Microbiology: A Systems Approach, 2nd ed. Chapter 17: Diagnosing Infections

17.1 Preparation for the Survey of Microbial Diseases Methods used to identify bacteria to the level of genus and species Phenotypic methods Morphology Physiology or biochemistry Immunologic method Serological analysis Genotypic techniques More and more often used as a sole resource for identifying bacteria

Phenotypic Methods Microscopic morphology Macroscopic morphology Physiological/Biochemical characteristics Chemical analysis

Microscopic Morphology Cell shape and size Gram stain reaction Acid fast reaction Special structures

Macroscopic Morphology Colony appearance Speed of growth Patterns of growth

Physiological/Biochemical Characteristics Traditional mainstay of bacterial identification Diagnostic tests for determining the presence of specific enzymes and assessing nutritional and metabolic activities Examples Fermentation of sugars Capacity to metabolize complex polymers Production of gas Presence of enzymes Sensitivity to antimicrobic drugs

Chemical Analysis Analyzing the types of specific structural substances that the microorganism contains Examples Chemical composition of peptides in the cell wall Lipids in membranes

Genotypic Methods Primary advantage over phenotypic methods: actually culturing the microorganisms is not always necessary Also are increasingly automated with results obtained very quickly

Immunologic Methods Antibody response to antigens Blood testing- often easier than testing for the microbe itself Laboratory kits available for immediate identification of a number of pathogens

17.2 On the Track of the Infectious Agent: Specimen Collection The success of identification and treatment depends on how specimens are collected, handled, and stored General aseptic procedures must be used

Figure 17.1

Overview of Laboratory Techniques Direct tests using microscopic, immunologic, or genetic methods Cultivation, isolation, and identification of pathogens using a wide variety of general and specific tests Results of specimen analysis entered in a summary patient chart

Figure 17.2

Figure 17.3

17.3 Phenotypic Methods Immediate Direct Examination of Specimen Gram stain Acid-fast stain Direct fluorescence antibody (DFA) tests Direct antigen testing

Figure 17.4

Cultivation of Specimen Isolation media Biochemical testing Carbohydrate fermentation (acid and/or gas) Hydrolysis of gelatin, startch, and other polymers Enzyme actions such as catalase, oxidase, and coagulase By-products of metabolism

Figure 5

Figure 17.6

Miscellaneous Tests Phage typing Animal inoculation Antimicrobial sensitivity

Determining Clinical Significance of Cultures Is an isolate clinically important? How do you decide whether it is a contaminant or part of the normal biota? Possible criteria Number Repeated isolation of a relatively pure culture of any microorganism

17.4 Genotypic Methods DNA Analysis Using Genetic Probes Hybridization- can identify a bacterial species by analyzing segments of its DNA Small fragments of single-stranded DNA or RNA called probes Known to be complementary to the specific sequences of DNA from a particular microbe Unknown test DNA from cells is bound to blotter paper Add probes to blotter Observe for signs that the probes have become fixed to the test DNA

Figure 17.7

Nucleic Acid Sequencing and rRNA Analysis Comparison of the sequence of nitrogen bases in rRNA Effective for differentiating general group differences Can be fine-tuned to identify at the species level

Polymerase Chain Reaction Rapid identification of pathogens Developed for a wide variety of bacteria, viruses, protozoa, and fungi Biosensor

17.5 Immunologic Methods Characteristics of antibodies can reveal the history of a patient’s contact with microorganisms or other antigens Serological testing Serology: the branch of immunology that traditionally deals with in vitro diagnostic testing of the serum

Figure 17.8

General Features of Immune Testing Strategies Agglutination Precipitation Immunodiffusion Complement fixation Fluorescent antibody tests Immunoassay tests Specificity and sensitivity

Figure 17.9

Visualizing Antigen-Antibody Interactions Figure 17.10

Agglutination and Precipitation Reactions Agglutination: antigens are whole cells such as red blood cells or bacteria with determinant groups on the surface Precipitation: the antigen is a soluble molecule

Agglutination Testing Antibodies cross-link the antigens to form visible clumps Performed routinely to determine ABO and Rh blood types Widal test: tube agglutination test for diagnosing salmonelloses and undulant fever Rapid plasma regain (RPR) test: tests for antibodies to syph8ilis Weil-Felix reaction: diagnoses ricketsial infections Latex agglutination tests: tiny latex beads with antigens affixed

Precipitation Tests The soluble antigen is precipitated by an antibody Reaction is observable as a cloudy or opaque zone at the point of contact VDRL (Veneral Disease Research Lab) test Double diffusion (Ouchterlony) method Immunoelectrophoresis

Figure 17.11

Figure 17.12

The Western Blot for Detecting Proteins Involves electrophoretic separation of proteins followed by an immunoassay to detect those proteins Counterpart of the Southern blot test Test material is electrophoresed in a gel to separate out particular bands Gel transferred to a special blotter that binds the reactants in place Blot developed by incubating it with a solution of antigen or antibody labeled with radioactive, fluorescent, or luminescent labels

Figure 17.13

Complement Fixation Lysin or cytolysin: an antibody that requires complement to complete the lysis of its antigenic target cell

Figure 17.14

Miscellaneous Serological Tests Treponema pallidum immobilization (TPI) test Toxin neutralization tests Serotyping Quellung test

Flurorescent Antibodies and Immunofluorescence Testing Direct testing: an unknown test specimen or antigen is fixed to a slide and exposed to a fluorescent antibody solution of known composition Indirect testing: the fluorescent antibodies are antibodies made to react with the Fc region of another antibody

Figure 17.15

Immunoassays Extremely sensitive methods that permit rapid and accurate measurement of trace antigen or antibody Radioactive isotope labels Enzyme labels Sensitive electronic sensors

Radioimmunoassay (RIA) Antibodies or antigens labeled with a radioactive isotope used to pinpoint minute amounts of a corresponding antigen or antibody Compare the amount of radioactivity present in a sample before and after incubation with a known, labeled antigen or antibody Large amounts of a bound radioactive component indicate that the unknown test substance was not present Radioimmunosorbent test (RIST) Radioallergosorbent test (RAST)

Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme-antibody complex that can be used as a color tracer for antigen-antibody reactions Indirect Direct

Figure 17.16

Tests that differentiate T Cells and B Cells Mix with untreated sheep red blood cells T cells bind RBCs into a rosette formation Figure 17.17

In Vivo Testing Tuberculin test Other diagnostic skin tests

A Viral Example Figure 17.18