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P896 Comparison of NucliSens easyMAG and Qiagen nucleic acid extraction using screening broths for the detection of MRSA
S232 P894 Characterisation of insertion sequence, conjugative transferase gene and mupA gene of mupR plasmid from MuH methicillin-resistant staphylococci J.I. Yoo, G.T. Chung, E.S. Shin, Y.S. Lee (Seoul, KR) Objectives: Mupirocin has been used to eliminate the carriage of S. aureus and prevent staphylococcal infections. However, mupirocinresistant S. aureus has been increased and the high level mupirocin resistance (MuH) is associated with an isoleucyl tRNA synthetase that is encoded by a novel plasmid-encoded gene, mupA. In this study, we investigated transferable mupirocin resistance element containing insertion sequence (IS), trs and mupA gene of mupR plasmid from MuH methicillin resistant staphylococci isolates. Methods: Against 680 staphylococci isolates from tertiary hospitals, antimicrobial susceptibility test including mupirocin was done by disk diffusion and MIC of mupirocin was determined by E-test method. The plasmid-encoded mupA gene was detected by PCR. The mupA-IS and trsLM-mupA specific primers were used to identify the presence and genetic location of IS element, trs and mupA gene. Results: 12 MuH-staphylococci isolates with different PFGE patterns were selected from hospitals (680 isolates) and long-term care facilities (407 isolates) in Korea. MuH MRSA (8 isolates), MuH S. haemolyticus (2 isolates), MuH S. hominis (1 isolates) and MuH S. epidermidis (1 isolates) were identified. The trsLM-mupA PCR showed 4 different products such as 7,033 bp (6 isolates), 5,024 bp (3 isolates), 4,530 bp (2 isolates) and 6,105 bp (1 isolates). IS sequence of all 12 isolates identified to have IS257 sequences except 2 nucleotide substitution (A174G, A536G) and one thymine nucleotide deletion at 644 nucleotide. The mupA was flanked by two directly repeated IS257 like sequences. The sequences of trsLM were also different in 68 nucleotide site compared to previously reported trsLM sequences. Conclusion: The high-level mupirocin resistant staphylococci isolated from Korea hospitals had different IS257 sequences and trsLM sequences. Because sequences of all MuH isolates showed the same sequences, the IS257 like sequences and trsLM sequences is typical in Korean isolates. According to the trsLM-mupA sequence analysis, there was no specific difference between MRSA and MRCNS. It indicated the possibility of mupA transfer from MRSA to MRCNS and vice versa. And also the sequence relation of trs, IS and mupA against MRCNS is the first report in the world.
Molecular diagnostics for staphylococci P895 Evaluation of a real-time PCR assay and an multiplex-reverse hybridisation system for the detection of methicillin-resistant Staphylococcus aureus M. Ieven, M. Michiels, H. Jansens, H. Goossens (Edegem, BE) Objective: To evaluate the performance of a commercial multiplex PCR – reverse hybridisation system, Hyplex StaphyloResist® (BAG Germany distributed in the Benelux by AlphaOmega instruments), and a real-time PCR assay for the detection of MRSA from 24 hours enrichment broths compared to the routine culture method. Methods: A total of 500 enrichment broths from MRSA screenings were included in the study. On arrival in the lab, swabs were cultured by direct plating and subsequently put in MRSA enrichment broth. After 24 hours incubation the broth was subcultured on routine media and MRSA was identified by conventional methods. A broth sample was taken for DNA extraction and two PCR assays. The Hyplex StaphyloResist® consists of a multiplex PCR amplifying simultaneously mecA gene, coagulase gene and a “housekeeping” gene. Specimens were processed according to the manufacturer’s instructions. For the real-time PCR assay specimens were extracted using the Qiagen blood mini kit with consecutive detection of mecA, nuc and SCCmec genes, according to a modified protocol by Huletsky et al (JCM 2004; 42: 1875−84). PCR results were considered positive if confirmed by culture or the second PCR.
