EPIDEMIOLOGICAL MARKERS

Dr. Soumya Dr. Savitha

Overview

Introduction

Qualities of epidemiological markers

Commonly used epidemiological markers

Definitions

Typing

What is typing

Scope of typing

Objective of typing

Reasons for typing

Criteria for validation of typing methods

Phenotypic typing methods

Genotypic typing methods

Applications

Summary

References

Introduction

Quickly & reliably differentiate related isolates

Microbial epidemiology is the study of the dissemination of human pathogens, including their transition patterns, risk factors for & control of infectious disease in human populations.

Epidemiological markers are biological markers which are used to characterize microorganisms or discriminate between genomes based on genetic variation among microbial isolates.

Essential in research including taxonomy, microbial epidemiology, population genetics & evolutionary biology.

Basic 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.

Utility of a particular marker related to its stability within a strain & its diversity within the species.

Diversity is due to evolutionary genetic divergence arising from random, non-lethal mutations that accumulate over time.

Such 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).

Qualities

Stable across generations to discriminate related & unrelated strains

Should be resistant to environmental perturbations & high frequency genomic reorganization

Widely available & common among strains

Easy to detect

Should provide data that reflects genetic distance at the level necessary for answering the questions

Definitions

Clone: 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.

Fingerprint

A 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.

Genotype

Genetic constitution of an organism as assessed by a molecular method

Phenotype

Observable characteristic of an isolate

What is typing?

Phenotypic 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.

Facilitate determination of relatedness among isolates to support or reject the hypothesis that the isolates came from a single source

Applies distinct labels to isolates

In practice, isolates from a cluster of infection – different primary types – distinct strains – not examined further.

Same primary type – further typing by secondary system

Third method – molecular methods

Types

Comparative: Outbreak related & unrelated strains compared

Library/Definitive typing: Strains from current outbreak compared with previous strains

Criteria for validation

Performance criteria

Stability: epidemiological marker should remain stable for each isolate after its primary isolation & during laboratory storage & subculture – across generations

Typeability: assign type to all isolates tested by it.

Non typable isolates are those for which typing yields either a null or a non-interpretable result.

Discriminatory power: ability to assign a different type to 2 unrelated strains sampled randomly from the population of a given species

Simpson’s index

N = total number of strains in the sample population

S = total number of types described

nj = number of strains belonging to the jth type

Ideally 1.00, but in practice should be at least 0.95

5% probability of error accepted.

Typing methods exploring polymorphism at multiple sites – more discriminatory

N =100, s = 20, n1 = 40, n2 = 30, n3 and n4 = 7, n5 to n20 =1

D = 1-[(40 x 39 + 30 x 29 + 7 x 6 + 7 x 6 + 1 x 0 . . .1 x 0)]

(100 x 99)

= 1 - (2514/9900) = 0.746.

Epidemiological concordance: should reflect, agree with, & possible further illuminate the available epidemiological information

Reproducibility: to assign the same type to an isolate tested on independent occasions, separated in time or space.

Convenience criteria

Flexibility: range of species that are typable with minimal modifications of the method.

Rapidity: total time required to get from the isolates to the final typing results.

Accessibility: availability of reagents & equipment

Ease of use: technical simplicity, workload, suitability for processing large number of isolates, ease of scoring & interpreting the results

Cost

Amenability to computerized analysis & incorporation of typing results in electronic databases

Typing methods

Phenotypic methods

Phenotype reflects genotype

Expression of genes is affected by environmental changes& reversible phenotypic switching.

In addition phage & plasmid can be transmitted horizontally

Biotyping

Biochemical characteristics that are known to vary within a given species

Typeability – excellent

Discriminatory power – variable

Stability dependent on species & characteristic

Variation in gene expression is the most common reason that isolates of same strain differ in one or more biochemical reactions

Random mutations may confound the interpretation of these data

Technically easy & inexpensive, data generated easy to score & interpret

Commercial systems available

Distinguish strains among species

Reproducibility organism &

character dependent.

Antibiogram typing

Drug diffusion on solid growth media or drug dilution in liquid media

Can be applied to most species

Discrimination dependent on diversity, stability & relative prevalence of detectable acquired resistance mechanism

Number of antimicrobials

Utility varies according to stability of resistance patterns, which can be insufficient for use as a clonal marker.

Plasmid borne determinants – readily lost in the absence of selective conditions

Resistance expression can be under the influence of complex regulatory systems

Quantitative antibiogram typing

similarity analysis of disk zone diameters

Euclidean distance

The Euclidean distance between A and B is E= √(7- 12)2 + (17 - 20)2 + (16 - 15)2 = 5.9.

