1.4.

1 Typing Methods
Among the genotypic typing methods a rough distinction can be made between
band-based methods (e.g. MLVA, AFLP, PFGE) and sequence based methods (e.g. Spa, MLST,
whole genome sequencing). In general, the band based methods
were considered faster (except for PFGE), as PCR is the underlying technique and the
procedure can be finished within a day. This makes these techniques ideal when a
rapid result is needed, for example in an outbreak investigation in a hospital.
However, the reproducibility of some of these techniques tends to pose a problem and
the portability is not as good. Recently, the introduction of automated band separation
on capillary sequencers has increased the reproducibility and portability remarkably.
In the sequence based methods, the results are straightforward, increasing the
portability and reproducibility. Sequencing used to be more time-consuming and
costly then band-based methods, but the advances in whole genome sequencing
methods and equipment has remarkably decreased the costs. The bioinformatics and
data-analysis now seem to be the main limiting factor, but rapid advancements in
automated data-analyses are currently being made to address this.

1.4.2 Pulsed-Field Gel Electrophoresis (PFGE)

In PFGE, genomic DNA is isolated and cleaved with a restriction enzyme that cuts
infrequently, resulting in large fragments of DNA. These fragments are separated
on an agarose gel by pulsed field electrophoresis, where the orientation of the
electric field changes periodically. In PFGE a large portion of the genome is
assessed and large genomic events, such as recombination, insertion or deletion of
large mobile elements will result in a change in the PFGE pattern. In addition, large
plasmids can also be seen on the gel. Pulsed field gel electrophoresis has been
considered the ‘gold standard’ of molecular typing methods for a wide spectrum of
clinically relevant pathogens. PFGE has been the primary approach for outbreak
characterization and the analysis of transmission events, but dependent on the
causative organism, other higher throughput methods, like MLST, MLVA and next
gen sequencing are replacing this method more and more. However, as it is a
relatively inexpensive, but time-consuming method, with high discriminatory
power, good epidemiological concordance and excellent reproducibility, it will
remain the method of choice in low-resource settings until other methods become
less expensive. In addition, standardized protocols (through Pulsenet and Harmony)
and quality control panels are available for some bacterial species, increasing the
portability and quality control.