DETECTION AND IDENTIFICATION OF MICROORGANISMS.PPTVIP

Chapter 12 Detection and Identification of Microorganisms 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. 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 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 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 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 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 PCR Quality Control: Internal Con