Since SARS-CoV-2-based disease (COVID-19) spreads as a pandemic, the necessity of a highly sensitive molecular diagnosis that can drastically reduce false negatives reverse transcription PCR (rtPCR) results, raises as a major clinical need. Here we evaluated the performance of a ddPCR-based assay to quantify SARS-CoV-2 titer in 55 suspected COVID-19 cases with negative rtPCR results thanks to in-house ddPCR assay (targeting RdRp and host RNaseP). Samples were collected at ASST-GOM Niguarda between February and May 2020 at hospital admission. Clinical and imaging data were obtained for clinical staging and definition of disease severity. Patients were mainly female (45.5%) with a median age of 73 (57-84) years. ddPCR-based assay detected SARS-CoV-2 genome in nasopharyngeal samples of 19 (34.5%) patients (median viral-load: 128 copies/mL, IQR: 72-345). In 15 of them (78.9%), chest CT showed a classical COVID-19 bilateral interstitial pneumonia; 14 patients (73.7%) showed severe COVID-19 manifestations. ddPCR did not identify any trace of SARS-CoV-2 genome in the respiratory samples of the remaining 36 patients. The serological assay performed in a subgroup of 34 patients at the later stage of illness (from 3 days to 90 days after) confirmed the presence of SARS-CoV-2 antibodies in all patients tested positive for SARS-CoV-2 in ddPCR (100%). Contrariwise, negative tests were observed in 95.0% ddPCR negative patients (P<0.001). Thanks to a ddPCR-based assay, we achieved a rapid and accurate SARS-CoV-2 diagnosis in rtPCR-negative respiratory

From February to April 2020, Lombardy (Italy) reported the highest numbers of SARS-CoV-2 cases worldwide. By analyzing 346 whole SARS-CoV-2 genomes, we demonstrate the presence of seven viral lineages in Lombardy, frequently sustained by local transmission chains and at least two likely to have originated in Italy. Six single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. The seven lineages, and the presence of local transmission clusters within three of them, revealed that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy.

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From February to April, 2020, Lombardy (Italy) was the area who worldwide registered the highest numbers of SARS-CoV-2 infection. By extensively analyzing 346 whole SARS-CoV-2 genomes, we demonstrated the simultaneous circulation in Lombardy of two major viral lineages, likely derived from multiple introductions, occurring since the second half of January. Seven single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. These two lineages, and the presence of two well defined clusters inside Lineage 1, revealed that a sustained community transmission was ongoing way before the first COVID-19 case found in Lombardy.

Since the start of SARS-CoV-2 pandemic, children aged ≤ 12 years have always been defined as underrepresented in terms of SARS-CoV-2 infections’ frequency and severity. By correlating SARS-CoV-2 transmission dynamics with clinical and virological features in 612 SARS-CoV-2 positive patients aged ≤ 12 years, we demonstrated a sizeable circulation of different SARS-CoV-2 lineages over the four pandemic waves in paediatric population, sustained by local transmission chains. Age < 5 years, highest viral load, gamma and delta clades positively influence this local transmission. No correlations between COVID-19 manifestations and lineages or transmission chains are seen, except for a negative correlation between B.1.1.7 and hospitalization.