ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections

Marcolungo, Luca; Beltrami, Cristina; Esposti, Chiara Degli; Lopatriello, Giulia; Piubelli, Chiara; Mori, Antonio; Pomari, Elena; Deiana, Michela; Scarso, Salvatore; Bisoffi, Zeno; Grosso, Valentina; Cosentino, Emanuela; Maestri, Simone; Lavezzari, Denise; Iadarola, Barbara; Paterno, Marta; Segala, Elena; Giovannone, Barbara; Gallinaro, Martina; Rossato, Marzia; Delledonne, Massimo

doi:10.1016/j.ygeno.2021.04.008

Cited by 10 publications

(16 citation statements)

References 63 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…COVID-19 diagnosis was performed by reverse-transcriptase real time PCR (RT-qPCR) on nasopharyngeal swab according to WHO guidelines (available at https://www.who.int/publications/ i/item/diagnostic-testing-for-sars-cov-2) and applying the CDC 2019-nCoV rRT-PCR Diagnostic Panel assay and protocol (available at https://www.fda.gov/media/134922/download) (21). IgG anti-SARS-CoV2 nucleocapsid protein (IgG-N) and IgM anti-SARS-CoV2 spike protein (IgM-S), were measured in patients' serum using a chemiluminescent microparticle immunoassays (CMIA) (Abbott, Ireland) following manufacturer's instructions.…”

Section: Sars-cov-2 Molecular and Serological Testsmentioning

confidence: 99%

Regulatory T Cells as Predictors of Clinical Course in Hospitalised COVID-19 Patients

et al. 2021

Self Cite

View full text Add to dashboard Cite

BackgroundThe host immune response has a prominent role in the progression and outcome of SARS-CoV-2 infection. Lymphopenia has been described as an important feature of SARS-CoV-2 infection and has been associated with severe disease manifestation. Lymphocyte dysregulation and hyper-inflammation have been shown to be associated with a more severe clinical course; however, a T cell subpopulation whose dysfunction correlate with disease progression has yet to be identify.MethodsWe performed an immuno-phenotypic analysis of T cell sub-populations in peripheral blood from patients affected by different severity of COVID-19 (n=60) and undergoing a different clinical evolution. Clinical severity was established based on a modified WHO score considering both ventilation support and respiratory capacity (PaO2/FiO2 ratio). The ability of circulating cells at baseline to predict the probability of clinical aggravation was explored through multivariate regression analyses.ResultsThe immuno-phenotypic analysis performed by multi-colour flow cytometry confirmed that patients suffering from severe COVID-19 harboured significantly reduced circulating T cell subsets, especially for CD4+ T, Th1, and regulatory T cells. Peripheral T cells also correlated with parameters associated with disease severity, i.e., PaO2/FiO2 ratio and inflammation markers. CD4+ T cell subsets showed an important significant association with clinical evolution, with patients presenting markedly decreased regulatory T cells at baseline having a significantly higher risk of aggravation. Importantly, the combination of gender and regulatory T cells allowed distinguishing between improved and worsened patients with an area under the ROC curve (AUC) of 82%.ConclusionsThe present study demonstrates the association between CD4+ T cell dysregulation and COVID-19 severity and progression. Our results support the importance of analysing baseline regulatory T cell levels, since they were revealed able to predict the clinical worsening during hospitalization. Regulatory T cells assessment soon after hospital admission could thus allow a better clinical stratification and patient management.

show abstract

Section: Sars-cov-2 Molecular and Serological Testsmentioning

confidence: 99%

Regulatory T Cells as Predictors of Clinical Course in Hospitalised COVID-19 Patients

et al. 2021

Self Cite

View full text Add to dashboard Cite

show abstract

“…RNA from 10 SARS-CoV-2 positive swab samples was extracted with STARMag 96 x 4 viral DNA/RNA 200 C kit * (Seegene, Seoul, Korea) and included in the ARTIC Network suggested v2 protocol ( 1 ) for viral genome sequencing. For each sample, 5 μl of purified RNA has been used for first-strand cDNA synthesis using SuperScript IV reverse transcriptase (Invitrogen, CA, USA) with modifications according to ( 9 ). Briefly, after the denaturation (65°C for 5 min followed by 1 min in ice) of the first mix - containing Random Examer (Invitrogen), dNTPs (Invitrogen), and the RNA - a second mix containing RT Buffer, 100 mM DTT, SuperScript-IV RT Enzyme, RNaseOUT (Invitrogen), and DEPC-treated water (Invitrogen) was added in a final volume of 20 μl.…”

