Y CONCLUSIONS Epidemiology, the study of the distribution and determinants of disease, is critical for recognition and control of emerging infectious diseases. Emerging infectious diseases are those that are increasing in incidence, whether due to the appearance of a new agent, pattern of resistance, or geographic spread. Communicable diseases differ from noncommunicable diseases in their propensity to cause both endemic disease and pandemics. Infections may be clinically inapparent or may cause disease. Those with subclinical disease can be important propagators of the infectious agent. Transmission can be vertical (mother to fetus or infant) or horizontal (direct or indirect person to person). Routes of horizontal transmission include respiratory, salivary, eye, skin, genital, fecal-oral, bloodborne, and vector-borne or zoonotic. The propensity for epidemic spread of an infection depends on agent, host, and environmental factors. Surveillance is a key to recognition and thereby to control. ++ Epidemiologic study is essential to identify, characterize, and control infectious diseases. Combating emerging infections requires recognizing new agents and patterns of disease, understanding their nature and spread, and then instituting control measures. The latter may involve prompt treatment of cases, prevention through selective chemoprophylaxis or immunization, implementation of environmental controls, and public education, depending on the specific agent. However, application of epidemiologic principles is essential for the health of both individuals and communities.

Various transmissible infections may be acquired from others by direct contact, indirectly through contaminated inanimate objects or materials, or by aerosol transmission of infectious secretions. Some infections, such as malaria, dengue, and chikungunya, involve an animate insect vector. These routes of spread are often referred to as horizontal transmission, in contrast to vertical or perinatal transmission—from mother to fetus or infant. +++ Vertical or Perinatal Transmission ++ Some infections can spread from mother to fetus through the placenta, during childbirth, or during breastfeeding. For example, rubella virus may cause birth defects when transmitted from the mother’s bloodstream across the placenta during the first trimester of pregnancy. Neonatal infections with group B streptococci, Chlamydia trachomatis, and Neisseria gonorrhoeae can occur following passage through the birth canal. Cytomegalovirus (CMV) can be acquired prenatally (across the placenta) or perinatally (from passage through an infected cervix, contact with blood, or through breast milk). ++ Vertical transmission = mother to fetus ++ Important effects of perinatal infection include prematurity, intrauterine growth retardation (IUGR) and low birth weight, developmental abnormalities, congenital disease, and persistent perinatal infection. Historically, the acronym TORCH was used to describe five clinically similar perinatal infections, including toxoplasmosis, other (syphilis), rubella, CMV, and herpes simplex. Now, however, the category “other” should include varicella-zoster virus, enteroviruses, parvovirus B19, and newly described Zika virus, the latter of which is unique in that it is transmitted by mosquitos (Aedes). ++ ❋ TORCH perinatal infections ++ The major routes of horizontal transmission of infectious diseases are summarized in Table 5–1 and discussed in the following text. ++Table Graphic Jump LocationTABLE 5–1Common Routes of Transmission of InfectionaView Table| Favorite Table |Download (.pdf) TABLE 5–1 Common Routes of Transmission of Infectiona ROUTE OF EXIT ROUTE OF TRANSMISSION EXAMPLE Respiratory Aerosol droplet inhalation Influenza virus; tuberculosis   Nose or mouth → hand or object → nose Common cold (rhinovirus) Salivary Direct salivary transfer (eg, kissing) Oral-labial herpes; Epstein-Barr virus, cytomegalovirus   Animal bite Rabies Eye Conjunctival Adenovirus Skin Skin discharge → air → respiratory tract Varicella, smallpox, or monkeypox   Skin to skin Human papillomavirus (warts); syphilis Genital secretions Urethral or cervical secretions Gonorrhea; herpes simplex; Chlamydia   Semen Cytomegalovirus Gastrointestinal Fecal–oral (Stool → hand → mouth and/or stool → object, water or food → mouth) Enterovirus; hepatitis A   Stool → water or food → mouth Salmonellosis; shigellosis Blood Transfusion or needle prick Hepatitis B; cytomegalovirus infection; malaria; HIV   Mosquito bite Malaria; arboviruses Urine Urine → hand → catheter Hospital-acquired urinary tract infections Zoonotic Animal bite Rabies   Contact with carcasses Tularemia   Tick bite Rickettsia; Lyme disease aThe examples cited are incomplete, and, in some cases, more than one route of transmission exists. An alternative classification is airborne (respiratory), food- or waterborne (fecal–oral), contact (skin, genital, eye, saliva), zoonotic or vector-borne, bloodborne, and perinatal. ++ ❋ Horizontal transmission = direct or indirect person to person +++ Respiratory Spread: Airborne, Droplet, or Contact with Respiratory Secretions ++ Many infections are transmitted by the respiratory route, often by aerosolization of respiratory secretions with subsequent inhalation by other persons. The efficiency of this process depends in part on the extent and method of propulsion of discharges from the mouth and nose, the size of the aerosol droplets, and the resistance of the infectious agent to desiccation and inactivation by ultraviolet light. The classic teaching is that in still air a particle 100 μm in diameter requires only seconds to fall the height of a room, a 10 m particle remains airborne for about 20 minutes, and smaller particles remain suspended even longer. When inhaled, particles with a diameter of 6 μm or more are usually trapped by the mucosa of the nasal turbinates, whereas particles of 0.6 to 5.0 μm attach to mucous sites at various levels along the upper and lower respiratory tract and may initiate infection. These “droplet nuclei” are most important in transmitting many respiratory pathogens (eg, M tuberculosis). Newer data suggest that humans with respiratory infections produce infectious aerosols comprising a wide range of particle sizes. SARS-CoV-2 coronavirus-2 is transmitted by both small and large particle aerosols; hence, surgical masks and physical “social” distancing (≥6 feet) are complementary approaches to preventing human-to-human transmission. ++ Droplet nuclei usually less than 6 μm in size ++ Respiratory secretions are often transferred on hands or inanimate objects (fomites) and risk of spread in these instances can be reduced best by handwashing. For example, spread of the common cold may involve transfer of infectious secretions from nose to hand by the infected individual, with transfer to others by hand-to-hand contact and then from hand to nose. Transmission of infectious secretions by direct contact with the nasal mucosa or conjunctiva often accounts for the rapid dissemination of agents, such as respiratory syncytial virus and adenovirus. ++ Handwashing is especially important to decrease transmission of the common cold +++ Salivary Spread: Kissing or Bite ++ Some infections, such as herpes simplex and infectious mononucleosis, can be transferred directly by contact with infectious saliva by drooling small children or through kissing. Saliva containing rabies virus can transmit rabies when the rabid animal bites. +++ Eye-to-Eye Transmission ++ Infections of the conjunctiva may occur in epidemic or endemic form. Epidemics of adenovirus and Haemophilus conjunctivitis may occur and are highly contagious. The major endemic disease is trachoma, caused by Chlamydia, which remains a common cause of blindness in developing countries. These diseases may be spread by direct contact via ophthalmologic equipment or by secretions passed manually or through fomites such as towels. ++ Fomites and unsterile ophthalmologic instruments are associated with transmission +++ Skin-to-Skin Transfer ++ Skin-to-skin transfer occurs with a variety of infections in which the skin is the portal of entry such as the spirochete of syphilis (Treponema pallidum), strains of group A streptococci that cause impetigo, and the dermatophyte fungi that cause ringworm and athlete’s foot. In most cases, an unapparent break in the epithelium is involved in infection. Other diseases may be spread indirectly from skin-to-skin through fomites such as shared towels and inadequately cleansed shower and bath floors. Skin-to-skin transfer usually occurs through abrasions of the epidermis, which may be unnoticed. ++ Syphilis, ringworm, and impetigo are examples +++ Genital Transmission ++ Disease transmission through the genital tract has been and remains one of the most common infections worldwide. Spread can occur between sexual partners or from the mother to the infant at birth. Major factors related to the persistence of these infections are high rates of asymptomatic carriage and the frequency of recurrence of organisms, such as C trachomatis, CMV, herpes simplex virus, and N gonorrhoeae. ++ Asymptomatic carriage and recurrence common +++ Foodborne or Waterborne Transmission: Fecal–Oral Spread ++ Fecal–oral spread involves direct or finger-to-mouth spread, the use of human feces as a fertilizer, or fecal contamination of food or water. Food handlers who are infected with an organism transmissible by this route constitute a special hazard, especially when they fail to wash their hands. Some viruses disseminated by the fecal–oral route infect and multiply in cells of the oropharynx and then disseminate to other body sites to cause infection. However, organisms that are spread in this way commonly multiply in the intestinal tract and may cause intestinal infections. They must, therefore, be able to resist the acid in the stomach, the bile, and the gastric and small intestinal enzymes. Many bacteria and enveloped viruses are rapidly killed by these conditions, but members of the Enterobacteriaceae and unenveloped viral intestinal pathogens (eg, enteroviruses) are more likely to survive. Even with these organisms, the infecting dose in patients with reduced or absent gastric hydrochloric acid is often much smaller than in those with normal stomach acidity. ++ ❋ Reduced gastric hydrochloric acid can facilitate the spread of enteric infections +++ Blood or Transfusion-Borne ++ Bloodborne transmission of infection through insect vectors requires a period of multiplication or alteration within an insect vector before the organism can infect another human host, as occurs with the female Anopheles mosquito and the malarial parasite. Direct transmission from human to human through blood has become increasingly important because of the use of blood transfusions and blood products and the increased self-administration of illicit drugs by intravenous or subcutaneous routes using shared nonsterile equipment. Hepatitis B and C viruses, as well as HIV, were frequently transmitted in this way before the institution of universal screening of blood. ++ ❋ Parenteral drug abuse, transfusion a major risk factor +++ Vector-borne and Zoonotic ++ Zoonotic infections are spread from animals, where they have their natural reservoir, to humans. Some zoonotic infections such as rabies are directly contracted from the bite of the infected animal, whereas others are transmitted by vectors, especially arthropods (eg, ticks, mosquitoes). Many infections contracted by humans from animals are dead-ended in humans, whereas others may be transferred between humans once the disease is established in a population. Plague, for example, has a natural reservoir in rodents. Human infections contracted from the bites of rodent fleas may produce pneumonia, which may then spread to other humans by the respiratory droplet route. Humans can contract Zika virus from the bite of a mosquito, vertically (from mother to fetus), or horizontally (sexual transmission). ++ ❋ Zoonotic = animals or vectors to humans ++ Classically the term vector was restricted to arthropods like ticks and mosquitoes; however, it is often used to refer to any animal that can transmit a pathogen to a human host. The probability of vector-borne transmission depends on the biology of the vector (mosquito, tick, snail, etc) and the infectivity of organism. ++ ❋ Vectorborne = vectors (e.g., mosquitos, ticks, snails) to humans

he incubation period is the time between the exposure to the organism/infection and the appearance of the first clinical manifestations of the disease. Organisms that multiply rapidly and produce local or systemic infections, such as gonorrhea and influenza, are associated with short incubation periods (eg, 2-4 days). Diseases such as typhoid fever, which depend on hematogenous spread and multiplication of the organism in distant target organs to produce symptoms, often have longer incubation periods (eg, 10 days to 3 weeks). Some diseases have even more prolonged incubation periods because of slow passage of the infecting organism to the target organ, as in rabies, or with slow growth of the organism, as in tuberculosis or leprosy. Incubation periods for one agent may also vary widely depending on route of acquisition and infecting dose; for example, the incubation period of hepatitis B virus infection may vary from a few weeks to several months. ++ ❋ Incubation periods range from a few days to several months ++ Communicability of a disease in which the organism is shed in secretions may occur primarily during the incubation period. In other infections, the disease course is short but the organisms can be excreted from the host for extended periods. In yet other cases, the symptoms are related to host immune response rather than the organism’s action and, thus, the disease process may extend far beyond the period in which the etiologic agent can be isolated or spread. Some viruses can integrate into the host genome or survive by replicating very slowly in the presence of an immune response. Such dormancy or latency is exemplified by the herpesviruses, and the organism may emerge long after the original infection and potentially infect others. ++ ❋ Transmission to others can occur before illness onset ++ The inherent infectivity and virulence of an agent are also important determinants of attack rates of disease in a community. In general, organisms of high infectivity spread more easily, and those of greater virulence are more likely to cause disease than subclinical infection. The infecting dose of an organism also varies with different organisms and, thus, influences the chance of infection and development of disease.

An important consideration in the study of the epidemiology of communicable organisms is the distinction between infection and disease. Infection involves multiplication of the organism in or on the host and may be clinically inapparent, such as during the incubation period or latency (when little or no replication is occurring, eg, with herpesviruses). Disease occurs when the infection becomes clinically apparent, that is, there is evidence of injury to the host as a result of the infection. With many communicable organisms, infection is much more common than disease, and asymptomatic infected individuals are important for propagation of the infectious agent. A recent example is Zika infection, which during the most recent epidemic was found to be nearly always clinically inapparent or mild, except for a developing fetus. Inapparent infections are termed subclinical, and the individual is sometimes referred to as a carrier. The latter term is also applied to situations in which an infectious agent establishes itself as part of a patient’s microbiota or causes low-grade chronic disease after an acute infection. For example, the clinically inapparent presence of S aureus in the anterior nares is termed carriage, as is chronic gallbladder infection with Salmonella serotype Typhi that can follow an attack of typhoid fever and result in fecal excretion of the organism for years. C difficile can colonize the gastrointestinal tract but cause severe disease only when associated with the production of a toxin. ++ ❋ Infection can result in little or no illness ++ With some infectious diseases such as measles, infection is almost invariably accompanied by clinical manifestations of the disease itself. These manifestations facilitate epidemiologic detection and control, because the existence and extent of infection in a community are readily apparent. Organisms associated with long incubation periods or high frequencies of subclinical infection, such as the human immunodeficiency virus (HIV-1), hepatitis B virus, or human papillomaviruses, may propagate and spread in a population for long periods before the extent of the problem is recognized. This makes epidemiologic control more difficult. ++ ❋ Carriers can be asymptomatic, but infectious to others ++ Illness severity reflects pathogen and host factors ++ The severity of infection reflects biologic characteristics of the organism as well as the host’s response. Infectivity reflects the secondary attack rate or the number of ill per number exposed. Pathogenicity reflects the ability of the agent to induce disease. Virulence is the severity of the disease after infection occurs. The expected severity of infection reflects pathogen factors (including infectivity, pathogenicity, and virulence) and host factors (eg, immune status, obesity, underlying illness, and age). Immunogenicity is the ability of a pathogen to produce a durable immune response to protect against reinfection with the same or a related organism. Some pathogens induce lifelong immunity whereas others are weakly immunogenic, so reinfections commonly occur.

Infectious diseases of humans may be caused by exclusively human pathogens such as Shigella, by environmental organisms such as Legionella pneumophila, or by organisms that have their primary reservoir in animals such as Salmonella. ++ Noncommunicable infections are those that are not transmitted from human to human and include: (1) infections related to the patient’s microbiota gaining access to a previously sterile site, such as peritonitis after rupture of the appendix; (2) infections caused by the ingestion of preformed toxins, such as botulism; and (3) infections caused by organisms found in the environment, such as clostridial gas gangrene. Some diseases transmitted from animals to humans (zoonotic infections), such as rabies and brucellosis, are not transmitted between humans, but others such as plague may be. Noncommunicable infections may still occur as common-source outbreaks, such as food poisoning from an enterotoxin-producing Staphylococcus aureus–contaminated chicken salad or multiple cases of pneumonia from extensive dissemination of Legionella through an air-conditioning system. Because these diseases are not transmissible to others, they do not lead to secondary spread. ++ Noncommunicable infections not spread person to person can occur as common-source outbreaks ++ Communicable infections require an organism to leave the body in a form that is either directly infectious or able to become so after development in a suitable environment. The respiratory spread of influenza virus is an example of direct communicability. In contrast, the malarial parasite requires a developmental cycle in a blood-feeding female anopheline mosquito before it can infect another human. Communicable infections can be endemic, present in the population at a low and constant level, or epidemic, present at a level of infection higher than that usually found in the community or population. With some infections, such as influenza, the infection can be endemic and persist at a low level from season to season; however, introduction of a new strain may result in epidemics, as illustrated in Figure 5–2. Communicable infections that are both widespread, for example, worldwide, and have high attack rates are termed pandemic. Pandemics have occurred throughout history, as illustrated in Figure 5–2, but have become increasingly frequent. Four new pandemics, three viruses with respiratory spread and one transmitted sexually were experienced in the 20th century. Just 20 years into the 21st century, we have now had five new pandemics, the latest and deadliest (SARS-CoV-2) of which has resulted in over 1 million deaths worldwide in its first 6 months and is still far from being controlled. ++ FIGURE 5–2. History of pandemics. (Reproduced with permisison from Visual Capitalists. www.visualcapitalist.com/history-of-pandemics-deadliest/). Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ ❋ Endemic = constant presence ❋ Epidemic = localized outbreak ❋ Pandemic = widespread regional or global epidemic

An emerging disease is an infectious disease whose incidence has increased in the past two decades and/or that threatens to increase soon. Emerging infectious diseases reflect the arrival of a new pathogen (newly emerging) or an old pathogen that is increasing in incidence, clinical or laboratory characteristics, or geographic range (re-emerging or resurging). An unusual third group is “deliberately emerging” infections, such as anthrax bioterrorism. The appearance of novel coronaviruses (eg, the severe acute respiratory syndrome [SARS] coronavirus and now SARS-CoV-2 [the cause of COVID-19]) are examples of new pathogens, multidrug-resistant Mycobacterium tuberculosis represents an old pathogen with new characteristics, and cholera and Zika in the Americas are examples of old pathogens with a new geographic range (Asia to South America). New methods of detection (eg, molecular) and surveillance (eg, global) have greatly improved our ability to detect and characterize emerging and reemerging infectious diseases. The fundamental methodologies of molecular epidemiology are described in Chapter 4, and their specific applications are discussed in many other chapters throughout this book. ++ Some factors that increase emergence or reemergence of infectious pathogens include: ++ Human and animal demographics and population movement with intrusion into new habitats (particularly tropical forests) Irrigation, especially primitive irrigation systems, which fail to control arthropods and enteric organisms Uncontrolled urbanization, with vector populations breeding in stagnant water Increased international commerce and travel with contact or transport of vectors and pathogens (globalization) Breakdown in public health measures, including sanitation, vector control, immunization programs related to social unrest, civil wars, and major natural disasters Ecological changes, including global climate change and deforestation, with farmers and their animals exposed to new arthropods, floods, and drought Microbial evolution whether related to indiscriminate use of anti-infective agents that leads to selection of multidrug-resistant strains (eg, methicillin-resistant staphylococci or carbapenem-resistant Enterobacteriaceae) or pathogens that mutate readily (eg, virulent strains of influenza A and HIV-1) ++ Zoonotic infections are disproportionately common as emerging pathogens. New, often unexpected, infectious diseases continue to emerge or reemerge despite public health efforts. Although mortality rates declined dramatically during much of the 20th century in the United States due to improved sanitation and the development of vaccines, the mortality rate from infectious diseases increased dramatically in the early 1980s with the introduction of HIV. The development of effective antiretroviral medications in the mid-1990s subsequently reversed HIV-AIDS-specific mortality in the United States that has persisted to the present; however, mortality from other infections, such as vector-borne diseases, drug-resistant pathogens, and Clostridioides (formerly Clostridium) difficile has increased over the same period such that overall infectious disease–related mortality in the United States is strikingly similar to 25 years ago. ++ Emerging and resurging infections on the rise globally include bacteria, viruses, and fungi that have outpaced us (antimicrobial resistance); emerging and resurging zoonotic and vector-borne diseases (including those newly emerging with global warming and human encroachment into previously uninhabited areas); global scourges that have eluded vaccine development (malaria and HIV); and infections for which action has trailed science (control measures exist but have not been effectively deployed). Tragically, much of the world has yet to experience the reduction in infectious diseases–related mortality enjoyed by wealthier countries owing to improved sanitation and the development and provision of effective vaccines. Measles has persisted in poor countries and reemerged in wealthy ones when deployment of effective vaccines is inadequate, or acceptance resisted. Control of HIV globally has been stymied not only by lack of a vaccine but also by inability to deploy known preventive measures and provide access to proven therapies. The global distribution of newly emerging and reemerging (resurging) infectious diseases is illustrated in Figure 5–1. ++ FIGURE 5–1. Global examples of emerging and re-emerging infectious diseases. C difficile, Clostridioides difficile; CRE, carbapenem-resistant Enterobacteriaceae; E coli, Escherichia coli; H3N2v, H3N2 variant; MRSA, multidrug-resistant Staphylococcus aureus; N. gonorrhoeae, Neisseria gonorrhoeae; SARS, severe acute respiratory syndrome; SFTSV, severe fever with thrombocytopenia syndrome virus; vCJD, variant Creutzfeldt-Jakob disease; XDR, extensively drug-resistant. (Reproduced with permission from Fauci AS. Infectious diseases: considerations for the 21st century, Clin Infect Dis 2001 Mar 1;32(5):675-685.) Graphic Jump Location

