Lecture 2 diagnostic molecular microbiology bls


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Lecture 2 diagnostic molecular microbiology bls

  1. 1. Diagnosis of Infectious Disease
  2. 2. <ul><li>Traditional methods of diagnosing infectious disease have limitations that influence their clinical utility </li></ul><ul><li>ISOLATION of the organism </li></ul><ul><ul><li>• may be time consuming </li></ul></ul><ul><ul><li>• needs viable organism </li></ul></ul><ul><ul><li>• long turn-around times for results (1d to 3 weeks) </li></ul></ul>
  3. 3. <ul><li>2. SEROLOGY </li></ul><ul><ul><ul><ul><ul><li>• retrospective </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>• lacks specificity and sensitivity </li></ul></ul></ul></ul></ul><ul><li>3. DIRECT detection (DFA, latex) </li></ul><ul><ul><li>• lacks sensitivity </li></ul></ul><ul><ul><li>• needs technical skill </li></ul></ul>
  4. 4. detecting the microbe detecting the response
  5. 5. Direct Detection ..going to the source
  6. 6. Applied Molecular Microbiology
  7. 7. <ul><li>For the last 50-100 years, Medical Microbiology has relied on these techniques to diagnose infections </li></ul><ul><li>Molecular methods have promised a change in traditional medical microbiology. </li></ul><ul><ul><ul><ul><ul><li>Faster results </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>More sensitive and specific </li></ul></ul></ul></ul></ul><ul><ul><ul><ul><ul><li>Adapted to instrumentation </li></ul></ul></ul></ul></ul>
  8. 8. <ul><li>Primarily involves the detection or manipulation of nucleic acids (DNA,RNA) </li></ul><ul><li>2 Main applications are </li></ul><ul><ul><ul><li>Detection of organism (diagnosis) </li></ul></ul></ul><ul><ul><ul><li>Characterisation (epidemiology) </li></ul></ul></ul><ul><li>Both may be done directly on the organism (eg bacteria) </li></ul><ul><li>More commonly involves amplification of part of the genome </li></ul>
  10. 10. MOLECULAR DIAGNOSTIC TESTING IN THE MICROBIOLOGY LABORATORY <ul><li>Evaluate the need </li></ul><ul><li>Identify the changes to be introduced </li></ul><ul><li>Develop suitable protocols </li></ul><ul><li>Provide adequate resources </li></ul><ul><li>Educate the staff and clients </li></ul><ul><li>Evaluate the procedure (long-term) </li></ul><ul><li>Continuous improvement </li></ul>
  11. 11. Evaluate the Need <ul><li>Some traditional procedures are cost-effective and clinically relevant </li></ul><ul><li>2. Identify areas where changes will improve existing procedures. </li></ul><ul><ul><li>Reduce turn-around times </li></ul></ul><ul><ul><li>Increased sensitivity/specificity </li></ul></ul><ul><ul><li>New organisms for which there are no existing tests </li></ul></ul><ul><ul><li>Results are more clinically relevant </li></ul></ul>
  12. 12. Evaluate the Need <ul><li>Organism significant by presence (clinical parameters may need to be considered) </li></ul><ul><li>If not - quantitative PCR necessary </li></ul><ul><li>Fastidious, slow growing and organisms which fail to grow </li></ul><ul><li>Transportation delays (viability) </li></ul><ul><li>Cost versus clinical utility </li></ul>
  13. 13. Evaluate the Need <ul><li>Example: </li></ul><ul><li>There is a need to better diagnose CMV in transplant patients. </li></ul><ul><ul><li>Infection with CMV in these patients can result in pneumonitis and death. </li></ul></ul><ul><li>Reason: </li></ul><ul><li>Present virus isolation can take up to 3 weeks for a result </li></ul><ul><li>Virus isolation is not sensitive </li></ul><ul><li>Virus in blood samples is viable for 24 hours only </li></ul><ul><li>CMV may be shed intermittently by healthy subjects </li></ul><ul><li>(poor clinical value of existing test) </li></ul>
  14. 14. Identify the changes to be introduced <ul><li>Collect EDTA blood not heparinized (inhibits PCR) </li></ul><ul><li>2. Transport specimen to laboratory at RT (not 4 o C) </li></ul><ul><li>3. Enter specimen test details on patient/result database </li></ul><ul><li>4. CMV DNA in specimen is extracted (staff) </li></ul><ul><li>5. Perform assay (new procedure) </li></ul><ul><li>6. Introduce appropriate quality control measures </li></ul><ul><li>7. Interpret results and clinical implications </li></ul><ul><li>8. Enter result on result database and generate a report </li></ul>
