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Evaluation of the fully automated DxN VERIS system for HIV-1 and HCV viral load monitoring
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Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
Abstract No: 1554 Presentation at ESCV 2015: Poster 2 Increasing frequency of HCMV seronegative unrelated donors in allogeneic hematopoietic stem cell transplantation in patients in IHBT during 1986–2015 S. Nemeckova 1 , M. Maly 2 , M. Stastna-Markova 1 1 Institute of Hematology and Blood Transfusion, Czech Republic 2 National Institutes of Health, Czech Republic
Background: Cytomegalovirus (HCMV) establishes lifelong latent infection accompanied by periodical subclinical reactiovations that can in immunosuppressed patients after hematopoietic stem cells transplantation (HSCT) result in severe life threatening disease. The HCMV seropositive transplanted patient who receives the cell graft from HCMV seronegative donor (R+/D−) is at high risk of developing recurrent HCMV reactivations after the day 100. Methods: To be able to assess the incidence of R+/D− combination further ahead, we evaluated retrospectively the frequency of HCMV seronegative donors (D−) for 626 allogeneic HSCT in IHBT in the years 1986–2015. Results: In total, we found 20% HCMV seronegative HSCT recipients, 21% HCMV seronegative related graft donors and 52% HCMV seronegative unrelated graft donors. The high risk combination R+/D− existed at 13% of transplantations of a graft from a relative and at 41% of cases if the graft was provided from an unrelated donor. HCMV seronegativity was found in 26% of unrelated donors from the Czech Republic, 63% unrelated donors from Germany and 52% of unrelated donors from other countries including US and UK. The proportion of HCMV seronegative grafts from unrelated donors during observed period continuously increased. The statistical analysis of the data will be shown. Conclusion: If the falling of HCMV seroprevalence of graft donors continues an increase of frequency of HCMV reactivations in our transplanted patients can be expected because of growing occurrence of the high risk R+/D− combination. This trend would increase the urgency of introduction the new therapeutic methods such as adoptive transfer of expanded or redirected HCMV specific T-lymphocytes. http://dx.doi.org/10.1016/j.jcv.2015.07.209 Abstract No: 1556 Presentation at ESCV 2015: Poster 2 Commutability of the 1st WHO International Standard for HCMV: Further comparisons in whole blood and plasma matrices J. Rehan Minhas Fryer, P. Minor, N. Almond, C. Morris Division of Virology, NIBSC, UK Human Cytomegalovirus (HCMV) is a ubiquitous Herpes virus with a high seroprevalence worldwide. It causes disease in the immunologically-naïve, such as newborns and infants, transplant recipients and AIDS patients. Clinical management including treatment with anti-herpetic agents is governed by the measurement of virus load by NAT assays. To support improved comparability between virus load measurements, the 1st International Standard (IS) for HCMV NAT assays was established by the WHO’s Expert Committee on Biological Standardisation in 2010 and comprises cell-free live virus preparation of HCMV strain, Merlin. Since, clinical diagnostic laboratories commonly test a wide range of sample
types for HCMV, the IS has been formulated in universal buffer allowing the end user to dilute the standard further in a matrix that is akin to the clinical samples under test. Ensuring that a reference material behaves in a manner analogous to clinical samples i.e. it is Commutable is critical for assay standardisation. For materials that may be used in a number of different matrices, this may present a challenge. Following the establishment there has been concern whether the standard behaves appropriately when it is diluted in whole blood matrix. As a result, we have undertaken further studies to investigate commutability of the HCMV IS. In our first study, we collaborated with the Royal Free Hospital London to analyse HCMV IS dilution series in whole blood or plasma along with 44 patient samples using a commercial and an in-house assay for HCMV NAT. The data indicates that whilst the IS appeared commutable when diluted in plasma, there was a significant (0.74 Log IU/mL) underreporting of IS concentration when diluted in whole blood and analysed by the commercial assay. This did not appear to be the case when the same samples were analysed by the in-house assay. To follow up these data we have undertaken a multicentre collaborative study, involving a