COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Eprobe mediated real-time PCR monitoring and melting curve analysis.

Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3' end-blocked Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to the same nucleotide and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Intercalation of dyes can further stabilize the DNA/DNA hybrid and increase the melting temperature compared to standard DNA oligonucleotides. Eprobes allow for specific real-time monitoring of amplification reactions by hybridizing to the amplicon in a sequence-dependent manner. Similarly, Eprobes allow for analysis of reaction products by melting curve analysis. The function of different Eprobes was studied using the L858R mutation in the human epidermal growth factor receptor (EGFR) gene, and multiplex detection was demonstrated for the human EGFR and KRAS genes using Eprobes with two different dyes. Combining amplification and melting curve analysis in a single-tube reaction provides powerful means for new mutation detection assays. Functioning as "sequence-specific dyes", Eprobes hold great promises for future applications not only in PCR but also as hybridization probes in other applications

Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays.

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download

Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism.

International audienceOBJECTIVES: To determine whether genotyping of HIV-1 by duplicate PCR amplification of the region encoding the V3 loop is more sensitive than single PCR for detecting CXCR4-using viruses. PATIENTS AND METHODS: The V3 genotypes of the HIV-1 infecting 152 patients enrolled in the multicentre GenoTropism ANRS study were determined by all the participating laboratories using a single PCR and V3 bulk sequencing. In parallel, one laboratory determined the V3 genotype using duplicate PCR and bulk sequencing of pooled amplicons. HIV tropism was predicted with the geno2pheno10 algorithm. The phenotypes of all samples were determined with the Trofile assay and the Toulouse tropism test (TTT) recombinant virus assay. RESULTS: Geno2pheno10 was 56.8% sensitive and 75.9% specific when compared with the Trofile assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and bulk sequencing of the pooled PCR amplicons increased the sensitivity to 68.2% and specificity to 79.6%. Geno2pheno10 was 64.1% sensitive and 77.0% specific when compared with the TTT assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and sequencing of the pooled PCR amplicons increased sensitivity to 76.9% and specificity to 80.5%. CONCLUSIONS: The genotypic determination of HIV-1 tropism can be improved by duplicate amplifications and sequencing the pooled PCR products. This is a good compromise between improved sensitivity and reasonable cost for the genotype-based determination of tropism

Eprobe mediated mutation detection.

<p>Asymmetric PCR was performed to enrich the reverse strand for mutation detection using different templates and Eprobes. For further details refer to Materials and Methods. A: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 5×10⁴ copies of plasmid DNA using Eprobe 205-13 wt TO. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature of asymmetric PCR experiments with 1 ng genomic DNA using Eprobe 215-21 wt TP. Wild-type to mutation ratios are indicated by colors (red: 100% wild-type, Yellow: 100% mutation, blue: 50% wild-type and 50% mutation, green: negative control).</p

Eprobes used for this study.

<p>(T_M values for TO-labeled Eprobes were estimated using the following settings: 0.05 M Na⁺, 0.002 M Mg²⁺, 0.2 µM Eprobe, 60°C Temperature; *T_M values for Eprobe 215-21 wt TP are actual values; in Eprobe name: TO = D514, TP = D570; Z indicating position of modified T).</p

Eprobes in multiplex detection.

<p>Regions of the human EGFR and KRAS genes were amplified from genomic DNA and amplification was monitored by specific Eprobes having different dyes. A: Amplification curves plotting random fluorescent units (RFU) obtained from LightCycler 480 against PCR cycle number (Red: genomic DNA, Green: negative control). B: Differential melting curve analysis by plotting –dF/dT against temperature (Red: genomic DNA, Green: negative control). Plots in yellow on the left show signals for KRAS using dye D514 (Eprobe KWT14m3 TO); plots in pink on the right show signals for EGFR using dye D570 (Eprobe 215-21 wt TP).</p

Eprobe derived background.

<p>A. Eprobe 215-21 wt TO labeled with D514 was digested as described in the Materials and Methods and fluorescence excitation spectra from 495 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). B. Eprobe 215-21 wt TP labeled with dye D570 was digested as described in the Materials and Methods and fluorescence excitation spectra from 550 nm to 800 nm were recorded before (blue) and after Eprobe digestion (red). C. Plotting random fluorescent units (RFU, mean values from triplicate data for each experiment) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against plasmid DNA template concentration (from 150 to 150,000,000 copies per reaction). Eprobe concentrations are indicated by the different colors (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). D. Plotting random fluorescent units (RFU) obtained from LightCycler 480 and Eprobe 205-13 wt TO at the beginning of the PCR reactions against Eprobe concentration (dark blue: 100 nM, light blue: 200 nM, red: 300 nM, purple: 400 nM, light green: 500 nM). For each data point, mean values plus error bars from triplicate values for each experiment and for all template concentrations are given in the graph.</p

Structure and function of Eprimer and Eprobe.

<p>A. Chemical structure of modified deoxythymidine carrying two dye moieties. B. Chemical structure of D514 “Thiazole Orange”. C. Chemical structure of D570 “Thiazole Pink”. D. Signal generation by an arbitrary Eprimer (ECHO), where the two dye moieties are paired in the single-stranded oligonucleotide for signal suppression. Upon hybridization to complementary strand, dye moieties get separated and intercalate into the double-strand leading to emission of strong fluorescence. E. Structure of an arbitrary Eprobe having a blocked 3′ end to prevent primer extension during PCR.</p

Agarose gel electrophoresis of PCR products.

<p>A: PCR products from real-time PCR experiments were analyzed on a 3% agarose gel. Lanes: 1: TaqMan Probe, 2:, Eprobe 203-10 wt TO 3: C3-blocked 203-10 wt oligonucleotide, 4: Eprobe 205-13 wt TO, 5: Eprobe205-13m TO, 6: C3-blocked 205-13 wt oligonucleotide, 7: Eprobe 215-21 wt TO, 8: Eprobe 215-21m TO, 9: C3-blocked 215-21 wt oligonucleotide, 10: Eprobe 215-21 wt TP, 11: SYBR Green I, M: marker. B: Partial sequence of human EGFR gene. Location of the primers (blue boxes) and the mutation L858R (red box) are indicated.</p