cEPIDEMIOLOGICAL MARKERS

cDr. Soumya Dr. Savitha

cOverview
cIntroduction
cQualities of epidemiological markers
cCommonly used epidemiological markers
cDefinitions
cTyping
ãcúhat is typing
ãcScope of typing
ãcObjective of typing
ãcReasons for typing
ãcCriteria for validation of typing methods
ãcPhenotypic typing methods
ãcGenotypic typing methods
cApplications
cSummary
cReferences
cIntroduction
cQuickly & reliably differentiate related isolates
cY
 
  is the study of the dissemination
of human pathogens, including their transition patterns,
risk factors for & control of infectious disease in human
populations.
c
 
 are biological markers which
are used to characterize microorganisms or discriminate
between genomes based on genetic variation among
microbial isolates.
cEssential in research including taxonomy, microbial
epidemiology, population genetics & evolutionary
biology.
c
cBasic premise ± epidemiologically related isolates are
derived from the clonal expansion of a single precursor
& thereby share characteristics or markers that differ
from epidemiologically unrelated strains.
cUtility of a particular marker related to its stability within
a strain & its diversity within the species.
c
cDiversity is due to evolutionary genetic divergence
arising from random, non-lethal mutations that
accumulate over time.
cSuch mutations are detectable if they occur at sites that
can be assayed ( gene coding for a metabolic enzyme or
restriction site that determines a DNA fingerprint
pattern).
cQualities
cStable across generations to discriminate related &
unrelated strains
cShould be resistant to environmental perturbations &
high frequency genomic reorganization
cúidely available & common among strains
cEasy to detect
cShould provide data that reflects genetic distance at the
level necessary for answering the questions
cDefinitions
cm< Bacterial isolates that, although have been
cultured independently from different sources in different
locations & different times, still have so many identical
phenotypic & genotypic traits that the most likely
explanation for this identity is a common origin within a
relevant time span.
c
cS   
ãcA special pattern (eg.< DNA banding pattern) or set
of marker scores (eg.< absorbance values) displayed
by any isolate on application of one or more typing
methods.
cV 
 ãcGenetic constitution of an organism as assessed by a
molecular method
cÊ  
ãcObservable characteristic of an isolate
ãcúhat is typing?
cPhenotypic or genetic analysis of isolates below the
species or subspecies level, performed in order to
generate strain or clone specific fingerprints that can be
used for example to detect or rule out cross-infection,
elucidate transmission patterns & find reservoirs &
source of infection in humans.
cFacilitate determination of relatedness among isolates to
support or reject the hypothesis that the isolates came
from a single source
cApplies distinct labels to isolates
c
cIn practice, isolates from a cluster of infection ± different
primary types ± distinct strains ± not examined further.
cSame primary type ± further typing by secondary system
cThird method ± molecular methods
cTypes
cComparative< Outbreak related & unrelated strains
compared
cLibrary/Definitive typing< Strains from current outbreak
compared with previous strains
cCriteria for validation
cPerformance criteria
c÷   < epidemiological marker should remain stable
for each isolate after its primary isolation & during
laboratory storage & subculture ± across generations
c
cÕ    < assign type to all isolates tested by it.
cNon typable isolates are those for which typing yields
either a null or a non-interpretable result.

c
c| 
     ability to assign a different type
to 2 unrelated strains sampled randomly from the
population of a given species
c÷   

c
cN = total number of strains in the sample population
cS = total number of types described
c= number of strains belonging to the jth type
cIdeally 1.00, but in practice should be at least 0.95
c5% probability of error accepted.
cTyping methods exploring polymorphism at multiple
sites ± more discriminatory
c
cN =100, s = 20, n1 = 40, n2 = 30, n3 and n4 = 7, n5 to
n20 =1
cD = 1-[(40 x 39 + 30 x 29 + 7 x 6 + 7 x 6 + 1 x 0 . . .1 x
0)]
(100 x 99)
c= 1 - (2514/9900) = 0.746.
c
c
 




 should reflect, agree with,
& possible further illuminate the available
epidemiological information
c› 

    to assign the same type to an isolate
tested on independent occasions, separated in time or
space.
cConvenience criteria
cS     range of species that are typable with
minimal modifications of the method.
c›
  total time required to get from the isolates to
the final typing results.
c„

     availability of reagents & equipment
c
c technical simplicity, workload, suitability
for processing large number of isolates, ease of scoring
& interpreting the results
cm
cAmenability to
  
