Droplet digital PCR (ddPCR) is a sensitive and reproducible technology widely used for quantitation of several viruses. The aim of this study was to evaluate the 2019-nCoV CDC ddPCR Triplex Probe Assay (BioRad) performance, comparing the direct quantitation of SARS-CoV-2 on nasopharyngeal swab with the procedure applied to the extracted RNA. Moreover, two widely used swab types were compared (UTM 3 mL and ESwab 1 mL, COPAN). A total of 50 nasopharyngeal swabs (n = 25 UTM 3 mL and n = 25 ESwab 1 mL) from SARS-CoV-2 patients, collected during the pandemic at IRCCS Sacro Cuore Don Calabria Hospital (Veneto Region, North-East Italy), were used for our purpose. After heat inactivation, an aliquot of swab medium was used for the direct quantitation. Then, we compared the direct method with the quantitation performed on the RNA purified from nasopharyngeal swab by automated extraction. We observed that the direct approach achieved generally equal RNA copies compared to the extracted RNA. The results with the direct quantitation were more accurate on ESwab with a sensitivity of 93.33% [95% CI, 68.05 to 99.83] and specificity of 100.00% for both N1 and N2. On the other hand, on UTM we observed a higher rate of discordant results for N1 and N2. The human internal amplification control (RPP30) showed 100% of both sensitivity and specificity independent of swabs and approaches. In conclusion, we described a direct quantitation of SARS-CoV-2 in nasopharyngeal swab. Our approach resulted in an efficient quantitation, without automated RNA extraction and purification. However, special care needs to be taken on the potential bias due to the conservation of samples and to the heating treatment, as we used thawed and heat inactivated material. Further studies on a larger cohort of samples are warranted to evaluate the clinical value of this direct approach.

Background: We assessed the sensitivity, specificity and positive and negative predictive value (PPV and NPV) of molecular and serological tests for the diagnosis of SARS-CoV-2 infection. Methods: A total of 346 patients were enrolled in the emergency room. We evaluated three Reverse Transcriptase-real time PCRs (RT-PCRs) including six different gene targets, five serologic rapid diagnostic tests (RDT) and one ELISA. The final classification of infected/non-infected patients was performed using Latent Class Analysis combined with clinical re-assessment of incongruous cases. Results: Out of these, 24.6% of patients were classified as infected. The molecular test RQ-SARS-nCoV-2 showed the highest performance with 91.8% sensitivity, 100% specificity, 100.0% PPV and 97.4% NPV respectively. Considering the single gene targets, S and RdRp of RQ-SARS-nCoV-2 had the highest sensitivity (94.1%). The in-house RdRp presented the lowest sensitivity (62.4%). The specificity ranged from 99.2% for in-house RdRp and N2 to 95.0% for E. The PPV ranged from 97.1% of N2 to 85.4% of E and the NPV from 98.1% of S to 89.0% of in-house RdRp. All serological tests had < 50% sensitivity and low PPV and NPV. VivaDiag IgM (RDT) had 98.5% specificity, with 84.0% PPV, but 24.7% sensitivity. Conclusion: Molecular tests for SARS-CoV-2 infection showed excellent specificity, but significant differences in sensitivity. Serological tests have limited utility in a clinical context.

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