An emerging disease is an infectious disease whose incidence has increased in the past two decades and/or that threatens to increase soon. Emerging infectious diseases reflect the arrival of a new pathogen (newly emerging) or an old pathogen that is increasing in incidence, clinical or laboratory characteristics, or geographic range (re-emerging or resurging). An unusual third group is “deliberately emerging” infections, such as anthrax bioterrorism. The appearance of novel coronaviruses (eg, the severe acute respiratory syndrome [SARS] coronavirus and now SARS-CoV-2 [the cause of COVID-19]) are examples of new pathogens, multidrug-resistant Mycobacterium tuberculosis represents an old pathogen with new characteristics, and cholera and Zika in the Americas are examples of old pathogens with a new geographic range (Asia to South America). New methods of detection (eg, molecular) and surveillance (eg, global) have greatly improved our ability to detect and characterize emerging and reemerging infectious diseases. The fundamental methodologies of molecular epidemiology are described in Chapter 4, and their specific applications are discussed in many other chapters throughout this book. ++ Some factors that increase emergence or reemergence of infectious pathogens include: ++ Human and animal demographics and population movement with intrusion into new habitats (particularly tropical forests) Irrigation, especially primitive irrigation systems, which fail to control arthropods and enteric organisms Uncontrolled urbanization, with vector populations breeding in stagnant water Increased international commerce and travel with contact or transport of vectors and pathogens (globalization) Breakdown in public health measures, including sanitation, vector control, immunization programs related to social unrest, civil wars, and major natural disasters Ecological changes, including global climate change and deforestation, with farmers and their animals exposed to new arthropods, floods, and drought Microbial evolution whether related to indiscriminate use of anti-infective agents that leads to selection of multidrug-resistant strains (eg, methicillin-resistant staphylococci or carbapenem-resistant Enterobacteriaceae) or pathogens that mutate readily (eg, virulent strains of influenza A and HIV-1) ++ Zoonotic infections are disproportionately common as emerging pathogens. New, often unexpected, infectious diseases continue to emerge or reemerge despite public health efforts. Although mortality rates declined dramatically during much of the 20th century in the United States due to improved sanitation and the development of vaccines, the mortality rate from infectious diseases increased dramatically in the early 1980s with the introduction of HIV. The development of effective antiretroviral medications in the mid-1990s subsequently reversed HIV-AIDS-specific mortality in the United States that has persisted to the present; however, mortality from other infections, such as vector-borne diseases, drug-resistant pathogens, and Clostridioides (formerly Clostridium) difficile has increased over the same period such that overall infectious disease–related mortality in the United States is strikingly similar to 25 years ago. ++ Emerging and resurging infections on the rise globally include bacteria, viruses, and fungi that have outpaced us (antimicrobial resistance); emerging and resurging zoonotic and vector-borne diseases (including those newly emerging with global warming and human encroachment into previously uninhabited areas); global scourges that have eluded vaccine development (malaria and HIV); and infections for which action has trailed science (control measures exist but have not been effectively deployed). Tragically, much of the world has yet to experience the reduction in infectious diseases–related mortality enjoyed by wealthier countries owing to improved sanitation and the development and provision of effective vaccines. Measles has persisted in poor countries and reemerged in wealthy ones when deployment of effective vaccines is inadequate, or acceptance resisted. Control of HIV globally has been stymied not only by lack of a vaccine but also by inability to deploy known preventive measures and provide access to proven therapies. The global distribution of newly emerging and reemerging (resurging) infectious diseases is illustrated in Figure 5–1. ++ FIGURE 5–1. Global examples of emerging and re-emerging infectious diseases. C difficile, Clostridioides difficile; CRE, carbapenem-resistant Enterobacteriaceae; E coli, Escherichia coli; H3N2v, H3N2 variant; MRSA, multidrug-resistant Staphylococcus aureus; N. gonorrhoeae, Neisseria gonorrhoeae; SARS, severe acute respiratory syndrome; SFTSV, severe fever with thrombocytopenia syndrome virus; vCJD, variant Creutzfeldt-Jakob disease; XDR, extensively drug-resistant. (Reproduced with permission from Fauci AS. Infectious diseases: considerations for the 21st century, Clin Infect Dis 2001 Mar 1;32(5):675-685.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt)

to bind to a sequence located between (internal to) the primers. D. Analysis of PCR amplified DNA. (1) The amplified sequence can be cloned into a plasmid vector. In this form, a variety of molecular manipulations or sequencing may be carried out. (2) Direct hybridizations usually make use of an internal probe. The example shows three specimens, each of which went through steps A and B. After amplification, each was bound to a separate spot on a filter (dot blot). The filter is then reacted with the internal probe to detect the PCR-amplified DNA. The result shows that only the middle specimen contained the target sequence. (3) The amplified DNA may be detected directly by agarose gel electrophoresis. The example shows detection of amplified fragments in two of three lanes on the gel. (4) The sensitivity of detection may be increased by use of the internal probe after Southern transfer. The example shows detection of a third fragment of the same size that was not seen on the original gel because the amount of DNA was too small. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ NAA replicates a genome segment ++ ❋ PCR uses temperature to manipulate primers and polymerases +++ Application of Nucleic Acid Methods to Infectious Diseases +++ DNA Probes ++ Probes may be recovered from NAA procedures or more commonly synthesized as a single chain of nucleotides (oligonucleotide probe) from known sequence data. They may contain a gene of known function or simply sequences empirically found to be useful for the application in question. When labeled with a fluorescent or chromogenic marker and used in hybridization reactions, they can detect the homologous sequences in unknown specimens (Figure 4–8). ++ Probes may be cloned or synthesized from known sequences ++ The diagnostic use of DNA probes is to detect or identify microorganisms by hybridization of the probe to homologous sequences in DNA extracted from the entire organism. A number of probes have been developed that can quickly and reliably identify organisms already isolated in culture. The application of probes for detection of infectious agents directly in clinical specimens such as blood, urine, and sputum is more difficult because only a small number of organisms may be present. This problem of sensitivity can be overcome by combining probes with NAA methods (see further text). This approach offers the potential for rapid diagnosis and the detection of characteristics not possible by routine methods. For example, a bacterial toxin gene probe can demonstrate both the presence of the related organism and its toxigenicity without the need for culture. ++ Probes can detect DNA of pathogen directly in clinical specimens +++ Applications of Polymerase Chain Reaction ++ The amplification power of the PCR offers a solution for the sensitivity problems inherent in the direct application of probes in clinical specimens. The nucleic acid segment amplified by PCR can be detected by direct hybridization with the probe (Figure 4–9C, D2) or for greater specificity after electrophoresis and Southern transfer (Figure 4–9D3,4). This approach has been successful for a wide range of infectious agents and awaits only further resolution of practical problems for wider use. ++ PCR plus probes gives greatest sensitivity ++ Another creative use of PCR has been in the study of infectious agents seen in tissue but not grown in culture. PCR primers derived from sequences known to be highly conserved among bacteria, such as ribosomal RNA, have been applied to tissue specimens. The amplification produces enough DNA to clone and sequence. This sequence can then be compared with sequences published for other organisms using computers. Thus, taxonomic relationships can be inferred for an organism that has never been isolated in culture. ++ PCR allows study of organisms that cannot be cultured ++ SUMMARY The application of some combination of the principles described in this chapter is appropriate to the diagnosis of any infectious disease. The recognition of specific etiologic agents as the causes of common infectious diseases and their detection in the laboratory in the last quarter of the 19th and early 20th centuries laid the foundation for the practice of medical microbiology for the next 100 years. The number of agents and their disease correlates continued to expand as did improved method for detection. The watershed discovery of the double helix twisted ladder structure of DNA by Watson and Crick in 1953 transformed future practice. The pace of change accelerated with discovery of PCR by Mullis in 1983. Contemporarily, a totally novel disease (AIDS) was recognized as subsequently was its cause (HIV-1) and methods for diagnosis (EIA/ELISA). Classic bacteriology as described in this chapter is still the bedrock of infectious disease diagnosis, but it too has been changed dramatically by the widespread use of NAA tests and MALDI-TOF MS in the past decade. All of the above (double helix, PCR, HIV-1, and MALDI-TOF) as well as below (HBV and HCV) resulted in Nobel Prizes for their discoverers. The advances molecular technologies have enabled in the past 40 years are nothing short of astounding: consider the discovery, methods for diagnosis, and therapeutic agents for HIV, HBV, and HCV despite their having never been grown in culture. There is no basis for expecting the pace of change to slow. There is every prospect that 16S rRNA gene cycle sequencing will become routine for identification of bacteria in the diagnostic laboratory. Whole-genome sequence typing has already supplanted previous methods for outbreak investigations of pathogens old and new. Metagenomic next-generation sequencing (mNGS) is on the way. Lest anyone doubt the need for continued research, education, and support in and of medical microbiology, consider the latest and on

