CMV DNA quantification in plasma using an automated real-time PCR assay

s: Exhibitors Symposium $7 Coefficient of variations of CMV real-time PCR assay varied from 1 to 12% for CMV DNA levels ranging between 4.0 to 1.3 log copies/ml. Comparative studies using 179 routine samples showed a concordance of 89%; 18 samples positive by CMV real-time PCR only and 2 samples positive by US Cobas. Discrepancies were only observed for samples with less than 300 copies/ml. The two assays showed high correlation (R = 0.93), and on average values obtained by CMV real-time PCR were 0.4 log higher than those of US Cobas. Successive samples of transplanted patients with evidence of CMV infection or reactivation revealed that CMV real-time PCR assay was positive earlier and for longer period of time after treatment initiation. Conclusion: Both assays had similar analytical performances, however the CMV real-time PCR assay has the advantages of automated extraction and higher dynamic range. On clinical samples there is a clear trend for a higher sensitivity of the CMV real-time PCR assay. Exhibitors Symposium l • T h e innovative qiasymphony system from Qiagen takes ease of use to a new level M.N. Kraak*. Qiagen Instruments, Switzerland Efficient purification of nucleic acids is critical for reliable results in downstream analyses. To address these needs, we are developing an innovative, modular system for medium-throughput sample prep and assay setup. The aim of this work is to provide an initial evaluation of the performance characteristics of the new QIAsymphony sample prep module for fully automated purification of pathogen nucleic acids from a range of samples. The QIAsymphony sample prep module takes ease of use to a new level and can be operated by anyone from the novice to the expert. Using proven magnetic-particle technology, a wide range of primary or secondary samples with input volumes up to 1 ml can be processed. You can perform all your molecular biology applications with one hardware configuration. Sample prep is even more economical since all your automated applications can be covered with just a few kits. High process safety is assured through automated bar code reading of samples and reagents, and a fully automated load check helps to prevent human error. For increased convenience and flexibility, the system allows in-process sample loading, enabling immediate processing of urgent samples. The QIAsymphony system will be available in 2007. The QIAsymphony system is under development and planned for general laboratory use. No claim or representation is intended for its use to provide information for the diagnosis, prevention, or treatment of a disease. I ~ Performance evaluation of real-time PCR based assays for the detection and quantitation of hepatitis B virus DNA J. Dannenberg*. QIAGEN Hamburg GmbH, KSnigstr. 4a, 22767 Hamburg, Germany Viral load determination for hepatitis B virus (HBV) is an essential marker for introducing and monitoring efficient therapy. artus HBV PCR Kits are real-time PCR based assays specifically developed for the use with the LightCycler | (Roche), the Rotor-Gene 3000 (Corbett Research), and the ABI Prism | Instruments (Applied Biosystems). The goal of this work was to evaluate the performance of artus HBV PCR Kits in combination with highly efficient viral DNA isolation systems: the silica membrane based QIAamp DSP Virus Kit and automated, magnetic-particle technology based BioRobot | Workstations (QIAGEN). Serial dilutions of plasma samples spiked with the HBV DNA 1st International Standard (WHO 97/746) were extracted using the QIAamp DSP Virus Kit and different BioRobot Workstations (QIAGEN). For the determination of the viral load the eluates were analyzed using artus HBV PCR Kits for the LightCycler | (Roche), the RotorGene 3000 (Corbett Research) and the ABI Prism Instruments (Applied Biosystems). Clinical sensitivity and specificity were evaluated and quantitative results were correlated by analyzing clinical samples using the artus HBV Kits in comparison with a reference method. The specificity of the artus HBV assays has been proven by testing the HBV Genotype Panel (Teragenix), in-house genotyped clinical samples, precore mutation (Teragenix) and seroconversion panels (Boston Biomedica Inc.). All viral DNA isolation systems showed highly sensitive and reliable results in combination with artus HBV PCR Kits for detection and quantitation of HBV. Quantitative results correlated strongly with a diagnostic reference method and all members of the HBV genotype panel were detected with comparable sensitivity. The combination of sample preparation using the QIAamp DSP Virus Kit or BioRobot ~ Workstations (QIAGEN) and artus HBV realtime PCR assays enables sensitive and highly reliable results for the detection and quantitation of HBV. l ~ N e w sensations in molecular diagnostics NucliSENS easyMAG extraction platform and NucliSENS EasyQ assays A. Troesch *. Director R&D Molecular Biology bioM~rieux, Grenoble, France bioM@ieux, through its NucliSENS range, offers a complete molecular diagnostic platform for extraction, amplification and real-time detection. This presentation will explain the proprietary core technologies as well as highlight some key products from the NucliSENS range. NucliSENS extraction is based on BOOM | technology, which is recognized as the Gold Standard in nucleic acid isolation. In the new platforms the technology has been further enhanced by the introduction of magnetic silica particles. Two systems are available for magnetic extraction, i.e. NucliSENS easyMAG, the automated platform and NucliSENS miniMAG, a more manual system ideally suited for lower throughput labs. NucliSENS amplification and detection is based on real-time NASBA | technology, which enables fast and accurate amplification and real-time detection using molecular beacons. This specific, isothermal method of nucleic acid amplification can be used for the amplification of RNA and DNA and is carried out on the NucliSENS EasyQ Analyzer. bioM@ieux is at the forefront of offering more sensitive molecularbased tests to respond to this major public health concern. An example is NucliSENS EasyQ HIV-1 v l .2, an established HIV-1 viral load assay that recently has been combined with the NucliSens easyMAG for maximum user convenience. Furthermore assays have been developed in the range of lower respiratory tract infections (LRTI), like NucliSENS EasyQ RSV A+B, NucliSENS EasyQ hMPV, NucliSENS EasyQ SARS-CoV, NucliSENS EasyQ Influenza H5 & N1, and CNS infections like NucliSENS EasyQ Enterovirus and NucliSENS EasyQ HSV 1/2. Other assays that will complement those panels are under development. 1 ~ Bayer Molecular: viral load testing today and tomorrow A.J. Uzgiris*. Bayer HealthCare LLC, Diagnostics Division, USA Viral load testing is an important component of patient management for those with HIV or hepatitis infection. Bayer HealthCare offers comprehensive viral testing solutions including VERSANT e HIV-1, HCV, and HBV viral load assays. The VERSANT TM 440 Molecular System will perform existing bDNA assays with walkaway automation providing greater laboratory efficiency. The first new assay for this system, an increased sensitivity HCV test, is in development.

