sops of antibiotic
TRANSCRIPT
ANTIBIOTIC SUSCEPTABILITY TESTING
(Kirby Bauer Disk Diffusion test)
PURPOSE:
It is used to asses the antibiotic sensitivity of a certain
bacterial isolate.
SPECIMEN :
Pure bacterial isolate from fresh culture plate.
MATERIALS :
1. Nutrient broth for fastidious organisms or sterile saline
for non fastidious organisms.
2. 0.5 Mc Farland standard for adjusting the turbidity of
the inoculums.
3. Vortex mixer for suspension of the inoculum.
4. View box for comparison of broth with standard
5. Mueller-Hinton agar plates unsupplemented for non
fastidious organisms or supplemented with RBCs in a
concentration of 5% for fastidious organisms (90-mm
diameter for seven disks; 150-mm diameter for a
maximum of 12 disks) from a lot that gives a
satisfactory quality control results. The PH must be 7.2
to 7.4, and the depth must be approximately 4 mm.
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6. Non-CO2 35- 37˚C incubator for non fastidious
organisms or 5% CO2 incubation for H.inf -
S.pneumoniae- N.meningititis
QUALITY CONTROL
Antibiotic discs for susceptibility testing are checked
weekly utilizing appropriate ATCC reference strains. In
addition, QC testing will be performed anytime when
antibiotic with a new lot number is used repeat the testing.
Document any corrective action in the QC log book. The
discs tested for QC must be the same discs used with the
patient specimens. Tolerance limits for antimicrobial potency
are based on CLSI guidelines. If the zone range limits are
exceed, the Lab Director must be immediately notified and
no sensitivity results will be reported.
E coli ATCC 25922Pseudomonas aeroginosae ATCC 27853S. aureus ATCC 29213
ALSO QC STRAINS FOR ESBL & FASTIDIOUS ORGANISMS MUST BE INCLUDED
QC ORGANISMS MAINTENANCE :
Avoid repeated subculture
Store stock isolates at -60C or below
Prepare working culture weekly & stored at -20C
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PROCEDURES
1. Preparation of inoculum :
a. With a sterile wire loop, touch the top of two to
five similar- appearing, well-isolated colonies on
an agar plate culture according to the size of
colonies as follows: large colonies as
citrobacter touch only the quarter of its size,
small colonies as strept touch five colonies,
while moderate sized colonies touch only
two colonies.
b. Emulsify them in 5mL of sterile physiological saline
or nutrient broth with the help of vortex.
c. The turbidity of the emulsification is adjusted to
0.5Mc Farland standard. Turbidity is matched
against a printed card or sheet of paper in a good
light.
d. Within 15 minutes of adjusting the turbidity of the
inoculums suspension, add the suspension to the
plate by pouring the suspension on the surface of
the agar plate, and then discard the excess in
waste container which contain a disinfectant
Replace the lid of the dish .Allow at least 5
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minutes but no longer than 15 minutes for the
surface to dry before adding the antibiotic disks
2. Testing of antibiotics:
a. Place the appropriate antimicrobial-impregnated
disks with specific concentration according to
(CLSI recommendation, age, pregnancy, inpatient
vs outpatient, type of specimens ) on the surface
of the agar, using forceps. Disks must be evenly
distributed on the agar so that they are no closer
than 25 mm from center to center and about 15
mm from the edge of the agar plate.
b. Gently tamp each disk down onto the agar to
provide uniform contact.
c. Within 15 minutes of applying the disks, invert the
plate and incubate it aerobically (ambient air) at
37˚C for 16-18 hours. Examine the plates after the
overnight incubation except for staph & strept up
to 24 hours
INTERPRETATION
With the use of a ruler or a template, the zones of
complete growth inhibition around each of the disks are
carefully measured to within the nearest millimeter; All 4
measurement are made by the unaided eye while viewing
the back of the petri dish with reflected light against a black,
non reflecting background. The plates should be viewed from
a directly vertical line of sight to avoid any parallax that may
result in misreading.
