nspcd laboratories -status and achievements dr. manish

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

Typing

May 2007

Typing methods for bacteriaTyping methods for bacteria


Learning objectivesLearning objectivesAt the end of the presentation, participants should:

• Identify situations when typing is relevant

• Know different methods of typing

• Understand problems that arise when using typing methods

Isolate versus StrainIsolate versus StrainIsolate: a pure culture derived from a single colony that is presumed to arise from a single organism/bacterium

Strain: a set of isolates, that when typed are indistinguishable from each other and can be differentiated from other isolates

OUTBREAK

OUTBREAK STRAIN

STRAIN 2

STRAIN 3

STRAIN 4

STRAIN 5

STRAIN 6Laboratory Training for Epidemiologists 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

A simple question ?A simple question ?Are these isolates the same or different?

Through a typing method we are looking for:

• Epidemiologically linked isolates that represent the clonal expansion of a single precursor

• Clonal isolates are the same type and unrelated isolates have a different type

Laboratory Training for Epidemiologists 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

Typing system evaluation criteriaTyping system evaluation criteria

Typeability Capacity to produce clearly interpretable results with most strains of the bacterial species

Reproducibility Capacity to repeatedly obtain the same typing profile result with the same bacterial strain

Discriminatory power

Ability to produce results that clearly allow differentiation between unrelated strains of the same bacterial species

Practicality (ease of performance & interpretation)

Method should be versatile, relatively rapid, inexpensive, technically simple and provide readily interpretable results


Teichoid acid

Lipoteichoic acid

Capsule

Polysaccharides

Fimbirae (M-protein)

Different Enzymes

Peptidoglycan

Phenotype & genotype bacteriaPhenotype & genotype bacteria

PHENOTYPEGENOTYPE

(Chromosomal & plasmid DNA)


Typing methodsTyping methodsPhenotypic

• Rely on expression of phenotypic characteristics (genetically coded)

– Antibiotic resistance, antigens etc.

Genotypic

• Analysis of the genetic material

– DNA, RNA

Laboratorz Training for Epidemiologists 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

Phenotypic techniquesPhenotypic techniquesSerotyping

Phage typing

Antimicrobial resistance monitoring

Multilocus enzyme electrophoresis (MLEE)

Other:

• Protein profiling – SDS PAGE, immunoblotting

• Based on nutritional requirement e.g. auxotyping

• Biotyping

• Bacteriocin typing


Phenotypic techniquesPhenotypic techniquesSerotyping

• Antigenetic determinants expressed on the cell surface

• Still widely used for Salmonella, Shigella, Neiseria, E. coli, V cholerae

• Slide/ tube agglutination

• LIMITATION: Requires extensive stock of absorbed/monoclonal sera (e.g. >2200 antisera required for definitive Salmonella typing)

Phage typing

• Viruses that infect and destroy bacterial cells –Bacteriophage

• The resistance or susceptibility of strains is used for differentiation

• LIMITATION: Technically demanding, time consuming, typeability is an issue


Phenotypic techniquesPhenotypic techniquesAntibiotic susceptibility testing

• Based on susceptibility of bacterial isolates to a panel of antimicrobial agents

• Routinely performed on clinical isolates

• A reasonable preliminary indicator to initiate epidemiological action

LIMITATIONS:

– Antibiotic resistance under extraordinary selective pressure

– Multiple mechanisms for a strain to become abruptly resistant


Phenotypic TechniquesPhenotypic TechniquesPhenotypic characteristics can vary in different conditions

• Antibiotic resistance can be expressed under antibiotic pressure

Methods are not very discriminatory



MLEEMLEECharacterizes the cellular proteins by electrophoreticallyseparating them in a gel matrix

Exposing the gel to chromogenic substrates (that reactwith the enzymes)

Limitation: Complexity of interpretation


Phenotypic typing system Phenotypic typing system characteristicscharacteristics

Typing system

Typeability Reproducibility Discrimination Ease of interpretation

Ease of performance

Serotyping Most Good Fair Good Fair

Phage typingMost Fair Fair Fair Poor

Antibiotic susceptibility

testingAll Fair Poor Excellent Excellent

MLEEAll Excellent Good Excellent Good


0

5

10

15

20

25

30 Phage type "1var3"Other phage types

Cases

July August September October November December

Phenotypic typing during an outbreakPhenotypic typing during an outbreakOutbreakOutbreak of Paratyphi B salmonellosis phage type 1var3 of Paratyphi B salmonellosis phage type 1var3

