Molecular typing is an essential tool to
monitor Clostridium difficile infections and outbreaks within healthcare
facilities. Molecular typing also plays a key role in defining the regional and
global changes in circulating C. difficile types. The patterns of C. difficile
types circulating within Europe (and globally) remain poorly understood,
although international efforts are under way to understand the spatial and
temporal patterns of C. difficile types. A complete picture is essential to
properly investigate type-specific risk factors for C. difficile infections (CDI) and track long-range transmission. Currently,
conventional agarose gel-based polymerase chain reaction (PCR) ribotyping is
the most common typing method used in Europe to type C. difficile. Although
this method has proved to be useful to study epidemiology on local, national
and European level, efforts are made to replace it with capillary
electrophoresis PCR ribotyping to increase pattern recognition, reproducibility
and interpretation. However, this method lacks sufficient discriminatory power
to study outbreaks and therefore multilocus variable-number tandem repeat
analysis (MLVA) has been developed to study transmission between humans,
animals and food. Sequence-based methods are increasingly being used for C.
difficile fingerprinting/typing because of their ability to discriminate
between highly related strains, the ease of data interpretation and
transferability of data. The first studies using whole-genome single nucleotide
polymorphism typing of healthcare-associated C. difficile within a clinically
relevant timeframe are very promising and, although limited to select
facilities because of complex data interpretation and high costs, these approaches
will likely become commonly used over the coming years.
Clostridium difficile is a gram-positive
rod-shaped anaerobic bacterium that is capable of forming spores. Since its
discovery as a cause of antibiotic-associated pseudomembranous colitis nearly
30 years ago , C. difficile has become the major cause of
antibiotic-associated diarrhoea. Antibiotics change the protective normal gut
flora, which enables C. difficile to colonise the colon. Clinical symptoms may
range from simple diarrhoea to severe colitis which can result in death .
Symptoms are primarily mediated by two virulence factors, toxins A (tcdA) and B
(tcdB), which are released in the gut upon colonisation by C. difficile [3-5].
In the past decade, the epidemiology of C. difficile has changed and a new type
emerged: polymerase chain reaction (PCR) ribotype (RT) 027/North American
pulsed (NAP)-field type 01. Besides the production of toxins A and B, the
binary C. difficile transferase toxin A/B (cdtA and cdtB) has probably
contributed to the increased virulence of this type in addition to still
unknown factors . Major outbreaks due to this strain were reported since
2004, first in Canada followed by North America and Europe [7-10]. In 2008, PCR
RT078/NAP07-08 was reported as an emerging strain .
To study the epidemiology of C. difficile,
several molecular typing methods have been introduced. Ideally, a typing method
must have sufficient discriminatory power, typeability (the ability to type
isolates unambiguously), reproducibility and transportability (the ability to
perform the method reproducibly in a fully compatible fashion in different
laboratories at different times) and must be relatively easy to perform .
In this review, we describe the most commonly used typing methods to characterise
C. difficile. In addition, we present the latest developments in typing of C.
difficile. Finally, we discuss the use of typing in surveillance studies, to
trace outbreaks and to study strain transmission from the environment to
Historical perspective of Clostridium
Molecular typing methods can be categorised
into two groups, phenotypic and genotypic methods. In the 1980s only phenotypic
techniques were available. Serotyping using slide agglutination was commonly
used in the mid-1980s. Initially, this assay was capable to differentiate six
serogroups , later this was improved to 15 serogroups . Other commonly
used methods in this period were autoradiography polyacrylamide gel electrophoresis
(radio PAGE)  and immunoblotting using rabbit antiserum prepared from
rabbits immunised with four different C. difficile strains . Phenotypic
assays had low reproducibility, low typeability and insufficient discriminatory
power to apply to epidemiological studies . Genotypic techniques with
better typeability and discriminatory power replaced phenotypic methods during
the 1990s . Genotypic methods are divided into band-based and
sequence-based methods. The most commonly used band-based methods were
restriction endonuclease analysis (REA), pulsed-field gel electrophoresis
(PFGE), capillary or conventional PCR ribotyping and multilocus variable-number
tandem repeat analysis (MLVA), whereas the most frequently used sequence-based
genotyping method was multilocus sequence typing (MLST). Recently whole genome
sequencing (WGS) has emerged as a promising sequence-based technique as it
allows the detection of variations between C. difficile strains by, for
example, single nucleotide polymorphisms (SNPs) analysis. Here we present a
brief summary of the current performance and costs of genotyping methods (Table
1 and 2), as a detailed description is beyond our scope and can be found in three
other reviews on molecular typing [12,17,18].
