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Is panton-valentine leukocidin (pvl) associated with the pathogenesis of Staphylococcus aureus bacteraemia in the UK?
Abstracts
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turned out to be S. aureus. While with the score of 2 or less, only 3/56 (5%) were found to be S. aureus. On the basis of scoring system, 26/82 (30%) of the blood cultures were telephoned. The sensitivity and specificity of the model to predict S. aureus in blood culture was 81% and 80%, respectively. Conclusion: The scoring system performed well to predict S. aureus in blood culture. It can potentially reduce clinical microbiologist’s workload and influence laboratory practice. Further evaluation of the scoring system is underway.
P 039
P 038
Background: Antibiotic prophylaxis is known to reduce surgical site infections and post-operative complications. For example, ß-lactam antibiotics are administered to patients who require emergency operations in the cardio-thoracic ward at Southampton General Hospital. MRSA infection is a particular risk in the case of operations in which prosthetic material is implanted. Therefore it is important to be able to identify patients colonised with MRSA rapidly, so that appropriate prophylaxis can be administered prior to the operation. Objectives and methods: A rapid molecular assay for direct MRSA detection delivering same-day results was therefore developed for application to high risk healthcare settings which require urgent answers as to MRSA status. The assay was designed so as to specifically detect the right-hand integration site of the staphylococcal chromosome cassette (SCCmec). The entire assay was based upon semi-automated DNA extraction direct from patient swabs followed by real-time PCR, utilising lysostaphin pre-treatment of the swab extract, the MagNA Pure LC (Roche) nucleic acids elution robot and the LightCycler 2.0 (Roche) real-time PCR machine. Results: DNA extraction and PCR protocols were optimised: Analytical sensitivity of the PCR reaction was 1 x 103 to 1 x 104 ng genomic DNA per reaction. Sensitivity of the complete process was 1 x 102 to 1 x 103 cfu per swab. Screening of isolates and clinical swabs enabled a comprehensive evaluation of the assay against an existing multiplex (mecA, femB) assay and culture-based bacteriology, with 93% negative predictive value and 90% sensitivity compared to bacteriological analyses. Sample-to-result turnaround for clinical swabs was four hours, facilitating the potential for the desired rapid service. Conclusions: The protocol may be used as a rapid service for urgent confirmation of MRSA-negative patients so as to inform clinical decisions regarding antimicrobial prophylaxis in high-risk patients.
IS PANTON-VALENTINE LEUKOCIDIN (PVL) ASSOCIATED WITH THE PATHOGENESIS OF STAPHYLOCOCCUS AUREUS BACTERAEMIA IN THE UK? Ellington Matthew J, Hope Russell, Ganner Mark, Ganner Marjorie, East Claire, Kearns Angela M Laboratory of Health Care Associated Infections, CfI, Health Protection Agency, 61 Colindale Avenue, London Background: PVL-positive MRSA combine resistance and enhanced virulence traits posing a public-health threat. Whilst such community associated strains are most often associated with skin and soft-tissue infections and necrotising pneumonia, concern has been expressed regarding the prevalence of PVL among isolates of S. aureus from life threatening disease, particularly bacteraemia. To investigate their contribution to the disease burden, we determined the prevalence of PVL amongst S. aureus bacteraemia isolates. Methods: Consecutive S. aureus isolated from bacteraemic patients (n ¼ 244), from 25 centres throughout the UK and Ireland, were screened for PVL and mecA by PCR. PVL-positive isolates were characterised by toxin gene profiling, PFGE, and spa sequence typing. MICs of a wide range antimicrobials of different classes were determined. Results: Four/244 isolates (1.6 %) were PVL-positive MSSA, no PVL-positive MRSA were detected. The four bacteraemic patients with PVL-positive MSSA (2 males; 25 and 30 years, 2 females; 62 and 80 years) had infection foci of: skin and soft tissue, gastrointenstinal, indwelling line, and surgical site, and were located at two centres in England and two in Ireland. All four isolates were susceptible to all antibiotics tested, excepting one English isolate found resistant to penicillin and fucidin. This latter isolate was distinct from the other English isolate by PFGE, agr and spa typing (spa types t127 and t383) and toxin gene compliment but was the same spa and PFGE type as previously identified among MLST ST1-like isolates from the UK. The two Irish isolates were spa type t021 and were indistinguishable from each other, suggesting a degree of clonal spread. Conclusion: Against a background of increased reports of PVL-positive MRSA we found no PVL-positive MRSA, and four PVL-positive MSSA amongst 244 S. aureus bacteraemia isolates, suggesting PVL to be of limited relevance for bloodstream infections.
DEVELOPMENT AND EVALUATION OF A RAPID DIRECT DNA EXTRACTION AND REAL-TIME PCR ASSAY FOR EARLY DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN HIGH-RISK PATIENTS Elston Lucy, Yam TatShing, Marsh Peter Health Protection Agency Southampton, Southampton General Hospital, Tremona Road, Southampton SO16 6YD
P 040 EVALUATION OF A NEW POLYMERASE CHAIN REACTION (PCR) ASSAY FOR THE DIRECT DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) FROM CLINICAL SAMPLES Meader Emma 1, Richards Judith 1, Sillis Margaret 1, Bull Victoria 2, Alcock Fiona 2
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turned out to be S. aureus. While with the score of 2 or less, only 3/56 (5%) were found to be S. aureus. On the basis of scoring system, 26/82 (30%) of the blood cultures were telephoned. The sensitivity and specificity of the model to predict S. aureus in blood culture was 81% and 80%, respectively. Conclusion: The scoring system performed well to predict S. aureus in blood culture. It can potentially reduce clinical microbiologist’s workload and influence laboratory practice. Further evaluation of the scoring system is underway.
