# Microbial Mechanisms of Pathogenicity

## Virulence Factors of Bacterial and Viral Pathogens

### Learning Objectives

By the end of this section, you will be able to:
1. Explain how virulence factors contribute to signs and symptoms of infectious disease
2. Differentiate between endotoxins and exotoxins
3. Describe and differentiate between various types of exotoxins
4. Describe the mechanisms viruses use for adhesion and antigenic variation
In the previous section, we explained that some pathogens are more virulent than others. This is due to the unique virulence factors produced by individual pathogens, which determine the extent and severity of disease they may cause. A pathogen’s virulence factors are encoded by genes that can be identified using molecular Koch’s postulates. When genes encoding virulence factors are inactivated, virulence in the pathogen is diminished. In this section, we examine various types and specific examples of virulence factors and how they contribute to each step of pathogenesis.

### Virulence Factors for Adhesion

As discussed in the previous section, the first two steps in pathogenesis are exposure and adhesion. Recall that an adhesin is a protein or glycoprotein found on the surface of a pathogen that attaches to receptors on the host cell. Adhesins are found on bacterial, viral, fungal, and protozoan pathogens. One example of a bacterial adhesin is type 1 fimbrial adhesin, a molecule found on the tips of fimbriae of enterotoxigenic E. coli (ETEC). Recall that fimbriae are hairlike protein bristles on the cell surface. Type 1 fimbrial adhesin allows the fimbriae of ETEC cells to attach to the mannose glycans expressed on intestinal epithelial cells.  lists common adhesins found in some of the pathogens we have discussed or will be seeing later in this chapter.

### Bacterial Exoenzymes and Toxins as Virulence Factors

After exposure and adhesion, the next step in pathogenesis is invasion, which can involve enzymes and toxins. Many pathogens achieve invasion by entering the bloodstream, an effective means of dissemination because blood vessels pass close to every cell in the body. The downside of this mechanism of dispersal is that the blood also includes numerous elements of the immune system. Various terms ending in –emia are used to describe the presence of pathogens in the bloodstream. The presence of bacteria in blood is called bacteremia. Bacteremia involving pyogens (pus-forming bacteria) is called pyemia. When viruses are found in the blood, it is called viremia. The term toxemia describes the condition when toxins are found in the blood. If bacteria are both present and multiplying in the blood, this condition is called septicemia.
Patients with septicemia are described as septic, which can lead to shock, a life-threatening decrease in blood pressure (systolic pressure <90 mm Hg) that prevents cells and organs from receiving enough oxygen and nutrients. Some bacteria can cause shock through the release of toxins (virulence factors that can cause tissue damage) and lead to low blood pressure. Gram-negative bacteria are engulfed by immune system phagocytes, which then release tumor necrosis factor, a molecule involved in inflammation and fever. Tumor necrosis factor binds to blood capillaries to increase their permeability, allowing fluids to pass out of blood vessels and into tissues, causing swelling, or edema (). With high concentrations of tumor necrosis factor, the inflammatory reaction is severe and enough fluid is lost from the circulatory system that blood pressure decreases to dangerously low levels. This can have dire consequences because the heart, lungs, and kidneys rely on normal blood pressure for proper function; thus, multi-organ failure, shock, and death can occur.

### Exoenzymes

Some pathogens produce extracellular enzymes, or exoenzymes, that enable them to invade host cells and deeper tissues. Exoenzymes have a wide variety of targets. Some general classes of exoenzymes and associated pathogens are listed in . Each of these exoenzymes functions in the context of a particular tissue structure to facilitate invasion or support its own growth and defend against the immune system. For example, hyaluronidase S, an enzyme produced by pathogens like , , and , degrades the glycoside hyaluronan (hyaluronic acid), which acts as an intercellular cement between adjacent cells in connective tissue (). This allows the pathogen to pass through the tissue layers at the portal of entry and disseminate elsewhere in the body ().
Pathogen-produced nucleases, such as DNAse produced by S. aureus, degrade extracellular DNA as a means of escape and spreading through tissue. As bacterial and host cells die at the site of infection, they lyse and release their intracellular contents. The DNA chromosome is the largest of the intracellular molecules, and masses of extracellular DNA can trap bacteria and prevent their spread. S. aureus produces a DNAse to degrade the mesh of extracellular DNA so it can escape and spread to adjacent tissues. This strategy is also used by S. aureus and other pathogens to degrade and escape webs of extracellular DNA produced by immune system phagocytes to trap the bacteria.
Enzymes that degrade the phospholipids of cell membranes are called phospholipases. Their actions are specific in regard to the type of phospholipids they act upon and where they enzymatically cleave the molecules. The pathogen responsible for anthrax, B. anthracis, produces phospholipase C. When B. anthracis is ingested by phagocytic cells of the immune system, phospholipase C degrades the membrane of the phagosome before it can fuse with the lysosome, allowing the pathogen to escape into the cytoplasm and multiply. Phospholipases can also target the membrane that encloses the phagosome within phagocytic cells. As described earlier in this chapter, this is the mechanism used by intracellular pathogens such as L. monocytogenes and Rickettsia to escape the phagosome and multiply within the cytoplasm of phagocytic cells. The role of phospholipases in bacterial virulence is not restricted to phagosomal escape. Many pathogens produce phospholipases that act to degrade cell membranes and cause lysis of target cells. These phospholipases are involved in lysis of red blood cells, white blood cells, and tissue cells.
Bacterial pathogens also produce various protein-digesting enzymes, or proteases. Proteases can be classified according to their substrate target (e.g., serine proteases target proteins with the amino acid serine) or if they contain metals in their active site (e.g., zinc metalloproteases contain a zinc ion, which is necessary for enzymatic activity).
One example of a protease that contains a metal ion is the exoenzyme collagenase. Collagenase digests collagen, the dominant protein in connective tissue. Collagen can be found in the extracellular matrix, especially near mucosal membranes, blood vessels, nerves, and in the layers of the skin. Similar to hyaluronidase, collagenase allows the pathogen to penetrate and spread through the host tissue by digesting this connective tissue protein. C. perfringens then uses toxins and a phospholipase to cause cellular lysis and necrosis. Once the host cells have died, the bacterium produces gas by fermenting the muscle carbohydrates. The widespread necrosis of tissue and accompanying gas are characteristic of the condition known as gas gangrene (). Although a rare event, collagenase can allow C. perfringens to spread to the bloodstream, causing a dangerous septicemia with a high mortality rate

