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

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...

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Published in:PloS one 2020-09, Vol.15 (9), p.e0236311-e0236311
Main Authors: Alteri, Claudia, Cento, Valeria, Antonello, Maria, Colagrossi, Luna, Merli, Marco, Ughi, Nicola, Renica, Silvia, Matarazzo, Elisa, Di Ruscio, Federica, Tartaglione, Livia, Colombo, Jacopo, Grimaldi, Chiara, Carta, Stefania, Nava, Alice, Costabile, Valentino, Baiguera, Chiara, Campisi, Daniela, Fanti, Diana, Vismara, Chiara, Fumagalli, Roberto, Scaglione, Francesco, Epis, Oscar Massimiliano, Puoti, Massimo, Perno, Carlo Federico
Format: Article
Language:English
Subjects:
Aged
Aged, 80 and over
Antibodies
Assaying
Betacoronavirus - genetics
Betacoronavirus - isolation & purification
Biology and life sciences
Computed tomography
Coronavirus Infections - diagnosis
Coronavirus Infections - pathology
Coronavirus Infections - virology
Coronaviruses
COVID-19
Diagnosis
Diagnostic tests
Disease transmission
Evaluation
Female
Genomes
Humans
Infectious diseases
Influenza
Intensive care
Laboratories
Limit of Detection
Male
Medical research
Medicine
Medicine and health sciences
Methods
Middle Aged
Molecular diagnostic techniques
Nasopharynx - virology
Oncology
Pandemics
Patients
Pneumonia
Pneumonia, Viral - diagnosis
Pneumonia, Viral - pathology
Pneumonia, Viral - virology
Polymerase chain reaction
Real-Time Polymerase Chain Reaction - methods
Regression analysis
Reverse transcription
Rheumatology
RNA polymerase
RNA, Viral - analysis
RNA, Viral - metabolism
SARS-CoV-2
Severe acute respiratory syndrome
Severe acute respiratory syndrome coronavirus 2
Severity of Illness Index
Subgroups
Viral diseases
Viral Load
Virology
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Summary: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
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0236311