Evaluation of the AdvanSure One-Stop COVID-19 Plus Kit for SARS-CoV-2 Detection Using a Streamlined RNA Extraction Method.

Real-time reverse transcription (rRT)-PCR, which is the reference standard for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, generally involves a time-consuming and costly RNA extraction step prior to amplification. We evaluated the performance of the AdvanSure One-Stop COVID-19 Plus Kit (LG Chem, Seoul, Korea), a novel rRT-PCR assay that can detect SARS-CoV-2 within 90 minutes using a streamlined RNA extraction method. In total, 509 nasopharyngeal swab (NPS) specimens (SARS-CoV-2 positive: N=205; SARS-CoV-2 negative: N=304) previously tested using the PowerChek SARS-CoV-2 Real-time PCR Kit (Kogene Biotech, Seoul, Korea) were tested using the AdvanSure assay. The limit of detection (LOD) of the AdvanSure assay was determined using serially diluted inactivated SARS-CoV-2. The positive and negative percent agreements between the AdvanSure and PowerChek assays were 99.5% (204/205) and 99.3% (302/304), respectively. The LODs of the AdvanSure assay for SARS-CoV-2 nucleocapsid and spike/RNA-dependent RNA polymerase genes were 672 and 846 copies/mL, respectively. The results show that the performance of the AdvanSure assay is comparable to that of the PowerChek assay used for routine SARS-CoV-2 testing, suggesting that the AdvanSure assay is a useful diagnostic tool for rapid and accurate detection of SARS-CoV-2 infection.

Direct SARS-CoV-2 Detection System Utilizing Simple-to-Use Capillary Gel Electrophoresis Sample-to-Result.

We present a Direct SARS-CoV-2 Detection System that achieves sample-to-results in less than two hours in three simple steps. The Detection System includes Direct one-step Reverse Transcription PCR (RT-PCR) reagents (Qexp-MDx kit), a portable thermal cycler (Qamp-mini) with a pre-programmed chip and a simple-to-use Capillary Gel Electrophoresis system (Qsep Series Bio-Fragment Analyzer) with high fluorescence detection sensitivity to solve the problems associated with traditional real-time PCR (qPCR) systems which produces inaccurate test results with high false negative and false positive rates. The proposed simple-to-use detection platform can provide high detection sensitivity (identify less than 20 copies), fast results (less than 120 minutes) and cost-effective results which should be suitable for decentralized testing application of COVID-19.

Relevance of cycle threshold values in mass screening by reverse-transcription-PCR during COVID-19 pandemic in Belgium: a decision-making support?

Aim: The Belgium's strategy against COVID-19 was partly based on mass screening. Here, we reported the results observed in a Belgian mass screening center. Materials & methods: Between October 2020 and February 2021, 32,089 samples were collected analyzed with reverse-transcription PCR (Thermo Fisher Scientific kits and apparatus). Patients were categorized according to their contagiousness (extrapolated from the cycle threshold [Ct] values and the recommendation of Sciensano). Results: We observed association between Ct values and age, with higher Ct observed in extreme age groups (<6 years and >75 years). Conclusion: The analysis of the evolution of the contagiousness of these patients tested twice within a 7-day period showed the relevancy of the recommendation edited by Sciensano.

Useful molecular tools for facing next pandemic events: Effective sample preparation and improved RT-PCR for highly sensitive detection of SARS-CoV-2 in wastewater environment.

Viral pandemics can be inevitable in the next future. Considering SARS-CoV-2 pandemics as an example, there seems to be a need to develop a surveillance system able to monitor the presence of potential pathogenic agents. The sewage and wastewater environments demonstrated to be suitable targets for such kind of analysis. In addition, it is important to have reliable molecular diagnostic tools and also to develop a robust detection strategy. In this study, an effective sample preparation procedure was selected from four options and combined with a newly developed improved RT-PCR. First, a model viral system was constructed, containing a fragment of the SARS-CoV-2 gene encoding for the Spike protein. The encapsidated S RNA mimic (ESRM) was based on the plum pox virus (PPV) genome with the inserted targeted gene fragment. ESRM was used for seeding wastewater samples in order to evaluate the viral recovery of four different viral RNA concentration/extraction methods. The efficiency of individual approaches was assessed by the use of a quantitative reverse transcription PCR (qRT-PCR) and by a one-step single-tube nested quantitative reverse transcription PCR (OSN-qRT-PCR). For the detection of viruses in wastewater samples with low viral loads, OSN-qRT-PCR assay produced the most satisfactory results and the highest sensitivity.

