part 2 lab methods overview

8/6/2019 Part 2 Lab Methods Overview

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Part 2Part 2Overview of laboratoryOverview of laboratory

methods used inmethods used inepidemiologic investigationsepidemiologic investigations


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Epidemiologic investigationsEpidemiologic investigations

Outbreak investigationsOutbreak investigations

SurveillanceSurveillanceResearchResearch


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Application of laboratory techniques toApplication of laboratory techniques to

epidemiologic investigationsepidemiologic investigations

DiagnosisDiagnosis

Gene probesGene probes

PCRPCR

Strain typing (subtyping)Strain typing (subtyping)

Growth and morphologicGrowth and morphologic

BiochemicalBiochemical

FunctionalFunctional

SerologicSerologic

GenotypicGenotypic


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Laboratory methods used to stratify dataLaboratory methods used to stratify data

related to infectious diseasesrelated to infectious diseases

Phenotypic methodsPhenotypic methods

Examine observable characteristics expressed by a cell orExamine observable characteristics expressed by a cell or

an organism (e.g., drug resistance, virulence,an organism (e.g., drug resistance, virulence,

morphology)morphology)

Genotypic methodsGenotypic methods

Examine genetic characteristics of a cell or an organismExamine genetic characteristics of a cell or an organism

according to its genome or specific genetic loci.according to its genome or specific genetic loci.


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Conventional laboratory techniques used toConventional laboratory techniques used to

subtype organismssubtype organisms

Growth and morphologic characteristicsGrowth and morphologic characteristics

Biochemical characteristicsBiochemical characteristics

Serologic or immunologic characteristicsSerologic or immunologic characteristics

Functional or physiologic characteristicsFunctional or physiologic characteristics antibiotic susceptibility testsantibiotic susceptibility tests

phage typingphage typing

colicin typingcolicin typing

cell culture assayscell culture assays toxigenicity assaystoxigenicity assays

in vitro survival characteristicsin vitro survival characteristics

Multilocus enzyme electrophoresis (MLEE)Multilocus enzyme electrophoresis (MLEE)


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Colony morphology


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From American Society of Microbiology,

Color Atlas of Bacteriology, 2007

Acid fast stain (AFB)

Gram-negative rods Gram-positive cocci

Gram-positive rods


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Staphylococcus aureus


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E. coli


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Leptospira interrogans


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Yeast

Hyphae

Fungi


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Biochemical testsBiochemical tests

e.g.,e.g., Triple sugar iron (TSI) slants (alkaline/acid/gas)Triple sugar iron (TSI) slants (alkaline/acid/gas)

Shigella spp. Salmonella typhimurium Escherichia coli


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API 20E system

From de la Maza et al, Color Atlas of

Medical Bacteriology, 2004

Biochemical testsautomated systems


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Serologic testsSerologic testse.g., grame.g., gram--negative bacterianegative bacteria

SerogroupingSerogroupingOO--polysaccharide (O) antigenspolysaccharide (O) antigens

SerotypingSerotypingO andO and flagellarflagellar (H) antigens(H) antigens

E. ColiO104:H4


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Strain typing by functional or physiologicStrain typing by functional or physiologic

characteristicscharacteristics

antibiotic susceptibility testsantibiotic susceptibility tests----antibiogramsantibiograms

phage typingphage typingphage typesphage types

colicin typingcolicin typingcolicin (or bacteriocin) typescolicin (or bacteriocin) types

cell culture assayscell culture assaysvirulence types (pathotypes)virulence types (pathotypes)

toxigenicity assaystoxigenicity assaysvirulence types (pathotypes)virulence types (pathotypes)

in vitro survival characteristicsin vitro survival characteristicsvirulence typesvirulence types


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Phagetyping


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E. coli K12 and HeLa cells


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Localized adherence (LA) ofEPEC on HeLa cells


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Diffuse-adherent E. coli (DAEC)


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Transmission EM of enteroinvasiveE. coli (EIEC)


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Genotyping techniques usedGenotyping techniques usedin epidemiologicin epidemiologicinvestigationsinvestigations


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Genotyping techniques used inGenotyping techniques used in

epidemiologic investigationsepidemiologic investigations

Electrophoretic bandingElectrophoretic banding

patternspatterns

Nucleic acid hybridizationNucleic acid hybridization

Nucleic acid sequencingNucleic acid sequencing


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Genotyping techniques used in epidemiologicGenotyping techniques used in epidemiologic

investigationsinvestigations

Plasmid profile analysisPlasmid profile analysis

Genomic fingerprintingGenomic fingerprinting

--chromosomal digestchromosomal digest

--restriction fragment length polymorphism (RFLP)restriction fragment length polymorphism (RFLP)--pulse field gel electrophoresis (PFGE)pulse field gel electrophoresis (PFGE)

--segmented RNA gel electrophoresissegmented RNA gel electrophoresis

--ribosomal RNA gel electrophoresisribosomal RNA gel electrophoresis

--PCRPCR--based methodsbased methods Nucleic acid hybridizationNucleic acid hybridization

