Evaluation of Four Strategies for SARS-CoV-2 Detection: Characteristics and Prospects.

The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed an enormous burden on the global public health system and has had disastrous socioeconomic consequences. Currently, single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests (POCTs), and rapid antigen tests are implemented in different scenarios to detect SARS-CoV-2, but a comprehensive evaluation of them is scarce and remains to be explored. In this study, 3 SARS-CoV-2 inactivated cell culture supernatants were used to evaluate the analytical performance of these strategies. Additionally, 5 recombinant SARS-CoV-2 nucleocapsid (N) proteins were also used for rapid antigen tests. For the wild-type (WT), Delta, and Omicron strains, the lowest inactivated virus concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68-142.86 ng/mL, respectively. This study provided helpful insights into the scientific deployment of tests and recommended the full-scale consideration of the testing purpose, resource availability, cost performance, result rapidity, and accuracy to facilitate a profound pathway toward the long-term surveillance of coronavirus disease 2019 (COVID-19). IMPORTANCE In the study, we reported an evaluation of 4 detection strategies implemented in different scenarios for SARS-CoV-2 detection: single sampling tests, 20-in-1 pooling tests, nucleic acid point-of-care tests, and rapid antigen tests. 3 SARS-CoV-2-inactivated SARS-CoV-2 cell culture supernatants and 5 recombinant SARS-CoV-2 nucleocapsid proteins were used for evaluation. In this analysis, we found that for the WT, Delta, and Omicron supernatants, the lowest concentrations to achieve 100% detection rates of single sampling tests ranged between 1.28 × 102 to 1.02 × 103, 1.28 × 102 to 4.10 × 103, and 1.28 × 102 to 2.05 × 103 copies/mL. The 20-in-1 pooling tests ranged between 1.30 × 102 to 1.04 × 103, 5.19 × 102 to 2.07 × 103, and 2.59 × 102 to 1.04 × 103 copies/mL. The nucleic acid POCTs were all 1.42 × 103 copies/mL. The rapid antigen tests ranged between 2.84 × 105 to 7.14 × 106, 8.68 × 104 to 7.14 × 106, and 1.12 × 105 to 3.57 × 106 copies/mL. For the WT, Delta AY.2, Delta AY.1/AY.3, Omicron BA.1, and Omicron BA.2 recombinant N proteins, the lowest concentrations to achieve 100% detection rates of rapid antigen tests ranged between 3.47 to 142.86, 1.74 to 142.86, 3.47 to 142.86, 3.47 to 142.86, and 5.68 to 142.86 ng/mL, respectively.

Effectiveness of SARS-CoV-2-inactivated vaccine and the correlation to neutralizing antibodies: A test-negative case-control study.

Severe acute respiratory syndrome coronavirus 2 breakthrough infection in highly vaccinated populations raises study on the effectiveness for inactivated vaccine, including effectiveness of the vaccine dose, the continuance of effectiveness, the effectiveness against severe/critical coronavirus disease 2019 and against secondary attacks. A population of 10 870 close contacts were investigated in a Delta variant's epidemic. The effectiveness of vaccination was estimated in a test-negative case-control study. In addition, serum was used to detect neutralizing antibodies, to explore their correlation to effectiveness. The vaccine effectiveness (VE) values were estimated for populations aged 12 years or older. The overall adjusted VE was 56.2% and a two-dose vaccine was more effective than a one-dose vaccine (56.7% vs. 43.8%). In addition, the population that got the second dose vaccine within 2 months showed higher VE than the population vaccinated for longer than 2 months (61.5% vs. 52.3%). Among the population who vaccinated 2 doses or within 2 months, a higher level of neutralizing antibodies was observed. For infected cases, vaccinated populations showed lower rates of transmission (2.63% vs. 4.36%). Further, those vaccinated cases, who were not found causing transmission, had a higher level of antibodies. The study provided a full view of the effectiveness of inactivated vaccines in a real-world setting. The time-related VE against infection and lower transmission of breakthrough vaccinated cases were observed, which may indicate that a necessity of a booster vaccine to maintain the effectiveness and high level of neutralizing antibody.

Evaluating the value of anti-SARS-CoV-2 antibody detection and neutralizing responses with euvirus: A population of 10776 close contacts in the epidemic of Fujian.