An interpretive correlate (susceptible, moderately
susceptible, intermediate or resistant) is provided by
reference to published CLSI guidelines.
LIMITATIONS
1- Do not move a disk once it has contacted the agar,
because some of the drug diffuses almost immediately.
2- Susceptibility plates prepared with blood must be
viewed from the agar surface and measurements made
with the cover of the Petri dish removed.
3- Zones that fall into the intermediate range should be
considered equivocal; if therapy with the drug is desired, a
dilution susceptibility test should be performed to clarify
the issue.
4- When testing staphylococci against methicillin or
oxacillin or enterococci against vancomycin, incubation
should be for 24 hours.
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5- Motile organisms such as Proteus mirabilis or P.vulgaris
may swarm when growing on agar surfaces, resulting in a
thin veil that may penetrate into the zones of inhibition
around antimicrobial agent susceptibility disks. This zone of
swarming should be ignored; the outer margin, which is
usually clearly outlined, should be measured. Similarly, with
sulfonamide disks, growth may not be completely inhibited
at the outer margin, resulting in a faint veil, where 80% or
more of the organisms are inhibited. The clear zone of ~
80% inhibition should be read as the zone diameter.
6- Presence of distinct colonies within the zone of inhibition
(2ry colonies) represent either mutant of the same species
that are more resistant to the antimicrobial agent than the
major portion of the bacterial strain being tested or the
culture is not pure and the separate colonies are of a
different species. If it is determined that the separate
colonies represent a variant of a mutant strain, the bacterial
species being tested must be considered resistant. If it is
determined to be a different species, return to the culture
Petri dish and realize whether it is a missed colony or a
contamination. If missed, do a separate antibiogram for the
isolate.
7- When there is overlapping between adjacent agent
zones, zones extend beyond the margin of the Petri dish
or oval(elliptical) zones;, the test must be repeated with
more careful placement of the antimicrobial agent disks
so that overlapping will not occur .When the plate is
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streaked poorly , this will lead to indistinct zones and the
test must be repeated
CHOICE OF ANTIBIOTICS IN ANTIBIOGRAM:
Drugs are listed by CLSI in 4 groups:
1- GROUP A: Testing & reporting against all isolate.
2- GROUP B: Testing when isolate is resistant to groupA.
3- GROUP C: Supplemental or alternative agent that can
be tested &reported in institutions that harbor resistant
strains.
4- GROUP U: Agents that should be tested & reported only
on isolates from urine.
5- Group O :
6- Group I :
Protocol of antibiotics choice in mic. lab
IN THE FIRST DAY GROUP A & B (GROUB 1) are tested in non urine isolates & (GROUP U) in urine isolates.
IN THE SECOND DAY
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GROUP C ( GROUP 2) for H.Infleuanza & Enterobacteriacae & antimicrobial combinations will be done .
IN THE THRID DAY Another combination will be tested in multi resistant strains
Antimicrobial agents with FDA clinical indication that should be considered for routine testing
Acinetobacter Fortum (CAZ) Tienam (IPM) or Meronam(MEM) Unasyn (SAM) Ciprofloxacin(CIP) or Levofloxacin(LEV)
Or ofloxacin ( OFX) Gentamycin (CN) or tobramycin (TOB) or
amikin (AK) SUTRIM ( SXT) Sulperazone (SCF) Cefotaxime (CTX) or Rocephine (CRO) Doxycycline (Do) or Tetracycline (TE) Tazocin (TZP)
Polymyxin local only in eye, ears
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H. Influenza Nisseria Gonorrhea Ampicillin (AMP) Sutrim (SXT) Unasyn (SAM) Cefuroxime (CXM) Cefotaxime (CTX) Rocephine (CRO) Fortum (CAZ) Azithromycin ( AZM) Augmentin ( AMC ) Cefopodoxime
( CPD) Ciprofloxacin ( CIP )
or Levofloxacin LEV Tienam or meronam
Cefopodixime( CPD) Cefotaxime( CTX) Rocephine (CRO) Fortum (CAZ) Ciprofloxacin( CIP) Ofloxacin (OFX)
Penicilin ( P) Tetracyclin (TE)
NB : Testing of B lactamase is mandatory for both isolates using either penicillin disc or nitrocefin sticks .