broader range of NAT-based assays. Each participant was asked to evaluate dilution series of the IS for HCMV alongside a panel of clinical samples that were supplied diluted into plasma, whole blood, amniotic fluid or urine matrices. We shall present the results of this second study to establish whether there is any evidence of systematic bias in results using the HCMV IS for NAT in any specific clinical matrix. The implications of these data and suitability of the IS to improve the quality and comparability of NAT based assays for HCMV will be discussed. http://dx.doi.org/10.1016/j.jcv.2015.07.210 Abstract No: 1561 Presentation at ESCV 2015: Poster 2 Evaluation of the fully automated DxN VERIS system for HIV-1 and HCV viral load monitoring A. Yates, G. Lucy Neill Kudesia, D. Whittaker Sheffield Teaching Hospitals NHSFT, United Kingdom The Northern General Hospital Virology Laboratory in Sheffield, England has evaluated the HIV-1 1000 L and 175 L volume (CE marked) assays, and HCV (CE marking pending) assay on the newly launched VERIS system by Beckman Coulter, a fully automated sample-to-answer system for the quantitative/qualitative analysis of molecular targets. Background: Viral load monitoring of HIV-1 and HCV is critical for effective patient management. Fully standardised reproducible and sensitive quantification assays are desirable. The introduction of the VERIS instrument allows the laboratory to perform true random access PCR testing for HIV-1/HCV, providing a single streamlined process direct from primary sample tube and a significant improvement in time to sample result. Methods: Controls and precision samples were run daily over a twenty day period. Six levels of precision samples were tested on the VERIS daily over a twenty day period for the HIV-1 1000 L and HCV assays. For the HIV-1, 175 L assay, five levels of precision samples were tested on the VERIS daily over a twenty day period. LOD was determined by running five levels (12 replicates of each level) of diluted 3rd WHO international standard (group M, subtype B) tested daily over three days. Method comparison of viral loads (93 plasma samples HIV-1 1000 L assay, 91 plasma samples HIV-1 175 L assay, and 88 plasma samples HCV assay) were determined using the VERIS and the Roche COBAS Ampliprep® /COBAS TaqMan® assays and compared. Dilution linearity was performed
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
on the VERIS for the HIV-1 1000 L (four replicates of seven dilution levels, and HCV assays (four replicates of six dilution levels). Results: The precision samples demonstrated a standard deviation of between 0.04 and 0.25 (combined inter and intra runs) across all assays. The limit of detection for the HIV-1 175 L assay was determined as128 copies/mL (108–162 copies/mL at 95% CI). Correlation between VERIS and Roche COBAS Ampliprep® /COBAS TaqMan ® assays was analysed using the Passing-Bablok regression model (0.922 for the HIV1 1000 L assay, 0.932 for the HIV1 175 L assay, and 0.94 for the HCV assay). Correlation between the VERIS HIV1 1000 L and HIV1 175 L assays was 0.973. The HIV-1 1000 L and HCV assays demonstrated linearity over the dilution range. Conclusions: The DxN VERIS offers sensitive assays for the reliable monitoring of HIV-1 and HCV viral loads. Random access and improved time to result should help in the treatment and management of patients. http://dx.doi.org/10.1016/j.jcv.2015.07.211 Abstract No: 1562 Presentation at ESCV 2015: Poster 2 Hepatitis B virus avidity testing; not always as it seems B. Haywood 1 , S. Ijaz 1 , R.S. Tedder 2 1
Public Health England, United Kingdom Public Health England, NHS Blood and Transplant, University College London, United Kingdom 2
Background: In 2013, the reported incidence of acute hepatitis B virus (HBV) infection was 0.77 per 100,000 within England. Continued misclassification of core IgM reactivity has resulted in incorrect assignation of acute hepatitis B in individuals, impacting both on patient management but also leading to an overestimation of the incidence of acute hepatitis B. Public Health England has offered hepatitis B core antibody (anti-HBc) avidity testing to aid discrimination between acute infections and chronic carriers. Using samples from patients with confirmed acute or chronic HBV infection, we aimed to investigate the validity of this assay for distinguishing between acute and chronic hepatitis B virus infections in a range of patient groups. Method: Samples from cases of acute and chronic HBV infection were identified. All were tested for full HBV serological markers using