S1 De voorjaarsbijeenkomst van de Nederlandse ... - NVMM

infections. In particular infections caused by methicillinresistant
S. aureus (MRSA) pose a major problem in health
care settings. In the Netherlands, the restricted use of
antibiotics and a ‘search and destroy’ policy have kept
incidence at a low level. However, in recent years there
seems to be a slow increase in incidence and the finding
that there is an animal reservoir has increased the interest
further. As a result, there is considerable interest in
molecular typing to study the epidemiology of MRSA.
S. aureus seems have a relatively stable core genome
resulting in a predominantly clonal genomic background.
Increased virulence and antibiotic resistance is acquired
through lateral transfer of DNA. The clonal nature of
S. aureus background enables the identification of certain
lineages through molecular typing. A plethora of typing
techniques have been used to characterise S. aureus, but
in recent years PFGE, MLST and spa-sequence typing
have become the most widely used techniques. The, until
recently, most frequently used technique, PFGE, is a band
based technique which is difficult to standardise and
unsuitable for (inter)national molecular typing databases.
The sequence based MLST is a robust technique yielding
unambiguous results that is extremely well suited for
electronic data exchange. However, MLST is both labor
intensive and expensive. Spa-sequence typing is rapidly
becoming the new molecular typing standard for S. aureus.
However, spa-sequence typing uses only a single genomic
locus for typing which may result in insufficient discriminatory
power. For this reason, we developed a MLVA
and compared typing capabilities with those of PFGE and
spa-sequence typing.
For MLVA, the number of repeats was determined by
amplifying the VNTRs of 8 different VNTR loci, including
spa, in 2 multiplex PCRs. The fluorescently labeled PCR
fragments were size on an automated DNA sequencer and
the number of repeats of each locus was combined to create
an 8-number numerical MLVA profile which was used for
clustering. Approximately 1680 S. aureus strains (predominantly
MRSA) isolated from humans were included in the
study. Although MLVA yielded many different genotypes
most types could be assigned to one of 11 large complexes
which were made up of single-locus MLVA variants. There
was 52% congruence between MLVA and spa-sequence
typing and 33% congruence between MLVA and PFGE.
Congruence between spa-sequence typing and PFGE
amounted 40%. Sensitive S. aureus strains (MSSA) were
present among all MLVA clusters and no MSSA-specific
MLVA types were found. MRSA strains belonging to the
PFGE non-typeable class, often referred to as pig strains all
belonged to single MLVA complex, confirming the clonal
nature of this type of strains.
MLVA appears to be a method that is suitable for molecular
typing of S. aureus. Its simplicity, robustness, relatively
low costs and unambiguous data make it an alternative
Ned Tijdschr Med Microbiol 2008;16:Supplement
S22
for MLST. The method is superior to PFGE and its data
are easier to use in clustering than those obtained by
spa-sequence typing.
O056
The epidemiology of Clostridium difficile PCR-ribotype 027
in The Netherlands since 2005
D.W. Notermans 1 , T.I.I. van der Kooi1 , A. Goorhuis2 ,
S.B. Debast3 , B.H.B. van Benthem1 , E.J. Kuijper2 1
Centre for Infectious Disease Control, National Institute
for Public Health and the Environment (RIVM), Bilthoven,
2 3 Leiden University Medical Centre, Leiden, Meander Medical
Centre, Amersfoort
Introduction: In 2005, outbreaks of Clostridium difficile
associated diarrhoea (CDAD) with the virulent PCR
ribotype 027/toxinotype III were first reported in the
Netherlands. C. difficile PCR ribotype 027 was already
causing considerable problems in hospitals in the United
States, Canada and England over the preceding years.
Several investigations have shown an increase in morbidity
and mortality and an increased relapsing rate for this type.
Studies on risk factors at individual patient level showed an
association with fluoroquinolones.
Methods: The Centre for Infectious Disease Control at the
National Institute for Public Health and the Environment
(RIVM) set up surveillance for CDAD in collaboration with
the Leiden University Medical Centre (LUMC). Hospitals
can send isolates or toxin positive faeces samples for typing,
if they suspect type 027 based on an increased CDAD
incidence or a severe clinical picture. From hospitals with
an outbreak with or transmission of type 027, information
is collected, including monthly CDAD incidence.
Results: Between February 2005 until half August 2007,
1886 samples have been typed, coming from 75 healthcare
institutions and laboratories. In 418 cases (22%), PCR
ribotype 027 was found. PCR ribotypes 001 (18%), 014
(7%) and 078 (8%) were also frequently found. To this
date, in 23 of in total of 97 Dutch hospitals, type 027 has
been detected. In 13 of the hospitals, the introduction
of 027 caused an increased incidence of CDAD, two of
which occurred since December, 2006. Ribotype 027
has also been detected in ten nursing homes. In eight of
11 hospitals where ribotype 027 was detected in 2005 or
2006 and outbreak occurred, no ribotype 027 has been
detected since April, 2007. Two hospitals that had the
epidemic well under control for a long time were faced
with a new increase in incidence. In most of the hospitals
where outbreaks occurred in 2005, the incidence decreased
substantially in 2006.
Conclusion: The spread of PCR ribotype 027 in the
Netherlands is still continuing. Outbreaks in new institutions
are limited compared to previous hospital epidemics in