Rapid adaptation of established high-throughput molecular testing infrastructure for detection of monkeypoxvirus (preprint)

Background Since May 2022, a rising number of monkeypox-cases has been reported in non-endemic countries of the northern hemisphere. In contrast to previous clusters, infections seem predominantly driven by human-to-human transmission, rather than animal sources. In this study, we adapted two published qPCR assays (non-variola orthopoxvirus and monkeypoxvirus specific) for use as a lab-developed dual-target monkeypoxvirus-test on widely used automated high-throughput PCR-systems (cobas5800/6800/8800). Methods Selected assays were checked for in-silico inclusivity and exclusivity in current orthopoxvirus sequences, as well as for multiplex compatibility. Analytic performance was determined by serial dilution of monkeypoxvirus reference material, quantified by digital PCR. Cross reactivity was ruled out through a clinical exclusivity set containing various bloodborne and respiratory pathogens. Clinical performance was compared to a commercial manual RUO-kit using clinical remnant samples. Results Analytic lower limit of detection (LoD) was determined as 4.795 dcp/ml (CI95%: 3.598 - 8.633 dcp/ml) for both assays combined, with a dynamic range of at least 5 log-steps. The assay showed 100% positive and negative agreement with the manual RUO orthopoxvirus PCR test kit in clinical swab samples. Discussion While the full extend of the ongoing monkeypox outbreak remains to be established, the WHO and local health authorities are calling for increased awareness and efforts to limit further spread. For this, timely and scalable PCR tests are an important prerequisite. The assay presented here allows streamlined high-throughput molecular testing for monkeypoxvirus on existing hardware, broadly established previously for SARS-CoV-2 diagnostics.

Evaluation of a fully automated high-throughput SARS-CoV-2 multiplex qPCR assay with build-in screening functionality for DelHV69/70- and N501Y variants such as B.1.1.7 (preprint)

1BackgroundNew SARS-CoV-2 variants with increased transmissibility, like B.1.1.7 from England or B1.351 from South Africa, have caused considerable concern worldwide. In order to contain the spread of these lineages, it is of utmost importance to have rapid, sensitive and high-throughput detection methods at hand. MethodsAnalytical sensitivity was assessed for both wild-type SARS-CoV-2 and B.1.1.7 lineage by serial dilution. A total of 141 clinical samples were subjected to the test and results compared to a commercial manual typing-PCR assay and NGS. ResultsThe multiplex assay is highly sensitive for detection of SARS-CoV-2 RNA in clinical samples, with an LoD of 25.82 cp/ml (CI: 11.61 - 57.48). LoDs are slightly higher for the HV68/70 deletion (111.36 cp/ml; CI: 78.16 - 158.67) and the N501Y SNP (2548.04 cp/ml, CI: 1592.58 - 4076.73). A total of 141 clinical samples were tested with the assay, including 16 samples containing SARS-CoV-2 of the B.1.1.7 lineage. Three non-B.1.1.7 samples contained a HV69/70 deletion. All were correctly identified by the multiplex assay. ConclusionWe describe here a highly sensitive, fully automated multiplex PCR assay for the simultaneous detection of del-HV69/70 and N501Y that can distinguish between lineages B.1.1.7 and B1.351. The assay allows for high-throughput screening for relevant variants in clinical samples prior to sequencing.

Clinical evaluation of a fully automated, lab developed multiplex RT-PCR assay integrating dual-target SARS-CoV-2 and Influenza-A/B detection on a high-throughput platform (preprint)

1BackgroundLaboratories worldwide face high demands for molecular testing during the SARS-CoV-2 pandemic that might be further aggravated with the upcoming influenza season in the northern hemisphere. Considering that symptoms of influenza are largely undistinguishable from COVID-19, both SARS-CoV-2 and the Influenza viruses require concurrent testing by RT-PCR in patients presenting with symptoms of respiratory tract infection. In this study, we adapted and evaluated a laboratory developed multiplex RT-PCR assay for simultaneous detection of SARS-CoV-2 (dual-target), Influenza-A and Influenza-B (SC2/InflA/InflB-UCT) on a fully automated high-throughput system (cobas6800). MethodsAnalytical performance was assessed by serial dilution of quantified reference material and cell culture stocks in transport medium, including pre-treatment for chemical inactivation. For clinical evaluation, residual portions of 164 predetermined patient samples containing SARS-CoV-2 (n=52), Influenza-A (n=43) or Influenza-B (n=19), as well as a set of negative samples was subjected to the novel multiplex assay. ResultsThe assay demonstrated analytical performance comparable to currently available commercial tests, with limits of detection of 94.9 cp/ml for SARS-CoV-2, 14.6 cp/ml for Influenza-A and 422.3 cp/ml for Influenza-B. Clinical evaluation showed excellent agreement with the comparator assays (sensitivity 98.1%, 97.7% and 100% for Sars-CoV-2, Influenza-A and -B respectively). ConclusionThe SC2/InflA/InflB-UCT allows for efficient high-throughput testing for all three pathogens and thus provides streamlined diagnostics while conserving resources during the Influenza-season. HighlightsO_LISimultaneous detection of highly pathogenic respiratory viruses Influenza-A/B and SARS-CoV-2 C_LIO_LIIncluding a dual-target assay for SARS-CoV-2 detection C_LIO_LIFull automation on the cobas6800 high-throughput platform C_LI

Pushing beyond specifications: Evaluation of linearity and clinical performance of a fully automated SARS-CoV-2 RT-PCR assay for reliable quantification in blood and other materials outside recommendations (preprint)

1BackgroundThe ongoing SARS-CoV-2 pandemic presents a unique challenge to diagnostic laboratories. There are preliminary studies correlating qRT-PCR results from different materials to clinical outcomes, yet, comparability is limited due to the plethora of different assays used for diagnostics. In this study we evaluate clinical performance and linear range for the SARS-CoV-2 IVD (cobas6800/8800 system, a fully automated sample-to-result platform) in different clinically relevant matrix materials outside official specifications. MethodsAssay performance was assessed in human plasma, BAL/BL and transport medium following chemical inactivation. For analytical evaluation, respective matrix materials were spiked with SARS-CoV-2 RNA in ten-fold dilution series. The efficacy of chemical inactivation by guanidine hydrochloride solution was confirmed in cell culture infectivity experiments. For correlation, a total of 235 predetermined clinical samples including respiratory swabs, plasma and BAL/BL were subjected to the SARS-CoV-2 IVD test and results were compared. ResultsThe SARS-CoV-2 IVD showed excellent linearity over five to seven log steps depending on matrix material. Chemical inactivation resulted in a reduction in plaque forming units of at least 3.5 log steps, while having no significant impact on assay performance. Inter-run consistency from three different testing sites demonstrated excellent comparability of RT-PCR results (maximum deviation was 1.53 CT). Clinical evaluation for respiratory swabs showed very good agreement with the comparator assay (Positive agreement 95.7%, negative agreement 98.9%). ConclusionThe SARS-CoV-2 IVD test for the cobas6800/8800 systems offers excellent linear range and inter-run consistency for quantification of SARS-CoV-2 RNA in different matrices outside official specifications. HighlightsO_LIEffective reduction of SARS-CoV-2 infectivity by chemical inactivation without affecting assay performance. C_LIO_LISARS-CoV-2 IVD for the cobas 6800/8800 is linear over up to seven log steps in different materials including human plasma. C_LIO_LIMinimal variance of CT values between testing sites indicates high comparability of quantification results. C_LI