Download 016 C-Reactive Protein Response Associated with

Abstracts of Posters Presented at
9th Tripartite Meeting of the Celtic
Microbiology Associations
(Hosted by the Welsh Microbiological Association)
Cardiff, 5-7th June 2009
www.wma-wales.org.uk
001 Identification of clinical isolates of alpha-haemolytic streptococci by 16S rRNA gene
amplification, Matrix-Assisted Laser Desorption Ionization coupled with Time Of Flight
analysis Mass Spectrometry (MALDI-ToF/MS) and conventional phenotypic methods – a
comparison.
S Hadfield1, M Reid1, WY Wan1, S Johnston1, N Berry1, K El-Bouri1, A Lewis1, D Mack1,2, AP
Davies1,2
1Molecular
Diagnostics Unit, NPHS Microbiology Swansea, Singleton Hospital, Abertawe-Bro
Morgannwg University NHS Trust, Swansea.
2Medical Microbiology and Infection, Institute of Life Science, School of Medicine, Swansea
University, Swansea.
Background
The alpha-haemolytic streptococci (other than Streptococcus pneumoniae) are notoriously difficult
to identify reliably in the clinical laboratory. They have important clinical significance, causing
serious infections such as endocarditis, brain and liver abscess and other deep-seated pyogenic
infections. Correct identification of these isolates is important clinically. For example, Abiotrophia sp
(previously referred to as nutritionally variant streptococci) require the use of gentamicin as well as
penicillin for therapy, and the milleri-group streptococci have a particular propensity for causing
pyogenic infection. Identification is currently attempted using phenotypic methods based on
biochemical reactions, such as API 20S or the automated BD Phoenix system. Other methods
such as MALDI-ToF/MS are becoming available but there is very little experience with this as yet.
Broad range 16S rRNA gene sequence analysis offers a genotypic method capable of definitive
identification.
Objectives
To identify clinically significant isolates of alpha-haemolytic streptococci using MALDI-ToF/MS, API
20S, BD Phoenix, and 16S rRNA gene sequence analysis to compare their reliability, and consider
the clinical implications.
Methods
Forty clinically significant isolates of alpha-haemolytic streptococci were tested by MALDI-ToF/MS,
API 20S, BD Phoenix, and 16S rRNA gene sequencing. Using 16S rRNA gene sequencing as the
gold standard, the relative reliability of each method was assessed.
Conclusions
Alpha-haemolytic streptococci pose well-known difficulties in laboratory identification. Currently,
universal 16S rRNA gene PCR and sequencing remains the best method and its application greatly
facilitates laboratory identification in a time-frame which aids clinical decision-making.
2
002 Dissociated Clindamycin Resistance in Staphylococci.
Katherine McBride1, Elaine McCulloch2, Leigh Williams1, Elizabeth Kilgour1, Craig Williams2, Alistair
Leanord1
1Microbiology
2Microbiology
Department, Monklands Hospital, Airdrie
Department, Yorkhill Hospital, Glasgow
Objective
Macrolide-lincosamide-streptogramin B group (MLSB) resistance can be caused by two
mechanisms. The msrA gene encodes for an efflux pump causing constitutive resistance, whilst the
erm genes confer resistance to the macrolide-lincosamide-streptogramin B group of antibiotics
(MLSB) by alteration of the ribosomal target. MLSB resistance may be inducible or constitutive.
Inducible MLSB resistance is not recognised using standard susceptibility testing methods,
including automated systems. Failure to identify this resistance mechanism may lead to clinical
failure of clindamycin therapy.
Previous work showed that 96% of our local S aureus population carried inducible MLSB
resistance. We undertook this study in order to establish the prevalence of genes coding for MLS B
resistance in a group of 55 MRSA, and 56 MSSA isolates and to discover which, if any standard
phenotypic laboratory techniques are efficient in detecting inducible resistance.
Methods
A resistance profile was built up using disk diffusion, Vitek automation and E tests. Real-time PCR
was used as the ‘gold standard’ in order to probe for resistance genes erm A, erm B, erm C and
msrA.
Results
All 74 strains (MSSA and MRSA) which phenotypically showed macrolide resistance carried a
resistance gene. Of the 35 clindamycin-susceptible, erythromycin-resistant MSSA strains 14 (40%)
carried erm A, 17 (49%) erm C, and 4 (11%) msrA. Of the 19 clindamycin-susceptible,
erythromycin-resistant MRSA strains 2 (11%) carried erm A and 17 (89%) erm C. All isolates that
contained an erm gene were D test positive. Of the 4 isolates that harboured msrA, all were D test
negative. Erythromycin MIC of >256ug/l was associated with the erm C gene in 90% of MRSA
strains but only 11% of MSSA.
Conclusions
In S aureus the D test is an effective method of identifying MLSBi resistance. In this strain
collection, erm A and erm C where equally distributed within MSSA, whilst MRSA had a
predominance of erm C. Although no phenotypic tests could reliably differentiate between the erm
genes, within MRSA, an erythromycin MIC of >256ug/l was associated with the erm C gene
3
003 Asymptomatic Carriage of Protozoan Parasites in Children in Day Care Centers in the
United Kingdom.
AP Davies1,2, B Campbell1, M R Evans3,4, A Bone5, A Roche5, RM Chalmers2
1Institute
of Life Sciences, School of Medicine, Swansea University
Cryptosporidium Reference Unit, National Public Health Service Microbiology Swansea
3National Public Health Service Communicable Disease Surveillance Centre, Cardiff
4Department of Primary Care and Public Health, Cardiff University
5South West London Health Protection Unit, Tooting, London
2UK
Background
Cryptosporidium and Giardia are protozoan parasites, endemic worldwide, which cause diarrhea in
healthy children. Most Cryptosporidium infections in the UK are caused by two species, C. hominis
and C. parvum, although occasionally other species or genotypes are detected. Previous studies
have found a significant association between infection with both Cryptosporidium hominis and
Giardia duodenalis and changing children’s nappies, suggesting that very young children may be
an important reservoir for these organisms in the community.
Objective
To investigate the prevalence of asymptomatic carriage of these parasites amongst infants and
preschool children attending day-care nurseries in two UK cities, using highly sensitive
immunomagnetic separation of Cryptosporidium oocysts and a commercially available ELISA kit to
detect Giardia antigens. The species/genotype of Cryptosporidium isolates was also defined.
Methods
Carers of healthy children attending daycare nurseries in Swansea and London were asked to
send faeces from soiled nappies for testing using immunomagnetic separation, an extremely
sensitive technique recently validated for use in human faecal samples at the UK Cryptosporidium
Reference Unit. Isolates were typed by PCR-sequencing ssu rDNA. The presence of Giardia was
also sought, using ELISA (Giardia II, Techlab®) and questionnaire data was collected on possible
risk factors.
Results
In total, 230 samples were collected. The point prevalence of both Cryptosporidium and Giardia
was 1.3% (95%CI 0.3 to 3.8%), with no dual infections. Only one of the three Cryptosporidium
isolates was C. hominis, the other two being Cryptosporidium skunk and cervine genotypes which
are rarely found in samples from symptomatic human cases.
Conclusions
The results do not lend support to the suggestion that young children are a significant reservoir of
Cryptosporidium and Giardia in the UK. The finding of genotypes of Cryptosporidium rarely
identified in samples from symptomatic patients raises the possibility that acquisition of nonhominis/parvum species may be relatively more likely to result in asymptomatic carriage than in
clinical disease, which may reflect lower pathogenicity
4
004 Comparison and evaluation of commercially available identification methods for
Candida albicans.
Catherine Price, Lorna Vale, Alan Paull
NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff
Candida species are known to cause superficial and systemic infections. Superficially they cause
irritation of the mucosal membranes or dermal rashes, but systemic candida infections may result
in death. Systemic infections are rated as the forth causative agent of mortality in the UK and third
in the US. Candida albicans is the most commonly isolated organism, accounting for over 75% of
all candidiasis.
Treating infections caused by Candida albicans is relatively straight forward as there is generally
very little acquired resistance to the commonly available antimycotics. Treatment failure is likely to
be a result of an infection by a non-albicans strain of Candida; some of which are intrinsically
resistant to the commonly used antimycotics.
Candida albicans is a commensal organism of the skin, oral and gastrointestinal flora, therefore
obtaining mixed cultures is very likely.
The performance of a germ tube test on a mixed culture of Candida species containing Candida
albicans would yield a positive result, which would confirm the presence of Candida albicans but
would mask the presence of the non-albicans species. The gold standard method of identifying
Candida albicans, thus differentiating it from non-albicans species is the germ tube method using
human plasma. However, due to risks of blood borne viruses with human plasma, animal sera or
solid media, such as Mueller-Hinton agar can be used as an alternative.
In this study four different methods of identifying Candida albicans were performed, evaluated and
compared to the Gold standard method, in order to determine which method would be a better
alternative to using human plasma. These methods were: germ tube test using horse serum and
Mueller-Hinton agar, “spiking” on heated horse blood agar and the chlamydospore test using
cornmeal/Tween 80 agar.
It was discovered that horse serum was superior for sensitivity and specificity; results were 100%.
However, use of Mueller-Hinton germ tube method, allowed for easier determination of true hyphae
and pseudohyphae.
