Week 4 Flashcards
2 Types of Microbiology Laboratory Methods
Direct methods
- detect the agent
Indirect methods
- detect the host response to the agent
Direct Methods
- Culture-based methods
- Macroscopic evaluation
- Direct microscopy
- Staining microscopy
- Electron microscopy
- Rapid tests
- Molecular methods
In vitro culturing normally
not a prerequisite for numbers 2 to 7
Culture-based Methods
Bacteria Primary culture Enrichment media Selective/differential media or non-selective media Culture conditions Length of incubation
Molecular Methods
Direct blotting
– no amplification step (where amount of DNA in specimen is sufficient for analysis)
• DNA of the microbial agent is extracted from the specimen
• The DNA is transferred onto a membrane and fixed
• The DNA is then hybridised with a radio-, chemiluminescent-, fluorescently-labelled probe targeting a specific sequence in the pathogen’s DNA
Polymerase chain reaction (PCR)
– amplification step used (where amount of DNA in specimen is not sufficient for analysis)
The Disk-Diffusion Method for determining the activity of
antibiotics against bacterial pathogens
Culture of bacterial species to be tested is spread onto agar
plates.
A disk of filter paper impregnated with a specific antibiotic is placed on the agar plate.
Sensitivity of the bacterial species to the antibiotic is seen as a zone of growth inhibition, the diameter of which can be
measured.
The Broth-Dilution Assay to determining the
Minimum Inhibitory Concentration (MIC) of Antibiotics
The minimum inhibitory concentration (MIC) is defined as the lowest concentration of an antibiotic that completely inhibits the growth of the test organism under a particular culture condition
Etest (Epsilometer test) for determining the
Minimum Inhibitory Concentration (MIC) of Antibiotics
The Etest (Epsilometer test) is a pre-defined gradient method of determining the antimicrobial susceptibility of antibiotics involving a “ready-to-use” strip.
Etest(Epsilometertest) for determining the Minimum Inhibitory Concentration (MIC) of Antibiotics
The Etest strip is placed on an agar surface which has been inoculated with the test bacterium and the antibiotic gradient on the strip transfers to the agar.
Bacterial growth becomes visible after incubation and inhibition is seen as a symmetrical ellipse centred along the strip.
The MIC value in μg/mL is read directly from the scale on the Etest strip at the point where the ellipse edge intersects the strip.
European Committee on Antimicrobial Susceptibility Testing
The main objectives of EUCAST are:
to harmonise antimicrobial breakpoints in Europe and
to act as the breakpoint committee for the European Medicines Agency during the registration
of new antimicrobial agents
what is a screening test EUCAST
A screening test uses an agent to predict resistance or susceptibility to one or more antimicrobial agents in the same class
Breakpoints EUCAST
Breakpoints in brackets distinguish between isolates without and with phenotypically detectable resistance mechanisms.
EUCAST disk diffusion method steps
Preparation and storage of media Preparation of inoculum Inoculation of agar plates Application of antimicrobial disks Incubation of plates Examination of plates after incubation Measurement of zones and interpretation of susceptibility
EUCAST reading guide for broth microdilution
broth microdilution is the reference method for antimicrobial susceptibility testing of rapidly growing aerobic bacteria, except for mecillinam and fosfomycin, where agar dilution is the reference method
Results are only valid when criteria are met:
Sufficient growth, Pure culture and correct inoculum of 5x10^5 CFU/mL
Growth appearance: Growth appears as turbidity or as a deposit of cells at the bottom of the well
Reading MIC (minimum inhibitory concentration) endpoints: results can be read manually and if automated reader is used then it must be calibrated to manual reading.
Interpretation of results: Make sure that MIC values for relevant quality control strains are within acceptable ranges and interpret MIC values into susceptibility categories.
Guideline for reporting of antimicrobials for NZ microbiology labs
Aim (to provide a framework for diagnostic microbiology labs within NZ)
Scope
Key Reporting strategies
1 Antimicrobial susceptibility reporting has a direct influence on prescribing practices by clinicians
2 Antimicrobial susceptibility reporting should be restricted to clinically relevant isolates
Carbonic acid equation
CO2 + H2O <–> H2CO3 <–> H + HCO3
Rapid detection of mycobacteria in clinical specimens by using the automated BACTEC 9000 MB System
For the growth and detection of AFB the BACTEC 9000 MB system differs from the BACTEC 9000 blood culture instrument in two ways: It uses an oxygen specific sensor and environmental parameters specific to mycobacteria have been accounted for
Nucleic Acid Amplification Techniques (NAAT)
Polymerase chain reaction (PCR)
-Detection of amplified gene segment used to confirm presence of pathogen
Quantitative real-time PCR (qPCR)
• Amplifies a pathogen-specific DNA sequence
• Uses a fluorescently labeled probe to provide real-time
quantification of PCR product
Reverse transcriptase PCR (RT-PCR)
• Reverse transcribes the RNA of interest into cDNA
• The target gene is then amplified by PCR
• Can be combined with qPCR to produce qRT-PCR
Nucleic Acid Amplification Techniques (NAAT)
No propagation: advantages/disadvantages
Advantages
• fast (turn-around-time can be <1 hour)
• normally inexpensive
Disadvantages
• limited sensitivity in some cases (resulting in false negatives)
• low amount of target template may be present in the specimen
• limited specificity in some cases (resulting in false positives)
• without an in vitro culture selection step, many other microorganisms may be present in the specimen
Nucleic Acid Amplification Techniques (NAAT)
Propagation: advantages/disadvantages
Advantages
• Allows phenotypic antimicrobial drug susceptibility testing to be performed
• Provides purified isolate for storage e.g. for research experimentation
Disadvantages
• Specimen must contain viable, in vitro culturable organisms
• Increases the turn-around-time of the laboratory diagnosis
Basis of a genotypic test for RMP^R
Approx. 95% of Rifampicin resistant clinical isolates of Mycobacterium tuberculosis have a mutation in
an 81 bp region known as Cluster 1 (codons 507-533) of the rpoB gene which encodes the beta chain
of the DNA-dependent RNA polymerase enzyme.
This core region of the rpoB gene forms the basis of a genotypic test for the detection of RMP^R
Mycobacterium tuberculosis developed by Cepheid Inc.
PCR is used to amplify the 81-bp Cluster 1 region of rpoB. Fluorescently-labeled hybridization probes are then used to distinguish between the conserved wild-type rpoB sequence, and mutations in the core region that are associated with Rifampicin resistance
when did NZ set up NAC
New Zealand established a National Antimicrobial Susceptibility Testing Committee (NAC) in
2017 “to provide expert advice to New Zealand diagnostic laboratories and to further the collaboration
and sharing of skill development in the area of antimicrobial susceptibility testing within New Zealand”
Susceptible (S) - Standard dosing regimen
A microorganism is categorised as “Susceptible, standard dosing regimen”, when there is a high likelihood of therapeutic success using a standard dosing regimen of the agent
I - Susceptible, increased exposure
A microorganism is categorised as “Susceptible, Increased exposure”, when there is a high likelihood of therapeutic success because exposure to the agent is increased by adjusting the dosing regimen or by its concentration at the site of infection
R - Resistant
A microorganism categorised as “resistant” when there is a high likelihood of therapeutic failure even when there is increased exposure
