 and 12 and is especially prevalent in the winter. Symptoms of glomerulonephritis may include hematuria, proteinuria, edema, and hypertension. Patients may also experience malaise, backache, and abdominal discomfort. Renal function is usually impaired because the glomerular filtration rate is reduced, but renal failure is not typical. The most widely accepted theory for the pathogenesis of poststreptococcal glomerulonephritis is that it results from deposition of immune complexes containing streptococcal antigens in the glomeruli. These immune complexes stimulate an inflammatory response that damages the kidneys and impairs function because of release of the lysosomal contents of leukocytes and activation of complement. Laboratory Diagnosis Culture Diagnosis of acute streptococcal infections typically is made by culture of the organism from the infected site. The specimen is plated on sheep blood agar and incubated. If GAS is present, small translucent colonies surrounded by a clear zone of β hemolysis will be visible (Fig. 20–12). Identification is made on the basis of susceptibility to bacitracin, testing for L-pyrrolidonyl-β- naphthylamide (PYR) activity, or through Lancefield typing. FIGURE 20-12 Throat culture plate showing a positive result for beta hemolytic Group A streptococci (S. pyogenes). Bacterial colonies not producing beta hemolysis represent indigenous microbiota of the oropharynx. (Courtesy of James Vossler.) Detection of Group A Streptococcal Antigens As an alternative to culture, rapid assays have been commercially developed to detect Group A streptococcal antigens directly from throat swabs. The Group A antigens are extracted by either enzymatic or chemical means, and the process takes anywhere from 2 to 30 minutes, depending on the particular technique. Lateral flow immunochromatographic assays (LFAs) are increasingly being used for the detection of bacterial, viral, fungal, and parasitic antigens in clinical samples. LFAs have largely replaced enzyme immunoassay (EIA) and LA assays to detect the antigens. LFAs are widely used in outpatient clinics, physician offices, and urgent care facilities for the rapid diagnosis of streptococcal pharyngitis. The assays are technically easy to perform, allow for singlesample testing because of the incorporation of an internal control, and in many cases are more sensitive than traditional laboratory methods and other antigen detection methods (see Fig. 11–11). An example of an LFA used for the detection of GAS from a throat swab is shown in Figure 20–13. Many of the assays require no more than 2 to 5 minutes of hands-on time. The specificity and sensitivity of the assays in many instances are higher than other methodologies. Although the assays have high sensitivities, cultures should be performed when rapid test results are negative. Molecular methods, including hybridization of specific rRNA sequences and real-time PCR, have also been developed as a means to rapidly detect Group A streptococcal infections. FIGURE 20-13 BinaxNOW lateral flow assay. The BinaxNOW Strep A Test immunochromatographic assay for the qualitative detection of Streptococcus pyogenes Group A antigen from throat swab specimens. To perform the test, a throat swab is inserted into the test card. Extraction reagents are added from dropper bottles. The test card is closed to bring the extracted sample in contact with the test strip. Strep A antigen captured by immobilized anti-strep A reacts to bind conjugated antibody. Immobilized species antibody captures the second visualizing conjugate. The test is interpreted by the presence or absence of visually detectable pink-to-purple colored lines. A positive result is indicated by production of both a sample line and a control line (shown on left), whereas a negative assay will produce only the control line (shown on right). (BinaxNOW is a trademark of the Alere Scarborough, Inc. Used with permission.) Detection of Streptococcal Antibodies Culture or rapid screening methods are extremely useful for diagnosis of acute pharyngitis. However, serological diagnosis must be used to identify acute rheumatic fever and post- streptococcal glomerulonephritis because the organism is unlikely to be present in the pharynx or on the skin at the time symptoms appear. Group A streptococci elaborate more than 20 exotoxins, and it is the antibody response to one or more of these that is used as documentation of nonsuppurative disease. Some of the exotoxin products include streptolysins O and S; deoxyribonucleases A, B, C, and D; streptokinase; NADase; hyaluronidase; diphosphopyridine nucleotidase; and pyrogenic exotoxins. The antibody response to these streptococcal products is variable because of several factors, such as age of onset, site of infection, and timeliness of antibiotic treatment. The most diagnostically important antibodies are the following: anti-streptolysin O (ASO), anti-DNase B, anti-NADase, and anti-hyaluronidase (AHase). Assays for the detection of these antibodies can be performed individually or through use of the streptozyme test, which detects antibodies to all of these products (see Streptozyme Testing later). During Group A streptococcal infections, other antibodies are made to cellular antigens, such as the Group A carbohydrate and the M protein. Generally, detection of these antibodies is done in research or reference laboratories because commercial reagents are not available. Serological evidence of disease is based on an elevated or rising titer of streptococcal antibodies. The onset of