The usefulness of using chromogenic agar as a primary plate, identification plate and for the
determination of mixed cultures in comparison to Sabouraud agar with chloramphenicol (SABC)
was also investigated in this study. Four commercially available chromogenic agars were used:
As an identification plate, the best agar was: ColorexTM Candida agar
As a primary plate the best agars were: SABC agar and ChromIDTM Candida agar (CANID2) by
Biomérieux
The best agar for determining mixed cultures was ChromIDTM Candida agar (CANID2) by
Biomérieux
5
005 Evaluation of Single and Multiplex PCR Assays for the Rapid Identification of
Dermatophytes from Nail and Skin Samples within Twenty Four Hours.
C.L.Alexander, L.Brown, R.Dunsmuir, G.S.Shankland.
Clinical Mycology, Microbiology Department, Yorkhill Hospital, Glasgow
Clinical Mycology, Glasgow receives over 10,000 specimens each year for the diagnosis and
identification of fungal infections from superficial sites. The causative fungi are known as
dermatophytes. Audits during 2007 and 2008 indicate Trichophyton rubrum is the most common
dermatophyte (84% of all positive superficial samples). The second is Trichophyton interdigitale
(14%).
Objectives:
To evaluate and validate rapid PCR-based methods with modifications based on studies by
Arabatzis et al* involving single and multiplex assays. To demonstrate prompt, accurate diagnosis
and identification of dermatophytes from nail and skin samples. The aim is to improve turnaround
times for dermatophyte identification which is currently 14 days by culture for Trichophyton rubrum
and Trichophyton interdigitale.
Method:
A total of 398 samples which included 362 nail samples and 36 skins were subjected to;
1) Microscopical analysis after 3 hours incubation in 20% KOH for the presence of fungal spores
and/or hyphae; 2) Culture on Sabourauds and Mycosel agar for 3 weeks. Identification is based
on colony morphology and micromorphology. 3) DNA extraction by BioRobot EZ1 then real-time
PCR using two separate assays to detect T. rubrum or T. interdigitale. Multiplex assays have also
been evaluated to detect Microsporum canis, Trichophyton tonsurans, Trichophyton violaceum,
Microsporum audouinii.
Results:
In 254 samples, 155 were microscopy, culture and PCR negative. The other 99 samples were
microscopy, culture and PCR positive (n=96 for T.rubrum, n=3 for T.interdigitale). T.rubrum was
detected in a further 55 samples by PCR which were microscopy positive but failed to grow any
organism on culture. Microscopy failed to detect fungi in 12 samples which grew T.rubrum and
which were PCR positive. 2 samples previously reported as T.interdigitale were PCR positive for
T.rubrum. PCR detected T.rubrum in a further 2 samples which yielded Scopulariopsis brevicaulis
or Candida albicans from culturing. 8 samples grew organisms which were not detected using
these assays.
Conclusions:
PCR successfully identified dermatophytes within 24 hours compared to 14 days by culturing. It
was more sensitive than culturing, detecting a greater number of infections and produced accurate
identifications.
Key Findings:
Identification of a range of dermatophytes from nail and skin samples is achievable within 24 hours
using this PCR methodology and samples negative for fungi can be reported within 24-48 hours
compared to 3 weeks.
References *Arabatzis et al, Br.J Dermatol Vol 157, pg 681-689.
6
006 Contamination of Laryngoscope Handles
David Williams1, Nidhika Berry2, John Dingley1, Ceri Jones2
1Deptartment
2NPHS
of Anaesthetics, Abertawe Bro Morganwwg NHS Trust, Swansea
Microbiology Swansea, Singleton Hospital, Swansea
Objectives
To assess the nature and extent of microbial contamination on the handles of laryngoscopes that
were considered to be clean and ready for use in the anaesthetic rooms within the operating
department of our hospital.
Background
Despite use of sterile or disposable laryngoscope blades for each patient, disinfection of
laryngoscope handles does not routinely occur. Handles may be contaminated by splashes and
contact with surfaces or the hands of the anaesthetist. Laryngoscope handles are typically knurled
to provide a good grip, however the fissures in this surface may harbour pathogens. The tip of the
blade usually makes contact with an area on the lower third of the handle when folded after use,
creating a potential route for transmission of pathogens between patients.
Method
Specimens collected from 64 laryngoscope handles which were deemed to be “ready for patient
use” were assessed semi-quantitatively for bacterial contamination. Further identification of all
isolates by routine methods and a MicroflexTM LT MALDI-TOF mass spectrometer was performed.
Samples were taken from three sites on each laryngoscope handle:
A. Hook mount (smooth)
B. Upper third (knurled)
C. Contact point (knurled)
Results
One or more species of bacteria were isolated from 61 (86%) of the handles. These included the
potential pathogens Enterococci, meticillin-sensitive Staphylococcus aureus, Klebsiella and
Acinetobacter. The cultures did not yield any anaerobes, fungi, meticillin-resistant Staphylococcus
aureus, vancomycin resistant enterococci or multiresistant gram negative bacilli.
192 samples yielded 130 positive cultures, of which Site A: 33 (25%); Site B: 44 (34%); Site C: 53
(41%). Site C was the only site to demonstrate heavy contamination, and the only site from which
Streptococcus viridans were isolated.
Conclusions & Key Findings
Contamination with aerobic bacteria, some of which were potentially pathogenic, was
demonstrated on the majority of laryngoscope handles studied.
A greater range of species and heavier growth were found on knurled surfaces (B,C) compared
with smooth surfaces (A). A greater range of species (including oral flora) and heavier growth were
found at the contact point where the tip of the laryngoscope blade made contact with the handle (C)
compared with other knurled surfaces (B), highlighting a potential route for transmission of
pathogens between patients.
Laryngoscope handles present a potential route for transmission of infection. Strategies to address
this include: revision of procedures for disinfection and storage prior to use, introduction of
disposable handles or sheaths, and re-design of handles to eliminate knurled surfaces and contact
points.
7
007 Improved laboratory diagnosis of herpes virus infections
Jenna Dawson, Jenny Bayliss, Michael Isaac, Nidhika Berry
Virology Department, NPHS Microbiology Swansea, Singleton Hospital, Swansea
Background
The traditional method of tissue culture confirmed by immunofluorescence (TC/ DIF) for diagnosis
of herpes and varicella infections has been replaced by molecular methods which have proved to
be more sensitive in other centres within the UK and elsewhere. Therefore, we compared the
sensitivity of our routine tissue culture method for diagnosis of herpes (HSV-1, HSV-2) and
varicella zoster (VZV) with a real time polymerase chain reaction.
Methods
Included in the 230 samples were 222 from genital sites, skin lesions (5) and CSF (3). The swabs
were transported in skimmed milk virus transport medium (SVTM), inoculated into MRC-5 human
fibroblast cells and incubated for up to 10 days. Cultures with a CPE were tested by DIF using
Chemicon HSV and VZV typing reagents.
A simultaneous detection system for HSV-1, HSV-2, and VZV DNA via multiplex real-time PCR
using different fluorophores was used. Viral DNA was extracted on a Corbett Xtractorgene using
Corbett VX reagent pack. A mastermix of Invitrogen Platinum Quantitative PCR SuperMix-UDG
and primer/ probes manufactured by Metabion and Applied Biosystems was added using the
Corbett CAS1200. Amplification was performed on the Rotorgene 6000 and results were read and
analysed. Discrepant specimens (TC/ DIF negative and PCR positive for HSV- 1 or HSV-2) were
referred to Bristol HPA laboratory for confirmation.
Results
Comparison of TC/DIF with RT-PCR (n=227) in swab specimens
PCR +ve
PCR-ve
HSV-1 +ve
36
0
TC/ DIF
HSV-1 –ve
7
184
HSV-2 +ve
49
0
HSV-2 -ve
11
167
For discrepant specimens referred for confirmation, complete agreement was seen with our in
house real time PCR for HSV-1 (4/4 of these specimens sent). 2 of the 11 HSV-2 discrepant
specimens did not confirm on repeat testing in our laboratory and were negative at Bristol.
VZV DNA was detected in 4/ 230 specimens, all of which were negative by TC/ DIF.
Conclusions
1. The detection rate of HSV-1, HSV-2 increased by 7.9%.
2. We were able to demonstrate VZV by real time PCR in clinically diagnosed chickenpox patients
and from other suspect lesions.
3. The turnaround time for the results was reduced to 24 hours using PCR.
4. We propose using this method as a timely, sensitive and reliable test for the diagnosis of HSV1, HSV-2 and VZV in genital and skin swabs.
5. Further comparison of CSF specimens will be carried out to enable us to use this test locally for
diagnosis of meningitis/ meningo-encephalitis.
8
008 The development of an in-house real-time PCR assay for Bordetella pertussis and
Bordetella parapertussis and its use in a tertiary referral paediatric hospital
Elaine McCulloch, Kathleen Harvey-Wood, Alison Balfour, Craig Williams
Department if Microbiology, Royal Hospital for Sick Children , Glasgow
B. pertussis, the cause of whooping cough, and B. parapertussis which causes a milder form of
disease, are responsible for significant disease burden, especially in those aged <1 year. Primary
vaccination against B. pertussis is in infancy, but immunity wanes and does not last lifelong.
