1. Molecular Diagnostics 1 Detection and Identification of Microorganisms Chapter 12

2. Molecular Diagnostics2 Applications of Molecular Based Testing in Clinical Microbiology Rapid or high-throughput identification of microorganisms  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

3. Molecular Diagnostics3 Applications of Molecular Based Testing in Clinical Microbiology Detection and analysis of resistance genes  mecA  oxacillin resistance in Staphylococcus aureus  vanA, vanB, and vanC  vancomycin resistance in Enterococcus  katG and inhA  isoniazid resistance in M. tuberculosis Genotyping  Mycobacterium, HCV, and HIV Reclassification of microorganisms for epidemiological purposes, and to predict therapeutic efficacy Discovery of new microorganisms

4. Molecular Diagnostics4 Specimen Collection Preserve viability/nucleic acid integrity of target microorganisms  Viability is not much critical for molecular testing  DNA and especially RNA can be damaged in lysed or nonviable cells Avoid contamination that could yield false-positive results  Due to the sensitivity of molecular testing Appropriate time and site of collection (blood, urine, other)  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 Diagnostics5 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 gram- negative 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 Diagnostics6 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 Molecular Diagnostics 6

7. 7 Quiz In order to increase the stringency of a PCR reaction we need to, a) Decrease the annealing temperature and increase the annealing time b) Increase the annealing temperature and increase the annealing time c) Decrease the annealing temperature and decrease the annealing time d) Increase the annealing temperature and decrease the annealing time

8. Molecular Diagnostics8 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 Diagnostics9 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 extraction. Target sequence

10. Molecular Diagnostics10 PCR Quality Control: Internal 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. Molecular Diagnostics 10 Target sequence

11. Molecular Diagnostics11 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 Molecular Diagnostics 11 Target sequence

12. Molecular Diagnostics12 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 Diagnostics13 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 Diagnostics14 Selection of Sequence Targets for Detection of Microorganisms Molecular Diagnostics 14

15. 15 Molecular Diagnostics 15

16. 16 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 Diagnostics17 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 Diagnostics18 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 Diagnostics19 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 Diagnostics20 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 Diagnostics21 Criteria for PFGE Pattern Interpretation: Rule of Three CategoryGenetic differences* Fragment differences* Epidemiological interpretation Indistinguishable00Test isolate is the same strain as the outbreak strain. Closely related12–32–3Test isolate is closely related to the outbreak strain. Possibly related24–64–6Test isolate is possibly related to the outbreak strain. Different>3>3>6>6Test isolate unrelated to the outbreak. * Compared to the outbreak strain.

22. Molecular Diagnostics22 Arbitrarily Primed PCR: Random Amplification of Polymorphic DNA (RAPD) M = Molecular weight marker O = Outbreak strain Four isolates differ from the outbreak strain. M O

23. Molecular Diagnostics23 Interspersed Repetitive Elements PCR amplification priming outward from repetitive elements generates strain-specific products. Is the unknown (U) strain A or B? Repetitive extragenic palindromic Enterobacterial repetitive intergenic consensus

24. Molecular Diagnostics24 Comparison of Molecular Epidemiology Methods MethodTyping capacity Discriminatory power ReproducibilityEase of use Ease of interpretation Plasmid analysis Good HighGood PFGEHigh ModerateGood moderate Genomic RFLP HighGood HighModerate– poor RibotypingHigh GoodHigh PCR-RFLPGoodModerateGoodHigh RAPDHigh PoorHighGood–high AFLPHigh GoodModerateHigh Repetitive elements Good High SequencingHigh ModerateGood–high

25. Molecular Diagnostics25 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 Diagnostics26 Test Performance Features for Viral Load Measurement CharacteristicDescription SensitivityLowest level detected at least 95% of the time AccuracyAbility to determine true value PrecisionReproducibility of independently determined test results SpecificityNegative samples are always negative and positive results are true positives LinearityA serial dilution of standard curve closely approximates a straight line FlexibilityAccuracy of measurement of virus regardless of sequence variations

27. Molecular Diagnostics27 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.