Serotyping

Traditionally most important phenotypic method – developed from early days of microbiology

Still widely used

React with surface antigen

High throughput procedures using defined sets of polyclonal & monoclonal antibodies available

Typeability & discrimination variable – crossreactions

Discrimination improved by combining serotyping & SDS-PAGE: Western immunoblotting

Genetic instability, horizontal gene transfer – limit serotyping

Species with large number of antigenic variants – poor discriminatory power

Preparation of sera expensive

Kauffmann-White classification of Salmonella Spp. and the Lancefield grouping of Streptococci

Phage typing

Assess lytic patterns of test isolates that have been exposed to a defined set of bacteriophages

Isolates characterized by their susceptibility or

resistance by each member of a panel of bacteriophages.

Restricted to a limited number of

species for which such agents

have been identified –

Salmonella & S aureus

Discrimination variable, typeability partial, reproducibility poor.

Expertise, time consuming

There are at present 33 internationally recognized Vi-types of the typhoid bacillus distinguishable by specifically adapted preparations of Vi-phage II.

Bacteriocin Typing

Protein antibiotics that kill sensitive indicator strains

based upon;

Ability lyse a standard set of indicator strains

Sensitivity to bacteriocins produced by a set of standard strains

Originally used in reference laboratories for typing Klebsiella pneumoniae, Pseudomonas aeruginosa

1 + reaction - partial inhibition with confluent growth.

2+ reaction - partial inhibition showing patches of semiconfluent growth or more than 10 colonies.

3+ reaction - clear zone containing no more than 10 distinct colonies.

4 + reaction –

completely clear

zone of inhibition.

SDS-PAGE of cellular & extracellular components

Detects variation in structure of bacterial proteins

Highly discriminatory typing method with applications in taxonomy also

Laborious & requires experience.

Reagent & equipment relatively inexpensive.

Modified for LPS by incorporating Proteinase K.

Virtually all strains are typable.

S aureus & Clostridium defficile

Multilocus Enzyme Electrophoresis (MLEE)

Identifies 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

Reference method for defining phylogenetic structure of clonal lineages in bacterial populations

Electrophoretic mobility depends on net charge of the protein.

Approximately 15% amino acid changes can be resolved by MLEE

Although individual enzymes may be absent, evaluation of multiple metabolic enzymes ensure that all isolates are typable.

Mass Spectrometry

Matrix – Assisted Laser Desorption Ionization Time – of – Flight (MALDI-TOF).

Infra red or Raman Spectroscopy: Use focused illumination of bacterial biomass & emission spectra generated are recorded.

Each peak assigned to a sub molecular particle

Composite pattern allows comparison to be performed & types to be assigned

Genotypic methods

Assess variation in genomes with respect to composition, overall structure or precise nucleotide sequence.

Hybridization – mediated methods

Direct hybridization

Ribotyping

Direct hybridization

Immobilized DNA is probed with DNA molecules that are selective.

Southern & Colleagues – Southern Hybridization

Also used to define nature of mobile elements.

Spoligotyping: DR contains multiple conserved 36 bp repeats interspersed with nonrepetitive short spacer sequences of 34 – 41 bp.

Strains tested by hybridizing PCR amplified DR regions to a membrane that contains an array of 43 covalently bound oligonucleotides representing polymorphic spacers.

Eg.: Mycobacterium tuberculosis

Ribotyping

Variant of Southern hybridization – mediated assay that estimates number of ribosomal gene loci & their position in the chromosome.

Ribosomal sequences are highly conserved

Probe derived from E coli ribosomal operon containing 23S & 16S sequences can be used with wide range of bacterial species.

All bacteria carry these operons & therefore are typable

Organisms with multiple ribosomal operons, E coli, Klebsiella, Haemophilus, Staphylococcus – ribotyping have 10 - 15 bands.

Provide moderate to good discrimnatory power

Mycobacteria – single operon – only one or 2 bands – limited utility

Ribotypes relatively stable within a species & reproducible

Genome analysis by array hybridization

Several hundreds & thousands of DNA probes are immobilized per square centimeter of a solid matrix.

Probes may be PCR products of defined length or synthetic oligonucleotides.

Cost & accessibility –

problematic

Has been used to fingerprint

several bacteria(Salmonella,

N meningitidis), Viruses

(Influenza), parasites

(T gondii), fungi (C albicans)

Fragment based methods

Plasmid Typing

Restriction Fragment Length Polymorphism (RFLP)

PCR fingerprinting

Multilocus Variable Number Tandem Repeat (VNTR) analysis (MLVA)

Plasmid typing

The first DNA-based typing method for epidemiological studies of nosocomial infections (1988)

Assess number, size &/or restriction endonuclease digestion profiles, after agarose gel electrophoresis

Typeability & discrimination variable

Lack of stability – lost or acquired spontaneously

Combined with other genomic typing methods,

Used frequently in combination with antibiogram typing to assess whether an antibiotic resistance gene is plasmid – borne & can be transferred.