Infected adults with chronic cough not only suffer illness themselves, but also act as a source of
infection to partially or non-immunised infants, in whom considerable morbidity and mortality can
occur.
Early diagnosis is crucial as it allows effective treatment to be commenced, limits transmission and
reduces complications. Culture has poor sensitivity and it can take up to 5 days for growth to
appear on charcoal blood agar plates. In November 2006 we developed an in-house real-time PCR
(QPCR) assay for B. pertussis and B. parapertussis which initially was accessed only by paediatric
inpatients and those presenting at the Accident and Emergency Department of our tertiary referral
paediatric hospital. In July 2007 the service was extended to other hospitals, local GPs and
community based paediatricians.
Our specific objectives were:
o
To determine specimen suitability (ie recoverability from standard swabs in charcoal
transport medium, dry pernasal swabs with no transport medium, and naso-pharyngeal
aspirates)
o
To determine specimen stability (ie length of time specimens could be left prior to
successful nucleic acid recovery)
o
To compare turnaround times of standard culture versus QPCR
DNA was extracted from all specimens using an EZ1 Biorobot (QIAGEN) and a tissue extraction
kit. All specimens were then tested using specific B. pertussis and B. parapertussis primers and
probes. Amplification and detection was performed using ABI 7000 QPCR system. Culture was on
charcoal blood agar plates incubated in a moist atmosphere at 350C for 7 days.
Analysis of stability of specimens showed that all specimens regardless of time to processing were
positive by QPCR. There was some decrease in DNA recovered after 6 days, particularly when
testing for B. parapertussis. This was not deemed to be of significance provided samples were
processed within 7 days.
Over 200 clinical specimens have been analysed to date. The sensitivity of PCR versus culture has
improved by 50% and the turn-around time is just under 2 days for QPCR versus an average of 7
days for culture.
In conclusion, improved turn-around time using QPCR and a decrease in the reliance of culture has
allowed this service to be accessed by a wide range of clinicians.
9
009 Q-PCR screening of BK viraemia in paediatric renal transplant patients
Kathleen-Harvey-Wood, Alison Balfour, Craig Williams, Jim Beattie
Department if Microbiology, Royal Hospital for Sick Children, Glasgow
Polyomavirus BK virus (BKV) is part of the Polyomaviridae family, which includes JC virus (JCV)
and the simian virus SV40. Primary infection with BKV occurs in early childhood and leads to viral
latency. In immunosuppressed post renal transplant patients BKV may reactivate. BKV
nephropathy has increasingly been recognised as a significant cause of renal dysfunction affecting
1 –10% of renal transplant patients and graft loss after renal transplantation of between 10 –80 %.
Routine screening for the virus is recommended so that early diagnosis and appropriate treatment
strategies can be instituted.
Specific objectives


To validate a commercial Q-PCR kit which can analyse urine and blood specimens for the
presence of BKV
To develop an algorithm for screening patients post-transplant for BKV which would
complement our current screening strategy for EBV, CMV and Adenovirus
Methods
EDTA blood and urine samples were extracted using EZ1 BioRobot (Qiagen). Blood was extracted
as per our established protocol for CMV and EBV using the EZ1 DNA blood kit with an elution
volume of 200 ul. Urines were pre-treated and extracted using the EZ1 DNA Tissue kit with 50 ul
elution volume.
The Q-PCR assay was performed using the BKV Q-PCR Alert Kit (Nanogen) supplied by Inverness
Medical. Samples were run in duplicate and amplification was performed on an ABI 7500 (standard
mode). Water samples were extracted and analysed. QCMD BK/JC 2008 panel was examined.
The assay was validated to determine the optimal number of amplification cycles.
Results
In order to validate the BKV Q-PCR kit, the results were compared with Micropathology Ltd where
samples are currently sent for testing. 100 specimens were analysed to date. All 43 EDTA Blood
samples tested were negative and the internal positive control was positive. 49 Urine specimens
were tested : 27 positive, 18 negative and 4 equivcocal.
8 extracted water samples showed no contamination. EBV, CMV and Adenovirus positive samples
were negative showing specificity of the PCR reaction. QCMD BKV 2008 panel samples examined
: the lowest value ( 245 copies) was not detected.
Conclusions
Recent studies indicate detectable virus in the blood is more predictive of BKV nephropathy than
viruria alone. We have developed and implemented a cost-effective algorithm for screening
paediatric patients post-renal transplant for BKV which complements our already established
regimen for screening for other viruses.
10
010
Rapid
identification
of
microorganisms
using
Matrix-Assisted
Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)
Laser-
Eugene Rees, Stuart Johnston, K El Bouri
NPHS Microbiology Swansea, Singleton Hospital, Swansea
A MALDI-TOF-MS system is available commercially that offers analysis and identification of
bacterial cultures within 10 minutes of isolation. This offers major benefits to the clinical laboratory:
More rapid isolate identification will support earlier clinical management decisions regarding
appropriate treatment; Specific sensitivity testing could be undertaken in the context of
identification data; Control of infection alerts could be generated at an earlier stage.
Isolates
Over 2,000 sequential, clinically isolated organisms identified by conventional methods, were
investigated using MALDI-TOF. Gram negative bacteria were identified by either API20E,API20NE
or BD Phoenix. Staphylococcus aureus was identified by Staph latex, coagulase reaction, Dnase
reaction or BD Phoenix. Coagulase negative Staphylococci were speciated by BD Phoenix. ß
haemolytic streptococci were identified by Lancefield grouping . Other streptococci, either non
haemolytic or α haemolytic were identified by Optochin sensitivity testing and BD Phoenix.
Miscellaneous bacteria (Neisseria sp, Campylobacter sp, Haemophilus sp, Clostridium sp etc.)
were identified by conventional biochemical methods)
MALDI Biotyper
Part of a colony from each overnight culture was smeared in duplicate onto the MALDI-TOF steel
target plate, overlaid with 1цl of matrix solution and allowed to air dry. The plate was placed into the
MALDI-TOF-MS target receptor and an emission spectrum produced. Spectral analysis and
identification was undertaken using the system’s ‘MALDI Biotyper’ software. The MALDI Biotyper
typically collects 240 spectra from each well and compares the spectra against a library of known
reference bacterial spectra.
Results
The identification software reports findings as one of the following:
i.
Excellent genus identification, good species identification
ii.
Good genus identification
iii.
No reliable identification
iv.
No peaks (spectra) found
Data comparison (conventional versus MALDI-TOF)
When an identification level of (i) or (ii) were obtained, the MALDI TOF results were compared with
the conventional results follows
A. Excellent match of species
B. Match of genus, different species
C. No match
Results
Coliforms - There was an 85% ‘Excellent Match’ (A) from at least one of the two duplicate wells. Of
the rest 12% gave a B match. No match (C) was found in only 3% of the coliforms (all of these
were confirmed as either non-fermenters or ‘unusual’ isolates e.g. Achromobacter. Mucoid strains
of Klebsiella sp. Consistently yielded no emission spectrum using the smear method. These were
identified by the MALDI-TOF later using an extraction method.
Staphylococci – over 98% ‘Excellent Match’ (A) with S.aureus and the coagulase negative species.
Streptococci – 95% ‘Excellent Match’ (A) of groupable streptococci, and 60% match(A or B) with
greening or non haemolytic streptococci
Conclusion
MALDI-TOF MS is a reliable method for identification of clinically isolated
microrgansims, is extremely fast compared to conventional methods, and requires
significantly less staff time and lower consumable costs.
11
011 Disruption of Staphylococcus epidermidis biofilms by medicinal maggot Lucilia
sericata excretions/secretions
Llinos G Harris1, Alyson Bexfield2, Yamni Nigam3, Holger Rohde4, Norman Ratcliffe2, Dietrich
Mack1
1Medical
Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine,
Swansea University, Swansea
2Department of Biological Sciences, School of the Environment & Society, Swansea University,
Swansea
3School of Health Sciences, Swansea University, Swansea, SA2 8PP, UK
4Institut für Medizinische Mikrobiologie, Virologie und Hygiene, Universitätsklinikum HamburgEppendorf, Universität Hamburg, Germany
Objectives
Chronic infections frequently cause substantial morbidity and mortality, and are commonly
associated with biofilms formed by bacteria such as Staphylococcus epidermidis. Biofilms have
been shown to protect bacteria from phagocytosis and antibiotic treatments. Thus with the increase
in antibiotic resistant bacteria, maggot debridement therapy has been reintroduce for the treatment
of chronic wounds. Studies have suggested that the beneficial effect of Lucilia sericata larvae
(maggots) are present within their excretion/secretions (ES), which contain several different
proteases, a glycosidase and an antibacterial compound. This present study evaluated the effect of
L. sericata ES on formation and disruption of biofilms formed by two S. epidermidis strains
employing different mechanisms of intercellular adhesion namely polysaccharide intercellular
adhesin (PIA) or accumulation associated protein (AAP), respectively.
