INTERPRETATION OF ANTIBIOGRAMSTrends of Change

INTERPRETATION OF
ANTIBIOGRAMS
Trends of Change
Dr.T.V.Rao MD
3/24/2016Dr.T.V.Rao MD
1

The Program file summarises on New Trends of Change
in Antibiotic sensitivity and resistance changes
Problem with Inconsistent on
phenotype
Implications Good reminder of
intrinsic and exceptional resistance
Everything about Microbes changes
faster than we think limited science
2

The purpose of Antibiograms
The purpose of a
susceptibility test is to in
vitro determine the
sensitivity or resistance of
pathogenic aerobic and
facultative anaerobic
bacteria to various
antimicrobial compounds.
3

Requirements to create
AntibiogramsThe use of standardized
culture media and careful
control of all test conditions
are fundamental
requirements in the
microbiological assay of
antibiotics in order to
achieve satisfactory test
results.
4

Monitoring of Antibiograms is crucial in
Hospital practice
It is crucial to monitor
emerging trends in resistance
at the local level to support
clinical decision making,
infection-control interventions,
and antimicrobial-resistance
containment strategies
5

Various methods of antibiotic susceptibility testing
are:
1. Quantitative Methods
2. Qualitative Methods
3. Automated
Susceptibility Tests
4. Newer Non-Automated
Susceptibility Tests
5. Molecular Techniques
6

Quantitative Methods
In these tests, the minimum amount of antibiotic that
inhibits the visible growth of an isolate or minimum
inhibitory
concentration (MIC) is determined. Bacterial isolate is
subjected to various dilutions of antibiotics. The
highest dilution of antibiotic that has inhibited the
growth of bacteria is considered as MIC. These tests
can be performed on broth or agar.
7

Quantitative Methods
1. Broth dilution methods
a. Macro broth dilution MIC
tests
b. Micro broth dilution MIC
tests
2. Agar dilution methods
8

AST standards
MIC taken as standard
measure of susceptibility
Agar dilution
Broth dilution
Micro broth dilution
9

AST standards
MIC taken as standard measure of susceptibility
Agar dilution
Broth dilution
Micro broth dilution
S/I/R Breakpoints defined by BSAC/EUCAST/CLSI
10

ISO standard
Broth microdilution
Mueller-Hinton
Broth
Inoculum 5x105
cfu/mL
11

The rationale for automated systems
More reliable AST results
?closer to “standard”
Reduced scope for “user” error
Reproducible
Improved ID linked to AST
Embedded expert rules
Dr.T.V.Rao MD3/24/2016
12

What is a Antibiogram
Antibiograms are
collection of information
obtained from Culture and
Sensitivity performed in
the institution within a
given time frame
13

Why things go wrong in Antibiotic Sensitivity
testing
 Excessive pre-incubation before discs applied
 Excessive pre-diffusion before plates incubated
 Incorrect incubation temperature (350C for
cefoxitin vs Staphylococci)
 Incorrect incubation atmosphere (false
metronidazole”)
 Prepared by Trevor Winstanley for the BSAC
recommendations
15

Why things go wrong in Antibiotic Sensitivity testing
 Incorrect incubation time
(VanB resistance in enterococci)
 Inadequate illumination of plates when
reading
 Incorrect reading of zone edges (cefoxitin for
the detection of MRSAs)Incorrect template
(wrong agent or wrong version of the
guidelines)
 Prepared by Trevor Winstanley for the BSAC
recommendations
16

We work with The ClSI
M39-A2 Guideline or Newer updates
CLSI M39-A2 is intended for those
involved in the preparation and use
of cumulative antibiogram reports, as
well as for information technology
managers who are responsible for
designing and supporting the clinical
laboratory's data management needs
17

What the Document Contains
The document contains specific recommendations for the
collection, storage, analysis, and presentation of data and
includes sample templates that highlight the
recommendations. Critical issues addressed include the
recommended frequency of reporting, the number of
isolates to include in a statistic, and a mechanism for
eliminating multiple isolates of a given bacterial species
obtained from an individual patient
18