  15. 15. Determine a Suitable Assay Protocol <ul><li>Use PCR for this assay as it gives maximum sensitivity needed to give early prediction of disease. </li></ul><ul><li>However, CMV may exist in normal hosts (latent phase) and detection may not necessarily equate to disease </li></ul><ul><li>Develop a quantitative PCR assay, and determine the viral load that is clinically significant (ie leads to disease) </li></ul><ul><li>Can use a kit assay ($120 per test) or develop an “in house” assay </li></ul>
  16. 16. Determine a Suitable Assay Protocol Quantitative PCR for CMV • Identify a suitable extraction method • Identify primers and probes from sequence database • Determine specificity of the primers and probes • Optimise reaction conditions • Determine analytical sensitivity of test (control) • Determine clinical sensitivity (clinical samples) • Laboratory evaluation (in parallel with existing method) • Document assay method and evaluation data
  17. 17. Determine a Suitable Assay Protocol Quality Control for the Assay NB: CONTAMINATION WITH PREVIOUSLY AMPLIFIED PCR PRODUCT IS THE MAJOR HAZARD Assay Controls -Positive - Negative (5 or 10%) - Environmental Internal QC - Swabs of work area - QC samples (sensitivity) External QC -QC samples (specificity and sensitivity
  18. 18. Provide Adequate Resources <ul><li>LABORATORY SPACE </li></ul><ul><li>Need adequate facilities to perform PCR. Cannot do molecular diagnostics “on the cheap” </li></ul>Work Flow in MDU Reagent Preparation Specimen Extraction Amplification Detection Specimen Addition <ul><li>Each area has dedicated equipment and labcoats </li></ul><ul><li>Traffic is in one direction only (low to high DNA levels) </li></ul>1 2 Low DNA Level 3 High DNA Level 4 Low DNA Level 5 Clean Room
  19. 19. Provide Adequate Resources <ul><li>Molecular diagnostic assays need dedicated equipment. </li></ul><ul><li>(eg pipettes in each work area) </li></ul><ul><li>Amplification instruments may be costly </li></ul>
  20. 20. <ul><li>STAFFING </li></ul><ul><li>Management has to provide an adequate number of technically capable staff dedicated to the procedure </li></ul><ul><li>Staff have to be trained in the new procedures and be </li></ul><ul><li>made aware of the important issues. </li></ul><ul><li>Assay procedure and quality methods have to be </li></ul><ul><li>documented. Someone has to take responsibility </li></ul>Provide Adequate Resources
  21. 21. Educate the Clients <ul><li>Change in procedures has to be communicated to the clients using the service, ie the doctors requesting the </li></ul><ul><li>tests. </li></ul>There will be changes in - result interpretation - test costs - specimen requirements - turn-around times
  22. 22. Evaluate Long-term Performance <ul><li>Once the assay is accepted as routine procedure, monitor the results for a number of indicators </li></ul><ul><li>QC results </li></ul><ul><li>2. Clinical significance </li></ul><ul><li>3. Incidence or prevalence data in the population </li></ul><ul><li>Monitor long-term cost benefit against other assays </li></ul>
  23. 23. Continuous Improvements <ul><li>Examine alternatives for more cost-effective or clinically relevant application </li></ul><ul><li>Use QC data to identify problems and introduce procedures to correct these </li></ul><ul><li>Identify new instrumentation as it becomes available </li></ul><ul><li>Maintain the technical capabilities of staff through Training </li></ul><ul><li>Look what other labs are doing, and are they doing it better? (benchmarking) </li></ul>
  24. 24. Introducing a Molecular Assay Involves 5 Steps Specimen Collection Nucleic Acid Extraction PCR Detection Reporting results An appropriate specimen must be collected from the correct site during the “clinical” phase of the disease process
  25. 25. Purification and Isolation of Nucleic Acids
  26. 26. Purification and Isolation of Nucleic Acids <ul><li>The quality of a molecular test depends on the availability of pure/clean, intact DNA/RNA </li></ul><ul><li>Isolation and purification essentially consists of two parts: </li></ul><ul><ul><li>• lyse the cells and solubilise the NA </li></ul></ul><ul><ul><li>• remove contaminating proteins /other NA/ macromolecules </li></ul></ul><ul><li>Method used depends on specimen type </li></ul><ul><li>Large scale isolation is usually performed by caesium chloride centrifugation. </li></ul>