features that roughly reflect the progression of the disease: placentitis, oligohydramnios, hyperechogenic bowel, hepatosplenomegaly, IUGR, ventriculomegaly, intracerebral hyperechogenic foci or microcephaly. These primary markers can lead to the diagnosis of more complex associations of both systemic and CNS abnormalities in up to 20% of the cases.
Early experience with treatment of intrauterine cytomegalovirus infection appears promising following two approaches. Therapeutic Valacyclovir concentrations were achieved in maternal and fetal blood with maternal oral administration of this drug in cases of symptomatic infection. Maternal administration of hyper-immune immunoglobulins may help reverting some of the early and acute effects of the infection on the placenta and the fetus. A combination of these two approaches should be evaluated in a randomized controlled trial. E-mail: yville@wanadoo.fr International collaboration allows the possibility of linking disease registers to improve power for analysis of outcome studies. Cohort also can provide a useful starting point to develop clinical networks for future trials in rare diseases. Generally data from a smaller number of committed centres, with clear reasons for starting the register and understanding of potential bias are the best prognostic factors for the success of a register. Clear clinical and financial plans for the long term outcome of the register and its ultimate goal are also vital for its success. Web based registers now simplify data collection, but lead to complex questions of consent, which vary across the EU. ECCI could develop a simple disease register across the EU and the possible role and use for such a register will be discussed. Detection and quantification of plasma RNA load plays a key role in the evaluation and clinical management of infection with HIV-1 and hepatitis C virus (HCV). In the era of highly active antiretroviral therapy, HIV-1 plasma RNA load continues to play an import prognostic role as a determinant of the rate of CD4 count decline and an indicator of the risk of transmission. Most importantly, plasma viral load suppression to below the lower limit of detection is the indicator of successful antiretroviral therapy and main surrogate marker for immunological and clinical success. Detection of HCV RNA has become increasingly important in the diagnosis and management of hepatitis C infection. Absolute viral load and the early log decline after starting treatment are clinically useful in predicting sustained virological responses and guiding whether therapy should be continued. Based on these considerations, the availability of reliable assays for HIV-1 and HCV RNA detection and quantification is critical in the routine diagnostic settings. At the Royal Free Hospital, over 2000 samples have been tested by the Abbott Real-time HIV-1 and HCV quantitative assays. The assays offer several advantages, including (a) availability of automated nucleic acid extraction; (b) turn-around time of 2 hours; and (c) wide dynamic ranges of quantification. Their use in clinical practice has demonstrated a low risk of carry-over contamination, high intra-assay and inter-assay reproducibility, good performance with a variety of HIV-1 subtypes and HCV genotypes, and strong correlation with results obtained by other assays in comparative studies (r >0.9). To accomplish this, a sequence alignment was performed which encompassed representative sequences for the various subtypes and genotypes of HBV. Next, a search for three conserved regions in close proximity to each other resulted in the identification of a highly conserved area in the HBV surface gene upstream from known HBsAg and Pol mutations. Putative PCR primer and probe sequences designed within this region were compared to all known HBV isolates to ensure that these sequences were optimal. Due to the high degree of conservation within the primer and probe regions selected, the PCR annealing and read steps can be performed at high temperature. Using high temperature anneal/read favors the formation of specific amplification product while also maintaining the ability of the assay to detect and quantitate diverse HBV genotypes. Studies were performed on the Abbott m2000 system to evaluate the RealTime HBV assay sensitivity, specificity and quantitation of patient specimens with HBV genotypes A through H. In addition, specimens containing known Pre-Core, Core and drug resistance mutations were tested in a blinded study and were compared to results from a comparator assay.

