Results of a SARS-CoV-2 virus genome detection external quality assessment round focusing on sensitivity of assays and pooling of samples

Abstract

Objectives Results of earlier external quality assessment (EQA) rounds suggested remarkable differences in the sensitivity of SARS-CoV PCR assays. Although the test systems are intended to detect SARS-CoV-2 in individual samples, screening is often applied to sample pools to increase efficiency and decrease costs. However, it is unknown to what extent these tests actually meet the manufacturer’s specifications for sensitivity and how they perform when testing sample pools. Methods The sensitivity of assays in routine use was evaluated with a panel of positive samples in a round of a SARS-CoV-2 virus genome detection EQA scheme. The panel consisted of samples at or near the lower limit of detection (“weakly positive”). Laboratories that routinely test sample pools were asked to also analyze the pooled EQA samples according to their usual pool size and dilution method. Results All participants could detect a highly positive patient-derived sample (>106 copies/mL). Most (96%) of the test systems could detect at least 1,000 copies/mL, meeting the minimum acceptable benchmark, and many (94%) detected the vRNA in a sample with lower concentration (500 copies/mL). The false negative ratio increased to 16 and 26% for samples with 100 and 50 copies/mL, respectively. Conclusions The performance of most assays met or exceeded their specification on sensitivity. If assays are to be used to analyze sample pools, the sensitivity of the assay and the number of pooled samples must be balanced.

Converting to an international unit system improves harmonization of results for SARS-CoV-2 quantification: results from multiple external quality assessments

“…We and others have observed through EQAs that there exists relatively high variability when reporting Ct values, and we presume this is also true outside of ring trials, for example, when measuring virus concentration in patient samples [5,8,9,11,12,19,21]. Our data suggested that converting Ct values to copies per mL (and reporting them as IU/mL) statistically improved precision for all samples.…”

“…Characterization also included determination of copy number by digital PCR (dPCR). Details of the second round were presented elsewhere [21].…”

Design of external quality assessment (EQA) schemes and definition of the roles of their providers in future epidemics

“…A mathematical model for the extent of the (relative) loss of sensitivity has been published earlier [52,53]. The expected loss of relative sensitivity was evident in the EQA round that reported on this [24]. Such information can only be obtained through a speci cally designed EQA round and needs collaboration of the participant test facility.…”

“…Literature on EQA is generally rare. We identi ed a total of 17 publications on EQA of SARS-CoV-2 in PubMed with the search terms "EQA" or "external quality assessment" or "pro ciency testing" or "PT" and "SARS-CoV-2" or "Covid-19" (search performed July 2022, period searched 01/2020 to 07/2022, see Table 1) [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. Information from each publication was aggregated (Table 1) and the subject criteria were derived from this.…”

“…Several EQA results from different providers at the beginning of the pandemic showed that the reported Cq/Ct values differ signi cantly between laboratories, the assays used and between selected target genes, so that a quantitative estimate of genome concentration from Cq/Ct values was unreliable [21][22][23][24]28,29,[34][35][36]. Therefore, in the above cited EQA scheme of April 2020 all four SARS-CoV-2-positive samples, which were from a 10fold dilution series, were quanti ed by digital droplet PCR (ddPCR), covering a linear concentration range between approx.…”