Burkholderia Stenotrophomonas Sutrim (SXT) Sutrim (SXT)
Fortum (CAZ ) levofloxacin
Meronam ( MEM) minocycline
minocycline
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. Haemolytic strept (pyogens) Strep. Viridance S. pneumoniae Ampicillin (AMP) Pencillin (P) Erythromycin(E)/ AZM Clindamycin(Cd) Maxipime (FEP) Cefotaxime(CTX) or
Rocephine (CRO) Vancomycin (VA) Levofloxacin(LEV) or
Ofloxacin (OFX) Bacitracin BC
Ampicillin(AMP) Pencillin P(MIC) only Maxipime (FEP) Cefotaxime(CTX) Rocephine(CRO) Vancomycin(VA) Erythromycin /AZM Clindamycin (Cd)
Erythromycin(E)/AZM Oxacillin (ox) testing
penicillin Sutrim (SXT) Clindamycin(Cd) Levofloxacin(LEV) Ofloxacian (OFX) Tetracycline (TE) Vancomycin (VA) Optochine OP
N.B If oxacillin sensitive S.pneumoniae report blindly all penicillins & cephalosporins sensitive but if resistant MIC for 3rd generation cephalosoprins is mandatory
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Enterococci ( non urine) Staph ( non urine ) Penicillin(P) Ampicillin (AMP) Vancomycin (VA) CN 120µg (high level
screen) or Streptomycin Erythromycin or
Azithromycin Tetracycline ( TE)
Cefoxitin (Fox 30g) Penicillin (P) if sensitive
report all penicillins cephalospoines & carbapenems are sensitive approved by FDA
Sutrim (SXT) Clindamycin(Cd) test of MLS
resistance is recommended Azithromycin(AZM)or
Erythromycin (E) Vancomycin(VA) CIP or OFX or LEV in MSSA
only DO or TE Gentamycin ( CN) Caphalothin ( CF )
Enterococci( urine )
Ciprofloxacin(CIP) Levofloxacin Norfloxacin
Furadantin (F) Tetracycline(TE) P AMP Vancomycin
Staph (urine )
Norfloxacin(NOR) Ofloxacin(OFX) Levofloxacin FOX P VA Furadantin (F) Sutrim (SXT)
Quinolones are not recommended in ttt of MRSA
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ESBL confirmation in Klebsiella , Ecoli & Proteus (blood isolates only) :
DISC APPROXIMATION TEST
AMC better or SAM ( at the center of the plate)
CPD alone or CAZ& CTX together ( 2.5cm around AMC from center to center)
FOX 30µg ( beside cephalosporin)
TZP OR SCF
FEP ( mandatory)
IPM OR MEM ( beside cephalosporin )
OFX OR LEV / NOR in urine only
CN OR AK
In urine add furadantin
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CHART FOR ESBL SCREENING & CONFIRMATION Furadantin in urine only
FOX30µg Or
FEP
OFX OR LEV
/NOR in urine only
CN or AK/Furadantin
in urine
TZP or SCF
IPM OR MEM
CPD
CAZ CTX, CFP
AMC MAINLY OR SAM OR
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Proteus & Enterobacter , Citrobacter
Pseudomonas
Gentamycin (CN) or Tobramycin (TOB) or Amikin (AK)
Unasyn (SAM) or Augmentin (AMC) Tazocin(TZP) Cefepime ( FEP ) cefotrioxne (CRO)or
Cefotaxime (CTX) Cefobid (CFP) Sulperazone (SCF) Ciprofloxacin ofloxacin or Levofloxacin (LEV) Tienam (IPM)or
Meronam (MEM) Sutrim (SXT) Tetracycline (TE)
Fortum (CAZ) Gentamycin (CN) orAmikin (AK) or Tobramycin Tazacin (TZP) Cefobid (CFP) CIP or LEV IPM or MEM SCF FEP
NB: fortum is 3rd generation cephalosporin with strong antipseudomonal activity while rocephine / claforan have weak antipseudomonal activity .