Provides Information on Resistance and Sensitivity
of the tested isolate
Provides the % of
samples for a given
organism which were
sensitive or resistant
to certain antibiotics
19

Potential sources of error in disc diffusion
antimicrobial susceptibility testing:
antimicrobial discs
Wrong agent or content used
Labile agent possibly deteriorated
Light sensitive agent left in light
Incorrect storage leading to deterioration
Disc containers opened before reachingroom temperature
Incorrect labelling of disc dispensers
Expiry date exceeded
 Prepared by Trevor Winstanley for the BSAC recommendations 3/24/2016Dr.T.V.Rao MD
20

Potential sources of error in disc diffusion
antimicrobial susceptibility testing: control
strainsContamination
Mutation
Incorrect inoculum density
Uneven inoculation
Old culture used not properly maintained
Prepared by Trevor Winstanley for the BSAC recommendations
21

Antibiograms are Important to Clinicians to
make decisions
An antibiogram is an
essential tool for any
clinician when treated an
infection empirically
• Antibiogram can serve as
an alternative to a C&S
report until the results of a
C&S are available
22

Antibiograms are Important to Clinicians to
make decisions
Antibiogram can
serve as an
alternative to a C&S
report if no organism
is grown out of a
C&S despite high
clinical suspicion of
an infection
23

Disk diffusion test
In this method the standardized bacterial isolate is spread
on an agar plate and then paper disc containing specific
concentration of antibiotics are placed and incubated at
37oC overnight. If the isolate is susceptible to the
antibiotic, it does not grow around the disk thus forming a
zone of inhibition. Strains resistant to an antibiotic grow
up to the margin of disk. The diameter of zone of
inhibition must be measured and result read from the
Kirby Bauer chart as sensitive, intermediate or resistant
25

Clinical utility
The greater the number of
isolates, the more accurate
the sensitivity results for the
given organism – The greater
the number of isolates, the
higher probability of
isolating the given organism
26

Parts of the Antibiogram % Susceptible
Percentage of
isolates of the
given organism
which is
susceptible
(sensitive) to the
given Antibiotic
27

Resistance
 Reflects the % of organism which are resistant
to certain Abx • Clinical Utility: – Assists in
determining if coverage for MDR organisms in
the empiric therapy are necessary • Information
can be obtained from the % susceptibility chart
• Important for MRSA, VRE, ESBL, and KPC
28

Antimicrobial agents to analyse.
Only results for antimicrobial agents that are routinely
tested and clinically useful should be presented to
clinicians. To avoid biases introduced by selective
reporting practices (e.g., reporting broad-spectrum agents
only for bacteria with resistance to primary agents), the
analysis database should include the results for all
antimicrobials tested, including those agents that may not
be routinely reported to clinicians.
29

Do not do disc contain test with Vancomycin for
Staph aureus
 Disc testing should NOT be used.
 • An MIC based method should be used.
 – Whichever method is used, a control
strain
 (ATCC 25923 or ATCC 29213) should be
tested in parallel with each run of test
organisms
 – Monitor performance of QC (even within
acceptable range
30

Antimicrobial agents to analyse.
Results for antimicrobials tested
only against drug-resistant strains
as part of reserve or second-line
testing panels are generally biased
towards higher rates of
antimicrobial resistance and
should not be considered to be
representative.
31

Should be familiar with trends in Antibiotic Science
 Susceptibility testing requires
 – Reliable/reproducible test
 – Relationship between test and clinical outcome
 • B. cepacia
 – Testing poorly reliable/reproducible
 – Poor definition of relationship with outcome
 • S. maltophila
 – Testing poorly reliable/reproducible
 – Poor definition of relationship with outcome
 – Co-trimoxazole AST remains challenging
32

