EPIDEMIOLOGICAL MARKERS Dr. Soumya 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 Dr. Savitha
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 Simpsons 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= (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 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 Vitypes 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 genom