[Tracking analysis of viral nucleic acid Ct value in patients with re-positive SARS-CoV-2 infection].

OBJECTIVE: To track analysis of viral nucleic acid test results in patients with re-positive SARS-CoV-2 infection, and provide clinical reference for nucleic acid test of re-positive cases. METHODS: A retrospective study was conducted. The multiple nucleic acid results of 96 cases with SARS-CoV-2 infection tested by medical laboratory of Shenzhen Luohu Hospital Group from January to September in 2022 were analyzed. The test dates and cycle threshold (Ct) values of detectable positive virus nucleic acid in the 96 cases were summarized and analyzed. RESULTS: A total of 96 patients with SARS-CoV-2 infection were retested re-sampled for nucleic acid testing at least 12 days after the initial positive screening. Among them, 54 cases (56.25%) had Ct value of < 35 for nucleocapsid protein gene (N) and/or open reading frame 1ab gene (ORF 1ab), 42 cases (43.75%) had Ct value ≥ 35. In the re-sampling of infected patients, N gene titers were 25.08 to 39.98 Ct cycles, and ORF 1ab gene titers were 23.16 to 39.56 Ct cycles. Compared with the positive results of the initial screening, the Ct values of N gene and/or ORF 1ab gene positive were increased in 90 cases (93.75%). Among them, the patients with the longest duration of nucleic acid positive could still be positive for double targets (the Ct value of N gene was 38.60, and the Ct value of ORF 1ab gene was 38.11) at an interval of 178 days after the initial positive screening. CONCLUSIONS: Patients infected with SARS-CoV-2 can be sustained or repeatedly tested positive for nucleic acid for a long period of time, and most of them had Ct values < 35. But whether it is infectious needs to be comprehensively evaluated by combining epidemiology, variant type, samples with the alive virus, and clinical symptoms and signs.

Protective antigenic epitopes revealed by immunosignatures after three doses of inactivated SARS-CoV-2 vaccine.

Background: SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected millions of people around the world. Vaccination is a pillar in the strategy to control transmission of the SARS-CoV-2 spread. Immune responses to vaccination require elucidation. Methods: The immune responses to vaccination with three doses of inactivated SARS-CoV-2 vaccine were followed in a cohort of 37 healthy adults (18-59 years old). Blood samples were collected at multiple time points and submitted to peptide array, machine learning modeling, and sequence alignment analyses, the results of which were used to generate vaccine-induced antibody-binding region (VIABR) immunosignatures (Registration number: ChiCTR2200058571). Results: Antibody spectrum signals showed vaccination stimulated antibody production. Sequence alignment analyses revealed that a third vaccine dose generated a new highly represented VIABR near the A570D mutation, and the whole process of inoculation enhanced the VIABR near the N501Y mutation. In addition, the antigen conformational epitopes varied between short- and long-term samples. The amino acids with the highest scores in the short-term samples were distributed primarily in the receptor binding domain (RBD) and N-terminal domain regions of spike (S) protein, while in the long-term samples (12 weeks after the 2nd dose), some new conformational epitopes (CEs) were localized to crevices within the head of the S protein trimer. Conclusion: Protective antigenic epitopes were revealed by immunosignatures after three doses of inactivated SARS-CoV-2 vaccine inoculation. A third dose results in a new top-10 VIABR near the A570D mutation site of S protein, and the whole process of inoculation enhanced the VIABR near the N501Y mutation, thus potentially providing protection from strains that have gained invasion and immune escape abilities through these mutation.

Performance comparison of two nucleic acid amplification systems for SARS-CoV-2 detection: A multi-center study.

