Evaluation of a Rapid and Accessible RT-qPCR Approach for SARS-CoV-2 Variant of Concern Identification

The ability to distinguish between SARS-CoV-2 variants of concern (VOCs) is of ongoing interest due to differences in transmissibility, response to vaccination, clinical prognosis, and therapy. Although detailed genetic characterization requires whole-genome sequencing (WGS), targeted nucleic acid amplification tests can serve a complementary role in clinical settings, as they are more rapid and accessible than sequencing in most laboratories. We designed and analytically validated a two-reaction multiplex reverse transcription quantitative PCR (RT-qPCR) assay targeting spike protein mutations L452R, E484K, and N501Y in Reaction 1, and del69-70, K417N, and T478K in Reaction 2. This assay had 95-100% agreement with WGS in 502 upper respiratory swabs collected between April 26 and August 1, 2021, consisting of 43 Alpha, 2 Beta, 20 Gamma, 378 Delta, and 59 non-VOC infections. Validation in a separate group of 230 WGS-confirmed Omicron variant samples collected in December 2021 and January 2022 demonstrated 100% agreement. This RT-qPCR-based approach can be implemented in clinical laboratories already performing SARS-CoV-2 nucleic acid amplification tests to assist in local epidemiological surveillance and clinical decision-making.

Multiplex SARS-CoV-2 Genotyping PCR for Population-Level Variant Screening and Epidemiologic Surveillance

BackgroundEmergence of SARS-CoV-2 variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for pandemic response, but many laboratories do not have the resources to support population-level sequencing. We hypothesized that a spike genotyping nucleic acid amplification test (NAAT) could facilitate high-throughput variant surveillance. MethodsWe designed and analytically validated a one-step multiplex allele-specific reverse transcriptase polymerase chain reaction (RT-qPCR) to detect three non-synonymous spike protein mutations (L452R, E484K, N501Y). Assay specificity was validated with next-generation whole-genome sequencing. We then screened a large cohort of SARS-CoV-2 positive specimens from our San Francisco Bay Area population. ResultsBetween December 1, 2020 and March 1, 2021, we screened 4,049 unique infections by genotyping RT-qPCR, with an assay failure rate of 2.8%. We detected 1,567 L452R mutations (38.7%), 34 N501Y mutations (0.84%), 22 E484K mutations (0.54%), and 3 (0.07%) E484K+N501Y mutations. The assay had near-perfect (98-100%) concordance with whole-genome sequencing in a validation subset of 229 specimens, and detected B.1.1.7, B.1.351, B.1.427, B.1.429, B.1.526, and P.2 variants, among others. The assay revealed rapid emergence of L452R in our population, with a prevalence of 24.8% in December 2020 that increased to 62.5% in March 2021. ConclusionsWe developed and clinically implemented a genotyping RT-qPCR to conduct high-throughput SARS-CoV-2 variant screening. This approach can be adapted for emerging mutations and immediately implemented in laboratories already performing NAAT worldwide using existing equipment, personnel, and extracted nucleic acid. Summary / Key PointsEmergence of SARS-CoV-2 variants with concerning phenotypes is of public health interest. We developed a multiplex genotyping RT-qPCR to rapidly detect L452R, E484K, and N501Y with high sequencing concordance. This high-throughput alternative to resource-intensive sequencing enabled surveillance of L452R emergence.

Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens

BackgroundSeveral point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for diagnosis of SARS-CoV-2. The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. ObjectivesThe aim of this study was to assess test performance of the Accula SARS-CoV-2 test. Study designThe performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT) targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohens kappa coefficient. ResultsOverall percent agreement between the assays was 84.0% (95% confidence interval [CI] 75.3 to 90.6%), PPA was 68.0% (95% CI 53.3 to 80.5%) and the kappa coefficient was 0.68 (95% CI 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test, and showed low viral load burden with a median cycle threshold value of 37.7. NPA was 100% (95% CI 94.2 to 100%). ConclusionCompared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings, and for confirmatory testing in individuals with moderate to high pre-test probability of SARS-CoV-2 who test negative on Accula.

