1. From crisis to routine – standardization of SARS-CoV-2 genome detection by enhanced EQA schemes in a scientific pandemic networkMartin Kammel, Hans-Peter Grunert, Anika Zimmermann, Annemarie Martin, Vanessa Lindig, Mojca Milavec, 2025, original scientific article Abstract: In the beginning of 2020, the outbreak of the COVID-19 pandemic led to a crisis in which diagnostic methods for the genome detection of SARS-CoV-2 were urgently needed. Based on the very early publication of the basic principles for a diagnostic test for the genome detection of SARS-CoV-2, the first noncommercial laboratory-developed tests (LDTs) and commercial tests were introduced. As there was considerable uncertainty about the reliability and performance of different tests and different laboratories, INSTAND established external quality assessment (EQA) schemes for the detection of SARS-CoV-2 starting in April 2020. In close partnership in a scientific network, the EQA schemes were enhanced, especially the April, June and November 2020 terms. The enhancement included: (i) immediate provision of suitable virus including variants of concern at the beginning of the pandemic outbreak, (ii) short frequency of EQA schemes, (iii) concentration dependency of the testing and sensitivity check, achieved by using SARS-CoV-2-positive samples from a 10-fold dilution series of the same starting material, (iv) specificity check of the testing, achieved by using SARS-CoV-2-negative samples containing human coronaviruses or MERS CoV, (v) revealed samples for orientation on test performance during an ongoing or at the start of an EQA scheme using a pre-quantified SARS-CoV-2-positive EQA sample with a low viral RNA load of only 1 570 copies/mL assigned by digital PCR (dPCR) in June 2020 and (vi) quantified reference materials based on the experiences of the first two EQA schemes with dPCR-assigned values in copies/mL beginning in November 2020 for self-evaluation of the applied test system. This manuscript summarizes the results of a total of 13 EQA schemes for the detection of SARS-CoV-2 between April 2020 and June 2023 in which a total of 1 413 laboratories from 49 countries participated. The qualitative results for the detection of SARS-CoV-2-positive samples were between 95.8 % and 99.7 % correct positive, excluding extremely low concentration samples. For all SARS-CoV-2-negative EQA samples, the qualitative success rates ranged from 95.1 % to 99.4 % correct negative results. The widely varying values for the cycle threshold (Ct)/crossing point (Cq) reported for the different target genes and test systems were striking. A few laboratories reported quantitative results in copies/mL for several VOCs with an acceptable rate of over 93 % correct positive results in the majority of cases. The description of the enhanced EQA schemes for SARS-CoV-2 detection in terms of timing and scope can serve as a blueprint for the rapid development of a quality assessment of diagnostics for an emerging pathogen.
Keywords: COVID-19 pandemic, SARS-CoV-2, virus genome detection tests, reference materials, external quality assessment, laboratory medicine, epidemiology
Published in DiRROS: 18.06.2025; Views: 497; Downloads: 445
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2. 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: 1217; Downloads: 727
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3. Digital PCR method for detection and quantification of specific antimicrobial drug-resistance mutations in human cytomegalovirusAlexandra Bogožalec Košir, Tašja Cvelbar, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Mojca Milavec, 2020, original scientific article Abstract: Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methods; e.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantification; and qPCR quantification is hampered by a lack of reference materials for standard curves. In recent years, digital PCR has been introduced, which provides absolute quantification without the need for reference materials for standard curves. Using digital PCR, we have developed a rapid, sensitive and accurate method for genotyping and quantification of the most prevalent mutations that cause human cytomegalovirus resistance to ganciclovir.
Keywords: digital PCR, antimicrobial-drug resistance, HCMV, polymerase chain reaction, viruses
Published in DiRROS: 22.07.2024; Views: 1007; Downloads: 540
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4. An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1Samreen Falak, Rainer Macdonald, Eloise J. Busby, Denise M. O'Sullivan, Mojca Milavec, Annabell Plauth, Martin Kammel, Heinz Zeichhardt, Hans-Peter Grunert, Andreas Kummrow, Jim F. Huggett, 2022, original scientific article Abstract: Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.
Published in DiRROS: 16.07.2024; Views: 1002; Downloads: 501
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5. 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: 1043; Downloads: 547
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