Inherent resistance we mostly
agree on - non-fermenters
Pseudomonas, Acinetobacter - ampicillin, cephs except
ceftazidime, tetracycline, chloramphenicol, ertapenem…
 S. maltophilia - all -lactams except ticar-clav
,aminoglycosides, trimethoprim alone
 Burkholderia - aminoglycosides & colistin
 Elizabethkingella - -lactams except pip-tazo
33

Exceptional
resistance
Enterobacteriaceae –
Meropenem / imipenem
 P. aeruginosa, Acinetobacter –
colistin
 Bacteroides – metronidazole &
carbapenems
34

Disc testing unreliable Colistin
 Colistin AST
 – Disc testing unreliable
 – MIC testing remains challenging (method
development)
 • Vancomycin AST for staphylococci
 – Disc testing unreliable
 – MIC testing remains challenging (BP close
to wild-type popln
35

B. cepacia complex
Multiple resistance mechanisms
(?variable expression)
• No reliable gold-standard test
• No defined relationship between MIC &
clinical outcome
• Wide MIC distribution across PKPD BP
 No AST method
36

B. cepacia complex:
a reliable/reproducible test?
Gold standard
–MIC performed to ISO
20776-1 (2006)
–Micro broth dilution
–Mueller-Hinton Broth
37

Stenotrophomonas
maltophilia
 Multiple resistance mechanisms
 (?variable expression)
 • No gold standard test
 –Results markedly affected by
 incubation temperature, culture
 medium and technique
 • V little data to relate test to clinical
 outcome
38

All the Microbiologists should be familiar
with
For the seriously-ill patient, fast
microbiology is more valuable than
precise microbiology…
And, mostly, microbiology moves
at the pace
it did in Fleming’s time: 48h from
specimen to susceptibility result
39

The truth about our Microbiology Departments
Far more potential to
accelerate seeking
resistance genes than
phenotypes
It means we detect many
matters later than the
Bactria have evolved
40

Does the absolute truth exists in
Antibiotic Sensitivity testing
Why we think EUCAST (& CLSI) ESBL advice is wrong
Pharmacokinetics more variable than we admit
 Outcomes are inconsistent vs. low-MIC ESBL &carbapenemase
producers Inoculum effect (TW)
 Susceptibility tests less precise than we wish
 Mechanism detection will become faster than susceptibility testing
Ref Livermore et al. JAC 2012;67:1569
41

Improved speed of results
Increased number of agents tested
Automated systems: an overview 3/24/2016Dr.T.V.Rao MD
42

Increased number of agents
tested
Laboratory workflow
Staff skill mix
Epidemiological software
Automated systems: an overview 3/24/2016Dr.T.V.Rao MD
43

Available systems
Micros can Walkaway
Biomerieux Vitek 2
BD Phoenix
44

Micro Scan
96 well plates
AST based on conventional
micro broth dilution MIC
ID currently biochemical
45

Micro scan Conventional panels
• Enterobacteriaceae, gram-negative non-fermenters,
staphylococci, enterococci
• Combo, MIC-only, ID-only
 Rapid panels
 2 - 2.5 hour ID (pre-formed enzymes)
 AST for Gram negative organisms (MIC > 4 hrs)
 Synergies plus panels
 2 - 2.5 hour ID (pre-formed enzymes)
 Broth microdilution “Read-when-ready” MIC
 Resistance detection flagged in as few as 4.5 hours
 All results finalized in 16/18 hours
 Additional
 Yeast ID, Anaerobe ID, HNID
 MICroSTREP plus ® - dry format
 ESβL plus ® -ESβL Confirmatory Panel
• Panels read by
– WalkAway® systems
– autoSCAN-4
– Manual
46

Vitek 2
AST based on broth
susceptibility with specific
cards
Growth/reactions
“continuously” monitored
47

Vitek 2
 ID Cards available for
Gram positives
Enterobacteriaceae
Non-Enterobacteriaceae
Neisseria/Haemophilus etc
Anaerobes
Yeasts
 AST Cards available for
Gram positive cocci
Gram negative bacilli
Yeasts
• Up to 22
antibiotics tested
on each card
48

