Microbiology: A Systems Approach, 2 nd ed.

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

2. 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

3. Phenotypic Methods • Microscopic morphology • Macroscopic morphology • Physiological/Biochemical characteristics • Chemical analysis

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

5. Macroscopic Morphology • Colony appearance • Speed of growth • Patterns of growth

6. 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

7. 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

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

9. 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

10. 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

11. Figure 17.1

12. 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

13. Figure 17.2

14. Figure 17.3

15. 17.3 Phenotypic Methods • Immediate Direct Examination of Specimen • Gram stain • Acid-fast stain • Direct fluorescence antibody (DFA) tests • Direct antigen testing

16. Figure 17.4

17. 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

19. Figure 5

20. Figure 17.6

21. Miscellaneous Tests • Phage typing • Animal inoculation • Antimicrobial sensitivity

22. 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

23. 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

24. Figure 17.7

25. 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

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

27. 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

28. Figure 17.8

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

30. Figure 17.9

31. Visualizing Antigen-Antibody Interactions Figure 17.10

32. 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

33. 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

34. 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

35. Figure 17.11

36. Figure 17.12

37. 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

38. Figure 17.13

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

40. Figure 17.14

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

42. 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

43. Figure 17.15

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

45. 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)

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

47. Figure 17.16

48. 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

49. In Vivo Testing • Tuberculin test • Other diagnostic skin tests

50. A Viral Example Figure 17.18