the Abbott Architect (Abbott UK). The avidity of the anti-HBc present in these samples was then assessed using a commercial anti-HBc with a modified protocol utilising guanidine hydrochloride as a dissociation agent. Results: Low avidity antibody was observed in five of the nine acute hepatitis B cases; it was not possible to accurately determine avidity status in the remaining four and these cases were assigned as being indeterminate. Seven samples from patients classified with chronic HBV infection that were core IgM negative all displayed high avidity antibodies. Similar high avidity profiles were noted in five out of six chronic carriers with detectable anti-HBc IgM with the remaining one assigned as being indeterminate. A range of avidity profiles were observed in the 10 HIV-infected individuals with chronic HBV (five with high avidity antibody, three with low avidity antibody and two with indeterminate avidity antibody). Of interest, low avidity antibody was persistently observed in one HIV-infected individual with acute hepatitis B over a period of 119 weeks. Patients undergoing HBV reactivation also displayed a range of avidity profiles. High avidity antibody was observed in two patients who remained anti-HBc IgM negative throughout the period of monitoring. In contrast, low avidity antibody was noted
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in two further patients both with detectable anti-HBc IgM during follow-up. Conclusion: While in immunocompetent individuals anti-HBc avidity can be a useful tool to aid distinction between acute and chronic infection, in some immunosuppressed individuals chronically infected with HBV the anti-HBc avidity results may be unreliable. Caution is required and results must be interpreted in light of all available serological and molecular testing data. http://dx.doi.org/10.1016/j.jcv.2015.07.212 Abstract No: 1563 Presentation at ESCV 2015: Poster 2 Detection of viral DNA by isothermal NASBA amplification and chemiluminescence gene probe hybridization assay in a microfluidic cartridge F. Bonvicini 1 , M. Mirasoli 2 , M. Zangheri 2 , A. Nascetti 3 , G. De Cesare 4 , D. Caputo 4 , A. Roda 2 , G. Gallinella 1 1 Department of Pharmacy and Biotechnology, University of Bologna, Italy 2 Department of Chemistry, University of Bologna, Italy 3 Department of Astronautics, Electrical and Energy Engineering, Sapienza University of Rome, Italy 4 Department of Information, Electronic and Telecommunication Engineering, Sapienza University of Rome, Italy
Background: Detection of viral nucleic acids in biological samples is most often based on molecular amplification followed by sequence-specific detection of amplicons. Isothermal nucleic acid amplification strategies are particularly suited for their implementation in lab-on-chip devices, owing to the simplicity of the protocol and instrumentation required. Among optical detection strategies, chemiluminescence offers the advantages of high detectability in low volumes and no requirements for lamps, filters, or specific optical arrangements. Recently, on-chip integration of photosensors has been proposed to enable highly sensitive detection of chemiluminescence reactions, thus prompting the development of innovative portable analytical devices with enhanced sensitivity and multiplexed capabilities. Methods: A method for amplification of Parvovirus B19 DNA by isothermal nucleic acid sequence-based amplification (NASBA), followed by on-chip hybridisation and chemiluminescence-based detection of amplicons in a portable microfluidic cartridge with integrated photosensors has been developed. Results: Isothermal NASBA for Parvovirus B19 DNA allowed specific amplification of a genotype consensus target, with a limit of detection threshold of 10 genome copies per reaction, equivalent to 100 IU/mL in serum samples. Amplification was followed by on-chip hybridisation and detection of amplicons in a portable microfluidic cartridge, composed of polydimethylsiloxane microfluidic layer coupled with a glass slide bearing an array of thin film a-Si:H photosensors deposited on one side and streptavidin functionalized surface on the opposite side. Biotin-labelled B19 NASBA amplicons were captured and then detected by means of sequence-specific digoxigenin labelled probes and then hybrids were detected by means of an anti-digoxigenin antibody labelled with horseradish peroxidase and chemiluminescence reaction. A linear correlation was obtained between amount of viral target and light emission intensity, with a limit of quantitation of 100 genome copies per reaction.