Table 1. Performance characteristics of
various genotyping methods for Clostridium difficile
Table 2. Techniques, time and costs
associated with various genotyping methods for Clostridium difficile
Currently used typing methods for
In Europe PCR ribotyping is presently the
most frequently used typing method of C. difficile. This method was first
applied by Gurtler et al.  and exploits the variability of the intergenic
spacer region (ISR) between the 16S and 23S ribosomal DNA (rDNA), which is type-dependent.
The variability, in combination with multiple copies of rDNA present in the
genome, results in various amplicons after PCR amplification. These amplicons
are separated by common agarose gel electrophoresis. The obtained banding
patterns are referred to as PCR RTs. Two different sets of primers have been
developed for typing of C. difficile [22,23]. The O’Neill primers described by
Stubbs et al.  seem to have better discriminatory power than the Bidet
primers . The discriminatory power (D) of a typing method is its ability to
distinguish between unrelated strains, this D-value is based on Simpson's index
of diversity . PCR ribotyping is currently capable of identifying more than
400 distinct PCR RTs.
In North-America, PFGE is commonly used.
PFGE of C. difficile involves digestion of genomic DNA
with an infrequent cutting restriction enzyme, for example SmaI . PFGE
allows separation of large DNA fragments which is not possible with
conventional agarose gel electrophoresis. The obtained DNA fragments are
separated using agarose gel electrophoresis with an electric field orientation repeatedly
switching in three different directions (pulsed-field); one direction is
through the central axis of the gel, whereas the other two are at an angle of
60 degrees on either side. The pulse time of the direction is linearly
increased during the run so that progressively larger fragments are able to
migrate forward through the gel, resulting into separation based on fragment
size. The obtained banding patterns are referred to as NAP-field types.
Unfortunately, standardisation of protocols and validation of PFGE for C.
difficile have never progressed as they did for other food-borne pathogens on
PulseNet at the United States (US) Centers for Disease
Control and Prevention (CDC) .
It has been reported that PFGE displays
better discriminatory power than PCR ribotyping with D-values
of 0.843 and 0.688, respectively . In contrast, preliminary results of a
study comparing different typing techniques on 39 of the most frequently found
PCR RTs in Europe demonstrate that only 16 NAP-field types were obtained of 39
PCR RTs (personal communications, M Mulvey and D McCannel, 2011). A common
concern with all band-based typing methods is the difficult interpretation of DNA banding patterns, especially when a DNA banding pattern differs marginally from the
reference patterns. Consequently, appropriate definitions are required to
identify new types with both PFGE and PCR ribotyping. In Europe, the Cardiff
collection of Jon Brazier and Val Hall serves as a reference collection and new
PCR RTs are always validated using this database. Currently, a clinical
collection of 20 different C. difficile PCR RTs (European Centre for Disease
Prevention and Control (ECDC)-Brazier
collection) isolated from various European countries is available to distribute
among all reference laboratories in Europe who participate in the European C.
difficile infection study network (ECDISnet)
. The usage of two different standard typing
methods in Europe and America has resulted into different nomenclatures, making
interlaboratory exchange of data difficult. Already in 1994 Brazier et al. 
emphasised the need for a unified nomenclature.
