Chapter 11

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Copyright ©2019 F.A. Davis Company Chapter 11 Detection and Identification of Microorganisms
Copyright ©2019 F.A. Davis Company Objectives Identify advantages and disadvantages of using molecular-based methods as compared with traditional culture-based methods in the detection and identification of microorganisms. Explain the value of controls, particularly amplification controls, in ensuring the reliability of P C R results.
Copyright ©2019 F.A. Davis Company Objectives (continued) List the genes involved in the emergence of antimicrobial resistance that can be detected by nucleic acid amplification methods. Interpret pulse-field gel electrophoresis patterns to determine whether two isolates are related to or different from each other. Compare and contrast the molecular methods that are used to type bacterial strains in epidemiological investigations.
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Copyright ©2019 F.A. Davis Company Target Microorganisms for Molecular- Based Testing Those that are difficult or time-consuming to isolate for example, Mycobacteria Hazardous organisms for example, Histoplasma, Coccidioides Those without reliable testing methods for example, HIV, hepatitis C virus (H C V) High-volume tests for example, S. pyogenes, N. gonorrhoeae, C. trachomatis
Copyright ©2019 F.A. Davis Company Applications of Molecular-Based Testing Rapid or high-throughput identification of microorganisms Detection and analysis of resistance genes Genotyping Classification Discovery of new microorganisms
Copyright ©2019 F.A. Davis Company 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
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Copyright ©2019 F.A. Davis Company Specimen Collection (continued) The Clinical and Laboratory Standards Institute (C L S I) has guidelines for proper specimen handling
Copyright ©2019 F.A. Davis Company Sample Preparation Consider the specimen type (for example, stool, plasma, cerebrospinal fluid [C S F]) 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 C S F) Inactivate R N ases
Copyright ©2019 F.A. Davis Company P C R Detection of Microorganisms: Target Selection
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Copyright ©2019 F.A. Davis Company P C R Detection of Microorganisms: Quality Control P C R 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
Copyright ©2019 F.A. Davis Company P C R Quality Control: Internal Controls Homologous extrinsic Controls for amplification Heterologous extrinsic Controls for extraction and amplification Heterologous intrinsic Human gene control
Copyright ©2019 F.A. Davis Company Quality Control: False Positives Contamination: check reagent blank Dead or dying organisms: retest 3 to 6 weeks after antimicrobial therapy Detection of less than clinically significant levels
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Copyright ©2019 F.A. Davis Company Quality Control: False Negatives Improper collection, specimen handling Extraction/amplification failure: check internal controls Technical difficulties with chemistry or instrumentation: check method and calibrations
Copyright ©2019 F.A. Davis Company Viruses “Classic methods” of detection include antibody detection, antigen detection, and culture Molecular methods of detection include target, probe, and signal amplification Test are designed for identification of virus, determination viral load (number of viruses per milliliter of fluid), and genotyping by sequence analysis
Copyright ©2019 F.A. Davis Company Viral Load Number of viruses per milliliter of body fluid Measured by quantitative polymerase chain reaction (q P C R), digital P C R (d P C R), or other methods Measurements by different methods may not be equivalent Technical variability Biological variability
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Copyright ©2019 F.A. Davis Company Test Performance Features for Viral Load Measurement Characteristic Description Sensitivity Lowest level detected at least 95% of the time Accuracy Ability to determine true value Precision Reproducibility of independently determined test results Specificity Negative samples are always negative, and positive results are true positives Linearity A serial dilution of standard curve closely approximates a straight line Flexibility Accuracy of measurement of virus regardless of sequence variations
Copyright ©2019 F.A. Davis Company Antimicrobial Agents Inhibit growth (- static ), for example, bacteriostatic, fungistatic Kill organisms (- cidal ), for example, bacteriocidal, fungicidal, viricidal Antimicrobial agents are classified by: 1. -static/-cidal 2. Mode of action 3. Chemical structure
Copyright ©2019 F.A. Davis Company Sites of Action of Antimicrobial Agents
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Copyright ©2019 F.A. Davis Company 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
Copyright ©2019 F.A. Davis Company 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
Copyright ©2019 F.A. Davis Company 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
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Copyright ©2019 F.A. Davis Company 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
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Copyright ©2019 F.A. Davis Company Molecular Epidemiology (continued) Phenotypic analysis measures biological characteristics of organisms Molecular epidemiology is a genotypic analysis targeting genomic or plasmid D N A Species-, strain-, or type-specific D N A sequences are the sources of genotype information
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Copyright ©2019 F.A. Davis Company Summary Molecular-based methods offer sensitive and direct detection of microorganisms Specimen collection and handling are critical for successful molecular analysis Quantitative viral load testing requires proper validation for accuracy Due to high sensitivity and specificity, proper quality control is critical for molecular testing
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Copyright ©2019 F.A. Davis Company Summary (continued) Molecular methods are used to type bacterial strains in epidemiological investigations
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