For an infectious disease to develop in a host, the organism must penetrate the skin or mucous membrane barrier (first line of defense) and survive other natural and adaptive body defense mechanisms (see Chapter 1). These mechanisms include phagocytosis, antibody and cell-mediated immunity or complement activation, and associated interacting effector mechanisms. Phagocytosis and complement activation may be initiated within minutes of invasion by a microorganism; however, unless primed by previous contact with the same or similar antigen, antibody and cell-mediated responses do not become activated for several days. Complement and antibodies are the most active constituents against microorganisms free in the blood or tissues, whereas cell-mediated responses are most active against microorganisms associated with cells.

The most effective mechanism of body defense in a healthy host depends on factors such as an appropriate portal of entry and the characteristics of each microorganism. The routes of infection or portals of entry can include transmission through oral routes (e.g., foodborne or water-borne contamination), maternal-fetal transmission, insect vectors, sexual transmission, parenteral routes (e.g., injection or transfusion of infected blood), and respiratory transmission. Development of an infectious disease occurs only if a microorganism can evade, overcome, or inhibit normal body defense mechanisms.

Bacterial Diseases

The presence of key substances (e.g., lysozyme) and the process of phagocytosis represent major immunologic defense mechanisms against bacteria. A microorganism, however, can survive phagocytosis if it possesses a capsule that impedes attachment or a cell wall that interferes with the digestion and release of exotoxins, which damage phagocytic and other cells. Most capsules and toxins are strongly antigenic, but antibodies can overcome many of their effects; this is the basis of most antibacterial vaccines.

Examples of representative bacterial diseases of importance in the study of immunology and serology are presented in Chapters 17 and 18.

Parasitic Diseases

Parasites are relatively large, may have resistant body walls, and may avoid being phagocytized because of their ability to migrate away from an inflamed area. These differences set parasitic infections apart from bacterial and viral infections to which some forms of natural and adaptive immunity afford protection. (Toxoplasmosis, a representative disease, is discussed in Chapter 20.)

Immune responses (effectors) to parasitic infections include immunoglobulins, complement, antibody-dependent, cell-mediated cytotoxicity, and cellular defenses such as eosinophils and T cells. Some cestodes, especially in their larval stages, may be eradicated by complement-fixing immunoglobulin G (IgG) antibodies. In addition, some antibodies may cross-react with other parasitic antigens. Increased levels of IgE may be noted in many helminth infections. Activation of the classic and alternate complement pathways may occur in some cases of schistosomiasis, and the alternate pathway of complement activation may kill larvae in the absence of antibody (see Chapter 5).

Phagocytosis may have some direct activity against parasitic organisms, but the most effective protection in some parasitic infections is provided by antibody-dependent, cell-mediated cytotoxicity. Macrophages, neutrophils, and eosinophils may demonstrate direct toxicity or phagocytosis toward parasites. The actual attachment of the cytotoxic cells is usually mediated by IgG, although IgE may be effective. The role of eosinophils is complex. They may phagocytize immune complexes and act as effector cells in mediating local (type I) reactions, primarily in tissue stage parasites. T cells are frequently involved in body defenses against parasites. Sequestration of microorganisms is a classic T-cell–dependent hypersensitivity response. In addition, helper T cells may sensitize B cells to specific parasitic antigens.

Other nonspecific factors (e.g., nonstimulated monocytes) are a major protective mechanism against parasites such as Giardia spp. Natural killer (NK) cells also have a direct activity against cancer cells and some parasites. Delayed hypersensitivity may be helpful in preventing some parasitic infections but may cause disease in other cases. Deposition of antigen-antibody complexes, demonstrated by Raji cell assays, is responsible for severe pathologic lesions in some parasitic infections. In addition, high levels of circulating IgE may cause hypersensitivity reactions in helminth and cestode infections. Anaphylaxis is a clear risk in echinococcal infections, especially with spontaneous or surgical rupture of a hydatid cyst.

Fungal Diseases

Fungal, or mycotic, infections are normally superficial, but a few fungi can cause serious systemic disease, usually entering through the respiratory tract in the form of spores. Disease manifestation depends on the degree and type of immune response elicited by the host. Fungi are common and harmless inhabitants of skin and mucous membranes under normal conditions (e.g., Candida albicans). In immunocompromised hosts, Candida spp. and other fungi become opportunistic agents that take advantage of the host’s weakened resistance. Manifestations of fungal disease may range from unnoticed respiratory episodes to rapid, fatal dissemination of a violent hypersensitivity reaction.

