Limitations Flashcards
SEROLOGY LIMITATIONS
- Agglutination tests should be performed from growth from a
non-selective media.
*Must perform biochemical tests to prove organism is
Salmonella, Shigella, or E.coli 0157 before doing serology, since
other organisms can cross react
*Some organisms can auto agglutinate, giving false positive results. Perform a normal saline control with every test to ensure the specificity of the reaction
- Salmonella should be both O & H positive. If H is positive & O is negative then the organism may have a Vi
antigens masking the O antigens - If the organism agglutinates with Vi antisera and not O antisera, make a suspension of the organism in saline, boil for 15 min
STAPHAUREX limitation
- A Gram stain must be performed to ensure that only organisms with staphylococcal morphology are tested –
because some streptococci and other organisms like E. coli can cross react in the Staphaurex test. - Some MRSA may test falsely negative with kits that target only clumping factor and Protein A and not the antibody that targets Staph aureus
PATHODX limitation
- False-negative results can occur if not enough colonies used for extraction
- False-positive results can occur if too many colonies used for extraction
- Only beta-hemolytic colonies on sheep blood agar from 18 to 24 hour colonies should be tested
- Make sure the colony is catalase negative before testing
- Listeria monocytogenes which is BH exhibits similar antigenicity
with the Group B and G streptococci & will X react - But is catalase positive
TUBE COAGULASE limitation
- Test results can be positive at 4 hours and then revert to negative after 24 hours.
- If negative at 4 hours, incubate at room temperature overnight and check again for clot formation.
- Coagulase testing cannot be performed from growth on mannitol salt agar
SLIDE COAGULASE TEST limitation
- Saline control must show no clumping for the test to be valid (no auto agglutination)
- All negative slide tests must be confirmed using the tube coagulase
- If Auto-agglutination positive: the tests will be invalid
- Test these organisms using the tube test
OXACILLIN SCREEN FOR MRSA
Media: Use 2 Mueller Hinton agar plates (control & test)
Control plate: MHA + 4% NaCl
Test plate: MHA + 4 % NaCl + 6ug/ml of oxacillin
* NaCl is added to media because S. aureus is halophilic (salt loving)
* Test with oxacillin because methicillin in unstable & results unreliable
Inoculum:
* Make a 0.5 Mcfld standard of organism to be tested
* Use a loop to inoculate the standard to a pre-labelled spot of the control plate and test plate
Quality Control:
* Include control organisms MRSA and MSSA on both the control and test Mueller Hinton agar plates.
Incubation:
* Incubate for a full 24 hours in a 35 0C aerobic incubator.
* Temperature must not exceed 35 0C
Results:
* More than one colony of growth on the plate with oxacillin indicates resistance.
* Organism must also show growth on the control plate
OXACILLIN SCREEN LIMITATIONS
- When reading the plates inspect carefully
- Detection of resistance, is improved by using a larger inoculum use a micropipette and inoculate the plate with 10µl – because all colonies may not be resistant (heterogenous)
- Any isolate showing oxacillin R should be confirmed quantitatively by doing an MIC or by molecular methods to
detect the mecA gene - Do not use the OX test for the detection of methicillin/oxacillin
resistant coagulase-negative staphylococci.
VANCOMYCIN SCREEN
Media: Use 2 BHI agar (control & test)
Control plate: BHI agar
Test plate: BHI agar + 6ug/ml vancomycin
Inoculum:
* Make a 0.5 Mcfld standard of organism to be tested
* Dip a swab in the standard and touch end of swab on prelabelled spot of the control plate and test plate
Quality Control: Include control organisms VRE and VSE on
both the control and test BHI agar plates.
Incubation: A full 24 hours in a 350C aerobic incubator.
