UNASSIGNED: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a life-threatening disease with largely unknown intraocular pathogenesis. Herein, we determined the presence of SARS-CoV-2-specific ribonucleic acid (RNA) and virus-associated antibodies in the vitreous humor of people who have recently recovered from SARS-CoV-2 infection.
UNASSIGNED: This cross-sectional study included 33 patients (33 eyes) who have recently recovered from SARS-CoV-2 infection. Vitreous humor and blood serum samples were tested for the SARS-CoV-2 RNA and virus-associated antibodies.
UNASSIGNED: Among 33 participants, blood serum and vitreous humor were all tested negative for SARS-CoV-2 RNA. SARS-CoV-2-specific IgM was detected in 87.88 % (29/33) patients in blood serum and 6.10 % (2/33) in vitreous humor; SARS-CoV-2-specific IgG was detected in 96.97 % (32/33) patient in blood serum and 81.82 % (27/33) in vitreous humor. Statistical significance was found for IgM expression between blood serum and vitreous humor (P < 0.01), while IgG was not (P = 0.11). The days after recovery were statistically longer both in IgM-positive blood serum samples group and IgG-positive vitreous humor samples group compared with negative samples of each group (P < 0.01). Additionally, no statistical difference could be detected in antibody expression in vitreous humor between different groups divided on the condition of the risk of blood-retina-barrier (BRB) failure (P = 0.49 for IgM; P = 0.37 for IgG).
UNASSIGNED: After recovering from COVID-19, no SARS-CoV-2 RNA was detected in vitreous humor, but anti-CoV-2 IgM was detected in 6.1 % and IgG in approximately 80 % of vitreous humor samples of participants. We also found that the positivety rate of SARS-CoV-2-specific antibodies in the blood serum and vitreous humor were both correlated with the days after recovery since the infection.

The occurrence of the COVID-19 pandemic caused by the SARS-CoV-2 virus since the end of 2019 has significantly affected the entire world. Now SARS-CoV-2 diagnostic tests are not only required for screening of suspected infected people for their medical treatment, but have also become a routine diagnosis for all people at a place where new cases have emerged in order to control spread of the disease from that region. For these reasons, sensitive methods for detection of SARS-CoV-2 are highly needed in order to avoid undetected infections. In addition, sample pooling that uses pooled specimens has been routinely employed as a time- and cost-effective strategy for community monitoring of SARS-CoV-2. In this regard, the content of each viral RNA sample of an individual will be further diluted in detection; therefore, higher detection sensitivity would be rather preferred. Among nucleic acid-based detection methods, isothermal nucleic acid amplifications are considered quite promising because they typically take less time to complete the test (even less than 20 min) without the need of thermal cycles. Hence, it does not necessitate the use of highly costly real-time PCR machines. According to recently published isothermal nucleic acid amplification methods, the reverse transcription recombinase polymerase amplification (RT-RPA) approach shows outstanding sensitivity with up to single-copy sensitivity in a test reaction. This chapter will mainly focus on how to employ RT-RPA technology to sensitively detect SARS-CoV-2 RNA. Besides, recently published RT-RPA based detection methods will be summarized and compared regarding their detection parameters and the primers and probes being used. In addition, we will also highlight the key considerations on how to design an ultrasensitive RT-RPA assay and the precautions needed to conduct the assay. Moreover, based on our recent report, we will also detail the methods we developed to detect SARS-CoV-2 RNA using modified RT-RPA, or RT-ERA, with single-copy sensitivity and the possible extensions beyond this method.

Nucleic acid detection is conducive to preventing the spread of COVID-19 pandemic. In this work, we successfully designed a soft interface confined DNA walker by anchoring hairpin reporter probes on cell membranes for the detection of SARS-CoV-2 variants. In the presence of target RNA, the cyclic self-assembly reaction occurred between hairpin probes H1 and H2, and the continuous walking of target RNA on cell membranes led to the gradual amplification of fluorescence signal. The enrichment of H1 on membranes and the unique fluidity of membranes promoted the collision efficiency between DNA strands in the reaction process, endowing this method with high sensitivity. In addition, the double-blind test of synthetic RNA in 5% normal human serum demonstrated the good stability and anti-interference in complex environment of this method, which exhibited great potential in clinical diagnostics.