TIME (hours)
0 1 2 3 4 5 6 7 8 9 10 11 12 1413 1615 17 18
Raw Data
% Change
Slope
Area
SLPAR
Interpretation of Raw Data
Growth Parameters
MIC
VITEK 2 – MIC Interpretation
49

Vitek 2
Expert rules within
the Advanced Expert
System (AES)
Extensive Knowledge
Base
>20,000 MIC
distributions
>2,500 Phenotypes
>200 Resistance
Mechanisms
50

Vitek 2
CARD Type Time to Result
GN ID 2 to  10 hours
GP ID 2 to  8 hours
NH ID 6 hours
ANC ID 6 hours
YST ID 18 hours
GN AST 2 to 18 Hours
GP AST 2 to 18 Hours
AST-YS01 6 hours
51

Phoenix
AST based on broth
susceptibility with specific
cards
Growth/reactions
“continuously” monitored
52

Phoenix
AST only or Combo panels available for
GN (Enterobacteriaceae/
Nonfermenters)
GP (Staphylococci/
Enterococci/ Streptococci)
Streptococci ID/AST
53

Phoenix
 Readings for each well taken and calculated every 20 minutes
 ID uses red, green, blue and UV light sources
 AST measures turbidity and color change
 Each well divided into pixels
 Readings taken for each pixel
 Entire process takes 7 minutes
 Pixel data analyzed for each well
 Readings for each well compared to previous reading and baseline
Well is complete when specific amount of change is not seen
Substrate/antimicrobic algorithm dependent
 Instrument will post results when enough data collected
54

PhoenixExpert rules within
BDXpert based on
CLSI/SFM/DIN/EUCAST
rules bases
55

Comparison of systems
Microscan Vitek 2 Phoenix
ID available
GPOS
GNEG NH
AnO2
Yeast
GPOS
GNEG
NH
AnO2
Yeast
GPOS
GNEG
AST available
GPOS
GNEG
GPOS
GNEG
Yeast
GPOS
GNEG
Reading
Continuous automated
or manual
Continuous automated Continuous automated
MIC Y +/- +/-
Expert system Published rules Phenotypic comparison Published rules
Biochemical ID   
Plans for integration with non-
biochemical ID systems
  
Epidemiological software   
56

Performance ID
Correct to genus >95%
Correct to Species>90%
Difficulties with difficult organisms
Non-fermenters
Shigella etc
 AST
Concordant with other methods >95%
Specific issues with specific drug-bug combinations & certain instruments
VISA/VRSA
Beta-lactams & Pseudomonas
57

Performance
ID/AST performance relies on
correct ID, correct AST plus
appropriate expert interpretation
Incorrect ID can lead to incorrect AST
Up to 5% errors mean that results
should be examined critically
58

Advantages of Automated systems in
AST
Automated systems can provide
Reliable AST integrated with ID & expert rules
Caution about staff skill mix and interpretation of
results
Microscan/ Vitek 2 /Phoenix have similar
performance overall
Results should be examined critically
59

References
1Why some tests are no longer recommended Robin A Howe, Cardiff, UK Public
health wales
2ANTIBIOTIC SUSCEPTIBILITY TESTING by Dr Sridhar Rao PN Open web resource
3Expert rules for Gram-negatives Trevor Winstanle Sheffield Teaching Hospitals
Presented on behalf of David Livermore University of East Anglia & Health
Protection Agency
4 Internal Quality Assurance Jenny Andrews Secretary of the BSAC Working Party
on Susceptibility Testing
5Automated systems: an overview Robin A Howe Cardiff Public health wales
60

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61

Program Created by Dr.T.V.Rao MD for
Benefit of Medical and Paramedical
Professionals in the Developing World
Created from World Wide Resources
Email
doctortvrao@gmail.com
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