Despite widespread use of nucleic acid diagnostic procedures, cultures remain essential in clinical diagnostic laboratories. Isolation in pure culture is required for identification and most phenotypic antimicrobial susceptibility testing. ++ Growth on artificial media, isolation, and identification of the infecting agent is usually the most sensitive and specific means for an etiologic diagnosis of common bacterial and fungal pathogens. Theoretically, the presence of a single live organism in the specimen can yield a positive result. Most bacteria and fungi can be grown in a variety of artificial media, but strictly intracellular microorganisms (eg, Chlamydia, Rickettsia, and viruses) can be isolated only in cultures of living eukaryotic cells. Consequently, molecular methods have replaced culture for these pathogens. +++ Isolation and Identification of Bacteria and Fungi ++ Almost all medically important bacteria can be cultivated outside the host in artificial culture media. A single bacterium placed in the proper culture conditions multiplies to quantities sufficient to be seen by the naked eye. Bacteriologic media are broth recipes prepared from digests of animal or vegetable protein supplemented with nutrients such as glucose, yeast extract, serum, or blood to meet the metabolic requirements of the organism. Their chemical composition is complex, and their success depends on matching the nutritional requirements of most heterotrophic living things. The same approaches are used for growing fungi. ++ Bacteria grow in broth and on solid media ++ Growth in media prepared in the fluid state (broth) is apparent when bacterial numbers are sufficient to produce turbidity or macroscopic clumps. Turbidity results from reflection of transmitted light by the bacteria; depending on the size of the organism, from 105 to 106 bacteria per milliliter of broth are required. The addition of a gelling agent to a broth medium allows its preparation in solid form in Petri dishes. The universal gelling agent for diagnostic bacteriology is agar—a polysaccharide extracted from seaweed. Agar has the convenient property of becoming liquid at approximately 95°C but not returning to the solid gel state until cooled to less than 50°C. This allows the addition of a heat-labile substance such as blood to the medium before it sets. At temperatures used in the diagnostic laboratory (37°C or lower), broth–agar exists as a smooth, solid, nutrient gel. This medium, usually termed agar, may be qualified with a description of any supplement (eg, blood agar). ++ Large numbers of bacteria produce turbidity Agar is used to solidify media ++ A useful feature of agar plates is that the bacteria can be separated by spreading a small sample of the specimen over the surface. Bacterial cells that are well separated from others grow as isolated colonies, often reaching 2 to 3 mm in diameter after overnight incubation. This allows isolation of bacteria in pure culture because the colony is assumed to arise from a single organism (Figure 4–5). Colonies vary greatly in size, shape, texture, color, and other features called colonial morphology. Colonies from different species or genera often differ substantially, whereas those derived from the same strain are usually consistent. Differences in colonial morphology are very useful for separating bacteria in mixtures and as clues to their identity. ++ FIGURE 4–5. Bacteriologic plate streaking. Plate streaking is essentially a dilution procedure. A. (1) The specimen is placed on the plate with a swab, loop, or pipette and evenly spread over approximately part of plate surface with a sterilized bacteriologic loop (2-5). The loop is flamed to remove residual bacteria, and a series of overlapping streaks are made flaming the loop between each one. B. After overnight incubation, heavy growth is seen in the primary areas followed by isolated colonies. More than one organism is present because both a red and a clear colony are seen. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Bacteria separated in isolated colonies Colonies may have characteristic features +++ Culture Media ++ Over the last 100 years, countless media have been developed by microbiologists to aid in the isolation and identification of medically important bacteria and fungi. Only a few have found their way into routine use in clinical laboratories. These may be classified as nutrient, selective, or indicator media. +++ Nutrient Media ++ The nutrient component of a medium is designed to satisfy the growth requirements of the organism to permit isolation and propagation. For medical purposes, the ideal medium would allow rapid growth of all agents. No such medium exists; however, several suffice for good growth of most medically important bacteria and fungi. These media are prepared with enzymatic or acid digests of animal or plant products, such as muscle, milk, or soybeans. The digest reduces the native protein to a mixture of polypeptides and amino acids that also includes trace metals, coenzymes, and various undefined growth factors. For example, one common broth contains a digest of casein (milk curd) and a digest of soybean meal. To this nutrient base, salts, vitamins, or body fluids such as serum may be added to provide pathogens with the conditions needed for optimum growth. All cultures of blood use this type of medium. ++ Media are prepared from animal or plant products +++ Selective Media ++ Selective media are used when specific pathogenic organisms are sought in sites with an extensive microbiota (eg, Campylobacter species in fecal specimens). In these cases, other bacteria may overgrow the suspected etiologic species in simple nutrient media, either because the pathogen grows more slowly or because it is present in much smaller numbers. Selective media usually contain dyes, other chemical additives, or antimicrobial agents at concentrations designed to inhibit contaminating flora but not the suspected pathogen. ++ Contaminants inhibited with chemicals or antimicrobials +++ Indicator Media ++ Indicator media contain substances designed to demonstrate biochemical or other features characteristic of specific pathogens or organism groups. The addition to the medium of one or more carbohydrates and a pH indicator is frequently used. A color change in a colony indicates the presence of acid products and thus of fermentation or oxidation of the carbohydrate by the organism. The addition of red blood cells (RBCs) to plates allows the hemolysis produced by some organisms to be used as a differential feature. In practice, nutrient, selective, and indicator properties are often combined to various degrees in the same medium. It is possible to include an indicator system in a highly nutrient medium and also make it selective by adding appropriate antimicrobials. Some examples of culture media commonly used in diagnostic microbiology are listed in Appendix 4–1, and more details of their constitution and application are provided in Appendix 4–2. ++Table Graphic Jump LocationAPPENDIX 4–1Some Media Used for Isolation of Bacterial PathogensView Table| Favorite Table |Download (.pdf) APPENDIX 4–1 Some Media Used for Isolation of Bacterial Pathogens MEDIUM USES General-purpose Media Nutrient broths (eg, soybean–casein digest broth) Most bacteria, particularly when used for blood culture Thioglycolate broth Anaerobes, facultative bacteria Blood agar Most bacteria (demonstrates hemolysis) and fungi Chocolate agar Most bacteria, including fastidious species (eg, Haemophilus) and fungi Selective Media   MacConkey agar Nonfastidious Gram-negative rods Hektoen enteric agar Salmonella and Shigella Selenite F broth Salmonella enrichment Sabouraud agar Isolation of fungi, particularly dermatophytes Special-purpose Media   Löwenstein–Jensen medium, Middlebrook agar M tuberculosis and other mycobacteria (selective) Martin–Lewis medium Neisseria gonorrhoeae and Neisseria meningitidis (selective) Tinsdale agar C diphtheriae (selective) Regan-Lowe charcoal agar Bordetella pertussis (selective) Buffered charcoal–yeast extract agar Legionella species (nonselective) Campylobacter blood agar Campylobacter jejuni (selective) Thiosulfate-citrate-bile-sucrose agar (TCBS) Vibrio cholerae and Vibrio parahaemolyticus (selective) ++Table Graphic Jump LocationAPPENDIX 4–2Characteristics of Commonly Used Bacteriologic MediaView Table| Favorite Table |Download (.pdf) APPENDIX 4–2 Characteristics of Commonly Used Bacteriologic Media Nutrient broths. Some form of nutrient broth is used for culture of blood and all direct tissue samples from sites that are normally sterile to obtain the maximum culture sensitivity. Selective or indicator agents are omitted to prevent inhibition of more fastidious organisms. Blood agar. The addition of defibrinated blood to a nutrient agar base enhances the growth of some bacteria, such as streptococci. This often yields distinctive colonies and provides an indicator system for hemolysis. Two major types of hemolysis are seen: β-hemolysis, a complete clearing of red cells from a zone surrounding the colony; and α-hemolysis, which is incomplete (ie, intact red cells are still present in the hemolytic zone), but shows a green color caused by hemoglobin breakdown products. The net effect is a hazy green zone extending 1 to 2 mm beyond the colony. A third type, α’-hemolysis, produces a hazy, incomplete hemolytic zone similar to that caused by α-hemolysis, but without the green coloration. Chocolate agar. If blood is added to molten nutrient agar at approximately 80°C and maintained at this temperature, the red cells are gently lysed, hemoglobin products are released, and the medium turns a chocolate brown color. The nutrients released permit the growth of some fastidious organisms such as H influenzae, which fail to grow on blood or nutrient agars. This quality is particularly pronounced when the medium is further enriched with vitamin supplements. Given the same incubation conditions, any organism that grows on blood agar also grows on chocolate agar. Martin–Lewis medium. A variant of chocolate agar, Martin–Lewis medium is a solid medium selective for the pathogenic Neisseria (N gonorrhoeae and N meningitidis). Growth of most other bacteria and fungi in the genital or respiratory flora is inhibited by the addition of antimicrobial agents. One formulation includes vancomycin, colistin, trimethoprim, and anisomycin. MacConkey agar. This agar is both a selective and an indicator medium for Gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas. In addition to a peptone base, the medium contains bile salts, crystal violet, lactose, and neutral red as a pH indicator. The bile salts and crystal violet inhibit Gram-positive bacteria and the more fastidious Gram-negative organisms, such as Neisseria and Pasteurella. Gram-negative rods that grow and ferment lactose produce a red (acid) colony, often with a distinctive colonial morphology. Hektoen enteric agar. The Hektoen medium is one of many highly selective media developed for the isolation of Salmonella and Shigella species from stool specimens. It has both selective and indicator properties. The medium contains a mixture of bile, thiosulfate, and citrate salts that inhibits not only Gram-positive bacteria, but members of Enterobacteriaceae other than Salmonella and Shigella that appear among the normal flora of the colon. The inhibition is not absolute; recovery of Escherichia coli is reduced 1000- to 10,000-fold relative to that on nonselective media, but there is little effect on growth of Salmonella and Shigella. Carbohydrates and a pH indicator are also included to help to differentiate colonies of Salmonella and Shigella from those of other enteric Gram-negative rods. Anaerobic media. In addition to meeting atmospheric requirements, isolation of some strictly anaerobic bacteria on blood agar is enhanced by reducing agents such as L-cysteine and by vitamin enrichment. Sodium thioglycolate, another reducing agent, is often used in broth media. Plate media are made selective for anaerobes by the addition of aminoglycoside antibiotics, which are active against many aerobic and facultative organisms but not against anaerobic bacteria. The use of selective media is particularly important with anaerobes because they grow slowly and are commonly mixed with facultative bacteria in infections. Highly selective media. Media specific to the isolation of almost every important pathogen have been developed. Many allow only a single species to grow from specimens with a rich normal flora (eg, stool). The most common of these media are listed in Appendix 4–1; they are discussed in greater detail in following chapters. ++ Metabolic properties demonstrated by indicator systems +++ Atmospheric Conditions +++ Aerobic ++ After inoculation, cultures of most aerobic bacteria are placed in an incubator with temperature maintained at 35°C to 37°C. Slightly higher or lower temperatures are used occasionally to selectively favor a certain organism or organism group. Most bacteria that are not obligate anaerobes grow in air; however, CO2 is required by some and enhances the growth of others. Incubators that maintain a 2% to 5% concentration of CO2 in air are frequently used for primary isolation, because this level is not harmful to any bacteria and improves isolation of some. Some bacteria (eg, Campylobacter) require a microaerophilic atmosphere with reduced oxygen (5%) and increased CO2 (10%) levels to grow. This can be achieved by using a commercially available packet that is placed in a jar which is then sealed similar to the anaerobic system described further. ++ Incubation temperature, atmosphere vary +++ Anaerobic ++ Strictly anaerobic bacteria do not grow under the conditions just described, and many die when exposed to atmospheric oxygen or high oxidation–reduction potentials. Most medically important anaerobes grow in the depths of liquid or semisolid media containing any of a variety of reducing agents, such as cysteine, thioglycollate, ascorbic acid, or even iron filings. An anaerobic environment for incubation of plates can be achieved by replacing air with a gas mixture containing hydrogen, CO2, and nitrogen and allowing the hydrogen to react with residual oxygen on a catalyst to form water. A convenient commercial system accomplishes this chemically in a packet that is added before the jar is sealed. Specimens suspected to contain significant anaerobes should be processed under conditions designed to minimize exposure to atmospheric oxygen at all stages. ++ Anaerobes require reducing conditions, no oxygen +++ Clinical Microbiology Procedures ++ Routine laboratory procedures for processing specimens from various sites are needed because no single medium or atmosphere is ideal for all bacteria. Combinations of broth and solid-plated media and aerobic, CO2, and anaerobic incubation must be matched to the organisms expected at any particular site or clinical circumstance. Examples of such routines are shown in Table 4–1. In general, it is not practical to routinely include specialized media for isolation of rare organisms, such as C diphtheriae or Legionella pneumophila. For detection of these and other uncommon organisms, the laboratory must be specifically informed of their possible presence by the physician. Appropriate media and special procedures can then be included. ++Table Graphic Jump LocationTABLE 4–1Routine Use of Gram Smear and Isolation Systems for Selected Clinical SpecimensaView Table| Favorite Table |Download (.pdf) TABLE 4–1 Routine Use of Gram Smear and Isolation Systems for Selected Clinical Specimensa SPECIMEN MEDIUM (INCUBATION) BLOOD CEREBROSPINAL FLUID WOUND, PUS GENITAL, CERVIX THROAT SPUTUM URINE STOOL Gram smear   × × ×   ×     Soybean–casein digest broth (CO2)a ×               Blood agar (CO2)   × ×   ×b × ×   Chocolate agar (CO2)   × × × PCR preferred   ×     Blood agar (anaerobic)     ×           MacConkey agar (air)     ×     × × × Hektoen agar (air)               × Selenite F broth (air)               × Campylobacter agar (CO2, 42°C)c               × Martin–Lewis agar (CO2)       × PCR preferred         aThe added sensitivity of a nutrient broth is used only when contamination by normal flora is unlikely. Exact media and protocols may vary between laboratories.bAnaerobic incubation used to enhance hemolysis by β-hemolytic streptococci.cIncubation in a reduced oxygen atmosphere. ++ Designed to detect the most common organisms +++ Identification ++ When growth is detected in any medium, the process of identification begins. Identification involves methods for obtaining pure cultures from single colonies, followed by tests designed to characterize and identify the isolate. The exact tests and their sequences vary with different groups of organisms, and the taxonomic level (genus, species, subspecies, etc.) of identification needed varies according to the medical usefulness of the information. In some cases, only a general description or the exclusion of particular organisms is important. For example, a report of “mixed oral flora” in a sputum specimen or “No Salmonella, Shigella, or Campylobacter isolated” in a fecal specimen may provide all the information needed. MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectroscopy has become the foremost tool used for the rapid identification of microorganisms already isolated in pure culture and has reduced time to identification and reporting substantially from a day or more to minutes. Although a major advance, MALDI-TOF complements but does not replace fully the need for traditional methods. The scope and accuracy of MALDI-TOF depend on the quality of the data based used for comparisons. As depicted schematically in Figure 4-6, ionized microorganisms are separated by mass-charge-ratio (effectively by molecular weight), collide under vacuum with an ion detector, and thereby generate a mass spectrum for comparative analysis. The net result is rapid identification of bacterial or fungal, especially yeasts, isolates. ++ FIGURE 4–6. MALDI-TOF mass spectrometer. As depicted schematically, ionized microorganisms are separated by mass-charge-ratio (effectively by molecular weight), collide under vacuum with an ion detector, and thereby generate a mass spectrum for comparative analysis. (From Patel R. Matrix-assisted laser desorption ionization-time of flight mass spectrometry in clinical microbiology. Clin Infect Dis. 2013 Aug;57(4):564–72; used with permission of Mayo Foundation for Medical Education and Research, all rights reserved.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Extent of identification is linked to medical relevance +++ Features Used to Classify Bacteria and Fungi +++ Cultural Characteristics ++ Cultural characteristics include the demonstration of properties such as unique nutritional requirements, pigment production, and the ability to grow in the presence of certain substances (sodium chloride, bile) or on certain media (MacConkey, nutrient agar). Demonstration of the ability to grow at a particular temperature or to cause hemolysis on blood agar plates is also used. For fungi, growth as a yeast colony or a mold is the primary separator. For molds, the morphology of the mold structures (hyphae, conidia, etc.) is the primary means of identification. ++ Growth under various conditions +++ Biochemical Characteristics ++ Traditionally, the ability to attack various substrates or to produce particular metabolic products has broad application to the identification of bacteria and yeast. The most common properties examined are listed in Appendix 4–3. Biochemical and cultural tests for bacterial identification are analyzed by reference to tables that show the reaction patterns characteristic of individual species. In fact, advances in computer analysis have now been applied to identification of many bacterial and fungal groups. These systems use the same biochemical principles together with computerized databases to determine the most probable identification from the observed test pattern. In many laboratories, MALDI-TOF mass spectrometry has replaced these biochemical approaches except for a few rapid colorimetric spot tests, eg, indole and PYR. When identification of bacteria remains elusive after biochemical and MALDI-TOF have been attempted, the isolates usually are sent to reference laboratories for 16S rRNA or other sequencing methods if the clinical importance warrants. ++Table Graphic Jump LocationAPPENDIX 4–3Common Biochemical Tests for Microbial IdentificationView Table| Favorite Table |Download (.pdf) APPENDIX 4–3 Common Biochemical Tests for Microbial Identification Carbohydrate breakdown. The ability to produce acidic metabolic products, fermentatively or oxidatively, from a range of carbohydrates (eg, glucose, sucrose, and lactose) has been applied to the identification of most groups of bacteria. Such tests are crude and imperfect in defining mechanisms, but have proved useful for taxonomic purposes. More recently, gas chromatographic identification of specific short-chain fatty acids produced by fermentation of glucose has proved useful in classifying many anaerobic bacteria. Catalase production. The enzyme catalase catalyzes the conversion of hydrogen peroxide to water and oxygen. When a colony is placed in hydrogen peroxide, liberation of oxygen as gas bubbles can be seen. The test is particularly useful in differentiation of staphylococci (positive) from streptococci (negative), but also has taxonomic application to Gram-negative bacteria. Citrate utilization. An agar medium that contains sodium citrate as the sole carbon source may be used to determine ability to use citrate. Bacteria that grow on this medium are termed citrate-positive. Coagulase. The enzyme coagulase acts with a plasma factor to convert fibrinogen to a fibrin clot. It is used to differentiate Staphylococcus aureus from other, less pathogenic staphylococci. Decarboxylases and deaminases. The decarboxylation or deamination of the amino acids lysine, ornithine, and arginine is detected by the effect of the amino products on the pH of the reaction mixture or by the formation of colored products. These tests are used primarily with Gram-negative rods. Hydrogen sulfide. The ability of some bacteria to produce H2S from amino acids or other sulfur-containing compounds is helpful in taxonomic classification. The black color of the sulfide salts formed with heavy metals such as iron is the usual means of detection. Indole. The indole reaction tests the ability of the organism to produce indole, a benzopyrrole, from tryptophan. Indole is detected by the formation of a red dye after addition of a benzaldehyde reagent. A spot test can be done in seconds using isolated colonies. Nitrate reduction. Bacteria may reduce nitrates by several mechanisms. This ability is demonstrated by detection of the nitrites and/or nitrogen gas formed in the process. O-Nitrophenyl-β-D-galactoside (ONPG) breakdown. The ONPG test is related to lactose fermentation. Organisms that possess the β-galactoside necessary for lactose fermentation but lack a permease necessary for lactose to enter the cell are ONPG-positive and lactose-negative. Oxidase production. The oxidase tests detect the c component of the cytochrome–oxidase complex. The reagents used change from clear to colored when converted from the reduced to the oxidized state. The oxidase reaction is commonly demonstrated in a spot test, which can be done quickly from isolated colonies. Proteinase production. Proteolytic activity is detected by growing the organism in the presence of substrates, such as gelatin or coagulated egg. Pyrrolidonyl arylamidase activity (PYR test) is a rapid colorimetric test for preliminary identification and screening of certain Gram-positive bacteria (eg, group A streptococci, enterococci, and Staphylococcus lugdenensis). A positive PYR test is color change from pink to red. Urease production. Urease hydrolyzes urea to yield two molecules of ammonia and one of CO2. This reaction can be detected by the increase in medium pH caused by ammonia production. Urease-positive species vary in the amount of enzyme produced; bacteria can thus be designated as positive, weakly positive, or negative. Voges–Proskauer test. The Voges–Proskauer test detects acetylmethylcarbinol (acetoin), an intermediate product in the butene glycol pathway of glucose fermentation. ++ Biochemical reactions give identification probability Toxin production and pathogenicity ++ Molecular assays have been developed for some toxins (eg, Clostridiodes difficile as an alternative to enzyme immunoassay [EIA]) for use in the clinical laboratory. Neutralization of a toxic effect in a test animal with specific antitoxin is the method used to confirm the identity of Clostidium botulinum (Chapter 29) toxin and is available only in public health reference laboratories. ++ Detection of specific toxin may define disease +++ Antigenic Structure ++ Viruses, bacteria, fungi, and parasites possess many antigens, such as capsular polysaccharides, surface proteins, and cell wall components. Serology involves the use of antibodies of known specificity to detect antigens present on whole organisms or free in extracts (soluble antigens). The methods used for demonstrating antigen–antibody reactions are discussed in Antibody Detection (Serology). ++ Antigenic structure demonstrated with antisera +++ Genomic Structure ++ Nucleic acid–sequence relatedness as determined by homology and direct sequence comparisons have become a primary determinant of taxonomic decisions. They are discussed later in the section on Methods of Nucleic Acid Analysis. +++ Isolation and Identification of Viruses +++ Cell and Organ Culture ++ Virtually no clinical microbiology laboratory still retains the capacity to do viral isolation by cell or organ culture. The classical techniques are done, if at all, in research or public health laboratories. The extensive repertoire of molecular assays now available for most human viral pathogens has far better sensitivity and specificity than traditional methods. The appropriate use of these NAATs in viral diagnosis is discussed for each virus in Chapters 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 in PART II, Pathogenic Viruses.

The diagnosis of a microbial infection begins with an assessment of the clinical and epidemiologic features and formulation of a diagnostic hypothesis. Anatomic localization of the infection depends on physical and radiologic findings (eg, right lower lobe pneumonia, subphrenic abscess). This clinical diagnosis suggests a number of possible etiologic agents based on knowledge of infectious syndromes and their courses. The specific cause or etiologic diagnosis is then established by the application of methods described in this chapter. A combination of science and art on the part of both the clinician and laboratory worker is required: The clinician must select the appropriate tests and specimens to be processed and, where appropriate, suggest the suspected etiologic agents to the laboratory. The laboratory scientist must use the methods that will demonstrate the probable agents and be prepared to explore other possibilities suggested by the clinical situation or by the findings of the laboratory examinations. The best results are obtained when communication between the clinician and laboratory is optimal. ++ ❋ Clinical diagnosis guides approach to etiologic diagnosis ++ Behind every clinical specimen submitted to the diagnostic laboratory should be a question. Does my patient have, can I exclude, does the result confirm the disease? Answers to such questions depend on understanding, whether articulated specifically or not, the characteristics of the tests ordered and performed. These characteristics are sensitivity (the test’s ability to rule out [snout] a disease because there are few false-negative results and thus fewer cases missed) and specificity (the test’s ability to rule in [spin] or confirm an etiology because there are few false-positive results). Ideally, a test would have both excellent sensitivity and specificity, but traditional methods often involved a trade-off between the two, which only emphasizes the need to know the clinical question or reason for ordering a test. Molecular methods, however, tend to have improved sensitivity as well as specificity, which is dramatically so for viral etiologic diagnoses. ++ ❋ Sensitivity is capacity of test to rule OUT a diagnosis ❋ Specificity is ability of test to rule IN or confirm diagnosis ++ Predictive value of a test is determined by its sensitivity and specificity and the prevalence of disease in a population or the likelihood thereof in a patient based on the history, clinical findings, and epidemiology of the infectious disease agent being considered. The more sensitive a test, the greater its negative predictive value (NPV), thus a patient with a negative test is very unlikely to have the disease. A positive result with a more specific test makes a diagnosis more likely or has a higher positive predictive value (PPV) and basically confirms an etiologic diagnosis. When the prevalence of a disease is exceedingly low or the likelihood is virtually nil based on the history, clinical findings, and epidemiology, even tests with high sensitivity and specificity may have a low PPV. This reality highlights the importance of the clinical diagnosis in guiding the approach to making an etiologic diagnosis based on the question(s) posed to the diagnostic microbiology laboratory. ++ The general approaches to laboratory diagnosis vary with different microorganisms and infectious diseases. However, the types of methods are usually some combination of direct microscopic examinations, culture, antigen detection, and antibody detection (serology). Nucleic acid amplification (NAA) assays that enable direct detection of genomic components of pathogens are now essential in clinical microbiology laboratories, especially for viral infections. Multiplexed polymerase chain reaction (PCR) platforms that enable rapid, direct detection of multiple potential pathogens in appropriate specimens are now available for respiratory, gastrointestinal, and central nervous system pathogens and for identification of positive blood cultures. Despite such progress, however, traditional methods remain important and complementary, since isolation of microorganisms by culture is needed for most antimicrobial susceptibility testing. Not all pathogens are detected in these panels, and only known pathogens are sought. Therefore, this chapter considers the principles of infectious disease laboratory diagnosis and the methods available with an emphasis on bacterial and fungal infections. Details about particular agents are discussed in the relevant chapters and in the section about infectious disease syndromes and etiologies at the back of the book. All diagnostic approaches begin with some kind of specimen collected from the patient. ++ ❋ Microscopic, culture, antigen, antibody detection classic ❋ NAA foremost for viral pathogens

Infection control is the sum of all the means used to prevent nosocomial infections. Historically, such methods have been developed as an integral part of the study of infectious diseases, often serving as key elements in the proof of infectious etiology. In the 19th century, Joseph Lister achieved a dramatic reduction in surgical wound infections by infusion of a phenolic antiseptic into wounds. This local destruction of organisms was known as antisepsis. As it became recognized that contamination of wounds was not inevitable, the emphasis gradually shifted to preventing contact between microorganisms and susceptible sites—a concept called asepsis. Asepsis, which combines containment with the methods of sterilization and disinfection previously discussed, is the central approach of infection control. The measures taken to achieve asepsis vary, depending on whether the circumstances and environment are the operating room, hospital ward, or outpatient clinic. ++ Antisepsis attacks contaminating organisms ++ ❋ Asepsis prevents contamination +++ Asepsis +++ Operating Room ++ The surgical suite and operating room represent the most controlled and rigid application of aseptic principles. The procedure begins with the use of an antiseptic scrub of the skin over the operative site and the hands and forearms of all who will have contact with the patient. The use of sterile drapes, gowns, and instruments serves to prevent spread through direct contact, and caps and face masks reduce airborne spread from personnel to the wound. The level of bacteria in the air is generally increased by the number of persons and amount of movement in the operating room more than any change in the incoming air. The net effect of these procedures is to draw a sterile curtain around the operative site, thus minimizing contact with microorganisms. Surgical asepsis is also used in other areas where invasive special procedures such as cardiac catheterization are carried out. ++ Sterile drapes and instruments prevent contact of organisms with wound ++ ❋ Airborne bacteria are associated with personnel in operating room +++ Hospital Ward ++ Although theoretically desirable, strict aseptic procedures as used in the operating room are impractical in the ward setting. Asepsis is practiced by the use of sterile needles, medications, dressings, and other items that could serve as transmission vehicles if contaminated. A “no touch” technique for examining wounds and changing dressings eliminates direct contact with any nonsterile item. Invasive procedures such as catheter insertion and lumbar punctures are carried out under aseptic precautions similar to those used in the operating room. In all circumstances, handwashing between patient contacts is the single most important aseptic precaution. ++ Handwashing is the most important measure +++ Outpatient Clinic ++ The general aseptic practices used on the hospital ward are also appropriate to the outpatient situation as preventive measures. Patients who may be infected should be segregated whenever possible using techniques similar to those of hospital ward isolation. The examining room may be used in a manner analogous to the private rooms on a hospital ward. Although this approach is difficult because of patient turnover, it should be attempted for infections that would require strict or respiratory isolation in the hospital. ++ Waiting areas present a risk +++ Isolation Procedures ++ Patients with infections pose special problems because they may transmit their infections to other patients either directly or by contact with a staff member. This additional risk is managed by the techniques of isolation, which place barriers between the infected patient and others on the ward. Because not every infected patient presents with suspect signs and/or symptoms, some precautions should be taken with all patients. In the system recommended by the Centers for Disease Control and Prevention, these are called standard precautions and include the use of gowns and gloves when in contact with patient blood or secretions. These are particularly directed at protecting healthcare workers from HIV and hepatitis infection. For those with suspected or proven infection, additional precautions are taken, the nature of which is determined by the known mode of transmission of the organism. These transmission-based precautions are divided into those directed at airborne, droplet, and contact routes. The airborne transmission precautions are for infections known to be transmitted by extremely small (<5 μm) particles suspended in the air. This requires that the room air circulation be maintained with negative pressure relative to the surrounding area and be exhausted to the outside. Those entering the room must wear surgical masks, and in the case of tuberculosis, specially designed respirators. Droplet precautions are for infections in which the organisms are suspended in larger droplets, which may be airborne, but generally do not travel more than 3 ft from the patient who generates them. These can be contained by the use of gowns, gloves, and masks when working close to the patient. Contact precautions are used for infections that require direct contact with organisms on or that pass in secretions of the patient. Diarrheal infections are of special concern because of the extent to which they contaminate the environment. Details of the precautions and examples of the typical infectious agents are summarized in Table 3–3. ++Table Graphic Jump LocationTABLE 3–3Precautions for Prevention of Nosocomial InfectionsView Table| Favorite Table |Download (.pdf) TABLE 3–3 Precautions for Prevention of Nosocomial Infections PRECAUTION ROOM HANDWASHINGa GLOVES GOWNS MASKb TYPICAL DISEASES Standard   After removing gloves, between patients Blood, fluid contact, touching skin Blood, fluid contact, during procedures During procedures All Transmission-based Airborne Private, negative pressurec After removing gloves, between patients Room entry Room entry Room entry or respiratord Measles, chickenpox, tuberculosisd Droplet Privatee After removing gloves, between patients Blood, fluid contact Blood, fluid contact Within 3 ft of patient Meningitis, pertussis, plague, influenza Contact Privatee After removing gloves, between patients Room entry Patient contact — Infectious diarrhea,f Staphylococcus aureus wounds aUsing a disinfectant soap.bStandard surgical mask, goggles.cRoom pressure must be negative in relation to surrounding area and the circulation exhausted outside the building.dFor patients with diagnosed or suspect tuberculosis, a specially filtered respirator/mask must be worn.eDoor may be left open and patients with the same organism may share a room.fParticularly Clostridium difficile, Escherichia coli O:157, Shigella, and incontinent patients shedding rotavirus or hepatitis A. ++ Standard precautions protect healthcare workers from HIV infection ++ *Transmission precautions block airborne, droplet, and contact routes +++ Prevention ++ The prevention of nosocomial infections is contingent on basic and applied knowledge drawn from all parts of this book. Applied with common sense, these principles can both prevent disease and reduce the costs of medical care.

