Multicenter international assessment of a SARS-CoV-2 RT-LAMP test for point of care clinical application

Continued waves, new variants, and limited vaccine deployment mean that SARS-CoV-2 tests remain vital to constrain the COVID-19 pandemic. Affordable, point-of-care (PoC) tests allow rapid screening in non-medical settings. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is an appealing approach. A crucial step is to optimize testing in low/medium resource settings. Here, we optimized RT-LAMP for SARS-CoV-2 and human {beta}-actin, and tested clinical samples in multiple countries. "TTTT" linker primers did not improve performance, and while guanidine hydrochloride, betaine and/or Igepal-CA-630 enhanced detection of synthetic RNA, only the latter two improved direct assays on nasopharygeal samples. With extracted clinical RNA, a 20 min RT-LAMP assay was essentially as sensitive as RT-PCR. With raw Canadian nasopharygeal samples, sensitivity was 100% (95% CI: 67.6% - 100%) for those with RT-qPCR Ct values [≤] 25, and 80% (95% CI: 58.4% - 91.9%) for those with 25 < Ct [≤] 27.2. Highly infectious, high titer cases were also detected in Colombian and Ecuadorian labs. We further demonstrate the utility of replacing thermocyclers with a portable PoC device (FluoroPLUM). These combined PoC molecular and hardware tools may help to limit community transmission of SARS-CoV-2.

Immunogenicity of convalescent and vaccinated sera against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron variants

The omicron variant of concern (VOC) of SARS-CoV-2 was first reported in November 2021 in Botswana and South Africa. Omicron has evolved multiple mutations within the spike protein and the receptor binding domain (RBD), raising concerns of increased antibody evasion. Here, we isolated infectious omicron from a clinical specimen obtained in Canada. The neutralizing activity of sera from 65 coronavirus disease (COVID-19) vaccine recipients and convalescent individuals against clinical isolates of ancestral SARS-CoV-2, beta, delta, and omicron VOCs was assessed. Convalescent sera from unvaccinated individuals infected by the ancestral virus during the first wave of COVID-19 in Canada (July, 2020) demonstrated reduced neutralization against beta and omicron VOCs. Convalescent sera from unvaccinated individuals infected by the delta variant (May-June, 2021) neutralized omicron to significantly lower levels compared to the delta variant. Sera from individuals that received three doses of the Pfizer or Moderna vaccines demonstrated reduced neutralization of the omicron variant relative to ancestral SARS-CoV-2. Sera from individuals that were naturally infected with ancestral SARS-CoV-2 and subsequently received two doses of the Pfizer vaccine induced significantly higher neutralizing antibody levels against ancestral virus and all VOCs. Importantly, infection alone, either with ancestral SARS-CoV-2 or the delta variant was not sufficient to induce high neutralizing antibody titers against omicron. This data will inform current booster vaccination strategies, and we highlight the need for additional studies to identify longevity of immunity against SARS-CoV-2 and optimal neutralizing antibody levels that are necessary to prevent infection and/or severe COVID-19.

Real-time RT-PCR Allelic Discrimination Assay for Detection of N501Y Mutation in the Spike Protein of SARS-CoV-2 Associated with Variants of Concern

The N501Y amino acid mutation caused by a single point substitution A23063T in the spike gene of SARS-CoV2 is possessed by the three most common variants of concern - B.1.1.7, B.1.351, and P.1. A rapid screening tool using this mutation is important for surveillance during the COVID-19 pandemic. We developed and validated a single nucleotide polymorphism real-time reverse transcription polymerase chain reaction assay using allelic discrimination of the spike gene N501Ymutation to screen for potential variants of concern and differentiate them from wild-type SARS-CoV-2. A total of 160 clinical specimens positive for SARS-CoV-2 were characterized as mutant (N501Y) or wild-type by Sanger sequencing and were subsequently tested with the N501Y single nucleotide polymorphism real time reverse transcriptase polymerase chain reaction assay. Our assay compared to sequencing, the gold standard for SNP detection and lineage identification, demonstrated clinical sensitivity of 100% for all 57 specimens displaying N501Y mutant, which were confirmed by Sanger sequencing to be typed as A23063T, including one specimen with mixed signal for wildtype and mutant. Clinical specificity was 100% in all 103 specimens typed as wild-type, with A23063 identified as wild-type by Sanger sequencing. The identification of circulating SARS-CoV-2 lineages carrying an N501Y mutation is critical for surveillance purposes. Current identification methods rely primarily on Sanger sequencing or whole genome sequencing which are time-consuming, labor-intensive and costly. The assay described herein is an efficient tool for high-volume specimen screening for SARS-CoV-2 VOCs and for selecting specimens for confirmatory Sanger or whole genome sequencing.