Section: Methodsmentioning

confidence: 99%

Genomic Snapshot of SARS-CoV-2 in Migrants Entering Through Mediterranean Sea Routes

Grandi

Paglietti

Cusano

et al. 2022

Front. Public Health

View full text Add to dashboard Cite

In December 2019, a novel coronavirus emerged in Wuhan, China, rapidly spreading into a global pandemic. Italy was the first European country to experience SARS-CoV-2 epidemic, and one of the most severely affected during the first wave of diffusion. In contrast to the general restriction of people movements in Europe, the number of migrants arriving at Italian borders via the Mediterranean Sea route in the summer of 2020 had increased dramatically, representing a possible, uncontrolled source for the introduction of novel SARS-CoV-2 variants. Importantly, most of the migrants came from African countries showing limited SARS-CoV-2 epidemiological surveillance. In this study, we characterized the SARS-CoV-2 genome isolated from an asymptomatic migrant arrived in Sardinia via the Mediterranean route in September 2020, in comparison with SARS-CoV-2 isolates arrived in Sicily through the Libyan migration route; with SARS-CoV-2 isolates circulating in Sardinia during 2020; and with viral genomes reported in African countries during the same summer. Results showed that our sequence is not phylogenetically related to isolates from migrants arriving in Sicily, nor to isolates circulating in Sardinia territory, having greater similarity to SARS-CoV-2 genomes reported in countries known for being sites of migrant embarkation to Italy. This is in line with the hypothesis that most SARS-CoV-2 infections among migrants have been acquired prior to embarking to Italy, possibly during the travel to or the stay in crowded Libyan immigrant camps. Overall, these observations underline the importance of dedicated SARS-CoV-2 surveillance of migrants arriving in Italy and in Europe through the Mediterranean routes.

show abstract

“…The products were purified using a 0.9× ratio of AMPureXP beads (Beckman Coulter, Brea, CA, USA) and amplicons were eluted in 15 µL water. The positive control sample was a SARS-CoV-2 cDNA generated using primers 96F (5'-GCCAACAACAACAAGGCCAAAC-3') and 96R (5'-TAGGCTCTGTTGGTGGGAATGT-3') from the ARTIC v3 protocol [49]. We amplified 3ng cDNA in triplicate 50 µL reactions containing 10µM of each primer using Q5 High Fidelity DNA Polymerase (New England Biolabs).…”

Section: Amplicon Generationmentioning

confidence: 99%

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

et al. 2022

Self Cite

View full text Add to dashboard Cite

Rapid and sensitive assays for the identification of plant pathogens are necessary for the effective management of crop diseases. The main limitation of current diagnostic testing is the inability to combine broad and sensitive pathogen detection with the identification of key strains, pathovars, and subspecies. Such discrimination is necessary for quarantine pathogens, whose management is strictly dependent on genotype identification. To address these needs, we have established and evaluated a novel all-in-one diagnostic assay based on nanopore sequencing for the detection and simultaneous characterization of quarantine pathogens, using Xylella fastidiosa as a case study. The assay proved to be at least as sensitive as standard diagnostic tests and the quantitative results agreed closely with qPCR-based analysis. The same sequencing results also allowed discrimination between subspecies when present either individually or in combination. Pathogen detection and typing were achieved within 13 min of sequencing owing to the use of an internal control that allowed to stop sequencing when sufficient data had accumulated. These advantages, combined with the use of portable equipment, will facilitate the development of next-generation diagnostic assays for the efficient monitoring of other plant pathogens.

show abstract

ACoRE: Accurate SARS-CoV-2 genome reconstruction for the characterization of intra-host and inter-host viral diversity in clinical samples and for the evaluation of re-infections

Cited by 10 publications

References 63 publications

Regulatory T Cells as Predictors of Clinical Course in Hospitalised COVID-19 Patients

Regulatory T Cells as Predictors of Clinical Course in Hospitalised COVID-19 Patients

Genomic Snapshot of SARS-CoV-2 in Migrants Entering Through Mediterranean Sea Routes

Real-Time On-Site Diagnosis of Quarantine Pathogens in Plant Tissues by Nanopore-Based Sequencing

Contact Info

Product

Resources

About