   & incorporation of
typing results in electronic

cTyping methods
cPhenotypic methods
cPhenotype reflects genotype
cExpression of genes is affected by environmental
changes& reversible phenotypic switching.
cIn addition phage & plasmid can be transmitted
horizontally
cBiotyping
cBiochemical characteristics that are known to vary
within a given species
cTypeability ± excellent
cDiscriminatory power ± variable
cStability dependent on species & characteristic
cVariation in gene expression is the most common reason
that isolates of same strain differ in one or more
biochemical reactions
 c
cRandom mutations may confound the interpretation of
these data
cTechnically easy & inexpensive, data generated easy to
score & interpret
cCommercial systems available
cDistinguish strains among species
cReproducibility organism &
character dependent.
ccc
ccc
cAntibiogram typing
cDrug diffusion on solid growth media or drug dilution in
liquid media
cCan be applied to most species
cDiscrimination dependent on diversity, stability &
relative prevalence of detectable acquired resistance
mechanism
c
cNumber of antimicrobials
cUtility varies according to stability of resistance patterns,
which can be insufficient for use as a clonal marker.
cPlasmid borne determinants ± readily lost in the absence
of selective conditions
cResistance expression can be under the influence of
complex regulatory systems
c
cQuantitative antibiogram typing
ãcsimilarity analysis of disk zone diameters
ãcEuclidean distance
ãcThe Euclidean distance between A and B is E= ¥(7-
12)2 + (17 - 20)2 + (16 - 15)2 = 5.9.
cSerotyping
cTraditionally most important phenotypic method ±
developed from early days of microbiology
cStill widely used
cReact with surface antigen
c
cHigh throughput procedures using defined sets of
polyclonal & monoclonal antibodies available
cTypeability & discrimination variable ± crossreactions
cDiscrimination improved by combining serotyping &
SDS-PAGE< úestern immunoblotting
c
cGenetic instability, horizontal gene transfer ± limit
serotyping
cSpecies with large number of antigenic variants ± poor
discriminatory power
cPreparation of sera expensive
cKauffmann-úhite classification of Salmonella Spp. and
the Lancefield grouping of Streptococci
cPhage typing
cAssess lytic patterns of test isolates that have been
exposed to a defined set of bacteriophages
cIsolates characterized by their susceptibility or
resistance by each member of a panel of bacteriophages.
cRestricted to a limited number of
species for which such agents
have been identified ±
Salmonella & S aureus
c
cDiscrimination variable, typeability partial,
reproducibility poor.
cExpertise, time consuming
cThere are at present 33 internationally recognized Vi-
types of the typhoid bacillus distinguishable by
specifically adapted preparations of Vi-phage II.
c

c„

c

cProtein antibiotics that kill sensitive indicator strains
cbased upon;
ãcAbility lyse a standard set of indicator strains
ãcSensitivity to bacteriocins produced by a set of
standard strains
ãcOriginally used in reference laboratories for typing
Klebsiella pneumoniae, Pseudomonas aeruginosa
c
c1 + reaction - partial inhibition with confluent growth.
c2+ reaction - partial inhibition showing patches of
semiconfluent growth or more than 10 colonies.
c3+ reaction - clear zone containing no more than 10
distinct colonies.
c4 + reaction ±
completely clear
zone of inhibition.
cSDS-PAGE of cellular & extracellular components
cDetects variation in structure of bacterial proteins
cHighly discriminatory typing method with applications in
taxonomy also
cLaborious & requires experience.
c
c
cReagent & equipment relatively inexpensive.
cModified for LPS by incorporating Proteinase K.
cVirtually all strains are typable.
cS aureus & Clostridium defficile
cMultilocus Enzyme Electrophoresis (MLEE)
cIdentifies electrophoretic variants of a set of
housekeeping enzymes encoded by different alleles of
the same gene by small but detectable variations in
protein size & charge
cReference method for defining phylogenetic structure of
clonal lineages in bacterial populations
c
cElectrophoretic mobility depends on net charge of the
protein.

