Generally, the direct assays are most useful for the diagnosis of an active infection because they involve the direct measurement of microbial components. Furthermore, because direct assays can be quantitative, they also can be used to monitor the level of the infecting microorganism and to assess the course of the infection and the response to antimicrobial treatment.

Although assays that measure the immune response to an infecting organism are useful in many situations, several advantages are inherent in assays that directly assess the presence of microbial organisms in body fluids. Until recently, the direct detection of microbial pathogens was accomplished by immuno-assays designed to detect antigenic components of the microorganism. These assays are similar in design to the immunoassays used for measuring antibodies in that they measure the interaction between antigens and antibodies. However, antigen detection assays differ because they involve the interaction of antigens in a sample with an antibody of known specificity. This interaction generally is accomplished by labeling the antibody with a suitable marker such as a radioactive isotope, enzyme, or latex particle (see Table 147.1). In general, the enzyme-based assays offer the highest levels of sensitivity and specificity; however, the particle-based assays can be useful in situations outside clinical laboratories in which rapid results are needed. For example, particle agglutination assays are used widely in clinical settings to detect antigens from group A streptococci rapidly.

Assays for the direct detection of microbial agents offer numerous advantages in establishing diagnoses of acute infections. Because they do not require the generation of an active immune response, they can be used to ascertain the presence of an infectious process before the patient has had sufficient time to generate detectable antibodies. Furthermore, these assays can be used for diagnosing infections in immunocompromised individuals, neonates, and other patients who would not be expected to generate a predictable immune response to infection. The principal drawback of antigen detection assays is that they are limited by the kinetics of the antigen-antibody reactions used to make the diagnostic measurement. Although this limitation can be overcome partially by selecting antigens that are present in multiple copies in microbial organisms (e.g., the capsular polysaccharide of Haemophilus influenzae), many important pathogens do not contain antigens that can be used for this purpose. In such cases, the presence of more than 1,000 organisms may be required before a detectable signal could be obtained, meaning that the assays would not be useful for the diagnosis of infection early in the course of disease, when the antigen load is low. In addition, the generation of an effective immune response to infection generally leads to the production of antibodies that bind to the microbial antigens that
are the targets of the immunoassay reagents. Such antibodies can interfere with the sensitivity of the immunoassay, thus decreasing the utility of these assays late in the course of a chronic infectious process.

A principal limitation of direct assays is that the levels of the infecting microorganism in some disease states may be quite low, in which case only assays capable of detecting small quantities of microorganisms would be able to establish accurate diagnoses in all infected patients. Another limitation is that direct assays require the presence of the organism or its antigens in an accessible body site for a diagnosis to be made. Hence, diagnosing some infections, such as those caused by hepatitis A virus, Mycoplasma pneumoniae, and human immunodeficiency virus (HIV), in which symptoms generally occur after microbial replication has reached its peak, is difficult.

Many different assays are available for the detection of antibodies to infectious agents. Although these assays are named for the method that is used for the measurement of the antigen-antibody reaction, all the assays make use of a similar basic reaction, namely, the binding of the patient’s immunoglobulin to a defined microbial antigen.

Some of the problems of direct detection are overcome by the use of assays that measure the immune response to microbial infection. In such assays, serum (or, in some cases, urine or another body fluid) is tested for the presence of immunoglobulins directed at specific microbial organisms or at microbial components. The advantage of this procedure is that the organism need not be present in the sampled site when the measurement of an immune response is performed. Furthermore, because even a small quantity of infecting antigen can give rise to a large number of activated B cells, high degrees of sensitivity generally are not required for the detection of specific antibodies. Finally, because the immune response to an infecting microorganism persists for an extended period, antibody detection assays allow for the diagnosis of an infection after microbial replication has declined. These assays thus can be used for the diagnosis of infections that lead to chronic disease processes, such as infections with hepatitis C virus, dengue viruses, Borrelia burgdorferi, and Treponema pallidum.

In the case of lifelong infections such as those caused by HIV, the detection of antibody (in the absence of maternal antibody derived prenatally) is diagnostic for the disease process. In many other cases, this persistence of detectable antibody constitutes one of the principal limitations of antibody detection assays, in that the antibody can be present long after the infectious process is completed or the patient has undergone successful treatment. For this reason, the detection of antibody
often cannot, by itself, be considered diagnostic of a current infection. For example, a child with antibodies to enteroviruses may have acute bacterial meningitis at the time of testing, a patient with antibodies to Epstein-Barr virus may have acute streptococcal pharyngitis, and a patient with a high antibody titer to histoplasmin antigen may have tuberculosis. This problem can be overcome partially by measuring antibody classes that are associated with an early immune response to infection, such as immunoglobulin M (IgM) and (IgA). Such measurements are useful particularly in the diagnosis of infections that occur during the prenatal period. Because IgM and IgA class antibodies do not cross the normal placenta in appreciable quantities, the detection of these antibodies in the infant can be used to distinguish maternal from fetal infections. This method has been used to detect perinatal infections with cytomegalovirus, rubella virus, Toxoplasma and, more recently, HIV. Of note is that in the case of infections that occur later in life, the persistence of these acute-phase antibodies varies, rendering their detection unreliable as specific indicators of recent infection, particularly when one uses more sensitive assays, which detect small concentrations of IgM antibody long after the initial infection occurs. The possibility of false-positive results caused by the presence of rheumatoid factors and other autoimmune reactants also limits the specificity of these reactions for the definitive diagnosis of the infectious process.

One approach to improving the specificity of antibody detection assays for the diagnosis of recent infectious diseases takes advantage of the finding that certain components of infecting microorganisms often are selected by the immune system as the initial targets of the immune response. The measurement of antibodies to such early antigens is thus a reflection of a recent infection, especially if it occurs in the absence of antibodies to other components of the organism that develop later in the course of infection (late antigens). Such assays are useful particularly for the diagnosis of infections caused by cytomegalovirus, Epstein-Barr virus, and other herpesviruses, in which the viral targets of an early and late immune response have been well characterized. The recent characterization of the timing of the immune response to B. burgdorferi also indicates that the measurement of antibodies to different microbial components can be used as a more accurate way to characterize the status of patients presumed to have Lyme disease. As the immune response to other microbial organisms becomes better characterized, the measurement of early antigens will play a more important role in the diagnosis of a recent infection in children.

Most antibody assays involve the measurement of immunoglobulins in blood or serum specimens. Recently, measuring antibodies in mucosal body sites has become possible. Studies have indicated that antibodies to infectious antigens can be measured accurately in mucosal sites such as saliva and urine. These antibodies can arise either from transudation from the systemic circulation or in response to antigenic stimulation at the mucosal surface. In either case, the measurement of antibodies at the mucosal site provides an assessment of the immune response to the infecting microorganism.

Being able to measure a range of antibodies in readily accessible body fluids would be extremely helpful in diagnosing infections in young infants and in monitoring immune responses in a clinical practice setting. Table 147.1 presents examples of the antibody detection assays that may be useful to the pediatric practitioner. Current assay methods vary considerably in their ability to detect specific antibodies and hence in their sensitivity and specificity. In general, the solid-phase immunoassays [exemplified by solid-phase enzyme immunoassays, also known as enzyme-linked immunosorbent assays (ELISA)] offer the highest degree of sensitivity while allowing for objective quantitation and the inclusion of controls for maintaining specificity. The explosion of knowledge of the antigenicity of microbial proteins and techniques for their cloning and production in recombinant forms should lead to development of more sensitive and specific solid-phase assays for detecting antibodies to a wide range of infecting microorganisms.