1. Chapter 12 Detection and Identification of Microorganisms

2. Objectives • Identify the advantages and disadvantages of using molecular-based methods as compared to traditional culture-based methods. • Explain the value of controls, in particular amplification controls, in ensuring the reliability of PCR results. • Compare and contrast the molecular methods that are used to type bacterial strains in epidemiological investigations.

3. Target Microorganisms for Molecular-Based Testing • Those that are difficult or time-consuming to isolate • e.g.,Mycobacteria • Hazardous organisms • e.g.,Histoplasma, Coccidiodes • Those without reliable testing methods • e.g.,HIV, HCV • High-volume tests • e.g.,S. pyogenes, N.gonorrhoeae, C. trachomatis

4. Applications of Molecular Based Testing in Clinical Microbiology • Rapid or high-throughput identification of microorganisms • Detection and analysis of resistance genes • Genotyping • Classification • Discovery of new microorganisms

5. Specimen Collection • Preserve viability/nucleic acid integrity of target microorganisms • Avoid contamination • Appropriate time and site of collection (blood, urine, other) • Use proper equipment (coagulant, wood, or plastic swab shafts) • Commercial collection kits are available • The Clinical and Laboratory Standards Institute (CLSI) has guidelines for proper specimen handling

6. Sample Preparation • Consider the specimen type (stool, plasma, CSF) • More rigorous lysis procedures are required to penetrate cell walls • Consider the number of organisms in the sample • Inactivate inhibitors (acidic polysaccharides in sputum or polymerase inhibitors in CSF) • Inactivate RNases

7. PCR Detection of Microorganisms: Quality Control • PCR and other amplification methods are extremely sensitive and very specific. For accurate test interpretation, use proper controls. • Positive control: positive template • Negative control: negative template • Amplification control: omnipresent template unrelated to target • Reagent blank: no template present

8. Homologous extrinsic Controls for amplification Heterologous extrinsic Controls for extraction and amplification Heterologous intrinsic Human gene control PCR Quality Control: Internal Controls Target sequence

9. Quality Control: False Positives • Contamination: check reagent blank • Dead or dying organisms: retest 3–6 weeks after antimicrobial therapy • Detection of less than clinically significant levels

10. Quality Control: False Positives • Improper collection, specimen handling • Extraction/amplification failure: check internal controls • Technical difficulties with chemistry or instrumentation: check method and calibrations

11. Antimicrobial Agents • Inhibit growth (-static); e.g., bacteriostatic, fungistatic • Kill organisms (-cidal); e.g., bacteriocidal, fungicidal, viricidal • Antimicrobial agents are classified by: 1. static/-cidal 2. mode of action 3. chemical structure

12. Sites of Action of Antimicrobial Agents

13. Mechanisms for Development of Resistance to Antimicrobial Agents • Enzymatic inactivation of agent • Altered target • Altered transport of agent in or out • Acquisition of genetic factors from other resistant organisms

14. Advantages of Molecular Detection of Resistance to Antimicrobial Agents • Mutated genes are strong evidence of resistance • Rapid detection without culturing • Direct comparison of multiple isolates in epidemiological investigations

15. Molecular Epidemiology • Epidemic: rapidly spreading outbreak of an infectious disease • Pandemic: a disease that sweeps across wide geographical areas • Epidemiology: collection and analysis of environmental, microbiological, and clinical data

16. Molecular Epidemiology • Phenotypic analysis measures biological characteristics of organisms. • Molecular epidemiology is a genotypic analysis targeting genomic or plasmid DNA. • Species, strain, or type-specific DNA sequences are the sources of genotype information.

17. O = Outbreak strain 1-6 = Isolates = Changes from outbreak strain Pulsed-field Gel Electrophoresis (PFGE) M O 1 2 3 4 5 6 M O 1 2 3 4 5 6

18. Criteria for PFGE Pattern Interpretation: Rule of Three *Compared to the outbreak strain.

19. M O Arbitrarily Primed PCR: Random Amplification of Polymorphic DNA (RAPD) M = Molecular weight marker O = Outbreak strain Four isolates differ from the outbreak strain.

20. Interspersed Repetitive Elements REP sequence inverted repeat ERIC sequence inverted repeat PCR amplification priming outward from repetitive elements generates strain-specific products. Isolate A Isolate B M A B M A B U Is the unknown (U) strain A or B?

21. Other Genotypic Methods Used to Type Organisms • Plasmid fingerprinting with restriction enzymes • RFLP analysis • Amplified Fragment Length Polymorphism (AFLP) • Interspersed repetitive elements • Ribotyping • spa typing • Multilocus sequence typing

22. Comparison of Molecular Epidemiology Methods

23. Viruses • “Classical methods” of detection include antibody detection, antigen detection, or culture. • Molecular methods of detection include target, probe, and signal amplification. • Tests are designed for identification of viruses, determination of viral load (number of viruses per ml of fluid), and genotyping by sequence analysis.

24. Test Performance Features for Viral Load Measurement

25. Viral Genotyping • Viral genes mutate to overcome antiviral agents. • Gene mutations are detected by sequencing. • Primary resistance mutations affect drug sensitivity but may slow viral growth. • Secondary-resistance mutations compensate for the primary-resistance growth defects.

26. Summary • Molecular-based methods offer sensitive and direct detection of microorganisms. • Due to high sensitivity and specificity, proper quality control is critical for molecular testing. • Several molecular methods are used to type bacterial strains in epidemiological investigations. • Target, probe, or signal amplification procedures are also used to determine viral load.