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

PLoS One. 2020 Sep 8;15(9):e0236311. doi: 10.1371/journal.pone.0236311. eCollection 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Betacoronavirus / genetics*
  • Betacoronavirus / isolation & purification
  • Coronavirus Infections / diagnosis*
  • Coronavirus Infections / pathology
  • Coronavirus Infections / virology
  • Female
  • Humans
  • Limit of Detection
  • Male
  • Middle Aged
  • Nasopharynx / virology*
  • Pandemics
  • Pneumonia, Viral / diagnosis*
  • Pneumonia, Viral / pathology
  • Pneumonia, Viral / virology
  • RNA, Viral / analysis*
  • RNA, Viral / metabolism
  • Real-Time Polymerase Chain Reaction / methods*
  • Severity of Illness Index
  • Viral Load

Substances

  • RNA, Viral

Supplementary concepts

  • COVID-19
  • severe acute respiratory syndrome coronavirus 2

Grant support

This work was financially supported by an unrestricted grant from AVIRALIA foundation.