Development of a Test System to Detect the Omicron Variant of SARS-CoV-2 and the Frequency of Its Detection in Patients.

We developed a new test system to detect the omicron variant of SARS-CoV-2 using allele-specific reverse transcription PCR and estimated the frequency of its detection in patients living in the Novosibirsk Region. Clinical samples were divided into 3 groups: samples collected from December 1 to December 30, 2021 (group 1; n=66), from December 30, 2021 to January 10, 2022 (group 2; n=20), and from January 11 to January 22, 2022 (group 3; n=101). Based on the identification of 5 mutations specific to SARS-CoV-2 (B.1.1.529), two systems of oligonucleotide primers and probes were developed for detecting this coronavirus genotype in clinical samples. Limit of detection (LOD95) was 4×103 genome equivalents per 1 ml of clinical sample for the first test system and 2×103 for the for the second test system. The omicron variant of SARS-CoV-2 was absent in group 1 of studied samples, but was detected in 20% (4/20) of group 2 samples and 88% of group 2 samples collected within less than 2 weeks of January 2022. Using developed test system, we showed that in less than 2 weeks the omicron variant has become dominant in patients, which confirms previously published data on its exceptional contagiousness.

SARS-CoV-2 Antigen Test Results to Infer Active or Non-Active Virus Replication Status in COVID-19 Patients.

We used nasopharyngeal swab samples of patients with a symptomatic (n = 82) or asymptomatic (n = 20) coronavirus disease 2019 (COVID-19) diagnosis to assess the ability of antigen detection tests to infer active (potentially transmissible) or inactive (potentially non-transmissible) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the subgenomic RNA (sgRNA) as an active replication marker of SARS-CoV-2, 48 (76.2%), 56 (88.9%), and 63 (100%) of 63 samples with sgRNA positive results tested positive with the SD BIOSENSOR STANDARD Q COVID-19 Ag (Standard Q), the SD BIOSENSOR STANDARD F COVID-19 Ag FIA (Standard F), or the Fujirebio LUMIPULSE G SARS-CoV-2 Ag (Lumipulse) assay, respectively. Conversely, 37 (94.9%), 29 (74.4%), and 7 (17.9%) of 39 samples with sgRNA negative results tested negative with Standard Q, Standard F, or Lumipulse, respectively. Stratifying results by the number of days of symptoms before testing revealed that most antigen positive/sgRNA positive results were among samples tested at 2-7 days regardless of the assay used. Conversely, most antigen negative/sgRNA negative results were among samples tested at 16-30 days only when Standard Q or Standard F were used. In conclusion, based on our findings, a negative antigen test, especially with the Lumipulse assay, or a positive antigen test, especially with the Standard F assay, may suggest, respectively, the absence or presence of replication-competent SARS-CoV-2.

Recurrent SARS-CoV-2 RNA Detection after COVID-19 Illness Onset during Pregnancy.

The Surveillance for Emerging Threats to Mothers and Babies Network conducts longitudinal surveillance of pregnant persons in the United States with laboratory-confirmed severe acute respiratory syndrome coronavirus 2 infection during pregnancy. Of 6,551 infected pregnant persons in this analysis, 142 (2.2%) had positive RNA tests >90 days and up to 416 days after infection.

Empowering Academic Labs and Scientists to Test for COVID-19

The lack of widespread COVID-19 testing and the prevalence of asymptomatic infections have been major factors in the current pandemic. Despite the improvements in clinical testing, as we move toward reopening USA, widespread surveillance testing becomes critical. Academic (nonmedical) labs can help provide such testing;the CDC-approved guidelines for COVID-19 testing require routine equipment and protocols that are commonly used in academic research labs around the country. Faculty at the authors institution were successfully able to test asymptomatic students for COVID-19. By empowering nonmedical academic scientists with preexisting knowledge, expertise with the protocols, and access to the instruments, an additional 1.23.5 million COVID-19 tests could be processed each day at local universities and academic labs.

The evaluation of potential global impact of the N501Y mutation in SARS-COV-2 positive patients.

Rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 mutations are significant to control the contagion and spread rate of the virus. We aimed to evaluate the N501Y mutation rate in randomly chosen positive patients with the polymerase chain reaction (PCR). The evaluation and analysis of the data with a retrospective approach in cases with mutations, in terms of public health, will contribute to the literature on the global pandemic that affects our society. Public health authorities will take the necessary precautions and evaluate the current situation. The N501Y mutation was detected in patients with positive Covid-19 PCR test results. The positive samples were examined based on the 6-carboxy-fluorescein (FAM) channel in reverse transcription PCR (RT-PCR) quantitation cycle (Cq) values as low Cq (<25), medium Cq (25-32), and high Cq (32-38) groups. In the study, 2757 (19.7%) of 13 972 cases were detected as mutation suspects and 159 (5.8%) of them were found to have mutations. The ages of the cases with mutations ranged from 1 to 88 years (mean age of 40.99 ± 17.55). 49.7% (n = 79) of the cases with mutations were male, and 50.3% (n = 80) were female. When the RT-PCR-Cq results were examined, it was seen that it varied between 11.3 and 35.03, with an average of 20.75 ± 3.32.

Relationship of SARS-CoV-2 Antigen and Reverse Transcription PCR Positivity for Viral Cultures.

We assessed the relationship between antigen and reverse transcription PCR (RT-PCR) test positivity and successful virus isolation. We found that antigen test results were more predictive of virus recovery than RT-PCR results. However, virus was isolated from some antigen-negative and RT-PCR‒positive paired specimens, providing support for the Centers for Disease Control and Prevention antigen testing algorithm.

Retrospective quantitative detection of SARS-CoV-2 by digital PCR showing high accuracy for low viral load specimens.

INTRODUCTION: Accurate detection of severe acute respiratory syndrome coronavirus 2 is critical for diagnosis and disease status evaluation of Coronavirus disease 2019. We retrospectively evaluated the infection status and viral load of severe acute respiratory syndrome coronavirus 2 in Nantong city, China, using a quantitative digital polymerase chain reaction and reverse-transcription PCR. METHODOLOGY: A total of 103 clinical specimens from 31 patients were collected and tested by digital PCR and reverse-transcription PCR. RESULTS: The overall accuracy of digital PCR was 96.8%, which was higher than the overall accuracy of 87.1% for reverse-transcription PCR. 4 (3.88%) specimens for ORF1ab and 22 (21.36%) specimens for N gene were negative by reverse-transcription PCR but positive by digital PCR. 3 (2.91%, 3/103) specimens of ORF1ab were positive by reverse-transcription PCR but negative by digital PCR. The digital PCR assay exhibited higher sensitivity to measure the N gene than the ORF1ab gene (p < 0.01). CONCLUSIONS: Our results showed that digital PCR assay provides more reliable detection of Coronavirus disease 2019 than reverse-transcription PCR, especially for low viral load specimens.

Saliva Quantification of SARS-CoV-2 in Real-Time PCR From Asymptomatic or Mild COVID-19 Adults.

The fast spread of COVID-19 is related to the highly infectious nature of SARS-CoV-2. The disease is suggested to be transmitted through saliva droplets and nasal discharge. The saliva quantification of SARS-CoV-2 in real-time PCR from asymptomatic or mild COVID-19 adults has not been fully documented. This study analyzed the relationship between salivary viral load on demographics and clinical characteristics including symptoms, co-morbidities in 160 adults diagnosed as COVID-19 positive patients recruited between September and December 2020 in four French centers. Median initial viral load was 4.12 log10 copies/mL (IQR 2.95-5.16; range 0-10.19 log10 copies/mL). 68.6% of adults had no viral load detected. A median load reduction of 23% was observed between 0-2 days and 3-5 days, and of 11% between 3-5 days and 6-9 days for the delay from onset of symptoms to saliva sampling. No significant median difference between no-symptoms vs. symptoms patients was observed. Charge was consistently similar for the majority of the clinical symptoms excepted for headache with a median load value of 3.78 log10 copies/mL [1.95-4.58] (P < 0.003). SARS-CoV-2 RNA viral load was associated with headache and gastro-intestinal symptoms. The study found no statistically significant difference in viral loads between age groups, sex, or presence de co-morbidity. Our data suggest that oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays.

