Lecture 2 diagnostic molecular microbiology bls


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Lecture 2 diagnostic molecular microbiology bls

  1. 1. Diagnosis of Infectious Disease
  2. 2. Traditional methods of diagnosing infectious disease have limitations that influence their clinical utility1. ISOLATION of the organism • may be time consuming • needs viable organism • long turn-around times for results (1d to 3 weeks)
  3. 3. 2. SEROLOGY • retrospective • lacks specificity and sensitivity3. DIRECT detection (DFA, latex) • lacks sensitivity • needs technical skill
  4. 4. detecting the response detecting the microbe
  5. 5. Direct Detection..going to the source
  6. 6. Applied Molecular Microbiology
  7. 7. •For the last 50-100 years, Medical Microbiology hasrelied on these techniques to diagnose infections•Molecular methods have promised a change intraditional medical microbiology. Faster results More sensitive and specific Adapted to instrumentation
  8. 8. Primarily involves the detection or manipulation ofnucleic acids (DNA,RNA)2 Main applications are  Detection of organism (diagnosis)  Characterisation (epidemiology)Both may be done directly on the organism (egbacteria)More commonly involves amplification of part ofthe genome
  10. 10. MOLECULAR DIAGNOSTIC TESTING IN THE MICROBIOLOGY LABORATORYEvaluate the needIdentify the changes to be introducedDevelop suitable protocolsProvide adequate resourcesEducate the staff and clientsEvaluate the procedure (long-term)Continuous improvement
  11. 11. Evaluate the Need1. Some traditional procedures are cost-effective and clinically relevant2. Identify areas where changes will improve existing procedures.  Reduce turn-around times  Increased sensitivity/specificity  New organisms for which there are no existing tests  Results are more clinically relevant
  12. 12. Evaluate the NeedOrganism significant by presence (clinicalparameters may need to be considered)If not - quantitative PCR necessaryFastidious, slow growing and organisms which fail togrowTransportation delays (viability)Cost versus clinical utility
  13. 13. Evaluate the NeedExample:There is a need to better diagnose CMV in transplantpatients. Infection with CMV in these patients can result in pneumonitis and death.Reason:•Present virus isolation can take up to 3 weeks for a result•Virus isolation is not sensitive•Virus in blood samples is viable for 24 hours only•CMV may be shed intermittently by healthy subjects(poor clinical value of existing test)
  14. 14. Identify the changes to be introduced1. Collect EDTA blood not heparinized (inhibits PCR)2. Transport specimen to laboratory at RT (not 4oC)3. Enter specimen test details on patient/result database4. CMV DNA in specimen is extracted (staff)5. Perform assay (new procedure)6. Introduce appropriate quality control measures7. Interpret results and clinical implications8. Enter result on result database and generate a report
  15. 15. Determine a Suitable Assay ProtocolUse PCR for this assay as it gives maximumsensitivity needed to give early prediction of disease.However, CMV may exist in normal hosts (latentphase) and detection may not necessarily equate todiseaseDevelop a quantitative PCR assay, and determinethe viral load that is clinically significant (ie leads todisease)Can use a kit assay ($120 per test) or develop an“in house” assay
  16. 16. Determine a Suitable Assay ProtocolQuantitative 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 AssayAssay Controls -Positive - Negative (5 or 10%) - EnvironmentalInternal QC - Swabs of work area - QC samples (sensitivity)External QC -QC samples (specificity and sensitivityNB: CONTAMINATION WITH PREVIOUSLY AMPLIFIEDPCR PRODUCT IS THE MAJOR HAZARD
  18. 18. Provide Adequate Resources LABORATORY SPACE•Need adequate facilities to perform PCR. Cannot domolecular diagnostics “on the cheap” Work Flow in MDU 1 2 3 4 5 Low High Low DNA Clean DNA DNA Level Room Level Level Specimen Amplification Specimen Reagent Extraction Detection Addition Preparation •Each area has dedicated equipment and labcoats •Traffic is in one direction only (low to high DNA levels)
  19. 19. Provide Adequate ResourcesMolecular diagnostic assays need dedicated equipment.(eg pipettes in each work area)Amplification instruments may be costly
  20. 20. Provide Adequate Resources STAFFINGManagement has to provide an adequate number oftechnically capable staff dedicated to the procedureStaff have to be trained in the new procedures and bemade aware of the important issues.Assay procedure and quality methods have to bedocumented. Someone has to take responsibility
  21. 21. Educate the ClientsChange in procedures has to be communicated to theclients using the service, ie the doctors requesting thetests. There will be changes in - result interpretation - test costs - specimen requirements - turn-around times
  22. 22. Evaluate Long-term PerformanceOnce the assay is accepted as routine procedure, monitorthe results for a number of indicators 1. QC results 2. Clinical significance 3. Incidence or prevalence data in the populationMonitor long-term cost benefit against other assays
  23. 23. Continuous ImprovementsExamine alternatives for more cost-effective or clinicallyrelevant applicationUse QC data to identify problems and introduce proceduresto correct theseIdentify new instrumentation as it becomes availableMaintain the technical capabilities of staff through TrainingLook what other labs are doing, and are they doing itbetter? (benchmarking)
  24. 24. Introducing a Molecular Assay Involves 5 Steps Specimen Collection An appropriate specimen must be collected from the correct site during the “clinical” phase of the disease process Nucleic Acid Extraction PCR Detection Reporting results
  25. 25. Purification and Isolation of Nucleic Acids
  26. 26. Purification and Isolation of Nucleic AcidsThe quality of a molecular test depends on the availability ofpure/clean, intact DNA/RNAIsolation and purification essentially consists of two parts: • lyse the cells and solubilise the NA • remove contaminating proteins /other NA/ macromoleculesMethod used depends on specimen typeLarge scale isolation is usually performed by caesiumchloride centrifugation.