In 2004, MLST was introduced to study the
population structure and global epidemiology of C. difficile . This
sequence-based typing method relies on sequencing of DNA fragments approximately
ranging between 300 and 500 bp representing seven housekeeping genes (MLST
7HG). Sequence variants for each housekeeping gene are assigned with a distinct
allele number and the combination of seven allele numbers (allelic profile)
provides a sequence type (ST). MLST generates high-throughput sequence data
that can be uploaded from laboratories worldwide to a common web database .
This facilitates ST calling as well as studying the population structure and
global epidemiology of C. difficile. Two different typing schemes have been
proposed in literature to characterise C. difficile isolates [30,32]. Both
typing schemes consist of seven housekeeping genes of which three are shared (triosephosphate
isomerase (tpi), recombinase A (recA) and superoxide dismutase A (soda). In
contrast to the scheme published by Griffiths et al. , the MLST scheme
described by Lemee et al.  was not widely adopted. This can be partially
explained by the presence of a null allele on the D-alanine--D-alanine ligase (ddl)
locus of the Lemee scheme which failed to amplify in certain strains .
Recently, this locus in the Lemee scheme was replaced by the groEL gene .
It has been reported that the
discriminatory power of MLST and PCR ribotyping is comparable [18,32]. For
studying outbreaks at a local level, a typing method should have higher
discriminatory power than PCR ribotyping and MLST. For instance an increase in
incidence of a PCR RT or MLST ST in a hospital can provide us with a clue for
an outbreak and is useful data for monitoring changes in type prevalence rates,
but does not necessarily proves clonal spread of one strain.
MLST is an appropriate tool for studying
the phylogeny of C. difficile. Compared to a band-based typing method, such as
PCR ribotyping, MLST is less vulnerable to recombination events. Recombination
in a housekeeping gene would change the allelic profile on a single locus only.
Even though the consequence would be a change of ST, this new ST would still be
closely related to the original ST maintaining the phylogenetic link.
Recombination of repeats present in the ISR between the 16S and 23S rDNA 
might lead to the formation of a novel PCR RT without a clear phylogenetic link.
However, the rate at which these recombination events occur and the
predisposing factors are unknown. Phylogeny reconstruction with MLST revealed
that C. difficile diversified into at least five well separated lineages during
evolution [32,35,36] and possibly a sixth monophyletic lineage . The
majority of STs were assigned to lineage 1 with no major subdivisions (Figure
1), but this result could be due to an unfortunate choice of housekeeping
genes. Changing the housekeeping genes or adding housekeeping genes to the
current MLST scheme might provide a better resolution of lineage 1.
Figure 1. Phylogenetic structure of Clostridium
A major advantage of sequence-based typing
methods like MLST is the ease of interpretation of the generated data. Sequence
data are unambiguous and therefore objective, highly reproducible and easily
exchangeable between laboratories. Moreover, many laboratories have submitted
their sequences to a freely accessible C. difficile MLST database .
Currently (last updated: 21 Nov 2012), 176 different STs have been identified.
A practical disadvantage of MLST remains the relatively high cost of sequencing
multiple targets, which could partially explain why MLST has not replaced
conventional PCR ribotyping in many European laboratories.
MLVA is a highly discriminatory molecular
typing method that has been introduced to study outbreaks and identify routes
of transmission between patients and hospitals [11,38–42]. MLVA relies on the
amplification of short tandem repeats that vary in size and are dispersed
throughout the genome. The obtained amplicons are separated with capillary
electrophoresis followed by automated fragment analysis. Initially, two
different typing schemes were published which both contain seven loci of which
four are identical [41,42]. Each of the seven loci is designated with a number
that corresponds to the sum of repeats present on that locus. A minimum
spanning tree (MST) can be constructed, in which the summed tandem repeat
difference (STRD) is used as a
measure of genetic difference (Figure 2). Clonal clusters are defined by an STRD of =2, and genetically related clusters are
defined by an STRD of =10 [11,41].