BACKGROUND: Many rapid nucleic acid testing systems have emerged to halt the development and spread of COVID-19. However, so far relatively few studies have compared the diagnostic performance between these testing systems and conventional detection systems. Here, we performed a retrospective analysis to evaluate the clinical detection performance between SARS-CoV-2 rapid and conventional nucleic acid detection system. METHODS: Clinical detection results of 63,352 oropharyngeal swabs by both systems were finally enrolled in this analysis. Sensitivity (SE), specificity (SP), and positive and negative predictive value (PPV, NPV) of both systems were calculated to evaluate their diagnostic accuracy. Concordance between these two systems were assessed by overall, positive, negative percent agreement (OPA, PPA, NPA) and κ value. Sensitivity of SARS-CoV-2 rapid nucleic acid detection system (Daan Gene) was further analyzed with respect to the viral load of clinical specimens. RESULTS: Sensitivity of Daan Gene was slightly lower than that of conventional detection system (0.86 vs. 0.979), but their specificity was equivalent. Daan Gene had ≥98.0% PPV and NPV for SARS-CoV-2. Moreover, Daan Gene demonstrated an excellent test agreement with conventional detection system (κ = 0.893, p = 0.000). Daan Gene was 99.31% sensitivity for specimens with high viral load (Ct < 35) and 50% for low viral load (Ct ≥ 35). CONCLUSIONS: While showing an analytical sensitivity slightly below than that of conventional detection system, rapid nucleic acid detection system may be a diagnostic alternative to rapidly identify SARS-CoV-2-infected individuals with high viral loads and a powerful complement to current detection methods.

Protective antigenic epitopes revealed by immunosignatures after three doses of inactivated SARS-CoV-2 vaccine

Background SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) has infected millions of people around the world. Vaccination is a pillar in the strategy to control transmission of the SARS-CoV-2 spread. Immune responses to vaccination require elucidation. Methods The immune responses to vaccination with three doses of inactivated SARS-CoV-2 vaccine were followed in a cohort of 37 healthy adults (18–59 years old). Blood samples were collected at multiple time points and submitted to peptide array, machine learning modeling, and sequence alignment analyses, the results of which were used to generate vaccine-induced antibody-binding region (VIABR) immunosignatures (Registration number: ChiCTR2200058571). Results Antibody spectrum signals showed vaccination stimulated antibody production. Sequence alignment analyses revealed that a third vaccine dose generated a new highly represented VIABR near the A570D mutation, and the whole process of inoculation enhanced the VIABR near the N501Y mutation. In addition, the antigen conformational epitopes varied between short- and long-term samples. The amino acids with the highest scores in the short-term samples were distributed primarily in the receptor binding domain (RBD) and N-terminal domain regions of spike (S) protein, while in the long-term samples (12 weeks after the 2nd dose), some new conformational epitopes (CEs) were localized to crevices within the head of the S protein trimer. Conclusion Protective antigenic epitopes were revealed by immunosignatures after three doses of inactivated SARS-CoV-2 vaccine inoculation. A third dose results in a new top-10 VIABR near the A570D mutation site of S protein, and the whole process of inoculation enhanced the VIABR near the N501Y mutation, thus potentially providing protection from strains that have gained invasion and immune escape abilities through these mutation.

Performance evaluation of an automatic chemiluminescence immune platform for SARS-CoV-2 neutralizing antibody after vaccination in real world.

OBJECTIVE: Reliable high-throughput serological assays for SARS-CoV-2 antibodies present an important role in the strength and duration of immunity after vaccination. The study investigated the analytical and clinical performances of neutralizing antibodies (NTAb) assay by chemiluminescent (CLIA), and SARS-CoV-2 neutralizing antibody after vaccination in real world. METHODS: The analytical performances of CLIA for SARS-CoV-2 NTAb were evaluated, followed by the sensitivity and specificity identified with a PRNT test from 50 volunteers. Then, a cohort of vaccine recipients (n = 37) were tracked with SARS-CoV-2 NTAb assay at prior to vaccination, one, three and six months post two doses. In real world, a total of 737 cases were recruited from physical examination center in Shenzhen Luohu People's Hospital (from Jun to August 2021) to analyze vaccination status. RESULTS: Serological assays on the CLIA were found with excellent characteristics including imprecision, repeatability and linearity. Besides, it was robust to icterus, lipemia and hemolysis. The good sensitivity and specificity were obtained at 98% and 100%, respectively. NTAb results showed a high correlation with PRNT50 titers (r 0.61). Until July 2021, the BBIBP-CorV (76.3%) and Sinovac CoronaVac (20.5%) were the predominant vaccines injection in Shenzhen, China. Adolescent less than 18 years was the main unvaccinated group (52.1%). The seropositive rate of inactive SRAR-CoV-2 vaccines exceeded 97% after inoculation. The NTAb generated by Sinovac CoronaVac with the schedule of 0-56 days was found significantly lower than that by BBIBP-CorV (P < 0.001). The follow-up of NTAb changes in a cohort and the dynamic variation of NTAb in real world disclosed steep downward by almost three times for NTAb level occurred at three months post twice vaccinations. The seropositive ratio was at least 50% over 6 months. CONCLUSIONS: SARS-CoV-2 neutralizing antibodies assay show excellent analytical and clinical performances, and a high correlation with neutralizing activity. Anti-epidemic measures and the urgent trial of SARS-CoV-2 vaccine was calling for adolescents.