Results: Any amount of growth on the plate with vancomycin indicates resistance. Organism must also show
growth on the control plate
VANCOMYCIN SCREEN LIMITATIONS
- Some Enterococci are intrinsically resistant to vancomycin any species of Enterococcus growing on the vancomycin plate should be identified – E. gallinarum & E. casseliflavus are intrinsically resistant so not important
- Only E. faecium or E. faecalis have acquired R to Vanc
- Confirm a VANC screen pos test with an MIC to determine the precise concentration of vancomycin that the isolate is resistant to
- Molecular testing to determine the gene responsible for the resistance to vancomycin (VanA, VanB or VanC)
What to do when things go wrong…. MRSA/VRE
Control Organisms → In order for the VRE or MRSA screen to be considered valid:
* QC organisms must perform as expected
* QC organisms must grow on the control plate (media without antibiotic)
* The entire test must be repeated & don’t report patient results
Patient/Test Organisms→ to be able to report Patient/Test Results
* QC organisms must be valid & Patient/Test organisms must grow on the media without antibiotics
If controls are valid and only 1 patient/test is not growing on the control plate – do not report that patient’s results
* Repeat the screen for only the one patient with new controls
* You can report every other patient/test from those plates
* If control/s are not valid all must be repeated
KIRBY BAUER (DISK DIFFUSION) SUSCEPTIBILITY
TESTING
In General:
* 0.5 McFarland standardized suspension of bacteria
* Swab onto Mueller Hinton agar plate = Lawn of growth AFTER 15 MINS WHY
* Placement of antibiotic disks (with specific concentration) AFTER DRYING 3-5 MINS
* WITHIN 15 MINS WHY Incubation overnight – antibiotic diffuses into the media to form a circular zone around the disk
* A blood agar plate must be inoculated from the standard you made to check for purity
Rate of diffusion depends on:
* Agar depth
* Concentration of agar
* pH
KB RESULTS
- Check that your BA purity plate is pure befor measuring zone sizes
-Hold the plate a few inches above a black, non reflecting surface illuminated with reflected light - Colonies do not grow where there is sufficient antimicrobial to inhibit growth -MEASURE ZONES FROM BACK OF PLATE
- Zone of inhibition = diameter of obvious margin of no growth(mm) including disk -Susceptible, Intermediate or Resistant (CLSI)
-if there is one colony ignore and read the plate if more than double check the purity plate
-if there is a haze read within the lighter margin - hetero resistant population
-IGNORE HAZES FOR PROTEUS - With trimethoprimsulfamethoxazole (SXT), antagonists inthe medium may allow some slight growth; therefore, disregard slight growth (20% or less of the lawn of growth)
- If haze is 80% or more when compared to the growth of the lawn: measure from the haze margin
Interpretive Criteria
Susceptible ( S )
Intermediate ( I )
Resistant (R)
Non Susceptible
Susceptible ( S )-Isolates are inhibited by the usually achievable concentrations of antimicrobial agent when the recommended dosage is used for the site of infection
Intermediate ( I )-Isolates that respond to the antimicrobial agent but the response rate is lower than those of the susceptible isolates
Resistant (R)-Implies that isolates are not inhibited by drug as used at the recommended dosage level. microbial resistance mechanisms are likely
Non Susceptible -Isolate with only a susceptible interpretation because resistant strains are rare. Show high MIC levels upon testing. AST must be confirmed
KB Possible Errors With Kirby Bauer
Inoculum:
If your suspension is not a 0.5 Mcfld
* Too heavy = smaller zone sizes & false interpretation of intermediate or resistant
* Too light = larger zone sizes & false interpretation of susceptible
* Using organism from old growth > 1 day old = organism that may not grow or perform properly
* Touching mixed colonies will result in more than 1colony type on the plate so results are not accurate for only the organism you were testing- Must repeat the Kirby Bauer
KB Possible Errors Continued
Media:
If you don’t use the right type of media for the organism you are testing, or something wrong with media:
Examples: Mueller Hinton, Mueller Hinton with 5% sheep blood (Streptococci), Haemophilus Test Media (HTM) for Haemophilus
* Media must be the right depth (4mm)
* If too thin <3mm antibiotic diffuses too much = false susceptible
* If too thick > 5mm antibiotic can’t diffuse as much = false resistance
* Media must be in date and have passed QC
* Expired or invalid media is not reliable and will not give accurate results
Something wrong with media ingredients: CA 25mg/l -MG 12.5mg/l
* Increased Ca or Mg2+ concentrations result in DECREASED activity of aminoglycocides and tetracyclines
* Decreased Ca2+ or Mg2+ concentrations result in increased
activity of aminoglycosides & tetracyclines
* Thymidine should be minimal or absent
* Excessive concentrations can result in false resistance in sulfonamides and trimethoprim
* Media pH should be 7.2-7.4