Infections occurring during any hospitalization could be either community-acquired or nosocomial. Community-acquired infections are defined as those present or incubating at the time of hospital admission. All others are considered nosocomial. For example, a hospital case of chickenpox could be community-acquired if it erupted on the fifth hospital day (incubating) or nosocomial if hospitalization was beyond the limits of the known incubation period (20 days). Infections appearing shortly after discharge (2 weeks) are considered nosocomial, although some could have been acquired at home. Infectious hazards are inherent to the hospital environment; it is there that the most seriously infected and most susceptible patients are housed and often cared for by the same staff. ++ Community-acquired infections are acquired before admission Nosocomial infections are acquired in hospital ++ The infectious agents responsible for nosocomial infections arise from various sources, including patients’ own microbiota. In addition to any immunocompromising disease or therapy, the hospital may impose additional risks by treatments that breach the normal defense barriers. Surgery, urinary or intravenous catheters, and invasive diagnostic procedures all may provide opportunistic microbes with access to usually sterile sites. Infections in which the source of organisms is the hospital rather than the patient include those derived from hospital personnel, the environment, and medical equipment. ++ Endogenous infections are part of hospital risk +++ Hospital Personnel ++ Physicians, nurses, students, therapists, and any others who come in contact with the patient may transmit infection. Transmission from one patient to another is called cross-infection. The vehicle of transmission is most often the inadequately washed hands of a medical attendant. Another source is the actively infected medical attendant. Many hospital outbreaks have been traced to hospital personnel, particularly physicians, who continue to care for patients despite an overt infection. Transmission is usually by direct contact, although airborne transmission is also possible. A third source is the person who is not ill, but asymptomatically carrying a virulent strain. For Staphylococcus aureus and group A streptococci, nasal carriage is most important, but sites such as the perineum have also been involved in outbreaks. An occult carrier is less often the source of nosocomial infection than a physician covering up a boil or a nurse minimizing “the flu.” ++ ❋ Cross-infection is usually by direct contact ❋ Infected medical attendants are particularly dangerous ++ Infection from carriers can transmit to patients +++ Environment ++ The hospital air, walls, floors, linens, and the like are not sterile and, thus, could serve as a source of organisms causing nosocomial infections, but the importance of this route has generally been exaggerated. With the exception of the immediate vicinity of an infected individual or a carrier, transmission through the air or on fomites is much less important than that caused by personnel or equipment. Notable exceptions are when the environment becomes contaminated with Mycobacterium tuberculosis from a patient or Legionella pneumophila in the water supply. These events are most likely to result in disease when the organisms are numerous or the patient is particularly vulnerable (eg, after heart surgery or bone marrow transplantation). ++ Environmental contamination is relatively unimportant M tuberculosis and Legionella are risks +++ Medical Devices ++ Much of the success of modern medicine is related to medical devices that support or monitor basic body functions. By their very nature, devices such as catheters, implants, and respirators carry a risk of nosocomial infection because they bypass normal defense barriers, providing microorganisms access to normally sterile fluids and tissues. Most of the recognized causes are bacterial or fungal. The risk of infection is related to the degree of debilitation of the patient and various factors concerning the design and management of the device. Any device that crosses the skin or a mucosal barrier may allow microbes in the patient or environment to gain access to deeper sites beyond the outside surface. Possible access inside the device (eg, in the lumen) adds another and, sometimes, greater risk. In some devices, such as urinary catheters, contamination is avoidable; in others, such as respirators, complete sterility is either impossible or impractical to achieve. ++ ❋ Equipment that crosses epithelial barriers provides microbial access ++ The risk of contamination leading to infection is increased if organisms that gain access can multiply within the system. The availability of water, nutrients, and a suitable temperature largely determine which organism will survive and multiply. Many of the Gram-negative rods such as Pseudomonas, Acinetobacter, and members of Enterobacteriaceae can multiply in an environment containing water and little else. Gram-positive bacteria generally require more physiologic conditions. ++ Conditions for bacterial growth increase risk ++ Even with proper growth conditions, many hours are required before contaminating organisms multiply to numbers sufficient to cause disease. Detailed studies of catheters and similar devices show that the risk of infection begins to increase after 24 to 48 hours of use and is cumulative even if the device is changed or disinfected at intervals. It is, thus, important to discontinue transcutaneous procedures as soon as medically indicated. The medical devices most frequently associated with nosocomial infections are listed in the following text. The infectious risk of others can be estimated from the principles discussed previously. New devices are constantly being introduced into medical care, occasionally, without adequate consideration of their potential to cause nosocomial infection. ++ Transcutaneous and indwelling devices should be changed as soon as possible +++ Urinary Catheters ++ Urinary tract infection (UTI) accounts for 40% to 50% of all nosocomial infections, and at least 80% of these are associated with catheterization. The infectious risk of a single urinary catheterization has been estimated at 1%, and indwelling catheters carry a risk that may be as high as 10%. The major preventive measure is maintenance of a completely closed system through the use of valves and aspiration ports designed to prevent bacterial access to the inside of the catheter or collecting bag. Unfortunately, breaks in closed systems eventually occur when the system is in place for more than 30 days. The urine itself serves as an excellent culture medium once bacteria gain access. ++ Closed urinary drainage systems are still violated +++ Vascular Catheters ++ Needles and plastic catheters placed in veins for fluid administration, monitoring vital functions, or diagnostic procedures are a leading cause of nosocomial bacteremia. These sites should always be suspected as a source of organisms whenever blood cultures are positive with no apparent primary site for the bacteremia. Contamination at the insertion site is generally staphylococcal, with continued growth in the catheter tip. Organisms may gain access somewhere in the lines, valves, bags, or bottles of intravenous solutions proximal to the insertion site. The latter circumstance usually involves Gram-negative rods. Preventive measures include aseptic insertion technique and appropriate care of the lines, including changes at regular intervals. ++ ❋ Skin is primary source for intravenous contamination +++ Respirators ++ Machines that assist or control respiration by pumping air directly into the trachea have a great potential for causing nosocomial pneumonia if the aerosol they deliver becomes contaminated. Bacterial growth is significant only in the parts of the device that contain water; in systems using nebulizers, bacteria can be suspended in water droplets small enough to reach the alveoli. The organisms involved include Pseudomonas, Enterobacteriaceae, and a wide variety of environmental bacteria such as Acinetobacter. The primary control measure is periodic changing and disinfection of the tubing, reservoirs, and nebulizer jets. ++ Changing controls nebulizer contamination +++ Blood and Blood Products ++ Infections related to contact with blood and blood products are generally more a risk for healthcare workers rather than patients. Manipulations ranging from phlebotomy and hemodialysis to surgery carry the varying risks of blood containing an infectious agent reaching mucous membranes or skin of the healthcare worker. The major agents transmitted in this manner are hepatitis B, hepatitis C, and HIV. Control requires meticulous attention to procedures that prevent direct contact with blood, such as the use of gloves, eyewear, and gowns. Cuts and needle sticks among healthcare workers carry a risk approaching 2%. Identification of hepatitis virus and HIV carriers is a part of a protective process that must be balanced by patient privacy considerations. Healthcare facilities all have established policies concerning serologic surveillance of patients and the procedures to follow (eg, testing, prophylaxis) when blood-related accidents occur. Similarly, products for transfusion undergo extensive screening to protect the recipient. ++ Risk of hepatitis B, hepatitis C, and HIV is related to blood manipulation Screen is determined by institutional policy

Some risk of infection exists in all healthcare settings. Hospitalized patients are particularly vulnerable, and the hospital environment is complex. Infection control is the proper matching of the principles and procedures described here to general and specialized situations, together with aseptic practices to reduce these risks. “Nosocomial” is a medical term for “hospital-associated.” Nosocomial infections are complications that arise during hospitalizations. The morbidity, mortality, and costs associated with these infections are preventable to a substantial degree. The purpose of hospital infection control is prevention of nosocomial infections by application of epidemiologic concepts and methods. +++ History: Semmelweis and Childbed Fever ++ The shining example of the fundamental importance of epidemiology in detection and control of nosocomial infections is the work of Ignaz Semmelweis, which preceded the microbiologic discoveries of Pasteur and Koch by a decade. Semmelweis was assistant obstetrician at the Vienna General Hospital, where more than 7000 infants were delivered each year. Childbed fever (puerperal endometritis), which we now know is caused primarily by group A streptococci, was a major problem accounting for 600 to 800 maternal deaths per year. By careful review of hospital statistics between 1846 and 1849, Semmelweis clearly showed that the death rate in one of the two divisions of the hospital was 10 times that in the other. Division I, which had the high mortality rate, was the teaching unit in which all deliveries were by obstetricians and students. In division II, all deliveries were by midwives. No similar epidemic existed elsewhere in the city of Vienna, and the mortality rate was very low in mothers delivering at home. ++ Childbed fever was associated with obstetricians on teaching unit Midwife and home births had lower rates ++ Semmelweis postulated that the key difference between divisions I and II was participation of the physicians and students in autopsies. One or more cadavers were dissected daily, some from cases of childbed fever and other infections. Handwashing was perfunctory, and Semmelweis believed this allowed the transmission of “invisible cadaver particles” by direct contact between the mother and the physician’s hands during examinations and delivery. In 1847, as a countermeasure, he required handwashing with a chlorine solution until the hands were slippery and the odor of the cadaver was gone. The results were dramatic. The full effect of the chlorine handwashing can be seen by comparing mortality rates in the two divisions for 1846 and 1848 (Table 3–2). The mortality rate in division I was reduced to that of division II, and both were lower than 2%. ++Table Graphic Jump LocationTABLE 3–2Childbed Fever at the Vienna General HospitalView Table| Favorite Table |Download (.pdf) TABLE 3–2 Childbed Fever at the Vienna General Hospital DIVISION I (TEACHING UNIT) DIVISION II (MIDWIFE UNIT) YEAR BIRTHS MATERNAL DEATHS PERCENTAGE BIRTHS MATERNAL DEATHS PERCENTAGE 1846a 4010 459 11.4 3754 105 2.7 1848b 3556  45  1.3 3219  43 1.3 aNo handwashing.bFirst full year of chlorine handwashing. ++ Transmission from cadavers was suspected Disinfectant handwashing reduced the infection rates

Killing of bacteria by heat, radiation, or chemicals is usually exponential with time; that is, a fixed proportion of survivors are killed during each time increment. Thus, if 90% of a population of bacteria are killed during each 5 minutes of exposure to a weak solution of a disinfectant, a starting population of 106/mL is reduced to 105/mL after 5 minutes, to 103/mL after 15 minutes, and theoretically to one organism (100)/mL after 30 minutes. Exponential killing corresponds to a first-order reaction or a “single-hit” hypothesis in which the lethal change involves a single target in the organism, and the probability of this change is constant with time. Thus, plots of the logarithm of the number of survivors against time are linear (Figure 3–1A); however, the slope of the curve varies with the effectiveness of the killing process, which is influenced by the nature of the organism, lethal agent, concentration (in the case of disinfectants), and temperature. In general, the rate of killing increases exponentially with arithmetic increases in temperature or in concentrations of disinfectant. If the microbial population includes a small proportion of more resistant forms (spores), the later stages of the curve may be flattened (Figure 3–1B), and extrapolations from the exponential phase of killing may underestimate the time needed for achieving complete sterility. ++ FIGURE 3–1. Kinetics of bacterial killing. A. Exponential killing is shown as a function of population size and time. B. Deviation from linearity, as with a mixed population, extends the time. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Bacterial killing follows exponential kinetics Heterogeneous micr

From the time of debates about the germ theory of disease, killing microbes before they reach patients has been a major strategy for preventing infection. In fact, Ignaz Semmelweis successfully applied disinfection principles decades before bacteria were first isolated. This chapter discusses the most important methods used for this purpose in modern medical practice. Understanding how these methods work has become of increasing importance in an environment that includes immunocompromised patients, transplantation, indwelling devices, and Covid-19. + DEFINITIONS Download Section PDF Listen +++ ++ Death/killing as it relates to microbial organisms is defined in terms of how we detect them in culture. Operationally, it is a loss of ability to multiply under any known conditions. This is complicated by the fact that organisms that appear to be irreversibly inactivated may, sometimes, recover when appropriately treated. For example, ultraviolet (UV) irradiation of bacteria can result in the formation of thymine dimers in the DNA with loss of ability to replicate. A period of exposure to visible light may then activate an enzyme that breaks the dimers and restores viability by a process known as photoreactivation. In addition, mechanisms exist for repair of the damage without light. Such considerations are of great significance in the preparation of safe vaccines from inactivated virulent organisms. ++ Absence of growth does not necessarily indicate sterility ++ Sterilization is an absolute term. It means complete killing, or removal, of all living organisms from a particular location or material. It can be accomplished by incineration, nondestructive heat treatment, certain gases, exposure to ionizing radiation, some liquid chemicals, and filtration. ++ ❋ Sterilization is killing of all living forms ++ Pasteurization is the use of heat at a temperature sufficient to inactivate important pathogenic organisms in liquids such as water or milk, but at a temperature lower than that needed to ensure sterilization. For example, heating milk at a temperature of 74°C for 3 to 5 seconds or 62°C for 30 minutes kills the vegetative forms of most pathogenic bacteria that may be present without altering its quality. Obviously, spores are not killed at these temperatures. ++ ❋ Pasteurization uses heat to kill vegetative forms of bacteria ++ Disinfection is a less precise term. It implies the destruction of pathogenic microorganisms by processes that fail to meet the criteria for sterilization. Pasteurization is a form of disinfection, but the term is most commonly applied to the use of liquid chemical agents known as disinfectants, which usually have some degree of selectivity. Bacterial spores, organisms with waxy coats (eg, mycobacteria), and some viruses may show considerable resistance to the common disinfectants. Antiseptics are disinfecting agents that can be used on body surfaces, such as the skin or vaginal tract, to reduce the numbers of pathogenic agents in the local microbiota. They have lower toxicity than disinfectants used environmentally, but are usually less active in killing vegetative organisms. Sanitization is an even less precise term with a meaning somewhere between disinfection and cleanliness. It is used primarily in housekeeping and food preparation contexts. ++ Disinfection uses chemical agents to kill pathogens with varying efficiency Spores are particularly resistant ++ Asepsis describes working systems designed to prevent microorganisms from reaching a protected environment. It is manifest in the multiple procedures used in the operating room, in the preparation of therapeutic agents, and in technical manipulations in the microbiology laboratory. An essential component of aseptic techniques is the prior sterilization of all materials and equipment to be used. ++ Asepsis applies sterilization and disinfection to create a protective environment

NATURAL IMMUNITY TO INFECTION ++ The majority of encounters with microorganisms including pathogens end favorably for the host. The heightened immunologic responses following infection usually provide immunity, often for life. This is called natural immunity. In some instances, the gauntlet is long because a pathogen of the same name may exhibit diverse antigenic profiles. Because of the specificity of the adaptive immune response, immunity must be developed individually for each antigenic type. Development of natural immunity need not require a clinical infection. There is ample evidence from population studies that individuals with no history or recollection of infection have evidence of immunity in the form of specific antibody. From the time of birth forward, we have many encounters with infectious agents, most of which lead to immunity without disease. ++ Natural infection often confers life-long immunity Clinical disease is not required +++ PASSIVE IMMUNITY ++ Passive immunity is the transfer of antibodies from one person to another. Because the antibody was not made by the recipient, this antibody is transient and lasts only a few weeks or months. This is a natural process in the case of IgG transferred transplacentally from mother to fetus. The protection provided by this antibody is limited to the immunologic experience of the mother, but covers a particularly vulnerable time in life, lasting as long as 6 months after birth. Passive immunity can also be provided as a therapeutic product in which specific antibodies are infused. Such antisera are available for only a limited number of diseases such as rabies, botulism, and tetanus. ++ Transplacental IgG protects the fetus +++ VACCINES ++ Vaccines artificially stimulate immunity through exposure to an antigenic substance. The early vaccines such as Jenner’s for smallpox and Pasteur’s for anthrax (in animals) were live attenuated strains with the ability to produce a true, if mild, infection. We later learned how to kill the agent in a way that retained its antigenicity. These killed vaccines are practical if the number of antigens present is limited as with a virus (polio) or bacterial toxin (diphtheria), but usually too crude if whole bacteria are used. Progress with killed bacterial vaccines required knowledge of just which antigenic component provides protective immunity. This allowed inactivation followed by purification of the selected component. This approach with bacterial polysaccharide capsules has produced a dramatic reduction (>95%) in childhood meningitis. Genomic approaches are now aimed at producing a protective antigen without growth of the organism itself. For each of the 57 chapters in this book devoted to specific infectious agents, vaccines and the immunologic mechanisms involved are carefully examined.

e immune system is no different from any other human system. In balance, we do not even know it is there, but in an exaggerated state called hypersensitivity, it can cause injury and even chronic disease. Hypersensitivity reactions have been placed into four classes on the basis of their mechanism of immunologic injury. Type I or allergic reactions relate to the action of IgE and the release of powerful mediators, such as histamine from mast cells. Type II or cytotoxic reactions are created when IgG or IgM antibodies are misdirected to host cells. Type III or immune complex reactions are created when an excess of antigen–antibody complexes are deposited and followed by complement-mediated inflammation. Type IV reactions are cell-mediated and often called delayed-type hypersensitivity (DTH) because of the time delay in invoking the TH1 response. The hypersensitivity diseases include allergy, anaphylaxis, asthma, transfusion reactions, rheumatoid arthritis, and type 1 diabetes. Infectious diseases are a relatively small part of this spectrum, but involve three of the four mechanisms (II, III, and IV). ++ Mechanisms I-IV involve antibody and cell-mediated injury Allergy, asthma, and diabetes due to hypersensitivity Infection a small part +++ ANTIBODY-MEDIATED (TYPE II) HYPERSENSITIVITY ++ Type II hypersensitivity is antibody-dependent cytotoxicity that occurs when antibody binds to antigens on host cells, leading to phagocytosis, cytotoxic T-cell activity, or complement-mediated lysis. The cells to which the antibody is specifically bound, as well as the surrounding tissues, are damaged because of the inflammatory amplification. In the best-understood situations related to infection, the mechanism of antibody stimulation is molecular mimicry. That is, the antibody stimulated by an epitope on the pathogen, unfortunately, also binds to a similar epitope on host cells. In rheumatic fever, the infectious epitope is in a surface protein of the group A streptococcus and the host epitope in the myocardium of the heart (see Chapter 25). The streptococcal protein and cardiac myosin share similar amino acid sequences; therefore, it is a cross-reaction. The result is acute myocarditis. ++ Antibody against microbe epitope also reacts with host cells Rheumatic fever is caused by molecular mimicry +++ IMMUNE COMPLEX (TYPE III) HYPERSENSITIVITY ++ When IgG is mixed in appropriate proportions with multivalent antigen molecules (ie, bearing multiple epitopes), aggregates of many antigen and antibody molecules may form. These antigen–antibody complexes can occur in infection when sufficient amounts of specific antibody and free antigen from an infecting microorganism combine to form an immune complex. These complexes are usually removed by cells of the monocyte-macrophage system, but, in excess, can circulate and become deposited in blood vessels, kidneys, or joints. When deposited, they bind complement and stimulate an inflammatory reaction that may injure the local tissue. This is postulated to be the mechanism of poststreptococcal acute glomerulonephritis (see Chapter 25), and is suspected to be responsible for some of the manifestations when microorganisms circulate in the bloodstream. ++ Excess antigen–antibody complexes are deposited in tissues ++ In the past, an immune complex disease called serum sickness used to follow the infusion of antibodies (antisera) produced in horses to combat infection. Human antibody to the foreign horse immunoglobulin was formed. These diseases (diphtheria, tetanus) are now prevented by vaccines that stimulate antibody against the same epitopes in humans. When passive immunization is used, human sources of antibody are now available. ++ Complement-mediated inflammation causes injury Serum sickness is reaction to animal immunoglobulin +++ DELAYED-TYPE (TYPE IV) HYPERSENSITIVITY ++ Type IV DTH is a cell-mediated immune reaction. The delay is the time required after initiation of a TH1 response for antigen to be processed, cytokines produced, and T cells to migrate and accumulate at the antigen site. At the site, cytotoxic T cells, macrophages, and other inflammatory mediators directed at cells containing the antigen also produce injury in the surrounding tissue. The purest form of DTH is the intradermal skin test for tuberculosis. In persons already sensitized to the antigens of M tuberculosis, it takes 1 to 2 days for induration to be produced at the site of inoculation of a standardized antigen called tuberculin. This is a useful diagnostic test, but, in infectious disease, DTH is also the hypersensitivity mechanism that causes the most injury. This occurs in diseases in which immunity is cell-mediated with little or no effective antibody component. If these responses are successful in containing the infection at an early stage, there is little destruction. If they are not successful enough to contain growth of the pathogen, increasing amounts of antigen stimulate continuing DTH-mediated destructive inflammation. This is the primary mechanism of injury in tuberculosis, fungal infections, and many parasitic diseases. ++ DTH requires time for TH1 response to develop