### Toxins

In addition to exoenzymes, certain pathogens are able to produce toxins, biological poisons that assist in their ability to invade and cause damage to tissues. The ability of a pathogen to produce toxins to cause damage to host cells is called toxigenicity.
Toxins can be categorized as endotoxins or exotoxins. The lipopolysaccharide (LPS) found on the outer membrane of gram-negative bacteria is called endotoxin (). During infection and disease, gram-negative bacterial pathogens release endotoxin either when the cell dies, resulting in the disintegration of the membrane, or when the bacterium undergoes binary fission. The lipid component of endotoxin, lipid A, is responsible for the toxic properties of the LPS molecule. Lipid A is relatively conserved across different genera of gram-negative bacteria; therefore, the toxic properties of lipid A are similar regardless of the gram-negative pathogen. In a manner similar to that of tumor necrosis factor, lipid A triggers the immune system’s inflammatory response (see Inflammation and Fever). If the concentration of endotoxin in the body is low, the inflammatory response may provide the host an effective defense against infection; on the other hand, high concentrations of endotoxin in the blood can cause an excessive inflammatory response, leading to a severe drop in blood pressure, multi-organ failure, and death.
A classic method of detecting endotoxin is by using the . In this procedure, the blood cells (amebocytes) of the horseshoe crab (Limulus polyphemus) is mixed with a patient’s serum. The amebocytes will react to the presence of any endotoxin. This reaction can be observed either chromogenically (color) or by looking for coagulation (clotting reaction) to occur within the serum. An alternative method that has been used is an enzyme-linked immunosorbent assay (ELISA) that uses antibodies to detect the presence of endotoxin.
Unlike the toxic lipid A of endotoxin, exotoxins are protein molecules that are produced by a wide variety of living pathogenic bacteria. Although some gram-negative pathogens produce exotoxins, the majority are produced by gram-positive pathogens. Exotoxins differ from endotoxin in several other key characteristics, summarized in . In contrast to endotoxin, which stimulates a general systemic inflammatory response when released, exotoxins are much more specific in their action and the cells they interact with. Each exotoxin targets specific receptors on specific cells and damages those cells through unique molecular mechanisms. Endotoxin remains stable at high temperatures, and requires heating at 121 °C (250 °F) for 45 minutes to inactivate. By contrast, most exotoxins are heat labile because of their protein structure, and many are denatured (inactivated) at temperatures above 41 °C (106 °F). As discussed earlier, endotoxin can stimulate a lethal inflammatory response at very high concentrations and has a measured LD of 0.24 mg/kg. By contrast, very small concentrations of exotoxins can be lethal. For example, botulinum toxin, which causes botulism, has an LD50 of 0.000001 mg/kg (240,000 times more lethal than endotoxin).
The exotoxins can be grouped into three categories based on their target: intracellular targeting, membrane disrupting, and superantigens.  provides examples of well-characterized toxins within each of these three categories.
The intracellular targeting toxins comprise two components: A for activity and B for binding. Thus, these types of toxins are known as A-B exotoxins (). The B component is responsible for the cellular specificity of the toxin and mediates the initial attachment of the toxin to specific cell surface receptors. Once the A-B toxin binds to the host cell, it is brought into the cell by endocytosis and entrapped in a vacuole. The A and B subunits separate as the vacuole acidifies. The A subunit then enters the cell cytoplasm and interferes with the specific internal cellular function that it targets.
Four unique examples of A-B toxins are the diphtheria, cholera, botulinum, and tetanus toxins. The diphtheria toxin is produced by the gram-positive bacterium , the causative agent of nasopharyngeal and cutaneous diphtheria. After the A subunit of the diphtheria toxin separates and gains access to the cytoplasm, it facilitates the transfer of adenosine diphosphate (ADP)-ribose onto an elongation-factor protein (EF-2) that is needed for protein synthesis. Hence, diphtheria toxin inhibits protein synthesis in the host cell, ultimately killing the cell ().
Cholera toxin is an enterotoxin produced by the gram-negative bacterium  and is composed of one A subunit and five B subunits. The mechanism of action of the cholera toxin is complex. The B subunits bind to receptors on the intestinal epithelial cell of the small intestine. After gaining entry into the cytoplasm of the epithelial cell, the A subunit activates an intracellular G protein. The activated G protein, in turn, leads to the activation of the enzyme adenyl cyclase, which begins to produce an increase in the concentration of cyclic AMP (a secondary messenger molecule). The increased cAMP disrupts the normal physiology of the intestinal epithelial cells and causes them to secrete excessive amounts of fluid and electrolytes into the lumen of the intestinal tract, resulting in severe “rice-water stool” diarrhea characteristic of cholera.
Botulinum toxin (also known as botox) is a neurotoxin produced by the gram-positive bacterium . It is the most acutely toxic substance known to date. The toxin is composed of a light A subunit and heavy protein chain B subunit. The B subunit binds to neurons to allow botulinum toxin to enter the neurons at the neuromuscular junction. The A subunit acts as a protease, cleaving proteins involved in the neuron’s release of acetylcholine, a neurotransmitter molecule. Normally, neurons release acetylcholine to induce muscle fiber contractions. The toxin’s ability to block acetylcholine release results in the inhibition of muscle contractions, leading to muscle relaxation. This has the potential to stop breathing and cause death. Because of its action, low concentrations of botox are used for cosmetic and medical procedures, including the removal of wrinkles and treatment of overactive bladder.
Another neurotoxin is tetanus toxin, which is produced by the gram-positive bacterium . This toxin also has a light A subunit and heavy protein chain B subunit. Unlike botulinum toxin, tetanus toxin binds to inhibitory interneurons, which are responsible for release of the inhibitory neurotransmitters glycine and gamma-aminobutyric acid (GABA). Normally, these neurotransmitters bind to neurons at the neuromuscular junction, resulting in the inhibition of acetylcholine release. Tetanus toxin inhibits the release of glycine and GABA from the interneuron, resulting in permanent muscle contraction. The first symptom is typically stiffness of the jaw (lockjaw). Violent muscle spasms in other parts of the body follow, typically culminating with respiratory failure and death.  shows the actions of both botulinum and tetanus toxins.
Membrane-disrupting toxins affect cell membrane function either by forming pores or by disrupting the phospholipid bilayer in host cell membranes. Two types of membrane-disrupting exotoxins are hemolysins and leukocidins, which form pores in cell membranes, causing leakage of the cytoplasmic contents and cell lysis. These toxins were originally thought to target red blood cells (erythrocytes) and white blood cells (leukocytes), respectively, but we now know they can affect other cells as well. The gram-positive bacterium  produces streptolysins, water-soluble hemolysins that bind to the cholesterol moieties in the host cell membrane to form a pore. The two types of streptolysins, O and S, are categorized by their ability to cause hemolysis in erythrocytes in the absence or presence of oxygen. Streptolysin O is not active in the presence of oxygen, whereas streptolysin S is active in the presence of oxygen. Other important pore-forming membrane-disrupting toxins include alpha toxin of  and pneumolysin of .
Bacterial phospholipases are membrane-disrupting toxins that degrade the phospholipid bilayer of cell membranes rather than forming pores. We have already discussed the phospholipases associated with B. anthracis, L. pneumophila, and Rickettsia species that enable these bacteria to effect the lysis of phagosomes. These same phospholipases are also hemolysins. Other phospholipases that function as hemolysins include the alpha toxin of , phospholipase C of P. aeruginosa, and beta toxin of Staphylococcus aureus.
Some strains of S. aureus also produce a leukocidin called Panton-Valentine leukocidin (PVL). PVL consists of two subunits, S and F. The S component acts like the B subunit of an A-B exotoxin in that it binds to glycolipids on the outer plasma membrane of animal cells. The F-component acts like the A subunit of an A-B exotoxin and carries the enzymatic activity. The toxin inserts and assembles into a pore in the membrane. Genes that encode PVL are more frequently present in S. aureus strains that cause skin infections and pneumonia.V. Meka. “Panton-Valentine Leukocidin.” http://www.antimicrobe.org/h04c.files/history/PVL-S-aureus.asp PVL promotes skin infections by causing edema, erythema (reddening of the skin due to blood vessel dilation), and skin necrosis. PVL has also been shown to cause necrotizing pneumonia. PVL promotes pro-inflammatory and cytotoxic effects on alveolar leukocytes. This results in the release of enzymes from the leukocytes, which, in turn, cause damage to lung tissue.
The third class of exotoxins is the superantigens. These are exotoxins that trigger an excessive, nonspecific stimulation of immune cells to secrete cytokines (chemical messengers). The excessive production of cytokines, often called a cytokine storm, elicits a strong immune and inflammatory response that can cause life-threatening high fevers, low blood pressure, multi-organ failure, shock, and death. The prototype superantigen is the toxic shock syndrome toxin of S. aureus. Most toxic shock syndrome cases are associated with vaginal colonization by toxin-producing S. aureus in menstruating women; however, colonization of other body sites can also occur. Some strains of  also produce superantigens; they are referred to as the streptococcal mitogenic exotoxins and the streptococcal pyrogenic toxins.

