Diagnosing Infections

Chapter 17

Adapted from McGraw Hill by Dr. G Cornwall

17.1 Preparation for Survey of Microbial Diseases

• Sample collection • Phenotypic analyses • Morphology • Biochemistry • Chemical analyses • Genotype analyses • Immunological analyses

Phenotypic Methods • Microscopic morphology • cell shape and size • gram stain & acid fast reactions • special structures - endospores, granules, and capsules • Macroscopic morphology - traits assessed with naked eye • appearance of colonies (texture, size, shape, pigment, patterns of growth • Physiological/Biochemical characteristics • test for presence of specific enzymes • metabolic activities (fermentation of sugars, metabolism of polymers, production of gasses) • sensitivity to antimicrobial drugs • Chemical analysis - analyzing specific structures of substances • chemical composition of peptides in cell wall or lipids in membrane Genotypic Methods

• Primary advantage over phenotypic methods: actually culturing the microorganisms is not always necessary • Also are increasingly automated with results obtained very quickly • Scientist are beginning to realize there are more microbes that we cannot grow in culture (recently 30 new species in plaque alone)

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 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

Specimen Isolation and Identification

17.3 Phenotypic Methods

• Immediate direct examination of specimen • Gram stain • Acid-fast stain • Direct fluorescence antibody (DFA) tests • Direct antigen testing Cultivation of Specimen • Isolation media - selective media • Biochemical testing • Carbohydrate fermentation (acid and/or gas) • Hydrolysis of gelatin, starch, and other polymers • Enzyme actions such as catalase, oxidase, and coagulase • By-products of metabolism

Rapid Test

Common Classification Scheme 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

Concept Check

Which of the following is not a biochemical test used for identification of pathogens?

A. Carbohydrate fermentation B. Polymer degradation C. Enzyme activity D. Gram reaction

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 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 • FISH- fluorescent in situ hybridization • Hybridization with 16s RNA • Identification time reduced from 24h to 90 minutes

Polymerase Chain Reaction (PCR)

• Rapid identification of pathogens • Developed for a wide variety of bacteria, viruses, protozoa, and fungi

Concept Check

What type of identification is the FISH analysis?

A. Morphological B. Genetic C. Biochemical D. Immunological 17.5 Immunologic Methods • Characteristics of antibodies can reveal the history of a patient’s contact with microorganisms or other antigens - underlaying basis of serological testing • Serology: the branch of immunology that traditionally deals with in vitro diagnostic testing of the serum • antibodies have extreme specificity to antigens - fit like gloves • powerful tool for identifying and quantifying antibodies (or antigens)

General Feature of Immune Testing

• Types of tests: agglutination, precipitation, complement fixation, fluorescent antibody tests, and immunoassay tests • The most effective tests have a high degree of Specificity and Sensitivity • specificity = focus on only a certain antigen or antibody (no false positives) • sensitivity = test can detect even small amounts of antibody or antigen (detect every positive person)

Visualizing Antigen- Antibody Interaction • Primary basis of serology testing - binding of an antibody to a specific molecular site on an antigen • Endpoint reaction visible to the naked eye • Agglutination vs Precipitation Agglutination and Precipitation Reactions • Agglutination: antigen-antibody reaction produces visible clumps • ABO, Rh factor • Rapid Plasma Reagin test - syphilis • Weil-Felix reaction diagnoses rickettsial infections • Agglutination tests largely replaced by fluorescent or genetic methods • Precipitation: the antigen is a soluble molecule made insoluble by antibody • Seen in test tube - opaque zone forma at border of antiserum and antigen solution • Venereal Disease Research Laboratory test

The Western Blot for Detecting Proteins

• Involves electrophoretic separation of proteins followed by immunoassay to detect them • Counterpart of the Southern blot test (used for DNA identification) • 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

Complement Fixation • Lysin or cytolysin: an antibody that requires complement to complete the lysis of its antigenic target cell • When lysins and the complement system act on red blood cells, hemolysis occurs • Lysin-mediated hemolysis is called complement fixation (CF) Concept Check

If you perform a complement fixation test and the RBCs lyse, what is your conclusion? A. The patient’s serum has antibodies to the pathogen. B. The patient’s serum does not have antibodies to the pathogen.

Fluorescent 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

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 • The more of the bound radioactive component there is after incubation, the less of the unknown test substance there was in the sample

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

A Viral Example