Chapter 43 : Molecular Tools for Epidemiological Investigations into Infections

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Molecular Tools for Epidemiological Investigations into Infections, Page 1 of 2

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This chapter describes the theoretical framework within which typing networks can be established. The definitions used for identifying bacterial strains and clones will be provided, and the clinical value of typing will be illustrated as well. The chapter discusses the current tools available for molecular typing of and their reproducibility. Some of the current developments in the field of bacterial genetic identification are pinpointed, and ongoing and future developments in the field of establishing typing networks are summarized. The most common overall methodological distinction is the one between phenotyping and genotyping procedures. Genes can be specifically amplified and analyzed for sequence variability on the basis of restriction site polymorphism or variable length of the PCR products. Random amplification of polymorphic DNA (RAPD) analysis and arbitrary primed PCR are methods that are essentially based on random priming of PCR primers, often using quite relaxed reaction conditions. Amplification fragment length polymorphism (AFLP) analysis is another recent PCR-mediated procedure with which subsets of DNA restriction fragments can be successfully amplified. DNA sequencing technology has revolutionized microbiology in general because it enabled whole genome sequencing for a variety of microorganisms. Multilocus sequence typing (MLST) is one example: the sequence polymorphism detected in a number of slowly evolving genes allows for the categorization of strains on the basis of allelic diversity. Future technology improvement may increase the self-sustained development of exchangeable data subdirectories in individual laboratories, but we still have to go along way before we get there.

Citation: van Belkum A. 2002. Molecular Tools for Epidemiological Investigations into Infections, p 227-236. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch43

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Random Amplified Polymorphic DNA
Pulsed-Field Gel Electrophoresis
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RAPD analysis of reference strains and isolates obtained during water contamination or nosocomial outbreaks of infection. A combination of two primers (ERIC2 and BG2) was applied during amplification. Lanes: A, NCTC12008-OLDA; B, NCTC11404-Bellingham; C, Hospital B, clinical isolate; D, water isolate hospital A; E through H, patient isolates obtained in hospital A. Patients F, G, and ? were hospitalized simultaneously. The identity of the RAPD fingerprints suggests an ongoing outbreak of infections involving patients F, G, and H. Note, however, that not all patients who are epidemiologically linked are infected by the same strain (lane E).

Citation: van Belkum A. 2002. Molecular Tools for Epidemiological Investigations into Infections, p 227-236. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch43
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Image of FIGURE 2

Principle of AFLP (Keygene NV, Wageningen, The Netherlands) and example of its experimental output. Panel A delineates the various stages in the typing protocol. Initially, DNA is digested with the help of two different restriction enzymes. Specific linkers are coupled to the respective sticky ends and these linkers serve as specific anchor points for fragment amplification by PCR. Inclusion of selective bases (encoded in the neighboring region of the native restriction sites) in the primer sequences results in amplification of limited numbers of specific restriction fragments. These can be effectively separated by acryl amide gel electrophoresis and visualized by techniques for detection of haptens incorporated in primers or dNTPs used during amplification (panel B).

Citation: van Belkum A. 2002. Molecular Tools for Epidemiological Investigations into Infections, p 227-236. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch43
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Frequency of usage of various typing procedures for epidemiological analysis of isolates: a recent historical perspective

Citation: van Belkum A. 2002. Molecular Tools for Epidemiological Investigations into Infections, p 227-236. In Marre R, Abu Kwaik Y, Bartlett C, Cianciotto N, Fields B, Frosch M, Hacker J, Lück P (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555817985.ch43

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