TY - JOUR
T1 - Detection and quantification of SARS-CoV-2 by droplet digital PCR in real-time PCR negative nasopharyngeal swabs from suspected COVID-19 patients
AU - Alteri, Claudia
AU - Cento, Valeria
AU - Antonello, Maria
AU - Colagrossi, Luna
AU - Merli, Marco
AU - Ughi, Nicola
AU - Renica, Silvia
AU - Matarazzo, Elisa
AU - Ruscio, Federica Di
AU - Tartaglione, Livia
AU - Colombo, Jacopo
AU - Grimaldi, Chiara
AU - Carta, Stefania
AU - Nava, Alice
AU - Costabile, Valentino
AU - Baiguera, Chiara
AU - Campisi, Daniela
AU - Fanti, Diana
AU - Vismara, Chiara
AU - Fumagalli, Roberto
AU - Scaglione, Francesco
AU - Epis, Oscar Massimiliano
AU - Puoti, Massimo
AU - Perno, Carlo Federico
N1 - Funding Information:
This work was financially supported by an unrestricted grant from AVIRALIA foundation. We thank Bio-Rad Italia for providing technical support, particularly Dr. Laura Sard, Dr. Marilisa Marinelli and Dr. Marco Tronconi and Medical Affairs Bio-Rad team, for their valuable assistance in the implementation of the study. The authors also thank Dr. Silvia Nerini and all the staff of the Microbiology and Virology Laboratory of ASST Grande Ospedale Metropolitano Niguarda for outstanding technical support in processing swab samples, performing laboratory analyses and data management.
Publisher Copyright:
© 2020 Alteri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2020/9
Y1 - 2020/9
N2 - 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.
AB - 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.
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U2 - 10.1371/journal.pone.0236311
DO - 10.1371/journal.pone.0236311
M3 - Article
C2 - 32898153
AN - SCOPUS:85090723099
SN - 1932-6203
VL - 15
JO - PLoS One
JF - PLoS One
IS - 9 September
M1 - e0236311
ER -