France, 1993France, 1993

E P I D E M I C A L E R T A N D R E S P O N S E

DNA moleculeDNA molecule


Source: Wikipedia, created by Michael Ströck

Genotypic methodsGenotypic methodsPlasmid profiling

Restriction enzyme analysis (REA)

Restriction fragment length polymorphism (RFLP)

Ribotyping

Pulse Field Gel Electrophoresis (PFGE)

Random Amplified Polymorphic DNA (RAPD)

Nucleic acid sequencingLaboratory Training for Epidemiologists 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

The principleThe principleWithout amplification

• Cutting the DNA in pieces

• Visualizing the pieces

With amplification

• Amplifying (using PCR) parts of the DNA

• Visualizing the pieces


Genotypic typing methodsGenotypic typing methodsMethods without prior amplification

• Isolation of the pathogen

• Extraction of the DNA

• Cutting the DNA with Enzymes (restriction endonuclease enzymatically cuts/ “digests” DNA at a specific/ “restricted” nucleotide recognition sequence)

• Separation of the pieces by size using an electric field (Gel-Electrophoresis)

• Visualization


Example of molecular typingExample of molecular typing

Cutting locations

Gel-Electrophoresis

Size of fragments


Restriction Enzyme Analysis (REA)Restriction Enzyme Analysis (REA)•Extraction of plasmid or chromosomal DNA

•Digestion of the DNA at particular sites using specific restriction enzymes

•Hundreds of DNA fragments of various sizes (0.5-50Kb) separated by gel electrophoresis

•LIMITATION: Complex profiles with hundreds of unresolved or overlapping bands


Southern blot analysis of RFLP Southern blot analysis of RFLP & ribotyping& ribotyping

Better analysis of restriction enzyme patterns

• Specific parts of the pieces are detected by pieces of DNA as a probe - Southern Blot

• Variation in number & size of fragments detected by the markers are referred to as restriction fragment length polymorphism (RFLP)

• Ribotyping: when probes mark ribosomal operons

LIMITATIONS: technically complex, organisms with singlecopy of ribosomal operons


Southern/Northern blottingSouthern/Northern blotting


Separate DNA fragments on the agarose gel

"Blot" DNA to membran

Membrane imprinted with DNA bands

Add a labelled probe to the membrane

Visualization reveals a band where your probe bound to the target sequence

Visualization with a markerVisualization with a marker

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__

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_

_I I I_


Adapted from: http://lifesciences.asu.edu/resources/mamajis/southern/southern.html

Pulsed-field gel electrophoresis (PFGE)Pulsed-field gel electrophoresis (PFGE)

• Rare cutting enzymes

• Alternate current orientations allow separation of large DNA fragments

• Highly discriminatory and reproducible; currently the method of choice for typing a range of bacteria

LIMITATIONS: time consuming (≥2 days), expensive, specialized equipment


Pulsed-field gel electrophoresis Pulsed-field gel electrophoresis (PFGE)(PFGE)

RFLPs of VRE isolates as determined by PFGE; all appear identical

RFLPs of two strains (B & C) from a patient as determined by PFGE; both different implying mixed infection; lane A is marker

RFLPs of MRSA isolates with similar ABT ST profile as determined by PFGE; only isolates B & C are identical


Genotypic typing methodsGenotypic typing methodsMethods with prior amplification

• Extraction of the DNA, separation

• Target with primer

• Amplification of specific region

• Separation of amplicons according to size using an electric field (gel-electrophoresis)


Molecular typingMolecular typing

Primer-locations

Gel-Electrophoresis

Size of (amplified) fragments


Random Amplification of Polymorphic Random Amplification of Polymorphic DNA (RAPD )DNA (RAPD )

Uses short primers that find a lot of targets

Different size amplicons

Products separated by electrophoresis

LIMTATIONS:

• Identification of suitable primers

• Difficult to interpret differences in the intensity of bands

• Inefficient reactions

• Amplification of cryptic genetic material (prophages, bacteriophages)


RAPD-PCRRAPD-PCR

60 70 80 90 100% 10 Isolates, two clusters

(3 isolates each)


Nucleic acid sequencingNucleic acid sequencingEnumeration of individual nucleotide base pairs

Provides highly reliable and objective data suitable forsubsequent quantitative analysis

Necessary for virus typing

LIMITATIONS:

• Locus with sufficient sequence variability

• Sequencing of a single locus may not be reliable result

• Prohibitively expensive for most settings


Multi Locus sequence typing Multi Locus sequence typing (MLST)(MLST)

