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1.
From crisis to routine – standardization of SARS-CoV-2 genome detection by enhanced EQA schemes in a scientific pandemic network
Martin 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: 447
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2.
The application of digital PCR as a reference measurement procedure to support the accuracy of quality assurance for infectious disease molecular diagnostic testing
Samreen Falak, Denise M O’Sullivan, Megan H. Cleveland, Simon Cowen, Eloise J. Busby, Alison S. Devonshire, Esmeralda Valiente, Gerwyn M. Jones, Martin Kammel, Mojca Milavec, 2025, original scientific article

Abstract: BACKGROUND: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available. This study investigated digital PCR (dPCR) RMP delivery of traceability for NAAT external quality assessment (EQA). METHODS: Three National Metrology Institutes (NMIs) applied reverse transcription (RT)-dPCR as a candidate RMP to estimate the RNA quantity in 32 independent severe acute respiratory syndrome coronavirus 2 materials. The results were combined to value assign the respective materials: 21 materials were used in 6 rounds of EQA over 17 months for 61 laboratories for COVID-19 testing results compared with reference values. RESULTS: The agreement between the 3 NMIs showed <2-fold difference between laboratories. EQA laboratory reverse transcription quantitative PCR (RTqPCR) values estimation of viral RNA quantity showed good median agreement with RT-dPCR reference value; however, RT-qPCR differences were generally between 10- and 50-fold between laboratories. CONCLUSION: This work demonstrates how RT-dPCR can provide reference values for whole virus materials for NAAT quality assurance. RT-dPCR values guided EQA control material selection and provided EQA participants with traceability to RNA copy number delivered through the RMP. This approach can be used to support routine reference material use as well as to standardize quality assurance for NAATs where established reference materials are not available, such as in disease outbreaks.
Keywords: nucleic acid amplification tests, dPCR, reference measurement procedure, external quality assessment, SARS-CoV-2
Published in DiRROS: 14.04.2025; Views: 771; Downloads: 436
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