### Virulence Factors for Survival in the Host and Immune Evasion

Evading the immune system is also important to invasiveness. Bacteria use a variety of virulence factors to evade phagocytosis by cells of the immune system. For example, many bacteria produce capsules, which are used in adhesion but also aid in immune evasion by preventing ingestion by phagocytes. The composition of the capsule prevents immune cells from being able to adhere and then phagocytose the cell. In addition, the capsule makes the bacterial cell much larger, making it harder for immune cells to engulf the pathogen (). A notable capsule-producing bacterium is the gram-positive pathogen , which causes pneumococcal pneumonia, meningitis, septicemia, and other respiratory tract infections. Encapsulated strains of S. pneumoniae are more virulent than nonencapsulated strains and are more likely to invade the bloodstream and cause septicemia and meningitis.
Some pathogens can also produce proteases to protect themselves against phagocytosis. As described in Adaptive Specific Host Defenses, the human immune system produces antibodies that bind to surface molecules found on specific bacteria (e.g., capsules, fimbriae, flagella, LPS). This binding initiates phagocytosis and other mechanisms of antibacterial killing and clearance. Proteases combat antibody-mediated killing and clearance by attacking and digesting the antibody molecules ().
In addition to capsules and proteases, some bacterial pathogens produce other virulence factors that allow them to evade the immune system. The fimbriae of certain species of Streptococcus contain M protein, which alters the surface of Streptococcus and inhibits phagocytosis by blocking the binding of the complement molecules that assist phagocytes in ingesting bacterial pathogens. The acid-fast bacterium  (the causative agent of tuberculosis) produces a waxy substance known as mycolic acid in its cell envelope. When it is engulfed by phagocytes in the lung, the protective mycolic acid coat enables the bacterium to resist some of the killing mechanisms within the phagolysosome.
Some bacteria produce virulence factors that promote infection by exploiting molecules naturally produced by the host. For example, most strains of Staphylococcus aureus produce the exoenzyme coagulase, which exploits the natural mechanism of blood clotting to evade the immune system. Normally, blood clotting is triggered in response to blood vessel damage; platelets begin to plug the clot, and a cascade of reactions occurs in which fibrinogen, a soluble protein made by the liver, is cleaved into fibrin. Fibrin is an insoluble, thread-like protein that binds to blood platelets, cross-links, and contracts to form a mesh of clumped platelets and red blood cells. The resulting clot prevents further loss of blood from the damaged blood vessels. However, if bacteria release coagulase into the bloodstream, the fibrinogen-to-fibrin cascade is triggered in the absence of blood vessel damage. The resulting clot coats the bacteria in fibrin, protecting the bacteria from exposure to phagocytic immune cells circulating in the bloodstream.
Whereas coagulase causes blood to clot, kinases have the opposite effect by triggering the conversion of plasminogen to plasmin, which is involved in the digestion of fibrin clots. By digesting a clot, kinases allow pathogens trapped in the clot to escape and spread, similar to the way that collagenase, hyaluronidase, and DNAse facilitate the spread of infection. Examples of kinases include staphylokinases and streptokinases, produced by  and , respectively. It is intriguing that S. aureus can produce both coagulase to promote clotting and staphylokinase to stimulate the digestion of clots. The action of the coagulase provides an important protective barrier from the immune system, but when nutrient supplies are diminished or other conditions signal a need for the pathogen to escape and spread, the production of staphylokinase can initiate this process.
A final mechanism that pathogens can use to protect themselves against the immune system is called antigenic variation, which is the alteration of surface proteins so that a pathogen is no longer recognized by the host’s immune system. For example, the bacterium , the causative agent of Lyme disease, contains a surface lipoprotein known as VlsE. Because of genetic recombination during DNA replication and repair, this bacterial protein undergoes antigenic variation. Each time fever occurs, the VlsE protein in B. burgdorferi can differ so much that antibodies against previous VlsE sequences are not effective. It is believed that this variation in the VlsE contributes to the ability B. burgdorferi to cause chronic disease. Another important human bacterial pathogen that uses antigenic variation to avoid the immune system is , which causes the sexually transmitted disease gonorrhea. This bacterium is well known for its ability to undergo antigenic variation of its type IV pili to avoid immune defenses.

### Viral Virulence

Although viral pathogens are not similar to bacterial pathogens in terms of structure, some of the properties that contribute to their virulence are similar. Viruses use adhesins to facilitate adhesion to host cells, and certain enveloped viruses rely on antigenic variation to avoid the host immune defenses. These virulence factors are discussed in more detail in the following sections.

### Viral Adhesins

One of the first steps in any viral infection is adhesion of the virus to specific receptors on the surface of cells. This process is mediated by adhesins that are part of the viral capsid or membrane envelope. The interaction of viral adhesins with specific cell receptors defines the tropism (preferential targeting) of viruses for specific cells, tissues, and organs in the body. The spike protein hemagglutinin found on Influenzavirus is an example of a viral adhesin; it allows the virus to bind to the sialic acid on the membrane of host respiratory and intestinal cells. Another viral adhesin is the glycoprotein gp20, found on HIV. For HIV to infect cells of the immune system, it must interact with two receptors on the surface of cells. The first interaction involves binding between gp120 and the CD4 cellular marker that is found on some essential immune system cells. However, before viral entry into the cell can occur, a second interaction between gp120 and one of two chemokine receptors (CCR5 and CXCR4) must occur.  lists the adhesins for some common viral pathogens and the specific sites to which these adhesins allow viruses to attach.

### Antigenic Variation in Viruses

Antigenic variation also occurs in certain types of enveloped viruses, including influenza viruses, which exhibit two forms of antigenic variation: antigenic drift and antigenic shift (). Antigenic drift is the result of point mutations causing slight changes in the spike proteins hemagglutinin (H) and neuraminidase (N). On the other hand, antigenic shift is a major change in spike proteins due to gene reassortment. This reassortment for antigenic shift occurs typically when two different influenza viruses infect the same host.
The rate of antigenic variation in influenza viruses is very high, making it difficult for the immune system to recognize the many different strains of Influenzavirus. Although the body may develop immunity to one strain through natural exposure or vaccination, antigenic variation results in the continual emergence of new strains that the immune system will not recognize. This is the main reason that vaccines against Influenzavirus must be given annually. Each year’s influenza vaccine provides protection against the most prevalent strains for that year, but new or different strains may be more prevalent the following year.

### Key Concepts and Summary

1. Virulence factors contribute to a pathogen’s ability to cause disease.
2. Exoenzymes and toxins allow pathogens to invade host tissue and cause tissue damage. Exoenzymes are classified according to the macromolecule they target and exotoxins are classified based on their mechanism of action.
3. Bacterial toxins include endotoxin and exotoxins. Endotoxin is the lipid A component of the LPS of the gram-negative cell envelope. Exotoxins are proteins secreted mainly by gram-positive bacteria, but also are secreted by gram-negative bacteria.
4. Bacterial pathogens may evade the host immune response by producing capsules to avoid phagocytosis, surviving the intracellular environment of phagocytes, degrading antibodies, or through antigenic variation.
5. Viral pathogens use adhesins for initiating infections and antigenic variation to avoid immune defenses.
6. Influenza viruses use both antigenic drift and antigenic shift to avoid being recognized by the immune system.

### Multiple Choice

### Fill in the Blank

### Critical Thinking


# Microbial Mechanisms of Pathogenicity

## Virulence Factors of Eukaryotic Pathogens

### Learning Objectives

By the end of this section, you will be able to:
1. Describe virulence factors unique to fungi and parasites
2. Compare virulence factors of fungi and bacteria
3. Explain the difference between protozoan parasites and helminths
4. Describe how helminths evade the host immune system
Although fungi and parasites are important pathogens causing infectious diseases, their pathogenic mechanisms and virulence factors are not as well characterized as those of bacteria. Despite the relative lack of detailed mechanisms, the stages of pathogenesis and general mechanisms of virulence involved in disease production by these pathogens are similar to those of bacteria.

### Fungal Virulence

Pathogenic fungi can produce virulence factors that are similar to the bacterial virulence factors that have been discussed earlier in this chapter. In this section, we will look at the virulence factors associated with species of Candida, Cryptococcus, Claviceps, and Aspergillus.
 is an opportunistic fungal pathogen and causative agent of oral thrush, vaginal yeast infections, and cutaneous candidiasis. Candida produces adhesins (surface glycoproteins) that bind to the phospholipids of epithelial and endothelial cells. To assist in spread and tissue invasion, Candida produces proteases and phospholipases (i.e., exoenzymes). One of these proteases degrades keratin, a structural protein found on epithelial cells, enhancing the ability of the fungus to invade host tissue. In animal studies, it has been shown that the addition of a protease inhibitor led to attenuation of Candida infection.K. Fallon et al. “Role of Aspartic Proteases in Disseminated  Similarly, the phospholipases can affect the integrity of host cell membranes to facilitate invasion.
The main virulence factor for , a fungus that causes pneumonia and meningitis, is capsule production. The polysaccharide glucuronoxylomannan is the principal constituent of the Cryptococcus capsule. Similar to encapsulated bacterial cells, encapsulated Cryptococcus cells are more resistant to phagocytosis than nonencapsulated Cryptococcus, which are effectively phagocytosed and, therefore, less virulent.
Like some bacteria, many fungi produce exotoxins. Fungal toxins are called mycotoxins. Claviceps purpurea, a fungus that grows on rye and related grains, produces a mycotoxin called ergot toxin, an alkaloid responsible for the disease known as ergotism. There are two forms of ergotism: gangrenous and convulsive. In gangrenous ergotism, the ergot toxin causes vasoconstriction, resulting in improper blood flow to the extremities, eventually leading to gangrene. A famous outbreak of gangrenous ergotism occurred in Eastern Europe during the 5th century AD due to the consumption of rye contaminated with C. purpurea. In convulsive ergotism, the toxin targets the central nervous system, causing mania and hallucinations.
The mycotoxin aflatoxin is a virulence factor produced by the fungus , an opportunistic pathogen that can enter the body via contaminated food or by inhalation. Inhalation of the fungus can lead to the chronic pulmonary disease aspergillosis, characterized by fever, bloody sputum, and/or asthma. Aflatoxin acts in the host as both a mutagen (a substance that causes mutations in DNA) and a carcinogen (a substance involved in causing cancer), and has been associated with the development of liver cancer. Aflatoxin has also been shown to cross the blood-placental barrier.C.P. Wild et al. “In-utero exposure to aflatoxin in west Africa.”  A second mycotoxin produced by Aspergillus is gliotoxin. This toxin promotes virulence by inducing host cells to self-destruct and by evading the host’s immune response by inhibiting the function of phagocytic cells as well as the pro-inflammatory response. Like Candida, Aspergillus also produces several proteases. One is elastase, which breaks down the protein elastin found in the connective tissue of the lung, leading to the development of lung disease. Another is catalase, an enzyme that protects the fungus from hydrogen peroxide produced by the immune system to destroy pathogens.