THE ADAPTIVE (SPECIFIC) IMMUNE SYSTEM Download Section PDF Listen +++ ++ The adaptive immune system differs from the innate immune response in its discrimination between self and nonself and in the magnitude and diversity of the highly specific immune responses that are engendered (Table 2–3). In addition, it has a memory function, which is able to mount an accelerated response if an invader returns. The adaptive system operates via two broad arms—humoral immunity and cell-mediated immunity. Humoral immunity comes from bone marrow-derived B cells and is exemplified by antibodies that are produced to bind foreign molecules called antigens. Cell-mediated (cellular) immunity is mediated through T cells that mature in the thymus and respond to antigens by directly attacking infected cells or by secreting cytokines to activate other cells. As shown in Figure 2–8, B-cell and T-cell systems are interactive. ++Table Graphic Jump LocationTABLE 2–3Cells Involved in the Adaptive Immune SystemView Table| Favorite Table |Download (.pdf) TABLE 2–3 Cells Involved in the Adaptive Immune System CELL FUNCTION SPECIFIC RECEPTORS FOR ANTIGEN CHARACTERISTIC CELL-SURFACE MARKER SPECIAL CHARACTERISTICS B cells Production of antibody Surface immunoglobulin (IgM monomer) Fc and complement C3d receptors; MHC class II Differentiate into plasma cells Helper T lymphocytes (TH) Stimulate macrophages, eosinophils, PMNs, IgE production, B cells α/β T-cell receptor (TCR) CD4+ Presented by MHC class II, Three subsets (TH1, TH2, TH17) Cytotoxic T lymphocytes (CTLs) Lyse antigen-expressing cells such as virally infected cells or allografts α/β TCR CD8+ Presented by MHC class I Natural killer (NK) cells Spontaneous lysis of tumor and infected cells Inhibitory; activating Fc receptor for IgG Recognize MHC class I Macrophages (monocytes) Phagocytosis, secretion of cytokines to activate T cells (eg, IL-1) or other accessory cells such as polymorphonuclear neutrophils (PMNs)c None, but can be “armed” by antibodies binding to Fc receptors Macrophage surface antigens Express surface receptors for the activated third component of complement (C3), kill ingested bacteria by oxidative bursts Polymorphonuclear leukocytes (neutrophils, eosinophils) Phagocytosis killing None, but can be “armed” by antibodies   Protective in bacterial and parasitic (eosinophils) infections MHC, major histocompatibility complex. ++ FIGURE 2–8. Acquired immune system development. A. Lymphocyte stem cells develop into B- and T-cell precursors that migrate to the bone marrow or thymus, respectively. Mature B and T cells seed secondary lymphoid tissues. B. Lymphocyte receptor binding of antigen activates B and T cells to become effector cells. C. B lymphocytes develop into memory cells and antibody-secreting plasma cells. D. T cells develop into memory cells, helper T cells, and cytotoxic T cells. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) +++ Antigens and Epitopes ++ An antigen is any substance (usually foreign) with the ability to stimulate an immune response when presented in an effective fashion. They are usually large structurally complex molecules, such as proteins, polysaccharides, or glycolipids. Each antigen can contain many sub-regions that are the actual antigenic determinants or epitopes. These epitopes can consist of separate peptides, carbohydrates, or lipids of the correct size and three-dimensional configuration to fit the combining site of an antibody molecule or a T-cell receptor (TCR) (Figure 2–9). Approximately six amino acids or monosaccharide units provide a correctly sized epitope. Antigens presented by infectious agents typically contain multiple epitopes, including copies of the same epitope. Thus, a single microbially derived molecule presents multiple opportunities for diverse antibody binding. Other, smaller molecules that may ordinarily not stimulate an immune response (haptens) may do so if bound to a larger carrier, such as a protein. The specificity of the immune response may be generated for both the hapten and its larger carrier. ++ FIGURE 2–9. Epitopes. Schematic of epitope recognition by an immunoresponsive lymphocyte. Epitope B on the antigen binds to a complementary recognition site on the surface of the immunoresponsive cell. Antigens may have many different epitopes, but an immunoresponsive lymphocyte has receptors of only one specificity. In most cases, epitopes are recognized on the surface of macrophages that have processed the antigen. The receptor for antigens on B cells is the combining site of the surface immunoglobulin. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Antigens stimulate immune response Epitopes fit to the combining site of TCR and antibodies ++ A foreign antigen entering a human host may, by chance, encounter a B cell whose surface antibody is able to bind it. This interaction stimulates the B cell to multiply, differentiate, and produce more surface and soluble antibodies of the same specificity. Eventually, the process leads to production of enough antibody to bind more of the antigen. This mechanism is most likely to operate with antigens such as polysaccharides that have repeating subunits, thus improving the possibility that exposed epitopes are recognized. ++ B cells multiply and produce antibody ++ Large, complex antigens such as proteins and viruses must be processed before their epitopes can be effectively recognized by the immune system. This processing takes place in macrophages or specialized epithelial cells found in the skin and lymphoid organs, where they are adjacent to other immunoresponsive cells. The ingested antigen is degraded to peptides of 10 to 20 amino acids that are presented by major histocompatibility molecules on the host cell surface to be recognized by T cells (Figures 2–10, 2–11). ++ FIGURE 2–10. MHC class I and II molecules. A. The class I molecule is a heterodimer composed of the alpha protein, which is divided into three domains: α1, α2, and α3, and the protein β2 microglobulin. B. The class II molecule is a heterodimer composed of two distinct proteins called alpha and beta. Each is divided into two domains α1, α2 and β1, β2, respectively. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ FIGURE 2–11. Antigen processing and presentation. A. Antigens originating in the cytoplasm are digested by the proteasome to peptides. The peptides are bound to the MHC class I molecules in the endoplasmic reticulum (ER) and transported to the surface for presentation. B. Antigens originating outside the cell are endocytosed and digested in the phagolysosome. The digested peptides are bound to MHC class II molecules in the ER and transported to the surface for presentation. MHC, major histocompatibility complex. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Protein antigens must be processed first +++ Recognition of Foreignness ++ Distinguishing between self and nonself is obviously essential to maintaining organism integrity and homeostasis. The compendium of molecules that control these functions is called the major histocompatibility complex (MHC), and it is present on the surface of almost all human cells. Of interest in infection are MHC class I and II molecules (Figure 2–10). MHC class I molecules are in the membrane of almost all cells, but MHC class II molecules are present only on certain leukocytes such as macrophages, dendritic cells, and some T and B cells. ++ MHC gene complex codes surface molecules MHC II on macrophages, dendritic cells ++ Both MHC class I and class II participate in antigen processing but by distinctly different pathways (Figure 2–11). MHC class I molecules bind to products generated in the cytoplasm by a natural process or a viral infection. Viral proteins are digested to peptides in a cytoplasmic structure called the proteasome, and delivered to the endoplasmic reticulum. Here they find the binding site of the class I molecule and are transported to the surface for presentation of the peptide. MHC class II molecules bind to fragments that originally come from outside the cell, but have been taken into the endocytic vacuole of a phagocyte. After digestion in the phagolysosome, peptide fragments are combined with class II molecules and move to the surface for presentation. The presented MHC class I peptides are recognized by CD8+ T cells and the MHC class II by CD4+ T cells. ++ MHC I presents cytoplasmic peptides to CD8+ MHC II presents foreign peptides to CD4+ +++ THE T-CELL RESPONSE ++ T cells originate in the bone marrow and migrate to the thymus for differentiation. Those that recognize self are destroyed. Those that survive are mature but require activation. T cells have specific TCRs on their surface, with binding sites extending to the external milieu (Figure 2–12). The two major types of T cells are helper T (CD4+) and cytotoxic (CD8+) T cells. The major roles of T cells in the immune response are as follows: ++ Recognition of peptide epitopes presented by MHC molecules on cell surfaces. This is followed by activation and clonal expansion of T cells in the case of epitopes associated with class II MHC molecules. Production of cytokines that act as intercellular signals and mediate the activation and modulation of various aspects of the immune response and of nonspecific host defenses. Direct killing of foreign cells, of host cells bearing foreign surface antigens along with class I MHC molecules (eg, some virally infected cells), and of some immunologically recognized tumor cells. ++ FIGURE 2–12. T-cell receptor and helper T activation. A. Structure of the T-cell antigen receptor. B. An antigen-presenting cell begins the activation process by displaying a peptide antigen fragment in its MHC class II molecule. A helper T cell is activated after the variable region of its receptor (Vα, Vβ) reacts with the fragment. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) +++ CD4+ Helper T Lymphocytes ++ Helper T cells are stimulated by antigen in the context of MHC class II presentation and are further marked by the presence of the CD4 cell surface antigen. If T cells are of the proper MHC background to recognize the antigen specifically, T-cell activation occurs. The antigen–MHC complex presented to a specific T cell by the macrophage is the specific signal that induces the T cell to become activated and divide. At this point, the T helper (Th) cells differentiate into three major subsets of effector cells each with characteristic cytokines, target cells, and typical microbial pathogen profiles. Th1 cells produce IFN-γ, target macrophages, and are effective against intracellular pathogens like Mycobacterium tuberculosis. Th2 cells produce multiple IL, and promote IgE, mast cell, and eosinophil-mediated destruction of parasites. Th17 cells also produce IL including IL-17, stimulate neutrophils, and are active against extracellular bacterial and fungal pathogens. ++ Helper T cells activated by specific antigens Subsets active against intracellular, extracellular, and parasitic pathogens +++ CD8+ Cytotoxic T Lymphocytes ++ CD8+ cytotoxic T lymphocytes (CTLs) are a second class of effector T cells. They are lethal to cells expressing the epitope against which they are directed when the epitope is presented by class I MHC molecules. They too have specific epitope recognition sites, but they are characterized by the CD8 cell surface marker; thus, they are referred to as CD8+ cytotoxic T cells. These cells recognize the association of antigenic epitopes with class I MHC molecules on a wide variety of cells of the body. In the case of virally infected cells, cytotoxic CD8+ cells prevent viral production and release by eliminating the host cell before viral synthesis or assembly is complete (Figure 2–13). The destruction of the virally infected cell is accomplished through a complement-like action mediated by perforins, which also facilitates entry into the cell of enzymes (granzymes) that activate apoptosis. ++ FIGURE 2–13. Cytotoxic T-cell (CTL) destruction virus-infected cells. A. Naïve CD8+ T cells are activated when they are exposed to antigen within a class I MHC molecule on an antigen-presenting cell. Antigen activation leads to development of effector CTL and memory cells. Effector CTLs and their memory cells subsequently react with antigen expressed in class I MHC molecules of any host cell to destroy it. T-cell cytotoxicity often involves the perforin pathway and leads to apoptosis or cytolysis. MHC, major histocompatibility complex. B. CTL (left) contacting target cell (right). C. Perforins form pores in target cell membrane. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ CD8+ lymphocytes react with MHC I Eliminate virally infected cells +++ Superantigens ++ A group of antigens have been termed superantigens because they stimulate a much larger number of T cells than would be predicted based on the specificity of combining site diversity. This causes a massive cytokine release. The action of superantigens is based on their ability to bind directly to MHC proteins and to particular Vβ regions of the TCR without involving the antigen-combining site. Individual superantigens recognize exposed portions defined by framework of residues that are common to the structure of one or more Vβ regions. Any T cells bearing those Vβ sites may be directly stimulated. A variety of microbial products have been identified as superantigens. Superantigens are discussed further in Chapter 22 (see Figure 22–7) and in Chapters 24 and 25, describing their role in toxic shock syndromes caused by Staphylococcus aureus and group A streptococci. ++ Superantigens bind directly to MHC proteins and TCR Vβ region Higher proportion of T cells are stimulated +++ Cell-Mediated Immunity ++ In the control of infection, cell-mediated immunity is most important in response to obligate facultative intracellular pathogens. These include some slow-growing bacteria, such as the mycobacteria and fungi against which antibody responses appear to be ineffective. The mechanisms are complex and involve a number of cytokines with amplifying feedback mechanisms for their production. After the initial processing of antigen to stimulate activation of the antigen-recognizing CD4+ T cell, cytokine feedback from the CD4+ T cells to macrophages further increases their clonal expansion (including memory cells) and activates CD8+ (cytotoxic) T lymphocytes. Other cytokines from CD4+ T cells attract macrophages to the site of infection, and activate them to greatly enhance microbiocidal activity. The sum of the individual and collaborative activities of T cells, macrophages, and their products is a progressive mobilization of a range of host defenses to the site of infection and greatly enhanced macrophage activity. In the case of tuberculosis, IFN-γ inhibits the replication of the mycobacteria inside macrophages. In viral infections, CD8+ cytotoxic lymphocytes destroy their cellular habitat leaving already assembled virions accessible to circulating antibody. ++ Of primary importance with intracellular pathogens Helper and CTL interact Macrophages are mobilized and enhanced +++ B CELLS AND ANTIBODY RESPONSES ++ B lymphocytes are the cells responsible for antibody responses. They develop from precursor cells in the bone marrow before migrating to other lymphoid tissues. Each mature cell of this series carries a specific epitope recognition site on its surface. This B-cell receptor is actually a monomer of one form of antibody (IgM) oriented with its binding sites facing outward. Upon binding antigen, the receptor-antigen complex is internalized for initiation of antibody production by the stimulated B cell. In this process, the B lymphocytes multiply, differentiating into either memory or plasma cells. Plasma cells are end cells adapted for secretion of large amounts of antibodies. In addition to their essential role in antibody production, B cells can present antigen to T cells. ++ B cells carry epitope recognition sites on their surface Stimulated cells differentiate to form memory, plasma cells ++ There are two broad types of antigen triggering: T-dependent and T-independent. T-dependent reactions are those that use collaboration between helper T cells and B cells to initiate the process of antibody production. This is the mechanism evoked by proteins and haptens bound to proteins. The response is strong and includes memory cells; therefore, it can be boosted in the case of immunization. ++ T-dependent has memory ++ T-independent responses are those that do not require help from T cells to stimulate B-cell antibody production. It is evoked by large molecules with many repeating units such as polysaccharides which cannot bind to MHC molecules. At first glance, this independence may seem to be an advantage, but T-independent responses are not the same as T-dependent responses. The antibody generally has a lower affinity for its antigen and a shorter duration in circulation. Memory cells are not produced, and T-independent responses mature more slowly than T-dependent responses. This delay in maturation may contribute to the increased susceptibility to some bacterial infections in early life. It certainly contributed to the failure of the first batch of purified polysaccharide vaccines to effectively immunize children younger than 2 years. For use in children, these vaccines have been replaced with a hapten approach in which the polysaccharide is conjugated to protein. In this form, antibody generated by the T-dependent mechanism (protein carrier) still has specificity for the polysaccharide epitopes. ++ T-cell independent responses are weaker and lack memory Poor response under 2 years of age ++ After challenge with foreign antigen, there is a lag period of 4 to 6 days before antibody can be detected in serum. This period reflects the events involved in the recognition of the antigen, its processing, and the specific activation of the cells of the immune system. The first event is the clearance of antigen from the circulation by what is essentially a metabolic process in which the antigen is recognized in a nonspecific sense and ingested. The vast preponderance of antigen ends up in circulating phagocytes or in stationary macrophages. The macrophages process the antigen; therefore, those immunogenic moieties can be presented to T cells, which then cause the B cells to produce immunoglobulins. The antibody-forming system is a learning system that responds to challenge by foreign molecules by producing large amounts of specific antibody. In addition, the affinity of its binding to the specifically recognized antigen often increases with time or secondary challenge. ++ Antigen processing causes delay in antibody response Learning system increases affinity with time or secondary challenge +++ Antibody Structure ++ Antibodies belong to the immunoglobulin family of proteins, which appear in quantity in serum and on the surfaces of B cells. Of the five known structural types, three (IgG, IgM, and IgA) are involved in the defense against infection. The basic structure of an immunoglobulin is illustrated in Figure 2–14, which depicts an IgG molecule. Immunoglobulins have a basic tetrameric structure consisting of two light polypeptide chains and two heavy chains usually associated as light/heavy pairs by disulfide bonds. The two light/heavy pairs are covalently associated by disulfide bonds to form the tetramer. There are two types of light chains, κ and λ, which are the products of distinct genetic loci. The class or isotype of the immunoglobulin is defined by the type of heavy chain expressed. ++ FIGURE 2–14. Immunoglobulin G structure. The IgG molecule consists of two identical light chains and two identical heavy chains held together by disulfide bonds. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Immunoglobulin structure combines light and heavy chains Isotypes defined by type of heavy chain ++ The Y-shaped structure includes two antigen binding sites (Fab) formed by interaction of the variable domains of the heavy chain and the light chain. The stalk is called the Fc fragment. Antibodies carry out two broad sets of functions: the recognition function is the property of the Fab sites for antigen, and the effector functions are mediated by the constant regions of the heavy chains. Variations in the hypervariable region of the Fab-combining site due to mutations are called idiotypes. Antibodies combine with foreign antigens, but the actual destruction or removal of antigen requires the interaction of portions of the Fc fragment with other molecules such as complement components and phagocytes which have Fc receptors. ++ Fab sites bind antigen Fc fragment recognized by complement, phagocytes Combining site is idiotype ++ Figure 2–15 shows a schematic representation of a serum IgM immunoglobulin. This molecule consists of five subunits of the typical IgG molecule. The molecule occurs as a cyclic pentamer, and a J (joining) chain links the intact structure. When IgM is present on the surface of B cells where it serves as a primary receptor for antigen, it is present as a monomer. Other immunoglobulins showing a difference in arrangement from the typical IgG model are the IgA immunoglobulins. In serum, these immunoglobulins can occur as a monomer, but they can also occur in dimers in which the joining chain is required to stabilize the dimer. IgA molecules in the gut occur as dimers in which both the J chain and an additional polypeptide, termed the secretory component, are present in the complex. ++ FIGURE 2–15. Immunoglobulin M structure. The pentameric structure has disulfide bonds linking peptide chains shown in black; carbohydrate side chains are in red. The J chain links the molecule together. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Fab is antigen-binding region IgM has five subunits IgA a monomer or dimer +++ Functional Properties of Immunoglobulins +++ Immunoglobulin G ++ Immunoglobulin G (IgG) is the most abundant immunoglobulin in health and provides the most extensive and long-lived antibody response to the various microbial and other antigens that are encountered throughout life. Although at least four subclasses of IgG have been characterized, they are grouped together for the purpose of this chapter. The IgG molecule is bivalent with two identical and specific combining sites. The Fc region does not vary with differences in specificity of combining sites of different antibody molecules. The Fc fragment binding sites for phagocytic cells are made available when the variable region of the antibody molecule has reacted with specific antigen, leaving the Fc facing outward. ++ Bivalent with specific combining site and constant region Constant region binds phagocytes ++ IgG antibody is characteristically formed in large amounts during the secondary response to an antigenic stimulus, and usually follows production of IgM (see Immunoglobulin M) in the course of a viral or bacterial infection. Memory cells are programmed for rapid IgG response when another antigenic stimulus of the same type occurs later. IgG antibodies are the most significant antibody class for neutralizing bacterial exotoxins and viruses often by blocking their attachment to cell receptors. Accelerated IgG responses from memory cell expansion frequently confer lifelong immunity when directed against microbial antigens that are determinants of virulence. IgG is the only immunoglobulin class able to cross the placental barrier and, thus, it provides passive immune protection to the newborn in the form of maternal antibody. ++ Secondary response antibodies neutralize toxins, viruses Binding may block attachment receptor +++ Immunoglobulin M ++ Monomers of immunoglobulin M (IgM) constitute the specific epitope recognition sites on B cells that ultimately give rise to plasma cells producing one or another of the different immunoglobulin classes of antibody. Because of its many specific combining sites, IgM is particularly effective in agglutinating particles carrying epitopes against which it is directed. It also contains many sites for binding the first component of complement. These sites become available once the IgM molecule has reacted with antigen. IgM is particularly active in bringing about complement-mediated cytolytic damage to foreign antigen-bearing cells. It is less effective as an opsonizing antibody because its Fc portion is not available to phagocytes. ++ Effective agglutinating antibody Binds complement at multiple sites +++ Immunoglobulin A ++ Immunoglobulin A (IgA) has a special role as a major determinant of so-called local immunity in protecting epithelial surfaces from colonization and infection. Certain B cells in lymphoid tissues adjacent to, or draining surface epithelia of the intestines, respiratory tract, and genitourinary tract, are encoded for specific IgA production. After antigenic stimulus, the clone expands locally, and some of the IgA-producing cells also migrate to other viscera and secretory glands. At the epithelia, two IgA molecules combine with another protein, termed the secretory piece, which is present on the surface of local epithelial cells. The complex, then termed secretory IgA (sIgA), passes through the cells into the mucous layer on the epithelial surface or into glandular secretions, where it exerts its protective effect. The secretory piece not only mediates secretion but also protects the molecule against proteolysis by enzymes such as those present in the intestinal tract. ++ sIgA is produced at mucosal surfaces Secretory piece combines molecules, resists proteolysis ++ The major role of sIgA is to prevent attachment of antigen-carrying particles to receptors on mucous membrane epithelia. Thus, in the case of bacteria and viruses, it reacts with surface antigens that mediate adhesion and colonization and prevents the establishment of local infection or invasion of the subepithelial tissues. sIgA can agglutinate particles but has no Fc domain for activating the classic complement pathway; however, it can activate the alternative pathway. Reaction of IgA with antigen within the mucous membrane initiates an inflammatory reaction that helps mobilize other immunoglobulin and cellular defenses to the site of invasion. IgA response to an antigen is shorter lived than the IgG response. ++ Interferes with attachment of microbes to mucosal surfaces +++ Antibody Production ++ The major events characterizing the time course of antibody production are illustrated in Figure 2–16 and summarized as follows: Initial contact with a new antigen evokes the primary response, which is characterized by a lag phase of approximately 1 week between the challenge and the detection of circulating antibodies. In general, the length of the lag phase depends on the immunogenicity of the stimulating antigen and the sensitivity of the detection system for the antibodies produced. Once antibody is detected in serum, the levels rise exponentially to attain a maximal steady state in approximately 3 weeks. These levels then decline gradually with time if no further antigenic stimulation is given. The first antibodies synthesized in the primary immune response are IgM and, then in the latter phase, IgG antibodies arise and eventually predominate. This transition is termed the IgM/IgG switch. ++ FIGURE 2–16. Antibody production and kinetics. The four phases of a primary antibody response correlate to the clonal expansion of the activated B cell, differentiation into plasma cells, and secretion of the antibody protein. The secondary response is much more rapid, and total antibody production is nearly 1000 times greater than that of the primary response. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ After lag, primary response lasts for weeks, then declines IgM response switches to IgG ++ After a subsequent exposure or booster injection of the same antigen, a different sequence called the secondary response or anamnestic response ensues. This response involves memory. In the secondary response, the lag time between the immunization and the appearance of antibody is shortened, the rate of exponential increase to the maximum steady-state level is more rapid, and the steady-state level itself is higher, representing a larger amount of antibody. Another key factor of the secondary response is that the antibodies formed are predominantly of the IgG class. In addition to higher levels, the secondary IgG antibodies have a higher affinity for their antigen. Figure 2–16 shows the participation of memory T cells created during the primary response in these reactions. ++ Secondary response is primarily IgG Affinity for antigen is greater +

