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Encephalitis viruses detected by FilmArray® multiplex PCR versus real-time PCR
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
Abstract no: 267 Presentation at ESCV 2016: Poster 36 Comparison of respiratory and Meningitis/Encephalitis viruses detected by FilmArray® multiplex PCR versus real-time PCR Roger Koller ∗ , M.T. Barbani, A.U. Lüthi, S. Zürcher, J.F. Steinlin-Schopfer, S.L. Leib, M. Gorgievski-Hrisoho Institute for Infectious Diseases, University of Bern, Switzerland Introduction: Fast and reliable pathogen detection is important for adequate management of infections. Although real-time PCR (rtPCR) is usually the most sensitive method for direct pathogen detection, it requires experienced technicians, includes several working steps and has a turnaround time of multiple hours. Therefore this method is not ideal for emergency diagnostics. The FDA cleared, fully automated sample to answer, FilmArray® (FA) multiplex PCR system (BioFire/bioMérieux) detects a broad spectrum of pathogens in ∼70 min. To optimize our diagnostic services during weekends and off-peak times, we compared the FA Respiratory Panel (RP) and FA Meningitis/Encephalitis (ME) Panel to our routinely used rtPCR assay. The FA panels detect 20 respiratory pathogens (17 viruses, 3 bacteria) in nasopharyngeal swabs (NPS) and 14 M/E pathogens (7 viruses, 6 bacteria, Cryptococcus neoformans/gattii) in cerebrospinal fluids (CSF). Materials and methods: With FA we tested 84 retrospective samples (23 NPS, 29 broncheoalveolar lavages [BALs], 32 CSF) and 60 prospectively collected NPS that required urgent testing during the 2015/2016 flu season by FA and rtPCR. FA sample input volume was 300 ml for RP and 200 ml for ME. Commercial RP and ME quality control panels (MMQC Inc., Scarborough, USA), containing samples positive and negative for each analyte detected by the FA panels, were tested multiple times. For rtPCR, nucleic acids were extracted from 220 ml of sample and eluted in 55 ml using NucliSENS easyMAG (bioMérieux). Respiratory viruses were analyzed by real-time PCR using a combination of 7 duplex Respiratory Multi Well System r-geneTM (RG) assays (influenza A/B, RSV/hMPV, HRV&EV/cell control, ADV/HBoV, HCoV/HPIV1-4) (Argene/bioMerieux), according to manufacturer’s instructions. Additionally, we expanded FA RP testing to include (BALs), by implementing one additional sample preparation step. CSF was analyzed for virus using laboratory developed tests (LDTs) certified by the Swiss authorities. Results: RP and ME quality control panel results were 100% concordant with expected results. For all NPS, both tests, FA RP and RG, identified one or more viruses in 45/83 (54.2%) samples. FA RP and RG results correlated for 42/48 viruses detected (87.5%). FA RP detected an additional 3 HRV/EV and RG detected additionally 1 FluA, 1 ADV and 1 HRV/EV. Positive percent agreement (PPA) between RG (laboratory standard) and FA RP for NPS was 93.3% and negative percent agreement (NPA) was 92.7%. Overall correlation was 93.2%. Results from BALs yielded 92% PPA, 93.1% NPA and overall correlation of 92.4%. For FA ME testing, 31/33 CSF samples had identical FA ME and LDT results with an overall correlation of 94.4%. FA ME did not detect 2 parechovirus low level LDT positive samples (Ct 36.3 and 37.0). Using LDTs as the laboratory standard, FA ME PPA and NPA were 93.9% and 100%, respectively. Conclusion: Results obtained with the FilmArray® RP and ME panels were highly concordant with our currently used diagnostic methods, demonstrating excellent performance. The simplicity of the FilmArray® system, requiring less than 5 min of hands-on time, easy to read reports, and low sample volume allows for testing during off shifts and when urgent results are required. The compre-
