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

Objective To evaluate the SARS-CoV-2 anti-spike IgG antibodies production after the vaccination with BNT162b2 and the protection from symptomatic breakthrough infections in healthcare workers. Patients and methods This prospective observational study (RENAISSANCE) had as primary endpoint the evaluation of serologic response to BNT162b2 14-days after second dose. SARS-CoV-2 anti-spike IgG antibodies were evaluated with LIAISON® SARS-CoV-2 TrimericS IgG assay, able to detect presence of both binding and neutralizing antibodies for trimeric Spike glycoprotein. Subjects were recruited from February 1, 2021 to February 22, 2021. Occurrence of vaccine-breakthrough infections was assessed by RT-PCR on symptomatic/contact cases, up to June 6, 2021. Results Out of 2569 staff only were 4 non-responders (0.16%, 95%CI:0.04%-0.41%). All 4 non-responders were severely immunosuppressed and on treatment with mycophenolate mofetil/mycophenolic acid. At 14-days since 2° dose, the 67.5% of staff had anti-S IgG titers ≥2000 BAU/mL, 19.2% between 1500- 2000 BAU/ml, 9.8% between 1000-1500 BAU/ml, and 3.4% ≤1000 BAU/ml. Females had a higher probability of having higher titers than males (64.5% [1044/1618] vs. 58.3% [410/703]; p=0.005). This was confirmed after adjusting for age group (OR [95%CI]: 1.275 [1.062-1.531]; p=0.009). Four months after the end of vaccination program, only 13 subjects (0.26%) had experienced a breakthrough SARS-CoV-2 infection, including 1 non-responder. This subject was the only requiring hospitalization for severe COVID-19. Conclusion Vaccination campaign among healthcare workers at the ASST GOM Niguarda has resulted in a significative serologic response, and reduction of incident COVID-19 cases. Yet, the lack of protection should not be overlooked in immunocompromised subjects.

To complement RT-qPCR testing for diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, many countries have introduced the use of rapid antigen tests. As they generally display lower real-life performances than expected, their correct positioning as frontline screening is still controversial. Despite the lack of data from daily clinical use, third generation microfluidic assays (such as the LumiraDx SARS-CoV-2 Ag test) have recently been suggested to have similar performances to RT-qPCR and have been proposed as alternative diagnostic tools. By analyzing 960 nasopharyngeal swabs from 960 subjects at the emergency department admissions of a tertiary COVID-19 hospital, LumiraDx assay demonstrated a specificity of 97% (95% CI: 96–98), and a sensitivity of 85% (95% CI: 82–89) in comparison with RT-qPCR, which increases to 91% (95% CI: 86–95) for samples with a cycle threshold ≤ 29. Fifty false-negative LumiraDx-results were confirmed by direct quantification of genomic SARS-CoV-2 RNA through droplet-digital PCR (median (IQR) load = 5880 (1657–41,440) copies/mL). Subgenomic N and E RNAs were detected in 52% (n = 26) and 56% (n = 28) of them, respectively, supporting the presence of active viral replication. Overall, the LumiraDx test complies with the minimum performance requirements of the WHO. Yet, the risk of a misrecognition of patients with active COVID-19 persists, and the need for confirmatory RT-qPCR should not be amended.

Sequenicing cOronavirus and Variability Analysis (SCoVA) Study Group. The authors also thank all of the staff of the Microbiology and Virology Laboratory of ASST Grande Ospedale Metropolitano Niguarda for their outstanding technical support in processing swab samples and performing laboratory analyses and data management.Disclaimer. The funding source had no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the paper for publication.Financial support.