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 samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.

Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients / C. Alteri, V. Cento, M. Antonello, L. Colagrossi, M. Merli, N. Ughi, S. Renica, E. Matarazzo, F.D. Ruscio, L. Tartaglione, J. Colombo, C. Grimaldi, S. Carta, A. Nava, V. Costabile, C. Baiguera, D. Campisi, D. Fanti, C. Vismara, R. Fumagalli, F. Scaglione, O.M. Epis, M. Puoti, C.F. Perno. - In: PLOS ONE. - ISSN 1932-6203. - 15:9(2020), pp. e0236311.1-e0236311.10. [10.1371/journal.pone.0236311]

Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients

C. Alteri
;
V. Cento;M. Antonello;L. Colagrossi;S. Renica;L. Tartaglione;V. Costabile;C.F. Perno
2020

Abstract

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 samples of individuals with COVID-19 suspect, allowing the rapid taking care and correct management of these patients.
No
English
Settore MED/07 - Microbiologia e Microbiologia Clinica
Articolo
Esperti anonimi
Pubblicazione scientifica
2020
Public Library of Science
15
9
e0236311
1
10
10
Pubblicato
Periodico con rilevanza internazionale
Aderisco
info:eu-repo/semantics/article
Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients / C. Alteri, V. Cento, M. Antonello, L. Colagrossi, M. Merli, N. Ughi, S. Renica, E. Matarazzo, F.D. Ruscio, L. Tartaglione, J. Colombo, C. Grimaldi, S. Carta, A. Nava, V. Costabile, C. Baiguera, D. Campisi, D. Fanti, C. Vismara, R. Fumagalli, F. Scaglione, O.M. Epis, M. Puoti, C.F. Perno. - In: PLOS ONE. - ISSN 1932-6203. - 15:9(2020), pp. e0236311.1-e0236311.10. [10.1371/journal.pone.0236311]
open
Prodotti della ricerca::01 - Articolo su periodico
24
262
Article (author)
no
C. Alteri, V. Cento, M. Antonello, L. Colagrossi, M. Merli, N. Ughi, S. Renica, E. Matarazzo, F.D. Ruscio, L. Tartaglione, J. Colombo, C. Grimaldi, S....espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/803267
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