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hensiveness of the FilmArray® panels is ideal for diagnosing clinical syndromes where there are many potential causes. http://dx.doi.org/10.1016/j.jcv.2016.08.076 Abstract no: 273 Presentation at ESCV 2016: Poster 37 Cross-contamination and carry-over study results obtained with ELITe InGenius, a new sample-to-result solution for in vitro diagnostics C. Bittoto ∗ , S. Costa, M. Enrietto, S. Patanè, F. Gorreta, A. Estampes, C. Olivo, G. Stefanuto, N. Scarr, W. Mahoney ELITechGroup Molecular Diagnostics, United Kingdom Background: ELITe InGeniusTM (ELITechGroup Molecular Diagnostics) is a fully automated sample-to-result solution, designed for the in vitro molecular diagnostics and monitoring of infectious diseases. The system combines on a single platform: sample processing (extraction and purification), PCR set-up, real-time amplification and detection of multiple parameters for qualitative and quantitative analysis, and result interpretation. Absence of cross-contamination and cross-over was evaluated within robustness study. Material/methods: ELITe InGenius features a universal extraction in a unitary cassette based format (ELITe InGenius SP200) and multiple and independent Real-Time PCR with mixed parameters including CE-IVD Real-Time PCR assays (ELITe MGB line) and Laboratory Developed Tests. One to twelve patient samples can be processed in 12 parallel tracks within one run. Cross-contamination and cross-over study protocols were designed in accordance with FDA Draft Guidance for Industry and Food and Drug Administration Staff Establishing the Performance Characteristics of Nucleic Acid-Based In vitro Diagnostic Devices. The tests to evaluate the rate of false positive included: (1) 30 high positive MRSA samples (107 CFU/ml, prepared by dilution of MRSA BAA-1720 strain) tested alternating to 30 negative samples within five 12-sample runs with MRSA/SA ELITe MGB kit; (2) negative MRSA samples (n = 50) tested within five runs along with 1 positive and 1 negative MRSA/SA controls per run with MRSA/SA ELITe MGB kit, and (3) 30 high CMV positive samples (104 IU/ml, prepared by dilution of the “1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques”) tested alternating to 30 negative samples within five 12-sample runs with CMV ELITe MGB kit. All samples were tested carrying out the whole analysis procedure: extraction, amplification, detection and result interpretation with ELITe InGeniusTM in combination with ELITechGroup reagents. Results: All negative and all positive MRSA and CMV samples were correctly identified by the system. 100% of concordance with the expected results was obtained for all the samples tested. Conclusions: The results obtained demonstrated the total absence of carry-over and cross-contamination of the system even when high positive samples were tested. They confirm the robustness and the reliability of ELITe InGenius for in vitro Molecular Diagnostics testing. http://dx.doi.org/10.1016/j.jcv.2016.08.077
Abstract no: 267 Presentation at ESCV 2016: Poster 36 Comparison of respiratory and Meningitis/Encephalitis viruses detected by FilmArray® multiplex PCR versus real-time PCR Roger Koller ∗ , M.T. Barbani, A.U. Lüthi, S. Zürcher, J.F. Steinlin-Schopfer, S.L. Leib, M. Gorgievski-Hrisoho Institute for Infectious Diseases, University of Bern, Switzerland Introduction: Fast and reliable pathogen detection is important for adequate management of infections. Although real-time PCR (rtPCR) is usually the most sensitive method for direct pathogen detection, it requires experienced technicians, includes several working steps and has a turnaround time of multiple hours. Therefore this method is not ideal for emergency diagnostics. The FDA cleared, fully automated sample to answer, FilmArray® (FA) multiplex PCR system (BioFire/bioMérieux) detects a broad spectrum of pathogens in ∼70 min. To optimize our diagnostic services during weekends and off-peak times, we compared the FA Respiratory Panel (RP) and FA Meningitis/Encephalitis (ME) Panel to our routinely used rtPCR assay. The FA panels detect 20 respiratory pathogens (17 viruses, 3 bacteria) in nasopharyngeal swabs (NPS) and 14 M/E pathogens (7 viruses, 6 bacteria, Cryptococcus neoformans/gattii) in cerebrospinal fluids (CSF). Materials and methods: With FA we tested 84 retrospective samples (23 NPS, 29 broncheoalveolar lavages [BALs], 32 CSF) and 60 prospectively collected NPS that required urgent testing during the 2015/2016 flu season by FA and rtPCR. FA sample input volume was 300 ml for RP and 200 ml for ME. Commercial RP and ME quality control panels (MMQC Inc., Scarborough, USA), containing samples positive and negative for each analyte detected by the FA panels, were tested multiple times. For rtPCR, nucleic acids were extracted from 220 ml of sample and eluted in 55 ml using NucliSENS easyMAG (bioMérieux). Respiratory viruses were analyzed by real-time PCR using a combination of 7 duplex Respiratory Multi Well System r-geneTM (RG) assays (influenza A/B, RSV/hMPV, HRV&EV/cell control, ADV/HBoV, HCoV/HPIV1-4) (Argene/bioMerieux), according to