clinical symptoms of rheumatic fever or glomerulonephritis typically coincides with the peak of antibody response. If acute- and convalescent-phase sera are tested in parallel, a four-fold rise in titer is considered significant. The use of at least two tests for antibodies to different exotoxins is recommended because the production of detectable ASO does not occur in all patients. The most commonly used tests are those for ASO and anti-DNase B. Antistreptolysin O (ASO) Testing ASO tests detect antibodies to the streptolysin O enzyme produced by GAS. This enzyme is able to lyse RBCs. The presence of antibodies to streptolysin O indicates recent streptococcal infection in patients suspected of having acute rheumatic fever or poststreptococcal glomerulonephritis following a throat infection. The classic hemolytic method for determining the ASO titer was the first test developed to measure streptococcal antibodies. This test was based on the ability of antibodies in the patient’s serum to neutralize the hemolytic activity of streptolysin O. The traditional ASO titer involved preparing dilutions of patient serum to which a measured amount of streptolysin O reagent was added. These were allowed to combine during an incubation period, then reagent RBCs were added as an indicator. If enough antibodies were present, the streptolysin O was neutralized, and no hemolysis occurred. The titer was reported as the reciprocal of the highest dilution demonstrating no hemolysis. This titer could be expressed in either Todd units (by using a streptolysin reagent standard) or in international units (when using the World Health Organization international standard). The range of expected normal values varies, depending on the patient’s age, geographic location, and season of the year. ASO titers tend to be highest in school-age children and young adults. Thus, the upper limits of normal must be established for specific populations. Typically, however, a single ASO titer is considered to be moderately elevated if the titer is at least 240 Todd units in an adult and 320 Todd units in a child. Because of the labor-intensive nature of the traditional ASO titer test and because the streptolysin O reagent and the RBCs used are not stable, ASO testing is currently performed by nephelometric methods. Nephelometry has the advantage of being an automated procedure that provides rapid, quantitative measurement of ASO titers. The antigen used in this technique is purified recombinant streptolysin. When antibody-positive patient serum combines with the antigen reagent, immune complexes are formed, resulting in an increase in light scatter that the instrument converts to a peak rate signal. All results are reported in international units, which are extrapolated from the classic hemolytic method described previously. When using the nephelometric method, individual laboratories must establish their own upper limits of normal for populations of different ages. ASO titers typically increase within 1 to 2 weeks after infection and peak between 3 to 6 weeks following the initial symptoms (e.g., sore throat). However, an antibody response occurs in only about 85% of acute rheumatic fever patients within this period. Additionally, ASO titers usually do not increase in individuals with skin infections. Anti-DNase B (Anti-Deoxyribonuclease B) Testing Testing for the presence of anti-DNase B is clinically useful in patients suspected of having glomerulonephritis preceded by streptococcal skin infections because ASO antibodies often are not stimulated by this type of disease. In addition, antibodies to DNase B may be detected in patients with acute rheumatic fever who have a negative ASO test result. DNase B is mainly produced by Group A streptococci, so testing for anti-DNase B is highly specific for Group A streptococcal sequelae. Macrotiter, microtiter, EIA, and nephelometric methods have been developed for anti-DNase testing. The classic test for the measurement of anti-DNase B activity is based on a neutralization method. If anti-DNase B antibodies are present, they will neutralize reagent DNase B, preventing it from depolymerizing DNA. The presence of DNase is measured by its effect on a DNA-methyl-green conjugate. This complex is green in its intact form; however, when hydrolyzed by DNase, the methyl green is reduced and becomes colorless. An overnight incubation at 37°C is required in some tests to permit antibodies to inactivate the enzyme. Tubes are graded for color, with a 4+ indicating that the intensity of color is unchanged and a 0 indicating a total loss of color. The result is reported as the reciprocal of the highest dilution demonstrating a color intensity of between 2+ and 4+. Normal titers for children between the ages of 2 and 12 years range from 240 to 640 units. Nephelometry provides an automated means of testing that can be used for rapid quantitation of anti-DNase B and has largely replaced the enzyme-neutralization method. In this method, immune complexes formed between antibodies in patient serum and DNase B reagent generate an increase in light scatter. Results are extrapolated from values from the classic method and are reported in international units per mL. Streptozyme Testing The streptozyme test is a slide agglutination screening test for the detection of antibodies against streptococcal antigens. The streptozyme test measures antibodies against five extracellular streptococcal antigens: ASO, anti-hyaluronidase (AHase), anti-streptokinase (ASKase), anti- nicotinamide-adenine dinucleotide (anti-NAD), and anti-DNAase B antibodies. The streptozyme test is positive in 95% of patients with acute poststreptococcal glomerulonephritis because of GAS pharyngitis. In this test, sheep RBCs are coated with streptolysin, streptokinase, hyaluronidase, DNase, and NADase so that antibodies to any of the streptococcal antigens can be detected. Reagent RBCs are mixed with a 1:100 dilution of patient serum. Hemagglutination represents a positive test, indicating that antibodies to one or more of these antigens are present. The test is rapid and simple to perform, but it appears to be less reproducible than other antibody tests. In addition, more false positives and false negatives have been reported for this test than for the ASO and anti-DNase B assays. Because a larger variety of antibodies are included in this test, the potential is higher for the detection of streptococcal antibodies. However, single-titer determinations are not as significant as several titrations performed at weekly or biweekly intervals following the onset of symptoms. The streptozyme test should be used in conjunction with the ASO or anti- DNase B tests when sequelae of Group A streptococcal infection are suspected and is especially useful when negative or borderline ASO results are obtained. (See the streptozyme test laboratory exercise online at DavisPius.) Helicobacter pylori First isolated from humans in 1982, Helicobacter pylori is now recognized as a major cause of both gastric and duodenal ulcers. The organism resides in the mucus layer, the gastric epithelium, and occasionally the duodenal epithelium. This gram-negative, microaerophilic spiral bacterium is observed in 30% of the population in developed countries and more than 90% of the population in developing countries. In developing countries, more than 70% carry H. pyiori by age 10, with carriage being nearly universal by the age of 20. Conversely, in the United States, there is little colonization during childhood. The rates gradually increase during adulthood, reaching a prevalence of 50% among persons older than 60 years. The high incidence of colonization in developing countries where living conditions are more crowded and sanitation conditions are suboptimal suggests that fecal–oral transmission is the most likely route. Since 1994, the National Institutes of Health has recommended that individuals with gastric or duodenal ulcers caused by H. pylori be given antibiotic treatment along with anti-ulcer medications. If untreated, H. pylori infection will last for the patient’s life and may lead to gastric carcinoma. Helicobacter pylori Virulence Factors A major virulence factor of H. pylori is the production of the protein CagA, which is highly immunogenic. The organism has the ability to inject the CagA protein into the gastric epithelial cells. Once the CagA protein is in the epithelial cells, changes occur in the function of the cell’s signal transduction pathways and in the structure of the cytoskeleton. A second virulence factor is vacuolating cytotoxin, or VacA. The VacA gene codes for a toxin precursor. Epidemiological studies have shown that if the CagA and VacA genes are present in the strain of bacteria infecting the individual, there is a higher risk of developing gastric or peptic ulcers or gastric carcinoma. Pathology and Pathogenesis Unlike most other bacteria, H. pylori is able to survive and multiply in the gastric environment. This occurs because of several characteristics of the bacteria. Its spiral shape and flagella help the organism to be highly motile and to penetrate the viscous mucus layer in the stomach. The organism produces large amounts of urease, providing a buffering zone around the bacteria that protects it from the effects of the stomach acid. In addition, the acid-labile flagella are coated with a flagellar sheath, protecting them from the acidic environment of the stomach. The pathology and mechanism of action leading to tissue damage are not clearly understood. Neutrophil-induced mucosal damage may be the result of the ammonia produced by urease. The ammonia has been shown to induce the release of chlorinated toxic oxidants from the neutrophils, which causes inflammation and damage to the mucosal cells. Once established below the mucosal layer, the organism does not invade the tissue. More likely, the pathology represents the host’s response to extracellular products produced by the bacteria. Antibodies are formed against the signaling molecules CagA and VacA. Strains from individuals with stomach ulcers produce higher levels of VacA than strains from individuals without stomach ulcers. The outcomes associated with H. pylori exposure vary. Not all individuals harboring the organism go on to develop disease, suggesting that there may be a genetic predisposition and that the interaction between the host and the bacteria plays a role. In some hosts, the organism is not able to establish itself. In others, asymptomatic colonization may persist, or hyperacidity may result in duodenal ulceration. Treatment of H. pylori consists of triple therapy with bismuth salts, metronidazole, and amoxicillin. Although highly effective, treatment will fail in some individuals because of poor patient compliance or resistance to metronidazole. An increased risk of developing gastric carcinoma and mucosa-associated lymphoid tumors (MALTomas) has also been shown. Diagnosis of H. pylori Infection Detection of H. pylori may be achieved by the invasive techniques of endoscopy or biopsy or through noninvasive techniques, including