Objectives External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. Methods Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. Results A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (C t ∼35.9, ∼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of C t values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. Conclusions Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.
Objectives Mutation-specific PCR assays have quickly found their way into laboratory diagnostics due to their capacity to be a fast, easy to implement and high-throughput method for the detection of known SARS-CoV-2 variants of concern (VoCs). However, little is known about the performance of such assays in routine laboratory analysis. Methods The results reported in a recent round of an external quality assessment (EQA) scheme for SARS-CoV-2 mutation-specific PCR were retrospectively analyzed. For the determination of individual variant-specific sequences as well as for the interpretation results for certain virus variants, correct, incorrect, and unreported results were evaluated, and their possible causes were investigated. Results A total of 34 laboratories participated in this study. For five samples containing the VoC Alpha + E484K, Beta, Gamma, Delta, or B.1.1.318 (as a variant of interest), 848 results for SARS-2-CoV mutation detection were reported, 824 (97.2%, range per sample 88–100%) of which were correct. Melting curve assays gave 99% correct results, real-time RT-qPCR 94%, microarray-based assays 100%, and MALDI-TOF MS 96%. A total of 122/167 (73%) reported results for SARS-CoV-2 variant determination were correct. Of the 45 inconclusive or incorrect results, 33 (73%) were due to inadequate selection of targets that did not allow identification of contemporary VoC, 11 (24%) were due to incorrect results, and one (3%) was due to correct results of mutation-specific PCR. Conclusions Careful and up-to-date selection of the targets used in mutation-specific PCR is essential for successful detection of current SARS-CoV-2 variants.
Background : Distinctive genotypes of SARS-CoV-2 have emerged that are or may be associated with increased transmission, pathogenicity, and/or antibody escape. In many countries, clinical and diagnostic laboratories are under mandate to identify and report these so-called variants of concern (VOC). Objectives : We used an external quality assessment scheme to determine the scope, accuracy, and reliability of laboratories using various molecular diagnostic assays to identify current VOC (03 March 2021). Study design : Participant laboratories were sent the same five patient-derived samples and were asked to provide their variant detection methods, variant detection results and interpretation of results. Results : Twenty-five laboratories reported a range of RT-qPCR-based assays to identify specific variations in the SARS-CoV-2 spike protein that are characteristic of three VOC lineages. Laboratories that detected VOC-associated nucleotide mutations at four specific sites had the highest ratio of correct classification. Low template copy number and additional variation in target regions resulted in loss of confidence and accuracy in sample classification. Conclusions : Melting-curve-based assays to identify genomic variants are less time-consuming and require less bioinformatic analysis compared to partial or whole genome sequencing. However, our results suggest that correct classification of a given genotype/lineage (e.g., a VOC) relies on the ability to detect more than one variant site, adequate template in the sample (i.e., relatively high viral load/copy number) and results may be unclear in certain samples with additional genetic variations. These initial results suggest that some diagnostic laboratories may require additional training to interpret and report complex genetic information about a dynamic emerging virus.
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.
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