Survival mechanisms of fungi that successfully invade the body are similar to bacterial characteristics and include the following: (1) presence of an antiphagocytic capsule; (2) resistance to digestion within macrophages; and (3) destruction of phagocytes (e.g., neutrophils). Some types of yeast activate complement through the alternative pathway, but it is unknown whether this activation has any effect on the microorganism’s survival.

Fungal infections are increasing worldwide for a variety of reasons, including the use of immunosuppressive drugs and the development of diseases that result in an immunocompromised host (e.g., acquired immune deficiency syndrome [AIDS]). Serologic tests often play an important role in the diagnosis of these fungal infections (Table 15-1).

Table 15-1

Testing Methods for Fungal Disease

Disease	Procedure
Aspergillosis	Gel immunodiffusion, EIA; IgG to Aspergillus fumigatus (≤110 mg/L), 85% of farmers and some persons with no evidence of disease
Blastomycosis	Complement fixation (>50% positive in proven cases); immunodiffusion (test is positive in about 80% of cases)
Coccidioidomycosis	Complement fixation using coccidioidin (blood, CSF)
Cryptococcosis	Latex agglutination (serum, CSF), EIA, immunofluorescence assay
Histoplasmosis	Complement fixation, immunodiffusion, PCR (sputum, blood, tissue); Histoplasma capsulatum antigen by EIA (urine); nucleic acid probe
Sporotrichosis	Latex particle agglutination

EIA, Enzyme immunoassay; CSF, cerebrospinal fluid; PCR, polymerase chain reaction.

Several species of fungi are associated with respiratory disease in human beings. These diseases are acquired by inhaling spores from exogenous reservoirs, including dust, bird droppings, and soil.

Viral, Rickettsial, and Mycoplasmal Diseases

The characteristic process associated with viral infections is cellular replication, which may or may not lead to cell death. Interferon plays a major role in body defenses against viral infections. Antibodies are valuable in preventing the entry and bloodborne spread of some viruses, but the ability of other viruses to spread from cell to cell places the burden of adaptive immunity on the T cell system, which specializes in recognizing altered self histocompatibility antigens (histocompatibility leukocyte antigen [HLA]). Macrophages may also play a role in immunity. Some of the most virulent viruses for human beings are zoonoses (e.g., rabies). Other viruses, however, can persist for years without symptoms and can then be reactivated to cause serious disease, possibly including tumors.

New viruses can cause old diseases, and old viruses can cause new diseases (see Chapters 21 to 25 for representative examples of immunologically important viral diseases). The mutation rates of viruses, especially ribonucleic acid (RNA) viruses such as human immunodeficiency virus (HIV), are extraordinarily high. Consequently, RNA viruses evolve much more rapidly under selective conditions than their hosts and contemporary RNA viruses may have descended from a common ancestor only relatively recently. The survival of influenza A and B viruses as new viruses depends on a continual evolution of mutants. These mutant forms are not recognized by the body as being variations of past viral exposures. The most frequent cause of new viral infections is old viruses that are not natural infections of human beings, but rather are accidentally transmitted from other species as zoonoses.

Organisms intermediate between viruses and bacteria are obligatory intracellular organisms with cell walls (e.g., rickettsiae) and without cell walls but capable of extracellular replication (e.g., Mycoplasma). Immunologically, the former are closer to viruses and the latter are closer to bacteria.

Dengue Fever

The rapidly expanding global footprint of Dengue fever is a public health challenge. An estimated 50 million infections occur every year in about 100 countries with the potential to spread to further. According to the World Health Organization and the Centers for Disease Control and Prevention, Florida and the coastal areas of Texas are included in the geographic areas that have high suitability for Dengue transmission. The major areas of disease are endemic tropical and subtropical latitudes (e.g. India, Southeast Asia). The primary vector is the urban-adapted Aedes aegypti mosquito. Global trade, with the unintentional transport of mosquitoes, and increased travel by viremic people, urban crowding, and ineffective mosquito control are all factors in this modern pandemic.

Dengue can be caused by one of four single-stranded, positive-sense RNA viruses (serotypes dengue virus type 1 to dengue virus type 4) of the Flavivirus genus. After an incubation period of 3 to 7 days, signs and symptoms start suddenly and follow three phases—initial febrile phase, a critical phase at about the time that the fever subsides (defervescence), and the final spontaneous recovery phase.

