Validation of Rapid and Economic Colorimetric Nanoparticle Assay for SARS-CoV-2 RNA Detection in Saliva and Nasopharyngeal Swabs.

Even with the widespread uptake of vaccines, the SARS-CoV-2-induced COVID-19 pandemic continues to overwhelm many healthcare systems worldwide. Consequently, massive scale molecular diagnostic testing remains a key strategy to control the ongoing pandemic, and the need for instrument-free, economic and easy-to-use molecular diagnostic alternatives to PCR remains a goal of many healthcare providers, including WHO. We developed a test (Repvit) based on gold nanoparticles that can detect SARS-CoV-2 RNA directly from nasopharyngeal swab or saliva samples with a limit of detection (LOD) of 2.1 × 105 copies mL-1 by the naked eye (or 8 × 104 copies mL-1 by spectrophotometer) in less than 20 min, without the need for any instrumentation, and with a manufacturing price of <$1. We tested this technology on 1143 clinical samples from RNA extracted from nasopharyngeal swabs (n = 188), directly from saliva samples (n = 635; assayed by spectrophotometer) and nasopharyngeal swabs (n = 320) from multiple centers and obtained sensitivity values of 92.86%, 93.75% and 94.57% and specificities of 93.22%, 97.96% and 94.76%, respectively. To our knowledge, this is the first description of a colloidal nanoparticle assay that allows for rapid nucleic acid detection at clinically relevant sensitivity without the need for external instrumentation that could be used in resource-limited settings or for self-testing.

Monoclonal antibodies constructed from COVID-19 convalescent memory B cells exhibit potent binding activity to MERS-CoV spike S2 subunit and other human coronaviruses.

Introduction: The Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are two highly contagious coronaviruses causing MERS and COVID-19, respectively, without an effective antiviral drug and a long-lasting vaccine. Approaches for diagnosis, therapeutics, prevention, etc., particularly for SARS-CoV-2 that is continually spreading and evolving, are urgently needed. Our previous study discovered that >60% of sera from convalescent COVID-19 individuals, but <8% from general population, showed binding activity against the MERS-CoV spike protein, indicating that SARS-CoV-2 infection boosted antibodies cross-reactive with MERS-CoV. Methods: To generate antibodies specific to both SARS-CoV-2 and MERS-CoV, here we screened 60 COVID-19 convalescent sera against MERS-CoV spike extracellular domain and S1 and S2 subunits. We constructed and characterized monoclonal antibodies (mAbs) from COVID-19 convalescent memory B cells and examined their binding and neutralizing activities against human coronaviruses. Results and Discussion: Of 60 convalescent serum samples, 34 showed binding activity against MERS-CoV S2, with endpoint titers positively correlated with the titers to SARS-CoV-2 S2. By sorting single memory B cells from COVID-19 convalescents, we constructed 38 mAbs and found that 11 mAbs showed binding activity with MERS-CoV S2, of which 9 mAbs showed potent cross-reactivity with all or a proportion of spike proteins of alphacoronaviruses (229E and NL63) and betacoronaviruses (SARS-CoV-1, SARS-CoV-2, OC43, and HKU1). Moreover, 5 mAbs also showed weak neutralization efficiency against MERS-CoV spike pseudovirus. Epitope analysis revealed that 3 and 8 mAbs bound to linear and conformational epitopes in MERS-CoV S2, respectively. In summary, we have constructed a panel of antibodies with broad-spectrum reactivity against all seven human coronaviruses, thus facilitating the development of diagnosis methods and vaccine design for multiple coronaviruses.

Opportunities of Digital Infrastructures for Disease Management-Exemplified on COVID-19-Related Change in Diagnosis Counts for Diabetes-Related Eye Diseases.