High frequency of SARS-CoV-2 RNAemia and association with severe disease

BackgroundDetection of SARS-CoV-2 RNA in the blood, also known as RNAemia, has been reported, but its prognostic implications are not well understood. This study aimed to determine the frequency of SARS-CoV-2 RNA in plasma and its association with the clinical severity of COVID-19. MethodsAn analytical cross-sectional study was performed in a single-center tertiary care institution in northern California and included consecutive inpatients and outpatients with COVID-19 confirmed by detection of SARS-CoV-2 RNA in nasopharyngeal swab specimens. The prevalence of SARS CoV-2 RNAemia and the strength of its association with clinical severity variables were examined and included the need for transfer to an intensive care unit (ICU), mechanical ventilation and 30-day all-cause mortality. ResultsPaired nasopharyngeal and plasma samples were included from 85 patients. The overall median age was 55 years, and individuals with RNAemia were older than those with undetectable SARS-CoV-2 RNA in plasma (63 vs 50 years; p=0.001). Comorbidities were frequent including obesity (37.7%), hypertension (30.6%) and diabetes mellitus (22.4%). RNAemia was detected in a total of 28/85 (32.9%) individual patients, including 22/28 (78.6%) who required hospital admission. RNAemia was detected more frequently in individuals who developed severe disease including the need for ICU transfer (32.1% vs 14.0%; p=0.05), mechanical ventilation (21.4% vs 3.5%; p=0.01) and 30-day all-cause mortality (14.3% vs 0%; p=0.01). No association was detected between RNAemia and estimated levels of viral RNA in the nasopharynx. An additional 121 plasma samples from 28 individuals with RNAemia were assessed longitudinally, and RNA was detected for a maximum duration of 10 days. ConclusionThis study demonstrated a high proportion of SARS-CoV-2 RNAemia, and an association between RNAemia and clinical severity suggesting the potential utility of plasma viral testing as a prognostic indicator for COVID-19.

Retrospective Pooled Screening for SARS-CoV-2 RNA in late 2019

Reports have emerged documenting earlier SARS-CoV-2 cases than previously recognized. To investigate this possibility in the Bay Area, we retrospectively tested 1,700 samples from symptomatic individuals for the last 2 months of 2019. No SARS-CoV-2 positive pools were identified, consistent with limited transmission in this population at this time.

Occurrence and Timing of Subsequent SARS-CoV-2 RT-PCR Positivity Among Initially Negative Patients

BackgroundSARS-CoV-2 reverse transcriptase polymerase chain reaction (RT-PCR) testing remains the cornerstone of laboratory-based identification of patients with COVID-19. As the availability and speed of SARS-CoV-2 testing platforms improve, results are increasingly relied upon to inform critical decisions related to therapy, use of personal protective equipment, and workforce readiness. However, early reports of RT-PCR test performance have left clinicians and the public with concerns regarding the reliability of this predominant testing modality and the interpretation of negative results. In this work, two independent research teams report the frequency of discordant SARS-CoV-2 test results among initially negative, repeatedly tested patients in regions of the United States with early community transmission and access to testing. MethodsAll patients at the University of Washington (UW) and Stanford Health Care undergoing initial testing by nasopharyngeal (NP) swab between March 2nd and April 7th, 2020 were included. SARS-CoV-2 RT-PCR was performed targeting the N, RdRp, S, and E genes and ORF1ab, using a combination of Emergency Use Authorization laboratory-developed tests and commercial assays. Results through April 14th were extracted to allow for a complete 7-day observation period and an additional day for reporting. ResultsA total of 23,126 SARS-CoV-2 RT-PCR tests (10,583 UW, 12,543 Stanford) were performed in 20,912 eligible patients (8,977 UW, 11,935 Stanford) undergoing initial testing by NP swab; 626 initially test-negative patients were re-tested within 7 days. Among this group, repeat testing within 7 days yielded a positive result in 3.5% (4.3% UW, 2.8% Stanford) of cases, suggesting an initial false negative RT-PCR result; the majority (96.5%) of patients with an initial negative result who warranted reevaluation for any reason remained negative on all subsequent tests performed within this window. ConclusionsTwo independent research teams report the similar finding that, among initially negative patients subjected to repeat SARS-CoV-2 RT-PCR testing, the occurrence of a newly positive result within 7 days is uncommon. These observations suggest that false negative results at the time of initial presentation do occur, but potentially at a lower frequency than is currently believed. Although it is not possible to infer the clinical sensitivity of NP SARS-CoV-2 RT-PCR testing using these data, they may be used in combination with other reports to guide the use and interpretation of this common testing modality.