Lecture 2 diagnostic molecular microbiology bls

Diagnosis of Infectious Disease

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)

2. SEROLOGY

• retrospective

• lacks specificity and sensitivity

3. DIRECT detection (DFA, latex)

• lacks sensitivity

• needs technical skill

detecting the microbedetecting the response

Direct Detection..going to the source

Applied Molecular Microbiology

•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

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

MOLECULAR DIAGNOSTICS

MOLECULAR DIAGNOSTIC TESTING INTHE 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

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

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

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)

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

Determine a Suitable Assay ProtocolUse 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

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

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

Provide Adequate Resources

LABORATORY SPACE•Need adequate facilities to perform PCR. Cannot do molecular diagnostics “on the cheap”

Work Flow in MDU

1 2

Low

DNA

Level

3

High

DNA

Level

4

Low DNA

Level

5

Clean

Room

Reagent Preparation

Specimen Extraction

Amplification Detection

Specimen Addition

•Each area has dedicated equipment and labcoats•Traffic is in one direction only (low to high DNA levels)

Provide Adequate Resources

Molecular diagnostic assays need dedicated equipment.(eg pipettes in each work area)

Amplification instruments may be costly

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 bemade aware of the important issues.

Assay procedure and quality methods have to bedocumented. Someone has to take responsibility

Provide Adequate Resources

Educate the Clients

Change in procedures has to be communicated to the clients using the service, ie the doctors requesting thetests.

There will be changes in

- result interpretation

- test costs

- specimen requirements

- turn-around times

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

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)

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 siteduring the “clinical” phase of thedisease process

Purification and Isolation of Nucleic Acids

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.

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 intheir sedimentation rate (size & shape)

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

Detection of product by agarose gel electrophoresis

Bottom of tube is punctured

0.5 mLfractionscollected

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)

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

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

DIRECT METHODS OF DETECTING NUCLEIC ACIDS

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

PFGE Profile of Acinetobacter Isolates

Restriction enzyme patternof multi resistant strain shows different profile from antibiotic sensitive Isolates MR S

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)

DNA Hybridisation

5’-AAAGGGTTACGAACGACGCC-3’3’-TTTCCCAATGCTTGCTGCGG-5’

Double StrandedDNA

Target DNA

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

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)