Week 4

Question 1

2 Types of Microbiology Laboratory Methods

Answer 1

Direct methods
- detect the agent

Indirect methods
- detect the host response to the agent

Question 2

Direct Methods

Answer 2

1. Culture-based methods
2. Macroscopic evaluation
3. Direct microscopy
4. Staining microscopy
5. Electron microscopy
6. Rapid tests
7. Molecular methods

In vitro culturing normally
not a prerequisite for numbers 2 to 7

Question 3

Culture-based Methods

Answer 3

Bacteria
Primary culture
Enrichment media
Selective/differential media or non-selective media
Culture conditions
Length of incubation

Question 4

Molecular Methods

Answer 4

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)

Question 5

The Disk-Diffusion Method for determining the activity of

antibiotics against bacterial pathogens

Answer 5

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.

Question 6

The Broth-Dilution Assay to determining the

Minimum Inhibitory Concentration (MIC) of Antibiotics

Answer 6

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

Question 7

Etest (Epsilometer test) for determining the

Minimum Inhibitory Concentration (MIC) of Antibiotics

Answer 7

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.

Question 8

European Committee on Antimicrobial Susceptibility Testing

Answer 8

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

Question 9

what is a screening test EUCAST

Answer 9

A screening test uses an agent to predict resistance or susceptibility to one or more antimicrobial agents in the same class

Question 10

Breakpoints EUCAST

Answer 10

Breakpoints in brackets distinguish between isolates without and with phenotypically detectable resistance mechanisms.

Question 11

EUCAST disk diffusion method steps

Answer 11

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

Question 12

EUCAST reading guide for broth microdilution

Answer 12

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.

Question 13

Guideline for reporting of antimicrobials for NZ microbiology labs

Answer 13

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

Question 14

Carbonic acid equation

Answer 14

CO2 + H2O <–> H2CO3 <–> H + HCO3

Question 15

Rapid detection of mycobacteria in clinical specimens by using the automated BACTEC 9000 MB System

Answer 15

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

Question 16

Nucleic Acid Amplification Techniques (NAAT)

Answer 16

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

Question 17

Nucleic Acid Amplification Techniques (NAAT)

No propagation: advantages/disadvantages

Answer 17

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

Question 18

Nucleic Acid Amplification Techniques (NAAT)

Propagation: advantages/disadvantages

Answer 18

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

Question 19

Basis of a genotypic test for RMP^R

Answer 19

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

Question 20

when did NZ set up NAC

Answer 20

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”

Question 21

Susceptible (S) - Standard dosing regimen

Answer 21

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

Question 22

I - Susceptible, increased exposure

Answer 22

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

Question 23

R - Resistant

Answer 23

A microorganism categorised as “resistant” when there is a high likelihood of therapeutic failure even when there is increased exposure