Nucleic acids in biofluids are emerging biomarkers for the molecular diagnostics of diseases, but their clinical use has been hindered by the lack of sensitive detection assays. Herein, we report the development of a sensitive nucleic acid detection assay named SPOT (sensitive loop-initiated DNAzyme biosensor for nucleic acid detection) by rationally designing a catalytic DNAzyme of endonuclease capability into a unified one-stranded allosteric biosensor. SPOT is activated once a nucleic acid target of a specific sequence binds to its allosteric module to enable continuous cleavage of molecular reporters. SPOT provides a highly robust platform for sensitive, convenient and cost-effective detection of low-abundance nucleic acids. For clinical validation, we demonstrated that SPOT could detect serum miRNAs for the diagnostics of breast cancer, gastric cancer and prostate cancer. Furthermore, SPOT exhibits potent detection performance over SARS-CoV-2 RNA from clinical swabs with high sensitivity and specificity. Finally, SPOT is compatible with point-of-care testing modalities such as lateral flow assays. Hence, we envision that SPOT may serve as a robust assay for the sensitive detection of a variety of nucleic acid targets enabling molecular diagnostics in clinics.

Improving the detection capability and efficiency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA specimens is very important for the prevention and control of the outbreak of Coronavirus disease 2019 (COVID-19). In this study, we evaluated the detection capability and efficiency of two outbreaks of COVID-19 before and after the process re-engineering in April and July 2022.
This retrospective cross-sectional study involved 359,845 SARS-CoV-2 RNA specimens 2 weeks before and 2 weeks after the two outbreaks of COVID-19 in April and July. The number, transportation time and detection time of specimens, and the number of reports of more than 24 h were analyzed by SPSS software.
While 16.84% of people chose nasopharyngeal swabs (NPS) specimens, 83.16% chose oropharyngeal swabs (OPS) specimens to detect SARS-CoV-2 RNA. There were significant upward trends in the percentage of 10 sample pooling (P-10) from April before process re-engineering to July after process re-engineering (p < 0.001). Compared with April, the number of specimens in July increased significantly not only 2 weeks before but also 2 weeks after the outbreak of COVID-19, with an increase of 35.46 and 93.94%, respectively. After the process re-engineering, the number of reports more than 24 h in the 2 weeks before and after the outbreak of COVID-19 in July was significantly lower than that in April before process re-engineering (0% vs. 0.06% and 0 vs. 0.89%, both p < 0.001).
The present study shows that strengthening the cooperation of multi-departments in process re-engineering, especially using the P-10 strategy and whole process informatization can improve the detection capability and efficiency of SARS-CoV-2 RNA specimens.

Studies on nonhuman primates, wild-type and transgenic mice have shown the presence of SARS-CoV-2 RNA components in the brains. Despite the Blood-Brain Barrier (BBB) provides protection there are less evidences on how the SARS-CoV-2 crosses the BBB. Given that there is an increase of Omicron reinfection rates, transmissibility rate and involvement to cause neurological dysfunctions, we hypothesized to investigate how the Omicron variant (B.1.1.529) binds structurally to key BBB-maintaining proteins and thus can possibly challenge the integrity and transportation to the brain. By using molecular dynamics simulation approaches we examined the interaction of Omicron variant (B.1.1.529) with different structural and transporter proteins located at the BBB. Our results show that in Zona Ocludin 1-RBD complex, we observe a distinct pattern. Omicron demonstrates a docking score of -88.9 ± 6.8 kcal/mol and six interactions, while the wild type (WT) presents a higher score of -94.0 ± 2.3 kcal/mol, forming eight interactions. Comparing affinities, the WT-RBD displays a stronger preference for Claudin-5, boasting a docking score of -110.2 ± 3.0 and nine interactions, versus Omicron-RBD\'s slightly reduced engagement, with a docking score of -105.6 ± 0.2 and seven interactions. Interestingly, the Omicron variant exhibits heightened stability in interactions with Glucose Transporter and ABC transporters, registering docking scores of -110.6 ± 1.9 and -112.0 ± 3.6 kcal/mol, respectively. This surpasses the WT\'s respective scores of -95.2 ± 2.2 and -104.0 ± 6.2 kcal/mol, reflecting a unique interaction profile. Rigorous molecular dynamics simulations validate our findings. Our study emphasizes the Omicron variant\'s increased affinity towards transporter proteins, illuminating potential implications for BBB integrity and brain transportation. While these insights offer a valuable framework, comprehensive experimental validation is indispensable for a comprehensive understanding.