Coryneform bacteria (Diphtheroid)o Sutrimo Penicillino Levo/ofloxo Erythromycin/azythromycino Vancomycino Tetracycline/doxyyclineo Augmentin/ unasyn
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Gram negative bacilli other than Ecoli & Klebsiella in urine :
Norfloxacin (NOR) OFX/LEV/CIP Furadantin (F) Lomefloxacin Sutrim (SXT) Cefobid ( CFP) Augmentin (AMC ) Gentamycin ( CN ) Carbencillin Tazocin FEP SCF
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CLSI recommendations:
1. The following antimicrobial agent may be appear active in vitro but are not
effective clinically and should not reported as susceptible:
Salmonella and Shigella
1st and 2nd generation cephalosporins and aminoglycoside
MRSA
Penicillin resistant oxacillin sensitive staph
All . Lactam, carpenams,
All penicillins except B lactamase inhibitors, cephms, & carbapenems
Enterococcus Aminoglycoside (except high conc.) cephalosporins, clindamycin, SXT
Yersinia . Lactam
Listeria
ESBL
Ampicillin resistant enteroccoci
Penicillin (Oxacillin) resistant pneumoniae
Cephalospornis
Penicillin ,cephalosporins & azactam
Penicillin ,B lactamase inhibitor ( AMC, SAM) & carbapenems the mechanism is altered PBPs
Penicillins ,cephalosporins ,carpenams except 3rd generation cephalosporins must do MIC
2. Warning (CSF): The following antimicrobial agent
should not be routinely reported:
a. Agents ad. By oral routes.b. 1st and 2nd gener ceph. except (CXM).c. Clindamycin.
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d. MacroLides.e. Tetracyclines.f. Fluroquinolones.
3. Susceptibility testing of penicillins and other lactams
approved by FDA for treatment of strep. Pyogenes and
agalactiae is not necessary for clinical purposes.
Recommendation P/AMP/cefazolin/clindamycin/
erythromycin.
4. P.mirabilis should be added to E.coli and
K.pneumoniae in screening for ESBL in bacterimic
isolates only (blood) because reports of ESBL in non
bacterimic isolates have been relatively rare due to low
frequency of plasmid conjugate.
5. Levofloxacin should be used for stenotrophomonas
with SXT.
6. Susceptibility testing is not recommended for
S.saprophyticus in urinary isolates NOV (R) <16mm.
7. Lab should identify S.lugdunensis (an uncommon) but
one cause of endocarditis: (PYR test +ve and ornithine
de carboxylase +ve).
8. Screening of MRSA by cefoxitin 30 or 10g by disc
diffusion while by oxacillin MIC by E test or broth dilution.
For CONS, the cefoxitin disk test has greater specificity
than oxacillin and equal sensitivity, although it may miss
some strains of mecA-detection and the latex test for
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PBP2a are the most accurate predictors of mecA-
mediated resistance.
9. For organisms (campy, corynebacterium, bacillus
spp.) consultation with an infectious disease specialist is
recommended for guidance in determining the need for
susceptibility testing and in the interpretation of results,
published reports in medical literature and current
consensus recommendations for therapy of uncommon
isolates, may obviate the need for testing. If necessary a
dilution method usually will be the most appropriate
testing method and this may require submitting the
organism to a reference lab.