Broukhanski et al.  observed that two MLVA loci (F3 and H9) were
invariable, indicating that loci F3 and H9 did not contribute to the
discriminatory power. In addition, Bakker et al.  reported that MLVA locus
A6 is a null allele in PCR RT078 and that for several other loci the PCR
settings had to be optimised for PCR RT078. Invariance of MLVA loci requires
optimisation and validation of MLVA for individual PCR RTs. Currently, MLVA has
been implemented as useful typing method to investigate C. difficile 027
outbreaks in the Netherlands, France and the United Kingdom (UK) [38,45,46]. In
England, C. difficile infection (CDI)
cases that are potentially linked, i.e. caused by isolates that share the same
PCR RT and which are related in time and place, are investigated using MLVA.
Notably, almost half of such presumed clusters are shown actually either to
consist of unrelated isolates or a mixture of related and distinct strains .
Figure 2. Minimum spanning tree
illustrating distinct local Clostridium difficile outbreaks
Recent developments in typing of
Variant multilocus variable-number tandem
repeat analysis typing schemes
Recently, a modified MLVA (mMLVA) was
developed, combining MLVA with PCR detection of several toxin genes (tcdA and tcdB,
cdtB; and deletions in the toxin C gene (tcdC)) . In addition, the number
of MLVA loci was restricted to five excluding the invariable loci F3 and H9.
Although the combination with toxin gene detection can be informative, it is
not yet possible to correlate these data with specific C. difficile types, like
PCR RT027/NAP01. This is partially because the presence of binary toxin genes
combined with the 18 bp tcdC deletion is not restricted to PCR RT027 strains [37,47].
In a study by Manzoor et al.  the
number of MLVA loci was increased to 15. This extended MLVA (eMLVA) scheme was
able to discriminate clinically significant clusters while maintaining a good
concordance with PCR ribotyping. Typing schemes containing only seven loci
showed in contrast poor association with PCR ribotyping [41,42]. These seven
loci schemes can only be used as a subtyping method together with PCR
ribotyping, whereas the extended MLVA can potentially replace both. It should
be noted, however, that increasing the number of loci makes the method more
laborious and increases the difficulty of data interpretation.
Wei et al.  screened 40 MLVA loci for
developing an MLVA typing scheme that has a good concordance with PCR
ribotyping and provides satisfactory data for studying outbreaks. From this
study, it was concluded that typing schemes consisting of MLVA loci with low
allelic diversity maintained a high correlation with PCR ribotyping, whereas
typing schemes using MLVA loci with high allelic diversity were required to
study outbreaks. To fulfil both purposes two different typing schemes were
proposed comprising 10 loci with limited allelic diversity and four loci with highly
variable allelic diversity.
Capillary polymerase chain reaction ribotyping
Although PCR ribotyping has become widely
used in many European laboratories for C. difficile surveillance, issues with
pattern interpretation and limited access to a well standardised database are
important limitations. The adaptation of PCR ribotyping to high resolution
capillary gel electrophoresis (CE) PCR ribotyping has greatly improved pattern
reproducibility and interpretation. For instance, using conventional agarose
gel-based PCR ribotyping, it is difficult to differentiate types 014 and 020.
In contrast, CE-PCR ribotyping can discriminate type 014 and type 020 and
distinguish subtypes within type 014 . However, the need for protocol
standardisation remains evident. C. difficile surveillance laboratories from
the CDC in the US, Public Health Agency of Canada (PHAC) in Canada, Leiden
University Medical Center (LUMC) in the Netherlands and Leeds Teaching
Hospitals NHS Trust in the UK are collaborating to develop and validate a
standardised protocol for the DNA extraction, primer sets, PCR cycling
conditions, and reference standards for CE-PCR ribotyping. The standardised
consensus protocol is tested on a well characterised collection of 70 different
PCR RTs  distributed to each of the four laboratories. Preliminary results
show consistent fingerprints between the laboratories. Peakfile-based analysis
is currently being optimised and validated, with a conclusion available by mid-2013.