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
Abstract No: 1554 Presentation at ESCV 2015: Poster 2 Increasing frequency of HCMV seronegative unrelated donors in allogeneic hematopoietic stem cell transplantation in patients in IHBT during 1986–2015 S. Nemeckova 1 , M. Maly 2 , M. Stastna-Markova 1 1 Institute of Hematology and Blood Transfusion, Czech Republic 2 National Institutes of Health, Czech Republic
Background: Cytomegalovirus (HCMV) establishes lifelong latent infection accompanied by periodical subclinical reactiovations that can in immunosuppressed patients after hematopoietic stem cells transplantation (HSCT) result in severe life threatening disease. The HCMV seropositive transplanted patient who receives the cell graft from HCMV seronegative donor (R+/D−) is at high risk of developing recurrent HCMV reactivations after the day 100. Methods: To be able to assess the incidence of R+/D− combination further ahead, we evaluated retrospectively the frequency of HCMV seronegative donors (D−) for 626 allogeneic HSCT in IHBT in the years 1986–2015. Results: In total, we found 20% HCMV seronegative HSCT recipients, 21% HCMV seronegative related graft donors and 52% HCMV seronegative unrelated graft donors. The high risk combination R+/D− existed at 13% of transplantations of a graft from a relative and at 41% of cases if the graft was provided from an unrelated donor. HCMV seronegativity was found in 26% of unrelated donors from the Czech Republic, 63% unrelated donors from Germany and 52% of unrelated donors from other countries including US and UK. The proportion of HCMV seronegative grafts from unrelated donors during observed period continuously increased. The statistical analysis of the data will be shown. Conclusion: If the falling of HCMV seroprevalence of graft donors continues an increase of frequency of HCMV reactivations in our transplanted patients can be expected because of growing occurrence of the high risk R+/D− combination. This trend would increase the urgency of introduction the new therapeutic methods such as adoptive transfer of expanded or redirected HCMV specific T-lymphocytes. http://dx.doi.org/10.1016/j.jcv.2015.07.209 Abstract No: 1556 Presentation at ESCV 2015: Poster 2 Commutability of the 1st WHO International Standard for HCMV: Further comparisons in whole blood and plasma matrices J. Rehan Minhas Fryer, P. Minor, N. Almond, C. Morris Division of Virology, NIBSC, UK Human Cytomegalovirus (HCMV) is a ubiquitous Herpes virus with a high seroprevalence worldwide. It causes disease in the immunologically-naïve, such as newborns and infants, transplant recipients and AIDS patients. Clinical management including treatment with anti-herpetic agents is governed by the measurement of virus load by NAT assays. To support improved comparability between virus load measurements, the 1st International Standard (IS) for HCMV NAT assays was established by the WHO’s Expert Committee on Biological Standardisation in 2010 and comprises cell-free live virus preparation of HCMV strain, Merlin. Since, clinical diagnostic laboratories commonly test a wide range of sample
types for HCMV, the IS has been formulated in universal buffer allowing the end user to dilute the standard further in a matrix that is akin to the clinical samples under test. Ensuring that a reference material behaves in a manner analogous to clinical samples i.e. it is Commutable is critical for assay standardisation. For materials that may be used in a number of different matrices, this may present a challenge. Following the establishment there has been concern whether the standard behaves appropriately when it is diluted in whole blood matrix. As a result, we have undertaken further studies to investigate commutability of the HCMV IS. In our first study, we collaborated with the Royal Free Hospital London to analyse HCMV IS dilution series in whole blood or plasma along with 44 patient samples using a commercial and an in-house assay for HCMV NAT. The data indicates that whilst the IS appeared commutable when diluted in plasma, there was a significant (0.74 Log IU/mL) underreporting of IS concentration when diluted in whole blood and analysed by the commercial assay. This did not appear to be the case when the same samples were analysed by the in-house assay. To follow up these data we have undertaken a multicentre collaborative study, involving a broader range of NAT-based assays. Each participant was asked to evaluate dilution series of the IS for HCMV alongside