Methods
Native excretions/secretions (ES) were collected from third instar L. sericata larvae (ZooBiotic Ltd.,
Bridgend, UK ). A semiquantitative biofilm assay was used and the OD 550 of stained adherent
bacterial biofilms was quantitated with a FLUOstar OPTIMA microplate reader. The presence of a
biofilm was defined to have a mean OD550 greater than 0.2. 10µl ES was either added to test wells
immediately after inoculation with bacteria (nascent biofilm formation) or after 24h of culturing (preformed biofilm). The effect of culturing for different times and temperatures, stability of ES by
boiling the extracts, and 3 different ES fractions were analysed applied to nascent biofilms and
pre-formed biofilms. The results were substantiated by immunolabelling using specific antibodies
against PIA and AAP.
Results
S. epidermidis biofilm formation was inhibited in the presence of ES. Whilst ES also disrupted preformed biofilms, the extent of disruption differed depending on the mechanisms of biofilm
accumulation employed by the respective S. epidermidis strain. The inhibitory activities were
temperature and time dependent and were inactivated by heat treatment. The active compounds in
ES against S. epidermidis biofilms were >10kDa in size, and appeared to have protease or
glucosaminidase activity, respectively. ES disintegrated cell aggregates by degrading PIA or AAP
as substantiated by the immunolabelling results.
Conclusions
In summary, ES interferes with S. epidermidis biofilm formation by specifically degrading factors
employed in biofilm accumulation, which are similarly employed by Staphylococcus aureus. In a
purified form ES-factors may have general applicability for the treatment or prevention of chronic
biofilm infections caused by staphylococci.
12
012 Molecular epidemiology of extended-spectrum beta-lactamase (ESBL) carrying
Enterobacteriaceae in Swansea
Caron Jones1, Llinos G. Harris1, Eugene Rees2, Khalid El-Bouri2, Angharad P. Davies1, Dietrich
Mack1
1Medical
Microbiology and Infectious Diseases, Institute of Life Science, School of Medicine,
Swansea University, Swansea
2NPHS Microbiology Swansea, Singleton Hospital, Swansea
Extended-spectrum beta-lactamases (ESBL) mediate resistance to 3rd generation cephalosporins
and aztreonam in Enterobacteriaceae and pose major clinical problems. Enterobacteria were
collected from Swansea NPHS Microbiology laboratory and ESBL status confirmed by BSAC
phenotypic methods. Of 167 isolates 118 were Escherichia coli, 38 Klebsiella spp., 5 Enterobacter
spp., 2 Citrobacter spp., 2 Pseudomonas spp., 1 Morganella morganii and 1 Acinetobacter
baumannii. Primer sets were used to specifically amplify TEM, SHV, CTX-M, and plasmid-encoded
ampC genes. The most predominant ESBLs were CTX-M (n=139). 128, 5, 4, and 1 ESBLs
belonged to CTX-M groups-1, -9, -25/26, and -2, respectively. 66/128 CTX-M group 1-positive
isolates (exclusively E. coli) generated a 400 bp fragment of insertion sequence IS26-CTX-M-15
link region characteristic for epidemic E. coli strain A, which were all clonally related by PFGE.
Nucleotide sequencing revealed 7 TEM-116 and 1 TEM-52 ESBLs, 1 TEM-33 (IRT), and 45 nonESBL TEM-1. 5 SHV-2 ESBLs were found, while 17 SHV-89, 6 SHV-11 and 10 SHV-1 genes, all
non-ESBL, were present. 6 isolates carried ampC genes (2 CIT, 1 DHA, 1 EBC, 2 Fox). 83% of
isolates carried CTX-M ESBLs, of which 92% belonged to CTX-M group 1. ESBL carrying E. coli
outnumber the other Enterobacteriaceae species studied.
13
013 Evaluation of glycopeptide discs for Staphlococcus aureus with reduced susceptibility
to glycopeptides (hGISA/GISA)
Leanne Davies1, Mandy Wootton1, Robin Howe2
1SACU,
2NPHS
NPHS Microbiology Cardiff, University Hospital of Wale, Cardiff
Microbiology Cardiff, University Hospital of Wale, Cardiff
OBJECTIVES
GISA/hGISA strains are not detected by current laboratory disc diffusion testing. The GISA/hGISA
detection method using Population Area Profile-Area Under Curve is proven to be accurate
however it is time consuming. Disc testing with a range of vancomycin (V) and teicoplanin (T)
concentrations was evaluated with Glycopeptide susceptible S aureus (GSSA), hGISA and GISA
strains.
METHOD
83 strains from a worldwide collection were tested in triplicate including; 14 meticillin resistant
GSSA, 49 hGISA and 20 GISA. Vancomycin discs containing 5µg and 30µg, and teicoplanin discs
containing 5µg, 10µg, 20µg and 30µg were tested on IsoSensitest (ISO) and Mueller Hinton Agar
(MHA). Variation in zone diameter (ZD) and reproducibility according to media and phenotype
were assessed.
RESULTS
ISO
MHA
GISA
hGISA
GSSA
GISA
hGISA
GSSA
V5
Mean
13.9
17.1
18.6
14.1
17.2
19.1
SE
0.34
0.09
0.19
0.32
0.08
0.16
V30
Mean
SE
21.1
0.15
22.1
0.09
23
0.24
21.1
0.15
22.2
0.09
22.9
0.25
T5
Mean
11.3
14.1
17.1
10.8
13.7
16.6
SE
0.32
0.19
0.13
0.32
0.19
0.15
T10
Mean
SE
14.2
0.26
16.5
0.17
18.4
0.15
13.8
0.26
15.9
0.18
18.4
0.15
T20
Mean
SE
16.9
0.22
18.9
0.15
20.5
0.18
16.2
0.24
18.1
0.15
20
0.15
T30
Mean
SE
18.7
0.17
20
0.14
21.2
0.15
17.9
0.21
19.3
0.15
20.7
0.17
Mean ZDs (mm) and standard error (SE) are given in the table. ZD was related to the level of
susceptibility for all discs. Differentiation was greatest for low content discs. T5 on ISO/MHA and
V5 on MHA distinguished between the GSSA and GISA strains. No disc could differentiate hGISA
from GISA and GSSA.
CONCLUSIONS
Low concentrations of vancomycin and Teicoplanin can differentiate between GISA and hGISA
strains. None of the glycopeptides concentrations tested were adequate for the accurate
identification of hGISA.
14
014 Blood Culture Results in Welsh Special Care Baby Units
SJA Froude1, RA Howe1, M Heiginbothom1, S Kotecha2
1NPHS
Microbiology Cardiff, University Hospital of Wale, Cardiff
of Child Health, School of Medicine, UHW Cardiff
2Department
Introduction:
Neonates are especially vulnerable to infection which can lead to significant morbidity and
mortality. The All Wales Perinatal Survey (2006) reported that 11% of neonatal mortality was as a
consequence of infection. Serious infections often manifest as bacteraemia, and the analysis of
blood culture (BC) isolates presented here allows a better understanding of the patterns of serious
infections presenting in Special Care Baby Units (SCBUs) across Wales.
Methods:
Data from all blood cultures submitted from SCBUs across Wales for the period 2005-07 were
downloaded from the microbiology DataStore repository. The numbers of live births (LB) for each
unit were obtained from the All Wales Perinatal Survey and used as a denominator of clinical
activity.
Results:
For 2005-7, 10,314 blood cultures were taken. Overall sampling rates in 2007 were 85 BC/1000LB
(being highest in level 3 units) with a positivity rate of 8%. The most frequent isolates were
Coagulase Negative Staphylococci (CNS) followed by Group B Streptococci (GBS),
Staphylococcus aureus, Escherichia coli, and Enterococci. CNS and coryneforms accounted for
71% of positive cultures. GBS incidence increased from 0.37 – 0.60/1000LB for 2005-07,
remaining lower than published UK rates (0.72/1000LB).
Conclusions:
Traditional neonatal pathogens (eg GBS) were cultured at rates in keeping with UK experience.
However the commonest isolates were CNS and coryneforms that, although sometimes implicated
in line-associated infections, frequently may be “contaminants”. Strategies to improve the taking of
blood cultures and assessment of potential contaminants should be developed to avoid
unnecessary antimicrobial therapy.
15
015 Antibiotic Usage in Welsh Special Care Baby Units
SJA Froude1, RA Howe1, M Heiginbothom1, S Kotecha2
1NPHS
Microbiology Cardiff, University Hospital of Wale, Cardiff
of Child Health, School of Medicine, UHW Cardiff
2Department
Introduction:
Neonates, particularly those admitted to Special Care Baby Units (SCBUs), are especially
vulnerable to infection. Since early effective antibiotic therapy is critical in reducing morbidity and
mortality, and signs of infection are often non-specific, SCBUs usually have clear empiric therapy
guidance. However there is little data available on antibiotic use on SCBUs and inter-unit variability.
This study provides such data.
Methods:
Ward stock data for antibiotics was obtained from the MEDUSA data repository for 11 of 13 SCBUs
in Wales. Data was transformed into WHO Defined Daily Doses (DDDs) to facilitate inter-unit
comparison. Occupied bed days (BD) data obtained from Health Solutions Wales was used as a
denominator of clinical activity.
Results:
Antibiotic use varied from 4 DDDs/100BD (level 1 unit) to 18 DDDs/100BD (level 3 unit), with more
different agents used (23) in level 3 than in level 1 (14). Of 9 units with complete data, 4 clearly
used benzylpenicillin as the primary beta-lactam, while 4 used amoxicillin. Two of the 3 highest
users of glycopeptides (up to 3 DDDs/100BD) were level 2 units that used predominantly
teicoplanin; other units used vancomycin.
