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

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.

SARS-CoV-2 infects carotid arteries: implications for vascular disease and organ injury in COVID-19

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.

Low and high infection dose transmission of SARS-CoV-2 in the first COVID-19 clusters in Northern Germany

ObjectivesWe used viral genomics to deeply analyze the first SARS-CoV-2 infection clusters in the metropolitan region of Hamburg, Germany. Epidemiological analysis and contact tracing together with a thorough investigation of virus variant patterns revealed low and high infection dose transmissions to be involved in transmission events. MethodsInfection control measures were applied to follow up contract tracing. Metagenomic RNA- and SARS-CoV-2 amplicon sequencing was performed from 25 clinical samples for sequence analysis and variant calling. ResultsThe index patient acquired SARS-CoV-2 in Italy and after his return to Hamburg transmitted it to 2 out of 132 contacts. Virus genomics and variant pattern clearly confirms the initial local cluster. We identify frequent single nucleotide polymorphisms at positions 241, 3037, 14408, 23403 and 28881 previously described in Italian sequences and now considered as one major genotype in Europe. While the index patient showed a single nucleotide polymorphism only one variant was transmitted to the recipients. Different to the initial cluster, we observed in household clusters occurring at the time in Hamburg also intra-host viral species transmission events. ConclusionsSARS-CoV-2 variant tracing highlights both, low infection dose transmissions suggestive of fomites as route of infection in the initial cluster and high and low infection dose transmissions in family clusters indicative of fomites and droplets as infection routes. This suggests (1) single viral particle infection can be sufficient to initiate SARS-CoV-2 infection and (2) household/family members are exposed to high virus loads and therefore have a high risk to acquire SARS-CoV-2.

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

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

Clinical evaluation of a SARS-CoV-2 RT-PCR assay on a fully automated system for rapid on-demand testing in the hospital setting

1BackgroundThe ongoing SARS-CoV-2 pandemic presents a unique challenge for diagnostic laboratories around the world. Automation of workflows in molecular diagnostics are instrumental for coping with the large number of tests ordered by clinicians, as well as providing fast-tracked rapid testing for highly urgent cases. In this study we evaluated a SARS-CoV-2 LDT for the NeuMoDx 96 system, a fully automated device performing extraction and real-time PCR. MethodsA publicly available SARS-CoV-2 RT-PCR assay was adapted for the automated system. Analytical performance was evaluated using in-vitro transcribed RNA and clinical performance was compared to the cobas 6800-based reference assay within the lab. ResultsThe NeuMoDx-sarbeco-LDT displayed good analytical performance with an LoD of 95.55 cp/ml and no false positives during evaluation of cross-reactivity. A total of 176 patient samples were tested with both the Sarbeco-LDT and the reference assay. Positive and negative agreement were 100% and 99.2% respectively. Invalid-rate was 6.3%. ConclusionThe NeuMoDx-sarbeco-LDT showed analytical and clinical performance comparable to the cobas6800-based reference assay. Due to its random-access workflow concept and rapid time-to-result of about 80 minutes, the device is very well suited for providing fast-tracked SARS-CoV-2 diagnostics for urgent clinical samples in the hospital setting. HighlightsO_LIA publicly available SARS-CoV-2 RT-PCR assay was adapted and evaluated on the open mode of the NeuMoDx 96 system (Qiagen) C_LIO_LIThe assay showed comparable analytical and clinical performance to the reference assay C_LIO_LIFast turn-around times (80 minutes) and random-access workflow of the system makes the assay well suited for urgent clinical samples. C_LI

Targeting the catecholamine-cytokine axis to prevent SARS-CoV-2 cytokine storm syndrome

In Coronavirus disease 2019 (COVID-19), the initial viral-replication phase is often followed by a hyperinflammatory reaction in the lungs and other organ systems that leads to acute respiratory distress syndrome (ARDS), the need for mechanical ventilation, and death despite maximal supportive care. As no antiviral treatments have yet proven effective, efforts to prevent progression to the severe stages of COVID-19 without inhibiting antiviral immune responses are desperately needed. We have previously demonstrated that a common, inexpensive, and well-tolerated class of drugs called alpha-1 adrenergic receptor (1-AR) antagonists can prevent hyperinflammation ("cytokine storm") and death in mice. We here present clinical data that supports the use of 1-AR antagonists in the prevention of severe complications of pneumonia, ARDS, and COVID-19.