serological analysis, fecal antigen detection, and demonstration of urease production with urea breath tests. The most specific test to detect H. pylori infection is culture, but the sensitivity is usually lower than other methods because the organism is not evenly distributed throughout the gastric tissue. Endoscopy and Biopsy Endoscopy and biopsy are the most expensive and invasive methods for diagnosing an infection with H. pylori. However, histological examination of the tissue may reveal a great deal of information regarding the lesion. One method of testing for H. pylori involves the detection of urease from a biopsy taken from the antrum of the stomach. The antrum is the portion of the stomach before the pyloric sphincter or valve responsible for releasing stomach contents into the intestines. An example of a test that detects urease in a tissue biopsy is the CLOtest. The CLOtest (for Campylobacter-like organisms) detects urease activity in gastric mucosal biopsies. During the endoscopy, a small biopsy is taken (1 to 3 mm). The specimen is placed in the test cassette, resealed following the manufacturer’s instructions, and sent to the laboratory. If urease is present, the yellow gel will turn a hot-pink color because of an increase in pH in the presence of urease. If urease is not present, the gel will remain yellow (Fig. 20–14). A majority of the tests will turn positive within 20 minutes; however, the test should be held and reexamined after 24 hours to allow time for the detection of a low-level infection. The test is easy to use, and results can be detected in a short period of time, making it ideal for rapid diagnosis of H. pylori infections. Noninvasive Detection Methods Procedures for detecting H. pylori that do not require the use of endoscopy include urea breath testing, enzyme or lateral flow immunoassays for the detection of bacterial antigens in the feces, molecular tests for H. pylori DNA, and serological testing. In the urea breath test, the patient ingests urea labeled with radioactive carbon (14C) or, in newer tests, a nonradioactive 13C urea is broken down by the urease enzyme of H. pylori, producing ammonia and bicarbonate. The bicarbonate is excreted in the breath, and the labeled carbon dioxide is measured by detection of radioactivity for 14C or MS analysis for 13C. The breath technique has excellent sensitivity and specificity and is helpful in determining if the bacteria have been eradicated by antimicrobial therapy. Because of the potential for treatment failure, analysis of stool samples before and after antimicrobial therapy for H. pylori antigens is done to determine if the bacteria have been eliminated following treatment. ELISA tests as well as LFA methods are available. Because of the potential for asymptomatic carriage of the organism, stool antigen testing for initial diagnosis of H. pylori infection is not recommended. FIGURE 20-14 CLOtest. The CLOtest rapid urease method uses a tissue biopsy to detect Helicobacter pylori. The test consists of a well of indicator gel sealed inside a plastic cassette. The gel contains urea, phenol red, buffers, and a bacterial static agent to prevent the growth of contaminating urease-positive organisms. If the urease from H. pylori is present in the tissue sample, it changes the gel from yellow (bottom cassette) to bright magenta (top cassette). A majority of positive tests change color within 20 minutes. The test is reviewed after 24 hours because a low-level positive infection may not become detectable until then. (Courtesy of Halyard Health, Inc., Irvine, CA. Used with permission.) Researchers also have developed molecular testing to detect H. pylori DNA. However, PCR- based methods, which detect the presence of the organism in fecal samples, cannot distinguish between living and dead H. pylori. Real-time PCR technology has been developed to determine the patient’s bacterial load and has shown good correlation with the urea breath test. At the time of this writing, FDA-approved molecular assays for H. pylori are not available for clinical use. Detection of H. pylori Antibodies Serological testing is a primary screening method of determining infection with H. pylori. Infections from this organism result in production of IgG, IgA, and IgM antibodies. Most serological tests in clinical use detect H. pylori–specific antibodies of the IgG class. Although IgM antibody is produced in H. pylori infections, testing for its presence lacks clinical value because most infections have become chronic before diagnosis. Thus, IgG is the primary antibody found. IgA testing has a lower sensitivity and specificity than IgG testing, but it may increase the sensitivity of detection when used in conjunction with IgG testing. The presence of antibodies in the blood of an untreated patient indicates an active infection because the bacterium does not spontaneously clear. Antibody levels in untreated individuals remain elevated for years. In treated individuals, the antibody concentrations decrease after about 6 months to approximately 50% of the level the patient had during the active infection. Therefore, convalescent testing should be performed 6 months to a year after treatment, which requires that the acute serum sample be stored for up to a year. A decrease in antibody titer of more than 25% must occur for treatment to be considered successful. Measurement of the antibodies may be done with several techniques, including ELISA, immunoblot, and rapid tests using LA or LFA. Several LFAs are approved for CLIA-waived