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

被引：92

作者：

Alteri, Claudia ^{[1
]}

Cento, Valeria ^{[2
]}

Antonello, Maria ^{[1
]}

Colagrossi, Luna ^{[3
]}

Merli, Marco ^{[4
]}

Ughi, Nicola ^{[5
]}

Renica, Silvia ^{[1
]}

Matarazzo, Elisa ^{[2
]}

Di Ruscio, Federica ^{[2
]}

Tartaglione, Livia ^{[2
]}

Colombo, Jacopo ^{[6
]}

Grimaldi, Chiara ^{[7
]}

Carta, Stefania ^{[7
]}

Nava, Alice ^{[7
]}

Costabile, Valentino ^{[8
]}

Baiguera, Chiara ^{[4
]}

Campisi, Daniela ^{[7
]}

Fanti, Diana ^{[7
]}

Vismara, Chiara ^{[7
]}

Fumagalli, Roberto ^{[9
]}

Scaglione, Francesco ^{[1
]}

Epis, Oscar Massimiliano ^{[5
]}

Puoti, Massimo ^{[4
]}

Perno, Carlo Federico ^{[1
,7
]}

机构：

[1] Univ Milan, Dept Oncol & Hematooncol, Milan, Italy

[2] Univ Milan, Residency Microbiol & Virol, Milan, Italy

[3] Bambino Gesu Pediat Hosp, Dept Labs, Rome, Italy

[4] ASST Grande Osped Metropolitano Niguarda, Infect Dis, Milan, Italy

[5] ASST Grande Osped Metropolitano Niguarda, Rheumatol Unit, Milan, Italy

[6] ASST Grande Osped Metropolitano Niguarda, Dept Cardiotoracovasc Anesthesia & Intens Care, Milan, Italy

[7] ASST Grande Osped Metropolitano Niguarda, Dept Lab Med, Milan, Italy

[8] Univ Milan, Dept Pathophysiol & Transplantat, Milan, Italy

[9] ASST Grande Osped Metropolitano Niguarda, Dept Anesthesiol Crit Care & Pain Med, Milan, Italy

来源：

PLOS ONE | 2020年 / 15卷 / 09期

关键词：

POLYMERASE-CHAIN-REACTION; CORONAVIRUS; DIAGNOSIS;

D O I：

10.1371/journal.pone.0236311

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页数：10

共 18 条

[1] Quantification of HIV-DNA and residual viremia in patients starting ART by droplet digital PCR: Their dynamic decay and correlations with immunological parameters and virological success [J].