INNATE (NATURAL) IMMUNITY Download Section PDF Listen +++ ++ Innate immunity acts through a series of specific and nonspecific mechanisms, all working to create a series of hurdles for the pathogen to navigate (Table 2–1). The first are mechanical barriers such as the tough multilayered skin or the softer but fused mucosal layers of internal surfaces. As discussed in Chapter 1, the microbiota on these surfaces present formidable competition for space and nutrients. Turbulent movement of the mucosal surfaces and enzymes or acid secreted on their surface make it difficult for an organism to efficiently colonize. Organisms that are able to pass the mucosa encounter a population of cells with the ability to engulf and destroy them. In addition, body fluids contain chemical agents such as complement, which can directly injure the microbe. The entire process has cross-links to the adaptive immune system. The endpoint of phagocytosis and digestion in a macrophage is the presentation of the antigen on its surface, the first step in specific immune recognition. ++Table Graphic Jump LocationTABLE 2–1Features of Innate Immunity in InfectionView Table| Favorite Table |Download (.pdf) TABLE 2–1 Features of Innate Immunity in Infection   LOCATION ACTIVITY AGAINST PATHOGENS Cells     Macrophage Circulation, tissues Phagocytosis, digestion Dendritic cell Tissues Phagocytosis, digestion Polymorphonuclear neutrophil (PMN) Circulation, tissues (by migration) Phagocytosis, digestion M cell Mucous membranes Endocytosis and delivery to phagocytes Surface Receptors     Lectin Phagocyte Recognize carbohydrates Arginine-glycine-arginine (RGD) Phagocyte Recognize arginine-glycine-aspartic acid sequence Toll-like receptor (TLR) Phagocyte Recognizes PAMP, such as bacterial LPS (TLR-4), peptidoglycana (TLR-2) Inflammation     Selectins Endothelium Attract and attach PMNs Integrins PMNs Attach to selectins Kallikrein Extracellular fluid Release bradykinin, prostaglandins Chemical Mediators     Cathelicidin PMNs, macrophages, epithelial cells Ionic membrane pores Defensins PMN granules Ionic membrane pores Complement (classical, alternative, lectin) Serum, extracellular fluid Membrane pores, phagocyte receptors LPS, of gram-negative bacterial outer membrane; PAMP, pathogen-associated molecular pattern.aCell wall component of gram-positive and gram-negative bacteria. ++ Skin, mucosa are barriers Cells engulf, digest, and present antigens from microbes +++ PHYSICAL BARRIERS ++ The thick layers of the skin containing insoluble keratins present the most formidable barrier to infection. The mucosal membranes of the alimentary and urogenital tract are not as tough but, often, are bathed in secretions inhospitable to invaders. Lysozyme is an enzyme that digests peptidoglycan—a unique structural component of the bacterial cell wall. Lysozyme is secreted onto many surfaces and is particularly concentrated in conjunctival tears. The acid pH of the vagina and particularly the stomach makes colonization difficult for most organisms. Only small particles (5-10 μm) can be inhaled deep into the lung alveoli because the lining of the respiratory tract includes cilia that trap and move them back toward the pharynx. ++ Lysozyme digests bacterial walls Cilia move particles away from pulmonary alveoli ++ The skin and mucosal surfaces of the intestinal and respiratory tract also contain concentrations of lymphoid tissue within or just below their surfaces, which provide a next-level defense for invaders surviving the above-described barriers. These lymphoid collections are designed to entrap and deliver invaders to some of the phagocytes described in the following text. For example, in the intestine, M cells (Figure 2–2) that lack the villous brush border of their neighbors endocytose bacteria and then release them into a pocket containing macrophages and lymphocytic components (T and B cells) of the adaptive immune system. The enteric pathogen Shigella flexneri exploits this receptiveness of the M cell to attack the adjacent enterocytes from the side. ++ FIGURE 2–2. M cell. An M cell is shown between two epithelial cells in a mucous membrane. It has endocytosed a pathogen and released it into a pocket containing macrophages and other immune cells. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ M cells deliver to macrophages and lymphocytes +++ IMMUNORESPONSIVE CELLS AND ORGANS ++ Not all the cells shown in Figure 2–1 are involved in the immune system; of those that are, not all respond to infection. What the immune-responsive cells have in common is derivation from hematopoietic stem cells in the bone marrow, which create the myeloid and lymphoid series followed by further differentiation into their mature cell types. Of the types shown, the erythroblast and megakaryocyte do not participate in immune reactions. In the myeloid series, basophils and mast cells are primarily involved in allergic reactions rather than infection. Immuno-responsive cells are found throughout the body in the circulation or at fixed locations in tissues. They are concentrated in the lymph nodes and spleen, and form a unified filtration network designed as a sentinel warning system. In the lymphoid series, cells destined to become T cells mature in the thymus (the source of their name). Thus, the thymus, spleen, and lymph nodes might be thought of as the organs of the immune system. These are collectively referred to as the lymphoid tissues. ++ Stem cells differentiate to myeloid and lymphoid series Thymus, spleen, and lymph nodes are immune organs +++ Cellular Receptors for Microbes ++ Fixed and circulating phagocytes express surface receptors which recognize a limited array of uniquely microbial structures based on the pattern of their molecular structure. These Pathogen-Associated Molecular Patterns (PAMPs) include bacterial cell wall peptidoglycan, the lipopolysaccharide (LPS, also called endotoxin) of Gram-negative bacteria, mannose and other glycoproteins, lipids, and polysaccharides. Nucleic acids are also recognized such as the double-stranded RNA found in many viruses. These PAMP-recognizing receptors may be found on the surface of phagocytes, dendritic cells, and specialized compartments called toll-like receptors (TLRs), of which 10 types have been described in humans and 12 in mice. The engagement of TLR receptors potentiates a signaling cascade that ultimately elicits the production of diverse antimicrobial cytokines specific to the TLR type. ++ Surface receptors recognize uniquely microbial PAMPs Cytokine production triggered by TLRs +++ Antimicrobial Peptides ++ Antimicrobial peptides (AMPs) are small peptide molecules with natural antimicrobial effects. In mammals there are two major families of AMPs called cathelicidins and defensins. They are produced by multiple cell types including leukocytes, mast cells, dendritic cells, and platelets in response to tissue damage. They exhibit broad-spectrum activity against bacteria, fungi, parasites, and some enveloped viruses. Their antimicrobial action is by electrostatic interaction with microbial outer membranes, cytoplasmic membranes, and cell walls; these interactions result in membrane rupture, electrostatic potential disruption, and consequently, cell death. Some AMPs may also disrupt metabolic processes like nucleic acid and protein synthesis. ++ Cathelicidins and defensins bind and disrupt microbial surfaces +++ Cells Responding to Infection +++ Monocytes ++ Monocyte is a general morphologic term for cells that include or quickly (within hours) differentiate into macrophages or dendritic cells. These are the cells of the immune system that both phagocytose invaders and process them for presentation to the adaptive immune system. Macrophages are found in the circulation and tissues, where they are sometimes given regional names such as alveolar macrophage. They possess surface receptors rich in mannose and fructose, which nonspecifically recognize components commonly found on pathogens and more specialized receptors able to recognize unique components of microbes such as the LPS of Gram-negative bacteria. They also have receptors that recognize antibody and complement. ++ Macrophages in circulation or tissues Surface receptors recognize pathogens ++ Dendritic cells have a distinctive star-like morphology, and are present in the skin and in the mucous membranes of the respiratory and intestinal tracts. Similar to macrophages, they phagocytose and present foreign antigens. They can also recognize PAMPs. After binding and phagocytosis, dendritic cells migrate to lymphoid tissues where specific adaptive immune responses are triggered. This interaction involving lymphocytes and T cells functions as a bridge between the innate and adaptive immune systems. ++ Star-like tissue phagocytes recognize PAMPs In lymphoid tissues interact with adaptive immunity +++ Granulocytes ++ Of the cells in the granulocyte series, the most active is the polymorphonuclear neutrophil or PMN. These cells have a distinctive multilobed nucleus and cytoplasmic granules that contain lytic enzymes and antimicrobial substances including peroxidase, lysozyme, defensins, collagenase, and cathelicidins. PMNs have surface receptors for antibody and complement and are active phagocytes. In addition to the digestive enzymes, PMNs have other oxygen-dependent and oxygen-independent pathways for killing microorganisms. Unlike macrophages, they are largely circulatory and are not present in tissues except via migration as part of an acute inflammatory response. ++ PMNs have digestive and killing pathways In circulation unless they migrate in inflammation ++ Eosinophils are nonphagocytic cells that participate in allergic reactions along with basophils and mast cells. Eosinophils are also involved in the defense against infectious parasites by releasing peptides and toxic reactive oxygen intermediates into the extracellular fluid, which are postulated to be damaging to parasite membranes. ++ Eosinophils damage parasites +++ Lymphocytes ++ Lymphocytes are the primary effector cells of the adaptive immune system. They are produced from a lymphocyte stem cell in the bone marrow and leave in a static state marked to become T, B, or null cells after further differentiation (Figure 2–3). This requires activation mediated by surface binding, which then stimulates further replication and differentiation. ++ FIGURE 2–3. B and T lymphocytes. B cells and T cells arise from the same cell lineage but diverge into two functional types. Immature B cells and T cells are indistinguishable by morphology. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ T, B, and null cells initially static ++ B cells mature in the bone marrow and then circulate in the blood to lymphoid organs. At these sites, they may become activated to a form called a plasma cell, which produces antibodies. T cells mature in the thymus and then circulate awaiting activation. Their activation results in production of cytokines, which are effector molecules for multiple immunocytes and somatic cells. Some of the uncommitted null cells become natural killer (NK) cells, which have the capacity to directly kill cells infected with viruses by secreting IFN-γ. ++ B cells make antibody T cells secrete cytokines +++ Phagocytosis ++ Phagocytosis is one of the most important defenses against microbial invaders (Figure 2–4). The major cells involved are PMNs, macrophages, and dendritic cells. For all, the process begins with surface–pathogen recognition mechanisms, which may be either dependent on opsonization of the organism with complement or antibody or independent of opsonization. At this point, only the opsonin-independent mechanisms are considered. These use the nonspecific mechanisms already described and hydrophobic interactions between bacteria and the phagocyte surface. More powerful killing mechanisms are mediated by lectins, which bind carbohydrate moieties, and protein–protein interactions based on a specific peptide sequence (arginine-glycine-aspartic-acid or RGD). These RGD receptors are present on virtually all phagocytes. ++ FIGURE 2–4. Phagocytosis. A. Drawing shows receptors on a phagocytic cell, such as a macrophage, and the corresponding PAMPs participating in phagocytosis. The schematic depicts the process of phagocytosis showing ingestion. B. Participation of primary and secondary granules. C. O2-dependent killing events. D. Intracellular digestion. E. Endocytosis LPS receptor, lipopolysaccharide receptor; TLRs, toll-like receptors; MHCI, class I major histocompatibility protein; MHC II, class II major histocompatibility protein; PAMPs, pathogen-associated molecular patterns. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Opsonization not required Carbohydrate and peptide sequence recognized ++ Bound organisms are taken inside the phagocyte in a membrane-bound phagosome destined to fuse with lysosomes inside to form a phagolysosome. This is the main killing ground of the phagocyte. The lysosomal enzymes include hydrolases and proteases that have maximum activity at the acidic pH inside the phagolysosome. In addition, the hostile intra-phagocyte environment includes oxidative killing mechanisms generated by enzymes that produce reactive oxygen intermediates (superoxide, hydrogen peroxide, singlet oxygen) driven by metabolic respiratory bursts in the cell cytoplasm. These mechanisms are particularly used for killing bacteria. Bacterial pathogens whose pathogenesis involves multiplication rather than destruction inside the phagocyte have mechanisms to block one or more of the preceding steps. For example, some pathogens (eg, M tuberculosis or Brucella sp) are able to block fusion of the phagosome with the lysosome; others (Listeria sp) interfere with the acidification of the phagolysosome, and many organisms employ both mechanisms. ++ Enzymes digest in acidic phagolysosome Reactive oxygen driven by respiratory burst ++ Another mechanism effective with some viruses, fungi, and parasites is the formation of reactive nitrogen intermediates (nitric oxide, nitrate, and nitrite) delivered into a vacuole or in the cytoplasm. PMN granules contain a variety of other antimicrobial substances, including peptides called defensins. Defensins act by permeabilizing membranes and, in addition to bacteria, are active against enveloped viruses. ++ Reactive nitrogen affects viruses +++ INFLAMMATION ++ Inflammation encompasses a series of events in which the above-mentioned cells are deployed in response to an injury—such as a new microbial invader. At the first insult, chemical signals mobilize cells, fluids, and other mediators to the site to contain, combat, and heal. In acute inflammation, the first events may be noticed in minutes, and the entire process resolved over a matter of days to a couple of weeks. Chronic inflammation may follow the incomplete resolution of an acute process or arise as a slow insidious process of its own. The natural history of infections such as tuberculosis, which follow this pattern, runs for months, years, even decades. ++ Acute = hours to days Chronic = weeks to months ++ The first event in acute inflammation is the release of chemical signals (chemokines) that act on adhesion molecules (selectins) in local capillaries. This slows the movement of passing PMNs and activates adhesive integrins on their surface. This leads to tight adhesion to the endothelium followed by squeezing past the endothelial wall to the tissues below. There, chemotactic factors released by the bacteria lead them to the primary site. Increasing acidity of local fluids releases enzymes (kallikrein, bradykinin) that open junctions in capillary walls and allow increased flow of fluids and more leukocytes. Histamine (from mast cells), arachidonic acid, and prostaglandin release complete the phenotype of swelling and pain. ++ PMNs migrate from capillaries Enzymes and chemical mediators facilitate swelling ++ Chronic inflammation bridges the innate and adaptive immune responses. An acute phase, if present, is usually not noticed, and the cellular infiltrate is composed of lymphocytes and macrophages with relatively few PMNs. It is generally associated with slower-growing pathogens such as mycobacteria, fungi, and parasites in which cell-mediated immunity is the primary adaptive defense. Many of these pathogens have mechanisms that allow them to multiply in nonactivated macrophages. If the macrophages are effectively activated by T cells, the multiplication ceases and the inflammation and injury are minimal. If not, multiplication and chronic inflammation continue sometimes in the form of a granuloma, which is an indication of a destructive hypersensitivity component to the inflammation. ++ Lymphocytes and macrophages predominate Granulomas indicate failure to resolve by adaptive cellular mechanisms +++ CHEMICAL MEDIATORS ++ Chemical mediators of innate immunity that have direct antimicrobial activity include cationic proteins and complement. The cationic proteins (cathelicidin, defensins) act on bacterial plasma membranes by the formation of ionic pores, which alter membrane permeability. The complement system is a series of glycoproteins, which can directly insert in bacterial membranes or act as receptors for antibody. Cytokines are proteins or glycoproteins released by one cell population that act as signaling molecules for another. They are generally thought of in the context of the adaptive immune system, but they can be stimulated directly by microorganisms. ++ Peptides alter membrane permeability +++ The Complement System ++ The complement system consists of more than 30 distinct components and several other precursors. All are in the plasma of healthy individuals in inactive forms that must be enzymatically cleaved to become active. When this happens, a cascade of reactions is triggered, which activates the various components in a fixed sequence (Figure 2–5). The difference between the pathways is in the mechanisms for their initiation. Once started, any pathway can produce the same effects on pathogens, which include enhancing phagocytosis, activation of leukocytes, and lysis of bacterial cell walls. An important step in the process is coating of the organism with serum components, a process called opsonization. The coatings may be mannose-binding proteins, complement components, or antibody. There is no immunologic specificity in complement activation or in its effects. ++ FIGURE 2–5. Components and action of complement. Complement activation involves a series of enzymatic reactions that culminate in the formation of C3 convertase, which cleaves complement component C3 into C3b and C3a. The production of the C3 convertase is where the three pathways converge. C3a is a peptide mediator of local inflammation. C3b binds covalently to the bacterial cell membrane and opsonizes the bacteria, enabling phagocytes to internalize them. C5a and C5b are generated by the cleavage of C5 by a C5 convertase. In addition, C5a is a powerful peptide mediator of inflammation. C5b promotes the terminal components complement to assemble into a membrane-attack complex. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Multiple components activated in cascade Differ in initiation mechanism Opsonization is serum coating of pathogens +++ Alternative Pathway ++ The alternative pathway is activated by bacterial cell wall components with repetitive surface structures such as LPS. The multiple components come together in the formation of the membrane-attack complex, which inserts directly into bacterial membranes (Figure 2–6), particularly the outer membrane of Gram-negative bacteria. This not only injures the organism but also enhances phagocytosis because the other end of the molecule has receptors for phagocytes. Gram-positive bacteria are less affected because they have no exposed membrane (see Chapter 21). These actions are particularly important for the effectiveness of innate immunity in the early stages of acute infections before the adaptive immune system has time to act. The key complement component for alternate pathway activity is C3b. C3b activation and degradation are regulated by a number of serum factors (factors B, D, and H) that can modulate its activity. A major mechanism for pathogens to block alternate pathway attack is by binding factor H to their surface. This is accomplished by bacterial capsules and surface proteins. This concentration of factor H causes local degradation of C3b (see Chapter 22, Figure 22–4). ++ FIGURE 2–6. Complement membrane-attack complex. The membrane-attack complex (MAC) is a tubular structure that forms a transmembrane pore in the target cell’s plasma membrane. The subunit architecture of the MAC shows that the transmembrane channel is formed by multiple polymerized molecules. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Activation by pathogen surfaces Membrane-attack complex inserts, provides phagocyte receptors Factor H binding accelerates C3b degradation +++ Lectin Pathway ++ Another means of activating the complement system is based on the carbohydrate building of lectins. In this case, the lectins bind to mannose—a common surface component of bacteria, fungi, and some virus envelopes. This binding opsonizes the pathogen and enhances phagocytosis. Thus, as in the alternative pathway, the activation comes from pathogen surfaces and proceeds through the same C3 convertase (Figure 2–5). ++ Lectins bind mannose on pathogens +++ Classic Pathway ++ The classic complement pathway is initiated by the binding of antibodies formed during the adaptive immune response (as described further) with their specific antigens on the surface of a pathogen. This binding is highly specific but amounts to another case of opsonization activating the complement cascade. In this case, specific sites on the Fc portion of immunoglobulin molecules bind and activate the C1 component of complement to start the process. The pathway and sequence of individual complements are characteristics of the classic pathway, but it still reaches C3b, the common point for microbial-directed action. As with the alternative pathway, this creates the membrane-attack complex, the mediators of inflammation, and receptors for phagocytes on C3b. ++ Antigen–antibody reaction exposes complement sites C3b has phagocyte receptors +++ Cytokines ++ Cytokine is a broad term referring to molecules released from one cell population destined to have an effect on another cell population (Table 2–2). As these proteins and glycoproteins have been discovered, they have been named and classified in relation to biologic effects observed initially only to discover that they have multiple other actions. For infectious diseases, the operative subcategories are chemokines, which are cytokines chemotactic for inflammatory cell migration, and interleukins (IL-1, 2, 3, etc), which regulate growth and differentiation between monocytes and lymphocytes. Tumor necrosis factor (TNF), so named for its cytotoxic effect on tumor cells, can also induce apoptosis (programmed cell death) in phagocytes—a useful feature for pathogens they have taken in. Interferons (INF-α, -β, and -γ) were originally named for their interference with viral replication (Figure 2–7), but are now known to be central to activation of T cells and macrophages. Unless commanded to understand specific situations, cytokine is used to represent all these mediators in these pages. ++Table Graphic Jump LocationTABLE 2–2Some Cytokines Acting in InfectionView Table| Favorite Table |Download (.pdf) TABLE 2–2 Some Cytokines Acting in Infection   CELL SOURCE FUNCTIONS Interleukins (IL) IL-1 Macrophages, endothelium, fibroblasts, epithelial Differentiation and function of immune effectors, PMN response (TH17) IL-2 T cells (TH1) T-cell proliferation, cytolytic activity of natural killer (NK) cells IL-4 T cells (TH2), macrophages, B cells Differentiation of naïve T cells to helper T cells, proliferation of B cells IL-5 T cells (TH2) Eosinophil activation IL-8 Macrophages, endothelial, T cells, keratinocytes, PMNs Chemoattractant for PMNs and T cells, PMN degranulation, migration of PMNs IL-17 T cells (TH17) Inflammation, PMN response IL-22 T cells (TH17) Antimicrobial peptides Interferons (IFN) IFN-α/β T cells, B cells, macrophages, fibroblasts Antiviral activity, stimulates macrophages, MHC class I expression IFN-γ T cells (TH1, CTLs), NK cells T-cell activation, macrophage activation, PMNs, NK cells, antiviral, MHC class I and II expression Tumor Necrosis Factor (TNF) TNF-α T cells, macrophages, NK cells Expression of multiple cytokines, (growth and transcription factors), stimulates inflammatory response, cytotoxic for tumor cells TNF-β T cells, B cells Same as TNF-α MHC, major histocompatibility complex; PMN, polymorphonuclear neutrophil. ++ FIGURE 2–7. Antiviral action of interferon. Interferon (IFN) synthesis and release are often induced by a virus infection. IFN binds to a ganglioside receptor on the plasma membrane of a second cell and triggers the production of enzymes that render the cell resistant to virus infection. The two most important such enzymes are oligo (A) synthetase and a special protein kinase. When an IFN-stimulated cell is infected, viral protein synthesis is inhibited by an active endoribonuclease that degrades viral RNA. An active protein kinase phosphorylates and inactivates the initiation factor elf-2 required for viral protein. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ ILs, IFNs, TNF, chemokin

