Transcript Antimicrobial resistance Antimicrobial resistance and susceptibility testing May 2007 P I D E M I C A L E R T Laboratory Training for FieldEEpidemiologists A.

Antimicrobial resistance
Antimicrobial resistance
and susceptibility testing
May 2007
P I D E M I C A L E R T
Laboratory Training for FieldEEpidemiologists
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R E S P O N S E
Learning objectives
At the end of the presentation, participants should:
• Identify antimicrobial susceptibility testing needs
• Understand standard antimicrobial susceptibility testing
• Interpret antimicrobial susceptibility testing
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Leading global infectious diseases
S. pneumonia: Up to 55%
S. dyentariae: 90% resistance to
cotrimoxazole S.Typhi: Outbreaks of
resistance to penicillin in
some regions
HIV: Report of
resistance to all
marketed agents
4
Millions of deaths, worldwide, 1998
multi-resistant strains in 11 countries
M. tuberculosis:
Multi-drug resistant
tuberculosis
3
P. falciparum:
Chloroquine resistance in
81/92 countries
2
1
0
Respiratory infections
HIV
Diarrheal diseases
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Tuberculosis
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Malaria
Antibiotic resistant infections
Diseases
Agent
Resistances
Pneumonia
S pneumoniae
Penicillin
Dysentery
S dysenteriae
Multiple resistances
Typhoid
S typhi
Multiple resistances
Gonorrhea
N gonorrhoeae
Penicillin and
tetracycline
Tuberculosis
M tuberculosis
Rifampicine and INH
Nosocomial infections
S aureus
Methicillin, vancomycin
E species
Vancomycin
Klebsiella,
Pseudomonas
Multiple resistances
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Antimicrobial resistance
Results from misuse, overuse, under/ inadequate use of
antimicrobials
•
Costs money, lives and undermines effectiveness of
health delivery programs
•
Threat to global stability and national security
WHO Global Strategy for Containment of Antimicrobial
Resistance:
•
Intervention framework to slow emergence and reduce
the spread of antimicrobial resistant microorganisms
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Natural & acquired resistance
Natural resistance
• Chromosomic genetic support
• Affect almost all species strains
• Existed before antibiotic use (Enterobacter sp. - amoxicillin)
Acquired resistance (mutation)
• Chromosomic, plasmidic or transposon genetic support
• Affects a fraction of strains
• Increased with antibiotic use
(extended spectrum beta-lactamase producing E. coli)
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Different acquired resistances
Acquired to a population of strains in a given species
• Extremely frequent in nosocomial infections
Acquired under treatment; specific strain,specific patient
• Relatively uncommon except for certain species
(e.g., Enterobacter, Pseudomonas, Mycobacterium)
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Mechanisms of resistance
Prevent antibiotic from reaching its target
• Impaired cell membrane permeability
• Efflux phenomenon
Prevent the antibiotic from biding to its target
• Supplementary targets
• Decreased affinity by target modification
Inactivation before reaching the target
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Genetic exchange of
antimicrobial resistance genes
Staphylococci
Pseudomonas
Enterococci
Enterobacteriaceae
Vibrio cholerae
Pneumococci
Campylobacter
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Streptococci
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Antimicrobial susceptibility tests
Minimum inhibitory concentration [MIC]
• The smallest concentration of antibiotic that inhibits the
growth of organism
Liquid media (dilution) allows MIC estimation
Solid media (diffusion)
• Disk diffusion (Kirby-Bauer)
• E-tests
• Allows MIC estimation
Beta lactamase production: quick screening method
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Dilution in liquid broth
•
Tubes containing increasing antibiotic concentrations
•
Incubation during 18 hr at 37°C
•
Tedious
MIC
Bacterial growth
0 (Control) 0,25
0,50
1
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2
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Inhibition
4
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8
mg/l
Kirby-Bauer disc testing
Antibiotic-impregnated discs placed on an agar plate at the
interface between test organism and susceptible control organism
Resulting zones of inhibition compared, use of controls
Susceptibility is inferred (standard tables)
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E-test
Plastic strips with a predefined
gradient of
• One antibiotic
• One antifungal
Only one manufacturer
One strip per antibiotic
Wide range of antibiotics
Easy to use
Storage at -20°C
Short shelf life, expensive
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Reading E-tests
Ciprofloxacin for
Yersinia pestis
Resistant > 4 ug/ml
Intermediate 1-4 ug/ml
Susceptible < 1
Upper reading
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Antimicrobial susceptibility tests
Antimicrobial susceptibility testing is expensive
(costs include all supplies)
Kirby-Bauer
• 12 discs panel = $1.35
E-test (Performed only in certain situations)
• One strip = $2.50
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Different standards
Use standardized reference
National Committee for Clinical Laboratory
Standards (USA)
Other norms
• Canadian
• Chinese
• National
Do not confuse the different tables
Choose one for everything
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Critical points in quality assurance
1.