manufacturer’s instructions. Additionally, we expanded FA RP testing to include (BALs), by implementing one additional sample preparation step. CSF was analyzed for virus using laboratory developed tests (LDTs) certified by the Swiss authorities. Results: RP and ME quality control panel results were 100% concordant with expected results. For all NPS, both tests, FA RP and RG, identified one or more viruses in 45/83 (54.2%) samples. FA RP and RG results correlated for 42/48 viruses detected (87.5%). FA RP detected an additional 3 HRV/EV and RG detected additionally 1 FluA, 1 ADV and 1 HRV/EV. Positive percent agreement (PPA) between RG (laboratory standard) and FA RP for NPS was 93.3% and negative percent agreement (NPA) was 92.7%. Overall correlation was 93.2%. Results from BALs yielded 92% PPA, 93.1% NPA and overall correlation of 92.4%. For FA ME testing, 31/33 CSF samples had identical FA ME and LDT results with an overall correlation of 94.4%. FA ME did not detect 2 parechovirus low level LDT positive samples (Ct 36.3 and 37.0). Using LDTs as the laboratory standard, FA ME PPA and NPA were 93.9% and 100%, respectively. Conclusion: Results obtained with the FilmArray® RP and ME panels were highly concordant with our currently used diagnostic methods, demonstrating excellent performance. The simplicity of the FilmArray® system, requiring less than 5 min of hands-on time, easy to read reports, and low sample volume allows for testing during off shifts and when urgent results are required. The compre-
S39
hensiveness of the FilmArray® panels is ideal for diagnosing clinical syndromes where there are many potential causes. http://dx.doi.org/10.1016/j.jcv.2016.08.076 Abstract no: 273 Presentation at ESCV 2016: Poster 37 Cross-contamination and carry-over study results obtained with ELITe InGenius, a new sample-to-result solution for in vitro diagnostics C. Bittoto ∗ , S. Costa, M. Enrietto, S. Patanè, F. Gorreta, A. Estampes, C. Olivo, G. Stefanuto, N. Scarr, W. Mahoney ELITechGroup Molecular Diagnostics, United Kingdom Background: ELITe InGeniusTM (ELITechGroup Molecular Diagnostics) is a fully automated sample-to-result solution, designed for the in vitro molecular diagnostics and monitoring of infectious diseases. The system combines on a single platform: sample processing (extraction and purification), PCR set-up, real-time amplification and detection of multiple parameters for qualitative and quantitative analysis, and result interpretation. Absence of cross-contamination and cross-over was evaluated within robustness study. Material/methods: ELITe InGenius features a universal extraction in a unitary cassette based format (ELITe InGenius SP200) and multiple and independent Real-Time PCR with mixed parameters including CE-IVD Real-Time PCR assays (ELITe MGB line) and Laboratory Developed Tests. One to twelve patient samples can be processed in 12 parallel tracks within one run. Cross-contamination and cross-over study protocols were designed in accordance with FDA Draft Guidance for Industry and Food and Drug Administration Staff Establishing the Performance Characteristics of Nucleic Acid-Based In vitro Diagnostic Devices. The tests to evaluate the rate of false positive included: (1) 30 high positive MRSA samples (107 CFU/ml, prepared by dilution of MRSA BAA-1720 strain) tested alternating to 30 negative samples within five 12-sample runs with MRSA/SA ELITe MGB kit; (2) negative MRSA samples (n = 50) tested within five runs along with 1 positive and 1 negative MRSA/SA controls per run with MRSA/SA ELITe MGB kit, and (3) 30 high CMV positive samples (104 IU/ml, prepared by dilution of the “1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques”) tested alternating to 30 negative samples within five 12-sample runs with CMV ELITe MGB kit. All samples were tested carrying out the whole analysis procedure: extraction, amplification, detection and result interpretation with ELITe InGeniusTM in combination with ELITechGroup reagents. Results: All negative and all positive MRSA and CMV samples were correctly identified by the system. 100% of concordance with the expected results was obtained for all the samples tested. Conclusions: The results obtained demonstrated the total absence of carry-over and cross-contamination of the system even when high positive samples were tested. They confirm the robustness and the reliability of ELITe InGenius for in vitro Molecular Diagnostics testing. http://dx.doi.org/10.1016/j.jcv.2016.08.077