a panel of clinical samples that were supplied diluted into plasma, whole blood, amniotic fluid or urine matrices. We shall present the results of this second study to establish whether there is any evidence of systematic bias in results using the HCMV IS for NAT in any specific clinical matrix. The implications of these data and suitability of the IS to improve the quality and comparability of NAT based assays for HCMV will be discussed. http://dx.doi.org/10.1016/j.jcv.2015.07.210 Abstract No: 1561 Presentation at ESCV 2015: Poster 2 Evaluation of the fully automated DxN VERIS system for HIV-1 and HCV viral load monitoring A. Yates, G. Lucy Neill Kudesia, D. Whittaker Sheffield Teaching Hospitals NHSFT, United Kingdom The Northern General Hospital Virology Laboratory in Sheffield, England has evaluated the HIV-1 1000 L and 175 L volume (CE marked) assays, and HCV (CE marking pending) assay on the newly launched VERIS system by Beckman Coulter, a fully automated sample-to-answer system for the quantitative/qualitative analysis of molecular targets. Background: Viral load monitoring of HIV-1 and HCV is critical for effective patient management. Fully standardised reproducible and sensitive quantification assays are desirable. The introduction of the VERIS instrument allows the laboratory to perform true random access PCR testing for HIV-1/HCV, providing a single streamlined process direct from primary sample tube and a significant improvement in time to sample result. Methods: Controls and precision samples were run daily over a twenty day period. Six levels of precision samples were tested on the VERIS daily over a twenty day period for the HIV-1 1000 L and HCV assays. For the HIV-1, 175 L assay, five levels of precision samples were tested on the VERIS daily over a twenty day period. LOD was determined by running five levels (12 replicates of each level) of diluted 3rd WHO international standard (group M, subtype B) tested daily over three days. Method comparison of viral loads (93 plasma samples HIV-1 1000 L assay, 91 plasma samples HIV-1 175 L assay, and 88 plasma samples HCV assay) were determined using the VERIS and the Roche COBAS Ampliprep® /COBAS TaqMan® assays and compared. Dilution linearity was performed
Abstracts / Journal of Clinical Virology 70 (2015) S1–S126
on the VERIS for the HIV-1 1000 L (four replicates of seven dilution levels, and HCV assays (four replicates of six dilution levels). Results: The precision samples demonstrated a standard deviation of between 0.04 and 0.25 (combined inter and intra runs) across all assays. The limit of detection for the HIV-1 175 L assay was determined as128 copies/mL (108–162 copies/mL at 95% CI). Correlation between VERIS and Roche COBAS Ampliprep® /COBAS TaqMan ® assays was analysed using the Passing-Bablok regression model (0.922 for the HIV1 1000 L assay, 0.932 for the HIV1 175 L assay, and 0.94 for the HCV assay). Correlation between the VERIS HIV1 1000 L and HIV1 175 L assays was 0.973. The HIV-1 1000 L and HCV assays demonstrated linearity over the dilution range. Conclusions: The DxN VERIS offers sensitive assays for the reliable monitoring of HIV-1 and HCV viral loads. Random access and improved time to result should help in the treatment and management of patients. http://dx.doi.org/10.1016/j.jcv.2015.07.211 Abstract No: 1562 Presentation at ESCV 2015: Poster 2 Hepatitis B virus avidity testing; not always as it seems B. Haywood 1 , S. Ijaz 1 , R.S. Tedder 2 1
Public Health England, United Kingdom Public Health England, NHS Blood and Transplant, University College London, United Kingdom 2
Background: In 2013, the reported incidence of acute hepatitis B virus (HBV) infection was 0.77 per 100,000 within England. Continued misclassification of core IgM reactivity has resulted in incorrect assignation of acute hepatitis B in individuals, impacting both on patient management but also leading to an overestimation of the incidence of acute hepatitis B. Public Health England has offered hepatitis B core antibody (anti-HBc) avidity testing to aid discrimination between acute infections and chronic carriers. Using samples from patients with confirmed acute or chronic HBV infection, we aimed to investigate the validity of this assay for distinguishing between acute and chronic hepatitis B virus infections in a range of patient groups. Method: Samples from cases of acute and chronic HBV infection were identified. All were tested for full HBV serological markers using the Abbott Architect (Abbott