Dynamic observation of SARS-CoV-2 IgM, IgG, and neutralizing antibodies in the development of population immunity through COVID-19 vaccination.

BACKGROUND: Currently, mass vaccine inoculation against coronavirus disease-2019 (COVID-19) has been being implemented globally. Rapid and the large-scale detection of serum neutralizing antibodies (NAbs) laid a foundation for assessing the immune response against SARS-CoV-2 infection and vaccine. Additional assessments include the duration of antibodies and the optimal time for a heightened immune response. METHODS: The performance of five surrogate NAbs-three chemiluminescent immunoassay (CLIA) and two enzyme-linked immunosorbent assays (ELISAs)-and specific IgM and IgG assays were compared using COVID-19-vaccinated serum (n = 164). Conventional virus neutralization test (cVNT) was used as a criterion and the diagnostic agreement and correlation of the five assays were evaluated. We studied the antibody responses after the two-dose vaccine in volunteers up to 6 months. RESULTS: The sensitivity and specificity of five surrogate NAb assays ranged from 84% to 100%. Our cVNT results indicated great consistency with the surrogate assays. At 28 days after primary vaccination, the seropositivities of the NAbs, IgG, and IgM were 6%, 4%, and 13%, respectively. After the booster dose, seropositivities reached 14%, 65%, and 97%, respectively. Six months after receipt of the second dose, the NAb positive rate was eventually maintained at 66%. In all COVID-19 convalescents, patients were detected with 100% NAb sat three months after discharge. CONCLUSION: COVID-19 vaccine induced a humoral immune response lasting at least six months. Rapid serological detection was used as a proxy for identifying changes in immunity levels and as a guide to whether an individual may require a booster vaccination.

SARS-CoV-2 Host Receptor ACE2 Protein Expression Atlas in Human Gastrointestinal Tract.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells through interactions with its receptor, Angiotensin-converting enzyme 2 (ACE2), causing severe acute respiratory syndrome and death in a considerable proportion of people. Patients infected with SARS-CoV-2 experience digestive symptoms. However, the precise protein expression atlas of ACE2 in the gastrointestinal tract remains unclear. In this study, we aimed to explore the ACE2 protein expression pattern and the underlying function of ACE2 in the gastrointestinal tract, including the colon, stomach, liver, and pancreas. METHODS: We measured the protein expression of ACE2 in the gastrointestinal tract using immunohistochemical (IHC) staining with an ACE2-specific antibody of paraffin-embedded colon, stomach, liver, and pancreatic tissues. The correlation between the protein expression of ACE2 and the prognosis of patients with gastrointestinal cancers was analyzed by the log-rank (Mantel-Cox) test. The influence of ACE2 on colon, stomach, liver, and pancreatic tumor cell line proliferation was tested using a Cell Counting Kit 8 (CCK-8) assay. RESULTS: ACE2 presented heterogeneous expression patterns in the gastrointestinal tract, and it showed a punctate distribution in hepatic cells. Compared to that in parallel adjacent non-tumor tissues, the protein expression of ACE2 was significantly increased in colon cancer, stomach cancer, and pancreatic cancer tissues but dramatically decreased in liver cancer tissues. However, the expression level of the ACE2 protein was not correlated with the survival of patients with gastrointestinal cancers. Consistently, ACE2 did not affect the proliferation of gastrointestinal cancer cells in vitro. CONCLUSION: The ACE2 protein is widely expressed in the gastrointestinal tract, and its expression is significantly altered in gastrointestinal tumor tissues. ACE2 is not an independent prognostic marker of gastrointestinal cancers.

Performance evaluation of a domestic new coronavirus detection kit

Objective: To evaluate whether a domestic novel coronavirus (SARS-CoV-2) nucleic acid detection kit meets the requirements of clinical SARS-CoV-2 nucleic acid detection.

Three cases of asymptomatic SARS-CoV-2 infections diagnosed by combination of nucleic acid and specific igm/IgG antibodies

Objective: Clinical value of SARS-CoV-2 nucleic acid detection combined with specific IgM/IgG in the diagnosis of three asymptomatic COVID-19 infections was investigated.