### Protozoan Virulence

Protozoan pathogens are unicellular eukaryotic parasites that have virulence factors and pathogenic mechanisms analogous to prokaryotic and viral pathogens, including adhesins, toxins, antigenic variation, and the ability to survive inside phagocytic vesicles.
Protozoans often have unique features for attaching to host cells. The protozoan , which causes the intestinal disease giardiasis, uses a large adhesive disc composed of microtubules to attach to the intestinal mucosa. During adhesion, the flagella of G. lamblia move in a manner that draws fluid out from under the disc, resulting in an area of lower pressure that facilitates adhesion to epithelial cells. Giardia does not invade the intestinal cells but rather causes inflammation (possibly through the release of cytopathic substances that cause damage to the cells) and shortens the intestinal villi, inhibiting absorption of nutrients.
Some protozoans are capable of antigenic variation. The obligate intracellular pathogen  (one of the causative agents of malaria) resides inside red blood cells, where it produces an adhesin membrane protein known as PfEMP1. This protein is expressed on the surface of the infected erythrocytes, causing blood cells to stick to each other and to the walls of blood vessels. This process impedes blood flow, sometimes leading to organ failure, anemia, jaundice (yellowing of skin and sclera of the eyes due to buildup of bilirubin from lysed red blood cells), and, subsequently, death. Although PfEMP1 can be recognized by the host’s immune system, antigenic variations in the structure of the protein over time prevent it from being easily recognized and eliminated. This allows malaria to persist as a chronic infection in many individuals.
The virulence factors of , the causative agent of African sleeping sickness, include the abilities to form capsules and undergo antigenic variation. T. brucei evades phagocytosis by producing a dense glycoprotein coat that resembles a bacterial capsule. Over time, host antibodies are produced that recognize this coat, but T. brucei is able to alter the structure of the glycoprotein to evade recognition.

### Helminth Virulence

Helminths, or parasitic worms, are multicellular eukaryotic parasites that depend heavily on virulence factors that allow them to gain entry to host tissues. For example, the aquatic larval form of , which causes schistosomiasis, penetrates intact skin with the aid of proteases that degrade skin proteins, including elastin.
To survive within the host long enough to perpetuate their often-complex life cycles, helminths need to evade the immune system. Some helminths are so large that the immune system is ineffective against them. Others, such as adult roundworms (which cause trichinosis, ascariasis, and other diseases), are protected by a tough outer cuticle.
Over the course of their life cycles, the surface characteristics of the parasites vary, which may help prevent an effective immune response. Some helminths express polysaccharides called glycans on their external surface; because these glycans resemble molecules produced by host cells, the immune system fails to recognize and attack the helminth as a foreign body. This “glycan gimmickry,” as it has been called, serves as a protective cloak that allows the helminth to escape detection by the immune system.I. van Die, R.D. Cummings. “Glycan Gimmickry by Parasitic Helminths: A Strategy for Modulating the Host Immune Response?”
In addition to evading host defenses, helminths can actively suppress the immune system. S. mansoni, for example, degrades host antibodies with proteases. Helminths produce many other substances that suppress elements of both innate nonspecific and adaptive specific host defenses. They also release large amounts of material into the host that may locally overwhelm the immune system or cause it to respond inappropriately.

### Key Concepts and Summary

1. Fungal and parasitic pathogens use pathogenic mechanisms and virulence factors that are similar to those of bacterial pathogens
2. Fungi initiate infections through the interaction of adhesins with receptors on host cells. Some fungi produce toxins and exoenzymes involved in disease production and capsules that provide protection of phagocytosis.
3. Protozoa adhere to target cells through complex mechanisms and can cause cellular damage through release of cytopathic substances. Some protozoa avoid the immune system through antigenic variation and production of capsules.
4. Helminthic worms are able to avoid the immune system by coating their exteriors with glycan molecules that make them look like host cells or by suppressing the immune system.

### Multiple Choice

### Fill in the Blank

### Short Answer


# Disease and Epidemiology

## Introduction

In the United States and other developed nations, public health is a key function of government. A healthy citizenry is more productive, content, and prosperous; high rates of death and disease, on the other hand, can severely hamper economic productivity and foster social and political instability. The burden of disease makes it difficult for citizens to work consistently, maintain employment, and accumulate wealth to better their lives and support a growing economy.
In this chapter, we will explore the intersections between microbiology and epidemiology, the science that underlies public health. Epidemiology studies how disease originates and spreads throughout a population, with the goal of preventing outbreaks and containing them when they do occur. Over the past two centuries, discoveries in epidemiology have led to public health policies that have transformed life in developed nations, leading to the eradication (or near eradication) of many diseases that were once causes of great human suffering and premature death. However, the work of epidemiologists is far from finished. Numerous diseases continue to plague humanity, and new diseases are always emerging. Moreover, in the developing world, lack of infrastructure continues to pose many challenges to efforts to contain disease.


# Disease and Epidemiology

## The Language of Epidemiologists

### Learning Objectives

By the end of this section, you will be able to:
1. Explain the difference between prevalence and incidence of disease
2. Distinguish the characteristics of sporadic, endemic, epidemic, and pandemic diseases
3. Explain the use of Koch’s postulates and their modifications to determine the etiology of disease
4. Explain the relationship between epidemiology and public health
The field of epidemiology concerns the geographical distribution and timing of infectious disease occurrences and how they are transmitted and maintained in nature, with the goal of recognizing and controlling outbreaks. The science of epidemiology includes etiology (the study of the causes of disease) and investigation of disease transmission (mechanisms by which a disease is spread).

### Analyzing Disease in a Population

Epidemiological analyses are always carried out with reference to a population, which is the group of individuals that are at risk for the disease or condition. The population can be defined geographically, but if only a portion of the individuals in that area are susceptible, additional criteria may be required. Susceptible individuals may be defined by particular behaviors, such as intravenous drug use, owning particular pets, or membership in an institution, such as a college. Being able to define the population is important because most measures of interest in epidemiology are made with reference to the size of the population.
The state of being diseased is called morbidity. Morbidity in a population can be expressed in a few different ways. Morbidity or total morbidity is expressed in numbers of individuals without reference to the size of the population. The morbidity rate can be expressed as the number of diseased individuals out of a standard number of individuals in the population, such as 100,000, or as a percent of the population.
There are two aspects of morbidity that are relevant to an epidemiologist: a disease’s prevalence and its incidence. Prevalence is the number, or proportion, of individuals with a particular illness in a given population at a point in time. For example, the Centers for Disease Control and Prevention (CDC) estimated that in 2012, there were about 1.2 million people 13 years and older with an active human immunodeficiency virus (HIV) infection. Expressed as a proportion, or rate, this is a prevalence of 467 infected persons per 100,000 in the population.H. Irene Hall, Qian An, Tian Tang, Ruiguang Song, Mi Chen, Timothy Green, and Jian Kang. “Prevalence of Diagnosed and Undiagnosed HIV Infection—United States, 2008–2012.”  On the other hand, incidence is the number or proportion of new cases in a period of time. For the same year and population, the CDC estimates that there were 43,165 newly diagnosed cases of HIV infection, which is an incidence of 13.7 new cases per 100,000 in the population.Centers for Disease Control and Prevention. “Diagnoses of HIV Infection in the United States and Dependent Areas, 2014.”  The relationship between incidence and prevalence can be seen in . For a chronic disease like HIV infection, prevalence will generally be higher than incidence because it represents the cumulative number of new cases over many years minus the number of cases that are no longer active (e.g., because the patient died or was cured).
In addition to morbidity rates, the incidence and prevalence of mortality (death) may also be reported. A mortality rate can be expressed as the percentage of the population that has died from a disease or as the number of deaths per 100,000 persons (or other suitable standard number).