Impact of COVID-19 pre-test probability on positive predictive value of high cycle threshold SARS-CoV-2 real-time reverse transcription PCR test results

BackgroundPerformance characteristics of SARS-CoV-2 nucleic acid detection assays are understudied within contexts of low pre-test probability, including screening asymptomatic persons without epidemiological links to confirmed cases, or asymptomatic surveillance testing. SARS-CoV-2 detection without symptoms may represent resolved infection with persistent RNA shedding, presymptomatic or asymptomatic infection, or a false positive test. This study assessed clinical specificity of SARS-CoV-2 real-time reverse transcription polymerase chain reaction (rRT-PCR) assays by retesting positive specimens from five pre-test probability groups ranging from high to low with an alternate assay. Materials and MethodsA total of 122 rRT-PCR positive specimens collected from unique patients between March and July 2020 were retested using a laboratory-developed nested RT-PCR assay targeting the RNA-dependent RNA polymerase (RdRp) gene followed by Sanger sequencing. ResultsSignificantly less positive results in the lowest pre-test probability group (facilities with institution-wide screening having [≤] 3 positive asymptomatic cases) were reproduced with the nested RdRp gene RT-PCR assay than in all other groups combined (5/32, 15{middle dot}6% vs 61/90, 68%; p <0{middle dot}0001), and in each subgroup with higher pre-test probability (individual subgroup range 50{middle dot}0% to 85{middle dot}0%). ConclusionsA higher proportion of false-positive test results are likely with lower pre-test probability. Positive SARS-CoV-2 PCR results should be interpreted within the context of patient history, clinical setting, known exposure, and estimated community disease prevalence. Large-scale SARS-CoV-2 screening testing initiatives among low pre-test probability populations should be evaluated thoroughly prior to implementation given the risk of false positives and consequent potential for harm at the individual and population level.

Ultrasensitive assay for saliva-based SARS-CoV-2 antigen detection.

Widespread SARS-CoV-2 testing is highly valuable for identifying asymptomatic/pre-symptomatic individuals to slow community disease transmission. However, there remains a technological gap for highly reliable, easy, and quick SARS-CoV-2 diagnostic tests that are suitable for frequent mass testing. Compared to the conventional nasopharyngeal (NP) swab-based tests, saliva-based methods are attractive due to easier and safer sampling protocols. Despite its merits in rapid turn-around-time and high throughput compared to traditional PCR-based technologies, the widespread use of saliva-based SARS-CoV-2 rapid antigen tests is hindered by limited analytical sensitivity of current methods. Here, we report the first ultrasensitive, saliva-based SARS-CoV-2 antigen assay with an analytical sensitivity of < 0.32 pg/ml, corresponding to 4 viral RNA copies/{micro}l, which is comparable to that of PCR-based tests. Using the novel electrochemiluminescence (ECL)-based S-PLEX immunoassay, we measured the SARS-CoV-2 nucleocapsid (N) antigen concentration in 105 saliva samples obtained from non-COVID-19 and COVID-19 patients. Our assay displayed absolute specificity and high sensitivity (90.2%), where it correctly identified samples with viral loads up to 35 CT cycles by saliva-based PCR. Paired NP swab-based PCR results were also obtained for 86 cases for comparison. Our assay showed high concordance with saliva-based and NP swab-based PCR in samples with negative (< 0.32 pg/ml) and strongly positive (> 2 pg/ml) N antigen concentrations. Our study unveiled the ultrasensitivity and specificity of the saliva-based S-PLEX assay, demonstrating its clinical value as a high throughput, complementary alternative to PCR-based techniques. The novel technique is especially valuable in cases where compliance to frequent swabbing may be problematic (e.g. schools, nursing homes, etc.).

