Digital PCR for high sensitivity viral detection in false-negative SARS-CoV-2 patients

Abstract

Patients requiring diagnostic testing for coronavirus disease 2019 (COVID-19) are routinely assessed by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) amplification of Sars-CoV-2 virus RNA extracted from oro/nasopharyngeal swabs. Despite the good specificity of the assays certified for SARS-CoV-2 molecular detection, and a theoretical sensitivity of few viral gene copies per reaction, a relatively high rate of false negatives continues to be reported. This is an important challenge in the management of patients on hospital admission and for correct monitoring of the infectivity after the acute phase. In the present report, we show that the use of digital PCR, a high sensitivity method to detect low amplicon numbers, allowed us to correctly detecting infection in swab material in a significant number of false negatives. We show that the implementation of digital PCR methods in the diagnostic assessment of COVID-19 could resolve, at least in part, this timely issue.

Figure 1

(a–d) Chest CT axial and coronal projections at the level of tracheal bifurcation in a SARS-CoV-2 positive patient (a,c) and patient #15 (b,d), admitted to our Center. (e) Correlation between the copies of N1/N2 amplicons (expressed as log₁₀ copies/mL) detected by dPCR and the Ct values of the N gene by the single primer present in the diagnostic test. It is evident a better correlation of the data below ~ 36 Ct (evidenced by the dotted line in the graph). (f) Individual results of digital PCR analysis of the 18 patients scoring negative in the conventional diagnostic test. In patients 1–7, the digital PCR test was unable to detect the virus in the diagnostic eluted RNA. Patients 8–18 exhibited a low copy number of N1, N2 amplicons, or both. The line labeled with LOD indicates the lower detection limit of ~ 2.2 copies/mL, calculated as described in materials and methods section. (g) Patient #15 was tested in four consecutive swabbing procedures and was invariantly negative wih the diagnostic assay. Re-testing by digital PCR of the eluted RNA showed a clear SARS-CoV-2 positivity by N2 sequence amplification. (h) The ACE2 mRNA was detected by conventional RT-PCR using the eluate RNA extracted from the swab material. ACE2 expression levels showed a decreased trend in negative vs. positive patients (P = 0.0515 by t test). (i) no differences were observed in the levels of ACE2 expression in the true-negative (TN) and false-negative (FN) subjects. In panels (h) and (i) data are reported as average ± standard deviation.