### Patterns of Incidence

Diseases that are seen only occasionally, and usually without geographic concentration, are called sporadic diseases. Examples of sporadic diseases include tetanus, rabies, and plague. In the United States, , the bacterium that causes tetanus, is ubiquitous in the soil environment, but incidences of infection occur only rarely and in scattered locations because most individuals are vaccinated, clean wounds appropriately, or are only rarely in a situation that would cause infection.Centers for Disease Control and Prevention. “Tetanus Surveillance—United States, 2001–2008.”  Likewise in the United States there are a few scattered cases of plague each year, usually contracted from rodents in rural areas in the western states.Centers for Disease Control and Prevention. “Plague in the United States.” 2015. http://www.cdc.gov/plague/maps. Accessed June 1, 2016.
Diseases that are constantly present (often at a low level) in a population within a particular geographic region are called endemic diseases. For example, malaria is endemic to some regions of Brazil, but is not endemic to the United States.
Diseases for which a larger than expected number of cases occurs in a short time within a geographic region are called epidemic diseases. Influenza is a good example of a commonly epidemic disease. Incidence patterns of influenza tend to rise each winter in the northern hemisphere. These seasonal increases are expected, so it would not be accurate to say that influenza is epidemic every winter; however, some winters have an usually large number of seasonal influenza cases in particular regions, and such situations would qualify as epidemics ( and ).
An epidemic disease signals the breakdown of an equilibrium in disease frequency, often resulting from some change in environmental conditions or in the population. In the case of influenza, the disruption can be due to antigenic shift or drift (see Virulence Factors of Bacterial and Viral Pathogens), which allows influenza virus strains to circumvent the acquired immunity of their human hosts.
An epidemic that occurs on a worldwide scale is called a pandemic disease. For example, HIV/AIDS is a pandemic disease and novel influenza virus strains often become pandemic.

### Etiology

When studying an epidemic, an epidemiologist’s first task is to determinate the cause of the disease, called the etiologic agent or causative agent. Connecting a disease to a specific pathogen can be challenging because of the extra effort typically required to demonstrate direct causation as opposed to a simple association. It is not enough to observe an association between a disease and a suspected pathogen; controlled experiments are needed to eliminate other possible causes. In addition, pathogens are typically difficult to detect when there is no immediate clue as to what is causing the outbreak. Signs and symptoms of disease are also commonly nonspecific, meaning that many different agents can give rise to the same set of signs and symptoms. This complicates diagnosis even when a causative agent is familiar to scientists.
Robert Koch was the first scientist to specifically demonstrate the causative agent of a disease (anthrax) in the late 1800s. Koch developed four criteria, now known as Koch’s postulates, which had to be met in order to positively link a disease with a pathogenic microbe. Without Koch’s postulates, the Golden Age of Microbiology would not have occurred. Between 1876 and 1905, many common diseases were linked with their etiologic agents, including cholera, diphtheria, gonorrhea, meningitis, plague, syphilis, tetanus, and tuberculosis. Today, we use the molecular Koch’s postulates, a variation of Koch’s original postulates that can be used to establish a link between the disease state and virulence traits unique to a pathogenic strain of a microbe. Koch’s original postulates and molecular Koch’s postulates were described in more detail in How Pathogens Cause Disease.

### The Role of Public Health Organizations

The main national public health agency in the United States is the Centers for Disease Control and Prevention (CDC), an agency of the Department of Health and Human Services. The CDC is charged with protecting the public from disease and injury. One way that the CDC carries out this mission is by overseeing the National Notifiable Disease Surveillance System (NNDSS) in cooperation with regional, state, and territorial public health departments. The NNDSS monitors diseases considered to be of public health importance on a national scale. Such diseases are called notifiable diseases or reportable diseases because all cases must be reported to the CDC. A physician treating a patient with a notifiable disease is legally required to submit a report on the case. Notifiable diseases include HIV infection, measles, West Nile virus infections, and many others. Some states have their own lists of notifiable diseases that include diseases beyond those on the CDC’s list.
Notifiable diseases are tracked by epidemiological studies and the data is used to inform health-care providers and the public about possible risks. The CDC publishes the , which provides physicians and health-care workers with updates on public health issues and the latest data pertaining to notifiable diseases.  is an example of the kind of data contained in the MMWR.

### Key Concepts and Summary

1. Epidemiology is the science underlying public health.
2. Morbidity means being in a state of illness, whereas mortality refers to death; both morbidity rates and mortality rates are of interest to epidemiologists.
3. Incidence is the number of new cases (morbidity or mortality), usually expressed as a proportion, during a specified time period; prevalence is the total number affected in the population, again usually expressed as a proportion.
4. Sporadic diseases only occur rarely and largely without a geographic focus. Endemic diseases occur at a constant (and often low) level within a population. Epidemic diseases and pandemic diseases occur when an outbreak occurs on a significantly larger than expected level, either locally or globally, respectively.
5. Koch’s postulates specify the procedure for confirming a particular pathogen as the etiologic agent of a particular disease. Koch’s postulates have limitations in application if the microbe cannot be isolated and cultured or if there is no animal host for the microbe. In this case, molecular Koch’s postulates would be utilized.
6. In the United States, the Centers for Disease Control and Prevention monitors notifiable diseases and publishes weekly updates in the Morbidity and Mortality Weekly Report.

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### Critical Thinking


# Disease and Epidemiology

## Tracking Infectious Diseases

### Learning Objectives

By the end of this section, you will be able to:
1. Explain the research approaches used by the pioneers of epidemiology
2. Explain how descriptive, analytical, and experimental epidemiological studies go about determining the cause of morbidity and mortality
Epidemiology has its roots in the work of physicians who looked for patterns in disease occurrence as a way to understand how to prevent it. The idea that disease could be transmitted was an important precursor to making sense of some of the patterns. In 1546, Girolamo Fracastoro first proposed the germ theory of disease in his essay De Contagione et Contagiosis Morbis, but this theory remained in competition with other theories, such as the miasma hypothesis, for many years (see What Our Ancestors Knew). Uncertainty about the cause of disease was not an absolute barrier to obtaining useful knowledge from patterns of disease. Some important researchers, such as Florence Nightingale, subscribed to the miasma hypothesis. The transition to acceptance of the germ theory during the 19th century provided a solid mechanistic grounding to the study of disease patterns. The studies of 19th century physicians and researchers such as John Snow, Florence Nightingale, Ignaz Semmelweis, Joseph Lister, Robert Koch, Louis Pasteur, and others sowed the seeds of modern epidemiology.

### Pioneers of Epidemiology

John Snow () was a British physician known as the father of epidemiology for determining the source of the 1854 Broad Street cholera epidemic in London. Based on observations he had made during an earlier cholera outbreak (1848–1849), Snow proposed that cholera was spread through a fecal-oral route of transmission and that a microbe was the infectious agent. He investigated the 1854 cholera epidemic in two ways. First, suspecting that contaminated water was the source of the epidemic, Snow identified the source of water for those infected. He found a high frequency of cholera cases among individuals who obtained their water from the River Thames downstream from London. This water contained the refuse and sewage from London and settlements upstream. He also noted that brewery workers did not contract cholera and on investigation found the owners provided the workers with beer to drink and stated that they likely did not drink water.John Snow.  Second, he also painstakingly mapped the incidence of cholera and found a high frequency among those individuals using a particular water pump located on Broad Street. In response to Snow’s advice, local officials removed the pump’s handle,John Snow. “The Cholera near Golden-Wquare, and at Deptford.”  resulting in the containment of the Broad Street cholera epidemic.
Snow’s work represents an early epidemiological study and it resulted in the first known public health response to an epidemic. Snow’s meticulous case-tracking methods are now common practice in studying disease outbreaks and in associating new diseases with their causes. His work further shed light on unsanitary sewage practices and the effects of waste dumping in the Thames. Additionally, his work supported the germ theory of disease, which argued disease could be transmitted through contaminated items, including water contaminated with fecal matter.
Snow’s work illustrated what is referred to today as a common source spread of infectious disease, in which there is a single source for all of the individuals infected. In this case, the single source was the contaminated well below the Broad Street pump. Types of common source spread include point source spread, continuous common source spread, and intermittent common source spread. In point source spread of infectious disease, the common source operates for a short time period—less than the incubation period of the pathogen. An example of point source spread is a single contaminated potato salad at a group picnic. In continuous common source spread, the infection occurs for an extended period of time, longer than the incubation period. An example of continuous common source spread would be the source of London water taken downstream of the city, which was continuously contaminated with sewage from upstream. Finally, with intermittent common source spread, infections occur for a period, stop, and then begin again. This might be seen in infections from a well that was contaminated only after large rainfalls and that cleared itself of contamination after a short period.
In contrast to common source spread, propagated spread occurs through direct or indirect person-to-person contact. With propagated spread, there is no single source for infection; each infected individual becomes a source for one or more subsequent infections. With propagated spread, unless the spread is stopped immediately, infections occur for longer than the incubation period. Although point sources often lead to large-scale but localized outbreaks of short duration, propagated spread typically results in longer duration outbreaks that can vary from small to large, depending on the population and the disease (). In addition, because of person-to-person transmission, propagated spread cannot be easily stopped at a single source like point source spread.
Florence Nightingale’s work is another example of an early epidemiological study. In 1854, Nightingale was part of a contingent of nurses dispatched by the British military to care for wounded soldiers during the Crimean War. Nightingale kept meticulous records regarding the causes of illness and death during the war. Her recordkeeping was a fundamental task of what would later become the science of epidemiology. Her analysis of the data she collected was published in 1858. In this book, she presented monthly frequency data on causes of death in a wedge chart histogram (). This graphical presentation of data, unusual at the time, powerfully illustrated that the vast majority of casualties during the war occurred not due to wounds sustained in action but to what Nightingale deemed preventable infectious diseases. Often these diseases occurred because of poor sanitation and lack of access to hospital facilities. Nightingale’s findings led to many reforms in the British military’s system of medical care.
Joseph Lister provided early epidemiological evidence leading to good public health practices in clinics and hospitals. These settings were notorious in the mid-1800s for fatal infections of surgical wounds at a time when the germ theory of disease was not yet widely accepted (see Foundations of Modern Cell Theory). Most physicians did not wash their hands between patient visits or clean and sterilize their surgical tools. Lister, however, discovered the disinfecting properties of carbolic acid, also known as phenol (see Using Chemicals to Control Microorganisms). He introduced several disinfection protocols that dramatically lowered post-surgical infection rates.O.M. Lidwell. “Joseph Lister and Infection from the Air.”  He demanded that surgeons who worked for him use a 5% carbolic acid solution to clean their surgical tools between patients, and even went so far as to spray the solution onto bandages and over the surgical site during operations (). He also took precautions not to introduce sources of infection from his skin or clothing by removing his coat, rolling up his sleeves, and washing his hands in a dilute solution of carbolic acid before and during the surgery.