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human ß2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

Diagnostic, Prognostic, and Therapeutic Value of Droplet Digital PCR (ddPCR) in COVID-19 Patients: A Systematic Review.

Accurate detection of SARS-CoV-2, the pathogen causing the global pandemic of COVID-19, is essential for disease surveillance and control. Quantitative reverse transcription PCR (RT-qPCR) is considered the reference standard test for the diagnosis of SARS-CoV-2 by the World Health Organization and Centers for Disease Control and Prevention. However, its limitations are a prompt for a more accurate assay to detect SARS-CoV-2, quantify its levels, and assess the prognosis. This article aimed to systematically review the literature and assess the diagnostic performance of droplet digital PCR (ddPCR), also to evaluate its potential role in prognosis and management of COVID-19 patients. PubMed and Scopus databases were searched to identify relevant articles published until 13 July 2021. An additional PubMed search was performed on 21 October 2021. Data from the 39 eligible studies were extracted and an overall 3651 samples from 2825 patients and 145 controls were used for our qualitative analysis. Most studies reported ddPCR was more accurate than RT-qPCR in detecting and quantifying SARS-CoV-2 levels, especially in patients with low viral loads. ddPCR was also found highly effective in quantifying SARS-CoV-2 RNAemia levels in hospitalized patients, monitoring their disease course, and predicting their response to therapy. These findings suggest ddPCR could serve as a complement or alternative SARS-CoV-2 tool with emerging diagnostic, prognostic, and therapeutic value, especially in hospital settings. Additional research is still needed to standardize its laboratory protocols, also to accurately assess its role in monitoring COVID-19 therapy response and in identifying SARS-CoV-2 emerging variants.

Implementation and Accuracy of BinaxNOW Rapid Antigen COVID-19 Test in Asymptomatic and Symptomatic Populations in a High-Volume Self-Referred Testing Site.

Rapid antigen tests are simple to perform and provide results within 15 min. We describe our implementation and assess performance of the BinaxNOW COVID-19 Antigen Test (Abbott Laboratories) in 6,099 adults at a self-referred walk-up testing site. Participants were grouped by self-reported COVID-19 exposure and symptom status. Most (89%) were asymptomatic, of whom 17% reported potential exposure. Overall test sensitivity compared with reference laboratory reverse-transcription [RT] PCR testing was 81% (95% confidence interval [CI] 75%, 86%). It was higher in symptomatic (87%; 95% CI 80%, 91%) than asymptomatic (71%; 95% CI 61%, 80%) individuals. Sensitivity was 82% (95% CI 66%, 91%) for asymptomatic individuals with potential exposure and 64% (95% CI 51%, 76%) for those with no exposure. Specificity was greater than 99% for all groups. BinaxNOW has high accuracy among symptomatic individuals and is below the FDA threshold for emergency use authorization in asymptomatic individuals. Nonetheless, rapid antigen testing quickly identifies positive among those with symptoms and/or close contact exposure and could expedite isolation and treatment. IMPORTANCE The BinaxNOW rapid antigen COVID-19 test had a sensitivity of 87% in symptomatic and 71% asymptomatic individuals when performed by health care workers in a high-throughput setting. The performance may expedite isolation decisions or referrals for time-sensitive monoclonal antibody treatment in communities where timely COVID PCR tests are unavailable.

Structure-Based Primer Design Minimizes the Risk of PCR Failure Caused by SARS-CoV-2 Mutations.

The coronavirus disease 2019 (COVID-19) has caused and is still causing tremendous damage to the global economy and human health. Qualitative reverse transcription-PCR (RT-qPCR) is the golden standard for COVID-19 test. However, the SARS-CoV-2 variants may not only make vaccine less effective but also evade RT-qPCR test. Here we suggest an innovative primer design strategy for the RT-qPCR test of SARS-CoV-2. The principle is that the primers should be designed based on both the nucleic acid sequence and the structure of the protein encoded. The three nucleotides closest to the 3' end of the primer should be the codon which encodes the tryptophan in the structure core. Based on this principle, we designed a pair of primers targeting the nucleocapsid (N) gene. Since tryptophan is encoded by only one codon, any mutation that occurs at this position would change the amino acid residue, resulting in an unstable N protein. This means that this kind of SARS-CoV-2 variant could not survive. In addition, both our data and previous reports all indicate that the mutations occurring at other places in the primers do not significantly affect the RT-qPCR result. Consequently, no SARS-CoV-2 variant can escape detection by the RT-qPCR kit containing the primers designed based on our strategy.