testing by physicians’ office laboratories. The method of choice for the detection of H. pylori antibodies is the ELISA technique because it is reliable and accurate. Tests employing antigens from a pooled extract from multiple and genetically diverse strains yield the best sensitivity because H. pylori is so heterogeneous. Very few, if any, patients produce antibodies to all of the H. pylori antigens; most patients produce antibodies against the CagA and VacA proteins. Antibodies to these two proteins indicate a more severe case of gastritis or an increased risk of developing gastric carcinoma. When compared with other techniques for antibody detection of H. pylori, ELISA tests are sensitive, specific, and cost effective for determining the organism’s presence in untreated individuals. However, because antibodies are not rapidly cleared after treatment, antibody testing is not as well suited for determining eradication of infection as are other methods. Additionally, individuals who are immunocompromised (the elderly or immunosuppressed individuals) may have a false-negative result with antibody testing. Rapid assays for the detection of H. pylori antibodies are also available. It is recommended that samples with positive rapid test results be tested by an ELISA method for confirmation. Mycoplasma pneumoniae Mycoplasma is a member of a unique group of organisms that belong to the class Mollicutes. Mycoplasmas represent the smallest known free-living life forms (150–250 nm) and have a small genome. Various members colonize plants, animals, and insects in addition to being human pathogens. These extracellular parasites attach to and exist on the surface of host cells using attachment organelles and adhesion molecules specific for their host cells. They absorb their nutrients from the host cells to which they are attached. The organisms lack cell walls (thus lacking peptidoglycan), have sterols in their cell membrane, and have complex growth requirements, making culture difficult and time consuming. Mycoplasma pneumoniae Pathogenesis The best-known Mycoplasma is M. pneumoniae, which is a leading cause of respiratory infections worldwide. M. pneumoniae infections are found in all age groups, with a majority of the infections involving the upper respiratory tract. M. pneumoniae is spread from one person to another by respiratory droplets. Relatively close association with an infected individual appears to be necessary for transmission of the organism. Unlike most respiratory infections, the incubation period is 1 to 3 weeks. The infection has an insidious onset, which differs from the acute onset observed with respiratory viruses such as influenza and adenovirus. Typically, there is development of a fever, along with headache, malaise, and a cough—the clinical hallmark of a M. pneumoniae infection. Depending on the age, approximately 5% to 10% of individuals progress to tracheobronchitis or pneumonia. Originally, pneumonia caused by M. pneumoniae was referred to as “atypical pneumonia” because the infection could not be treated with penicillin. This is because the organism lacks the cell wall to which penicillin is directed against. In many cases, the pneumonia is mild, oftentimes appearing as a cold, and symptoms are generally mild enough that bedrest or hospitalization is not required. The infection has been referred to as “walking pneumonia” because individuals often do not stay home from work or school and still participate in their daily activities. Although many infections are mild, M. pneumoniae accounts for 20% of all hospitalizations for pneumonia in the United States. M. pneumoniae may remain in the respiratory tract for several months after resolution of the infection, causing chronic inflammation and a lingering cough. Based on nucleic acid detection of the organism, there is increasing evidence that M. pneumoniae may initiate or exacerbate asthma. Dermatological Manifestations Up to 7% of individuals with M. pneumoniae develop Stevens–Johnson syndrome, or erythema multiforme major, a condition in which the top layer of the skin dies and sheds. The syndrome is considered a medical emergency that usually requires hospitalization. The conjunctivae as well as the joints and various organs in the genitourinary and gastrointestinal tract may also be involved. The basis of Stevens–Johnson syndrome is not clearly known, but it may be caused by the immune response of the host or by augmented sensitivity to antibiotics while being treated for M. pneumoniae. Another manifestation of M. pneumoniae infection is Raynaud syndrome, which is a transient vasospasm of the digits in which the fingers turn white when exposed to the cold. Although the exact cause is unclear, it may be related to the development and action of cold agglutinins in the body (see Chapter 14). Other extrapulmonary manifestations of Raynaud syndrome include arthritis, meningoencephalitis, pericarditis, and peripheral neuropathy. Immunology of Mycoplasma pneumoniae Infection In addition to stimulating the production of many proinflammatory and anti-inflammatory cytokines and chemokines, several classes of antibodies are produced in the course of a M. pneumoniae infection. As with any infection in which there is a humoral response, the body produces antibodies that neutralize the microorganism. M. pneumoniae also induces the production of autoantibodies, including agglutinins directed against the lungs, brain, cardiolipins, and smooth muscle. The cold isoagglutinins observed with M. pneumoniae infection are among