  27. 27. CsCl gradient centrifugation Direction of migration • Centrifugation in CsCl which has high density • Sample is layered on top of CsCl gradient together with Etbr • Tube is centrifuged in ultra centrifuge (4hr at 300000g) • Particles separate through differences in their sedimentation rate (size & shape)
  28. 28. CsCl gradient centrifugation • Centrifugation in CsCl which has high density • Sample is layered on top of CsCl gradient together with Etbr • Tube is centrifuged in ultra centrifuge (4hr at 300000g) • Particles separate through differences in their sedimentation rate (size & shape) • NA of given sedimentation coefficient migrate down as a zone Direction of migration
  29. 29. Detection of product by agarose gel electrophoresis Bottom of tube is punctured 0.5 mL fractions collected
  30. 30. Purification and Isolation of Nucleic Acids <ul><li>Other Extraction Procedures: </li></ul><ul><ul><li>phenol/chloroform mixture (DNA) </li></ul></ul><ul><ul><li>2. guanidinium isothiocyanate (RNA) </li></ul></ul>Disadvantages • Time consuming/labour intensive • Involves the use of noxious chemicals • Phenol oxidises DNA/RNA resulting in loss of target NA Other Methods • Anion exchange chromatography • Boom process (silica particles)
  31. 32. Purification and Isolation of Nucleic Acids Advantages of the Boom method • Fast • No dangerous chemicals used • High recovery of pure NA • Can be used for both DNA and RNA <ul><li>Commercial kits developed using Boom technology </li></ul><ul><ul><li>• QIAGEN </li></ul></ul><ul><ul><li>• ROCHE MOLECULAR BIOCHEMICALS </li></ul></ul>
  32. 33. Detection and Characterisation of DNA <ul><li>After extraction, the nucleic acid is used for diagnosis or </li></ul><ul><li>characterisation of the organism </li></ul>Direct Methods • Agarose gel electrophoresis • Pulse field gel electrophoresis • Restriction fragment length polymorphism • Capillary electrophoresis • Hybridisation with NA probes Indirect Methods • After amplification of NA target • Real-time detection
  34. 35. Pulsed Field Gel Electrophoresis <ul><li>Genotyping by restriction fragment-length polymorphism (RFLP) analysis of genomic DNA by Pulsed-Field Gel Electrophoresis (PFGE) </li></ul><ul><li>Considered to be the “gold” standard for strain typing in epidemiological analysis of bacteria </li></ul>
  35. 36. PFGE Profile of Acinetobacter Isolates <ul><li>Restriction enzyme pattern </li></ul><ul><li>of multi resistant strain shows different profile from antibiotic sensitive Isolates MR S </li></ul>
  36. 37. Hybridisation Analysis <ul><li>Principles of hybridisation analysis is that single stranded DNA or RNA molecules of defined sequence (probe), can base-pair to a second DNA or RNA molecule that contains a complementary sequence (target). </li></ul><ul><li>The stability of the hybridisation product depends on the extent that of base pairing that has occurred. </li></ul><ul><li>Hybrid stability is expressed as the melting temperature (Tm) </li></ul>
  37. 39. DNA Hybridisation 5’-AAAGGGTTACGAACGACGCC-3’ 3’-TTTCCCAATGCTTGCTGCGG-5’ Double Stranded DNA Target DNA
  38. 40. Hybridisation Analysis <ul><li>The probe is usually labeled with a marker and the target DNA has been immobilised. </li></ul><ul><li>Markers </li></ul><ul><ul><li>- radioactivity </li></ul></ul><ul><ul><li>- fluorescence </li></ul></ul><ul><ul><li>- protein (detected by antibody conjugate) </li></ul></ul><ul><li>Immobilised </li></ul><ul><ul><li>– Nitrocellulose/ nylon </li></ul></ul><ul><ul><li>- Plastic plates </li></ul></ul>
  39. 41. Southern blotting – large DNA fragments <ul><li>Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support. </li></ul><ul><li>2. The DNA is immobilised by UV irradiation (cross-linking) </li></ul><ul><li>3. Membrane is subjected to hybridisation with a labeled DNA probe </li></ul><ul><li>4. Band of homology with the probe are detected. (radioactivity, chemiluminescence or colour) </li></ul>