cApproximately 15% amino acid changes can be resolved

by MLEE
cAlthough individual enzymes may be absent, evaluation
of multiple metabolic enzymes ensure that all isolates are
typable.
cMass Spectrometry
cMatrix ± Assisted Laser Desorption Ionization Time ± of
± Flight àY„ |
ÕS

c
cInfra red or Raman Spectroscopy< Use focused
illumination of bacterial biomass & emission spectra
generated are recorded.
cEach peak assigned to a sub molecular particle
cComposite pattern allows comparison to be performed &
types to be assigned
cGenotypic methods
cAssess variation in genomes with respect to composition,
overall structure or precise nucleotide sequence.
cHybridization ± mediated methods
ãcDirect hybridization
ãcRibotyping
ãcDirect hybridization
cImmobilized DNA is probed with DNA molecules that
are selective.
c÷ m ± Southern Hybridization
cAlso used to define nature of mobile elements.
c
c÷    DR contains multiple conserved 36 bp
repeats interspersed with nonrepetitive short spacer
sequences of 34 ± 41 bp.
cStrains tested by hybridizing PCR amplified DR regions
to a membrane that contains an array of 43 covalently
bound oligonucleotides representing polymorphic
spacers.
cEg.< Mycobacterium tuberculosis
cRibotyping
cVariant of Southern hybridization ± mediated assay that
estimates number of ribosomal gene loci & their position
in the chromosome.
cRibosomal sequences are highly conserved
cProbe derived from E coli ribosomal operon containing
23S & 16S sequences can be used with wide range of
bacterial species.
cAll bacteria carry these operons & therefore are typable
c
cOrganisms with multiple ribosomal operons, E coli,
Klebsiella, Haemophilus, Staphylococcus ± ribotyping
have 10 - 15 bands.
cProvide moderate to good discrimnatory power
cMycobacteria ± single operon ± only one or 2 bands ±
limited utility
cRibotypes relatively stable within a species &
reproducible
cGenome analysis by array hybridization
cSeveral hundreds & thousands of DNA probes are
immobilized per square centimeter of a solid matrix.
cProbes may be PCR products of defined length or
synthetic oligonucleotides.
cCost & accessibility ±
problematic
cHas been used to fingerprint
several bacteria(Salmonella,
N meningitidis), Viruses
(Influenza), parasites
(T gondii), fungi (C albicans)
cFragment based methods
cPlasmid Typing
cRestriction Fragment Length Polymorphism (RFLP)
cPCR fingerprinting
cMultilocus Variable Number Tandem Repeat (VNTR)
analysis (MLVA)
cPlasmid typing
cThe first DNA-based typing method for epidemiological
studies of nosocomial infections (1988)
cAssess number, size &/or restriction endonuclease
digestion profiles, after agarose gel electrophoresis
cTypeability & discrimination variable
cLack of stability ± lost or acquired spontaneously

c
cCombined with other genomic typing methods,
cUsed frequently in combination with antibiogram typing
to assess whether an antibiotic resistance gene is plasmid
± borne & can be transferred.
cMost effective in studies that are restricted in terms of
time & place ± those involving acute outbreaks within a
single hospital.
cRestriction Fragment Length Polymorphism
cIn divergence of strains within a species restriction sites
change, leading to changes in the length of the DNA
sequences between them
cDifferences in banding patterns of two isolates of same
species are due to difference in fragment sizes that can
occur as a result of changes in restriction site sequences,
secondary modifications of restriction sites, deletion or
insertion of sequences between restriction sites
cThese changes accumulate as strains diverge ± sum of
the changes provides an indicator of evolutionary
distance
cRestriction enzymes
cNatural part of the bacterial defense system
cRestrict ability of foreign DNA (such as bacteriophage
DNA) to infect/invade the host bacterial cell by cutting it
up

ãcEcoRI - from Escherichia coli - 5¶GAATTC3¶

ãcBamHI - from Bacillus amyloliquefaciens ±
5¶GGATCC3¶
ãcHindIII - from Haemophilus influenzae ±
5¶GANTC3¶
ãcPstI - from Providencia stuartii
ãcSau3AI - from Staphylococcus aureus
ãcAvaI - from Anabaena variabilis
c
cRestriction Endonuclease analysis (REA)<
 ãcEach restriction endonuclease enzyme cuts
(³digests´) DNA at particular (³restricted´)
nucleotide recognition sequence.
ãcNumber & size of restriction fragments generated
by digestion of a given piece of DNA reflect the
frequency & distribution of such restriction sites.
ãcFragments are separated by gel electrophoresis into
complex patterns
ãcPattern detected by staining gel with Ethydium
Bromide & photographing it under UV light.
ãc
ãcAll isolates are typable by REA
ãcRapid, reproducible, high discriminatory power
ãcMajor limitation is difficulty of interpretation of
complex profiles consisting of hundreds of bands
that may be unresolved & overlapping.
ãcFurther confounded by presence of plasmids ±
úhose DNA can readily contaminate genomic
DNA preparations
ãcSimplified by adding Southern blot & hybridization
steps
ãcIS 6110 Typing & IS 200 Typing

