Transcript Document 7154075

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Performance Standards for
Antimicrobial Susceptibility testing
Dr S. Hekmat MD. CAP
Reference Laboratory of Iran
Dept Of Microbiology
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Antimicrobial Susceptibility Testing
( AST )
Indications for Performing AST
Methods of AST
Reagents for the Disk Difusion Test
Procedure for Performing Disk Difusion Test
Factors influencing AST
General comments and warning
Fastidious and problem organisms
Quality control in AST
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METHODS OF AST
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Diffusion
• Kirby – Bauer method
Dilution
MIC
1) Broth dilution
2) Agar dilution
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Diffusion &
Dilution
E – Test method
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Reagents for the Disk Diffusion Test
• 1- Mueller Hinton Agar medium
• 2- Preparation of Turbidity Standard
• 3- Antimicrobial Disks
• 4- Storage of antimicrobial discs
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Mueller Hinton Agar Medium
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Preparation of Mueller Hinton Agar
PH
Moisture
Effects of Thymidine or Thymine
Effects of Variation in Divalent Cations
Testing Strains That Fail to Grow Satisfactorily
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Turbidity Standard
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0.5 McFarland standard
• ( 0.5 ml of 0.048 mol/ml BaCl2 added to
• 99.5 ml of 0.18 mol/L H2SO4(1% v/v)
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density in 625 nm = 0.08 - 0.1
• Keep in 4-6 ml aliquots in screw cap
• tubes in dark , room temperature
• Replace or verify density monthly
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Procedure for Performing Disk Diffusion Test
• Inoculum Preparation
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Growth Method
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Direct Colony Suspension Method
• Inoculation of Test Plates
• Application of Disks to Inoculated Plates
• Reading Plates & Interpreting Results
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Fastidious or Problem Organisms
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Antibiotic susceptibility of Streptococcus . Pneumoniae
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Antibiotic susceptibility of other Streptococcus
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Antibiotic susceptibility of Enterococcus
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spp.
spp.
Antibiotic susceptibility of Staphylococcus spp.
Antibiotic susceptibility of Haemophilus spp.
Antibiotic susceptibility of Neisseria gonorrhoeae
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Antibiotic susceptibility of S. pneumonia
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Media : Mulller- Hinton with 5 % Sheep blood agar
inoculum : Direct colony suspension in normal saline
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Inoculated plates containing disks are incubated at 35C 20–24h and % 5 CO2.
The zone measurement is from the top of plate with the lid removed.
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Amoxicillin , Ampicillin , cefepime , cefotaxime, ceftriaxone , imipenem , meropenem may be used
treat pneumococcal infections , but they should be determined by MIC ( not DDM )
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Isolates of S. pneumonia with oxacillin disk zone ≥ 20mm ( MIC < 0.06 ) are susceptible to
Penicillin , and can be considered susceptible to : ampicillin , amoxicillin , clavulanic – acid ,
cephems , imipenem, meropenem and these agents need not to be tested.
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Oxacillin zone sizes ≤ 19 mm occur with penicillin – resistant , intermediate strains , so isolates
should not be reported as penicillin – resistant or intermediate until Penicillin , ampicillin ,
cephems , imipenem, meropenem MICs be determind.
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Penicillin , cefotaxime or ceftriaxone , and meropenem should be tested by MIC Method and
reported routinely for CSF isolates of S. pneumonia . These isolates should be tested for
vancomycin also.
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Antibiotic susceptibility of other Streptococcus spp
• Medium : Mueller Hinton agar
• Inoculum ; Growth or direct method
• Incubation ; 35C , ambient air , 16-18 h
• Susceptibility testing of penicillins and other β - lactams for treatment
of Streptococcus pyogenes or Streptococcus agalactiae is
not routinely necessary.
• Disk diffusion for penicilin , ampicillin , quinolones are for reporting
isolates of beta-hemolytic streptococci only.
• Viridans streptococci isolated from blood ,CSF ,.. Should be tested
for penicilin , ampicillin susceptibility testing bu MIC method.
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Antibiotic susceptibility of Enterococcus spp.
• Medium ; Mueller- Hinton agar
• Inoculum : Direct or growth method
• Incubation : 35 c, ambient air , 16-18h ,24h for vancomycin.
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For Enterococcus spp. Cephems , aminoglycosides (except for highlevel ), clindamycin , SXT , may be active invitro ,but are not clinically
effective ,and should not be reported susceptible.
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Penicillin susceptibility may be used to predict susceptibility to other
penicillins , for non - β - lactamase producing enterococci.
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A β - lactamase test is recommended for blood and CSF isolates. A
positive test predicts resistance to penicillin as well as amino ,carboxy –
and ureidopenicillins.