Background: Retrospective research on real-world data provides the ability to gain evidence on specific topics especially when running across different sites in research networks. Those research networks have become increasingly relevant in recent years; not least due to the special situation caused by the COVID-19 pandemic. An important requirement for those networks is the data harmonization by ensuring the semantic interoperability. Aims: In this paper we demonstrate (1) how to facilitate digital infrastructures to run a retrospective study in a research network spread across university and non-university hospital sites; and (2) to answer a medical question on COVID-19 related change in diagnostic counts for diabetes-related eye diseases. Materials and methods: The study is retrospective and non-interventional and runs on medical case data documented in routine care at the participating sites. The technical infrastructure consists of the OMOP CDM and other OHDSI tools that is provided in a transferable format. An ETL process to transfer and harmonize the data to the OMOP CDM has been utilized. Cohort definitions for each year in observation have been created centrally and applied locally against medical case data of all participating sites and analyzed with descriptive statistics. Results: The analyses showed an expectable drop of the total number of diagnoses and the diagnoses for diabetes in general; whereas the number of diagnoses for diabetes-related eye diseases surprisingly decreased stronger compared to non-eye diseases. Differences in relative changes of diagnoses counts between sites show an urgent need to process multi-centric studies rather than single-site studies to reduce bias in the data. Conclusions: This study has demonstrated the ability to utilize an existing portable and standardized infrastructure and ETL process from a university hospital setting and transfer it to non-university sites. From a medical perspective further activity is needed to evaluate data quality of the utilized real-world data documented in routine care and to investigate its eligibility of this data for research.

CRISPR/Cas12a-based technology: A powerful tool for biosensing in food safety.

BACKGROUND: In the context of the current pandemic caused by the novel coronavirus, molecular detection is not limited to the clinical laboratory, but also faces the challenge of the complex and variable real-time detection fields. A series of novel coronavirus events were detected in the process of food cold chain packaging and transportation, making the application of molecular diagnosis in food processing, packaging, transportation, and other links urgent. There is an urgent need for a rapid detection technology that can adapt to the diversity and complexity of food safety. SCOPE AND APPROACH: This review introduces a new molecular diagnostic technology-biosensor analysis technology based on CRISPR-Cas12a. Systematic clarification of its development process and detection principles. It summarizes and systematically organizes its applications in viruses, food-borne pathogenic bacteria, small molecule detection, etc. In the past four years, which provides a brand-new and comprehensive solution for food detection. Finally, this article puts forward the challenges and the prospects for food safety. KEY FINDINGS AND CONCLUSIONS: The novel coronavirus hazards infiltrated every step of the food industry, from processing to packaging to transportation. The biosensor analytical technology based on CRISPR-Cas12a has great potential in the qualitative and quantitative analysis of infectious pathogens. CRISPR-Cas12a can effectively identify the presence of the specific nucleic acid targets and the small changes in sequences, which is particularly important for nucleic acid identification and pathogen detection. In addition, the CRISPR-Cas12a method can be adjusted and reconfigured within days to detect other viruses, providing equipment for nucleic acid diagnostics in the field of food safety. The future work will focus on the development of portable microfluidic devices for multiple detection. Shao et al. employed physical separation methods to separate Cas proteins in different microfluidic channels to achieve multiple detection, and each channel simultaneously detected different targets by adding crRNA with different spacer sequences. Although CRISPR-Cas12a technology has outstanding advantages in detection, there are several technical barriers in the transformation from emerging technologies to practical applications. The newly developed CRISPR-Cas12a-based applications and methods promote the development of numerous diagnostic and detection solutions, and have great potential in medical diagnosis, environmental monitoring, and especially food detection.

Stressful events induce long-term gut microbiota dysbiosis and associated post-traumatic stress symptoms in healthcare workers fighting against COVID-19.

OBJECTIVE: The microbiota-gut-brain axis is a key pathway perturbed by prolonged stressors to produce brain and behavioral disorders. Frontline healthcare workers (FHWs) fighting against COVID-19 typically experience stressful event sequences and manifest some mental symptoms; however, the role of gut microbiota in such stress-induced mental problems remains unclear. We investigated the association between the psychological stress of FHW and gut microbiota. METHODS: We used full-length 16S rRNA gene sequencing to characterize the longitudinal changes in gut microbiota and investigated the impact of microbial changes on FHWs' mental status. RESULTS: Stressful events induced significant depression, anxiety, and stress in FHWs and disrupted the gut microbiome; gut dysbiosis persisted for at least half a year. Different microbes followed discrete trajectories during the half-year of follow-up. Microbes associated with mental health were mainly Faecalibacterium spp. and [Eubacterium] eligens group spp. with anti-inflammatory effects. Of note, the prediction model indicated that low abundance of [Eubacterium] hallii group uncultured bacterium and high abundance of Bacteroides eggerthii at Day 0 (immediately after the two-month frontline work) were significant determinants of the reappearance of post-traumatic stress symptoms in FHWs. LIMITATIONS: The lack of metabolomic evidence and animal experiments result in the unclear mechanism of gut dysbiosis-related stress symptoms. CONCLUSION: The stressful event sequences of fighting against COVID-19 induce characteristic longitudinal changes in gut microbiota, which underlies dynamic mental state changes.