### Types of Epidemiological Studies

Today, epidemiologists make use of study designs, the manner in which data are gathered to test a hypothesis, similar to those of researchers studying other phenomena that occur in populations. These approaches can be divided into observational studies (in which subjects are not manipulated) and experimental studies (in which subjects are manipulated). Collectively, these studies give modern-day epidemiologists multiple tools for exploring the connections between infectious diseases and the populations of susceptible individuals they might infect.

### Observational Studies

In an observational study, data are gathered from study participants through measurements (such as physiological variables like white blood cell count), or answers to questions in interviews (such as recent travel or exercise frequency). The subjects in an observational study are typically chosen at random from a population of affected or unaffected individuals. However, the subjects in an observational study are in no way manipulated by the researcher. Observational studies are typically easier to carry out than experimental studies, and in certain situations they may be the only studies possible for ethical reasons.
Observational studies are only able to measure associations between disease occurrence and possible causative agents; they do not necessarily prove a causal relationship. For example, suppose a study finds an association between heavy coffee drinking and lower incidence of skin cancer. This might suggest that coffee prevents skin cancer, but there may be another unmeasured factor involved, such as the amount of sun exposure the participants receive. If it turns out that coffee drinkers work more in offices and spend less time outside in the sun than those who drink less coffee, then it may be possible that the lower rate of skin cancer is due to less sun exposure, not to coffee consumption. The observational study cannot distinguish between these two potential causes.
There are several useful approaches in observational studies. These include methods classified as descriptive epidemiology and analytical epidemiology. Descriptive epidemiology gathers information about a disease outbreak, the affected individuals, and how the disease has spread over time in an exploratory stage of study. This type of study will involve interviews with patients, their contacts, and their family members; examination of samples and medical records; and even histories of food and beverages consumed. Such a study might be conducted while the outbreak is still occurring. Descriptive studies might form the basis for developing a hypothesis of causation that could be tested by more rigorous observational and experimental studies.
Analytical epidemiology employs carefully selected groups of individuals in an attempt to more convincingly evaluate hypotheses about potential causes for a disease outbreak. The selection of cases is generally made at random, so the results are not biased because of some common characteristic of the study participants. Analytical studies may gather their data by going back in time (retrospective studies), or as events unfold forward in time (prospective studies).
Retrospective studies gather data from the past on present-day cases. Data can include things like the medical history, age, gender, or occupational history of the affected individuals. This type of study examines associations between factors chosen or available to the researcher and disease occurrence.
Prospective studies follow individuals and monitor their disease state during the course of the study. Data on the characteristics of the study subjects and their environments are gathered at the beginning and during the study so that subjects who become ill may be compared with those who do not. Again, the researchers can look for associations between the disease state and variables that were measured during the study to shed light on possible causes.
Analytical studies incorporate groups into their designs to assist in teasing out associations with disease. Approaches to group-based analytical studies include cohort studies, case-control studies, and cross-sectional studies. The cohort method examines groups of individuals (called cohorts) who share a particular characteristic. For example, a cohort might consist of individuals born in the same year and the same place; or it might consist of people who practice or avoid a particular behavior, e.g., smokers or nonsmokers. In a cohort study, cohorts can be followed prospectively or studied retrospectively. If only a single cohort is followed, then the affected individuals are compared with the unaffected individuals in the same group. Disease outcomes are recorded and analyzed to try to identify correlations between characteristics of individuals in the cohort and disease incidence. Cohort studies are a useful way to determine the causes of a condition without violating the ethical prohibition of exposing subjects to a risk factor. Cohorts are typically identified and defined based on suspected risk factors to which individuals have already been exposed through their own choices or circumstances.
Case-control studies are typically retrospective and compare a group of individuals with a disease to a similar group of individuals without the disease. Case-control studies are far more efficient than cohort studies because researchers can deliberately select subjects who are already affected with the disease as opposed to waiting to see which subjects from a random sample will develop a disease.
A cross-sectional study analyzes randomly selected individuals in a population and compares individuals affected by a disease or condition to those unaffected at a single point in time. Subjects are compared to look for associations between certain measurable variables and the disease or condition. Cross-sectional studies are also used to determine the prevalence of a condition.

### Experimental Studies

Experimental epidemiology uses laboratory or clinical studies in which the investigator manipulates the study subjects to study the connections between diseases and potential causative agents or to assess treatments. Examples of treatments might be the administration of a drug, the inclusion or exclusion of different dietary items, physical exercise, or a particular surgical procedure. Animals or humans are used as test subjects. Because experimental studies involve manipulation of subjects, they are typically more difficult and sometimes impossible for ethical reasons.
Koch’s postulates require experimental interventions to determine the causative agent for a disease. Unlike observational studies, experimental studies can provide strong evidence supporting cause because other factors are typically held constant when the researcher manipulates the subject. The outcomes for one group receiving the treatment are compared to outcomes for a group that does not receive the treatment but is treated the same in every other way. For example, one group might receive a regimen of a drug administered as a pill, while the untreated group receives a placebo (a pill that looks the same but has no active ingredient). Both groups are treated as similarly as possible except for the administration of the drug. Because other variables are held constant in both the treated and the untreated groups, the researcher is more certain that any change in the treated group is a result of the specific manipulation.
Experimental studies provide the strongest evidence for the etiology of disease, but they must also be designed carefully to eliminate subtle effects of bias. Typically, experimental studies with humans are conducted as double-blind studies, meaning neither the subjects nor the researchers know who is a treatment case and who is not. This design removes a well-known cause of bias in research called the placebo effect, in which knowledge of the treatment by either the subject or the researcher can influence the outcomes.

### Key Concepts and Summary

1. Early pioneers of epidemiology such as John Snow, Florence Nightingale, and Joseph Lister, studied disease at the population level and used data to disrupt disease transmission.
2. Descriptive epidemiology studies rely on case analysis and patient histories to gain information about outbreaks, frequently while they are still occurring.
3. Retrospective epidemiology studies use historical data to identify associations with the disease state of present cases. Prospective epidemiology studies gather data and follow cases to find associations with future disease states.
4. Analytical epidemiology studies are observational studies that are carefully designed to compare groups and uncover associations between environmental or genetic factors and disease.
5. Experimental epidemiology studies generate strong evidence of causation in disease or treatment by manipulating subjects and comparing them with control subjects.

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# Disease and Epidemiology

## Modes of Disease Transmission

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the different types of disease reservoirs
2. Compare contact, vector, and vehicle modes of transmission
3. Identify important disease vectors
4. Explain the prevalence of nosocomial infections
Understanding how infectious pathogens spread is critical to preventing infectious disease. Many pathogens require a living host to survive, while others may be able to persist in a dormant state outside of a living host. But having infected one host, all pathogens must also have a mechanism of transfer from one host to another or they will die when their host dies. Pathogens often have elaborate adaptations to exploit host biology, behavior, and ecology to live in and move between hosts. Hosts have evolved defenses against pathogens, but because their rates of evolution are typically slower than their pathogens (because their generation times are longer), hosts are usually at an evolutionary disadvantage. This section will explore where pathogens survive—both inside and outside hosts—and some of the many ways they move from one host to another.

