EPIDEMIOLOGICAL MARKERS Dr. Soumya
Dr. Savitha
Overview Introduction Qualities of epidemiological markers Commonly used epidemiological markers Definitions Typing hat is typing
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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. E pidemiological 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 idely available & common among strains
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Easy to detect Should provide data that reflects genetic distance at the level necessary for answering the questions Definitions lone: Bacterial C lone
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.
ingerprint F ingerprint
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 Phenotype
Observable characteristic of an isolate hat is typing?
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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 tability: epidemiological S tability
marker should remain stable for each isolate after its primary isolation & during laboratory storage & subculture ± across generations
T ypeability: 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 Discriminatory power: ability
to assign a different type to 2 unrelated strains sampled randomly from the population of a given species impson¶s index S impson¶s
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. should reflect, agree with, & possible further illuminate the available epidemiological information
pidemiological concordance: E pidemiological
to assign the same type to an isolate tested on independent occasions, separated in time or space.
Reproducibility: Reproducibility:
Convenience criteria lexibility: range of species that F lexibility:
are typable with minimal modifications of the method.
total time required to get from the isolates to the final typing results.
Rapidity: Rapidity:
Accessibility: Accessibility: availability
of reagents & equipment
ase of use: technical simplicity, workload, suitability E ase
for processing large number of isolates, ease of scoring & interpreting the results ost C ost
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 sp ecies 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 Euclid ean distance between A and B is E= ¥(7¥(712)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 charact erized by their susceptibility susceptibi lity 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 Vitypes of the typhoid t yphoid 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 ( M ALDI-TO F). 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. out hern & C olleagues olleagues ± S out
Southern Hybridization
Also used to define nature of mobile elements. S poligotyping: 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 variable Lack of stability ± lost or acquired spontaneously
Combined with other genomic typing methods, Used frequently in combination with antibiogram ant ibiogram 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 abilit y of foreign DNA (such as bacteriophage bacteriop hage DNA) to infect/invade the host bacterial cell by cutting it up
EcoRI - from Escherichia Escherich ia 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 (³digests´) DNA at particular (³restricted´) 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 E thydium 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 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 restriction fragments are singled out by 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 ith use of random primers of approximately 10 bases in length, amplicons throughout genome are amplified by PCR W
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. hen several repeat loci are analyzed per isolate, several such digits are obtained, resulting in a multi-digit specific strain code. W
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 S pyogenes.. Multiple Locus Sequence Typing (MLST) Assesses DNA sequence variation among alleles of housekeeping genes Also non-housekeeping genes & combination of both pneumop hila. ila. Epidemiological Epidemiological typing t yping of Legionella Legionella pneumoph
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 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:
S j = 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 Intra-species population structure ±phylogenetic ±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. 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.
