1. Survey results on nucleic acid tests of infectious diseases : present status and need for rapid and near-patient diagnosticsJörg Neukammer, Martin Hussels, Andreas Kummrow, Alison S. Devonshire, Carole A. Foy, Jim F. Huggett, Helen C. Parkes, Jana Žel, Mojca Milavec, Heinz Schimmel, Wolfgang Unger, Müslüm Akgöz, Timothy D. McHugh, Viktorija Tomič, Hans-Peter Grunert, Heinz Zeichhardt, 2015, original scientific article Abstract: This survey discusses current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for near-patient diagnostics.
To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire.
Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were methicillin-resistant Staphylococcus aureus (MRSA), norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acids to identify infectious pathogens.
Keywords: nucleic acid tests, infectious diseases, virus detection, bacteria detection, isothermal nucleic acid amplification, status report, questionnaire, NAT, PCR
Published in DiRROS: 26.07.2024; Views: 200; Downloads: 277
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2. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNAJernej Pavšič, Alison S. Devonshire, Andrej Blejec, Carole A. Foy, Fran Van Heuverswyn, Gerwyn M. Jones, Heinz Schimmel, Jana Žel, Jim F. Huggett, Nicholas Redshaw, Maria Karczmarczyk, Erkan Mozioglu, Sema Akyürek, Müslüm Akgöz, Mojca Milavec, 2017, original scientific article Abstract: Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.
Keywords: digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials
Published in DiRROS: 24.07.2024; Views: 114; Downloads: 94
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3. The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four yearsMojca Milavec, Jernej Pavšič, Alexandra Bogožalec Košir, Gerwyn M. Jones, Denise M. O'Sullivan, Alison S. Devonshire, Fran Van Heuverswyn, Maria Karczmarczyk, Jannika Neeb, Annabell Plauth, Philippe Corbisier, Heinz Schimmel, Andreas Kummrow, Jörg Neukammer, Carole A. Foy, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Jim F. Huggett, 2022, original scientific article Published in DiRROS: 15.07.2024; Views: 213; Downloads: 61
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