Conclusions:
There is predictable variability in antibiotic use with greater use in level 2 & 3 units. Differences in
beta-lactam selection are presumably due to local preference rather than specific infection-related
requirements. The high use of glycopeptides (teicoplanin) in level 2 units may warrant further
investigation. Given the variance in antibiotic use, consideration could be given to ‘All Wales’
guidance for antibiotic prescribing in SCBUs.
16
016 C-Reactive Protein Response Associated with Candidaemia
K.Marden1, S.Schelenz2, R.A.Barnes1
1NPHS
Microbiology Cardiff, University Hospital of Wales, Cardiff
and Norwich University Hospital
2Norfolk
Background
The literature relating C-reactive protein (CRP) levels in response to candidaemia is small. In
clinical practice one might suspect candidaemia in a high risk patient with an elevated CRP when
no other cause for an acute phase response is evident.
Objectives
To determine the relationship between CRP levels and candidaemia in a hospital case series.
Method
Cases included were a consecutive series of positive Candida species blood cultures identified in
Cardiff and Vale NHS Trust between 2000 and 2006. Source hospital unit and contemporaneous
CRP, white blood cells (WBC) and liver function tests (LFT) were recorded. The CRP taken at the
shortest interval to the positive blood culture, up to a maximum of +/- 5 days, was recorded. All
cases with concurrent bacteraemia or without a contemporaneous CRP were excluded.
Results
There were a total of 290 candidaemia episodes. After exclusion criteria were applied 118 episodes
were eligible for study.
The majority (51%) of the isolates were Candida albicans with C.glabrata and C.parapsilosis
contributing 17% and 15% respectively.
CRP in all the candidaemia episodes ranged from <6 (normal) to 476 mg/l. 7% of CRP values were
normal. CRP was elevated in 93% of episodes, 41% were between 6 and 99 and 52% were 100.
44% of all candidaemia episodes occurred during a stay on an intensive care unit (n=52), be it
adult general (GITU), cardiothoracic, paediatric or neonatal.
For episodes occurring on an adult ITU, 2% of CRP values were normal, 36% were between 6 and
99 and 62% were 100. In adult haematology (n=22) all CRP values were elevated, 27% were
between 6 and 99 and 73% were 100.
There were 25 candidaemia episodes in children, including 11 in paediatric oncology, 6 in neonates
and 8 from general paediatrics. CRP was normal in 17% of neonatal episodes and 45% of
paediatric oncology episodes. CRP values did not rise above 89 in all paediatric oncology
episodes.
There was no relationship with CRP and LFT. The correlation with WBC will be described.
Conclusion
In most cases candidaemia is associated with a markedly elevated CRP. However in paediatric
oncology and neonates CRP was normal in a substantial minority. This may suggest these
Candida isolates were contaminants or that CRP response is absent in some cases. Further
studies are warranted.
17
017 Evaluation of IQ200 Sprint urine analyser as a primary screen for bacteriuria
Julie Varga, Teresa Peach, Peter James
NPHS Microbiology Cardiff, University Hospital of Wales, Cardiff
Introduction:
The IQ200 Sprint analyser employs real-time video flow imaging and patented Automated
Intelligent Microscopy (AIM) technology for automating urine microscopy. It is validated here as a
primary screen for bacteriuria.
Objectives:
To evaluate the utility of selected parameters measured by the IQ200 Sprint analyser to screen
urine samples for bacteriuria. To compare the negative predictive value (NPV) of the analyser
when compared to two culture methods (CLED and UTI clear, Oxoid), both independently and
collectively. A comparison of RBC and WBC counts from the analyser and from inverted manual
microscopy is also presented.
Methods:
1020 urine samples were processed using the IQ200 Sprint Urine analyser. The samples were
inoculated onto CLED (Oxoid) and inverted light microscopy performed as part of the routine
screening procedure according to the laboratory’s Standing Operating Procedure. Samples were
also inoculated onto UTI clear (Oxoid) using 1uL sterile disposable loops. Comparisons were made
between WBC and RBC counts obtained by the two methods. A linearity check was performed by
selecting two urine samples with large numbers of RBCs and WBCs respectively and processing
doubling dilutions of each sample individually and mixed together to reveal any quenching effects
on either cell type.
Results:
246 organisms (24% positivity), were reported using CLED agar compared with 294 organisms
(29% positivity) using chromogenic agar. There was complete agreement for 186 organisms, 58
organisms were grown on CLED agar only, and 106 organisms were grown on chromogenic agar
only. The NPV of the IQ200 Sprint was 94.9% when compared with chromogenic agar and 94.0%
when compared with CLED agar. This compares favourably with the NPV of chromogenic agar
when compared with CLED agar (94.7%) and CLED when compared with chromogenic agar
(93.3%). There was at least a four-fold increase in average RBC counts determined by the
automated analyser in comparison with manual counts. In samples where the WBC count was
<100, more than 50% of samples had RBC counts <1 per uL by manual microscopy, whereas only
6% had RBC counts <1 per uL. WBC counts, though variable between methods were broadly
comparable. There was no distortion of RBC counts by large numbers of WBCs and visa versa
obtained from the urine analyser and counts were linear over a wide dilution range.
Conclusions:
The IQ200 Sprint urine analyser performs (using the parameters described here) at least as well as
culture when used as a primary negative screen for bacteriuria. The heightened sensitivity of the
IQ200 Sprint analyser for RBCs requires careful introduction of this technology to the clinical
setting. We recommend continuation of culture on all samples from critical patient groups such as
paediatrics and ICU patients.
18
018 A review of Staphylococcus aureus bacteraemia in a Welsh district general hospital
A Towers1, A Omrani2
1Department
2Department
of Medicine, Royal Glamorgan Hospital, Llantrisant
of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant
Aim:
To review diagnosis, management and follow up of patients with Staphylococcus aureus
bacteraemia with a view to creating a management pathway
Method:
Data was collected from Royal Glamorgan Hospital in South Wales. Patients were identified using
MSSA and MRSA positive blood cultures. A proforma was designed to collect relevant data. The
case notes and medication charts were reviewed and data collected. There are no evidence-based
UK or European guidelines on the management of S aureus bacteraemia. For the purpose of this
study, a minimum of 14 days of effective antibiotic therapy in addition to adequate assessment for
a source of sepsis and, where applicable, source control were considered minimal standards of
care. Furthermore, follow up blood cultures and regular clinical monitoring were assessed.
Results:
There were 65 MSSA/MRSA positive blood cultures in 2008. Notes were available for 32 patients.
The majority of the study population were over 50 years of age and had a medical underlying
diagnosis. Furthermore, 13% were on haemodialysis, 3% were intravenous drug users, 22%
immunocompromised, 13% were known to have a heart murmur, 16% had diabetes mellitus and
13% had an underlying malignancy. The overall in-hospital mortality was 44% with all deaths
occurring in the over 50 years age groups. MRSA-associated in-hospital mortality was 64%,
compared with 28% in patients with MSSA bacteraemia. The majority of cases were healthcareassociated (59%). The relapse rate was low at 6%. In the majority of cases the source of
bacteraemia was respiratory, intravascular catheter or uncertain. Of those cases with an
intravascular catheter source, approximately 70% were MRSA-associated. Two thirds of the
patients received the minimum standard of care outlined above. In those who were considered to
have received suboptimal therapy, half received less than 14 days of effective antibiotic therapy.
Seventy eight per cent were considered to have had adequate monitoring of inflammatory markers
and regular clinical reviews by the team in charge of their care. However, only 31% of cases had
repeat blood cultures within 72 hours of the first episode of S aureus bacteraemia. Advice from
Microbiology & Infectious Diseases was documented on the blood culture reports, but
documentation of such advice in the clinical notes was poor.
Conclusion:
In-hospital mortality associated with S aureus bacteraemia was high despite adequate treatment in
most cases. This is probably because most patients had significant co-morbidities. A considerable
proportion of S aureus bacteraemia was nosocomial. Perhaps focussing on prevention and rapid
identification of cases would be a more effective management strategy. One possible strategy to
improve documentation in clinical notes could be insertion of alert stickers in the patients’ notes
once a diagnosis of S aureus bacteraemia is made, with advice on further assessment, monitoring
and therapy.
19
019 Audit of Compliance with Antimicrobial Therapy Guidelines for Patients at Increased
Risk of C difficile Infection at Cwm Taf NHS Trust (South)
Taha Akhtar1, Natalie Collings2, Huma Changez1, Ali Omrani3
1Department
of Medicine, Royal Glamorgan Hospital, Llantrisant
University School of Medicine, Cardiff
3Department of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant
2Cardiff
Introduction:
Similar to many hospitals in UK, an increase in Clostridium difficile infection (CDI) rate was noted in
our institution, in association with a general excessive use of antibiotics. The increase was
particularly notable in medical wards in the acute and rehabilitation hospitals. Along with increased
infection control precautions and a review of cleaning procedures, new antimicrobial therapy
guidelines were introduced. The guidelines were only applicable to patients at increased risk of
CDI, defined as those aged over 65 years, were on long-term haemodialysis or had history of
previous CDI. The guidelines discouraged the use of cephalosporins, fluoroquinolones and
Clindamycin for the target patient group. Tetracyclines, sulphonamides (co-trimoxazole) and betalactam/beta-lactamase inhibitors (co-amoxiclav & piperacillin/tazocin) were utilised, were indicated.