Whole-genome single nucleotide polymorphism
High-throughput, WGS of bacterial pathogens
has reached a scale and reliability to accurately define the natural history
and global population structures of virulent and epidemic lineages [51–55].
Phylogenetic and comparative genome analysis of hundreds (soon to be thousands)
of genomes can identify precise genetic changes, often linked to virulence and
antibiotic resistance phenotypes, that can quickly inform about the pathogen’s
biology. Whole genome sequencing can also distinguish between strains at the
single nucleotide level, by comparing genomes in terms of single nucleotide
polymorphisms, and therefore drastically improves the discriminatory power over
conventional genetic typing methods. Thus, WGS has also (i.e. besides
phylogeny) practical value for clinical microbiology and public health epidemiology
by defining the selective forces that precipitate pathogen emergence and also
by tracking transmission events (, Figure 3).
Figure 3. General sequencing and analysis
strategy used to track genomic variants of Clostridium difficile at local and
WGS approaches represent the ultimate
pathogen typing method and, although its use and application remains limited to
select facilities, we believe WGS will become a commonly used tool for C.
difficile surveillance and epidemiology in the coming years. Although the cost
of WGS is relatively high compared to traditional typing methods, sequencing
costs are falling rapidly [19,57]. In addition, the ability to extrapolate
MLST, PFGE, resistance gene, toxin gene sequence and other data from the same
test could balance the cost-benefit analysis. Standardised computational
pipelines are emerging for C. difficile genome data quality control and
subsequent downstream analysis associated with informatics, phylogeny and
phylogeography (Figure 3). Improved high-quality draft genomes  for the
most common C. difficile variants causing disease in human and animal
populations  serve as references to map next generation sequence data in
order to detect variation within the core genome (genes shared by all
organisms) or the accessory genome (genes present in only some organisms) .
The first description of C. difficile PCR
RT027 phylogeny using high-throughput WGS demonstrated that 25 PCR RT027
isolates from the US and Europe could be further discriminated into 25 distinct
genotypes based on SNP analysis . Furthermore, this study demonstrated that
isolates from different regions of the US and Europe occupy distinct
evolutionary lineages and harbour unique antibiotic resistance genes. More
recently, it was demonstrated that PCR RT027 isolates emerged through two
distinct epidemic lineages after acquiring the same antibiotic resistance mutation;
moreover these two lineages displayed different patterns of global spread .
The routine use of WGS in diagnostics and epidemiology is nicely reflected by the
study of Koser et al. . In this study it was reported that whole-genome SNP
typing can be mainly used for monitoring outbreaks and recognition of pathogen
transmission pathways. Current methods for monitoring C. difficile hospital
associated outbreaks, such as PCR ribotyping, have too limited discriminatory
power to characterise potential outbreak strains as the same bacterial clone.
Sequencing of whole genomes offers the optimal discriminatory power allowing
laboratories to detect transmission pathways between hospitals, hospital wards
and patients on the same ward.
In addition, Eyre et al.  demonstrated
that WGS can produce practical, clinically relevant data in a time frame that
can influence patient management and infection control practice during an
outbreak. Moreover, this study demonstrated that a cluster of
healthcare-associated C. difficile cases caused by the same ST was in fact a
number of unrelated sub-lineages, therefore allowing to rule out in
patient-to-patient transmission. Furthermore, WGS combined with comparative
genomics is an effective approach to identify novel genetic markers that are
potentially linked to virulence. This is an important advantage above
conventional typing methods that use existing markers for characterisation of
isolates. Whole genome sequencing is not likely to replace routine diagnostic
techniques in reference laboratories. For example, matrix-assisted laser
desorption/ionisation (MALDI) time-of-flight (TOF), which is rapid and easy to
perform, is currently used in the Dutch reference laboratory for primary
detection of pathogens.
