A set of training materials for professionals working in intervention epidemiology, public health microbiology and infection control and hospital hygiene.
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1. Molecular epidemiology of infectious diseases.
Different definitions of molecular epidemiology can be found in the literature. In the field of infectious diseases, molecular epidemiology has been defined as the use of molecular typing methods for infectious agents in order to study the distribution, dynamics, and determinants of health and disease in human populations [1,2]. In particular, molecular epidemiology of infectious diseases combines traditional epidemiological methods with analysis of genome polymorphisms of pathogens over time, place and person across human populations and relevant reservoirs, to study host–pathogen interactions and infer hypotheses about host-to-host or source-to-host transmission [3,4,5].
Molecular typing methods are used to study the genomic organisation and evolution of pathogens, to identify patterns of infection and sources of transmission, as standard component of epidemiological surveillance of infectious diseases and to support outbreak investigations. Of particular interest is the application of these typing methods to study markers associated with pathogenicity and antibiotic resistance (see Roles for microbial typing in HCAI prevention and control and Antimicrobial Stewardship).Notably, the results of studies using molecular typing methods (such as laboratory-based surveillance) do not substitute for a comprehensive epidemiological investigation (such as patient-based surveillance), but laboratory studies and epidemiological studies should be analyzed in parallel and results should be integrated as complementary components of the overall investigation .
Traditional phenotypic typing systems, based on microbiological, biochemical, serological, and physiological characters, have been widely used (see Phenotypic typing), but genotyping methods that examine the relatedness of isolates at a molecular level have changed the ability to differentiate among bacterial types or subtypes and have been used all over the world (see Overview of molecular typing methods).
2. Comparative and library typing systems.
Usually, typing methods can be described as comparative or library typing systems:
in the first approach (comparative), mainly used for outbreak investigation, a set of outbreak-related and unrelated isolates are tested to identify outbreak-related strains and to distinguish epidemic from endemic or sporadic isolates. In the long term, comparison between outbreak-related isolates and other isolates collected at different times, from the past or future, is not relevant. Generally, comparative systems produce significant results only in a local context for delineation of isolates closely related from those significantly different in genomic backgrounds;
Comparative or library epidemiological typing systems are not to be considered as intrinsic characters of each method but an alternative way of use of it. As such, for example, PFGE may be used as comparative typing in outbreak investigations and as library typing in surveillance of infectious diseases [3,4,7]
3. Overview of molecular typing methods.
Over the last years different molecular typing methods have been developed and used all over the world. The selection of an appropriate molecular typing method depends essentially on the problem to explain - particularly for the epidemiological surveillance of infectious diseases, including healthcare associated infections, and for outbreak investigation - and on the level at which typing is being used, the epidemiological context, and the time and geographical scale of its use. Typing methods need to be evaluated and validated with respect to a number of criteria (see Criteria for assessing microbial typing systems). Guidelines and general criteria have been proposed to interpret the obtained results [3,4,8].
Based on recent published reviews [8,9] the Table below reports the characteristics, advantages, and limits of the main molecular typing methods, currently used in outbreak investigations and in epidemiological surveillance studies. Furthermore, some useful links to web site and/or to online databases for bacterial typing are reported. There has also been the modification of the STROBE tools to improve the reporting of molecular epidemiology for infectious diseases .
Table 1.Main molecular typing methods.
Molecular typing method
Pulsed-field gel electrophoresis (PFGE)
Whole genome restriction polymorphism
Excellent discriminatory power
High intra- and inter-laboratory reproducibility
High epidemiological concordance
Limited ease of use
Low resolution for similar fragments size
Amplified fragment length polymorphism (AFLP)
Selective PCR amplification of a subset of restriction fragments
Random Amplification of Polymorphic DNA (RAPD)
PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence
Ease of use
Low discriminatory power
Low intra-laboratory reproducibility
Repetitive-element polymerase chain reaction (rep-PCR)
PCR amplification of non coding intergenic
High discriminatory power
Low inter- laboratory reproducibility (improved by semi-automated commercial systems)
Variable-Number Tandem Repeat (VNTR) typing and Multilocus VNTR analysis
PCR amplification of polymorphisms of genomic variable number tandem repeat elements
Moderate inter-laboratory reproducibility
Single Locus Sequence Typing (SLST)
Sequencing of single
High discriminatory power for some species (e.g. spa-typing for S. aureus)
Potential misclassification of particular types, due to recombination and/or homoplasy
Multilocus sequence typing (MLST)
Sequencing of allelic variants of 7 housekeeping genes.