17th ECCMID / 25th ICC, Posters Results: In total 78 (16%) of the 500 MRSA enrichment broths tested were culture and both PCRs positive, 6 were culture negative and both PCRs positive, 1 was culture positive and both PCRs negative. 6 broths were culture positive and one PCR negative (5 real-time PCR assay, 1 Hyplex StaphyloResist® assay) and 28 broths were culture negative and one PCR negative (14 real-time PCR assay, 14 Hyplex StaphyloResist® assay). The sensitivity, specificity and negative and positive preditive values of the Hyplex StaphyloResist® assay for the detection of MRSA from broth were 98%, 96%, 99% and 83% and 93%, 96%, 98% and 83% for the real-time PCR assay, respectively. Both PCR assays provided same day results on 24 hours enrichment broths compared to 48 to 96 hours for routine culture method. The time needed to complete the Hyplex StaphyloResist® and real-time PCR assays were 3 hrs 25 min and 2 hrs 30 min with a hands-on time of 1 hrs 30 min and 1 hrs 15 min, respectively. Conclusion: There is no significant difference in sensitivity and specificity between the Hyplex StaphyloResist® assay and the real-time assay for the detection of MRSA from enrichment broth. More than 90% of MRSA positives can be identified after one day by either PCR if performed on MRSA enrichment broth. P896 Comparison of NucliSens easyMAG and Qiagen nucleic acid extraction using screening broths for the detection of MRSA M. Ieven, M. Michiels, K. Bergs, D. Ursi, H. Goossens (Edegem, BE) Background: The NucliSens easyMAG platform (bioM´erieux) is a second generation system for automated isolation of nucleic acids (NA) from clinical samples, based upon silica extraction technology. Currently, no data are available on the NucliSens easyMAG for the extraction of MRSA DNA from screening samples. Objectives: To evaluate the performance and user convenience of the NucliSens easyMAG platform for NA extraction from MRSA enrichment broths compared to manual Qiagen extraction. Materials and Methods: 500 enrichment broths from MRSA screenings were included in the study: on arrival in the lab, swabs were cultured by direct plating and enrichment cultures. 200 ml broth was used for NA extraction using the Qiagen blood mini kit and 1 ml broth was frozen. NAs extracts were analysed by real-time PCR targeting the mecA, nuc and SCCmec genes for the detection of MRSA DNA. 60 broths proven positive by culture and/or PCR were retrospectively extracted using the NucliSens easyMAG protocol on 200 ml of the frozen aliquots. An equal number of negative samples were analysed by the same extraction and PCR protocol. Results: The real-time PCR detected MRSA DNA in 60 enrichment broths as well after Qiagen as after easyMAG extraction. In 49, 48 and 48 of the extracted MRSA positive broths Icycler Ct values for detection of SCCmec, mecA and nuc genes respectively were lower after NucliSens easyMAG extraction compared to the values after Qiagen extraction with a mean Ct difference of 3.02, 1.79 and 2.04. If the Ct values after Qiagen extraction were lower than after NucliSens easyMAG extraction, which was the case in less than 10 of the extracted broths, the mean Ct difference was minimal: 1.2, 1.3 and 1.2 for the detection of SCCmec, mecA and nuc genes, respectively. For all 60 negative broths, both NucliSens easyMAG and Qiagen produced clear cut negative results. The time needed for the Nuclisens easyMAG extraction procedure was 40 minutes for 24 samples, with a hands-on time of less than 20 min, compared to 85 minutes for Qiagen extraction. Conclusion: In this study the Nuclisens easyMAG extracted more efficiently the MRSA DNA of the enrichment broths by showing on average lower and higher Ct values for respectively MRSA positive and negative samples in the real-time PCR assays. The instrument features user-friendly, intuitive software, and delivers high throughput capabilities with 40 minutes turn-around-times.
Molecular diagnostics for staphylococci P895 Evaluation of a real-time PCR assay and an multiplex-reverse hybridisation system for the detection of methicillin-resistant Staphylococcus aureus M. Ieven, M. Michiels, H. Jansens, H. Goossens (Edegem, BE) Objective: To evaluate the performance of a commercial multiplex PCR – reverse hybridisation system, Hyplex StaphyloResist® (BAG Germany distributed in the Benelux by AlphaOmega instruments), and a real-time PCR assay for the detection of MRSA from 24 hours enrichment broths compared to the routine culture method. Methods: A total of 500 enrichment broths from MRSA screenings were included in the study. On arrival in the lab, swabs were cultured by direct plating and subsequently put in MRSA enrichment broth. After 24 hours incubation the broth was subcultured on routine media and MRSA was identified by conventional methods. A broth sample was taken for DNA extraction and two PCR assays. The Hyplex StaphyloResist® consists of a multiplex PCR amplifying simultaneously mecA gene, coagulase gene and a “housekeeping” gene. Specimens were processed according to the manufacturer’s instructions. For the real-time PCR assay specimens were extracted using the Qiagen blood mini kit with consecutive detection of mecA, nuc and SCCmec genes, according to a modified protocol by Huletsky et al (JCM 2004; 42: 1875−84). PCR results were considered positive if confirmed by culture or the second PCR.
