1. Understanding the Clinical Microbiology Laboratory Carol R. Quinter Ph.D. January, 2007

3. Is This Possible?!!

4. The Role of the Clinical Microbiology Laboratory Clarify the presence of infection Clarify the presence of infection Specify the etiology Specify the etiology Motivate the appropriate selection of antimicrobic Motivate the appropriate selection of antimicrobic Promote the appropriateness of use of antimicrobic Promote the appropriateness of use of antimicrobic Assist in the identification, control, and prevention of nosocomial infections Assist in the identification, control, and prevention of nosocomial infections Assist in the identification, control, and prevention of infections in the community Assist in the identification, control, and prevention of infections in the community The quality microbiology laboratory will lead a physician to a specific diagnosis by requiring quality specimens which result in relevant information which when communicated in a timely manner results in appropriate therapy.

5. Responsibility Accurate, Clinically Relevant, Timely Data Garbage In—Garbage Out 250 million annually is wasted by the processing of improperly obtained and clinically irrelevant specimens, which results in: 250 million annually is wasted by the processing of improperly obtained and clinically irrelevant specimens, which results in:

6. Bad Information Becomes part of a patient’s medical record Becomes part of a patient’s medical record Has little value in patient care Has little value in patient care Perpetuates the misuse and overuse of antimicrobials Perpetuates the misuse and overuse of antimicrobials Contributes to the morbidity and mortality of nosocomial infections Contributes to the morbidity and mortality of nosocomial infections Has an adverse effect on length of stay Has an adverse effect on length of stay Has a negative effect on quality of care and patient outcome Has a negative effect on quality of care and patient outcome

7. Function of the Microbiology Laboratoy Determine the Etiology of the Infectious Process Microscopic Examination Microscopic Examination Culture Culture Immunochemical Immunochemical Molecular Molecular

8. Microscopic Examination Gram-stained Smear: Timely Timely Presumptive identification of etiology Presumptive identification of etiology Drives empiric therapy Drives empiric therapy Assesses quality of specimen and predicts clinical relevance of culture results Assesses quality of specimen and predicts clinical relevance of culture results Guide to the determination of colonization vs. infection Guide to the determination of colonization vs. infection

9. Interpretation of Gram-stained Smear Adequacy of specimen measured by presence of inflammation (WBC, RBC, Necrotic Debris) Adequacy of specimen measured by presence of inflammation (WBC, RBC, Necrotic Debris) Presence of epithelial elements may indicate specimen inadequacy. Look for ratio of 2:1, WBC:Epi Presence of epithelial elements may indicate specimen inadequacy. Look for ratio of 2:1, WBC:Epi Morphologic diversity of organisms present and their relative numbers is important and can provide important clinical clues Morphologic diversity of organisms present and their relative numbers is important and can provide important clinical clues Focus on predominant morphologic type(s) Focus on predominant morphologic type(s) Alert to unusual pathogen Alert to unusual pathogen

10. Normal Flora Specimens which are obtained from or through areas where normal flora exists, will grow normal flora. Normal flora is predictable and changes within 24 hours of hospitalization to hospital flora. All infectious agents establish themselves at portals of entry, prior to producing infection, hence become: “normal flora” Accessible sites to culture usually have large numbers of normal flora.

12. Dilemma of Colonization vs. Infection All data must be interpreted clinically.

13. Culture Historic, remains gold standard Historic, remains gold standard Not timely Not timely Laboratory is adverse environment Laboratory is adverse environment Host is adverse environment Host is adverse environment Depends on the laboratory’s ability to provide the organism with favorable conditions to grow Depends on the laboratory’s ability to provide the organism with favorable conditions to grow

14. Immunochemical Monoclonal Antibodies Substances (antigens) foreign to the body invoke the production of antibodies which are highly specific. Substances (antigens) foreign to the body invoke the production of antibodies which are highly specific. Monoclonal antibody technology allows us to produce large amounts of pure antibody which reacts to a specific antigen. Monoclonal antibody technology allows us to produce large amounts of pure antibody which reacts to a specific antigen.

15. Principal Tools Direct Fluorescent Antibody Direct Fluorescent Antibody Indirect Fluorescent Antibody Indirect Fluorescent Antibody ELISA ELISA Sensitivity—The ability to detect Sensitivity—The ability to detect Specificity—The ability to detect precisely Specificity—The ability to detect precisely

16. How Does It Work?

17. Pearls A positive is a postive A positive is a postive A negative means further testing A negative means further testing

18. Molecular Probes Probes Amplification—Gene or Signal Amplification—Gene or Signal Pulse Gel Pulse Gel

20. Usefulness of Molecular Technology Rapid diagnosis of infection, particularly in sterile body sites and particularly where numbers of organisms may be low and culture difficult Rapid diagnosis of infection, particularly in sterile body sites and particularly where numbers of organisms may be low and culture difficult Characterization of strains for epidemiologic purposes Characterization of strains for epidemiologic purposes Rapid identification of isolates which otherwise require complex procedures which are costly and take too much time Rapid identification of isolates which otherwise require complex procedures which are costly and take too much time Appropriate designation of genus and species of organisms Appropriate designation of genus and species of organisms Detection of resistance Detection of resistance

21. Susceptibility Testing Minimum Inhibitory Concentration Minimum Inhibitory Concentration MIC - Lowest Concentration of antimicrobic which will inhibit in vitro growth of microorganism. Minimum Bacteriocidal Concentration Minimum Bacteriocidal Concentration MBC -Lowest Concentration of antimicrobic which will kill a microorganism in vitro.

22. Tolerance The relationship of MIC to MBC in any drug/bug combination.

25. Interpretation of MIC Based on achievable concentration of antimicrobic following IM injection in healthy subjects. In general, the achievable in vivo concentration should be 2 to 4 times the MIC for an organism to test susceptible to the antimicrobic. The Therapeutic Index is the relationship of MIC to antimicrobic level.

30. The Future Infectious diseases continue to impose a huge global public health burden, accounting for more than one-quarter of all deaths annually and similar fraction of morbidity. Infectious diseases continue to impose a huge global public health burden, accounting for more than one-quarter of all deaths annually and similar fraction of morbidity.

31. Challenges New and changing pathogens New and changing pathogens Global eradication of old pathogens Global eradication of old pathogens Developing diagnostics Developing diagnostics Developing treatments and preventions and managing resistance Developing treatments and preventions and managing resistance Control of infections within institutions and other closed settings and designing effective surveillance strategies Control of infections within institutions and other closed settings and designing effective surveillance strategies Control of infectious disease outbreaks Control of infectious disease outbreaks Understanding the role of animal and insect reservoirs Understanding the role of animal and insect reservoirs Prevention of bioterrorism Prevention of bioterrorism