UK). The avidity of the anti-HBc present in these samples was then assessed using a commercial anti-HBc with a modified protocol utilising guanidine hydrochloride as a dissociation agent. Results: Low avidity antibody was observed in five of the nine acute hepatitis B cases; it was not possible to accurately determine avidity status in the remaining four and these cases were assigned as being indeterminate. Seven samples from patients classified with chronic HBV infection that were core IgM negative all displayed high avidity antibodies. Similar high avidity profiles were noted in five out of six chronic carriers with detectable anti-HBc IgM with the remaining one assigned as being indeterminate. A range of avidity profiles were observed in the 10 HIV-infected individuals with chronic HBV (five with high avidity antibody, three with low avidity antibody and two with indeterminate avidity antibody). Of interest, low avidity antibody was persistently observed in one HIV-infected individual with acute hepatitis B over a period of 119 weeks. Patients undergoing HBV reactivation also displayed a range of avidity profiles. High avidity antibody was observed in two patients who remained anti-HBc IgM negative throughout the period of monitoring. In contrast, low avidity antibody was noted
S91
in two further patients both with detectable anti-HBc IgM during follow-up. Conclusion: While in immunocompetent individuals anti-HBc avidity can be a useful tool to aid distinction between acute and chronic infection, in some immunosuppressed individuals chronically infected with HBV the anti-HBc avidity results may be unreliable. Caution is required and results must be interpreted in light of all available serological and molecular testing data. http://dx.doi.org/10.1016/j.jcv.2015.07.212 Abstract No: 1563 Presentation at ESCV 2015: Poster 2 Detection of viral DNA by isothermal NASBA amplification and chemiluminescence gene probe hybridization assay in a microfluidic cartridge F. Bonvicini 1 , M. Mirasoli 2 , M. Zangheri 2 , A. Nascetti 3 , G. De Cesare 4 , D. Caputo 4 , A. Roda 2 , G. Gallinella 1 1 Department of Pharmacy and Biotechnology, University of Bologna, Italy 2 Department of Chemistry, University of Bologna, Italy 3 Department of Astronautics, Electrical and Energy Engineering, Sapienza University of Rome, Italy 4 Department of Information, Electronic and Telecommunication Engineering, Sapienza University of Rome, Italy
Background: Detection of viral nucleic acids in biological samples is most often based on molecular amplification followed by sequence-specific detection of amplicons. Isothermal nucleic acid amplification strategies are particularly suited for their implementation in lab-on-chip devices, owing to the simplicity of the protocol and instrumentation required. Among optical detection strategies, chemiluminescence offers the advantages of high detectability in low volumes and no requirements for lamps, filters, or specific optical arrangements. Recently, on-chip integration of photosensors has been proposed to enable highly sensitive detection of chemiluminescence reactions, thus prompting the development of innovative portable analytical devices with enhanced sensitivity and multiplexed capabilities. Methods: A method for amplification of Parvovirus B19 DNA by isothermal nucleic acid sequence-based amplification (NASBA), followed by on-chip hybridisation and chemiluminescence-based detection of amplicons in a portable microfluidic cartridge with integrated photosensors has been developed. Results: Isothermal NASBA for Parvovirus B19 DNA allowed specific amplification of a genotype consensus target, with a limit of detection threshold of 10 genome copies per reaction, equivalent to 100 IU/mL in serum samples. Amplification was followed by on-chip hybridisation and detection of amplicons in a portable microfluidic cartridge, composed of polydimethylsiloxane microfluidic layer coupled with a glass slide bearing an array of thin film a-Si:H photosensors deposited on one side and streptavidin functionalized surface on the opposite side. Biotin-labelled B19 NASBA amplicons were captured and then detected by means of sequence-specific digoxigenin labelled probes and then hybrids were detected by means of an anti-digoxigenin antibody labelled with horseradish peroxidase and chemiluminescence reaction. A linear correlation was obtained between amount of viral target and light emission intensity, with a limit of quantitation of 100 genome copies per reaction.