cPulse field gel electrophoresis

cMade it possible to separate large DNA fragments in
agarose gels by periodic alteration of angle of the electric
field.
cDNA fragments generated with restriction endonuclease
with 6 or more base pair recognition sites yielding fewer
than 30 large fragments, normally ranging in size
between 20 & 600 kbp.
c
cIsolates with patterns differing by 1 to 4 bands ±
subtypes of the same type
cIsolates with patterns differing by 5 or more bands ±
distinct types
cRemarkable discriminatory power & reproducibility
c2-4 days required, expensive equipment
cGels need to be analyzed closely & carefully
cPCR Fingerprinting
cEssential feature is ability to amplify rapidly &
exponentially a particular DNA sequence ± template,
typically 0.5kb to 2.0kb.
cReaction requires a DNA polymerase, only a minute
amount of template & oligonucleotides ± primers,
typically 18-20 base pair length corresponding to
sequences on the template
cEg.< BOX for ÷  or IS256 for ÷
cReadily detectable amount of product generated in less
than a few hours.
cFlexibility, technical simplicity, wide availability of
equipment & reagents, rapid
c
cSeveral modifications for epidemiological typing<
cPCR product digested with restriction endonuclease,
resulting restriction fragments analyzed
cUsed in S aureus & Cryptococcus neoformans
cARDRA< Amplified Ribosomal DNA Restriction
Analysis
c
cArbitrarily primed PCR< Employs a single, short
(typically 10bp length) primer whose nucleotide is not
directed at known genetic locus.
cResult in amplification of one or more unpredictable loci,
& PCR reaction will generate a set of fragments
cNumber & size of the fragment ± basis of typing the
isolate
cIdentifying primers that provide consistent reproducible
results ± difficult, discriminatory power - uncertain
cAmplified Fragment Length Polymorphism (AFLP)
analysis<
 ãcSelectively amplifies subsets of genomic fragments
generated with one or two restriction enzymes.
ãcRandom restriction fragments are singled out by
using a specific base sequence at the 3¶ end of the
primers.
c
cElongation will only take place if a nucleotide
complementary to the selective base in the primer
sequence is present in the fragment.
cProducts separated in agarose gel
cHighly reproducible.
cNearly whole genome coverage can be obtained.
cInter-repeat PCR
cVariable length segments found between consecutive
repeat elements, rather than repeat elements themselves
are amplified.
cS pneumoniae, Pseudomonas aeruginosa
cRandom Amplification of Polymorphic DNA
cMost frequent method of DNA fingerprinting of
eukaryotic organisms
cúith use of random primers of approximately 10 bases
in length, amplicons throughout genome are amplified by
PCR
cProducts separated on agarose gel & visualized by
ethydium bromide staining.
cReproducibility poor
cPolymorphisms arise when distances between primer
hybridization sites change or when primer sites appear,
disappear or change location due to insertion, deletion or
recombination.
cElectrophoretic Karyotyping
cFingerprinting eukaryotic pathogens
cNucleic acid released, electrophoresed.
cChromosomal DNA visualized with Ethydium Bromide
cIdentified by Southern Blot Hybridization with
chromosome specific probes
cMultilocus Variable Number Tandem Repeat (VNTR)
analysis (MLVA)
cCapitalizes on inherent variability encountered in many
regions of repetitive DNA
cVTNRs are very short tandem repetitive elements found
within genomes of both prokaryotes & eukaryotes
cRepetitive DNA is often incorrectly copied in bacterial
species, through slipped strand mispairing (SSM),
resulting in shortening or lengthening of repeat units.
c
cFor each repeat locus a digit can be assigned.
cúhen several repeat loci are analyzed per isolate, several
such digits are obtained, resulting in a multi-digit
specific strain code.
cIsolates within an outbreak have identical MLVA profile
cP falciparum
cSequence based methods
cComparing multiple isolates by sequencing the same
locus from each
cSingle Locus Sequence Typing (SLST)
cMultiple Locus Sequence Typing (MLST)
cSNP genotyping
cSingle Locus Sequence Typing (SLST)
cA single genetic locus is analyzed
cHighly variable gene sequences are selected
cemm typing for ÷ 
cMultiple Locus Sequence Typing (MLST)
cAssesses DNA sequence variation among alleles of
housekeeping genes
cAlso non-housekeeping genes & combination of both
cEpidemiological typing of    
cLimited accessibility, high cost
cSNP genotyping
cDetermination of nucleotide base that is present in a
given isolate at defined nucleotide positions known to be
