ddPCR increases detection of SARS-CoV-2 RNA in patients with low viral loads

Abstract

RT-qPCR detection of SARS-CoV-2 RNA still represents the method of reference to diagnose and monitor COVID-19. From the onset of the pandemic, however, doubts have been expressed concerning the sensitivity of this molecular diagnosis method. Droplet digital PCR (ddPCR) is a third-generation PCR technique that is particularly adapted to detecting low-abundance targets. We developed two-color ddPCR assays for the detection of four different regions of SARS-CoV-2 RNA, including non-structural (IP4-RdRP, helicase) and structural (E, N) protein-encoding sequences. We observed that N or E subgenomic RNAs are generally more abundant than IP4 and helicase RNA sequences in cells infected in vitro, suggesting that detection of the N gene, coding for the most abundant subgenomic RNA of SARS-CoV-2, increases the sensitivity of detection during the highly replicative phase of infection. We investigated 208 nasopharyngeal swabs sampled in March-April 2020 in different hospitals of Greater Paris. We found that 8.6% of informative samples (n = 16/185, P < 0.0001) initially scored as “non-positive” (undetermined or negative) by RT-qPCR were positive for SARS-CoV-2 RNA by ddPCR. Our work confirms that the use of ddPCR modestly, but significantly, increases the proportion of upper airway samples testing positive in the framework of first-line diagnosis of a French population.

Fewer non‐COVID‐19 respiratory tract infections and gastrointestinal infections during the COVID‐19 pandemic

“…15 This observation could also be a result of a reporting bias in cases where the initial diagnosis of viral pneumonia in association with a suspected COVID-19 infection was established, but not revised promptly after ruling out SARS-CoV-2 by means of polymerase chain reaction testing. 16 One previous investigation reports that the number of consultations in gastroenterological practices and corresponding diagnoses related to gastroenterological illness decreased gradually during the COVID-19 pandemic. 17 Jördens and colleagues reported a reduction of 19% in the rate of gastro-intestinal infections.…”

Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review

“…An overall of 3651 samples, from 2825 patients and 145 controls, were extracted from the 39 included studies. The studies were conducted worldwide; 16 in Asia [ 12 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ], 14 in Europe [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ], and 9 in North America [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ]. A minority of studies (8) included control samples [ 21 , 28 , 29 , 33 , 38 , 47 , 54 , 56 ].…”

“…Moreover, digital PCR was able to resolve a few ambiguous RT-qPCR results, detecting the SARS-CoV-2 virus in 17 cases of SARS-CoV-2 reported as equivocal (16/17) or negative (1/17) from 29 close contacts [ 22 ]. Marchio et al also demonstrated the ability of ddPCR to clarify RT-qPCR ambiguous cases, reporting as positive 2/8 equivocal samples [ 40 ].…”

“…For instance, it could be the primary testing method in the hospital setting, especially in samples of low or fluctuating viral loads, or when the clinical suspicion is high. Furthermore, ddPCR could aid in resolving the undetermined results reported by RT-qPCR or in confirming negative results of patients before their discharge, leading to improved surveillance and control and reduction of false-negative interpretations [ 40 ]. In the community setting, ddPCR could be useful for the early detection of SARS-CoV-2, especially when RT-qPCR testing is negative in individuals at risk of carrying the virus, shortening the window period.…”

“…As mentioned before, ddPCR has several advantages over other commercial tests used for COVID-19 diagnostics, such as RT-qPCR. However, because of certain disadvantages—such as its limited availability, higher cost, relatively low throughput, and longer turnaround times compared to RT-qPCR—it could be unfeasible to regularly perform ddPCR in the community setting, instead of rapid antigen or RT-qPCR testing [ 34 , 40 ]. Some of these disadvantages can yet be overcome.…”