Abbott Molecular
The resulting assay detects below 50 copies/ML up to at least 1E9 copies/ML on HBV serum or plasma specimens using automated sample preparation and covers genotypes A-H with excellent specificity.

I~CMV
DNA quantification in plasma using an automated real-time PCR assay Objectives: We evaluated a new sensitive PCR assay coupled with an automated extraction system (CMV real-time PCR, Abbott) by direct comparison with the US Cobas.
Results: Using limiting dilutions of CMV DNA positive plasma, the two assays had a similar detection threshold of 20-40 copies/ml. Abstracts: Exhibitors Symposium $7 Coefficient of variations of CMV real-time PCR assay varied from 1 to 12% for CMV DNA levels ranging between 4.0 to 1.3 log copies/ml. Comparative studies using 179 routine samples showed a concordance of 89%; 18 samples positive by CMV real-time PCR only and 2 samples positive by US Cobas. Discrepancies were only observed for samples with less than 300 copies/ml. The two assays showed high correlation (R = 0.93), and on average values obtained by CMV real-time PCR were 0.4 log higher than those of US Cobas. Successive samples of transplanted patients with evidence of CMV infection or reactivation revealed that CMV real-time PCR assay was positive earlier and for longer period of time after treatment initiation. Conclusion: Both assays had similar analytical performances, however the CMV real-time PCR assay has the advantages of automated extraction and higher dynamic range. On clinical samples there is a clear trend for a higher sensitivity of the CMV real-time PCR assay.