P 039
P 038
Background: Antibiotic prophylaxis is known to reduce surgical site infections and post-operative complications. For example, ß-lactam antibiotics are administered to patients who require emergency operations in the cardio-thoracic ward at Southampton General Hospital. MRSA infection is a particular risk in the case of operations in which prosthetic material is implanted. Therefore it is important to be able to identify patients colonised with MRSA rapidly, so that appropriate prophylaxis can be administered prior to the operation. Objectives and methods: A rapid molecular assay for direct MRSA detection delivering same-day results was therefore developed for application to high risk healthcare settings which require urgent answers as to MRSA status. The assay was designed so as to specifically detect the right-hand integration site of the staphylococcal chromosome cassette (SCCmec). The entire assay was based upon semi-automated DNA extraction direct from patient swabs followed by real-time PCR, utilising lysostaphin pre-treatment of the swab extract, the MagNA Pure LC (Roche) nucleic acids elution robot and the LightCycler 2.0 (Roche) real-time PCR machine. Results: DNA extraction and PCR protocols were optimised: Analytical sensitivity of the PCR reaction was 1 x 103 to 1 x 104 ng genomic DNA per reaction. Sensitivity of the complete process was 1 x 102 to 1 x 103 cfu per swab. Screening of isolates and clinical swabs enabled a comprehensive evaluation of the assay against an existing multiplex (mecA, femB) assay and culture-based bacteriology, with 93% negative predictive value and 90% sensitivity compared to bacteriological analyses. Sample-to-result turnaround for clinical swabs was four hours, facilitating the potential for the desired rapid service. Conclusions: The protocol may be used as a rapid service for urgent confirmation of MRSA-negative patients so as to inform clinical decisions regarding antimicrobial prophylaxis in high-risk patients.
IS PANTON-VALENTINE LEUKOCIDIN (PVL) ASSOCIATED WITH THE PATHOGENESIS OF STAPHYLOCOCCUS AUREUS BACTERAEMIA IN THE UK? Ellington Matthew J, Hope Russell, Ganner Mark, Ganner Marjorie, East Claire, Kearns Angela M Laboratory of Health Care Associated Infections, CfI, Health Protection Agency, 61 Colindale Avenue, London Background: PVL-positive MRSA combine resistance and enhanced virulence traits posing a public-health threat. Whilst such community associated strains are most often associated with skin and soft-tissue infections and necrotising pneumonia, concern has been expressed regarding the prevalence of PVL among isolates of S. aureus from life threatening disease, particularly bacteraemia. To investigate their contribution to the disease burden, we determined the prevalence of PVL amongst S. aureus bacteraemia isolates. Methods: Consecutive S. aureus isolated from bacteraemic patients (n ¼ 244), from 25 centres throughout the UK and Ireland, were screened for PVL and mecA by PCR. PVL-positive isolates were characterised by toxin gene profiling, PFGE, and spa sequence typing. MICs of a wide range antimicrobials of different classes were determined. Results: Four/244 isolates (1.6 %) were PVL-positive MSSA, no PVL-positive MRSA were detected. The four bacteraemic patients with PVL-positive MSSA (2 males; 25 and 30 years, 2 females; 62 and 80 years) had infection foci of: skin and soft tissue, gastrointenstinal, indwelling line, and surgical site, and were located at two centres in England and two in Ireland. All four isolates were susceptible to all antibiotics tested, excepting one English isolate found resistant to penicillin and fucidin. This latter isolate was distinct from the other English isolate by PFGE, agr and spa typing (spa types t127 and t383) and toxin gene compliment but was the same spa and PFGE type as previously identified among MLST ST1-like isolates from the UK. The two Irish isolates were spa type t021 and were indistinguishable from each other, suggesting a degree of clonal spread. Conclusion: Against a background of increased reports of PVL-positive MRSA we found no PVL-positive MRSA, and four PVL-positive MSSA amongst 244 S. aureus bacteraemia isolates, suggesting PVL to be of limited relevance for bloodstream infections.
DEVELOPMENT AND EVALUATION OF A RAPID DIRECT DNA EXTRACTION AND REAL-TIME PCR ASSAY FOR EARLY DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) IN HIGH-RISK PATIENTS Elston Lucy, Yam TatShing, Marsh Peter Health Protection Agency Southampton, Southampton General Hospital, Tremona Road, Southampton SO16 6YD
P 040 EVALUATION OF A NEW POLYMERASE CHAIN REACTION (PCR) ASSAY FOR THE DIRECT DETECTION OF METHICILLIN RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) FROM CLINICAL SAMPLES Meader Emma 1, Richards Judith 1, Sillis Margaret 1, Bull Victoria 2, Alcock Fiona 2