Most effective in studies that are restricted in terms of time & place – those involving acute outbreaks within a single hospital.

Restriction Fragment Length Polymorphism

In divergence of strains within a species restriction sites change, leading to changes in the length of the DNA sequences between them

Differences 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

These changes accumulate as strains diverge – sum of the changes provides an indicator of evolutionary distance

Restriction enzymes

Natural part of the bacterial defense system

Restrict ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host bacterial cell by cutting it up

EcoRI -  from Escherichia coli - 5’GAATTC3’

BamHI - from Bacillus amyloliquefaciens – 5’GGATCC3’

HindIII - from Haemophilus influenzae – 5’GANTC3’

PstI -  from Providencia stuartii

Sau3AI - from Staphylococcus aureus

AvaI -  from Anabaena variabilis

Restriction Endonuclease analysis (REA):

Each restriction endonuclease enzyme cuts (“digests”) DNA at particular (“restricted”) nucleotide recognition sequence.

Number & size of restriction fragments generated by digestion of a given piece of DNA reflect the frequency & distribution of such restriction sites.

Fragments are separated by gel electrophoresis into complex patterns

Pattern detected by staining gel with Ethydium Bromide & photographing it under UV light.

All isolates are typable by REA

Rapid, reproducible, high discriminatory power

Major limitation is difficulty of interpretation of complex profiles consisting of hundreds of bands that may be unresolved & overlapping.

Further confounded by presence of plasmids – Whose DNA can readily contaminate genomic DNA preparations

Simplified by adding Southern blot & hybridization steps

IS 6110 Typing & IS 200 Typing

Pulse field gel electrophoresis

Made it possible to separate large DNA fragments in agarose gels by periodic alteration of angle of the electric field.

DNA 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.

Isolates with patterns differing by 1 to 4 bands – subtypes of the same type

Isolates with patterns differing by 5 or more bands – distinct types

Remarkable discriminatory power & reproducibility

2-4 days required, expensive equipment

Gels need to be analyzed closely & carefully

PCR Fingerprinting

Essential feature is ability to amplify rapidly & exponentially a particular DNA sequence – template, typically 0.5kb to 2.0kb.

Reaction requires a DNA polymerase, only a minute amount of template & oligonucleotides – primers, typically 18-20 base pair length corresponding to sequences on the template

Eg.: BOX for S pneumoniae or IS256 for S aureus

Readily detectable amount of product generated in less than a few hours.

Flexibility, technical simplicity, wide availability of equipment & reagents, rapid

Several modifications for epidemiological typing:

PCR product digested with restriction endonuclease, resulting restriction fragments analyzed

Used in S aureus & Cryptococcus neoformans

ARDRA: Amplified Ribosomal DNA Restriction Analysis

Arbitrarily primed PCR: Employs a single, short (typically 10bp length) primer whose nucleotide is not directed at known genetic locus.

Result in amplification of one or more unpredictable loci, & PCR reaction will generate a set of fragments

Number & size of the fragment – basis of typing the isolate

Identifying primers that provide consistent reproducible results – difficult, discriminatory power - uncertain

Amplified Fragment Length Polymorphism (AFLP) analysis:

Selectively amplifies subsets of genomic fragments generated with one or two restriction enzymes.

Random restriction fragments are singled out by using a specific base sequence at the 3’ end of the primers.

Elongation will only take place if a nucleotide complementary to the selective base in the primer sequence is present in the fragment.

Products separated in agarose gel

Highly reproducible.

Nearly whole genome coverage can be obtained.

Inter-repeat PCR

Variable length segments found between consecutive repeat elements, rather than repeat elements themselves are amplified.

S pneumoniae, Pseudomonas aeruginosa

Random Amplification of Polymorphic DNA

Most frequent method of DNA fingerprinting of eukaryotic organisms

With use of random primers of approximately 10 bases in length, amplicons throughout genome are amplified by PCR

Products separated on agarose gel & visualized by ethydium bromide staining.

Reproducibility poor

Polymorphisms arise when distances between primer hybridization sites change or when primer sites appear, disappear or change location due to insertion, deletion or recombination.

Electrophoretic Karyotyping

Fingerprinting eukaryotic pathogens

Nucleic acid released, electrophoresed.

Chromosomal DNA visualized with Ethydium Bromide

Identified by Southern Blot Hybridization with chromosome specific probes

Multilocus Variable Number Tandem Repeat (VNTR) analysis (MLVA)

Capitalizes on inherent variability encountered in many regions of repetitive DNA

VTNRs are very short tandem repetitive elements found within genomes of both prokaryotes & eukaryotes

Repetitive DNA is often incorrectly copied in bacterial species, through slipped strand mispairing (SSM), resulting in shortening or lengthening of repeat units.

