Molecular Epidemiology CVSN 20uuu24.pptx

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1. Molecular Epidemiology CVSN

2. Epidemiology Refers to the science of the occurrence of diseases in a population. Disease occurrence is measured and related to the different characteristics of individuals or their environment

3. Molecular epidemiology of Infectious Diseases Is the study of the distribution and determinants of infectious diseases that use molecular biology methods. The target of analysis includes the organism and its interaction with the host and the environment in which it resides.

4. Conventional Typing Methods These are techniques that are based on phenotypic methods, which have limitations and may be unpredictably altered: Biotyping Antibiogram Phage typing Serotyping Bacteriocin typing

5. Biotyping Strains are differentiated based on a panel of biochemical tests Governed by pattern of activity of cellular metabolic enzymes May use conventional testing methods of miniaturized systems e.g. API 20E, Microbact , etc Profiles may be unstable due to plasmid loss, inoculum size, incubation condition, etc.

6. Antibiogram Refers to the susceptibility/resistance of organisms to a panel of antimicrobial agents Differences in susceptibility to a panel of people agents are determined to generate an antibiogram , where the antibiogram becomes a taxionomic unit or marker Problems include R factors, other mobile genetic elements, poor discrimination, etc.

7. Phage Typing Method classifies bacterial organisms according to susceptibilitiy to a panel of bacteriophages: Test bacteria is spread on agar surface and dried A drop of each bacteriophage (which are carefully selected for the species) is placed on plate Plate is incubated as determined A susceptible bacteria will yield a a zone of inhibition or plaque at position The pattern of plaque formation is compared to a chart Method is complex, laborious, too few/many phages in some species, etc

8. Serotyping It is based on differences in antigenic determinants including proteins, polysaccharides, lipopolysaccharides of species or subspecies , which are detected by antibodies raised in an infected host. Microorganisms differ in terms of expression of antigenic determinants on cell surfaces Serotyping is the detection of these differences by immunological methods using specific somatic, flagellar and capsular antisera especially in enteric bacteria, based on O or H antigens; E. coli has 181 distinct O antgens and 56 H antigens. Complex, expensive, laborious, poor discrimination, etc.

9. Bacteriocin / Colicin Typing Typing method determines susceptibility of bacterial strain to bacteriocins produced by other bacteria Colicins are proteins produced by some bacteria, that are active or can kill different strains Typing done by testing strains against a panel of colicins by a standard selected strains Test strains are spread on agar and a drop of each bacteriocin is placed to detect zones of inhibition Challenges: coding genes are mostly plasmid encoded

10. Genotypic Techniques DNA based methods; also protein or LPS characterization

11. Plasmid Profile Only applicable to bacteria Plasmids are extrachromosomal genetic materials located in the cytoplasm They confer selective advantage, but not essential for growth or survival (mitochondria may be used for typing of fungal or parasitic organisms). Plasmids are isolated, separated by agarose gel electrophoresis, stained, and photographed; include molecular weight marker Pattern will give number and sizes of plasmids harbored: each strain has a unique banding pattern/fingerprint/profile

12. Problems attendant to plasmid profile Test strain must harbor plasmids Plasmids can be spontaneously lost Difficult to isolate from some strains Plasmids may be isolated in three molecular forms Plasmid rearrangements may occur Plasmids are not sensitive in detecting small size differences Serotype associated plasmids are stable in size and not useful epidemiologically.

13. Plasmid Restriction Endonuclease Fingerprint Isolate and purify plasmids Select suitable restriction enzyme(s) and digest plasmid Separate on agarose gel, stain, view and photograph; include molecular size marker to cover expected fragment sizes Determine fingerprint by comparing number and sizes of fragments, termed RFLPs Plot log mol. size Vs mobility

14. Restriction Enzyme Analysis of Chromosomal DNA Isolate, purify and quantify chromosomal DNA Several methods of DNA isolation Choose restriction enzyme carefully to obviate problem stated below Generates hundreds of DNA fragments, poorly resolved, overlapping bands, and difficult to analyze or interpret May be confounded by presence of plasmids

15. Pulsed Field Gel Electrophoresis Widely applied epidemiological tool Principle based on using rare cutting enzymes to generate a limited no. (5-20) of large genomic DNA PFGE can resolve fragments in the range of 10-1000Kbps Major difference with conventional agarose gel electrophoresis is in the application of electrical current, in which the orientation of the electric field across the gel is changed periodically, which allows large DNA fragments to be resolved by retardation than sieving. Separate the fragments under set parameters voltage, buffers, temperature and duration Appropriate molecular size standards included e.g. concameters of λ fragments