1. Diagnosis of Infectious Disease

2. Traditional methods of diagnosing infectious
disease have limitations that influence their clinical
utility
1. ISOLATION of the organism
• may be time consuming
• needs viable organism
• long turn-around times for results (1d to 3 weeks)

3. 2. SEROLOGY
• retrospective
• lacks specificity and sensitivity
3. DIRECT detection (DFA, latex)
• lacks sensitivity
• needs technical skill

4. detecting the response detecting the microbe

5. Direct Detection
..going to the source

6. Applied Molecular Microbiology

7. •For the last 50-100 years, Medical Microbiology has
relied on these techniques to diagnose infections
•Molecular methods have promised a change in
traditional medical microbiology.
Faster results
More sensitive and specific
Adapted to instrumentation

8. Primarily involves the detection or manipulation of
nucleic acids (DNA,RNA)
2 Main applications are
 Detection of organism (diagnosis)
 Characterisation (epidemiology)
Both may be done directly on the organism (eg
bacteria)
More commonly involves amplification of part of
the genome

9. MOLECULAR DIAGNOSTICS

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. Evaluate the Need
1. Some traditional procedures are cost-effective and
clinically relevant
2. 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. Evaluate the Need
Organism significant by presence (clinical
parameters may need to be considered)
If not - quantitative PCR necessary
Fastidious, slow growing and organisms which fail to
grow
Transportation delays (viability)
Cost versus clinical utility

13. Evaluate the Need
Example:
There is a need to better diagnose CMV in transplant
patients.
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. Identify the changes to be introduced
1. Collect EDTA blood not heparinized (inhibits PCR)
2. Transport specimen to laboratory at RT (not 4oC)
3. Enter specimen test details on patient/result database
4. CMV DNA in specimen is extracted (staff)
5. Perform assay (new procedure)
6. Introduce appropriate quality control measures
7. Interpret results and clinical implications
8. Enter result on result database and generate a report

15. Determine a Suitable Assay Protocol
Use PCR for this assay as it gives maximum
sensitivity needed to give early prediction of disease.
However, CMV may exist in normal hosts (latent
phase) and detection may not necessarily equate to
disease
Develop a quantitative PCR assay, and determine
the viral load that is clinically significant (ie leads to
disease)
Can use a kit assay ($120 per test) or develop an
“in house” assay

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. Determine a Suitable Assay Protocol
Quality Control for the Assay
Assay Controls -Positive
- Negative (5 or 10%)
- Environmental
Internal QC - Swabs of work area
- QC samples (sensitivity)
External QC -QC samples (specificity and
sensitivity
NB: CONTAMINATION WITH PREVIOUSLY AMPLIFIED
PCR PRODUCT IS THE MAJOR HAZARD

18. Provide Adequate Resources
LABORATORY SPACE
•Need adequate facilities to perform PCR. Cannot do
molecular 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. Provide Adequate Resources
Molecular diagnostic assays need dedicated equipment.
(eg pipettes in each work area)
Amplification instruments may be costly

20. Provide Adequate Resources
STAFFING
Management has to provide an adequate number of
technically capable staff dedicated to the procedure
Staff have to be trained in the new procedures and be
made aware of the important issues.
Assay procedure and quality methods have to be
documented. Someone has to take responsibility

21. Educate the Clients
Change in procedures has to be communicated to the
clients using the service, ie the doctors requesting the
tests.
There will be changes in
- result interpretation
- test costs
- specimen requirements
- turn-around times

22. Evaluate Long-term Performance
Once the assay is accepted as routine procedure, monitor
the 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. Continuous Improvements
Examine alternatives for more cost-effective or clinically
relevant application
Use QC data to identify problems and introduce procedures
to 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 it
better? (benchmarking)

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. Purification and Isolation of Nucleic Acids

26. Purification and Isolation of Nucleic Acids
The quality of a molecular test depends on the availability of
pure/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 caesium
chloride centrifugation.

27. CsCl gradient centrifugation
• Centrifugation in CsCl which has high
density Direction of migration
• 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. CsCl gradient centrifugation
• Centrifugation in CsCl which has Direction of migration
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

29. Detection of product by agarose gel
electrophoresis
Bottom of tube is
punctured
0.5 mL
fractions
collected

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 NA
Other Methods
• Anion exchange chromatography
• Boom process (silica particles)

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
Commercial kits developed using Boom technology
• QIAGEN
• ROCHE MOLECULAR BIOCHEMICALS

33. Detection and Characterisation of DNA
After extraction, the nucleic acid is used for diagnosis or
characterisation 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. DIRECT METHODS OF DETECTING NUCLEIC ACIDS

35. Pulsed Field Gel Electrophoresis
Genotyping by restriction fragment-length polymorphism
(RFLP) analysis of genomic DNA by Pulsed-Field Gel
Electrophoresis (PFGE)
Considered to be the “gold” standard for strain typing in
epidemiological analysis of bacteria

36. PFGE Profile of
Acinetobacter Isolates
Restriction enzyme pattern
of multi resistant strain shows
different profile from antibiotic
sensitive Isolates MR S

37. Hybridisation Analysis
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).
The stability of the hybridisation product depends on the
extent that of base pairing that has occurred.
Hybrid stability is expressed as the melting temperature (Tm)

39. DNA Hybridisation
Double Stranded 5’-AAAGGGTTACGAACGACGCC-3’
DNA 3’-TTTCCCAATGCTTGCTGCGG-5’
Target DNA

40. Hybridisation Analysis
The probe is usually labeled with a marker and the target
DNA has been immobilised.
Markers
- radioactivity
- fluorescence
- protein (detected by antibody conjugate)
Immobilised
– Nitrocellulose/ nylon
- Plastic plates

41. Southern blotting – large DNA fragments
1. 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
probe
4. Band of homology with the probe are detected.
(radioactivity, chemiluminescence or colour)