Abstracts, 7th International Conference of the Hospital Infection Society, 10–13 October 2010, Liverpool, UK / Journal of Hospital Infection 76S1 (2010) S1–S90 S63

P21.04

Comparison of the analytical sensitivity of three commercial

real time PCR assays for the detection of MRSA

C. Pope, K. Capaldi. St Georges Hospital, United Kingdom

Background: Real time PCR is widely used to detect nasal carriage

of MRSA in hospitalised patients. However perforamnce of these

assays varies.

Aims/Objectives: The aims were to compare the sensitivity and

specificity of three commercial real time PCR MRSA assays from

diluted culture. The assays compared were Molecular Detection Inc

(MDI) Detect Ready MRSA, Becton Dickinson GeneOhm MRSA ACP

and Roche LightCycler MRSA Advanced Test.

Methods: Both the Detect Ready MRSA kit and the GeneOhm

MRSA ACP kit were run on the Cepheid SmartCycler platform

while the LightCycler MRSA Advanced test was run on the Roche

LightCycler 2.0. A colony from an overnight culture plate was

diluted to approximately the limit of detection. This equated to

adding approximately 102–103 CFU per reaction. Fifty strains of

staphylococci (15 MSSA, 11 CNS and 24 MRSA) with previously

confirmed identification and susceptibility profiles were cultured

and for each one colony was serially diluted in TE buffer. 10ml ofthis dilution was added to real time PCR reaction tubes for each

assay.

Results: The sensitivity of the LightCycler MRSA Advanced Test,

Detect Ready MRSA and GeneOhm MRSA ACP were 66.7%, 96% and

96.3% respectively. Therefore the Roche assay demonstrated the

lowest sensitivity. The specificity of the LightCycler MRSA Advanced

Test, Detect Ready MRSA and GeneOhm MRSA ACP were 100%, 100%

and 96.3% respectively. The BD assay detected one resistant CNS as

MRSA.

Conclusions: We have shown that the GeneOhm MRSA ACP assay

and the Detect Ready MRSA kit have a higher analytically sensitivity

than the LightCycler MRSA Advanced Test.

P21.05

Two step algorithm for the detection of Clostridium difficile

from stool samples

N. Clark1, M. Doughton1, A. Karas2, D. Enoch1. 1Peterborough &

Stamford Hospitals NHS Foundation Trust, United Kingdom; 2Health

Protection Agency, United Kingdom

Background: C. difficile is the commonest cause of hospital acquired

infectious diarrhoea in the developed world. Rapid and accurate

diagnosis is essential.

Aim: We sought to determine the optimal testing strategy in terms

of accuracy and cost effectiveness.

Methods: We prospectively analysed 322 diarrhoeal samples

from consecutive patients by enzyme immunoassay (EIA; VIDAS),

glutamate dehydrogenase (GDH) and cell cytotoxicity (CTN; HPA).

We retrospectively used PCR (GeneXpert) on samples with a

positive result by any method. CTN was used as the gold

standard. Cost analysis was performed using manufacturer’s quotes

(EIA = £5.50; GDH=£4; PCR=£25.35 per test). Testing 20 samples

per day took 60 min (EIA), 80 min (GDH) and 40 min (PCR) by a

band 4 (£10 per hour).

Results: 11 of 322 samples (3.4%) were positive by CTN. EIA detected

12, 304 were negative and 6 equivocal. The sensitivity (sens),

specificity (spec), positive predictive value (PPV) and negative

predictive value (NPV) were 70%, 98%, 59% & 98%. 44 were positive

by GDH (sens 100%, spec 89%, PPV 25% & NPV 100%). PCR was

performed on 106 samples. 17 were positive; 3 were invalid. PCR

had a sens, spec, PPV & NPV of 100%, 90%, 41% and 100%. Using a 2

step algorithm, the sens, spec, PPV and NPV were 70%, 94%, 78% and

91% for GDH/EIA and 100%, 77%, 47% and 100% for GDH/PCR. EIA

was the cheapest option (£1932 for 322 tests) followed by GDH/PCR

(£2631) and PCR alone (£8269).

Discussion: Using the 2 step GDH/PCR algorithm would have

detected 15 cases, all of which had clinical evidence of infection.

3 of these additional cases were positive by EIA on subsequent

samples. The low prevalence of C. difficile in this study affects the

PPV and NPV of all tests in this study. In terms of cost analysis,

EIA remains the cheapest with PCR being the most expensive. The

GDH/PCR algorithm is fast, accurate and cost effective.

P21.06

A United Kingdom audit of the laboratory diagnosis of

Clostridium difficile infection

R. Cooke1, A. Galloway2, A. Collins2, D. Holland2, G. Trigg2.1University Hospital Aintree, United Kingdom; 2Freeman Hospital,

United Kingdom

Background: A consistent UK approach to the laboratory diagnosis

of Clostridium difficile infection (CDI) is critically important

especially as surveillance is now linked ro national and local targets

on CDI reduction. However there have been previous concerns

about the ability of laboratories to comply with national standards

on CDI diagnosis. A national survey in England found that 19%

of microbiology laboratories reported toxin-positive results from

non-diarrhoeal samples.

Aims/Objectives: To undertake a UK wide audit of the laboratory

diagnosis of CDI. Such an audit should help to improve the quality

of data fed into the national CDI surveillance programme.

Methods: The audit was undertaken on behalf of the Association

of Clinical Pathologists (ACP) and the Royal College of Pathologist

(RCP), in collaboration with the National Pathology Benchmarking

Service of Keele University. A tick-box format questionnaire was

posted to all consultant Microbiologists listed on the ACP and RCP

databases as working in the UK. Audits standards were based upon

published recommendations from the Health Protection Agency

and the Department of Health.

Results: A total of 200 questionnaires were sent out of which 80

were returned and suitable for analysis. There was considerable

variation in laboratory practices. In particular, only 56% of

laboratories gave guidance on follow up testing of toxin negative

samples, only 68% provided a 7 day week diagnostic service and

only 66% would routinely communicate positive in-patient results

to ward nursing or medical staff.

Conclusions: To ensure a consistent approach to the diagnosis

of CDI, UK microbiology laboratories require more detailed and

prescriptive guidance than is currently available.

P21.07

Evaluation of the application of the Scottish Clostridium difficile

infection (CDI) testing protocol in a diagnostic laboratory

J. Coia1, A. Leanord2, W. Cowan2, A. Badriya2, G. McLean2,

D. Nelson3, C. Wiuff4, B. Adawi2. 1Scottish Salmonella, Shigella &

C. difficile Reference Laboratory, United Kingdom; 2Southern General

Hospital, United Kingdom; 3Glasgow Royal Infirmary, United Kingdom;4Health Protection Scotland, United Kingdom

Background: The gold standard for Clostridium difficile infection

(CDI) diagnosis is cell-culture cytotoxicity. This is not widely

available, and has been replaced by immunoassay (EIA) tests. These

miss true positives and generate false-positive results. Scottish

CDI diagnostic guidance recommends confirmatory testing using

a combination of tests in a diagnostic algorithm.

Aim: This study had three aims. Firstly, it assessed the use an

EIA test (Mini VIDAS) and combined toxin A/B and Glutamate

Dehydrogenase (GDH) antigen detection (QuikChek Complete) as

part of a diagnostic algorithm. Secondly, it assessed the potential

for resolving discrepant results arising from the initial tests by

centralised cell-culture cytotoxicity assay at a second laboratory in

the same Health Board area. Thirdly, it assessed on-site toxigenic

culture to resolve discrepant results.