Characteristics of SARS-CoV-2 Testing for Rapid Diagnosis of COVID-19 during the Initial Stages of a Global Pandemic

Accurate SARS-CoV-2 diagnosis is essential to guide prevention and control of COVID-19. From January 11 - April 22, 2020, Public Health Ontario conducted SARS-CoV-2 testing of 86,942 specimens collected from 80,354 individuals, primarily using real-time reverse-transcription polymerase chain reaction (rRT-PCR) methods. We analyzed test results across specimen types and for individuals with multiple same-day and multi-day collected specimens. Nasopharyngeal compared to throat swabs had a higher positivity (8.8% vs. 4.8%) and an adjusted estimate 2.9 Ct lower (SE=0.5, p<0.001). Same-day specimens showed high concordance (98.8%), and the median Ct of multi-day specimens increased over time. Symptomatic cases had rRT-PCR results with an adjusted estimate 3.0 Ct (SE=0.5, p<0.001) lower than asymptomatic/pre-symptomatic cases. Overall test sensitivity was 84.6%, with a negative predictive value of 95.5%. Molecular testing is the mainstay of SARS-CoV-2 diagnosis and testing protocols will continue to be dynamic and iteratively modified as more is learned about this emerging pathogen.

A Multiplexed, Next Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2

Population scale sweeps of viral pathogens, such as SARS-CoV-2, that incorporate large numbers of asymptomatic or mild symptom patients present unique challenges for public health agencies trying to manage both travel and local spread. Physical distancing is the current major strategy to suppress spread of the disease, but with enormous socio-economic costs. However, modelling and studies in isolated jurisdictions suggest that active population surveillance through systematic molecular diagnostics, combined with contact tracing and focused quarantining can significantly suppress disease spread1-3 and has significantly impacted disease transmission rates, the number of infected people, and prevented saturation of the healthcare system4-7. However, reliable systems allowing for parallel testing of 10-100,000s of patients in larger urban environments have not yet been employed. Here we describe "COVID-19 screening using Systematic Parallel Analysis of RNA coupled to Sequencing" (C19-SPAR-Seq), a scalable, multiplexed, readily automated next generation sequencing (NGS) platform8 that is capable of analyzing tens of thousands of COVID-19 patient samples in a single instrument run. To address the strict requirements in clinical diagnostics for control of assay parameters and output, we employed a control-based Precision-Recall and predictive Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of over 600 patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads and a sensitivity of > 95% on high viral loads associated with disease onset and peak transmissibility. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

Prospective Observational Study of Screening Asymptomatic Healthcare Workers for SARS-CoV-2 at a Canadian Tertiary Care Center

We screened three separate cohorts of healthcare workers for SARS-CoV-2 via nasopharyngeal swab PCR. A seroprevalence analysis using multiple assays was performed in a subgroup. The asymptomatic health care worker cohorts had a combined swap positivity rate of 29/5776 (0.50%, 95%CI 0.32-0.75) compared to the symptomatic cohort rate of 54/1597 (3.4%) (ratio of symptomatic to asymptomatic 6.8:1). Sequencing demonstrated several variants. The seroprevalence (n=996) was 1.4-3.4% depending on assay. Protein microarray analysis showed differing SARS-CoV-2 protein reactivities and helped define likely true positives vs. suspected false positives. Routine screening of asymptomatic health care workers helps identify a significant proportion of infections.

Comparison of SARS-CoV-2 Indirect and Direct Detection Methods

The COVID-19 pandemic caused by the SARS-CoV-2 virus has placed extensive strain on RNA isolation and RT-qPCR reagents. Rapid development of new test kits has helped to alleviate these shortages. However, comparisons of these new detection systems are largely lacking. Here, we compare indirect methods that require RNA extraction, and direct RT-qPCR on patient samples. For RNA isolation we compared four different companies (Qiagen, Invitrogen, BGI and Norgen Biotek). For detection we compared two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system proved most sensitive, generating comparable results to clinical diagnostic data, and identifying samples ranging from 65 copies - 2.1x105 copies of viral Orf1ab/l. However, the BGI detection system is [~]4x more expensive than other options tested here. With direct RT-qPCR we found that simply adding RNase inhibitor greatly improved sensitivity, without need for any other treatments (e.g. lysis buffers or boiling). The best direct methods were [~]10 fold less sensitive than indirect methods, but reduce sample handling, as well as assay time and cost. These studies will help guide the selection of COVID-19 detection systems and provide a framework for the comparison of additional systems.