Lecture 21: MALDI-TOF and Sensitire Flashcards
MALDI-TOF is used to accurately and rapidly identify ___ and ___from clinical specimen or other sources
Bacteria, fungi
MALDI-TOF is a ___technique that uses a laser energy absorbing matrix ___ to create ions from large molecules with minimal fragmentation
Ionization, HCCA
What is time of flight
Time required to travel a distance through a medium
How does MALDI-TOF identify bacteria
Measures highly abundant proteins found in microorganisms and compares them to database
What are the MALDI-TOF reagents and supplies
- Target plate
- Toothpicks
- Target position
- HCCA matrix
- BTS
What does HCCA matrix do
Lyses bacterial cell walls and extract proteins, absorbs layer energy and plays role in desorption and ionization
What is BTS
Bacterial test standard used for calibration and quality control
Use E. Coli
What is calibration
Process to maintain accuracy of instrument. Configures instrument to provide result for a sample within an acceptable range
What is quality control
Way to maintain standards in manufactured products by testing a sample against the specification. Measures test performance
What are the MALDI-TOF steps for bacterial identification
- Collect sample
- Culture and incubate
- Examine plate for growth
4a. If pure culture- proceed to MALDI-TOF
4b. If mixed culture- subculture and then proceed to MALDI-TOF
What are the 3 types of extraction (lysis) methods
- Direct smear
- Formic acid extraction (smear/formic acid/ HCCA)
- Tube extraction (methanol/ acetonitrile/ formic acid)
How do you extract bacteria that produce a capsule or have excess exopolysaccharide matrix
Short/ 5 minute formic acid-acetonitrile extraction and centrifugation step before application on target plate
What is the purpose of the extracted method
Helps release internal proteins that can’t be extracted by matrix solution (HCCA) allowing for more efficient ionization and thus higher quality identification
What does a MALDI-TOF range of 2-3 indicate and what color would it be on results sheet
High confidence identification
Color: green
What does a MALDI-TOF range of 1.7-1.99 mean and what color
Low confidence identification
Color: yellow
What does a MALDI-TOF range of 0-1.69 mean and what color
No organism identification possible
Color: red
What does a category A MALDI-TOF result mean
High consistency: best match is a high confidence match. Second best match is high confidence in which species is identical to best match. Low confidence identification in which species or genus is identical to best match, or non-identification
What does a MALDI-TOF category B result mean
Low consistency- requirements for high consistency not met. Best match is a high or low confidence identification. Second best match is a high or low confidence identification in which genus is identical to best match or non-identification
What is a category C MALDI-TOF result
No consistency- requirements for high or low consistency not met
What could have occurred to result in no organism identification possible
- Organism not in MALDI-TOF database
- Too much bacterial cells on target plate
- Too little bacterial cells on target plate
- Reagent failure
- No peaks found- problem with preparation
What are some advantages to MALDI-TOF
- Fast- 45 minutes
- Minimal sample preparation time
- No gram stain required
- Low cost
- Non-expert identification
What are some limitations of MALDI-TOF
- Data base dependent
- Unable to discriminate phyletically related microorganisms such as Shigella and E. Coli
T or F: MALDI-TOF low confidence interpretations (1.7-1.99) means there are matches in MALDI-TOF database but matches did not meet established criteria for high confidence identification
True
What is sensititre MIC system
Equipment used to perform anti microbial susceptibility tests based on MIC values
What is principle of MIC
Determine lowest concentration possible to inhibit growth of test bacteria
What are the materials needed for Sensitire MIC testing
- 0.5 McFarland polymer turbidity standard
- Water
- Mueller-Hinton broth
- Antibiotic panels
- Quality control organisms
- 18-24hr old pure culture of test bacteria
What is the 0.5 McFarland polymer turbidity standard used for
To assure that the number of bacteria to test will be within given range
What are the quality control organisms used for Sensititre MIC testing
E.coli, pseudomonas aerguinosa, S. Aureus, Enterococcus faecalis
What is the COMPG1F Sensitire antibiotic panel
Used against gram positive organisms in companion animals
25 drugs
What is the COMPGN1F sensititre antibiotic panel
Used against gram negative organisms in companion animals
19 drugs
What is the EQUIN7F sensititre antibiotic panel
Used in horses
20 drugs
What is the BOPO7F sensititre antibiotic panel
Used for cows and pigs
20 drugs
What is the AVIAN1F sensititre antibiotic panel
Used for avian species
19 drugs
What is the SMV1AMAF sensititre antibiotic panel
Used for mastitis
10 drugs
What does it mean for sensititre result to be sensitive
Unable to grow in presence of particular antimicrobial concentration
What does it mean for sensititre result to be resistant
Grows in presence of particular antimicrobial concentration or in the presence of a particular antimicrobial
What does it mean for sensititre result to be intermediate sensitive
Higher dose of antimicrobial is needed to prevent growth
What does it mean for sensitire result to be no interpretation
Insufficient pharmacological data is available to determine the break point for MIC test interpretation
What is a breakpoint
Chosen concentration of antibiotic that defines whether a species of bacteria is susceptible or resistant to antibiotic
Who determines breakpoint
US-FDA, center for drug evaluation and research
What does the clinical laboratory standard institute do
Develop standard for clinical laboratory testing and in-vitro diagnostic test systems
What are the advantages for Susceptibility testing
- Assure susceptibility of drug of choice and help select antimicrobial therapy
- Detect resistant
What are some limitations to susceptibility testing
- In vitro can’t duplicate host environment
- Can’t include all biological variables within patient body
- Variability of drug distribution to infection site not considered
- Panel may not contain some preferred drugs
T or F: no interpretation implies that the sensitire software was unable to interpret results and sign results to sensitive, resistant or intermediate
True