An enhanced method for nucleic acid detection with CRISPR-Cas12a using phosphorothioate modified primers and optimized gold-nanopaticle strip.

CRISPR-Cas12a system has been shown promising for nucleic acid diagnostics due to its rapid, portable and accurate features. However, cleavage of the amplicons and primers by the cis- and trans-activity of Cas12a hinders the attempts to integrate the amplification and detection into a single reaction. Through phosphorothioate modification of primers, we realized onepot detection with high sensitivity using plasmids of SARS-CoV-2, HPV16 and HPV18. We also identified the activated Cas12a has a much higher affinity to C nucleotide-rich reporter than others. By applying such reporters, the reaction time required for a lateral-flow readout was significantly reduced. Furthermore, to improve the specificity of the strip-based assay, we created a novel reporter and, when combined with a customized gold-nanopaticle strip, the readout was greatly enhanced owing to the elimination of the nonspecific signal. This established system, termed Targeting DNA by Cas12a-based Eye Sight Testing in an Onepot Reaction (TESTOR), was validated using clinical cervical scrape samples for human papillomaviruses (HPVs) detection. Our system represents a general approach to integrating the nucleic acid amplification and detection into a single reaction in CRISPR-Cas systems, highlighting its potential as a rapid, portable and accurate detection platform of nucleic acids.

Evaluation of clinical application of three chemiluminescence detection kits for detection of novel coronavirus (SARS-CoV-2) antibody

Objective: To evaluate the performance of three kinds of chemiluminescence immunoassay (CLIA) kits for novel Coronavirus (SARS-COV-2) antibody, and study the clinical application of SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) antibodies detection.

Rapid detection of SARS-CoV-2 with CRISPR-Cas12a.

The recent outbreak of betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is responsible for the Coronavirus Disease 2019 (COVID-19) global pandemic, has created great challenges in viral diagnosis. The existing methods for nucleic acid detection are of high sensitivity and specificity, but the need for complex sample manipulation and expensive machinery slow down the disease detection. Thus, there is an urgent demand to develop a rapid, inexpensive, and sensitive diagnostic test to aid point-of-care viral detection for disease monitoring. In this study, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) 12a-based diagnostic method that allows the results to be visualized by the naked eye. We also introduced a rapid sample processing method, and when combined with recombinase polymerase amplification (RPA), the sample to result can be achieved in 50 minutes with high sensitivity (1-10 copies per reaction). This accurate and portable detection method holds a great potential for COVID-19 control, especially in areas where specialized equipment is not available.

Comparison of three automatic chemiluminescent immunoassays for monitoring dynamic profile of SARS-CoV-2 IgG and IgM.

BACKGROUND: Seldom performance evaluation and diagnosis comparison studies were reported for different chemiluminescent immunoassay (CLIA) kits approved under an emergency approval program for SARS-CoV-2 infection. METHODS: A total of 100 and 105 serum separately from non-infected populations and COVID-19 patients were detected with SARS-CoV-2 IgM and IgG kits. The characteristics including precision, functional sensitivity, linearity, and accuracy were evaluated for Axceed, iFlash, and Maglumi CLIA kits. RESULTS: Maglumi and iFlash had the best analytical sensitivity for IgM and IgG, respectively. Axceed kits had a linearity response in the detected concentration. The clinical sensitivity of Axceed, iFlash, and Maglumi was 68.0%, 64.9%, and 63.9% with a specificity of 99.0%, 96.0%, and 100% for IgM, 85.6%, 97.9%, and 94.8% with a specificity of 97.0% for IgG. ROC analysis indicated all kits had a diagnostic power greater than 0.9. Notably, either IgM or IgG kits obtained a poor agreement (Kappa value from 0.397 to 0.713) with others. Among 38 recovered patients, 94.7% had an effective immune response, and both seropositive IgM and IgG accounted for the biggest proportion (medium, 42 days onset), then followed by the single seropositive IgG (medium, 50 days onset) in Ab profile. CONCLUSION: The performance of CLIA kits satisfied the diagnosis of SARS-CoV-2 infection. Both positive of IgG and IgM contributes to improve the specificity, and a positive one will enhance the sensitivity.

Analytical performance evaluation of five RT-PCR kits for severe acute respiratory syndrome coronavirus 2.