Within a very short period immunity has been placed in possession not only of a host of medical ideas of the highest importance, but also of effective means of combating a whole series of maladies of the most formidable nature in man and domestic animals. —Elie Metchnikoff, 1905 ++ The “maladies” Metchnikoff and the other pioneers of immunology were fighting were infections and, for decades, their field was defined in terms of the immune response to infection. We now understand that the immune system is as much a part of everyday human biologic function as the cardiovascular or renal systems. In its adaptive and disordered states, infectious diseases are only one of the major players along with cancer and autoimmune diseases. Students of medicine study immunology as a separate unit with its own textbook covering the field broadly. This chapter is not intended to fulfill that function, or, indeed, to be a shortened but comprehensive version of those sources. It is included as an overview of aspects related to infection for other students and as an internal reference for topics that reappear in later pages of this book. These include some of the greatest successes of medical science. The early and continuing development of vaccines that prevent and potentially eliminate diseases is but one example. In addition, knowledge of the immune response to infection is integral to understanding the pathogenesis of infectious diseases. It turns out that one of the main attributes of a successful pathogen is evading or confounding the immune system. ++ The immune response to infection is presented as two major components—innate immunity and adaptive immunity. The primary effectors of both are cells that are members of the white blood cell series derived from hematopoietic stem cells in the bone marrow (Figure 2–1). Innate immunity includes the role of physical, cellular, and chemical systems that are in place and that respond to all aspects of “foreignness.” These include mucosal barriers, phagocytic cells, and the action of circulating glycoproteins such as complement. The adaptive side is sometimes called specific immunity because it has the ability to develop new responses that are highly specific to molecular components of infectious agents, called antigens. These encounters trigger the development of new cellular responses and production of circulating antibodies, which have a component of memory if the invader returns. Artificially creating this memory is, of course, the goal of vaccines. ++ FIGURE 2–1. Human blood cells. Stem cells in the bone marrow divide to form two blood cell lineages: (1) the lymphoid stem cell gives rise to B cells that become antibody-secreting plasma cells, T cells that become activated T cells, and natural killer cells. (2) The common myeloid progenitor cell gives rise to granulocytes and monocytes that give rise to macrophages and dendritic cells. (Reproduced with permission from Willey JM: Prescott, Harley, & Klein’s Microbiology, 7th ed. New York, NY: McGraw Hill; 2008.) Graphic Jump Location

INFECTIOUS DISEASE Download Section PDF Listen +++ ++ Of the thousands of species of viruses, bacteria, fungi, and parasites, only a tiny portion is involved in disease of any kind. These are called pathogens. There are plant pathogens, animal pathogens, and fish pathogens, as well as the subject of this book, human pathogens. Among pathogens, there are degrees of potency called virulence, which sometimes makes drawing the dividing line between benign and virulent microorganisms difficult. Pathogens are associated with disease with varying frequency and severity. Yersinia pestis, the cause of plague, causes fulminant disease and death in 50% to 75% of persons who come in contact with it. Therefore, it is highly virulent. Understanding the basis of these differences in virulence is a fundamental goal of this book. The better students of medicine understand how a pathogen causes disease, the better they will be prepared to intervene and help their patients. ++ Pathogens are rare Virulence varies greatly ++ For any pathogen, the basic aspects of how it interacts with the host to produce disease can be expressed in terms of its epidemiology, pathogenesis, and immunity. Usually, our knowledge of one or more of these topics is incomplete. It is the task of the physician to relate these topics to the clinical aspects of disease and be prepared for new developments which clarify, or in some cases, alter them. We do not know everything, and not all of what we believe we know is correct. +++ EPIDEMIOLOGY ++ Epidemiology is the “who, what, when, and where” of infectious diseases. The power of the science of epidemiology was first demonstrated by Semmelweis, who by careful analysis of statistical data alone determined how streptococcal puerperal fever is transmitted. He even devised a means to prevent transmission (handwashing) decades before the organism itself (Streptococcus pyogenes) was discovered. Since then, each organism has built its own profile of vital statistics. Some agents are transmitted by air, some by food, and others by insects; many spread by the person-to-person route. Figure 1–5 presents some of the variables in this regard. Some agents occur worldwide, and others only in certain geographic locations or ecologic circumstances. Knowing how an organism gains access to its victim and spreads is crucial to understanding the disease. It is also essential in discovering the emergence of “new” diseases, whether they are truly new (HIV, Covid-19) or just recently discovered (Legionnaires disease). Solving mysterious outbreaks or recognizing new epidemiologic patterns have often pointed the way to the isolation of new agents. ++ FIGURE 1–5. Infection overview. The sources and potential sites of infection are shown. Infection may be endogenous from the internal flora or exogenous from the sources shown around the outside. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Each agent has its own mode of spread ++ Epidemic spread and disease are facilitated by malnutrition, poor socioeconomic conditions, natural disasters, and hygienic inadequacy. Epidemics, caused by the introduction of new organisms of unusual virulence, often result in high morbidity and mortality rates. We are currently witnessing a new and extended Covid-19 pandemic, but the prospect of recurrence of old pandemic infections (influenza, cholera) remains. Modern times and technology have introduced new wrinkles to epidemiologic spread. Air travel has allowed diseases to leap continents even when they have very short incubation periods. The efficiency of the food industry has sometimes backfired when the distributed products are contaminated with infectious agents. The outbreaks of hamburger-associated E coli O157:H7 bloody diarrhea and hemolytic uremic syndrome are examples. The nature of massive meat-packing facilities allowed organisms from infected cattle on isolated farms to be mixed with other meat and distributed rapidly and widely. By the time outbreaks were recognized, cases of disease were widespread, and tons of meat had to be recalled. In simpler times, local outbreaks from the same source might have been detected and contained more quickly. ++ Poor socioeconomic conditions foster infection Modern society may facilitate spread ++ Of course, the most ominous and uncertain epidemiologic threat of these times is not amplification of natural transmission but the specter of unnatural, deliberate spread. Anthrax is a disease uncommonly transmitted by direct contact with animals or animal products. Under natural conditions, it produces a nasty, but not usually life-threatening, ulcer. The inhalation of human-produced aerosols of anthrax spores could produce a lethal pneumonia on a massive scale. Smallpox is the only disease officially eradicated from the world. It took place sufficiently long ago that most of the population has never been exposed or immunized and is, thus, vulnerable to its reintroduction. We do not know whether infectious bioterrorism will work on the scale contemplated by its perpetrators; however, in the case of anthrax, we do know that sophisticated systems have been designed to attempt it. We hope never to learn whether bioterrorism will work on a large scale. ++ Anthrax and smallpox are new bioterrorism threats +++ PATHOGENESIS ++ When a potential pathogen reaches its host, features of the organism determine whether or not disease ensues. The primary reason pathogens are so few in relation to the microbial world is that being successful at producing disease is a very complicated process. Multiple features, called virulence factors, are required to persist, cause disease, and escape to repeat the cycle. The variations are many, but the mechanisms used by many pathogens have now been dissected at the molecular level. ++ Pathogenicity is multifactorial ++ The first step for any pathogen is to attach and persist at whatever site it gains access. This usually involves specialized surface molecules or structures that correspond to receptors on human cells. Because human cells were not designed to receive the microorganisms, the pathogens are often exploiting some molecule important for some other essential function of the cell. For some toxin-producing pathogens, this attachment alone may be enough to produce disease. For most pathogens, it just allows them to persist long enough to proceed to the next stage—invasion into or beyond the surface mucosal cells. For viruses, invasion of cells is essential, because they cannot replicate on their own. Invading pathogens must also be able to adapt to a new milieu. For example, the nutrients and ionic environment of the cell surface differ from those inside the cell or in the submucosa. Some of the steps in pathogenesis at the cellular level are illustrated in Figure 1–6. ++ FIGURE 1–6. Infection cellular view. Left. A virus is attaching to the cell surface but can replicate only within the cell. Middle. A bacterial cell attaches to the surface, invades, and spreads through the cell to the bloodstream. Right. A bacterial cell attaches and injects proteins into the cell. The cell is disrupted while the organism remains on the surface. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Pathogens have molecules that bind to host cells Invasion requires adaptation to new environments ++ Persistence and even invasion do not necessarily translate immediately to disease. The invading organisms must disrupt function in some way. For some, the inflammatory response they stimulate is enough. For example, a lung alveolus filled with neutrophils responding to the presence of S pneumoniae loses its ability to exchange oxygen. The longer a pathogen can survive in the face of the host response, the greater the compromise in host function. Most pathogens do more than this. Destruction of host cells through the production of digestive enzymes, toxins, or intracellular multiplication is among the more common mechanisms. Other pathogens operate by altering the function of a cell without injury. Diphtheria is caused by a bacterial toxin that blocks protein synthesis inside the host cell. Details of the molecular mechanism for this action are illustrated in Figure 1–7. Some viruses cause the insertion of molecules in the host cell membrane, which causes other host cells to attack it. The variations are diverse and fascinating. ++ FIGURE 1–7. Action of diphtheria toxin, molecular view. The toxin-binding (B) portion attaches to the cell membrane, and the complete molecule enters the cell. In the cell, the A subunit dissociates and catalyzes a reaction that ADP-ribosylates (ADPR) and, thus, inactivates elongation factor 2 (EF-2). This factor is essential for ribosomal reactions at the acceptor and donor sites, which transfer triplet code from messenger RNA (mRNA) to amino acid sequences via transfer RNA (tRNA). Inactivation of EF-2 stops building of the polypeptide chain. Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Inflammation alone can result in injury Cells may be destroyed or their function altered +++ IMMUNITY ++ Although the science of immunology is beyond the scope of this book, understanding the immune response to infection (see Chapter 2) is an important part of appreciating pathogenic mechanisms. In fact, one of the most important virulence attributes any pathogen can have is an ability to neutralize the immune response to it in some way. Some pathogens attack the immune effector cells, and others undergo changes that evade the immune response. The old observation that there seems to be no immunity to gonorrhea turns out to be an example of the latter mechanism. Neisseria gonorrhoeae, the causative agent of gonorrhea, undergoes antigenic variation of important surface structures so rapidly that antibodies directed against the bacteria become irrelevant. ++ Evading the immune response is a major feature of virulence ++ For each pathogen, the primary interest is whether there is natural immunity and, if so, whether it is based on cell-mediated (TH1, CMI) or humoral (TH2, antibody) mechanisms. Humoral and CMI responses are broadly stimulated with most infections, but the specific response to a particular molecular structure is usually dominant in mediating immunity to reinfection. For example, the repeated nature of strep throat (group A streptococcus) in childhood is not due to antigenic variation as described above for gonorrhea. The antigen against which protective antibodies are directed (M protein) is stable, but naturally exists in more than 80 types. Each type requires its own specific antibody. Thus, even with a strong immune response the gauntlet is great. Identifying the specific molecular structure against which the protective immune response is directed is particularly important for devising preventive vaccines. ++ Antibody or cell-mediated mechanisms may be protective +++ CLINICAL ASPECTS OF INFECTIOUS DISEASE +++ Manifestations ++ Fever, pain, and swelling are the universal signs of infection. Beyond this, the particular organs involved and the speed of the process dominate the signs and symptoms of disease. Cough, diarrhea, and mental confusion represent disruption of three different body systems. On the basis of clinical experience, physicians have become familiar with the range of behavior of the major pathogens. However, signs and symptoms overlap considerably. Skilled physicians use this knowledge to begin a deductive process leading to a list of suspected pathogens and a strategy to make a specific diagnosis and provide patient care. Through the probability assessment, an understanding of how the diseases work is a distinct advantage in making the correct decisions. ++ Body system(s) involved dictate clinical approach +++ Diagnosis ++ A major difference between infectious and other diseases is that the probabilities just described can be specifically resolved, often overnight. Most microorganisms can be isolated from the patient, grown in artificial culture, and identified. Others can be seen microscopically or detected by measuring the specific immune response to the pathogen. Preferred modalities for diagnosis of each agent have been developed and are available in clinics, hospitals, and public health laboratories all over the world. Empiric diagnosis made on the basis of clinical findings can be confirmed and the treatment plan modified accordingly. New methods which detect molecular or genomic markers of the agent are now realizing much greater application for rapid, specific diagnosis. ++ Disease-causing microbes can be identified by culture or genomics +++ Treatment ++ Over the past 80+ years, therapeutic tools of remarkable potency and specificity have become available for the treatment of bacterial infections. These include all the antibiotics and an array of synthetic chemicals that kill or inhibit the infecting organism but have minimal or acceptable toxicity for the host. Antibacterial agents exploit the structural and metabolic differences between microbial and human eukaryotic cells to provide the selectivity necessary for good antimicrobial therapy. Penicillin, for example, interferes with the synthesis of the bacterial cell wall, a structure that has no analog in human cells. There are fewer antifungal and antiprotozoal agents because the eukaryotic cells of the host and those of the parasite have metabolic and structural similarities. Nevertheless, hosts and parasites do have some significant differences, and effective therapeutic agents have been discovered or developed to exploit them. ++ Antibiotics are directed at structures of bacteria not present in host ++ Specific therapeutic attack on viral disease has posed more complex problems, because of the intimate involvement of viral replication with the metabolic and replicative activities of the cell. However, recent advances in molecular virology have identified specific viral targets that can be attacked. Scientists have developed successful antiviral agents, including those that interfere with viral attachment, the liberation of viral nucleic acid from its protective protein coat, or with the processes of viral nucleic acid synthesis and replication. The successful development of new agents for human immunodeficiency virus has involved targeting enzymes coded by the virus genome. ++ Antivirals target unique virus-coded enzymes ++ The success of the “antibiotic era” has been clouded by the development of resistance by the organisms. The mechanisms involved are varied but, most often, involve a mutational alteration in the enzyme, ribosome site, or other target against which the antimicrobial is directed. In some instances, organisms acquire new enzymes or block entry of the antimicrobial to the cell. Many bacteria produce enzymes that directly inactivate antibiotics. To make the situation worse, the genes involved are readily spread by promiscuous genetic mechanisms. New agents that are initially effective against resistant strains have been developed, but resistance by new mechanisms usually follows. The battle is by no means lost, but it has become a never-ending policing action. ++ Resistance complicates therapy Mechanisms include mutation and inactivation +++ Prevention ++ The goal of the scientific study of any disease is its prevention. In the case of infectious diseases, this has involved public health measures and immunization. The public health measures depend on knowledge of transmission mechanisms and on interfering with them. Water disinfection, food preparation, insect control, handwashing, and a myriad of other measures prevent humans from coming in contact with infections agents. Immunization relies on knowledge of immune mechanisms and designing vaccines that stimulate protective immunity. ++ Public health and immunization are primary preventive measures ++ Immunization follows two major strategies—live vaccines and inactivated vaccines. The former uses live organisms that have been modified (attenuated) so they do not produce disease, but still stimulate a protective immune response. Such vaccines have been effective, but they carry the risk that the vaccine strain itself may cause disease. This event has been observed with the live oral polio vaccine. Although this rarely occurs, it has caused a shift back to the original Salk inactivated vaccine. This issue has reemerged with a debate over strategies for the use of smallpox immunization to protect against bioterrorism. This vaccine uses vaccinia virus, a cousin of smallpox, and its potential to produce disease on its own has been recognized since its original use by Jenner in 1798. Serious disease would be expected primarily in immunocompromised individuals (eg, from cancer chemotherapy or AIDS), who represent a significantly larger part of the population than when smallpox immunization was stopped in the 1970s. Could immunization cause more disease than it prevents? Despite the claims of those who oppose the use of all vaccines as “unnatural,” the risk/benefit ratio of all currently licensed vaccines is greatly on the positive side. ++ Attenuated strains stimulate immunity Live vaccines rarely cause disease ++ The safest immunization strategy is the use of organisms that have been killed or, better yet, killed and purified to contain only the immunizing component. This approach requires much better knowledge of pathogenesis and immune mechanisms. Vaccines for meningitis use the polysaccharide capsule of the bacterium, and vaccines for diphtheria and tetanus use only a formalin-inactivated protein toxin. Pertussis (whooping cough) immunization has undergone a transition in this regard. The original killed whole-cell vaccine was effective, but it caused a significant incidence of side effects. A purified vaccine containing pertussis toxin and a few surface components has reduced side effects, but its efficacy compared with the previous vaccine is now in question. ++ Purified components are safe vaccines ++ The newest approaches for vaccines require neither live organisms nor killed, purified ones. As the entire genomes of more and more pathogens are being reported, an entirely genetic strategy is emerging. Armed with knowledge of molecular pathogenesis and immunity and the tools of genomics and proteomics, scientists can now synthesize an immunogenic protein without ever growing the organism itself. Two of the most successful new Covid-19 vaccines use coded messenger RNA (mRNA) which instructs human cells to produce the immunogen. Such ideas would have astonished even the great microbiologists of the last two centuries. ++ Vaccines can be genetically engineered ++ SUMMARY Infectious diseases remain as important and fascinating as ever. Where else do we find the emergence of new diseases, together with improved understanding of the old ones? At a time when the revolution in molecular biology and genetics has brought us to the threshold of new and novel means of infection control, the perpetrators of bioterrorism threaten us with diseases we have already conquered. Meeting this challenge requires a secure knowledge of the pathogenic organisms and how they produce disease, as well as an understanding of the clinical aspects of these diseases. In the collective judgment of the authors, this book presents the principles and facts required for students of medicine to understand the most important infectious diseases.

