1. Diagnosing Infections
Chapter 17
Diagnosing Infections

2. Survey of Microbial Disease
Methods of identifying unknown microbes fall into three categories:
phenotypic - observable microscopic and macroscopic characteristics (morphology) as well as bacterial physiology or biochemistry
genotypic – genetic make up. Data can produce a unique profile of each bacterium. Being used as a sole resource for identifying bacteria
immunological – serology (analysis of blood); antibody-antigen reactions

3. Phenotypic Methods
Microscopic morphology – fresh or stained microorganisms from specimen; shape, size, stain reaction, cell structures
Macroscopic morphology – colony appearance; texture, size, shape, pigment, growth requirements
Physiological/biochemical characteristics – detection of presence or absence of particular enzymes or metabolic pathways
Chemical analysis – analyze specific chemical composition; cell wall peptides, cell membrane lipids

4. Genotypic Methods Assess genetic make-up Culture is not necessary
Precise, automated methods, quick results

5. Immunological Methods
Specific antibodies are used to detect antigens

6. Specimen Collection and Laboratory Methods
Sampling body sites or fluids for suspected infectious agent
Results depend on specimen collection, handling, transport and storage
Aseptic procedures should be used

7. Insert figure 17.1
Sampling sites

10. Phenotypic Methods Observation – Direct antigen/antibody testing
macroscopic - cultivation – colony appearance, growth requirements, appropriate media
microscopic - differential and special stains – Gram, AFB, fluorescent antibody stains
Direct antigen/antibody testing
Biochemical testing – physiological reactions to nutrients as evidence of the absence or presence of enzymes

15. It is important to consider whether microbe recovered and identified is actually causing the disease or simply normal flora

16. Genotypic Methods DNA analysis
assess the proportion of G + C nucleotides relative to A + T content
determine DNA or ribosomal RNA sequences using probes and polymerase chain reactions

18. Immunological Methods
Serology – attempts to detect signs of infection in a patient by identifying specific antibodies in vitro
Visible reactions include precipitates, color changes, or the release of radioactivity
Tests can be used to identify and to determine the amount of antibody in serum – titer

20. Agglutination and Precipitation Reactions
Agglutination testing – antibody cross links whole-cell antigens, forming complexes that settle out and form visible clumps
blood typing, some bacterial and viral diseases
Precipitation tests – soluble antigen is made insoluble by an antibody
syphilis
Western blot – immunoelectrophoresis; separates antigens into bands
HIV

22. Cellular\molecular view
Insert figure 17.10
Cellular\molecular view

26. Complement Fixation
Detect antibodies that fix complement and lyse target cells
antigen, antibody, complement, and sensitized sheep RBCs
if complement is fixed by the Ag-Ab, the RBCs will not be lysed

28. Immunoassays
Extremely sensitive to detect trace antigens and antibodies
Radioimmunoassay (RIA) – antigens and antibodies labeled with radioactive isotopes
Enzyme-linked immunosorbent assay (ELISA) – enzyme-antibody complex produces a colored product when an enzyme-substrate reaction occurs

31. Tests That Differentiate T Cells and B Cells
Used to evaluate immune dysfunctions such as those in AIDS, immunodeficiencies, and cancer

33. In vivo Testing
Antigens are introduced directly into the body to determine the presence or absence of antibodies
tuberculin skin test, allergy testing

34. Viral Infection diagnosis