High-Throughput COVID-19 Testing of Naso-Oropharyngeal Swabs Using a Sensitive Extraction-Free Sample Preparation Method.

High-throughput diagnostic assays are required for large-scale population testing for severe acute respiratory coronavirus 2 (SARS-CoV-2). The gold standard technique for SARS-CoV-2 detection in nasopharyngeal swab specimens is nucleic acid extraction followed by real-time reverse transcription-PCR. Two high-throughput commercial extraction and detection systems are used routinely in our laboratory: the Roche cobas SARS-CoV-2 assay (cobas) and the Roche MagNA Pure 96 system combined with the SpeeDx PlexPCR SARS-CoV-2 assay (Plex). As an alternative to more costly instrumentation, or tedious sample pooling to increase throughput, we developed a high-throughput extraction-free sample preparation method for naso-oropharyngeal swabs using the PlexPCR SARS-CoV-2 assay (Direct). A collection of SARS-CoV-2-positive (n = 185) and -negative (n = 354) naso-oropharyngeal swabs in transport medium were tested in parallel to compare Plex to Direct. The overall agreement comparing the qualitative outcomes was 99.3%. The mean cycle of quantification (Cq) increase and corresponding mean reduction in viral load for Direct ORF1ab and RdRp compared to Plex was 3.11 Cq (-0.91 log10 IU/mL) and 4.78 Cq (-1.35 log10 IU/mL), respectively. We also compared Direct to a four-sample pool by combining each positive sample (n = 185) with three SARS-CoV-2-negative samples extracted with MagNA Pure 96 and tested with the PlexPCR SARS-CoV-2 assay (Pool). Although less sensitive than Plex or Pool, the Direct method is a sufficiently sensitive and viable approach to increase our throughput by 12,032 results per day. Combining cobas, Plex, and Direct, an overall throughput of 19,364 results can be achieved in a 24-h period. IMPORTANCE Laboratories have experienced extraordinary demand globally for reagents, consumables, and instrumentation, while facing unprecedented testing demand needed for the diagnosis of SARS-CoV-2 infection. A major bottleneck in testing throughput is the purification of viral RNA. Extraction-based methods provide the greatest yield and purity of RNA for downstream PCR. However, these techniques are expensive, time-consuming, and depend on commercial availability of consumables. Extraction-free methods offer an accessible and cost-effective alternative for sample preparation. However, extraction-free methods often lack sensitivity compared to extraction-based methods. We describe a sensitive extraction-free protocol based on a simple purification step using a chelating resin, combined with proteinase K and thermal treatment. We compare the sensitivity qualitatively and quantitatively to a well-known commercial extraction-based system, using a PCR assay calibrated to the 1st WHO international standard for SARS-CoV-2 RNA. This method entails high throughput and is suitable for all laboratories, particularly in jurisdictions where access to instrumentation and reagents is problematic.

Clinical associations of SARS-CoV-2 viral load using the first WHO International Standard for SARS-CoV-2 RNA.

SARS-CoV-2 viral load declines from the time of symptom onset; in some studies viral load is higher or persists longer in more severe COVID-19 infection, and viral load correlates with culture positivity. This was a retrospective cohort study of inpatients and outpatients during the first wave of COVID-19 infection in Western Australia, March to May 2020, of the relationship of SARS-CoV-2 viral load (using the First WHO International Standard for SARS-CoV-2 RNA) from symptom onset, by clinical subgroups determined from the public health database and hospital records, using regression analysis. We studied 320 samples from 201 COVID-19 cases: 181 mild, seven severe, 11 critical, and four cases who died (two were also critical cases). At symptom onset the mean viral load was 4.34 log10 IU/mL (3.92-4.77 log10 IU/mL 95% CI, cobas SARS-CoV-2 assay ORF1a Ct 28.9 cycles). The mean viral load change was -0.09 log10 IU/mL/day (-0.12 to -0.06 95% CI). R2 was 0.08 and residual standard deviation 2.68 log10 IU/mL. Viral load at symptom onset was higher for those reporting fever compared to those not reporting fever. Viral load kinetics were not different for gender, age, shortness of breath, or those requiring oxygen. Mean viral load at usual release from isolation at 14 days was 2.5 log10 IU/mL or day 20 was 1.8 log10 IU/mL. Variability in respiratory sample SARS-CoV-2 viral load kinetics suggests viral loads will only have a role supporting clinical decision making, and an uncertain role for prognostication.

Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay.

Accurate and rapid diagnostic tests are a critical component for the early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of the overall control strategy for the current pandemic. Nucleic acid amplification tests are the gold standard for diagnosis of acute SARS-CoV-2 infection, and many real-time PCR diagnostic assays have been developed. Mutations that occur within the primer/probe binding regions of the SARS-CoV-2 genome can negatively impact the performance of diagnostic assays. Here, we report two single-point mutations in the N gene of SARS-CoV-2 associated with N gene target detection failures in the Cepheid Xpert Xpress SARS-CoV-2 assay, the first a C to T mutation at position 29197, found in five patients, and the second a C to T mutation at position 29200, found in eight patients. By sequencing the Xpert amplicons, we showed both mutations to be located within the amplified region of the Xpert N gene target. This report highlights the necessity for multiple genetic targets and the continual monitoring and evaluation of diagnostic assay performance. IMPORTANCE This paper reports the identification of single-point mutations in the N gene of SARS-CoV-2 associated with a gene target failure by the Cepheid Xpert commercial system. In order to determine the mutation(s) responsible for the N gene detection failures, the genomic products from the Cepheid Xpert system were sequenced and compared to whole genomes of SARS-CoV-2 from clinical cases. This report is the first to our knowledge which characterizes the amplified PCR products of the Xpert system, confirming the mutations associated with the gene target failure. The mutations identified have previously been reported.

Evaluation of a PlexZyme-Based PCR Assay and Assessment of COVID-19 Surge Testing Throughput Compared to Cobas SARS-CoV-2.

Reliable high-throughput methods are required for the detection of severe acute respiratory coronavirus 2 (SARS-CoV-2). We evaluated the new research use only (RUO) SpeeDx PlexZyme SARS-CoV-2 components (Plex) compared to the Roche cobas SARS-CoV-2 assay (cobas). A collection of positive (n = 214) and negative samples (n = 201) was tested in parallel comparing Plex with cobas. The overall agreement comparing the qualitative outcomes was 96.9%. Using an in-house quantitative PCR method, correlation comparing Plex ORF1ab to cobas ORF1a was r2 = 0.95. The median Plex ORF1ab change in target copy number compared to cobas ORF1a was +0.48 log10 copies/mL respectively. Inter- and intra-assay reproducibility of each assay was compared, including a limit-of-detection study. Reproducibility was comparable; however cobas was more sensitive than Plex by 1-log dilution. Throughput was evaluated during a COVID-19 testing surge of 4324 samples in a 30-h period. Plex demonstrated less hands-on time per reportable result (19% decrease) and increased throughput (155% increase of 102 results/hour) compared to cobas (40 results/hour). Our study demonstrates good qualitative and quantitative correlation of Plex compared to cobas and that Plex is well-suited for high throughput testing.

Qualitative and quantitative effects of thermal treatment of naso-oropharyngeal samples before cobas SARS-CoV-2 testing.

To improve laboratory safety we thermally treated naso-oropharyngeal samples before testing with the cobas SARS-CoV-2 assay. This study aimed to determine if thermal treatment significantly affects the qualitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the quantitative measurement of cobas SARS-CoV-2 ORF1a and E-gene target copy number using an in-house quantitative method. A collection of positive (n = 238) and negative samples (n = 196) was tested in parallel comparing thermal treatment (75 °C for 15 minutes) to room-temperature. There were no significant differences in the final qualitative outcomes for thermal treatment versus room-temperature (99.8% agreement) despite a statistically significant reduction (P < 0.05) in target copy number following thermal treatment. The median ORF1a and E-gene reduction in target copy number was -0.07 (1.6%) and -0.22 (4.2%) log10 copies/mL respectively. The standard curves for both ORF1a and E-gene targets were highly linear (r2 = 0.99). Good correlation was observed for ORF1a (r2 = 0.96) and E-gene (r2 = 0.98) comparing thermal treatment to room-temperature control.