Most dengue virus infections are asymptomatic, with a wide variety of clinical manifestations. Signs and symptoms range from mild febrile illness to severe and fatal disease.

Laboratory diagnostic testing is by detection of viral components in serum or directly by serologic testing. Diagnostics tests are as follows:

• Viral component testing

• Detection of viral nucleic acid in serum by reverse-transcriptase polymerase-chain reaction (RT-PCR)

• Detection of soluble nonstructural protein 1 (NS1) by enzyme-linked immunosorbent assay (ELISA) or lateral-flow rapid testing

• Serologic testing

• Detection of IgM seroconversion by ELISA

• Lateral flow methods

• Detection of IgG in secondary infections by ELISA or lateral flow methods

Currently, no effective antiviral agents are available to treat dengue infection. Treatment is supportive. If patients have severe bleeding, a blood transfusion can be lifesaving. Clinical research with potential drugs or vaccines is ongoing.

Herpesviruses

Two members of the human herpesviruses, cytomegalovirus (CMV) and Epstein-Barr virus (EBV), are described in detail in Chapters 21 and 22. The following sections briefly describe other members of the human herpesvirus family, including herpes simplex, varicella-zoster, and human herpesvirus-6.

All the human herpesviruses are large, enveloped DNA viruses that replicate within the cell’s nucleus. The virus gains an envelope when the virus buds through the nuclear membrane, which has been altered to contain specific viral proteins.

The herpesviruses cause a number of clinical diseases, although they share the basic characteristic of being cell-associated, which may partly account for their ability to produce subclinical infections that can be reactivated under appropriate stimuli.

Herpes Simplex Virus

Herpes simplex virus (HSV) can be cultured from the oropharynx in about 1% of healthy adults and from the genital tract of slightly less than 1% of asymptomatic adult women who are not pregnant. HSV is widespread. Human beings are the only natural hosts or known reservoir of infection. The incubation period is 2 to 12 days. The incidence of seropositivity rises to almost 100% in some populations by the age of 45 years. Antibody prevalence in adults varies greatly with socioeconomic class; 30% to 50% of upper socioeconomic class adults have detectable antibody to HSV compared with 80% to 100% of adults in lower socioeconomic groups.

The most frequent manifestation of HSV infection is the common cold sore or fever blister. HSV has been shown to be related to a wide variety of clinical syndromes and to subclinical infection, occurring with primary or recurrent disease. Recurrent HSV disease usually results from the reactivation of latent virus resting in paraspinal or cranial nerve ganglia that innervate the site of primary infection. Distant sites may be involved. Activated virus presumably travels down the axon to the skin (or other site) and induces disease. In some cases, exogenous reinfection can occur. Recurrence with cell-to-cell spread of virus occurs in the presence of serum-neutralizing antibodies.

Two cross-reacting antigen types of HSV have been identified, type 1 (HSV-1) and type 2 (HSV-2). HSV-1 is generally found in and around the oral cavity and in skin lesions that occur above the waist. HSV-2 is isolated primarily to the genital tract and skin lesions below the waist.

Congenital and Neonatal Infection

Malnutrition, severe illness, many acute childhood illnesses, and prematurity predispose infants and young children to disseminated primary infection. Neonatal HSV infections may be acquired in the antenatal or perinatal period. Active lesions in the mother’s genital tract at birth present the greatest risk of infection to the newborn. The spectrum of disease in an infected newborn varies from subclinical to severe. In cases of overwhelming generalized infection, the infant may develop encephalitis and respiratory failure; hepatic failure, with increasing jaundice and adrenal insufficiency, may occur. Infants who survive severe infection are frequently left with some neurologic damage and may have recurrent vesicular skin lesions for many years.

Laboratory Diagnosis

Methods for the laboratory diagnosis of HSV include isolation of the virus and direct detection of antigen in tissues or cytologic preparation through the use of immunofluorescence or immunoenzyme methods. In addition, detection of the virus in body fluids (using monoclonal antibodies) can be performed with immunoassays or immunoblot techniques. Serologic diagnosis of primary infections can be demonstrated when a fourfold or higher positive-sense RNA viruses rise in titer occurs. Titers may rise significantly in early recurrent infection but usually become stable at moderately high levels after multiple recurrences.