The first aim of this study was to audit antimicrobial prescribing and its compliance with those
guidelines. The second was to review CDI rates before and after the introduction of the guidelines
and assess the relationship between any change in rates and antimicrobial therapy guidelines.
Methods:
The first part of the study was a point-prevalence study whereby all patients in a particular ward
had their medication charts reviewed. Notes of patients who were an increased risk of CDI and
were on antimicrobial agents were reviewed to assess the indication for prescribing and whether
that was consistent with the recommendations set out in the guidelines. The second part was a
retrospective review of computer records of CDI and a calculation of the CDI rates for individual
wards per 1,000 admissions. The rate was calculated for the months of October and November
2008, the period of the study, as well as the same two months in the two preceding years.
Results:
One hundred and forty two patients were receiving care on the acute medical wards at the point of
study. Fifty four patients (38%) were on antibiotics, of which thirty eight were considered to be at
higher risk of CDI (70%). Over all, two thirds of the antimicrobial therapy was consistent with the
guidelines. Twenty two out of one hundred and twenty nine patients (17%) in the non-acute
medical wards were on antibiotics, all of whom were at an increased risk of CDI. Compliance with
the recommendations was generally excellent (83%) in those wards. The situation was different in
the surgical wards, where eighteen (19%) of the ninety four patients were at higher risk of CDI and
on antibiotics. However, only half of the prescribing was compliant with antimicrobial therapy
guidelines (range 33-67%).Rates/1000 admissions in wards
Wards
Acute medical wards
Non acute medical wards
Surgical wards
October 06
5.6
101.7
4.6
October 07
17.5
89.3
0
October 08
3.6
52.6
7.27
Rates of CDI per 1000 admissions fell considerable following the introduction of the new guidelines
on medical wards. However in surgical wards, the rates increased. In medical wards, although
other interventions were introduced at the same time, the guidelines appear to have contributed to
the decline in CDI rates.
Conclusions:
Compliance with newly introduced antimicrobial therapy guidelines for patients at increased risk of
CDI is general good in medical wards where there historically higher rates of CDI. The guidelines
appear to have contributed to the fall in CDI rates. Further interventions are required to address
antimicrobial prescribing in the surgical wards in our hospital.
20
020 An Audit of Rubella Susceptibility Rates in the Cwm Taf (South) NHS Trust Over a Four
Year Period
LA Matthews1, L Lawrance2, S Gray2, D Gray2
1Virology
Laboratory, Department of Microbiology & Infectious Diseases, Royal Glamorgan
Hospital, Llantrisant
2University of the West of England, Bristol
Background
Rubella is an insignificant childhood disease that has devastating results in pregnancy.
Immunisation using Rubella single vaccine was replaced by Measles, Mumps and Rubella vaccine
(MMR) in 1988. Adverse publicity (1998) caused a fall in uptake of MMR. Anecdotal evidence
suggests that rubella susceptibility levels in pregnancy have subsequently increased.
Aims
Using data collected in one NHS Trust area in Wales:
To determine the rate of rubella susceptibility in antenatal women by age group, in first and
subsequent pregnancies and to compare with National figures.
Methods
A retrospective analysis (2005 – 2007) of the results of rubella immunity in ante-natal screening
tests was carried out. Information given on the request forms was also examined. Analysis was
also planned prospectively for another 3 years from 2008 to 2010. To date 3 years retrospective
and one year prospective have been analysed.
Results
The number and percentage of all women who are susceptible to rubella (as defined by an
antibody level of <10 IU/ml) has risen over the period 2005-2008. The susceptibility rate in the most
vulnerable group, namely women in their first pregnancy have increased from 6.6% to 9.5% in this
four year period. Data will presented by age group, first pregnancy and ethnicity.
Conclusions
Poor uptake in the MMR Immunisation programme has been associated with an increase in rubella
susceptibility rates particularly in the under twenties. The reduction in uptake of MMR may result in
the re-emergence of rubella and cases of congenital rubella.
21
021 Evaluation of the Early (2005) HAIN GenotypeR Staphylococcus to Identify MRSA from
Cultured Specimens in a District General Hospital
Kelly Ward
Department of Microbiology & Infectious Diseases, Royal Glamorgan Hospital, Llantrisant
The major reservoir of MRSA in the hospital setting is the infected or colonised patient (Wernitz et
al., 2005). The screening of MRSA at hospital admission offers an early opportunity to detect
MRSA and so aiding in the reduction of the noscomial spread of MRSA.
Molecular methods to detect MRSA are now increasingly common and a range of primers have
been designed to amplify species-specific targets. The aim of this project was to evaluate the use
of the HAIN GenoTypeR Staphylococcus to identify MRSA from cultured specimens in a District
General Hospital
The HAIN GenoTypeR Staphylococcus is a molecular genetic assay. The test is based on Line
Probe Assay DNA STRIP technology and permits the identification of a range of Staphylococcal
species. The strip also detects the methicillin resistance mediating mecA gene and the cytotoxic
virulence factor, Panton-Valentine Leukocidin (PVL).
The method consists of 3 steps. Step 1 is the isolation of the DNA from the bacterial cells. Step 2 is
an End Point PCR Amplification with Biotinylated primers to exponentially amplify the DNA using
Taq DNA Polymerase. The final step is a hybridisation process to allow the development of
coloured bands on nitrocellulose strips.
Seventeen isolates of S.aureus were confirmed as MRSA by the BD Phoenix analyser. The
GenoTypeR Staphylococcus also confirmed they were MRSA. The banding pattern on the
nitrocelluolose strips consisted of the two control bands which demonstrated the steps in the PCR
and Hybridisation worked correctly. They also all had the band for mecA which confirmed
methicillin resistance. They all contained bands 5 and 9 which are specific oligonucleotide bands
for S.aureus. None of the isolates were carrying the gene for PVL toxin however we were able to
demonstrate it using a laboratory control.
Due to the high cost of the molecular kit, only 1 kit (25 tests) was purchased. However this system
was excellent for the confirmation of MRSA and its ability to detect PVL was very useful. However
the cost and expertise required to perform the test makes it unlikely that it would be introduced into
a district general hospital for routine MRSA screening.
A disadvantage this test has is that it is based on confirmation using an already cultured organism.
The culture and incubation process still takes 24hrs so there would be no gain in the time it would
take to achieve a result compared to the current culture alone method.
22
022 Case Study: Toxic Shock Syndrome Associated with a Prior Sports Injury
S. Iyer1, B. Tan2, A. Gandhe3, T. Andarde3
1Department
of Microbiology, Royal Berkshire NHS Foundation Trust, Reading
of Pharmacy, Royal Berkshire NHS Foundation Trust, Reading
3Department of Orthopaedics, Royal Berkshire NHS Foundation Trust, Reading
2Department
Background:
The case study presented highlights the importance of obtaining a detailed clinical history and the
need for a multi-disciplinary approach in the management of unusual emergency admissions.
Case study:
A previously healthy 33 year-old female presented in Accident and Emergency with severe back
pain. One week prior to onset of pain she had fallen on her back whilst snowboarding but did not
report any obvious injury. On admission her temperature was 36.4 oC, RR 16/min, PR 88/min CRP
270 and WBC 6.33X109/L. There was no sensory loss or weakness but she had difficulty in flexion
of both hips. Nothing abnormal was visible on lumber X-ray. MRI showed a haemorrhagic cyst on
the right ovary. Ultrasound showed no obvious adenexal mass or cysts. 24 hours post admission
temperature spiked to >39oC; CRP increased to 360 and WBC decreased to 3.77. Blood culture
drawn at admission was positive for Staphylococcus aureus and, due to history of ‘penicillin
allergy’, IV teicoplanin 400 mg 3 loading doses followed by 400 mg once daily was initiated. The
patient continued to deteriorate and she became clinically septic. On CT scan an abscess was
found in front of the right iliac crest adjacent to the sacral joint. Frank pus was drained and sent for
culture that grew S. aureus. Clindamycin 1.2g 6h was added.The blood culture and pus isolates
subsequently on typing were confirmed to be toxic shock syndrome toxin 1 (TSST-1) positive. The
patient continued to deteriorate and developed right sided consolidation with pleural effusion. She
was transferred to high dependency care. IV linezolid 600mg 12h was added. 48h blood culture
was S. aureus positive, indicating continued bacteraemia. Teicoplanin was replaced with IV
daptomycin 8 mg/kg once daily. Blood cultures 48 and 96h post initiation of daptomycin were
culture negative and the patient improved clinically.
Discussion:
Patients with TSS exhibit a number of clinical symptoms, including fever, diarrhoea, inability to
maintain homeostasis and multiple-organ involvement, with the most severe cases being
fatal. In the presence of fever of unknown origin the early involvement of microbiology
and the need for molecular typing are highlighted by this case study. In vitro studies have
shown that daptomycin has high activity against toxin producing strains of S. aureus, and
the successful outcome in this case confirms its activity in clinical practice.