Genetic Diversity of SARS-CoV-2 among Travelers Arriving in Hong Kong.

We sequenced 10% of imported severe acute respiratory syndrome coronavirus 2 infections detected in travelers to Hong Kong and revealed the genomic diversity of regions of origin, including lineages not previously reported from those countries. Our results suggest that international or regional travel hubs might be useful surveillance sites to monitor sequence diversity.

Investigating the sequence variation in the influenza A matrix genes during the 2017-2018 and 2018-2019 seasons in samples from a local population in London.

Recent publications have highlighted the emergence of mutations in the M1 gene of both influenza A H1N1pdm09 and H3N2 subtypes affecting the performance of commercial RT-PCR assays. Respiratory samples from the 2018/2019 season positive by our in-house RT-PCR for influenza A were analysed for the prevalence and impact of any M1 gene mutations. Sequence information was used to re-design primers for our routine assay and their performance assessed. Forty-five samples, consisting of 11 H1N1pdm09 and 34 H3N2 subtypes, together with the NIBSC H1N1 control were sequenced. All samples displayed the core mutations for H1N1 M1(C154T; G174A and G238A) and for H3N2 M1(C153T; C163T and G189T); three of the H1N1pdm09 viruses also showed a small number of point mutations. None of the mutations appeared to affect either the sensitivity or efficiency of the RT-PCR when compared to the re-designed primers. Although the mutations we found agreed with those in the publications cited we did not encounter any problems with our routine diagnostic assay and no improvements were found when the primers were modified to suit those mutations. However, it is likely that the influenza A virus M1 gene will accumulate further mutations that could impact RT-PCR assays and, therefore, it would be prudent to implement routine sequencing of samples during the influenza seasons to ensure no loss in assay performance.

Probability-Based Estimates of Severe Acute Respiratory Syndrome Coronavirus 2 Seroprevalence and Detection Fraction, Utah, USA.

We aimed to generate an unbiased estimate of the incidence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 4 urban counties in Utah, USA. We used a multistage sampling design to randomly select community-representative participants >12 years of age. During May 4-June 30, 2020, we collected serum samples and survey responses from 8,108 persons belonging to 5,125 households. We used a qualitative chemiluminescent microparticle immunoassay to detect SARS-CoV-2 IgG in serum samples. We estimated the overall seroprevalence to be 0.8%. The estimated seroprevalence-to-case count ratio was 2.5, corresponding to a detection fraction of 40%. Only 0.2% of participants from whom we collected nasopharyngeal swab samples had SARS-CoV-2-positive reverse transcription PCR results. SARS-CoV-2 antibody prevalence during the study was low, and prevalence of PCR-positive cases was even lower. The comparatively high SARS-CoV-2 detection rate (40%) demonstrates the effectiveness of Utah's testing strategy and public health response.

Clinical Performance of the Standard Q COVID-19 Rapid Antigen Test and Simulation of its Real-World Application in Korea.

The rapid antigen test (RAT) for coronavirus disease (COVID-19) represents a potent diagnostic method in situations of limited molecular testing resources. However, considerable performance variance has been reported with the RAT. We evaluated the clinical performance of Standard Q COVID-19 RAT (SQ-RAT; SD Biosensor, Suwon, Korea), the first RAT approved by the Korean Ministry of Food and Drug Safety. In total, 680 nasopharyngeal swabs previously tested using real-time reverse-transcription PCR (rRT-PCR) were retested using SQ-RAT. The clinical sensitivity of SQ-RAT relative to that of rRT-PCR was 28.7% for all specimens and was 81.4% for specimens with RNA-dependent RNA polymerase gene (RdRp) threshold cycle (Ct) values ≤23.37, which is the limit of detection of SQ-RAT. The specificity was 100%. The clinical sensitivity of SQ-RAT for COVID-19 diagnosis was assessed based on the Ct distribution at diagnosis of 33,294 COVID-19 cases in Korea extracted from the laboratory surveillance system of Korean Society for Laboratory Medicine. The clinical sensitivity of SQ-RAT for COVID-19 diagnosis in the Korean population was 41.8%. Considering the molecular testing capacity in Korea, use of the RAT for COVID-19 diagnosis appears to be limited.