For each repeat locus a digit can be assigned.

When several repeat loci are analyzed per isolate, several such digits are obtained, resulting in a multi-digit specific strain code.

Isolates within an outbreak have identical MLVA profile

P falciparum

Sequence based methods

Comparing multiple isolates by sequencing the same locus from each

Single Locus Sequence Typing (SLST)

Multiple Locus Sequence Typing (MLST)

SNP genotyping

Single Locus Sequence Typing (SLST)

A single genetic locus is analyzed

Highly variable gene sequences are selected

emm typing for S pyogenes.

Multiple Locus Sequence Typing (MLST)

Assesses DNA sequence variation among alleles of housekeeping genes

Also non-housekeeping genes & combination of both

Epidemiological typing of Legionella pneumophila.

Limited accessibility, high cost

SNP genotyping

Determination of nucleotide base that is present in a given isolate at defined nucleotide positions known to be variable within the population

spa typing for S aureus

A 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.

Data analysis

Computing Similarity Coefficient (SABS)

Presence of band = 1

Absence of band = 0

nAB = number of common bands (1,1)

a = number of bands in A with no counterpart in B (1,0)

b = number of bands in B with no counterpart in A (0,1)

c = number of bands absent in A & B

Number of matches m = nAB + C

Number of mismatches u = a + b

Coefficient of Jaccard:

Sj = nAB

nAB + a + b

Coefficient of Dice:

SD = 2nAB

2 nAB + a + b

Band intensity

Generating Dendrograms

Matrix of values generated for every pair of isolates

Un-weighted Pair Group Method using Arithmetic averages (UPGMA)

SABS scanned for most similar isolates

If more than 1 group identified, first is arbitrarily taken as group 1.

Isolates are joined at appropriate positions along SABS axis.

Matrix 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.

Bootstrapping

Jackknifing

eBURST

Simple presentation of relationships

The 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

Strain with highest number

of single locus variants –

founder genotype

Phylogenetic tree

Example

Seq. A = A A C C G G T T

Seq. B = A A C C G G T G

Seq. C = A C C C G G T C

Seq. D = A C C C G G T A

Applications of typing

Surveillance of infectious diseases

Systematic, ongoing process of data collection, analysis, interpretation, dissemination of results & action taken, aimed at recording disease trends & designing ways in which to curb them.

Detection of clusters of pathogens with similar type – early warning of a potential outbreak

Serotyping, phage typing, PFGE, MLST

Outbreak investigation

Outbreak 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.

Generate & test hypothesis

Pathogenesis & course of infection

Progress of infection: endogenous microflora or an exogenous source.

Pathogenesis related markers: virulent or non-virulent

Antimicrobial Resistance Genotyping

Use of probe hybridization or DNA amplification techniques to detect various genes that encode for antimicrobial resistance factors, thus providing an antimicrobial resistance genotype.

Calibration of conventional susceptibility test

Choose antimicrobial therapy least likely to select resistant organisms

To track the spread of specific resistance gene within & among health care facilities & communities.

Bacterial population genetics

Intra-species population structure –phylogenetic hypothesis.

MLST

Valuable information concerning evolution & diversification of species

How commonly bacterial genomes undergo horizontal transfer

Selection of vaccine strains

Serotyping - Polio vaccine, S pneumoniae, H influenzae

Genotyping – Rotavirus G1 – G4 & P1A & P1B – globally, G5, G8, P2A [6]

Identification of genes encoding dominant immuogens in subunit & recombinant vaccines

Differentiate wild & vaccine strains

d marker

rct 40

MS

Mc Bride’s intratypic antigenic marker

Molecular: monoclonal antibodies specific for vaccine strains, oligonucleotide fingerprinting, nucleic acid sequencing

Summary

Epidemiological markers have played an important role in study of bacterial evolution, outbreak investigation, surveillance of infectious diseases.

Application of molecular techniques to identification & differentiation of isolates provided a powerful set of new tools that can augment both patient management & epidemiological investigations.

Supplement rather than replace clinical hypothesis.

Although there are limitations for each technique, use of combination of techniques provides good discriminatory power to assess if two isolates are related or not

References

Clinical Microbiology & Infectious Diseases, 13 (Suppl. 3), 1 – 46.

Clinical Infectious Disease; 1993; 17:153-164

Topley & Wilson, Bacteriology I

Pattrick Murray, Manual of Clinical Microbiology, 9th ed, Vol 1, 129 – 151

Various journals.