23
023 Highlighting a Clinical Conundrum – The Need for Microbiological Clinical Expertise
S. Iyer1, G. Boden2, T. Pollard3, R. Dodds3 , B. Tan4, A. Andrade3
1Department
of Microbiology, Royal Berkshire NHS Foundation Trust, Reading
of Paediatrics, Royal Berkshire NHS Foundation Trust, Reading
3Department of Orthopaedics, Royal Berkshire NHS Foundation Trust, Reading
4Department of Pharmacy, Royal Berkshire NHS Foundation Trust, Reading
2Department
Background:
This case study highlights the need for a team approach in usual presentations of infection of
unknown origin.
Case study:
A 16 year-old boy presented to the Paediatric Assessment Unit with fever, chills, diarrhoea and
vomiting of 3 days duration. He had right knee pain, progressively increasing in intensity. Two days
prior to the onset of symptoms he had been playing football, but could not remember being injured.
On examination his temperature was 39.5oC, RR 20/min, PR 120/min CRP 183 and WBC
10.34X109/L. His right knee was slightly swollen, with pain on rotation and standing. Initial
diagnosis was viral illness with reactive arthritis. Analgesics and anti-inflammatory drugs were
initiated. Over the next 48h the pain continued to worsen, his temperature was spiking WBC fell to
6X109/L and CRP increased to 300. Knee aspirate obtained at admission was culture-ve at 24h.
The knee was re-aspirated. Results from the blood sample obtained at admission were positive for
Gram-positive cocci in clusters (2/2). IV flucloxacillin 1g 6h and IV clindamycin 1.2g 6h were
commenced. He underwent arthroscopic washout of his knee that did not reveal any evidence of
synovitis. On day-3 he became afebrile but complained of chest pain on coughing and swollen left
ankle. The chest X-ray showed right midzone shadowing. The patient was admitted to the
paediatric HDU with clinical sepsis and respiratory distress. Antibiotics were continued and PVLtyping of S. aureus was requested by microbiology. At MRI, right proximal tibial and left distal tibial
osteomyelitis was confirmed. Surgical debridement of right tibia was performed. On day-8, he
developed multifocal lung infiltrates and lesions on chest X-ray and CT scan. The patient was
transferred to ITU. IV linezolid replaced flucloxacillin. On day-9 the patient had stabilised and was
transferred back to the HDU. IV Daptomycin 8mg/kg was added on day-10. On day-12 the isolate
was confirmed as PVL-+ve. By day-12 the patient was progressively improving and was blood
culture-ve by day-15. One month after admission the patient was discharged on oral clindamycin.
Discussion:
This case study of a child presenting with fever, chills, diarrhoea and vomiting highlights the need
for a team approach and the early involvement of microbiological services. In the presence of the
increasing incidence of PVL-producing strains clinicians need to maintain a high level of suspicion
for these organisms, especially in the presence of sports associated injuries.
24
024 Chromogenic Agar – a change of culture on the Urine Bench
Linda Sim, Glen Widdows, Dr Andrew Hay
Bacteriology Laboratory, Microbiology Department, Raigmore Hospital Inverness
Objectives
Traditionally, our laboratory identified all significant urinary isolates either by an automated system
(Vitek, Biomérieux UK Ltd) or other methods such as Lancefield grouping for
Streptococci/Enterococci. Although reliable, these methods are relatively time consuming and
expensive. The development of chromogenic agars for simultaneous primary isolation and initial
identification of specific urinary pathogens gave the opportunity to review our methods our ways.
The aims of this study are (i) to examine the primary isolation on chromogenic UTI medium (Oxoid
UK Ltd) as a means of reliably identifying E.coli and Enterococci, which account for two thirds of
our urinary isolates, (ii) produce an algorithm for identification of urinary isolates and (iii) to identify
potential cost savings.
Methods and Results
100 presumptive E.coli (pink colonies), from chromogenic agar, were identified through Vitek and
results compared. Similarly 100 presumptive Enterococci (small dark blue colonies) on
chromogenic agar were identified by Lancefield’s grouping (Pro Lab) and results compared.
Results show ≥ 98% agreement between standard methods of identification and morphological
appearance on chromogenic agar. This is considered acceptable for both E.coli and Enterococcus
species. From these results an algorithm for identification of urinary isolates was developed which
has realised significant savings in the laboratory.
Conclusions
This study showed that, in our laboratory, Oxoid chromogenic UTI medium can be used as a cost
effective identification method for E.coli isolates and Enterococcus species. It also realised
significant savings without compromising clinical care or epidemiological analysis.
25
025 Physiology of Clostridium difficile Spores:
Lovleen Joshi, Prof. Les Baillie
Welsh School of Pharmacy, Cardiff University, Cardiff
Clostridium difficile, an anaerobic, spore-forming bacterium, is commonly associated with
Clostridium difficile-associated diarrhoea and pseudomembranous colitis – both serious hospital–
acquired infections. In Wales there were over 3000 cases of C. difficile in patients over 65 between
July 2007 and June 2008. Literature and high infection rates have highlighted some of the spores’
complexities, such as their ability to remain viable on contaminated surfaces for months and their
resistance to biocides.
Objectives:
To characterise the physiological properties of Clostridium difficile spores to assist combat of
infection in the hospital environment, and to understand if these properties play a role in disease.
Methods:
Sporulation: 20 strains of C. difficile from the National Anaerobic Reference Unit, Cardiff, including
hyper-virulent ribotypes 001, 027 and 106, were compared for sporulation efficiency. Spores were
grown using methods described by Perez et al., 2005. Spore yield was determined by assessing
germination via Miles-Misra method on Brain-Heart Infusion agar. BHI agar was then
supplemented with 1% Sodium taurocholate to further deduce if there were differences in
sporulation between strains. Spore yield was determined on Columbia agar, with and without 1%
Sodium taurocholate, and germination assessed.
Hydrophobicity: Spores persist on surfaces for months, which is thought to be linked to their
surface charge. The relative hydrophobicity of 20 C. difficile strains was ascertained using the
microbial adhesion to hydrocarbon test, using Hexadecane as the organic solvent.
Transmission Electron Microscopy: Strains with the highest and lowest relative hydrophobicity
were further examined using TEM at the Electron Microscopy Unit, Cardiff University School of
Biosciences.
Results & Key Findings:
Germination & Sporulation: There was variation in germination between strains on BHI and
Columbia agar. Addition of 1% Sodium taurocholate to both media resulted in consistent yield with
little variation. Thus there is no difference in ability of Clostridium difficile strains to produce spores.
However there is a difference in germination ability according to media. Sodium taurocholate
appears to stabilise germination conditions for C. difficile spores.
Hydrophobicity: Downward trend in percentage hydrophobicity according to ribotype exhibited.
Strains with hyper-virulent ribotypes seem to be more hydrophobic than other strains.
TEM: TEM revealed significant physiological differences between highly hydrophobic DS1813
strain and least hydrophobic DS1748 strain. Some hydrophobic DS1813 spores exhibit what may
be an exosporium. This suggests that Clostridium difficile spores vary physiologically across
strains.
Conclusion:
This research is designed to underpin efforts to combat and eradicate C. difficile infection in
hospital environments.
26
026 MRSA in Companion Animals
Tracey Jolliffe
Ninewells Hospital, Dundee
Bacteria belonging to the genus Staphylococcus are catalase positive, Gram-positive cocci, which
form part of the normal flora of humans and animals, and in general have an innocuous association
with the host. They are, however, capable of causing opportunistic infection in the host species.
Staphylococcus aureus is the predominant coagulase positive Staphylococci found in humans. In
Methicillin* Resistant Staphylococcus aureus (MRSA), the presence of the mecA gene allows the
bacteria to produce the normal penicillin binding protein as well as an alternative penicillin binding
protein (PBP2a). Unlike the normal penicillin binding protein, PBP2a is not inhibited by antibiotics
such as the β-lactams, so can bypass the effect of these antibiotics. It is known that animals can be
infected and colonised by MRSA, but prevalence studies tend to focus on clinical infections and
animals presented at veterinary surgeries. In this study, I sampled 262 dogs and cats, both healthy
and unwell, to attempt to discover the prevalence of Staphylococcus aureus and MRSA carriage.
Questionnaires where given to the owners to establish possible risk factors such as recent
antibiotic use, recent in-patient stays at the veterinary surgery, owners who worked in the
healthcare professions, and households that had an MRSA positive person as a member. Nasal
swabs were taken and processed using an enrichment broth, and both chromogenic MRSA media
and Staphylococcal selective media. Identification used phenotypical and biochemical methods,
and MRSA positive isolates were sent to the Scottish MRSA reference laboratory for confirmation
and further tests. In total, twenty Staphylococcus aureus isolates were found, two of which were
MRSA. The two MRSA isolates, from one dog and one cat, had only one risk factor in common: the
owners of both animals were currently, or had been, MRSA positive.
27
027 The Effect of Health-Care Associated Infection Surveillance and the Introduction of Care
Bundles in a Regional Intensive Care Unit in Northern Ireland.
Hilda Crookshanks1, Colin Lavelle2, Gerard McIlvenny3, Irene Thompson2, Joanna McCormick2,
Gavin Lavery2, Anthony Stevens2, Edward T. M. Smyth1,2
1Northern
2Belfast
Ireland Healthcare-associated Infection Surveillance Centre, Belfast
HSC Trust, Belfast
Background:
Our Trust participated in the Safer Patient Initiative (SPI) project coordinated by the Institute of
Healthcare Improvement (IHI, USA) and the Health Foundation (UK).