High discriminatory power (not for all species)
Comparative genomic hybridisation (CGH): microarrays
Labelled cDNA/RNA, hybridized with specific probes
High throughput technique
Simultaneous genotyping and profiling
The intra- and inter-laboratory reproducibility of microarray data needs to be established prior to the application
Whole Genome - Next generation Sequencing (WG-NGS)
Sequencing of multiple, overlapped regions
4. Next Generation Sequencing (NGS).
In the last years, Next Generation Sequencing (NGS) technologies (also called second generation sequencing o high-throughput sequencing) have revolutionized molecular typing methods providing the possibility to obtain complete or nearly complete genome sequences (often approximately 90% of the entire genome) of thousands of strains (Whole Genome Sequencing, WGS).
WGS has already been used for the accurate identification of bacterial isolates, for the characterization of strains in large outbreaks at national/international levels and to reveal the global genetic diversity of pathogens. It is expected that in the near future, WGS will replace currently used typing methods. In particular, WGS has the potential to compare different genomes with a single-nucleotide resolution and this allows a precise characterisation of cross-transmission episodes and outbreaks. In addition WGS can also be useful for well defining phenotypic characteristics, such as the virulence or antibiotic resistance of a particular pathogen.
The strong advantage of NGS versus traditional Sanger sequencing is the ability to generate millions of reads in single runs at comparatively low costs. However, WGS is still too laborious and time-consuming to obtain useful data in routine surveillance and in small research and clinical laboratories.
Notably, the development of NGS technologies was accompanied by the generation of huge amounts of data leading to the need to develop web-based bioinformatics platforms for rapid data processing and analysis. The NGS revolution will not be extensively available to health professionals and the results cannot be applied in everyday clinical practice, until several bioinformatics challenges have been solved. The integration of genomic and epidemiological databases and NGS data will be the next frontier in bacterial epidemiology in order to empower stakeholders in public health decisions [7,8].
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Struelens MJ, De Gheldre Y, Deplano A. Comparative and library epidemiological typing systems: outbreak investigations versus surveillance systems. Infect Control Hosp Epidemiol 1998;19(8):565-9.
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Original contribution from:
Antonella Agodi, Dept of Medical and Surgical Sciences and Advanced Technologies “GF Ingrassia”, University of Catania, Catania, Italy.
Join the discussion about this article in the forum!
sarika_desai posted on 9/21/2010 10:45:08 AM:
This chapter is well-written, covers the subject area comprehensively and is easy to read. I have made a few minor editorial changes to the text and below are a few comments and suggestions on specific sections of the chapter.
1. Should biases be mentioned here with a link to the chapter on biases? It is an important consideration when selecting controls and maybe a sentence or two could then really highlight the role of controls.
1. In the text you have differentiated controls by random sampling and by matching and I think it would also be clearer if you made this separation at the beginning. Your options could be:
1. Unmatched controls/Randomly selected
a. Population etc
2. Matched controls
a. Neighbourhood etc
This way the text has the same chronological order as the above list.
2. I think it would be a shame not to include control selection in case-case, and case-cross over designs as these are used even if not as commonly as classical case control studies. Their inclusion would complete the picture of control selection.
3. Would it be useful to provide links/references to articles for each type of control selection? For case-case you could use
a. Aiken et al Risk of Salmonella infection with exposure to reptiles in England, 2004-2007. Euro Surveill. 2010; 15(22).
b. McCarthy and Giesecke. Case-case comparisons to study causation of common infectious diseases. Int J Epidemiol 1999; 28:764-8.
For case-crossover you could use
a. Soverow et al. Infectious disease in a warming world: how weather influenced West Nile virus in the United States (2001-2005). Environ Health Perspect. 2009; 117:1049-52.
There is an article by Grimes that might be nice to reference (Grimes DA and Schulz KF. Compared to what? Finding controls for case-control studies. Lancet. 2005;365:1429-33).
“Special considerations in control selection”
1. I think it would be useful to have links to other sections of the manual embedded into the text for “case cohort, traditional case control, density case control”.
“Developing a control definition”
1. I feel it would be more appropriate if this section came straight after the summary page as for me it is more logical to define controls and then determine how to select them.
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