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Enterococcal endocarditis requires combined therapy.
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Antibiotic susceptibility of Staphylococcus spp.
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Medium : Mueller – Hinton agar
Inoculum : Direct colony suspension
Incubation : 35C ambient air , 16-18h , 24h for oxacillin , methicillin ,
nafcillin ,and vancomycin.
• Penicillin- susceptible staph are also susceptible to other
penicilins, carbapenems, cephems.
• Penicillin- resistant , oxacillin – susceptible strains are
also resistant to β -lactamase –labile penicillins ,but susceptible
to other β -lactamase stable penicillins, β -lactamase inhibitors ,
cephems , carbapenems.
• Oxacillin – resistant are resistant to all β -lactam antibiotics.
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Susceptibility or resistance to β -lactam antibiotics may be deduced
from testing only penicillin and oxacillin ,routine testing of others is not
advised.
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• Penicillin- resistant , oxacillin – susceptible strains staphs produce β lactamase and testing of 10-unit penicillin disk instead of ampicillin is
preferred.
• Penicillin should be tested for all of β - lactamase –labile penicillins.
• From β - lactamase- stable penicillins oxacillin is the choice , because
is more resistant to degradation in storage, is able to detect
heteroresistant strains. Cloxacillin disks should not be used, because
they may not detect oxacillin – resistant strains.
• For intermediate results perform oxacilin – salt agar test.
• Routine testing of urine isolates of S. saprophyticus is not advised if
there is not complicated UTI. They usually respond to concentrations
achieved in urine by commonly used antibiotics (F/M, SXT, Quinolone,.. )
• MRS are often resistant to multiple classes of antibiotics , like :
Aminoglycosides , Quinolones , clindamycin, SXT, macrolides,
tetracycline ,....
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Antibiotic susceptibility of Haemophilus spp.
• Medium : Haemophilus Test Medium ( HTM )
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Muller- Hinton agar
15 µ g/ml β -NAD
15 µ g/ml bovine hematin
5 µ 9/ml yeast extract
• Inoculum : Direct colony suspension
• Incubation ; 35 C , % 5 CO2 , 16 – 18 h.
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Only results of ampicilin , one of 3rd generation cephems
,chloramphenicol , meropenem should be reported routinely for CSF
isolates of Haemophilus influenzae.
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Β – lactamase test can provide a rapid means for detecting
ampicilin,amoxicillin resistance.
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Antibiotic susceptibility of Neisseria gonorrhoeae
• Medium : GC agar
• Inoculum ; Direct colony suspension
• Incubation ; 35 C , 5% CO2 , 20-24h
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Disk diffusion tests with ampicillin , penicillin , rifampin are unreliable for
Nisseria meningitidis , MIC is necessary.
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Positive β - lactamase test predicts resistance to ampicillin , penicillin
,amoxicillin.
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Gonococci with 10-unit penicillin disk zone ≤ 19 mm are likely to be
β - lactamase producing strains.
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β - lactamase test remains preferable to other susceptibility methods for
rapid , accurate penicillin resistance.
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“ WARNING “
The following antimicrobial agent / organism combinations may be active invitro
But are not clinically effective and should not be reported as susceptible.
Organism
Antibiotics that should not be reported susceptible
MRS, MRSA
All penems , cephems , other β- Lactams :, tazobactam
,imipenem ,piperacillin.
Enterococcus
Aminoglycosides , cephems , clindamycin , SXT
spp
Salmonella ,
Shigella spp
CSF isolates
First , second – generation cephalosporins ,
aminoglycosides
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First , second – generation cephalosporins
Clindamycin , macrolides , tetracyclines, quinolones
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Quality Control Procedures
• Purpose
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Precision & Accuracy of AST procedure
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Performance of used reagents
Performance of responsible personel
• Reference Strains for QC
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To control Precision & Accuracy
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Stock Strains for Quality Control
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Enterococcus Faecalis
Escherichia Coli
Escherichia Coli
Staphylococcus
aureus
Strep. Pneumoniae
P. aeruginosa
Kleb.Pneumonia
Haem. influenzae
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ATCC 29212
ATCC 25922
ATCC 35218
ATCC 25923
• ATCC 49619
• ATCC 27853
• ATCC 700603
• ATCC 49247
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Quality Control Procedures
• Storing Quality Control strains
• Zone Diameter Quality Control Limits
• Frequency of Quality Control Testing
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Daily Testing
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Testing
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Corrective Action
• Out-Of Control Result due to an obvious error
–Use of the wrong disk
–Use of wrong control strain
–Obvious contamination of strain
–Wrong procedure
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