Varicella-Zoster Virus

Varicella-zoster virus (VZV) is the cause of two different types of clinical diseases resulting from the same virus infection. Primary infection with the virus results in the clinical manifestations of chickenpox. After a primary infection, the virus enters a latent phase, presumably within nuclei in the dorsal root ganglia. Reactivation of the virus results in the characteristic clinical manifestation of (zoster), known as shingles.

Epidemiology and Etiology

Human beings are the only natural hosts of VZV. Varicella primarily affects children age 2 to 5 years. The virus is endemic and highly contagious. Periodic epidemics do occur. The presumed route of transmission is through the respiratory tract.

Zoster is less communicable than varicella. This sporadic disease occurs most frequently in older individuals. Antibodies to varicella do not protect against reactivation or clinical zoster. The reactivation of VZV is associated with a depressed immune response. Patients with AIDS, older adults, and immunocompromised persons are at high risk of developing disease. In addition, manipulation of the spinal cord, local radiation therapy, and therapy that suppresses cellular immunity have been associated with triggering the onset of zoster.

Varicella has an incubation period of 14 to 17 days. There may be a 1- to 3-day prodromal period of fever, headache, and malaise. This precedes the eruption of the characteristic red macular rash, which progresses to papules, vesicles, and pustules that crust over and shed without scarring. Successive crops of lesions continue to appear for 2 to 6 days; therefore, multiple lesions in various stages of development are present at any one time.

The name of the virus reflects two associated diseases—varicella (chickenpox) and zoster (shingles). Primary infection with the virus results in the clinical manifestation of chickenpox. After this, the virus enters a latent phase, presumably within nuclei of neurons in dorsal root ganglia or cranial nerve sensory ganglia. The reactivity of the virus results in the clinical manifestations characteristic of zoster.

Signs and Symptoms

Complications of VZV include pneumonitis, encephalitic conditions, nephritis, hepatitis, myocarditis, arthritis, and Reye’s syndrome. Susceptible individuals who are immunosuppressed have a greater risk of complications after VZV exposure. Another complication can include febrile purpura, which can occur a few days after the onset of the rash and is seen in children and adults. This complication is characterized by thrombocytopenia and hemorrhage into the vesicles. Postinfection purpura, which begins 1 to 2 weeks after the appearance of the rash, is characterized by thrombocytopenia with gastrointestinal, genitourinary, cutaneous, and mucous membrane hemorrhage. More severe hemorrhagic complications include malignant varicella with purpura and purpura fulminans.

Zoster Infection

Zoster infection is characterized by vesicular eruptions, typically confined to one or two adjacent dermatomes. The viral replication follows the nerve fiber. Neuralgia accompanies the skin eruptions and can last for months after the skin heals. Persistent neuralgia can be severe and can last months or longer.

Neonatal Varicella Infection

Neonatal varicella may be acquired in utero or in the perinatal period and can result in congenital abnormalities. The infant is at greatest risk if the mother’s illness occurs 4 days or less before delivery.

Laboratory Diagnosis

The laboratory diagnosis of VZV is similar to HSV methods. Serologic methods include indirect immunofluorescence, which detects antibodies to specific membrane antigens, and EIA.

Rapid preliminary diagnosis can also be made by direct immunofluorescence to detect viral antigens in vesicular lesions. A smear of cells taken from lesions enables direct examination. A presumptive diagnosis can be made by examining scrapings from the base of a vesicular lesion and histologically observing multinucleated giant cells containing intranuclear inclusion bodies, or by observing virus particles on electron microscopy. The best way to confirm VZV infection is to recover the virus in human diploid fibroblast cell cultures.

Antibodies to varicella are detectable within several days of the onset of rash and peak at 2 to 3 weeks. Antibodies to zoster increase more rapidly and are detectable at the onset of clinical symptoms. Because of the rapid turnaround time and correlation with clinical symptoms, serologic methods are preferable to viral isolation methods. In addition, ELISA methods are valuable for assessing the immune status of adults.

Prevention

A vaccine is available for those in high-risk groups. The vaccine can be administered prior to an infection or to prevent reinfection because of waning immunity.

Human Herpesvirus 6

A new virus classified as a herpesvirus because of its shape, size, and in vitro behavior has been identified. Genomic analysis shows the virus to be molecularly unrelated to other human herpesviruses. Initially the virus was called B-lymphotropic virus, but subsequent studies indicated that T cells are the primary target of infection. This viral agent is classified as human herpesvirus 6 (HHV-6).