Culture media: Muller-Hinton
2.
Reagents: disks
3.
Size of the inoculums
4.
Incubation condition
5.
Control with reference strains
6.
Reading inhibition diameters (accurate measurement)
7.
Knowledge of staff
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Standard strains for
quality assurance
Precision and accuracy ensured through control strains
• Known susceptibility to antimicrobial agents
Standard strains include
• Staplylococcus aureus ATCC 25923
• Escherichia coli ATCC 25922
• Pseudomonas aeruginosa ATCC 27853
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Interpretation
The main concept is the “clinical categorisation"
•
Strains are sorted according to level of Minimal Inhibitory
Concentration (MIC) versus reference breakpoints
•
c and C are the minor and major breakpoints
Susceptible
MIC <
Intermediate
c
≤ MIC
<
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Resistant
C
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≤ MIC
Understanding breakpoints
Words of laboratory specialists
• It is not possible to work alone
• Breakpoints are the expression of a consensus among the
scientific community at a given time in a country
Breakpoints are determined using two approaches
• Pharmacological concept
• Epidemiological concept
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The epidemiological concept
for breakpoints
Inherited resistance
mechanism
Wild type
60
50
40
30
20
10
0
0.01
0.03
0.06
0.12
0.25
0.5
c
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1
2
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4
C
8
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16
32
64
128
MIC
The pharmacological concept
for breakpoints
The concentration range tested for a drug and the
interpretative criteria for various categories are based on
extensive studies that correlate with
• Serum achievable levels for each antimicrobial agent
• Particular resistance mechanisms
• Successful therapeutic outcome
In practice situations the entire range may not be used for
decision making and therefore the concept of breakpoint
concentration
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From breakpoints to interpretation
MIC ≤ c
Sensitive strain
MIC > C
Intermediate strain
c < MIC ≤ C
Resistant strain
Measuring antimicrobial sensitivity of a strain isolated
from a patient, to determine its status as S, I or R is
an individual problem
Defining the status of a bacterial species or genus is
an epidemiological problem distributed across time
and space that requires monitoring
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Host factors affecting treatment
Diffusion in tissues
Serum protein binding
Drug interactions
Immune system
Multiple simultaneous infections
Virulence of organism
Site and severity of infection
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Interpreting intermediate
resistance
Sometime the agent can still be used
• Higher doses required to ensure efficacy
• Agent may be efficacious if concentrated in vivo in an
infected body fluid (e.g., urine)
Sometimes there is uncertainty
• Intermediate resistance may represent a “buffer” zone
that prevents strains with borderline susceptibility from
being incorrectly categorized as resistant
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Common interpretation problems
Results depends on the technique used
Many factors influence results
• Lack of standardization of the inoculums
• Thickness and quality of the culture media
• Quality and conservation of the disks
• Wuality control with standardized strains
• Condition and duration of incubation
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Common interpretation problems
An agar gel that is too thick leads to smaller zones
Source: http://www.who.int/csr/resources/publications/drugresist/WHO_CDS_CSR_RMD_2003_6/en/
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Common interpretation problems
Problem with the size of the
inoculums
Solution:
•
Use McFarland 0.5 photometer
•
Scale -> same tubes
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Common interpretation problems
Contamination with another
organism
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Common interpretation problems
Bad manipulation
Inoculation of the Muller Hinton
• Swabbing
• Not by flooding
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Common interpretation problems
Problems with E-test reading
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Cost of anti-microbial resistance
Cheap antimicrobials become ineffective
Individual treatment failure
Prolonged illness, hospitalization
Need to switch to more expensive, complex drugs that are
often not even available in resource-poor settings
Need to develop new antimicrobials
Good antimicrobial susceptibility testing saves lives and
money
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WHO/CDS/CSR/APH/2000.4
Distr. : General
English only
WHO Global Principles For
The Containment of Antimicrobial
Resistance In Animals Intended
for
Food
Report of a WHO Consultation
with the participation of the Food and Agriulture Organization and the
Office International des Epizooties
Geneva, Switzerland
5 – 9 June 2000
Department for Communicable Diseases Surveillance and Response
World Health Organization
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Antimicrobial resistance
Developed by the Department of Epidemic and
Pandemic Alert and Response of the World Health
Organization with assistance from:
European Program for Intervention
Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur
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References
•
Manual for the laboratory Identification and
Antimicrobial Susceptibility Testing of Bacterial
Pathogens of Public Health Importance in the
Developing World
WHO/CDS/CSR/RMD/2003.6
http://www.who.int/csr/resources/publications/drugresi
st/WHO_CDS_CSR_RMD_2003_6/en/
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