variable within the population
cspa typing for S aureus
cA gene which is polymorphic due to 24-bp repeat
sequences that may vary in both number of repeats &
overall sequence in the polymorphic X or short sequence
repeat region.
cData analysis
cComputing Similarity Coefficient (SABS)
ãcPresence of band = 1
ãcAbsence of band = 0
ãcnAB = number of common bands (1,1)
ãca = number of bands in A with no counterpart in B
(1,0)
ãcb = number of bands in B with no counterpart in A
(0,1)
ãcc = number of bands absent in A & B
ãcNumber of matches m = nAB + C
ãcNumber of mismatches u = a + b
ãc
cCoefficient of Jaccard<
 ãcSj = nAB
nAB + a + b
cCoefficient of Dice<
ãcSD = 2nAB
2 nAB + a + b
cBand intensity
cGenerating Dendrograms
cMatrix of values generated for every pair of isolates
cUn-weighted Pair Group Method using Arithmetic
averages (UPGMA)
cSABS scanned for most similar isolates
cIf more than 1 group identified, first is arbitrarily taken
as group 1.
cIsolates are joined at appropriate positions along SABS
axis.
cMatrix scanned again for next most similar isolate or
group of isolates which is then connected along the SABS
matrix to the first group ± repeated over & over again
until all isolates are incorporated into the tree.
c
cBootstrapping
cJackknifing
c
ceBURST
ãcSimple presentation of relationships
ãcThe default setting in eBURST is the most exclusive
group definition, in which STs are included within
the same group only if they share identical alleles at
six or seven of the seven MLST loci with at least
one other ST in the group. Thus defined, each group
equates to a single clonal complex
ãcStrain with highest number
of single locus variants ±
founder genotype
cPhylogenetic tree
cExample
ãcSeq. A = A A C C G G T T
ãcSeq. B = A A C C G G T G
ãcSeq. C = A C C C G G T C
ãcSeq. D = A C C C G G T A
cApplications of typing
cSurveillance of infectious diseases
cSystematic, ongoing process of data collection, analysis,
interpretation, dissemination of results & action taken,
aimed at recording disease trends & designing ways in
which to curb them.
cDetection of clusters of pathogens with similar type ±
early warning of a potential outbreak
cSerotyping, phage typing, PFGE, MLST
cOutbreak investigation
cOutbreak can be defined as a temporal increase in the
incidence of infection (or colonization) by a certain
bacterial species, caused by enhanced transmission of a
specific strain.
cGenerate & test hypothesis
cPathogenesis & course of infection
cProgress of infection< endogenous microflora or an
exogenous source.
cPathogenesis related markers< virulent or non-virulent
cAntimicrobial Resistance Genotyping
cUse of probe hybridization or DNA amplification
techniques to detect various genes that encode for
antimicrobial resistance factors, thus providing an
antimicrobial resistance genotype.
cCalibration of conventional susceptibility test
cChoose antimicrobial therapy least likely to select
resistant organisms
cTo track the spread of specific resistance gene within &
among health care facilities & communities.
cBacterial population genetics
cIntra-species population structure ±phylogenetic
hypothesis.
cMLST
cValuable information concerning evolution &
diversification of species
cHow commonly bacterial genomes undergo horizontal
transfer
cSelection of vaccine strains
cSerotyping - Polio vaccine, S pneumoniae, H influenzae
cGenotyping ± Rotavirus G1 ± G4 & P1A & P1B ±
globally, G5, G8, P2A [6]
cIdentification of genes encoding dominant immuogens in
subunit & recombinant vaccines
cDifferentiate wild & vaccine strains
cd marker
crct 40
cMS
cMc Bride¶s intratypic antigenic marker
cMolecular< monoclonal antibodies specific for vaccine
strains, oligonucleotide fingerprinting, nucleic acid
sequencing
cSummary
cEpidemiological markers have played an important role
in study of bacterial evolution, outbreak investigation,
surveillance of infectious diseases.
cApplication of molecular techniques to identification &
differentiation of isolates provided a powerful set of new
tools that can augment both patient management &
epidemiological investigations.
cSupplement rather than replace clinical hypothesis.
cAlthough there are limitations for each technique, use of
combination of techniques provides good discriminatory
power to assess if two isolates are related or not
cReferences
cClinical Microbiology & Infectious Diseases, 13 (Suppl.
3), 1 ± 46.
cClinical Infectious Disease; 1993; 17<153-164
cTopley & úilson, Bacteriology I
cPattrick Murray, Manual of Clinical Microbiology, 9th
ed, Vol 1, 129 ± 151
cVarious journals.