the most studied agglutinins by researchers. They are oligoclonal IgM antibodies directed against the altered I antigens found on the surface of human RBCs. These antibodies can agglutinate the RBCs at temperatures below 37°C. Development of the antibodies is thought to result from cross-reaction of antibodies formed against M. pneumoniae and the I antigen on human RBCs, or from alteration of the RBC antigen by the organism. Laboratory Diagnosis of Mycoplasma pneumoniae Infection Laboratory means of detecting Mycoplasma infection may involve culturing of the organism, detection of M. pneumoniae-specific antibodies in serum, and detection of M. pneumoniae- specific antigens or nucleotide sequences directly in patient specimens. Detection of Mycoplasma pneumoniae by Culture Although culturing has been considered the gold standard for diagnosis, culturing for the organism is rarely carried out in the clinical laboratory because of the fastidious nature of the organism. Collection and transport of the specimen differs from traditional methods used for culturing other microorganisms. The transport media may be trypticase soy broth with 0.5% albumin, SP4 medium, or a viral transport medium. If the sample cannot be plated immediately, it should be frozen at –70°C. Culturing requires the use of specialized media designed for the recovery of Mycoplasma. Growth of the organism takes several weeks in most cases. If the culture is successfully performed, the growth produces a “mulberry” colony with a typical “fried egg” appearance. Because of the difficulty in culturing for the organism and the time required for the organism to grow, culturing for the organism is rarely used except in research settings. Detection of Antibodies to Mycoplasma pneumoniae Detection of M. pneumoniae-specific IgM immunoglobulin is the most useful diagnostic test because it likely indicates a recent infection. Enzyme-linked immunoassays have been the most widely used methods for antibodies and can detect IgM or IgG directed against M. pneumoniae. Although IgM is the primary immunoglobulin response to infection, testing for the presence of IgG antibodies is necessary; the reason is that adults may only elicit an IgG response because of reinfection with the organism. ELISA methods have a specificity of more than 99% and a sensitivity of 98%. Detection of Cold Agglutinins For many years, before the development of antigen-specific serological tests, laboratory diagnosis of M. pneumoniae involved testing for cold agglutinins. The agglutinins are capable of clumping RBCs at 4°C. The reaction is reversible when the samples are warmed to 37°C. Cold agglutinins develop in about 50% of patients with M. pneumoniae infection. These antibodies are produced early in the disease (7–10 days) and can typically be detected at the time the patient seeks medical attention. The titer peaks at 2 to 3 weeks, and antibodies are present for 2 to 3 months after infection. Although once considered the primary means of diagnosing Mycoplasma infection, the assay is not very specific (50% to 70%) nor is it very sensitive. Testing for cold agglutinins is no longer recommended because the development of cold agglutinins occurs in other circumstances, including some viral infections and collagen vascular diseases. However, a titer of 1:64 or greater, along with the clinical presentation of the patient, is suggestive of infection with M. pneumoniae. It should be noted that cold agglutinins may be found in patients with infections whose clinical presentations resemble M. pneumoniae infection, such as mononucleosis caused by Epstein-Barr virus (anti-i) and cytomegalovirus (anti-I). Molecular Diagnosis of Mycoplasma Infections Molecular methods will, in all likelihood, become the gold standard for the diagnosis of Mycoplasma infections. Before 2012, there were no FDA-approved assays available for the detection of M. pneumoniae. BioFire Diagnostics, Inc. (Salt Lake City, UT), now part of the BioMerieux corporation, received FDA approval in 2012 for its BioFire FilmArray Respiratory Panel. Using nested multiplex PCR, the assay is able to detect 20 respiratory viruses and bacteria, including B. pertussis, C. pneumoniae, and M. pneumoniae. The illumigene Mycoplasma Direct by Meridian Bioscience (Cincinnati, OH) detects M. pneumonia in throat swab specimens and became available in 2014. The illumigene uses Loop-Mediated Isothermal Amplification (LAMP) technology. As additional assays are developed and receive FDA approval, molecular testing will become more widespread and will likely replace serology as the primary means for the diagnosis of M. pneumoniae infections. Rickettsial Infections Members of the Rickettsiaceae family cause a variety of infections in man and animals. Because of advances in molecular technology, various members originally belonging to the genus Rickettsiae have been reclassified. These obligate intracellular, gram-negative bacteria now include the genera Rickettsia (rickettsiosis) and Orientia (Orientia tsutsugamushi causing scrub typhus). Additional members are the Ehrlichia group including Ehrlichia (ehrlichiosis), Anaplasma (anaplasmosis), Neorickettsia (associated with helminths), and Neoehrlichia. Agents of Rickettsia-Related Disease The genus Rickettsia is made up of two distinct groups: the spotted fever group (SFG) and the typhus group (TG). Each is responsible for a different set of diseases (Table 20–1). In the United States, the main Rickettsial disease is Rocky Mountain spotted fever (RMSF), caused by R. rickettsia (SFG), with approximately 2,500 cases reported each year. Epidemic typhus, caused by Rickettsia prowazekii (TG), is the most prevalent member of the TG globally. Typhus fever (also known as epidemic typhus) occurs in conditions of poor hygiene and overcrowding, such as in prisons and refugee camps. Epidemic typhus was responsible for more than 3 million deaths in World War I. Once prevalent throughout the globe in the first half of the 20th century, only a few foci of epidemic typhus still exist in the world today (Ethiopia, Burundi, Rwanda, part of Mexico). Individuals traveling to areas with large homeless populations and regions that have recently experienced war or natural disasters leading to poor hygiene and crowded conditions (such as refugee camps) are at risk of acquiring typhus fever. Table 20-1 Classification of Selected Rickettsiae Known to Cause Disease in Humans ANTIGENIC ANIMAL GEOGRAPHIC GROUP DISEASE SPECIES VECTOR RESERVOIR(S) DISTRIBUTION Anaplasma Human Anaplasma Tick Small mammals, Primarily United granulocytic phagocytophilum rodents, and deer States, worldwide anaplasmosis Ehrlichia Human Ehrlichia Tick Deer, wild and Common in United monocytic chaffeensis domestic dogs, States, probably ehrlichiosis domestic worldwide ruminants, and rodents Ehrlichiosis E. muris Tick Deer and rodents North America, Europe, Asia Ehrlichiosis E. ewingii Tick Deer, wild and North America, domestic dogs, and Cameroon, Korea rodents Neoehrlichia Human Neoehrlichia Tick Rodents Europe, Asia neoehrlichiosis mikurensis Neorickettsia Sennetsu fever Neorickettsia Trematode Fish Japan, Malaysia, sennetsu possibly other parts of Asia Scrub typhus Scrub typhus Orientia Larval mite Rodents Asia-Pacific region tsutsugamushi (chigger) from maritime Russia and China to Indonesia and North Australia to Afghanistan Spotted fever Rocky Mountain Rickettsia rickettsii Tick Rodents North, Central, and spotted fever South America Rickettsiosis Rickettsia Tick Unknown South Africa, aeschlimannii Morocco, Mediterranean shore Queensland tick Rickettsia australis Tick Rodents Australia, Tasmania typhus Boutonneuse Rickettsia conorii Tick Dogs, rodents Southern Europe, fever or southern and Mediterranean western Asia, spotted fever Africa, India Typhus fever Epidemic Rickettsia Human body Humans, flying Central Africa, Asia, typhus, prowazekii louse, flying squirrels Central America, sylvatic typhus squirrel North America, and ectoparasites, South America Amblyomma ticks Murine typhus Rickettsia typhi Flea Rodents Tropical and subtropical areas worldwide Adapted from Centers for Disease Control and Prevention. 2018 Yellow Book: Traveler’s Health. Nicholson WL and Paddock CD. Chapter 3. Rickettsial (Spotted & Typhus Fevers) & Related Infections, including Anaplasmosis & Ehrlichiosis. Online version at https://wwwnc.cdc.gov/travel/yellowbook/2018/infectious-diseases-related-to- travel/nckettsial-spotted-and-typhus-fevers-and-related-infections-including-anaplasmosis-and-ehrlichiosis#5251. Accessed May 31, 2019. Each of the species responsible for the various Rickettsial diseases has a variety of animal reservoirs. The vectors responsible for the transmission are arthropods (ticks, mites, lice, or fleas) that transmit the organism through its bite after feeding on an infected animal (Fig. 20–15). The one exception is typhus fever (epidemic louse-borne typhus), which is transmitted when an infected human body louse excretes R. prowazekii onto the skin while feeding and the individual becomes infected by rubbing louse fecal matter or crushed lice into the bite wound. Except for R. prowazekii (epidemic typhus), humans are accidental hosts for Rickettsia and Rickettsia-related organisms. Rickettsia and Rickettsia-related organisms have worldwide distribution; however, certain members have a specific geographic distribution. For example, Rickettsia japonica is found only in Japan, whereas R. rickettsii is found in the Western hemisphere. Some species, such as Rickettsia typhi, are found everywhere in the world. FIGURE 20-15 The Rocky Mountain wood tick, Dermacentor andersoni, is a known North American vector of Rickettsia rickettsii. (Courtesy of the CDC/Dr. Christopher Paddock and James Gathany, Public Health Image Library.) The members of the Rickettsia genus and Anaplasma and Ehrlichia cause several clinical diseases. Because of the prevalence of RMSF in North and South America, this chapter will focus on RMSF Rocky Mountain Spotted Fever Epidemiology RMSF is caused by R. rickettsii. The organism is transmitted to the human host by the bite of a tick. In the United States, the organism is transmitted by the American dog tick (Dermacentor variabilis), the Rocky Mountain wood tick (Dermacentor andersoni), and the brown dog tick (Rhipicephalus sanguineus). Although called Rocky Mountain spotted fever, five states—North Carolina, Oklahoma, Arkansas, Tennessee, and Missouri—account for more than 60% of RMSF cases in the continental United States. The occurrence of the disease has seasonal variation corresponding to tick activity, being most prevalent between May and September. The organism is transmitted transstadially in the tick (i.e., it remains present in the tick as the tick progresses from the nymph state to the adult) and is transmitted transovarially (from generation to generation through the eggs of the tick), allowing for maintenance of the organism in the tick population. Transmission occurs when the tick bites the host for a blood meal. When the tick has fed after 6 to 10 hours, the organism