### Reservoirs and Carriers

For pathogens to persist over long periods of time they require reservoirs where they normally reside. Reservoirs can be living organisms or nonliving sites. Nonliving reservoirs can include soil and water in the environment. These may naturally harbor the organism because it may grow in that environment. These environments may also become contaminated with pathogens in human feces, pathogens shed by intermediate hosts, or pathogens contained in the remains of intermediate hosts.
Pathogens may have mechanisms of dormancy or resilience that allow them to survive (but typically not to reproduce) for varying periods of time in nonliving environments. For example,  survives in the soil and in the presence of oxygen as a resistant endospore. Although many viruses are soon destroyed once in contact with air, water, or other non-physiological conditions, certain types are capable of persisting outside of a living cell for varying amounts of time. For example, a study that looked at the ability of influenza viruses to infect a cell culture after varying amounts of time on a banknote showed survival times from 48 hours to 17 days, depending on how they were deposited on the banknote.Yves Thomas, Guido Vogel, Werner Wunderli, Patricia Suter, Mark Witschi, Daniel Koch, Caroline Tapparel, and Laurent Kaiser. “Survival of Influenza Virus on Banknotes.”  On the other hand, cold-causing rhinoviruses are somewhat fragile, typically surviving less than a day outside of physiological fluids.
A human acting as a reservoir of a pathogen may or may not be capable of transmitting the pathogen, depending on the stage of infection and the pathogen. To help prevent the spread of disease among school children, the CDC has developed guidelines based on the risk of transmission during the course of the disease. For example, children with chickenpox are considered contagious for five days from the start of the rash, whereas children with most gastrointestinal illnesses should be kept home for 24 hours after the symptoms disappear.
An individual capable of transmitting a pathogen without displaying symptoms is referred to as a carrier. A passive carrier is contaminated with the pathogen and can mechanically transmit it to another host; however, a passive carrier is not infected. For example, a health-care professional who fails to wash his hands after seeing a patient harboring an infectious agent could become a passive carrier, transmitting the pathogen to another patient who becomes infected.
By contrast, an active carrier is an infected individual who can transmit the disease to others. An active carrier may or may not exhibit signs or symptoms of infection. For example, active carriers may transmit the disease during the incubation period (before they show signs and symptoms) or the period of convalescence (after symptoms have subsided). Active carriers who do not present signs or symptoms of disease despite infection are called asymptomatic carriers. Pathogens such as hepatitis B virus, herpes simplex virus, and HIV are frequently transmitted by asymptomatic carriers. Mary Mallon, better known as Typhoid Mary, is a famous historical example of an asymptomatic carrier. An Irish immigrant, Mallon worked as a cook for households in and around New York City between 1900 and 1915. In each household, the residents developed typhoid fever (caused by ) a few weeks after Mallon started working. Later investigations determined that Mallon was responsible for at least 122 cases of typhoid fever, five of which were fatal.Filio Marineli, Gregory Tsoucalas, Marianna Karamanou, and George Androutsos. “Mary Mallon (1869–1938) and the History of Typhoid Fever.”  See Eye on Ethics: Typhoid Mary for more about the Mallon case.
A pathogen may have more than one living reservoir. In zoonotic diseases, animals act as reservoirs of human disease and transmit the infectious agent to humans through direct or indirect contact. In some cases, the disease also affects the animal, but in other cases the animal is asymptomatic.
In parasitic infections, the parasite’s preferred host is called the definitive host. In parasites with complex life cycles, the definitive host is the host in which the parasite reaches sexual maturity. Some parasites may also infect one or more intermediate hosts in which the parasite goes through several immature life cycle stages or reproduces asexually.

### Transmission

Regardless of the reservoir, transmission must occur for an infection to spread. First, transmission from the reservoir to the individual must occur. Then, the individual must transmit the infectious agent to other susceptible individuals, either directly or indirectly. Pathogenic microorganisms employ diverse transmission mechanisms.

### Contact Transmission

Contact transmission includes direct contact or indirect contact. Person-to-person transmission is a form of direct contact transmission. Here the agent is transmitted by physical contact between two individuals () through actions such as touching, kissing, sexual intercourse, or droplet sprays. Direct contact can be categorized as vertical, horizontal, or droplet transmission. Vertical direct contact transmission occurs when pathogens are transmitted from mother to child during pregnancy, birth, or breastfeeding. Other kinds of direct contact transmission are called horizontal direct contact transmission. Often, contact between mucous membranes is required for entry of the pathogen into the new host, although skin-to-skin contact can lead to mucous membrane contact if the new host subsequently touches a mucous membrane. Contact transmission may also be site-specific; for example, some diseases can be transmitted by sexual contact but not by other forms of contact.
When an individual coughs or sneezes, small droplets of mucus that may contain pathogens are ejected. This leads to direct droplet transmission, which refers to droplet transmission of a pathogen to a new host over distances of one meter or less. A wide variety of diseases are transmitted by droplets, including influenza and many forms of pneumonia. Transmission over distances greater than one meter is called airborne transmission.
Indirect contact transmission involves inanimate objects called fomites that become contaminated by pathogens from an infected individual or reservoir (). For example, an individual with the common cold may sneeze, causing droplets to land on a fomite such as a tablecloth or carpet, or the individual may wipe her nose and then transfer mucus to a fomite such as a doorknob or towel. Transmission occurs indirectly when a new susceptible host later touches the fomite and transfers the contaminated material to a susceptible portal of entry. Fomites can also include objects used in clinical settings that are not properly sterilized, such as syringes, needles, catheters, and surgical equipment. Pathogens transmitted indirectly via such fomites are a major cause of healthcare-associated infections (see Controlling Microbial Growth).

### Vehicle Transmission

The term vehicle transmission refers to the transmission of pathogens through vehicles such as water, food, and air. Water contamination through poor sanitation methods leads to waterborne transmission of disease. Waterborne disease remains a serious problem in many regions throughout the world. The World Health Organization (WHO) estimates that contaminated drinking water is responsible for more than 500,000 deaths each year.World Health Organization. Fact sheet No. 391 Similarly, food contaminated through poor handling or storage can lead to foodborne transmission of disease ().
Dust and fine particles known as aerosols, which can float in the air, can carry pathogens and facilitate the airborne transmission of disease. For example, dust particles are the dominant mode of transmission of hantavirus to humans. Hantavirus is found in mouse feces, urine, and saliva, but when these substances dry, they can disintegrate into fine particles that can become airborne when disturbed; inhalation of these particles can lead to a serious and sometimes fatal respiratory infection.
Although droplet transmission over short distances is considered contact transmission as discussed above, longer distance transmission of droplets through the air is considered vehicle transmission. Unlike larger particles that drop quickly out of the air column, fine mucus droplets produced by coughs or sneezes can remain suspended for long periods of time, traveling considerable distances. In certain conditions, droplets desiccate quickly to produce a droplet nucleus that is capable of transmitting pathogens; air temperature and humidity can have an impact on effectiveness of airborne transmission.
Tuberculosis is often transmitted via airborne transmission when the causative agent, Mycobacterium tuberculosis, is released in small particles with coughs. Because tuberculosis requires as few as 10 microbes to initiate a new infection, patients with tuberculosis must be treated in rooms equipped with special ventilation, and anyone entering the room should wear a mask.

### Vector Transmission

Diseases can also be transmitted by a mechanical or biological vector, an animal (typically an arthropod) that carries the disease from one host to another. Mechanical transmission is facilitated by a mechanical vector, an animal that carries a pathogen from one host to another without being infected itself. For example, a fly may land on fecal matter and later transmit bacteria from the feces to food that it lands on; a human eating the food may then become infected by the bacteria, resulting in a case of diarrhea or dysentery ().
Biological transmission occurs when the pathogen reproduces within a biological vector that transmits the pathogen from one host to another (). Arthropods are the main vectors responsible for biological transmission (). Most arthropod vectors transmit the pathogen by biting the host, creating a wound that serves as a portal of entry. The pathogen may go through part of its reproductive cycle in the gut or salivary glands of the arthropod to facilitate its transmission through the bite. For example, hemipterans (called “kissing bugs” or “assassin bugs”) transmit Chagas disease to humans by defecating when they bite, after which the human scratches or rubs the infected feces into a mucous membrane or break in the skin.
Biological insect vectors include mosquitoes, which transmit malaria and other diseases, and lice, which transmit typhus. Other arthropod vectors can include arachnids, primarily ticks, which transmit Lyme disease and other diseases, and mites, which transmit scrub typhus and rickettsial pox. Biological transmission, because it involves survival and reproduction within a parasitized vector, complicates the biology of the pathogen and its transmission. There are also important non-arthropod vectors of disease, including mammals and birds. Various species of mammals can transmit rabies to humans, usually by means of a bite that transmits the rabies virus. Chickens and other domestic poultry can transmit avian influenza to humans through direct or indirect contact with avian influenza virus A shed in the birds’ saliva, mucous, and feces.