BACKGROUND: Nucleic acid detection represents limitations due to its false-negative rate and technical complexity in the COVID-19 pandemic. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests are widely spread all over the world presently. However, there is no report on the effectiveness of anti-SARS-CoV-2 antibody testing methods in China. METHODS: We gathered 10776 serum samples from close contacts of the SARS-CoV-2 infections in Fujian of China and used 2 chemiluminescence immunoassays (Wantai Bio., Yahuilong Bio.) and 2 lateral flow immunoassays (Lizhu Bio. and Dongfang Bio.) to perform the anti-SARS-CoV-2 antibody tests in China. RESULTS: The 4 antibody tests have great diagnostic value for infected or uninfected, especially in the neutralizing antibodies tests, the AUC can reach 0.939 (Wantai Bio.) and 0.916 (Yahuilong Bio.). Furthermore, we used pseudoviruses and euvirus neutralization assay to validate the effectiveness of these antibody test, the results of pseudoviruses neutralization assay or euvirus neutralization assay shows a considerable correlation with the 4 antibody detection respectively, particularly in euvirus neutralization assay, neutralizing antibodies detected by Wantai Bio. or Yahuilong Bio., the correlation can get the level of 0.93 or 0.82. CONCLUSIONS: The findings of this study demonstrate that the detections of antibodies have profound value in the diagnosis of COVID-19.

Comparison of analytical sensitivity of SARS-CoV-2 molecular detection kits.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a significant impact on global public health systems, making nucleic acid detection an important tool in epidemic prevention and control. Detection kits based on real-time reverse transcriptase PCR (rRT-PCR) have been used widely in clinics, but their analytical sensitivity (limit of detection, LOD) remains controversial. Moreover, there is limited research evaluating the analytical sensitivity of other molecular detection kits. METHODS: In this study, armored ribonucleic acid reference materials developed in-house were used to evaluate the analytical sensitivity of SARS-CoV-2 detection kits approved by the National Medical Products Administration. These were based on rRT-PCR and other molecular detection assays. RESULTS: The percentage retesting required with rRT-PCR kits was as follows: 0%, 7.69%, 15.38%, and 23.08% for samples with concentrations ranging from 50 000 to 781 copies/ml. In total, 93% of rRT-PCR kits had a LOD <1000 copies/ml. Only one kit had an LOD >1000 copies/ml. The LOD of other molecular detection kits ranged from 68 to 2264 copies/ml. CONCLUSIONS: The study findings can help pharmaceutical companies optimize and improve detection kits, guide laboratories in selecting kits, and assist medical workers in their daily work.

Nomogram Model for Prediction of SARS-CoV-2 Breakthrough Infection in Fujian: A Case-Control Real-World Study.

SARS-CoV-2 breakthrough infections have been reported because of the reduced efficacy of vaccines against the emerging variants globally. However, an accurate model to predict SARS-CoV-2 breakthrough infection is still lacking. In this retrospective study, 6,189 vaccinated individuals, consisting of SARS-CoV-2 test-positive cases (n = 219) and test-negative controls (n = 5970) during the outbreak of the Delta variant in September 2021 in Xiamen and Putian cities, Fujian province of China, were included. The vaccinated individuals were randomly split into a training (70%) cohort and a validation (30%) cohort. In the training cohort, a visualized nomogram was built based on the stepwise multivariate logistic regression. The area under the curve (AUC) of the nomogram in the training and validation cohorts was 0.819 (95% CI, 0.780-0.858) and 0.838 (95% CI, 0.778-0.897). The calibration curves for the probability of SARS-CoV-2 breakthrough infection showed optimal agreement between prediction by nomogram and actual observation. Decision curves indicated that nomogram conferred high clinical net benefit. In conclusion, a nomogram model for predicting SARS-CoV-2 breakthrough infection based on the real-world setting was successfully constructed, which will be helpful in the management of SARS-CoV-2 breakthrough infection.

Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Mediated Multiplexable and Portable Detection Platform for GII Genotype Porcine Epidemic Diarrhoea Virus Rapid Diagnosis

Porcine epidemic diarrhoea virus (PEDV) is a member of the genus Alphacoronavirus in the family Coronaviridae. It causes acute watery diarrhoea and vomiting in piglets with high a mortality rate. Currently, the GII genotype, PEDV, possesses a high separation rate in wild strains and is usually reported in immunity failure cases, which indicates a need for a portable and sensitive detection method. Here, reverse transcription–recombinase aided amplification (RT-RAA) was combined with the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas12a system to establish a multiplexable, rapid and portable detection platform for PEDV. The CRISPR RNA (crRNA) against Spike (S) gene of GII PEDV specifically were added into the protocol. This system is suitable for different experimental conditions, including ultra-sensitive fluorescence, visual, UV light, or flow strip detection. Moreover, it exhibits high sensitivity and specificity and can detect at least 100 copies of the target gene in each reaction. The CRISPR/Cas12a detection platform requires less time and represents a rapid, reliable and practical tool for the rapid diagnosis of GII genotype PEDV.

Impact of SARS-CoV-2 Variants on the Analytical Sensitivity of rRT-PCR Assays.

Emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility, pathogenicity, and immune escape ability have ravaged many countries and regions, which has brought substantial challenges to pandemic prevention and control. Real-time reverse transcriptase PCR (rRT-PCR) is widely used for SARS-CoV-2 detection but may be limited by the continuous evolution of the virus. However, the sensitivity of Chinese commercial rRT-PCR kits to critical SARS-CoV-2 variants remains unknown. In this study, contrived MS2 virus-like particles were used as reference materials to evaluate the analytical sensitivity of Daan, BioGerm, EasyDiagnosis, Liferiver, and Sansure kits when detecting six important variants (Alpha, Beta, Gamma, Delta, Omicron, and Fin-796H). The Beta and Delta variants adversely affected the analytical sensitivity of the BioGerm ORF1ab gene assay (9.52% versus 42.96%, P = 0.014, and 14.29% versus 42.96%, P = 0.040, respectively), whereas the N gene assay completely failed in terms of the Fin-796H variant. The Gamma and Fin-796H variants impeded the PCR amplification efficiency for the Sansure ORF1ab gene assay (33.33% versus 66.67%, P = 0.031, and 66.67% versus 95.24%, P = 0.040, respectively), and the Delta variant compromised the E gene assay (52.38% versus 85.71%, P = 0.019). The Alpha and Omicron variants had no significant effect on the kits. This study highlights the necessity of identifying the potential effect of viral mutations on the efficacy and sensitivity of clinical detection assays. It can also provide helpful insights regarding the development and optimization of diagnostic assays and aid the strategic management of the ongoing pandemic.

Chinese Expert Consensus on the Nucleic Acid Detection of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) has already become a pandemic wherein the infection's timely diagnosis has proven beneficial to patient treatment and disease control. Nucleic acid detection has been the primary laboratory diagnostic method for the detection of SARS-CoV-2. To ensure laboratory staff safety and quality nucleic acid testing, the Chinese Society of Laboratory Medicine formulated this consensus, based on the Chinese National Recommendations and previous literature for nucleic acid detection. A working group comprises 34 hospital professionals experience with real-time polymerase chain reactions (PCR) testing for SARS-CoV-2 drafted guidance statements during online discussions. A modified Delphi methodology was used in forming a consensus among a wider group of hospital professionals with SARS-CoV-2 detection experience. Guidance statements were developed for four categories: (I) specimen type, priority, collecting, transportation and receiving; (II) nucleic acid isolation and amplification; (III) quality control; (IV) biosafety management and decontamination. The modified Delphi voting process included a total of 29 guidance statements and final agreement. Consensus was reached after two rounds of voting. Recommendations were established for the detection of SARS-CoV-2 using real time PCR testing based on evidence and group consensus. The manuscript was evaluated against The Appraisal of Guidelines for Research & Evaluation Instrument (AGREE II) and was developed to aid medical laboratory staff in the detection of the ribonucleic acid (RNA) of SARS-CoV-2.

Immunologic Testing for SARS-CoV-2 Infection from the Antigen Perspective.

Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread globally as a severe pandemic. SARS-CoV-2 infection stimulates antigen-specific antibody responses. Multiple serologic tests have been developed for SARS-CoV-2. However, which antigens are most suitable for serological testing remains poorly understood. Specifically, which antigens have the highest sensitivity and specificity for serological testing and which have the least cross-reactivity with other coronaviruses are currently unknown. Previous studies have shown that the S1 domain of the spike (S) protein has very low cross-reactivity between epidemic coronaviruses and common human coronaviruses, whereas the S2 domain of the S protein and the nucleocapsid protein (N protein) show low-level cross-reactivity. Therefore, S1 is considered more specific than the native homotrimer of the S protein, and the receptor-binding domain as an antigen to test patient antibodies is more sensitive than the native N protein. In addition, an increasing number of studies have used multiantigen protein arrays to screen serum from convalescent patients with COVID-19. Antigen combinations demonstrated improved performance compared to each individual antigen. For rapid antigen detection, the sensitivity of the test is higher in the first week of onset of the disease with high viral loads. Highly sensitive and specific immunological diagnostic methods for antibodies or those that directly detect viral antigens in clinical samples would be beneficial for the rapid and accurate diagnosis of SARS-CoV-2 infection.

External Quality Assessment for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Clinical Laboratories.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Viral nucleic acid testing is the diagnostic gold standard and can play an important role in the prevention and control of this infection. In this study, bacteriophage MS2 virus-like particles encapsulating specific RNA sequences of SARS-CoV-2 and other coronaviruses were prepared by genetic engineering. The assessment panel, consisting of four positive samples with concentrations of 2.8, 3.5, 4.2, and 4.9 log10 copies/mL and five negative samples with other human coronaviruses, was prepared and distributed to evaluate the accuracy of routine viral RNA detection. Results of 931 panels from 844 laboratories were collected. The overall percentage agreement, positive percentage agreement (PPA), and negative percentage agreement, defined as the percentage of agreement between the correct results and total results submitted for all, positive, and negative samples were 96.8% (8109/8379), 93.9% (3497/3724), and 99.1% (4612/4655), respectively. For samples with concentrations of 4.9 and 4.2 log10 copies/mL, the PPAs were >95%. However, for 3.5 and 2.8 log10 copies/mL, the PPAs were 94.6% (881/931) and 84.9% (790/931), respectively. For all negative samples, the negative percentage agreement values were >95%. Thus, most laboratories can reliably detect SARS-CoV-2. However, further improvement and optimization are required to ensure the accuracy of detection in panel members with lower concentrations of viral RNA.

COVID-19: Insight into the asymptomatic SARS-COV-2 infection and transmission.

The existence of a substantial but unclear number of asymptomatic SARS-COV-2 patients worldwide has raised concerns among global public health authorities. In this review, according to the published literature, we provided the evidence that asymptomatic infections can result in person-to-person transmission. Four studies suggested that the virus can be transmitted by asymptomatic patients for at least two consecutive generations, indicating its strong infectivity. Asymptomatic infection tends to be, but is not only, identified among young people (<20 years old). The majority of asymptomatic patients appear to have a milder clinical course during hospitalization, but the severity of the symptoms of the secondary patients infected by SARS-COV-2 from asymptomatic patients varies with their physical constitution. The proportion of asymptomatic individuals among all confirmed cases widely differed (from 1.95% to 87.9%) according to the study setting and the populations studied. The increasing large-scale tests are expected to give more information about the true number of asymptomatic infections in the population. In China and other countries, various guidelines for management of asymptomatic cases have been issued. Importantly, early detection, early reporting, early isolation and early treatment of asymptomatic patients require the joint efforts of policy makers, clinicians, technicians, epidemiologists, virologists and patients.

Primer design for quantitative real-time PCR for the emerging Coronavirus SARS-CoV-2.

In December 2019, a new coronavirus disease (COVID-19) outbreak occurred in Wuhan, China. Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), which is the seventh coronavirus known to infect humans, is highly contagious and has rapidly expanded worldwide since its discovery. Quantitative nucleic acid testing has become the gold standard for diagnosis and guiding clinical decisions regarding the use of antiviral therapy. However, the RT-qPCR assays targeting SARS-CoV-2 have a number of challenges, especially in terms of primer design. Primers are the pivotal components of a RT-qPCR assay. Once virus mutation and recombination occur, it is difficult to effectively diagnose viral infection by existing RT-qPCR primers. Some primers and probes have also been made available on the WHO website for reference. However, no previous review has systematically compared the previously reported primers and probes and described how to design new primers in the event of a new coronavirus infection. This review focuses on how primers and probes can be designed methodically and rationally, and how the sensitivity and specificity of the detection process can be improved. This brief review will be useful for the accurate diagnosis and timely treatment of the new coronavirus pneumonia.