is injected into the host from the salivary glands. Pathogenesis Once introduced into the skin, the organisms spread via the lymphatic and circulatory system, where they attach to and invade their target cells, the vascular endothelium, by means of the OmpA and OmpB ligands. The organisms multiply by binary fission inside the endothelial cells, are released, and infect adjacent cells. This leads to hundreds of contiguous infected cells, producing the lesions and skin rash associated with the infection. The main pathophysiological event caused by the infection is endothelial cell damage, which leads to increased vascular permeability, resulting in edema, hypovolemia, hypotension, and hypoalbuminemia. Clinical Manifestations The symptoms observed with RMSF occur approximately 2 to 14 days (median 7 days) after a tick bite. Before the development of the hallmark rash, a large percentage of patients will experience a severe headache, nausea, vomiting, abdominal pain, diarrhea, and abdominal tenderness. The fever usually begins within the first 3 to 5 days after the onset of symptoms, and the rash usually appears 3 to 5 days after the onset of the fever. The rash typically starts on the hands and soles of the feet and proceeds to the trunk, although it may start on the trunk in some individuals (Fig. 20–16). With the classic form of RMSF, death occurs 7 to 15 days after the onset of symptoms if appropriate therapy is not provided. With the fulminant (i.e., severe and sudden onset) form of RMSF, death occurs within the first 5 days. The resolution or fatal outcome of the disease is largely related to the timeliness of initiating appropriate therapy. Immediate treatment with doxycycline reduces the severity of the infection. FIGURE 20-16 The characteristic spotted rash of Rocky Mountain spotted fever, the most severe and most frequently reported rickettsial illness in the United States. The disease is caused by Rickettsia rickettsii. (Courtesy of the CDC, Public Health Image Library.) Diagnosis of RMSF Initial diagnosis is often made clinically after ruling out a large variety of other conditions, including typhoid fever, measles, rubella, enteroviral infection, and respiratory tract infection. The overlapping symptoms, or clinical presentation, during the initial stages of the disease can make the diagnosis of RMSF extremely difficult. The diagnosis of fulminant RMSF is even more difficult because of its rapid course. The rash develops shortly before death, if at all; therefore, antibodies to R. rickettsii do not have time to develop. Serological and Molecular Diagnosis The organism infects the endothelial cells and does not circulate until the disease has severely progressed. Therefore, culturing for the organism and molecular methods are not always useful. If the patient has a rash, molecular diagnosis using DNA from the skin lesions is of value. Several assays using real-time PCR have been described in the literature, but at the time of this writing, there are no FDA-approved assays. Serology is the usual method for confirming the diagnosis of RMSF, but this is a retrospective diagnosis. Antibodies to R. rickettsia develop 7 to 10 days after the onset of symptoms, and a majority of patients do not have detectable antibodies during the first week of illness. For a successful outcome, therapy needs to be initiated before that time. The gold standard for the serological diagnosis of RMSF is the indirect immunofluorescence assay (IFA) with R. rickettsii antigen, performed on two paired serum samples to demonstrate a significant (four-fold) rise in antibody titers. For many years, antibodies produced in patients with Rickettsial infections were detected by an agglutination test known as the Weil-Felix test, which was based on cross-reactivity of the patient’s antibodies with polysaccharide antigens present on the OX-19 and OX-2 strains of Proteus vulgaris and the OX-K strain of Proteus mirabilis. This method lacks sensitivity and specificity and should not be relied on. SUMMARY The indigenous microbiota varies at different sites of the body. The symbiotic relationship that exists between bacteria and humans is beneficial in protecting against infection, stimulating the immune system, aiding in digestion of food, and producing various vitamins. Humans exist in a commensalistic relationship with the bacteria that comprise the human microbiome. The encounter with some microbial organisms results in a parasitic relationship in which there is harm to the host that may result in an infection. Pathogenicity refers to the ability of an organism to cause disease, virulence refers to the extent that a pathogen causes damage to the host, and infectivity refers to the ability of an organism to spread from one host to another. To be virulent, an organism must possess structural features or produce extracellular substances that allow it to invade or cause damage to the host. These are referred to as virulence factors. Live bacteria may produce exotoxins that are generally specific to a particular bacterial organism and have specific modes of action on the host. Exotoxins are highly immunogenic, and antibodies formed against them are protective. Endotoxin, or lipid A, is part of the gram-negative bacterial cell wall that is released from dead bacteria. Endotoxin has a broad range of systemic effects on the body because it induces the release of cytokines that can lead to septic shock. Endotoxin, although immunogenic, does not result in the production of protective antibodies. Innate immune def