Comprehensive evaluation of ACE2 expression in female ovary by single-cell RNA-seq analysis (preprint)

Pneumonia induced by severe acute respiratory coronavirus 2 (SARS-CoV-2) via ACE2 receptor may affect many organ systems like lung, heart and kidney. An autopsy report revealed positive SARS-Cov-2 detection results in ovary, however, the developmental-stage-specific and cell-type-specific risk in fetal primordial germ cells (PGCs) and adult women ovary remained unclear. In this study, we used single-cell RNA-sequencing (scRNA-seq) datasets spanning several developmental stages of ovary including PGCs and cumulus-oocyte complex (COC) to investigate the potential risk of SARS-CoV-2 infection. We found that PGCs and COC exhibited high ACE2 expression. More importantly, the ratio of ACE2-positive cells was sharply up-regulated in primary stage and ACE2 was expressed in all oocytes and cumulus cells in preovulatory stage, suggesting the possible risk of SARS-CoV-2 infection in follicular development. CatB/L, not TMPRSS2, was identified to prime for SARS-CoV-2 entry in follicle. Our findings provided insights into the potential risk of SARS-CoV-2 infection during folliculogenesis in adulthood and the possible risk in fetal PGCs.

Benign Clinical and Epidemiological Outcomes-Associated Factors of COVID-19 from a Solved Epidemic with a Low Case Fatality Rate (preprint)

Background: Since December 2019, COVID-19 has been confirmed in more than18.8 million patients and leads to 0.70 million deaths worldwide. The mortality and disease severity predictors of COVID-19 have been investigated in many studies. However, they are based on early or partial datasets from high epidemic areas. Here, we retrospect benign clinical and epidemiological outcomes-associated factors from a solved epidemic in a low epidemic area. Methods: All 98 laboratory-confirmed COVID-19 patients in a local epidemic (Zhuhai, China) from January 17, 2020 to March 10, 2020 were enrolled. Data were updated until all patients having final outcomes. Results: Patients were all hospitalized. The case fatality rate was 1.0%. There were no local secondary infection cases. The median age was 46.3 years. Underlying diseases were found in 33.7% patients. The severe/critical rate was 19.4%. The mean period from disease onset to admission was 4.4 days. Compared with serious/critical cases, mild/common cases on admission were much younger, lacks of comorbidities and normal in functions of vital organs and indicators of secondary bacterial infections. The lymphocyte counts in serious/critical cases began to be significantly lower 3 days before their identification dates. The absence of lymphopenia before the eighth day from disease onset can exclude the possibility of 78.5% to be serious/critical ill. Most patients (88.8%) received antiviral treatments. Early antiviral treatment significantly shortened the viral RNA-negative conversion time. The delayed antiviral treatment was associated with critical patients.Conclusions: Younger age, lack of aging-related diseases and early hospitalization of all patients to conduct antiviral treatment and prevention of secondary epidemic were the important benign clinical and epidemiological outcomes-associated factors of COVID-19. In combating COVID-19, the active intervention strategies are crucial in low epidemic areas and the continuous monitoring of lymphocytes may be useful to sort patients reasonably in high epidemic areas.

Investigation and Analysis of 108 Cases of Home Isolated Patients With Mild COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) began to spread across Wuhan, China, by the end of 2019, and patients were unable to be hospitalized because medical resources were limited. METHODS: A questionnaire survey was conducted among 108 participants with mild COVID-19 who have isolated at home under the guidance of doctors. The results of the questionnaire and outpatient data were integrated to evaluate participants' compliance with various epidemic prevention measures. RESULTS: During isolation, most participants were able to follow epidemic prevention measures under the guidance of doctors. After 14 d from the start of isolation, 45.37% of the participants recovered. Approximately half of the participants were relieved of symptoms, and most of them were transferred to mobile cabin hospitals to continue isolation. Three participants with worsening symptoms were transferred to the designated hospitals. There were no deaths of the participants, but there were 7 family members that were infected. CONCLUSIONS: During a period of home isolation under the guidance of a doctor, individuals can comply with epidemic prevention measures and symptoms can be improved. Scientific home isolation may be an effective way to relieve the strain of medical and social resources during the epidemic of COVID-19.