17th ECCMID / 25th ICC, Posters Results: In total 78 (16%) of the 500 MRSA enrichment broths tested were culture and both PCRs positive, 6 were culture negative and both PCRs positive, 1 was culture positive and both PCRs negative. 6 broths were culture positive and one PCR negative (5 real-time PCR assay, 1 Hyplex StaphyloResist® assay) and 28 broths were culture negative and one PCR negative (14 real-time PCR assay, 14 Hyplex StaphyloResist® assay). The sensitivity, specificity and negative and positive preditive values of the Hyplex StaphyloResist® assay for the detection of MRSA from broth were 98%, 96%, 99% and 83% and 93%, 96%, 98% and 83% for the real-time PCR assay, respectively. Both PCR assays provided same day results on 24 hours enrichment broths compared to 48 to 96 hours for routine culture method. The time needed to complete the Hyplex StaphyloResist® and real-time PCR assays were 3 hrs 25 min and 2 hrs 30 min with a hands-on time of 1 hrs 30 min and 1 hrs 15 min, respectively. Conclusion: There is no significant difference in sensitivity and specificity between the Hyplex StaphyloResist® assay and the real-time assay for the detection of MRSA from enrichment broth. More than 90% of MRSA positives can be identified after one day by either PCR if performed on MRSA enrichment broth. P896 Comparison of NucliSens easyMAG and Qiagen nucleic acid extraction using screening broths for the detection of MRSA M. Ieven, M. Michiels, K. Bergs, D. Ursi, H. Goossens (Edegem, BE) Background: The NucliSens easyMAG platform (bioM´erieux) is a second generation system for automated isolation of nucleic acids (NA) from clinical samples, based upon silica extraction technology. Currently, no data are available on the NucliSens easyMAG for the extraction of MRSA DNA from screening samples. Objectives: To evaluate the performance and user convenience of the NucliSens easyMAG platform for NA extraction from MRSA enrichment broths compared to manual Qiagen extraction. Materials and Methods: 500 enrichment broths from MRSA screenings were included in the study: on arrival in the lab, swabs were cultured by direct plating and enrichment cultures. 200 ml broth was used for NA extraction using the Qiagen blood mini kit and 1 ml broth was frozen. NAs extracts were analysed by real-time PCR targeting the mecA, nuc and SCCmec genes for the detection of MRSA DNA. 60 broths proven positive by culture and/or PCR were retrospectively extracted using the NucliSens easyMAG protocol on 200 ml of the frozen aliquots. An equal number of negative samples were analysed by the same extraction and PCR protocol. Results: The real-time PCR detected MRSA DNA in 60 enrichment broths as well after Qiagen as after easyMAG extraction. In 49, 48 and 48 of the extracted MRSA positive broths Icycler Ct values for detection of SCCmec, mecA and nuc genes respectively were lower after NucliSens easyMAG extraction compared to the values after Qiagen extraction with a mean Ct difference of 3.02, 1.79 and 2.04. If the Ct values after Qiagen extraction were lower than after NucliSens easyMAG extraction, which was the case in less than 10 of the extracted broths, the mean Ct difference was minimal: 1.2, 1.3 and 1.2 for the detection of SCCmec, mecA and nuc genes, respectively. For all 60 negative broths, both NucliSens easyMAG and Qiagen produced clear cut negative results. The time needed for the Nuclisens easyMAG extraction procedure was 40 minutes for 24 samples, with a hands-on time of less than 20 min, compared to 85 minutes for Qiagen extraction. Conclusion: In this study the Nuclisens easyMAG extracted more efficiently the MRSA DNA of the enrichment broths by showing on average lower and higher Ct values for respectively MRSA positive and negative samples in the real-time PCR assays. The instrument features user-friendly, intuitive software, and delivers high throughput capabilities with 40 minutes turn-around-times.