M.N. Kraak*. Qiagen Instruments, Switzerland
Efficient purification of nucleic acids is critical for reliable results in downstream analyses. To address these needs, we are developing an innovative, modular system for medium-throughput sample prep and assay setup. The aim of this work is to provide an initial evaluation of the performance characteristics of the new QIAsymphony sample prep module for fully automated purification of pathogen nucleic acids from a range of samples.
The QIAsymphony sample prep module takes ease of use to a new level and can be operated by anyone -from the novice to the expert. Using proven magnetic-particle technology, a wide range of primary or secondary samples with input volumes up to 1 ml can be processed. You can perform all your molecular biology applications with one hardware configuration. Sample prep is even more economical since all your automated applications can be covered with just a few kits.
High process safety is assured through automated bar code reading of samples and reagents, and a fully automated load check helps to prevent human error. For increased convenience and flexibility, the system allows in-process sample loading, enabling immediate processing of urgent samples.
The QIAsymphony system will be available in 2007. The QIAsymphony system is under development and planned for general laboratory use. No claim or representation is intended for its use to provide information for the diagnosis, prevention, or treatment of a disease. Clinical sensitivity and specificity were evaluated and quantitative results were correlated by analyzing clinical samples using the artus HBV Kits in comparison with a reference method.

I~ Performance evaluation of real-time PCR based assays for the detection and quantitation of hepatitis B virus DNA
The specificity of the artus HBV assays has been proven by testing the HBV Genotype Panel (Teragenix), in-house genotyped clinical samples, precore mutation (Teragenix) and seroconversion panels (Boston Biomedica Inc.).
All viral DNA isolation systems showed highly sensitive and reliable results in combination with artus HBV PCR Kits for detection and quantitation of HBV. Quantitative results correlated strongly with a diagnostic reference method and all members of the HBV genotype panel were detected with comparable sensitivity. The combination of sample preparation using the QIAamp DSP Virus Kit or BioRobot ~ Workstations (QIAGEN) and artus HBV realtime PCR assays enables sensitive and highly reliable results for the detection and quantitation of HBV.

l~New sensations in molecular diagnostics -NucliSENS easyMAG extraction platform and NucliSENS EasyQ assays
A. Troesch *. Director R&D Molecular Biology bioM~rieux, Grenoble, France bioM@ieux, through its NucliSENS range, offers a complete molecular diagnostic platform for extraction, amplification and real-time detection. This presentation will explain the proprietary core technologies as well as highlight some key products from the NucliSENS range.
NucliSENS extraction is based on BOOM | technology, which is recognized as the Gold Standard in nucleic acid isolation. In the new platforms the technology has been further enhanced by the introduction of magnetic silica particles. Two systems are available for magnetic extraction, i.e. NucliSENS easyMAG, the automated platform and NucliSENS miniMAG, a more manual system ideally suited for lower throughput labs.
NucliSENS amplification and detection is based on real-time NASBA | technology, which enables fast and accurate amplification and real-time detection using molecular beacons. This specific, isothermal method of nucleic acid amplification can be used for the amplification of RNA and DNA and is carried out on the NucliSENS EasyQ Analyzer.
bioM@ieux is at the forefront of offering more sensitive molecularbased tests to respond to this major public health concern. An example is NucliSENS EasyQ HIV-1 vl.2, an established HIV-1 viral load assay that recently has been combined with the NucliSens easyMAG for maximum user convenience. Furthermore assays have been developed in the range of lower respiratory tract infections (LRTI), like NucliSENS EasyQ RSV A+B, NucliSENS EasyQ hMPV, NucliSENS EasyQ SARS-CoV, NucliSENS EasyQ Influenza H5 & N1, and CNS infections like NucliSENS EasyQ Enterovirus and NucliSENS EasyQ HSV 1/2. Other assays that will complement those panels are under development.

1~ Bayer Molecular: viral load testing today and tomorrow
A.J. Uzgiris*. Bayer HealthCare LLC, Diagnostics Division, USA Viral load testing is an important component of patient management for those with HIV or hepatitis infection. Bayer HealthCare offers comprehensive viral testing solutions including VERSANT e HIV-1, HCV, and HBV viral load assays. The VERSANT TM 440 Molecular System will perform existing bDNA assays with walkaway automation providing greater laboratory efficiency. The first new assay for this system, an increased sensitivity HCV test, is in development.