Patients with serologic evidence of acute HHV-6 infection are reported to experience mild nonspecific symptoms and cervical lymphadenopathy. The same agent has been implicated as the cause of roseola infantum (exanthema subitum). Up to 75% of infants develop antibody to HHV-6 by age 10 to 11 months, which suggests a high rate of seropositivity in the general population.

Laboratory methods include direct examination by immunofluorescence or immunoperoxidase staining of cells taken from lesions. In addition, PCR, DNA probes, and serologic methods (e.g., ELISA, radioimmunoassay, indirect immunofluorescence, latex agglutination) can be used.

Culture methods include the cocultivation of the patient’s peripheral blood cells with cord blood mononuclear cells and examination of these cultures after 5 to 10 days by electron microscopy. Anticomplement immunofluorescence of infected cell culture has also been used for antibody detection and titration.

Laboratory Detection of Immunologic Responses

Because immunoglobulin M (IgM) is usually produced in significant quantities during the first exposure of a patient to an infectious agent, the detection of specific IgM can be of diagnostic significance (see Chapter 2). This immunologic characteristic is particularly important in diseases that do not manifest decisive clinical signs and symptoms (e.g., toxoplasmosis) or under conditions in which a rapid therapeutic decision may be required (e.g., rubella).

Antibody Significance

In many diseases, infected individuals show a spectrum of responses. Some patients may develop and manifest antibodies from a subclinical infection or after colonization of an agent without actually developing disease. In these patients, the presence of antibody in a single serum specimen or a comparative titer of antibody in paired specimens may merely indicate past contact with the agent; the presence of antibodies cannot be used for the accurate diagnosis of a recent disease. In comparison, some patients may respond to an antigenic stimulus by producing antibodies that can cross-react with other antigens. These antibodies are nonspecific and may lead to misinterpretation of serologic tests.

Serologic diagnosis of recent infection using acute and convalescent specimens is the method of choice. Except for the detection of IgM or in diseases with no chance of developing an immune response (e.g., rabies virus, botulism toxin), testing a single specimen is usually not recommended. In a number of circumstances, when only one specimen is tested to determine immune status, antibody to past infection or to immunization can be determined.

The testing protocols described in this chapter for the immunologic detection of representative infectious diseases are examples of the types of procedures typically encountered in the immunology-serology laboratory.

TORCH Testing

Procedures that specifically evaluate the presence of IgM or IgG are frequently used to detect CMV, herpesviruses (types 1 and 2), Toxoplasma gondii, and rubella. The names of the tests have been grouped under the acronym TORCH: Toxoplasma, other (viruses), rubella, CMV, and herpes (Tables 15-2 and 15-3).

Table 15-2

TORCH Antibodies: Immunoglobulin M

Infectious Agent	Interpretation of Assay
CMV	Positive—IgM antibody to CMV detected; may indicate current or recent infection; 1:10 IV or greater = positive
HSV-1, HSV-2	Positive (>1.10 IV)—IgM antibody to HSV detected (ELISA); may indicate current or recent infection
Rubella	Positive—1.10 IV or greater; IgM antibody to rubella detected; may indicate current or recent infection or immunization
Toxoplasma gondii	Positive: 1.10 IV or greater; significant level of antibody detected; may indicate current or recent infection

TORCH, Toxoplasma, other (viruses), rubella, CMV, herpes; CMV, cytomegalovirus; HSV, herpes simplex virus; ELISA, enzyme-linked immunosorbent assay.

Adapted from Associated Regional and University Pathologists: ARUP test reference guide, 2011 (http://www.aruplab.com/Testing-Information/lab-test-directory.jsp).

Table 15-3

TORCH Antibodies: Immunoglobulin G

Infectious Agent	Interpretation of Assay
CMV antibody	Positive—≥1:10; IgG antibody to CMV detected; may indicate current or previous CMV infection.
HSV-1, HSV-2	Positive—≥1:10; IgG antibody to HSV detected (ELISA); may indicate current or previous HSV infection.
Rubella	Positive—10 IU/mL or greater; IgG antibody to rubella detected; may indicate current or previous exposure/immunization to rubella.
Toxoplasma gondii	≥6 IU/mL, negative; ≥9 IU/mL, positive; results may indicate current or past infection.