Carryover Contamination-Controlled Amplicon Sequencing Workflow for Accurate Qualitative and Quantitative Detection of Pathogens: a Case Study on SARS-CoV-2.

Carryover contamination during amplicon sequencing workflow (AMP-Seq) put the accuracy of the high-throughput detection for pathogens at risk. The purpose of this study is to develop a carryover contaminations-controlled AMP-Seq (ccAMP-Seq) workflow to enable accurate qualitative and quantitative detection for pathogens. By using the AMP-Seq workflow to detect SARS-CoV-2, Aerosols, reagents and pipettes were identified as potential sources of contaminations and ccAMP-Seq was then developed. ccAMP-Seq used filter tips and physically isolation of experimental steps to avoid cross contamination, synthetic DNA spike-ins to compete with contaminations and quantify SARS-CoV-2, dUTP/uracil DNA glycosylase system to digest the carryover contaminations, and a new data analysis procedure to remove the sequencing reads from contaminations. Compared to AMP-Seq, the contamination level of ccAMP-Seq was at least 22-folds lower and the detection limit was also about an order of magnitude lower-as low as one copy/reaction. By testing the dilution series of SARS-CoV-2 nucleic acid standard, ccAMP-Seq showed 100% sensitivity and specificity. The high sensitivity of ccAMP-Seq was further confirmed by the detection of SARS-CoV-2 from 62 clinical samples. The consistency between qPCR and ccAMP-Seq was 100% for all the 53 qPCR-positive clinical samples. Seven qPCR-negative clinical samples were found to be positive by ccAMP-Seq, which was confirmed by extra qPCR tests on subsequent samples from the same patients. This study presents a carryover contamination-controlled, accurate qualitative and quantitative amplicon sequencing workflow that addresses the critical problem of pathogen detection for infectious diseases. IMPORTANCE Accuracy, a key indicator of pathogen detection technology, is compromised by carryover contamination in the amplicon sequencing workflow. Taking the detection of SARS-CoV-2 as case, this study presents a new carryover contamination-controlled amplicon sequencing workflow. The new workflow significantly reduces the degree of contamination in the workflow, thereby significantly improving the accuracy and sensitivity of the SARS-CoV-2 detection and empowering the ability of quantitative detection. More importantly, the use of the new workflow is simple and economical. Therefore, the results of this study can be easily applied to other microorganism, which has great significance for improving the detection level of microorganism.

SARS-CoV-2 might transmit through the skin while the skin barrier function could be the mediator.

During the coronavirus disease 2019 (COVID-19) pandemic, it were reported that COVID-19 patients could have cutaneous symptoms, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was observed on the skin of COVID-19 patients, which indicated that the skin is one target of SARS-CoV-2. Meanwhile, reports about SARS-CoV-2 transmission through food cold-chain overpacks emerged. With the fact that SARS-CoV-2 could survive on the skin for more than 9 h, the skin could be implicated in SARS CoV-2 transmission. Angiotensin-converting enzyme 2 (ACE2), a critical membrane protein for SARS-CoV-2 that enters a host cell, was recognized to be associated with the risk of SARS-CoV-2 infection. Therefore, tissues that express ACE2 might have the potential to be infected by and transmit SARS-CoV-2. The skin is one such tissue that expresses ACE2. However, unlike the lung that expresses ACE2 on the upper-most epithelial layer, the skin is composed of different layers of cells that function as a barrier, and cells under the top epidermal layer express ACE2. Since the skin barrier is the first line of protection, the typical position of ACE2-expressing cells in the skin implies that the skin barrier function could be the mediator of SARS-CoV-2. In our study, we found that ACE2 could be expressed in the skin, and its expression level is increased in psoriasis, an inflammatory disease of the skin with barrier dysfunction. Additionally, by applying the SARS-CoV-2 pseudovirus on mouse models with or without deteriorated skin barrier, we found that the SARS-CoV-2 pseudovirus could infect the skin and lungs of mouse models, and when the skin barrier was impaired, more SARS-CoV-2-infected cells could be found. Thus, we hypothesized that a deteriorated condition of the skin barrier might increase the risk of SARS-CoV-2 infection through the skin.

Prevention and control strategies for the post-pandemic era: finding a balance between COVID-19 and reviving medical service.

INTRODUCTION: Public life in China is gradually returning to normal with strong measures in coronavirus 2019 (COVID-19) control. Because of the long-term effects of COVID-19, medical institutions had to make timely adjustments to control policies and priorities to balance between COVID-19 prevention and daily medical services. METHODOLOGY: The framework for infection prevention and control in the inpatient department was effectively organized at both hospital and department levels. A series of prevention and control strategies was implemented under this leadership: application of rigorous risk assessment and triage before admission through a query list; classifying patients into three risk levels and providing corresponding medical treatment and emergency handling; establishing new ward visiting criteria for visitors; designing procedures for PPE and stockpile management; executing specialized disinfection and medical waste policies. RESULTS: Till June 2020, the bed occupancy had recovered from 20.0% to 88.1%. In total, 13045 patients were received in our hospital, of which 54 and 127 patients were identified as high-risk and medium-risk, respectively, and 2 patients in the high-risk group were eventually laboratory-confirmed with COVID-19. No hospital-acquired infection of COVID-19 has been observed since the emergency appeared. CONCLUSIONS: The strategies ensured early detection and targeted prevention of COVID-19 following the COVID-19 pandemic, which improved the recovery of medical services after the pandemic.

Practice and exploration of infection prevention and control measures based on risk management of surgical patients during the epidemic of corona virus disease 2019 (COVID-19).

BACKGROUND: An outbreak of corona virus disease 2019 (COVID-19) in Wuhan, China has spread quickly across the world, the World Health Organization (WHO) has declared this a pandemic. COVID-19 can be transmitted from human to human and cause nosocomial infection that has brought great challenges to infection control in medical institutions. Due to the professional characteristics, the research hospital still received a large number of trauma emergency tasks during the outbreak. It is urgent to establish a graded prevention and control guidance of surgery. METHODS: Review the implementation of surgical grading control measures in this hospital during the epidemic of COVID-19. RESULTS: The surgical prevention measures based on patients with different risks included prescreening and preoperative risk assessment, preparation of operating room, medical staff protection and environmental disinfection measures, etc. From January 20 to March 5, 2020, a total of 4,720 operations had been performed in this hospital, of which 1,565 were emergency operations and 22 for medium-risk and high-risk patients who may have the 2019 severe acute respiratory syndrome coronavirus 2 infection. And there is no medical staff exposed during the implementation of protective measures. CONCLUSIONS: Through the risk assessment of surgical patients and adopting surgical grading control measures, the risk of severe acute respiratory syndrome coronavirus 2 spread during the surgical process can be reduced greatly.