Inadequate sampling approaches to wastewater analyses can introduce biases, leading to inaccurate results such as false negatives and significant over- or underestimation of average daily viral concentrations, due to the sporadic nature of viral input. To address this challenge, we conducted a field trial within the University of Tennessee residence halls, employing different composite sampling modes that encompassed different time intervals (1 h, 2 h, 4 h, 6 h, and 24 h) across various time windows (morning, afternoon, evening, and late-night). Our primary objective was to identify the optimal approach for generating representative composite samples of SARS-CoV-2 from raw wastewater. Utilizing reverse transcription-quantitative polymerase chain reaction, we quantified the levels of SARS-CoV-2 RNA and pepper mild mottle virus (PMMoV) RNA in raw sewage. Our findings consistently demonstrated that PMMoV RNA, an indicator virus of human fecal contamination in water environment, exhibited higher abundance and lower variability compared to pathogenic SARS-CoV-2 RNA. Significantly, both SARS-CoV-2 and PMMoV RNA exhibited greater variability in 1 h individual composite samples throughout the entire sampling period, contrasting with the stability observed in other time-based composite samples. Through a comprehensive analysis of various composite sampling modes using the Quade Nonparametric ANCOVA test with date, PMMoV concentration and site as covariates, we concluded that employing a composite sampler during a focused 6 h morning window for pathogenic SARS-CoV-2 RNA is a pragmatic and cost-effective strategy for achieving representative composite samples within a single day in wastewater-based epidemiology applications. This method has the potential to significantly enhance the accuracy and reliability of data collected at the community level, thereby contributing to more informed public health decision-making during a pandemic.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may transmit through airborne route particularly when the aerosol particles remain in enclosed spaces with inadequate ventilation. There has been no standard recommended method of determining the virus in air due to limitations in pre-analytical and technical aspects. Furthermore, the presence of low virus loads in air samples could result in false negatives. Our study aims to explore the feasibility of detecting SARS-CoV-2 ribonucleic acid (RNA) in air samples using droplet digital polymerase chain reaction (ddPCR). Active and passive air sampling was conducted between December 2021 and February 2022 with the presence of COVID-19 confirmed cases in two hospitals and a quarantine center in Klang Valley, Malaysia. SARS-CoV-2 RNA in air was detected and quantified using ddPCR and real-time reverse transcriptase-polymerase chain reaction (RT-PCR). The comparability of two different digital PCR platforms (QX200 and QIAcuity) to RT-PCR were also investigated. Additionally negative staining transmission electron microscopy was performed to visualize virus ultrastructure. Detection rates of SARS-CoV-2 in air samples using ddPCR were higher compared to RT-PCR, which were 15.2% (22/145) and 3.4% (5/145), respectively. The sensitivity and specificity of ddPCR was 100 and 87%, respectively. After excluding 17 negative samples (50%) by both QX200 and QIAcuity, 15% samples (5/34) were found to be positive both ddPCR and dPCR. There were 23.5% (8/34) samples that were detected positive by ddPCR but negative by dPCR. In contrast, there were 11.7% (4/34) samples that were detected positive by dPCR but negative by ddPCR. The SARS-CoV-2 detection method by ddPCR is precise and has a high sensitivity for viral RNA detection. It could provide advances in determining low viral titter in air samples to reduce false negative reports, which could complement detection by RT-PCR.

UNASSIGNED: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been shown to be detectable in blood from infected individuals. Though RNAemia frequencies are typically low, the presence of potentially infectious virus potentially poses a transmission risk during blood transfusion.
UNASSIGNED: Archived plasma samples were collected from blood donors who later reported possible SARS-CoV-2 infection with the wild-type strain, Delta variant, or Omicron variant. This was based on either symptom onset or a positive test within 2 weeks from their donation. Donations were tested for SARS-CoV-2 RNA, and information on symptoms and testing results were gathered during postdonation interview.
UNASSIGNED: Of 518 archived plasma samples tested, 19 (3.7%) were found to have detectable levels of SARS-CoV-2 RNA. SARS-CoV-2 RNA was detected in donors who donated during the Delta (10/141 [7.1%]) and Omicron (9/162 [5.6%]) waves. SARS-CoV-2 RNA was not detected in donors who donated during the wild-type wave (0/215). Seventeen of 19 RNAemic donors reported symptom onset or a positive test within 2 days of donating. SARS-CoV-2 RNA was detected in asymptomatic or presymptomatic blood donors.
UNASSIGNED: Despite RNAemia being correlated with SARS-CoV-2 disease severity, RNAemia was detected in asymptomatic or presymptomatic blood donors.