Targets different DNA pieces and sequences them

Compares results with data banks

Pro: highly comparable

Con: expensive equipment

Laboratory Training for Epidemiologists

Genotypic typing system Genotypic typing system characteristics characteristics

Typing system Typeability Reproducibility Discrimination Ease of interpretation

Ease of performance

REA All Good Good Poor Excellent

Ribotyping All Excellent Fair Good Good

PFGE All Excellent Excellent Excellent Good

Restriction digests of PCR products

All Excellent Good Excellent Good

PCR based on repeated sequences

All Good Good Good Good

RAPD All Fair Good Fair Good

Nucleotide sequencing All Excellent Excellent Excellent Fair


Limitations of typing methodsLimitations of typing methods• Discriminatory function

• Type of material and pathogen

• Reproducibility , cost, technique, etc.

• No “gold standard”

RESULTS OF A TYPING SYSTEM SHOULD BE CONSIDERED RELATIVE TO THE AVAILABLE EPIDEMIOLOGICAL DATA OR TO THE RESULTS OF OTHER SYSTEMS

• The technique used needs to be adapted to the question


Llisteriosis outbreaks, France 1999-2000Llisteriosis outbreaks, France 1999-2000

0

2

4

6

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12

14 Other sporadic cases

Sporadic cases used as controls

Outbreak 2 (32 cases)

Outbreak 1 (11 cases)

October November December January February March 1999 2000

Cases


Interpretation of strain typing dataInterpretation of strain typing dataSeveral factors affect interpretation:

Natural biologic variation

• Epidemiologically related isolates of the same strain demonstrate minor typing differences due to phenotypic variations or actual genotypic alterations, when collected and examined over an extended interval

Technical variations

• Limited reproducibility and discriminatory power


Interpretation of typing resultsInterpretation of typing resultsGenetic relatedness assessed with clinical andepidemiologic relatedness

Restrict analysis to discrete set of isolates (≤30)

Identify “index isolate” as starting point for analysis that isdefined on the basis of:

• Epidemiological data (first case in an outbreak)

• Clinical data (initial isolate from patient with multiple infections)

• Strain typing data (most common strain type in the set)


Interpretation of typing resultsInterpretation of typing resultsMultiple isolates representing a single type are mostappropriately designated “indistinguishable”

No typing method confirms that entire genomes of twoorganisms are identical

Indistinguishable vs. closely related vs. possibly related

→Final assessment lies with integration of molecular and epidemiological analyses


Problems with result interpretationProblems with result interpretation

Types versus subtypes

Isolates assigned as different types if differ in some specified manner (2 or more band shift in S blot)

Isolates that differ but not sufficiently to be designated as distinct types are designated as subtypes of similar types

Restriction fragments of different sizes may represent same chromosomal DNA

Insertion or deletion of extra-chromosomal DNA such as bacteriophage DNA

DNA fragment data are not suitable for quantitating genetic relatedness among different isolates


Application of typing systemsApplication of typing systems

Detection of outbreaks:

Concept of “prior probability”; rigorous epidemiologicalinvestigation and data to avoid misleading results

Epidemiological investigation

Request for Molecular typing

• Increased prevalence

• Same bacteria species from a cluster of cases

• Multiple isolates with distinct biotype/ AST pattern

Typing technique with

good reproducibility & discriminatory

power


Applications of typing systemsApplications of typing systemsDistinguish relapse from re-infection

Identify types associated with increased transmission & virulence

Emergence of new types ; implications on control measures

Clonality of acute infection:

• Infection vs. colonization vs. sample contamination

• Pseudo-outbreaks related to:

– Clinician or clinical entity

– Laboratory

– Case finding

– Chance clustering


Phenotypic vs. genotypic typing of Phenotypic vs. genotypic typing of Staphylococcus aureusStaphylococcus aureus

Method Discriminatory index

Percentage Typeability

Ease of performance

Basic set up

Phage typing0.556 30 Reference

laboratory

ARM .880 100 ++++ All laboratories

Coagulase typing .659 100 ++++ All laboratories

Protien profiling – SDS PAGE, Immunoblotting

.978 100 +++Molecular

biology laboratory

Ribotyping .845 100 +

Molecular biology

laboratory

PFGE.986 100 ++

Molecular biology

laboratory


To summarizeTo summarizeTyping data are most appropriately evaluated in the context of a hypothesis and questions thoughtfully developed by the clinician or the epidemiologist. They should augment rather that replace those analyses

Typing is performed independently by the laboratory to avoid any bias but the results are considered collaboratively

Laboratory Training for Epidemiologists Laboratory Training for Epidemiologists


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

Typing

nspcd laboratories -status and achievements dr. manish

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