Nucleic acid sequencingNucleic acid sequencing


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Genotyping techniquesGenotyping techniques

Techniques that measureTechniques that measure microdiversitymicrodiversity

Analyzes changes that occur in part of the genomeAnalyzes changes that occur in part of the genomeof a microorganismof a microorganism

Techniques that measureTechniques that measure macrodiversitymacrodiversity

Analyzes the whole genomeAnalyzes the whole genome


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Gel electrophoresis: principlesGel electrophoresis: principles

ConventionalDNA electrophoresis:ConventionalDNA electrophoresis: can separatecan separateDNA


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Strain typing methods based on electrophoreticStrain typing methods based on electrophoretic

analysis of nucleic acid fragmentsanalysis of nucleic acid fragments

Restriction fragment length polymorphism (RFLP)Restriction fragment length polymorphism (RFLP)

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

RFLPRFLP--Southern blot hybridizationSouthern blot hybridization

Ribosomal RNA gel electrophoresis (ribotyping)Ribosomal RNA gel electrophoresis (ribotyping)

Segmented RNA gel electrophoresisSegmented RNA gel electrophoresis

PCRPCR--based methodsbased methods

All of these techniques were developed to overcome the problem ofAll of these techniques were developed to overcome the problem of

ease of interpretation of the pattern of nucleic acid restrictionease of interpretation of the pattern of nucleic acid restriction

fragments resolved by electrophoresis.fragments resolved by electrophoresis.


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Restriction fragment length polymorphism (RFLP)analysis of M. tuberculosis DNA (Ferrazoli et al)


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IS6110 Southern blot hybridization of RFLP analysis(Ferrazoli et al)


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Gel electrophoresis: principlesGel electrophoresis: principles--cont.cont.

Pulse field gel electrophoresis (PFGE): separatesPulse field gel electrophoresis (PFGE): separates

DNA up to 4DNA up to 4--Mb in lengthMb in length

movement depends on DNAs plasticity inmovement depends on DNAs plasticity in

electrical field applied in different directionselectrical field applied in different directions

separation depends on DNA reorienting itself inseparation depends on DNA reorienting itself in

the gel matrix as the electrical field orientationthe gel matrix as the electrical field orientation

changeschanges


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Obstacles that had to be overcome to doObstacles that had to be overcome to do

PFGEPFGE

DNA fragments of >25,000 are poorlyDNA fragments of >25,000 are poorly

resolved by agarose gel electrophoresisresolved by agarose gel electrophoresis

Autodigestion of large pieces of DNAAutodigestion of large pieces of DNA

generated by raregenerated by rare--cutter endonucleases.cutter endonucleases.


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Conventional gel electrophoresis PFGE

Principles of gel electrophoresis--


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Pulse field gel electrophoresis (PFGE): patternPulse field gel electrophoresis (PFGE): pattern

interpretationinterpretation

(Tenover et al.(Tenover et al.J Clin MicrobiolJ Clin Microbiol

. 1995; 33:2233). 1995; 33:2233)


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Criteria for interpreting PFGE patternsCriteria for interpreting PFGE patterns(Tenover et al, J Clin Microbiol 1995;33:2233)(Tenover et al, J Clin Microbiol 1995;33:2233)

Category Number of Typicalmutations number of

band differences

Indistinguishable 0 0

Closely related 1 2-3

Possibly related 2 4-6

Different >3 >7


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Criteria for interpreting PFGE patternsCriteria for interpreting PFGE patterns

cont.cont.

Important to identify an appropriate reference strain first;Important to identify an appropriate reference strain first;comparison of PFGE patterns must be made against such acomparison of PFGE patterns must be made against such areference strain.reference strain.

Comparison should not be made among subtype strainsComparison should not be made among subtype strainsthemselves.themselves.

Reference strainReference strain::

Recognized epidemic or outbreak strainRecognized epidemic or outbreak strain

Predominant strain in a limited geographic and temporalPredominant strain in a limited geographic and temporalsetting identified by a surveillance systemsetting identified by a surveillance system

WellWell--documented newly introduced strain (e.g., in adocumented newly introduced strain (e.g., in ahospital or community)hospital or community)


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Plasmid profile analysis


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Segmented RNAgel electrophoresis

From Rogers et al, 1981

Segmented RNAgel electrophoresis of rotaviruses from Melbourne,Australia


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Shigella dysenteriaetype 1 ribotyping(CDC, Wachsmuth)


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PFGE analysis ofE. coliisolates from cases of

urinary tract infection (Manges et al)

1 2 3 4 5 6 7 8 9 10


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Methods used to genotypeMethods used to genotype

M. tuberculosisM. tuberculosis

IS6110IS6110 RFLPRFLP

SpoligotypingSpoligotyping

MIRUMIRU--VNTRVNTR DeligotypingDeligotyping

Repetitive element PCRRepetitive element PCR

RAPD PCRRAPD PCR PFGEPFGE

part 2 lab methods overview

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