Porth's Essentials of Pathophysiology, 4e

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Infection and Immunity

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Serology Serology —literally, “the study of serum”—is an indi- rect means of identifying infectious agents by measur- ing serum antibodies in the diseased host. A tentative diagnosis can be made if the antibody level, also called antibody titer , against a specific pathogen rises during the acute phase of the disease and falls during conva- lescence. Serologic identification of an infectious agent is not as accurate as culture, but it may be a useful adjunct, especially for the diagnosis of diseases caused by pathogens such as the hepatitis B virus that cannot be cultured or diagnosis of past diseases. The measure- ment of antibody titers has another advantage in that specific antibody types such as IgM and IgG are pro- duced by the host during different phases of an infec- tious process. IgM-specific antibodies generally rise and fall during the acute phase of the disease, whereas the synthesis of the IgG class of antibodies increases during the acute phase and remains elevated until or beyond resolution. Measurements of class-specific antibodies are also useful in the diagnosis of congenital infections. IgM antibodies do not cross the placenta, but cer- tain IgG antibodies are transferred passively from mother to child during the final trimester of gestation. Consequently, an elevated level of pathogen-specific IgM antibodies in the serum of a neonate must have originated from the child and therefore indicates con- genital infection. A similarly increased IgG titer in the neonate does not differentiate congenital from maternal infection. The technology of direct antigen detection incorpo- rates features of culture and serology but reduces to a fraction the time required for diagnosis. In principle, this method relies on purified antibodies to detect anti- gens of infectious agents in specimens obtained from the diseased host. Common sources of these antibod- ies are hybridomas , cell lines created by fusing normal antibody-producing spleen cells from an immunized ani- mal with malignant myeloma cells. The resulting hybrid synthesizes large quantities of so-called monoclonal antibodies that are highly specific for a single antigen and a single pathogen. The antibodies are labeled with a substance that allows microscopic or overt detection when bound to the pathogen or its products. In general, the three types of labels used for this purpose are fluorescent dyes, enzymes, and particles such as latex beads. Fluorescent antibodies allow visualization of an infec- tious agent with the aid of fluorescence microscopy. Depending on the type of fluorescent dye used, the organism may appear bright green or orange against a black background, making detection extremely easy. Enzyme-labeled antibodies function in a similar man- ner. The enzyme is capable of converting a colorless compound into a colored substance, thereby permit- ting detection of antibody bound to an infectious agent without the use of a fluorescent microscope. Particles coated with antibodies clump together, or agglutinate, when the appropriate antigen is present in

a specimen. Particle agglutination is especially useful when examining infected body fluids such as urine, serum, or spinal fluid.

Protein Detection Mass spectrometry is a technique for determining the composition of a sample. It generates a protein-based profile or “fingerprint” from microbes that is unique to a given species. By analyzing the proteins that make up bacteria, yeast, or molds, clinical laboratories can quickly fingerprint these organisms and identify them based on the size and number of proteins detected. For example, analysis of bacteria such as S. aureus can often be accomplished by direct analysis of colony growth by the mass spectrometer within minutes of bacterial growth. DNA and RNA Detection Methods for identifying a pathogen by its unique DNA or RNA sequence are increasingly being used. Several techniques have been devised to accomplish this goal, each having different degrees of sensitivity regarding the number of organisms that need to be present in a speci- men for detection. The first of these methods is called DNA probe hybridization. Small fragments of DNA are cut from the genome of a specific pathogen and labeled with compounds (photo-emitting chemicals or antigens) that allow detection. The labeled DNA probes are added to specimens from an infected host. If the patho- gen is present, the probe attaches to the complementary strand of DNA on the genome of the infectious agent, permitting rapid diagnosis. The use of labeled probes has allowed visualization of particular agents within and around individual cells in histologic sections of tissue. A second and more sensitive method of DNA detec- tion is the polymerase chain reaction (PCR). This method allows technicians to tag a segment of patho- gen DNA—if present in the patient sample—and then multiply it to detectable levels. To perform the assay, a specimen containing the suspect pathogen is heated (Fig. 14-11). This causes the double-stranded DNA in the specimen to separate into single strands. It is then allowed to cool. Next, two short DNA sequences (usually less than 25 nucleotides long) called prim- ers are added to the specimen. These primers locate and bind only to the complementary target DNA of the pathogen in question. Then, a heat-stable DNA polymerase—an enzyme that catalyzes the synthesis of DNA—is added. It begins to replicate the DNA from the point at which the primers attached, similar to two trains approaching each other on separate but converging tracks. After the initial cycle, DNA polym- erization ceases at the point where the primers were located, producing two new strands of DNA. The specimen is heated again, and the process starts anew.