Abstracts of Posters Presented at 9th Tripartite Meeting of the Celtic Microbiology Associations
(Hosted by the Welsh Microbiological Association)
Cardiff, 5-7th June 2009
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.
002 Dissociated Clindamycin Resistance in Staphylococci.
Katherine McBride1, Elaine McCulloch2, Leigh Williams1, Elizabeth Kilgour1, Craig Williams2, Alistair Leanord1
1Microbiology Department, Monklands Hospital, Airdrie
2Microbiology 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 MLSB 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
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
2UK 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
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 non-hominis/parvum species may be relatively more likely to result in asymptomatic carriage than in clinical disease, which may reflect lower pathogenicity
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
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.
006 Contamination of Laryngoscope Handles
David Williams1, Nidhika Berry2, John Dingley1, Ceri Jones2
1Deptartment of Anaesthetics, Abertawe Bro Morganwwg NHS Trust, Swansea
2NPHS 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:
- Hook mount (smooth)
- Upper third (knurled)
- 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.
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
TC/ DIFHSV-1 +ve HSV-1 –ve HSV-2 +ve HSV-2 -ve
PCR +ve / 36 7 49 11
PCR-ve / 0 184 0 167
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 HSV-1, 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.
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.