N B :
Cephalothin CF/CF/CL is the disc
representative for the 1st, 2nd gener cephal
and cefopodoxime
Tetracycline: is representative for Do,
minocyclin.
Erythromycine: is representative for
macrohide.
10. Oxacillin screening disk diffusion used to detect high
rate of penicillin resistance in S.pneumoniae (>20mm
susceptible, <19mm (do MIC testing) and correlate
with the body site it is collected from.
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11. S.pneumoniae isolates from CSF, it is recommended
testing penicillin, cefotaxime ceftrioxone, meronam and
vancomycin by broth dilution method as disc diffusion
with carbapenems or cephalosporines for S.pneumoniae
do not exist. But for non life threatening infections,
agents to be consider are penicillin, erythromycin sutrim,
by broth and disc diffusion method.
12. Staph isolates that are resistant to erythromycin but
susceptible to clindamycin should be tested for inducible
resistnace to clindamycin (MLS) resistance mediated by
"erm" gene using the D-zone approximation test with
closely approximated erythromycin and clindamycin test.
13. Strept viridans any isolate from a sterile body site or
implicated in a serious infection as endocarditis should
be tested for penicillin susceptibility and cephalosporins
especi especially 3rd generation as some viridans may
exhibit relative resistance. Vancomycin is the
recommended alternative to lactam Abs.
14. For fecal isolates of salmonella and shigella: (ampicillin
, fluroquinolones , SXT (only).while in Extra intestinal
isolates : Chloramphenicol , 3rd generation cephalosporins.
15. As regards ESBL detection:
Due to variable affinity of these enzymes for
diffusion subs and inoculum effect, some ESBL 19
producing organism with 3rd generation cephalosporins
may result in clinical failure if infection is (outside the
urinary tract).
Testing of cephamycins is recommended in ESBL
producing isolates.
Cefpodoxime and ceftazidime have been proposed
as indictors of ESBL production as compared to
cefotaxime and ceftrioxone.
These enzymes can be induced by certain Abs,
AAs, or body fluids.
It is possible for one specimen to contain both
ESBL producing and non ESBL producing cells of the
same species. So, it must test several colonies for a
primary culture plate.
Latest guidelines recommended screening of ESBL
with a MIC 2mg/dL against cefpodoxime ceftazidime,
aztreonam, ceftaxime or ceftrixone.
Three indicators of ESBL:
An 8 fold reduction in MIC in the presence of
clavulonic acid by broth dilution method.
Potentiation of the inhibitor zone by clavulonic
acid >5mm in diameter of inhibition by disc
diffusion.
Disc approximation test by using of cefoxitin
(inducer) placed at a distance of 2.5cm from
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cephalosporin disc flattening of the zone of
inhibition of cephalosporin disc towards inducer
disc >1mm.
As regards treatment of ESBL carbapenens are the
most effective and reliable as they are highly
resistant to hydrolytic activity of all ESBL enzymes
due to trans-6-hydroxy ethyl group.
Meronam is the most active with MICs generally
lower than those of IPM (0.03-0.12mg/ml vs 0.06-
0.5mg/ml).
Also ESBL activity is inhibited by clavulonic acid,
the only infections that can be treated safely with
lactamase inhibitor are those involving the urinary
tract in which the concentration high enough to
counteract the hydrolytic activity of ESBL.
Clavulonic acid appears more efficient than
sulbactam it takes about eight times more to obtain
a protective similar to that by C.acid.
Plasmids responsible for ESBL production tend to be large and carry resistance to several agents an important limitation in the design of treatment. The most frequent co-resistance are aminoglycosides, flouroquinolones , TE, chloramphenicol and sutrim
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Issue No/Revision No:Ain Shams University
HospitalsIssue date:Revision date:Copy number:
Code No:Main LaboratoriesPage of
ANTIMICROBIAL COMBINATION BY DIFFUSION METHODS:
Disk approximation test :
Principle:
This method has been explored to assess primarily in a
qualitative fashion the interaction of antimicrobials as they
diffuse through agar plates seeded with a test organism.