n fact, from shortly after birth onward, it is universal; we harbor 10 times more microbial cells than human cells. This population, formerly called the normal flora, is now referred to as our microbiota or microbiome. These microorganisms, which are overwhelmingly bacteria, are frequently found colonizing various body sites in healthy individuals. The constituents and numbers of the microbiota vary in different areas of the body and, sometimes, at different ages and physiologic states. Their names are mostly unfamiliar because they have not (yet) been associated with disease. They comprise microorganisms whose morphologic, physiologic, and genetic properties allow them to colonize and multiply under the conditions that exist in particular sites, to coexist with other colonizing organisms, and to inhibit competing intruders. Thus, each accessible area of the body presents a particular ecologic niche, colonization of which requires a particular set of properties of the colonizing microbe. ++ Organisms of the microbiota may have a symbiotic relationship that benefits the host or may simply live as commensals with a neutral relationship to the host. A parasitic relationship that injures the host would not be considered “normal,” but, in most instances, not enough is known about the organism–host interactions to make such distinctions. Some have been characterized by genomic methods but not yet grown in culture. Like houseguests, the members of the microbiota may stay for highly variable periods. Residents are strains that have an established niche at one of the many body sites, which they occupy indefinitely. Transients are acquired from the environment and establish themselves briefly, but they tend to be excluded by competition from residents or by the host’s innate or immune defense mechanisms. The term carrier state is used when organisms known to be potentially pathogenic are involved, although its implication of risk is not always justified. For example, Streptococcus pneumoniae, a cause of pneumonia, and Neisseria meningitidis, a cause of meningitis, may be isolated from the throat of 5% to 40% of healthy people. Whether these bacteria represent transient flora, resident flora, or carrier state is largely semantic. The possibility that their presence could be the prelude to disease is presently impossible to determine in advance. ++ Flora may stay for short or extended periods If pathogens involved, the relationship is called the carrier state ++ It is important for students of medical microbiology and infectious disease to understand the role of the microbiota because of its significance both as a defense mechanism against infection and as a source of potentially pathogenic organisms. In addition, it is important for physicians to know the typical composition of the microbiota at various sites to avoid confusion when interpreting laboratory culture results. The following excerpt indicates that the English poet W.H. Auden understood the need for balance between the microbiota and its host. He was stimulated by a 1969 article by Mary J. Marples in Scientific American about the microbial flora of the skin. + On this day tradition allots to taking stock of our lives, my greetings to all of you, Yeasts, Bacteria, Viruses, Aerobics and Anaerobics: A Very Happy New Year to all for whom my ectoderm is as Middle Earth to me. For creatures your size I offer a free choice of habitat, so settle yourselves in the zone that suits you best, in the pools of my pores or the tropical forests of arm-pit and crotch, in the deserts of my fore-arms, or the cool woods of my scalp. Build colonies: I will supply adequate warmth and moisture, the sebum and lipids you need, on condition you never do me annoy with your presence, but behave as good guests should, not rioting into acne or athlete’s-foot or a boil. —W.H. Auden, “A New Year Greeting” +++ ORIGIN AND NATURE ++ The healthy fetus is sterile until the birth membranes rupture. During and after birth, the infant is exposed to the flora of the mother’s vagina and to other organisms in the environment. During the infant’s first few days of life, the microbiota reflects chance exposure to organisms that can colonize particular sites in the absence of competitors. Subsequently, as the infant is exposed to a broader range of organisms, those best adapted to colonize particular sites become predominant. Thereafter, the flora generally resembles that of other individuals in the same age group and cultural milieu. ++ Initial flora acquired during and immediately after birth ++ Local physiologic and ecologic conditions determine the microbial makeup of the microbiota. These conditions are sometimes highly complex, differing from site to site, and sometimes with age. Conditions include the amounts and types of nutrients available, pH, oxidation–reduction potentials, and resistance to local antibacterial substances, such as bile and lysozyme. Many bacteria have adhesin-mediated affinity for receptors on specific types of epithelial cells; this facilitates colonization and multiplication and prevents removal by the flushing effects of surface fluids and peristalsis. Various microbial interactions also determine their relative prevalence in the flora. These interactions include competition for nutrients and inhibition by the metabolic products of other organisms.

ROLES IN HEALTH AND DISEASE +++ Opportunistic Infection ++ Many species among the microbiota are opportunists in that they can cause infection when they reach protected areas of the body in sufficient numbers. For example, certain strains of E coli can reach the urinary bladder by ascending the urethra and cause acute urinary tract infection. Perforation of the colon from a ruptured diverticulum or a penetrating abdominal wound releases feces into the peritoneal cavity; this contamination may be followed by peritonitis or intraabdominal abscesses caused by members of the flora which have virulence factors allowing them to exploit this situation. There are now examples of the microbiota supplying a step in the pathogenesis of a classic pathogen. Attachment of Neisseria gonorrhoeae to the cervix has been shown to be enhanced when an enzyme produced by the cervicovaginal microbiota unmasks a crucial receptor. Caries and periodontal disease are caused by organisms that are members of the oral microbiota (see Chapter 41). ++ Flora that reach sterile sites may cause disease Virulence factors increase opportunity for invasion +++ Exclusionary Effect ++ Balancing the prospect of opportunistic infection is the tendency of the resident microbiota to produce conditions that compete with extraneous newcomers who happen to be pathogens and thus reduce their ability to establish a niche in the host. The microbiota in the colon of the breastfed infant produces an environment inimical to colonization by enteric pathogens, as does a vaginal flora dominated by lactobacilli. The benefit of this exclusionary effect has been demonstrated by what happens when it is removed. Antibiotic therapy, particularly with broad-spectrum agents, may so alter the microbiota of the gastrointestinal tract that antibiotic-resistant organisms multiply in the ecologic vacuum as in the C difficile toxic colitis discussed above. ++ Competing with pathogens has a protective effect Antibiotic therapy may provide advantage for pathogens +++ Priming of Immune System ++ Organisms of the microbiota play an important role in the development of immunologic competence. Animals delivered and raised under completely aseptic conditions (“sterile” or gnotobiotic animals) have a poorly developed reticuloendothelial system, low serum levels of immunoglobulins, and lack antibodies to antigens that often confer a degree of protection against pathogens. There is evidence of immunologic differences between children who are raised under usual conditions and those whose exposure to diverse flora is minimized. Some studies have found a higher incidence of immunopathologic states, such as asthma in the more isolated children. ++ Sterile animals have little immunity Low exposure correlates with asthma +++ PROMOTING A GOOD MICROBIOTA ++ The field of probiotics is based on the notion that we can manipulate the microbiota by promoting colonization with “good” bacteria. Elie Metchnikoff originally suggested this in his observation that the longevity of Bulgarian peasants was attributable to their consumption of large amounts of yogurt; the live lactobacilli in the yogurt presumably replaced the colonic flora to the general benefit of their health. This notion persists today in capsules containing freeze-dried lactobacilli sold by the sizable probiotics industry and by promotion of the health benefit of natural (unpasteurized) yogurt, which contains live lactobacilli. Because these lactobacilli are adapted to food and not the intestine, they are unlikely to persist, much less replace, the typical microbiota of the adult colon. In some clinical studies, administration of preparations containing a particular strain of Lactobacillus (Lactobacillus rhamnosus strain GG, LGG) has been shown to reduce the duration of rotavirus diarrhea in children. The use of similar preparations to prevent relapses of antibiotic-associated diarrhea caused by C difficile has shown little success, but fecal transplant (a whole new microbiota) has blocked recurrences of pseudomembranous colitis, the most serious form of this disease. ++ Intestinal lactobacilli may protect against diarrheal agents ++ Research into the role of the microbiota in health and disease is one of the most exciting topics in science. The Human Microbiome Project funded by the US National Institutes of Health is by no means limited to topics related to infectious disease. Currently, the most active areas involve mechanisms of obesity, autoimmune disorders (arthritis, asthma), and more subjective subjects like human cravings. Much of the work involves the interactions between multiple species many of which can only be detected by genomic methods. Obviously, it is going to take considerable time to sort these relationships out.

Parasites are the most diverse of all microorganisms. They range from unicellular amoebas of 10 to 12 μm to multicellular tapeworms 1 m long. The individual cell plan is eukaryotic, but organisms such as worms are highly differentiated and have their own organ systems. Most worms have a microscopic egg or larval stage, and part of their life cycle may involve multiple vertebrate and invertebrate hosts. Most parasites are free living, but some depend on combinations of animal, arthropod, or crustacean hosts for their survival.

Blood, Body Fluids, and Tissues ++ In health, the blood, body fluids, and tissues are sterile. Occasional organisms may be displaced across epithelial barriers as a result of trauma or during childbirth; they may be briefly recoverable from the bloodstream before they are filtered out in the pulmonary capillaries or removed by cells of the reticuloendothelial system. Such transient bacteremia may be the source of infection when structures such as damaged heart valves and foreign bodies (prostheses) are in the bloodstream. ++ Tissues, body fluids, blood are sterile +++ Skin ++ The skin surface provides a dry, slightly acidic, aerobic environment. It plays host to an abundant flora that varies according to the presence of its appendages (hair, nails) and the activity of sebaceous and sweat glands. The flora is more abundant on moist skin areas (axillae, perineum, and between toes). Staphylococci and members of the Propionibacterium genus occur all over the skin, and facultative Corynebacterium species are found in moist areas. Propionibacterium species are slim, anaerobic, or microaerophilic Gram-positive rods that grow in subsurface sebum and break down skin lipids to fatty acids. Thus, they are most numerous in the ducts of hair follicles and of the sebaceous glands that drain into them. Even with antiseptic scrubbing, it is difficult to eliminate bacteria from skin sites, particularly those bearing pilosebaceous units. Organisms of the skin flora are resistant to the bactericidal effects of skin lipids and fatty acids, which inhibit or kill many extraneous bacteria. The conjunctivae have a very scanty flora derived from the skin. The low bacterial count is influenced by the high lysozyme content of lachrymal secretions and the flushing effect of tears. ++ Propionibacteria, staphylococci dominant bacteria Skin flora is not easily removed +++ Intestinal Tract ++ The mouth and pharynx contain large numbers of facultative and anaerobic bacteria. Different species of streptococci predominate on the buccal and tongue mucosa because of different specific adherence characteristics. Other genera include Actinomyces, Bacteroides, Fusobacterium, and Corynebacterium. Strict anaerobes and microaerophilic organisms of the oral cavity have their niches in the depths of the gingival crevices surrounding the teeth and in sites such as tonsillar crypts, where anaerobic conditions can develop readily. The role of the oral microbiome in dental infections is addressed in Chapter 41. ++ Oropharynx has streptococci and anaerobes ++ The total number of organisms in the oral cavity is very high, and it varies from site to site. Saliva usually contains a mixed flora of about 108 organisms per milliliter, derived mostly from the various epithelial colonization sites. The genera include Actinomyces, Bacteroides, Prevotella, Streptococcus, and others. The stomach contains few, if any, resident organisms in health because of the lethal action of gastric hydrochloric acid and peptic enzymes on bacteria. One species, H pylori, long thought to be a common resident, is now known to be the primary cause of ulcers. The small intestine has a scanty resident flora, except in the lower ileum, where it begins to resemble that of the colon. ++ H pylori turned out to be a stomach pathogen Small intestinal flora is scanty but increases toward lower ileum ++ The colon carries the most abundant and diverse microbiota in the body. In the adult, feces are 25% or more bacteria by weight (about 1010 organisms per gram). More than 90% are anaerobes, predominantly members of the genera Bacteroides, Fusobacterium, Eubacterium, and Clostridium. The remainder of the flora is composed of facultative organisms, such as Escherichia coli, enterococci, yeasts, and numerous other species. There are considerable differences in adult flora depending on the diet of the host. Those whose diets include substantial amounts of meat have more Bacteroides and other anaerobic Gram-negative rods in their stools than those on a predominantly vegetable or fish diet. Due to its ability to form spores, Clostridioides difficile is able to survive and multiply in association with antimicrobial therapy, causing a life-threatening colitis. Recent studies have suggested the composition of the colonic microbiota could play a role in obesity. ++ Colonic flora predominantly anaerobic C difficile causes colitis +++ Respiratory Tract ++ The external 1 cm of the anterior nares has a flora similar to that of the skin. This is the primary site of carriage of a major pathogen, Staphylococcus aureus. Approximately 25% to 30% of healthy people carry this organism as either resident or transient flora at any given time. The nasopharynx has a flora similar to that of the mouth; however, it is often the site of carriage of potentially pathogenic organisms, such as pneumococci, Neisseria, and Haemophilus species. ++ S aureus is carried in anterior nares ++ The respiratory tract below the level of the larynx is protected in health by the action of the epithelial cilia and by the movement of the mucociliary blanket; thus, only transient inhaled organisms are encountered in the trachea and larger bronchi. The accessory sinuses are normally sterile and are protected in a similar fashion, as is the middle ear by the epithelium of the eustachian tubes. ++ Lower tract is protected by mucociliary action +++ Genitourinary Tract ++ The urinary tract is sterile in health above the distal 1 cm of the urethra, which has a scanty flora derived from the perineum. Thus, in health, the urine in the bladder, ureters, and renal pelvis is sterile. The vagina has a flora that varies according to hormonal influences at different ages. Before puberty and after menopause, it is mixed, nonspecific, and relatively scanty, and it contains organisms derived from the flora of the skin and colon. During the childbearing years, it is composed predominantly of anaerobic and microaerophilic members of the genus Lactobacillus, with smaller numbers of anaerobic Gram-negative rods, Gram-positive cocci, and yeasts (Figure 1–4) that can survive under the acidic conditions produced by the lactobacilli. These conditions develop because glycogen is deposited in vaginal epithelial cells under the influence of estrogenic hormones and metabolized to lactic acid by lactobacilli. This process results in a vaginal pH of 4 to 5, which is optimal for growth and survival of the lactobacilli but inhibits many other organisms. ++ FIGURE 1–4. Vaginal flora. Vaginal Gram smear showing budding yeast (long arrow), epithelial cells (short arrow), and a mixture of other bacterial morphologies. The long Gram-positive rods are most likely lactobacilli. (Reproduced with permission from Centers for Disease Control and Prevention [CDC].) Graphic Jump LocationView Full Size| Favorite Figure |Download Slide (.ppt) ++ Hormonal changes a

Parasites are the most diverse of all microorganisms. They range from unicellular amoebas of 10 to 12 μm to multicellular tapeworms 1 m long. The individual cell plan is eukaryotic, but organisms such as worms are highly differentiated and have their own organ systems. Most worms have a microscopic egg or larval stage, and part of their life cycle may involve multiple vertebrate and invertebrate hosts. Most parasites are free living, but some depend on combinations of animal, arthropod, or crustacean hosts for their surviv

FUNGI ++ Fungi exist in either yeast or mold forms. The smallest of yeasts are similar in size to bacteria, but most are larger (2-12 μm) and multiply by budding. Molds form tubular extensions called hyphae, which, when linked together in a branched network, form the fuzzy structure seen on neglected bread slices. Fungi are eukaryotic, and both yeasts and molds have a rigid external cell wall composed of their own unique polymers, called glucan, mannan, and chitin. Their genome may exist in a diploid or haploid state and replicate by meiosis or simple mitosis. Most fungi are free living and widely distributed in nature. Generally, fungi grow more slowly than bacteria, although their growth rates sometimes overlap.

Bacteria are the smallest (0.1-0 μm) independently living agents known. They have a cytoplasmic membrane surrounded by a cell wall; a unique interwoven polymer called peptidoglycan makes the wall rigid. The simple prokaryotic cell plan includes no mitochondria, lysosomes, endoplasmic reticulum, or other organelles (Table 1–2). In fact, most bacteria are approximately the size of mitochondria. Their cytoplasm contains only ribosomes and a single, double-stranded DNA chromosome. Bacteria have no nucleus, but all the chemical elements of nucleic acid and protein synthesis are present. Although their nutritional requirements vary greatly, most bacteria are free living if given an appropriate energy source. Tiny metabolic factories, they divide by binary fission and can be grown in artificial culture, producing progeny sometimes in a matter of hours. The Archaea are similar to bacteria but evolutionarily distinct. They are prokaryotic, but they differ in the chemical structure of their cell walls and other features. The Archaea (archebacteria) can live in environments humans consider hostile (eg, hot springs, high salt areas) but are not associated with disease.

VIRUSES ++ Viruses are strict intracellular parasites of other living cells, not only of mammalian and plant cells but also of simple unicellular organisms, including bacteria (the bacteriophages). Viruses are simple forms of replicating, biologically active particles that carry genetic information in either DNA or RNA molecules. Most mature viruses have a protein coat over their nucleic acid and, sometimes, a lipid surface membrane derived from the cell they infect. Because viruses lack the protein-synthesizing enzymes and structural apparatus necessary for their own replication, they bear essentially no resemblance to a true eukaryotic or prokaryotic cell. ++ Viruses contain little more than DNA or RNA ++ Viruses replicate by using their own genes to direct the metabolic activities of the cell they infect to bring about the synthesis and reassembly of their component parts. A cell infected with a single viral particle may, thus, yield thousands of viral particles, which can be assembled almost simultaneously under the direction of the viral nucleic acid. Infection of other cells by the newly formed viruses occurs either by seeding from or lysis of the infected cells. Sometimes, viral and cell reproduction proceed simultaneously without cell death, although cell physiology may be affected. The close association of the virus with the cell sometimes results in the integration of viral nucleic acid into the functional nucleic acid of the cell, producing a latent infection that can be transmitted intact to the progeny of the cell. ++ Replicatio

The discoveries of penicillin by Fleming in 1929 and of sulfonamides by Domagk in 1935 opened the way to great developments in chemotherapy. These gradually extended from bacterial diseases to fungal, parasitic, and finally viral infections. Almost as quickly, virtually all categories of infectious agents developed resistance to all categories of antimicrobial agents to counter these chemotherapeutic agents.

hen Sir William Osler, the great physician/humanist, wrote these words, fever (infection) was indeed the scourge of the world. Tuberculosis and other forms of pulmonary infection were the leading causes of premature death among the well-to-do and the less fortunate. The terror was due to the fact that, although some of the causes of infection were being discovered, little could be done to prevent or alter the course of disease. In the 20th century, advances in public sanitation and the development of vaccines and antimicrobial agents changed this (Figure 1–1), but only for the nations that can afford these interventions. As we move through the second decade of the 21st century, the world is divided into countries in which heart attacks, cancer, and stroke have surpassed infection as causes of premature death and those in which infection is still the leader. That is, unless there is a pandemic causing infection to again become the leading killer everywhere.