Laboratory Training for Field Epidemiologists Antimicrobial resistance and susceptibility testing Antimicrobial resistance May 2007

E P I D E M I C A L E R T A N D R E S P O N S E Laboratory Training for Field Epidemiologists

Antimicrobial resistance and susceptibility testing

Antimicrobial resistance

May 2007


Learning objectivesLearning objectives

At the end of the presentation, participants should:

• Identify antimicrobial susceptibility testing needs

• Understand standard antimicrobial susceptibility testing

• Interpret antimicrobial susceptibility testing


Leading global infectious diseasesLeading global infectious diseases

0

1

2

3

4

Respiratory infections HIV Diarrheal diseases Tuberculosis Malaria

Mil

lio

ns

of

dea

ths,

wo

rld

wid

e, 1

998

S. pneumonia: Up to 55% resistance to penicillin in

some regions

HIV: Report of resistance to all marketed

agents

S. dyentariae: 90% resistance to cotrimoxazole S.Typhi: Outbreaks

of multi-resistant strains in 11 countries

M. tuberculosis:Multi-drug resistant

tuberculosis

P. falciparum:Chloroquine resistance

in 81/92 countries


Antibiotic resistant infections Antibiotic resistant infections

Diseases Agent ResistancesPneumonia 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


Antimicrobial resistanceAntimicrobial resistanceResults from misuse, overuse, under/ inadequate use ofantimicrobials

• Costs money, lives and undermines effectiveness of health delivery programs

• Threat to global stability and national security

WHO Global Strategy for Containment of AntimicrobialResistance:

• Intervention framework to slow emergence and reduce the spread of antimicrobial resistant microorganisms


Natural & acquired resistanceNatural & acquired resistanceNatural 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)


Different acquired resistancesDifferent 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)


Mechanisms of resistanceMechanisms 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


Genetic exchange of antimicrobial Genetic exchange of antimicrobial resistance genesresistance genes

Enterobacteriaceae Enterococci

StaphylococciPseudomonas

Campylobacter

Vibrio cholerae Pneumococci

Streptococci


Antimicrobial susceptibility testsAntimicrobial susceptibility testsMinimum 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


Dilution in liquid brothDilution in liquid broth

• Tubes containing increasing antibiotic concentrations

• Incubation during 18 hr at 37°C

• Tedious

0 (Control) 0,25 0,50 1 2 4 8 mg/l

MICBacterial growth Inhibition


Kirby-Bauer disc testingKirby-Bauer disc testingAntibiotic-impregnated discs placed on an agar plate at theinterface between test organism and susceptible control organism

Resulting zones of inhibition compared, use of controls

Susceptibility is inferred (standard tables)


E-testE-testPlastic 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


Reading E-testsReading E-tests

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading


Antimicrobial susceptibility testsAntimicrobial susceptibility tests

Antimicrobial susceptibility testing is expensive Antimicrobial susceptibility testing is expensive (costs include all supplies)(costs include all supplies)

Kirby-Bauer

• 12 discs panel = $1.35

E-test (Performed only in certain situations)

• One strip = $2.50


Different standardsDifferent standardsUse standardized reference

National Committee for Clinical Laboratory Standards (USA)

Other norms

• Canadian

• Chinese

• National

Do not confuse the different tables

Choose one for everything


Critical points in quality assuranceCritical 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


Standard strains for Standard strains for quality assurancequality 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


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 Intermediate Resistant

MIC < c ≤ MIC <

C ≤ MIC

InterpretationInterpretation


Understanding breakpointsUnderstanding 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


0

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0.01 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 128

MICc

Wild type

Inherited resistance

mechanism

C

The epidemiological concept for The epidemiological concept for breakpoints breakpoints


The pharmacological concept for The pharmacological concept for breakpointsbreakpoints

The concentration range tested for a drug and theinterpretative criteria for various categories are based onextensive 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 fordecision making and therefore the concept of breakpointconcentration


From breakpoints to interpretationFrom breakpoints to interpretation

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

MIC ≤ c Sensitive strain

MIC > C Intermediate strain

c < MIC ≤ C Resistant strain


Host factors affecting treatmentHost factors affecting treatment

Diffusion in tissues

Serum protein binding

Drug interactions

Immune system

Multiple simultaneous infections

Virulence of organism

Site and severity of infection


Interpreting intermediate resistanceInterpreting 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


Common interpretation problemsCommon 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


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/



Problem with the size of the inoculums

Solution:

• Use McFarland 0.5 photometer

• Scale -> same tubes



Contamination with another organism



Bad manipulation

Inoculation of the Muller Hinton

• Swabbing

• Not by flooding


Problems with E-test reading



Cost of anti-microbial resistanceCost of anti-microbial resistance

Cheap antimicrobials become ineffective

Individual treatment failure

Prolonged illness, hospitalization

Need to switch to more expensive, complex drugs that areoften not even available in resource-poor settings

Need to develop new antimicrobials

Good antimicrobial susceptibility testing saves lives andmoney


WHO Global Principles For The Containment of AntimicrobialResistance In Animals Intended for Food

Department for Communicable Diseases Surveillance and Response

World Health Organization

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

WHO/CDS/CSR/APH/2000.4

Distr. : General

English only


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

Antimicrobial resistance


ReferencesReferences

• 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/drugresist/WHO_CDS_CSR_RMD_2003_6/en/

Documents

Laboratory Training for Field Epidemiologists Antimicrobial resistance and susceptibility testing Antimicrobial resistance May 2007