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

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

COVID‐19: Virology, biology and novel laboratory diagnosis

“…In view of the need to restrict (as far as possible) false‐negative results in COVID‐19 diagnosis, use of the ddPCR can provide a vital support. Even so, the ddPCR assay is still very rarely studied in clinical settings and there is currently no available evidence for European cases 118 …”

Molecular diagnostic assays for COVID-19: an overview

“…This advantage of digital PCR over RT-PCR was corroborated by other independent studies [ 41 , 42 ]. An investigation of 55 suspected COVID-19 cases who had had previous negative RT-PCR test results showed evidence of SARS-CoV-2 genome in the NP samples of 35% of the tested individuals when they were retested with digital PCR [ 43 ].…”

Accurate bulk quantitation of droplet digital PCR

“…While accurate ddPCR has been demonstrated in polydispersed emulsions when droplets are imaged and counted 32 , it is unclear whether this holds for bulk detection because, when cycled to endpoint, the number of amplicons generated in a droplet scales with its volume. Thus, the total number of amplicons in the recovered pool will depend on the volumes of the positive droplets, which will vary, = 1 1 1 (6) with 1 the amplicon concentration and 1 the volume produced by the ith positive droplet. In the limit of large % , however, the sum can be approximated in terms of the average , simplifying the expression to ≈ % (7) such that Eq.…”

“…By contrast, digital PCR (dPCR) subdivides the sample such that partitions contain one or no target molecule; after end-point amplification, positives are enumerated, yielding the target concentration [2][3][4] . Digital PCR affords numerous advantages over qPCR, including absolute quantitation and enhanced accuracy for small concentration changes, making it especially valuable for clinical applications [5][6][7] . It has secondary benefits, including increased resistance to inhibition [8][9] and the ability to differentiate intact from fragmented molecules [10][11] , which are valuable in the identification of viable pathogens in minimally processed samples [12][13] .…”