SARS-CoV-2 blood RNA load predicts outcome in critically ill COVID-19 patients

Background SARS-CoV-2 RNA loads in patient specimens may act as a clinical outcome predictor in critically ill patients with COVID-19. Methods We evaluated the predictive value of viral RNA loads and courses in the blood compared to the upper and lower respiratory tract loads of critically ill COVID-19 patients. Daily specimen collection and viral RNA quantification by RT-qPCR was performed in all consecutive 170 COVID-19 patients between March 2020 and February 2021 during the entire ICU stay (4145 samples analyzed). Patients were grouped according to their 90-days outcome as survivors (n=100) or non-survivors (n=70). Results In non-survivors, blood SARS-CoV-2 RNA loads were significantly higher at the time of admission to the ICU (p=0.0009). Failure of blood RNA clearance was observed in 33/50 (66 %) of the non-survivors compared to 12/64 (19 %) of survivors (p<0.0001). As determined by multivariate analysis, taking sociodemographic and clinical parameters into account, blood SARS-CoV-2 RNA load represents a valid and independent predictor of outcome in critically ill COVID-19 patients (OR [log10]: 0.23 [0.12 – 0.42], p<0.0001) with a significantly higher effect for survival compared to the respiratory tract SARS-CoV-2 RNA loads (OR [log10]: 0.75 [0.66 – 0.85], p<0.0001). Blood RNA loads exceeding 2.51 x 10 3 SARS-CoV-2 RNA copies/ml were found to indicate a 50% probability of death. Consistently, 29/33 (88%) of the non-survivors with failure of virus clearance exceeded this cut-off value constantly. Conclusion Blood SARS-CoV-2 load is an important independent outcome predictor and should be further evaluated for treatment allocation and patient monitoring.

Nested pool testing strategy for the diagnosis of infectious diseases

The progress of the SARS-CoV-2 pandemic requires the design of large-scale, cost-effective testing programs. Pooling samples provides a solution if the tests are sensitive enough. In this regard, the use of the gold standard, RT-qPCR, raises some concerns. Recently, droplet digital PCR (ddPCR) was shown to be 10–100 times more sensitive than RT-qPCR, making it more suitable for pooling. Furthermore, ddPCR quantifies the RNA content directly, a feature that, as we show, can be used to identify nonviable samples in pools. Cost-effective strategies require the definition of efficient deconvolution and re-testing procedures. In this paper we analyze the practical implementation of an efficient hierarchical pooling strategy for which we have recently derived the optimal, determining the best ways to proceed when there are impediments for the use of the absolute optimum or when multiple pools are tested simultaneously and there are restrictions on the throughput time. We also show how the ddPCR RNA quantification and the nested nature of the strategy can be combined to perform self-consistency tests for a better identification of infected individuals and nonviable samples. The studies are useful to those considering pool testing for the identification of infected individuals.

Combining Protein and RNA Quantification to Evaluate Promoter Activity by Using Dual-color Fluorescent Reporting Systems

Herein, a Broccoli/mCherry and an EGFP/mCherry dual-color fluorescent reporting systems have been established to quantify the promoter activity at transcription and translation level in eukaryotic cells. Based on those systems, four commonly used promoters (CMV and SV40 of Pol II and U6, H1 of Pol III) were accurately evaluated at both the transcriptional and translational levels by combining accurate protein and RNA quantification. Furthermore, we verified that Pol III promoters can induce proteins expression, and Pol II promoter can be applied to express RNA molecules with defined length by combining a self-cleaving ribozyme and an artificial poly(A) tail. The dual-color fluorescence reporting systems described here could play a significant role in evaluating other gene expression regulators for gene therapy.

RT-qPCR Diagnostics: The “Drosten” SARS-CoV-2 Assay Paradigm

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first assays targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level, and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail, and our findings reveal some limitations but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Nevertheless, whilst our data show that some errors can be tolerated, it is always prudent to confirm that the primer and probe sequences complement their intended target, since, when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case, it is unlikely that a mismatch will result in poor specificity or a significant number of false-positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

Effect of storage conditions on SARS-CoV-2 RNA quantification in wastewater solids

SARS-CoV-2 RNA in wastewater settled solids is associated with COVID-19 incidence in sewersheds and therefore, there is a strong interest in using these measurements to augment traditional disease surveillance methods. A wastewater surveillance program should provide rapid turn around for sample measurements (ideally within 24 hours), but storage of samples is necessary for a variety of reasons including biobanking. Here we investigate how storage of wastewater solids at 4 °C, −20 °C, and −80 °C affects measured concentrations of SARS-CoV-2 RNA. We find that short term (7 or 8 d) storage of raw solids at 4 °C has little effect on measured concentrations of SARS-CoV-2 RNA, whereas longer term storage at 4 °C (35–122 d) or freezing reduces measurements by 60%, on average. We show that normalizing SARS-CoV-2 RNA concentrations by concentrations of pepper mild mottle virus (PMMoV) RNA, an endogenous wastewater virus, can correct for changes during storage as storage can have a similar effect on PMMoV RNA as on SARS-CoV-2 RNA. The reductions in SARS-CoV-2 RNA in solids during freeze thaws is less than those reported for the same target in liquid influent by several authors.

RT-qPCR Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target, since when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

Rt-Qpcr Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target as when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.