In order to determine whether sequenced
isolates are part of an outbreak, it must be defined how many SNP differences
still represent ‘related’ isolates. For that reason, we should be informed on
the rate of SNP accumulation in C. difficile lifecycle (molecular clock),
although bacterial isolates with a hypermutator phenotype could complicate the
determination of such a threshold . The molecular clock rate of C.
difficile was reported at 2.3 SNPs/genome/year in the study done by Eyre et al.
. Further study is necessary to confirm this rate of C. difficile
Application of typing methods to study the
epidemiology of Clostridium difficile infections
An obvious reason to type C. difficile
isolates is to early detect and investigate outbreaks, which can be defined as ’a
temporal increase in the incidence of a bacterial species caused by
transmission of a certain strain‘ . In addition, typing methods contribute
to epidemiological surveillance on national, European or worldwide level and
can be used to report the incidence of various C. difficile types and recognise
newly emerging virulent types . Typing might also establish the local and
global spread of bacteria and elucidate routes of transmission.
the beginning of the 21st century, a worldwide increase in the incidence of CDI was seen. Soon thereafter, it was recognised that
a specific type of C. difficile, PCR RT027, was linked to this increase of
incidence [7,9]. PCR RT027 was associated with specific
predisposing factors, course and outcome of CDI.
In a large Canadian outbreak, fluoroquinolones were associated with PCR RT027
and mortality rates among patients with this type increased to 23% within 30
days of diagnosis [9,64]. In the Netherlands, molecular typing of C. difficile using PCR
ribotyping contributed to recognition of an outbreak of two simultaneously
occurring PCR RTs (027 and 017) . Again, patients
had PCR RT-specific risk factors and mortality rates. Numerous studies
demonstrated the increased virulence of PCR RT027 [6–10] and found that other
emerging types, such as PCR RT078, were also associated with specific risk
factors or complicated clinical course . Without results from typing
methods, these associations would have stayed unrecognised.
typing results can also be used to compare the distribution of various C.
difficile types isolated from animals, humans and food, which can hint towards
food-borne disease or zoonotic potential of specific PCR RTs. The emerging C. difficile PCR RT078 in humans is found in high numbers in
animals, especially piglets and calves [11,65–67]. Koene et al. 
investigated the presence and characteristics of C. difficile in seven
different animal species. PCR RTs 012, 014 and 078 were most frequently
isolated among these Dutch animals,
similar types were found among hospitalised patients in the Netherlands in
2009/2010. Meat consumption has also been suspected to contribute to
transmission of C. difficile. PCR RTs 001, 017, 012 and 087 have been isolated
from meat in Europe, however, isolation rates are low and might not be high
enough to exceed the infectious dose [65–69] . Although PCR RTs in animals,
meat and humans overlap, PCR ribotyping lacks discriminatory power to show
clonal spread of C. difficile isolates from humans to animals. New molecular
methods should be developed and applied. The optimised MLVA scheme developed by
Bakker et al.  showed relatedness between human and porcine PCR RT078
strains, although this could not always be confirmed with epidemiological data.
Hopefully, highly discriminative typing methods such as whole-genome SNP typing
can provide us with novel insights on zoonotic transmission.
Importance of molecular typing for national
surveillance by reference laboratories
In Europe and North America, surveillance
studies to monitor the incidence of CDI
and the spread of hypervirulent strains have been established at regional and
national levels since 2007 although reporting of CDI
is not mandatory in all European Union (EU) countries. To enhance surveillance
for CDI, the ECDC and the US CDC
advised to widely launch surveillance programmes for CDI
. Consequently, a European network to support capacity building for
standardised surveillance of CDI was
initiated by the ECDC .