Advantages:
Simple.
The use of readily available materials (discs and
Muller Hinton agar).
Disadvantages:
Qualitative method only.
Low sensitivity and specificity compared to dilution
methods.
i.e.: The results of this test may differ from results obtained when the same agents and organism are tested in liquid media.
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Procedure: This technique uses the same standard inoculums
and Muller-Hinton agar as a routine Bauer-Kirby
susceptibility test.
To assess possible interactions between two drugs
(A and B) disks containing these drugs are placed on a
plate that has been inoculated with a tested organism.
The distance by which the disks are separated
may be varied, but it should generally be equal to or
slightly greater than the sum of the radii of the zones of
inhibition of the drugs when examined alone (mostly
15mm from centre to centre).ONLY FIVE COMBINATION
ARE TESTED IN THE 100 mm PLATE
After overnight incubation (16-18hrs) at 37C the
plate are ready for examination.
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Issue No/Revision No:Urine Microbiological
ExaminationAin Shams University
Hospitals Issue date:Revision date:Copy number:
Code No:Main LaboratoriesPage of
Example of antibiotic combinations used for multi resistant organisms "by Dilution methods":1. Pseudomonas:
Bactericidal:
Ciprofloxacin and tienam (CIP and IPM).
Ciprofloxacin and Amikin (CIP and AK).
Ciprofloxacin and Azactam (CIP and ATM).
Ciprofloxacin and Fortum (CIP and CAZ).
Levofloxacin and Maxipime (LEV and FEP).
Ciprofloxacin and Maxipime (CIP and FEP).
Levofloxacin and Meronam (LEV and MEM).
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Ciprofloxacin and Tazocin (CIP and TZP).
Levofloxacin and Tazocin (LEV and TZP).
Levofloxacin and Gentamycin (LEV and CN).
Tazocin and Gentamycin (TZP and CN).important
Tazocin and Tienam (TZP and IPM).
Bacteriostatic:
Augmentine and Ampicillin (AMC and AMP).not used
Vanocomycin and Carbencillin (VA and Pip).not used
Azactam and Maxipime (ATM and FEP).
2. Acinetobacter:
Bactericidal :
Doxycyclin and Amikin (Do and AK).
Ciprofloxacin and Fortum (CIP and CAZ).
Ciprofloxacin and Meranam (CIP and MEM).
Ciprofloxacin and Azactam (CIP and ATM).
Tazocin and Gentamycin (TZP and CN).
Ciprofloxacin and Tazocin (CIP and TZP).
Bacteriostatic :
Tienam and Amikin (IPM and AK).
Unasyn and Amikin (SAM and AK).
3. Enterobacteriaceae:
o Tazocin and Amikin or Gentamycin (TZP and AK or
CN).
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o Cefotaxim and Amikin or Gentamycin (CTX and AK
or CN).
o Azactam and Tienam (ATM and APM).
o Azactam and Maxipime (ATM and FEP).
o Ceftazidime and Oflaxocin (CTZ and OFO).
o Cefoxitin and Amikin (FOX and AK).
o Ciprofloxacin and Fortum (CIP and CAZ).
o Ciprofloxacin and Tazocin (CIP and TZP).
4. Proteus:
o Tazocin and Amikin (TZP and AK).
o Tienam and Amikin (IPM and AK).
5. Enteroccoci:
.lactam (penicillin) and amino glycoside.
(gentamycin )
Glycopeptide (VA or TEC) and aminoglycoside.
Teinam and Teicoplanin (IPM and TEC).
Tazocin and Gentamycin (TZP and CN).
Tazocin and Ciprofloxacin (TZP and CIP).
Glycopeptide and .lactam (TEC and P).
Ciproflxocin and Vancomycin or Penicillin (CIP and
VA).
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Ciproflxocin and Ampicllin (CIP and AMP).
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