BACKGROUND: We aimed to evaluate the analytical performance of five commercial RT-PCR kits (Genekey, Daan, BioGerm, Liferiver, and Yaneng) commonly used in China, since such comparison data are lacking. METHODS: A total of 20 COVID-19 confirmed patients and 30 negative nasopharyngeal swab specimens were analyzed by five kits. The detection ability of five RT-PCR kits was evaluated with 5 concentration gradients diluted by a single positive sample. The limit of detection was evaluated by N gene fragment solid standard. Two positive clinical specimens were used to evaluate the repeatability and imprecision. Finally, we used six human coronaviruses plasmid and four respiratory pathogens plasmid to check for cross-reactivity. RESULTS: The positive detection rate was 100% for Genekey, Daan, and BioGerm,and 90% for Liferiver and Yaneng in 20 clinical SARS-CoV-2 infection. The coincidence rate of five kits in 10 negative samples was 100%. The detection rate of target genes for Daan, BioGerm, Liferiver, and Yaneng was 100% from Level 1 to Level 3. In Level 4, only Daan detection rate was 100%. In Level 5, five kits presented poor positive rate. The limit of detection declared by each manufacturer was verified. The repeatability for target genes was less than 5% and so did the total imprecision. There is no cross-reactivity of five kits with six human coronaviruses and four respiratory pathogens for ORF1ab and N gene. CONCLUSIONS: Five RT-PCR kits assessed in this study showed acceptable analytical performance characteristics and are useful tools for the routine diagnosis of SARS-CoV-2.

A CRISPR-Cas12a-based specific enhancer for more sensitive detection of SARS-CoV-2 infection.

BACKGROUND: Real-time reverse transcription-PCR (rRT-PCR) has been the most effective and widely implemented diagnostic technology since the beginning of the COVID-19 pandemic. However, fuzzy rRT-PCR readouts with high Ct values are frequently encountered, resulting in uncertainty in diagnosis. METHODS: A Specific Enhancer for PCR-amplified Nucleic Acid (SENA) was developed based on the Cas12a trans-cleavage activity, which is specifically triggered by the rRT-PCR amplicons of the SARS-CoV-2 Orf1ab (O) and N fragments. SENA was first characterized to determine its sensitivity and specificity, using a systematic titration experiment with pure SARS-CoV-2 RNA standards, and was then verified in several hospitals, employing a couple of commercial rRT-PCR kits and testing various clinical specimens under different scenarios. FINDINGS: The ratio (10 min/5 min) of fluorescence change (FC) with mixed SENA reaction (mix-FCratio) was defined for quantitative analysis of target O and N genes, and the Limit of Detection (LoD) of mix-FCratio with 95% confidence interval was 1.2≤1.6≤2.1. Totally, 295 clinical specimens were analyzed, among which 21 uncertain rRT-PCR cases as well as 4 false negative and 2 false positive samples were characterized by SENA and further verified by next-generation sequencing (NGS). The cut-off values for mix-FCratio were determined as 1.145 for positive and 1.068 for negative. INTERPRETATION: SENA increases both the sensitivity and the specificity of rRT-PCR, solving the uncertainty problem in COVID-19 diagnosis and thus providing a simple and low-cost companion diagnosis for combating the pandemic. FUNDING: Detailed funding information is available at the end of the manuscript.

RT-LAMP for rapid diagnosis of coronavirus SARS-CoV-2.

The pandemic coronavirus SARS-CoV-2 in the world has caused a large infected population suffering from COVID-19. To curb the spreading of the virus, WHO urgently demanded an extension of screening and testing; thus, a rapid and simple diagnostic method is needed. We applied a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) to achieve the detection of SARS-CoV-2 in 30 min. We designed four sets of LAMP primers (6 primers in each set), targeting the viral RNA of SARS-CoV-2 in the regions of orf1ab, S gene and N gene. A colorimetric change was used to report the results, which enables the outcome of viral RNA amplification to be read by the naked eye without the need of expensive or dedicated instrument. The sensitivity can be 80 copies of viral RNA per ml in a sample. We validated the RT-LAMP method in a hospital in China, employing 16 clinic samples with 8 positives and 8 negatives. The testing results are consistent with the conventional RT-qPCR. In addition, we also show that one-step process without RNA extraction is feasible to achieve RNA amplification directly from a sample. This rapid, simple and sensitive RT-LAMP method paves a way for a large screening at public domain and hospitals, particularly regional hospitals and medical centres in rural areas.