When methods and data on existing national
CDI surveillance systems in Europe were reviewed (personal communication, A
Kola, 2012), surveillance
of CDI was reported in 45% (14/31) of the European countries. Active surveillance of CDI is performed in Austria, Norway, Belgium,
Denmark, France, Germany, Ireland, Hungary, the Netherlands, Spain, Sweden,
Luxembourg and the UK [46,70–79]. Surveillance was mostly continuous and prospective, but only four surveillance
systems combined microbiological and epidemiological data (typing and
susceptibility testing results) on a regular basis. A second recently completed
survey in Europe (personal communication, D W Notermans, 2012) demonstrated
that the majority of the laboratories were able to culture, but only half had
access to typing. This limited typing capacity demonstrates the uncertainty of
the true incidence levels of C. difficile types across Europe and hampers
recognition of new emerging C. difficile types.
contribution of national reference laboratories to survey CDI on a national
level is illustrated by examples from the Netherlands and the UK. In 2005, soon
after the emergence of C. difficile PCR RT027, the Center for Infectious
Disease Control (CIb) of the National Institute for Public Health and the
Environment (RIVM) in the Netherlands started a national Reference Laboratory
for C. difficile. In 2009, this laboratory noticed an emergence of a new
virulent PCR RT078, which was the third most frequently found type in the
Netherlands among humans and was present in nearly all pig farms investigated [11,67].
Subsequently, this type was also found emerging in other European countries .
Recently, the reference laboratory noticed a re-emergence of C. difficile PCR
RT027 since 2010. In the period between May 2011 and May 2012, 289 samples from
26 healthcare facilities and laboratories in the Netherlands were submitted
because of severe CDI cases or outbreaks. PCR RTs 001 and 027 were the most
commonly found (both 15.0%). Interestingly, in contrast to a previous report of
declining PCR RT027 in hospitals in the Netherlands , type 027 was
frequently identified in long-term care facilities associated with exchange of
patients to neighbouring hospitals.
In the UK, the C. difficile Ribotyping
Network (CDRN) was established in
2007, as part of improved CDI
surveillance, to facilitate the detection and control of epidemic strains.
Between 2007 and 2010, the CDRN
received a large number of isolates (n=11,294) for PCR ribotyping. Typing
results indicated that almost all of the 10 most common PCR RTs changed
significantly during this time period . As the proportion of CDI caused by PCR RT027 declined (from 55% to 21%),
significant increases were observed in the prevalence of other C. difficile
types, especially PCR RTs 014/020, 015, 002, 078, 005, 023, and 016. In
addition, there was a 61% reduction in reports of C. difficile in England from
2008 to 2011, which occurred coincidently as the proportion of CDI caused by C. difficile PCR RT027 declined.
Notably, the large reduction in incidence of C. difficile PCR RT027 cases has
been paralleled by decreases in CDI
related mortality . The perceived success of the surveillance programme
means that currently approximately a third of all CDI
cases in England are referred to CDRN.
CDI control programs should ideally
include prospective access to C. difficile typing and analysis of risk factors
for CDI and outcomes.
In the last fifteen years molecular genotyping
methods have replaced some of the more traditional typing methods. WGS will
dominate the field of molecular typing in the next decade. However, before WGS
can be used as a routine tool for molecular typing some requirements need to be
fulfilled. First, WGS needs to be fast, preferentially within 48 hours.
Furthermore, the technical workflow including data analysis needs to be
simplified into an automatic pipeline. Finally, the costs for acquiring the
technical and organisational platform needed to perform WGS must be reduced.
Fulfilling, these requirements, which is in our opinion a matter of time, would
greatly increase the use of WGS worldwide.
This work was supported by ZonMw Grant
50-50800-98-079 from the Netherlands Organization for Scientific Research (NWO).
We would like to acknowledge the European
Study group of Clostridium difficile on behalf of the European Society for
Clinical Microbiology and Infectious Diseases (E.J.K.) for their contribution.
This work was funded by the European Centre
for Disease Prevention and Control (ECDC) through the call for tender OJ/2010/07/09-PROC/2010/035.
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