Objective:
To reduce ventilator associated pneumonia (VAP) and central line associated bloodstream
infection (CLABSI) rates in a 17-bedded, level 3 Regional Intensive Care Unit (RICU) by the
introduction of care bundles.
Methods:
In March 2007 care bundles recommended by the IHI were applied to patients requiring
mechanical ventilation and central line insertions. No care bundle was used for urinary catheters.
Ventilator care and central line insertion bundle compliance was recorded daily. To ascertain the
effect of the bundles on patient care, a dedicated surveillance nurse was employed to conduct
active, prospective surveillance of device-associated infections (including catheter associated
urinary tract infection [CAUTI]). Device associated infections were recorded using Centers for
Disease Control (CDC) definitions of infection. Denominator data was collected daily to record
device utilisation rates and the surveillance nurse actively participated in the microbiology round.
Data was recorded on a scannable form and data fed back monthly as per the SPI protocol.
Results:
When surveillance was implemented in the RICU in February 2007, care bundle compliance was
72% and the VAP rate was 8.99 per 1000 ventilator days. The CLABSI rate was 10.75 per 1000
catheter days. Bundle compliance has shown an upward trend reaching 95% compliance in
October 2007 and has remained consistent. In October 2008, the VAP rate was been 0.00 per
1000 ventilator days and the CLABSI rate was 6.5 per 1000 patient days. CAUTI rates were 4.17
per 1000 catheter days in March 2007 and have demonstrated a gradual upward trend to 10.02 per
1000 days by October 2008.
Ventilator-Figure 1 - Ventilator-associated pneumonia rate
100
90
14.00
RICU
12.00
NHSN
10.00
Bundle Compliance
80
70
60
8.00
50
6.00
40
4.00
30
2.00
20
0.00
Percentage compliance
VAP rate /1000 ventilator days
16.00
10
0
M
ar
0
Ap 7
r0
M 7
ay
0
Ju 7
n
07
Ju
l0
Au 7
g
0
Se 7
p
0
O 7
ct
0
N 7
ov
0
D 7
ec
0
Ja 7
n
0
Fe 8
b
0
M 8
ar
0
Ap 8
r0
M 8
ay
0
Ju 8
n
08
Ju
l0
Au 8
g
0
Se 8
p
0
O 8
ct
08
-2.00
Conclusions:
Where care bundles were implemented in the management of the prevention of VAPs and
CLABSIs, a steady improvement was recorded in the infection rate particularly in the occurrence of
VAPs (Figure 1). This improvement was associated with increasing care bundle compliance. No
improvement was observed in CAUTI rates where a care bundle had not been employed. A CAUTI
care bundle devised by the Department of Health (England) Saving Lives Campaign 2007 is
currently being reviewed for implementation in the unit. The care bundle concept and surveillance
system is currently being developed to enable regional participation.
28
028 Prevalence Survey of Healthcare Associated Infections (HCAI) In Argentina
Ricardo Durlach1, Gerard McIlvenny2, Geraldine Reid3, Lorraine Doherty4, Edward TM Smyth2,3
1Instituto
Técnico de Acreditación de Establecimientos de Salud, Buenos Aires, Argentina
HSC Trust, Belfast
3Northern Ireland Healthcare-associated Infection Surveillance Centre, Belfast
4Department of Health, Social Services and Public Safety, Northern Ireland
2Belfast
Background:
The use of scannable forms has now been used in many countries with minimal cost implications.
In collaboration with the Northern Ireland Healthcare Associated Infection Surveillance Centre an
healthcare-associated infection (HCAI) prevalence survey was carried out in hospitals in Argentina.
Objectives:
Determine prevalence rates of HCAIs and provide data, compatible for international comparisons,
which will assist in the prioritization of measures to reduce the occurance of HCAIs.
Methods:
The NHSN definitions of infections were used. Participation in the survey was organized by the
Instituto Técnico de Acreditación de Establecimientos de Salud. Between March and and June
2008, infection prevention & control (IP&C) staff with assistance from clinical colleagues across
Argentina, completed the survey. Data was collected for all types of HCAI and included specific
questions regarding MRSA. The dataset was identical to the recent Hospital Infection Society’s
HCAI prevalence survey of England, Wales, Northern Ireland and the Republic of Ireland.
Standardised methods and training were used and the CDC definitions of infection employed.
Questionnaires were returned to HISC in Northern Ireland for processing utilizing optical mark
reader technology (OMR). Feedback of data to individual hospitals will be by means of individual
reports.
Results:
The prevalence survey included 4 249 patients from 43 hospitals. There were 480 patients with one
or more healthcare associated infections giving a prevalence of 11.30% (95%CI 10.38 to 12.28).
Results for types of HCAI are given in the table below:
Table: Types and prevalence of healthcare-associated infection in Argentinian hospitals
HCAI
Primary bloodstream
infection
Pneumonia
Surgical site infection
Urinary tract infection
Skin and soft tissue
infection
MRSA infections
Number of
patients
Number of
infections
Prevalence
95%
Confidence
Interval
4 249
62
1.46
1.14 – 1.87
4 249
1 207
(Surgical
patients only)
4 249
141
3.32
2.82 – 3.90
123
10.19
8.61 – 12.03
133
2.76
2.22 – 3.42
4 249
51
1.20
0.91 – 1.57
4 249
48
1.13
0.85 – 1.49
Conclusions:
The initial results of the survey are encouraging. The methodology, definitions and use of the
scannable surveillance questionnaires proved highly acceptable and economical to those involved.
This survey has established a baseline for HCAI prevalence in Argentina.
29
029 Two Into One Won’t Go? Reporting Surgical Site Infections in Hip Arthroplasty and
Hemiarthroplasty of the Hip as Opposed to Hip Prosthesis
Geraldine Reid1, Hilda Crookshanks1, Gavan McAlinden2, Chris Andrews3, Gerard McIlvenny1,
Lorraine Doherty4, Edward T. M. Smyth1,5
1Northern
Ireland Healthcare-associated Infection Surveillance Centre, Belfast
Hospital Dundonald, Belfast & Musgrave Park Hospital, Belfast
3The Royal Hospitals, Belfast, & Musgrave Park Hospital, Belfast
4Department of Health Social Services & Public Safety, Northern Ireland
5Belfast HSC Trust, Belfast
2Ulster
Background/Objective:
In Northern Ireland SSI surveillance is performed on all orthopaedic procedures. We currently
report aggregate and country specific rates for arthroplasty and hemiarthroplasty of the hip as
separate categories. The National Healthcare Safety Network (NHSN) Atlanta, Georgia report rates
of SSI for the operative procedure hip prosthesis which includes both arthroplasty and
hemiarthroplasty of the hip. The objective was to compare the validity of reporting results of
arthroplasty and hemiarthroplasty against hip prosthesis.
Methods:
We compared the published rates for Northern Ireland with the NHSN published rates.
Comparisons were also made with the proportions of patients that fall into the different risk
categories
Results:
The SSI rates for arthroplasty of hip and hemiarthroplasty of hip in Northern Ireland (2003 – 2007)
are compared with the NHSN SSI rates (2007 – 2008) [Table]. The SSI rate for hemiarthroplasty of
hip with RI 0 of 2.05 was significantly higher than the rate of 0.48 for arthroplasty hip RI0 [OR 4.37,
p<0.001] and similarly, the SSI rate for hemiarthroplasty of hip with RI1 of 3.32 was significantly
higher than the rate of 1.47 for arthroplasty hip RI1 [OR 2.38, p<0.001]. Patients undergoing
arthroplasty of hip were predominately in risk index category 0 (74%) and the majority of
hemiarthroplasty of hip were in risk index category 1 (74%). However, patients in both operative
categories covered the range of risk index categories, i.e. 0 – 3.
Operative procedure category
Reporting
Number
Risk index
SSI rate
Northern Ireland
5676
0
0.48
Arthroplasty of hip
1764
1
1.47
Arthroplasty of hip
245
2,3
1.63
635
0
2.05
Hip hemiarthroplasty
1900
1
3.32
Hip hemiarthroplasty
26
2,3
-
17521
0
0.75
Hip prosthesis
22681
1
1.68
Hip prosthesis
5492
2,3
2.79
Arthroplasty of hip
Hemiarthroplasty of hip
Hip prosthesis
Northern Ireland
NHSN
Conclusions:
The NHSN operative procedure categories are groups of clinically similar operative procedures.
The purpose of the categories is to make it possible to compare SSI rates in groups of patients that
have similar procedures. Until 2004 NNIS reported the operative procedure category ’vascular
surgery’. In 2008 the NHSN subdivided the vascular surgery operative procedure category into
three separate operative procedure categories, i.e. abdominal aortic aneurysm repair, carotid
endartectomy and peripheral vascular bypass surgery. It may be appropriate to adopt this
approach for hip prosthesis and report the rates for arthroplasty and hemiarthroplasty separately,
due to the fact that the SSI rates for hemiarthroplasty of hip are significantly higher than the SSI
rates for arthroplasty of hip.
30