### Quarantining

Individuals suspected or known to have been exposed to certain contagious pathogens may be quarantined, or isolated to prevent transmission of the disease to others. Hospitals and other health-care facilities generally set up special wards to isolate patients with particularly hazardous diseases such as tuberculosis or Ebola (). Depending on the setting, these wards may be equipped with special air-handling methods, and personnel may implement special protocols to limit the risk of transmission, such as personal protective equipment or the use of chemical disinfectant sprays upon entry and exit of medical personnel.
The duration of the quarantine depends on factors such as the incubation period of the disease and the evidence suggestive of an infection. The patient may be released if signs and symptoms fail to materialize when expected or if preventive treatment can be administered in order to limit the risk of transmission. If the infection is confirmed, the patient may be compelled to remain in isolation until the disease is no longer considered contagious.
In the United States, public health authorities may only quarantine patients for certain diseases, such as cholera, diphtheria, infectious tuberculosis, and strains of influenza capable of causing a pandemic. Individuals entering the United States or moving between states may be quarantined by the CDC if they are suspected of having been exposed to one of these diseases. Although the CDC routinely monitors entry points to the United States for crew or passengers displaying illness, quarantine is rarely implemented.

### Healthcare-Associated (Nosocomial) Infections

Hospitals, retirement homes, and prisons attract the attention of epidemiologists because these settings are associated with increased incidence of certain diseases. Higher rates of transmission may be caused by characteristics of the environment itself, characteristics of the population, or both. Consequently, special efforts must be taken to limit the risks of infection in these settings.
Infections acquired in health-care facilities, including hospitals, are called nosocomial infections or healthcare-associated infections (HAI). HAIs are often connected with surgery or other invasive procedures that provide the pathogen with access to the portal of infection. For an infection to be classified as an HAI, the patient must have been admitted to the health-care facility for a reason other than the infection. In these settings, patients suffering from primary disease are often afflicted with compromised immunity and are more susceptible to secondary infection and opportunistic pathogens.
In 2011, more than 720,000 HAIs occurred in hospitals in the United States, according to the CDC. About 22% of these HAIs occurred at a surgical site, and cases of pneumonia accounted for another 22%; urinary tract infections accounted for an additional 13%, and primary bloodstream infections 10%.Centers for Disease Control and Prevention. “HAI Data and Statistics.” 2016. http://www.cdc.gov/hai/surveillance. Accessed Jan 2, 2016. Such HAIs often occur when pathogens are introduced to patients’ bodies through contaminated surgical or medical equipment, such as catheters and respiratory ventilators. Health-care facilities seek to limit nosocomial infections through training and hygiene protocols such as those described in Control of Microbial Growth.

### Key Concepts and Summary

1. Reservoirs of human disease can include the human and animal populations, soil, water, and inanimate objects or materials.
2. Contact transmission can be direct or indirect through physical contact with either an infected host (direct) or contact with a fomite that an infected host has made contact with previously (indirect).
3. Vector transmission occurs when a living organism carries an infectious agent on its body (mechanical) or as an infection host itself (biological), to a new host.
4. Vehicle transmission occurs when a substance, such as soil, water, or air, carries an infectious agent to a new host.
5. Healthcare-associated infections (HAI), or nosocomial infections, are acquired in a clinical setting. Transmission is facilitated by medical interventions and the high concentration of susceptible, immunocompromised individuals in clinical settings.

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### Short Answer

### Critical Thinking


# Disease and Epidemiology

## Global Public Health

### Learning Objectives

By the end of this section, you will be able to:
1. Describe the entities involved in international public health and their activities
2. Identify and differentiate between emerging and reemerging infectious diseases
A large number of international programs and agencies are involved in efforts to promote global public health. Among their goals are developing infrastructure in health care, public sanitation, and public health capacity; monitoring infectious disease occurrences around the world; coordinating communications between national public health agencies in various countries; and coordinating international responses to major health crises. In large part, these international efforts are necessary because disease-causing microorganisms know no national boundaries.

### The World Health Organization (WHO)

International public health issues are coordinated by the World Health Organization (WHO), an agency of the United Nations. Of its roughly $4 billion budget for 2015–16World Health Organization. “Programme Budget 2014–2015.” http://www.who.int/about/finances-accountability/budget/en., about $1 billion was funded by member states and the remaining $3 billion by voluntary contributions. In addition to monitoring and reporting on infectious disease, WHO also develops and implements strategies for their control and prevention. WHO has had a number of successful international public health campaigns. For example, its vaccination program against smallpox, begun in the mid-1960s, resulted in the global eradication of the disease by 1980. WHO continues to be involved in infectious disease control, primarily in the developing world, with programs targeting malaria, HIV/AIDS, and tuberculosis, among others. It also runs programs to reduce illness and mortality that occur as a result of violence, accidents, lifestyle-associated illnesses such as diabetes, and poor health-care infrastructure.
WHO maintains a global alert and response system that coordinates information from member nations. In the event of a public health emergency or epidemic, it provides logistical support and coordinates international response to the emergency. The United States contributes to this effort through the CDC. The CDC carries out international monitoring and public health efforts, mainly in the service of protecting US public health in an increasingly connected world. Similarly, the European Union maintains a Health Security Committee that monitors disease outbreaks within its member countries and internationally, coordinating with WHO.

### Emerging and Reemerging Infectious Diseases

Both WHO and some national public health agencies such as the CDC monitor and prepare for emerging infectious diseases. An emerging infectious disease is either new to the human population or has shown an increase in prevalence in the previous twenty years. Whether the disease is new or conditions have changed to cause an increase in frequency, its status as emerging implies the need to apply resources to understand and control its growing impact.
Emerging diseases may change their frequency gradually over time, or they may experience sudden epidemic growth. The importance of vigilance was made clear during the Ebola hemorrhagic fever epidemic in western Africa through 2014–2015. Although health experts had been aware of the Ebola virus since the 1970s, an outbreak on such a large scale had never happened before (). Previous human epidemics had been small, isolated, and contained. Indeed, the gorilla and chimpanzee populations of western Africa had suffered far worse from Ebola than the human population. The pattern of small isolated human epidemics changed in 2014. Its high transmission rate, coupled with cultural practices for treatment of the dead and perhaps its emergence in an urban setting, caused the disease to spread rapidly, and thousands of people died. The international public health community responded with a large emergency effort to treat patients and contain the epidemic.
Emerging diseases are found in all countries, both developed and developing (). Some nations are better equipped to deal with them. National and international public health agencies watch for epidemics like the Ebola outbreak in developing countries because those countries rarely have the health-care infrastructure and expertise to deal with large outbreaks effectively. Even with the support of international agencies, the systems in western Africa struggled to identify and care for the sick and control spread. In addition to the altruistic goal of saving lives and assisting nations lacking in resources, the global nature of transportation means that an outbreak anywhere can spread quickly to every corner of the planet. Managing an epidemic in one location—its source—is far easier than fighting it on many fronts.
Ebola is not the only disease that needs to be monitored in the global environment. In 2015, WHO set priorities on several emerging diseases that had a high probability of causing epidemics and that were poorly understood (and thus urgently required research and development efforts).
A reemerging infectious disease is a disease that is increasing in frequency after a previous period of decline. Its reemergence may be a result of changing conditions or old prevention regimes that are no longer working. Examples of such diseases are drug-resistant forms of tuberculosis, bacterial pneumonia, and malaria. Drug-resistant strains of the bacteria causing gonorrhea and syphilis are also becoming more widespread, raising concerns of untreatable infections.

### Key Concepts and Summary

1. The World Health Organization (WHO) is an agency of the United Nations that collects and analyzes data on disease occurrence from member nations. WHO also coordinates public health programs and responses to international health emergencies.
2. Emerging diseases are those that are new to human populations or that have been increasing in the past two decades. Reemerging diseases are those that are making a resurgence in susceptible populations after previously having been controlled in some geographic areas.

### Multiple Choice

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### Critical Thinking


# Innate Nonspecific Host Defenses

## Introduction

Despite relatively constant exposure to pathogenic microbes in the environment, humans do not generally suffer from constant infection or disease. Under most circumstances, the body is able to defend itself from the threat of infection thanks to a complex immune system designed to repel, kill, and expel disease-causing invaders. Immunity as a whole can be described as two interrelated parts: nonspecific innate immunity, which is the subject of this chapter, and specific adaptive host defenses, which are discussed in the next chapter.
The nonspecific innate immune response provides a first line of defense that can often prevent infections from gaining a solid foothold in the body. These defenses are described as nonspecific because they do not target any specific pathogen; rather, they defend against a wide range of potential pathogens. They are called innate because they are built-in mechanisms of the human organism. Unlike the specific adaptive defenses, they are not acquired over time and they have no “memory” (they do not improve after repeated exposures to specific pathogens).
Broadly speaking, nonspecific innate defenses provide an immediate (or very rapid) response against potential pathogens. However, these responses are neither perfect nor impenetrable. They can be circumvented by pathogens on occasion, and sometimes they can even cause damage to the body, contributing to the signs and symptoms of infection ().