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Transcript
Molecular Diagnostics Detection and Identification of Microorganisms Chapter 12 1 Molecular Diagnostics 2 Applications of Molecular Based Testing in Clinical Microbiology Rapid or high-throughput identification of microorganisms Those that are difficult or time-consuming to isolate Hazardous organisms e.g., Histoplasma, Coccidiodes Those without reliable testing methods e.g., Mycobacteria e.g., HIV, HCV High-volume tests e.g., S. pyogenes, N. gonorrhoeae, C. trachomatis 3 Molecular Diagnostics Applications of Molecular Based Testing in Clinical Microbiology Detection and analysis of resistance genes Genotyping mecA oxacillin resistance in Staphylococcus aureus vanA, vanB, and vanC vancomycin resistance in Enterococcus katG and inhA isoniazid resistance in M. tuberculosis Mycobacterium, HCV, and HIV Reclassification of microorganisms for epidemiological purposes, and to predict therapeutic efficacy Discovery of new microorganisms 4 Molecular Diagnostics Specimen Collection Preserve viability/nucleic acid integrity of target microorganisms Avoid contamination that could yield false-positive results Due to the sensitivity of molecular testing Appropriate time and site of collection (blood, urine, other) Viability is not much critical for molecular testing DNA and especially RNA can be damaged in lysed or nonviable cells Obtimize the likely presence of the infectious agent E.g., Salmonella typhi is initially present in peripheral blood but not in urine or stool until at least 2 weeks after infection Use proper equipment (coagulant, wood, or plastic swab shafts) E.g., Plastics are less adherent to the microorganisms and will not interfere with PCR reagents as do emanations from wooden shafted swabs 5 Molecular Diagnostics Sample Preparation Depending on the microorganism more rigorous lysis procedures may be required Mycobacteria and fungi have thick cell walls that are more difficult to lyse than other bacteria and parasites. Gram-positive bacteria cell wall is thicker than gramnegative bacteria Mycoplasma, lacks a cell wall, thus avoid spontaneous lysis of the cells and loss of nucleic acids The concentration of organisms within the clinical sample must be considered. Centrifuge to concentrate the fluid and the organisms within the fluid 6 Molecular Diagnostics Sample Preparation Inhibitors of enzymes used in molecular analysis may be present in clinical specimens Acidic polysaccharides in sputum or polymerase inhibitors in CSF if RNA is to be analyzed inactivation or removal of RNases in the sample and in all reagents and materials that come into contact with the sample 7 Molecular Diagnostics Quiz In order to increase the stringency of a PCR reaction we need to, a) b) c) d) Decrease the annealing temperature and increase the annealing time Increase the annealing temperature and increase the annealing time Decrease the annealing temperature and decrease the annealing time Increase the annealing temperature and decrease the annealing time Molecular Diagnostics 8 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 template control: negative template Amplification control: omnipresent template unrelated to target Reagent blank/contamination control: no template present 9 Molecular Diagnostics PCR Quality Control: Internal Controls Homologous extrinsic wild-type–derived control with a nontarget-derived sequence insert Added to every sample after nucleic acid extraction and before amplification Amplification occurs using the same primers as for the target Good for ensuring that amplification occurs in the sample does not control for target nucleic acid degradation during Target sequence extraction. 10 Molecular Diagnostics PCR Quality Control: Internal Target sequence Controls Heterologous extrinsic Nontarget-derived controls Added to every sample before nucleic acid extraction Will ensure that extraction and amplification procedures are acceptable A second set of primers must also be added to the reaction for this control to be amplified. The procedure must be optimized such that the amplification of the control does not interfere with the amplification of the target. 11 Molecular Diagnostics PCR Quality Control: Internal Controls Heterologous intrinsic Eukaryotic genes. ensures that human nucleic acid is present in the sample in addition to controlling for extraction and amplification Requires two amplification reactions for the sample, or the amplification procedure be multiplexed Target sequence 12 Molecular Diagnostics 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 13 Molecular Diagnostics Quality Control: False negative Improper collection, specimen handling Extraction/amplification failure: check internal controls Technical difficulties with chemistry or instrumentation: check method and calibrations 14 Molecular Diagnostics Selection of Sequence Targets for Detection of Microorganisms 15 Molecular Diagnostics 16 Molecular Diagnostics 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 17 Molecular Diagnostics 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 18 Molecular Diagnostics 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 19 Molecular Diagnostics 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. 20 Molecular Diagnostics Pulsed-field Gel Electrophoresis (PFGE) Organisms with large genomes or multiple chromosomes DNA is digested with infrequently cutting restriction enzymes Large fragments (hundreds of thousands of base pairs) are resolved by PFGE Patterns of organisms will differ depending on the chromosomal DNA sequence of the organisms O = Outbreak strain 1-6 = Isolates = Changes from outbreak strain 21 Molecular Diagnostics Criteria for PFGE Pattern Interpretation: Rule of Three Category Genetic differences* Fragment differences* Epidemiological interpretation Indistinguishable 0 0 Test isolate is the same strain as the outbreak strain. Closely related 1 2–3 Test isolate is closely related to the outbreak strain. Possibly related 2 4–6 Test isolate is possibly related to the outbreak strain. Different >3 >6 Test isolate unrelated to the outbreak. *Compared to the outbreak strain. 22 Molecular Diagnostics Arbitrarily Primed PCR: Random Amplification of Polymorphic DNA (RAPD) MO M = Molecular weight marker O = Outbreak strain Four isolates differ from the outbreak strain. 23 Molecular Diagnostics Interspersed Repetitive Elements Enterobacterial repetitive intergenic consensus Repetitive extragenic palindromic PCR amplification priming outward from repetitive elements generates strain-specific products. Is the unknown (U) strain A or B? Molecular Diagnostics 24 Comparison of Molecular Epidemiology Methods Method Typing capacity Discriminatory power Reproducibility Ease of use Ease of interpretation Plasmid analysis Good Good Good High Good PFGE High High High Moderate Good moderate Genomic RFLP High Good Good High Moderate– poor Ribotyping High High High Good High PCR-RFLP Good Moderate Good High High RAPD High High Poor High Good–high AFLP High High Good Moderate High Repetitive elements Good Good High High High Sequencing High High High Moderate Good–high 25 Molecular Diagnostics 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. 26 Molecular Diagnostics 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 27 Molecular Diagnostics 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.