  27. 27. CsCl gradient centrifugation• Centrifugation in CsCl which has highdensity Direction of migration• Sample is layered on top of CsClgradient together with Etbr• Tube is centrifuged in ultra centrifuge(4hr at 300000g)• Particles separate through differences intheir sedimentation rate (size & shape)
  28. 28. CsCl gradient centrifugation• Centrifugation in CsCl which has Direction of migrationhigh density• Sample is layered on top of CsClgradient together with Etbr• Tube is centrifuged in ultra centrifuge(4hr at 300000g)• Particles separate throughdifferences in their sedimentation rate(size & shape)• NA of given sedimentation coefficientmigrate down as a zone
  29. 29. Detection of product by agarose gel electrophoresisBottom of tube ispunctured 0.5 mL fractions collected
  30. 30. Purification and Isolation of Nucleic Acids Other Extraction Procedures: 1. phenol/chloroform mixture (DNA) 2. guanidinium isothiocyanate (RNA)Disadvantages• Time consuming/labour intensive• Involves the use of noxious chemicals• Phenol oxidises DNA/RNA resulting in loss of target NAOther Methods• Anion exchange chromatography• Boom process (silica particles)
  31. 31. Purification and Isolation of Nucleic AcidsAdvantages of the Boom method • Fast • No dangerous chemicals used • High recovery of pure NA • Can be used for both DNA and RNACommercial kits developed using Boom technology • QIAGEN • ROCHE MOLECULAR BIOCHEMICALS
  32. 32. Detection and Characterisation of DNAAfter extraction, the nucleic acid is used for diagnosis orcharacterisation of the organism 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. 34. Pulsed Field Gel ElectrophoresisGenotyping by restriction fragment-length polymorphism(RFLP) analysis of genomic DNA by Pulsed-Field GelElectrophoresis (PFGE)Considered to be the “gold” standard for strain typing inepidemiological analysis of bacteria
  35. 35. PFGE Profile ofAcinetobacter IsolatesRestriction enzyme patternof multi resistant strain showsdifferent profile from antibioticsensitive Isolates MR S
  36. 36. Hybridisation AnalysisPrinciples of hybridisation analysis is that single strandedDNA or RNA molecules of defined sequence (probe), canbase-pair to a second DNA or RNA molecule that contains acomplementary sequence (target).The stability of the hybridisation product depends on theextent that of base pairing that has occurred.Hybrid stability is expressed as the melting temperature (Tm)
  37. 37. DNA Hybridisation Double Stranded 5’-AAAGGGTTACGAACGACGCC-3’ DNA 3’-TTTCCCAATGCTTGCTGCGG-5’Target DNA
  38. 38. Hybridisation AnalysisThe probe is usually labeled with a marker and the targetDNA has been immobilised.Markers - radioactivity - fluorescence - protein (detected by antibody conjugate)Immobilised – Nitrocellulose/ nylon - Plastic plates
  39. 39. Southern blotting – large DNA fragments1. Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support.2. The DNA is immobilised by UV irradiation (cross-linking)3. Membrane is subjected to hybridisation with a labeled DNA probe4. Band of homology with the probe are detected. (radioactivity, chemiluminescence or colour)