Towards application of CRISPR-Cas12a in the design of modern viral DNA detection tools (Review).

Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.

iciHHV-6 in a Patient With Multisystem Inflammatory Syndrome in Children.

Multisystem inflammatory syndrome in children (MIS-C) is a serious, sometimes life-threatening late complication of coronavirus disease 2019 (COVID-19) with multiorgan involvement and evidence of immune activation. The pathogenesis of MIS-C is not known, nor is the pathogenesis of the severe organ damage that is the hallmark of MIS-C. Human herpesvirus 6 (HHV-6), the virus responsible for roseola, is a ubiquitous herpesvirus that causes close to universal infection by the age of 3 years. HHV-6 remains latent for life and can be activated during inflammatory states, by other viruses, and by host cell apoptosis. HHV-6 has been associated with end-organ diseases, including hepatitis, carditis, and encephalitis. In addition, ∼1% of people have inherited chromosomally integrated human herpesvirus 6 (iciHHV-6), which is HHV-6 that has been integrated into chromosomal telomeric regions and is transmitted through the germ line. iciHHV-6 can be reactivated and has been associated with altered immune responses. We report here a case of MIS-C in which an initial high HHV-6 DNA polymerase chain reaction viral load assay prompted testing for iciHHV-6, which yielded a positive result. Additional research may be warranted to determine if iciHHV-6 is commonly observed in patients with MIS-C and, if so, whether it may play a part in MIS-C pathogenesis.

Impact of Collection Volume and DNA Extraction Method on the Detection of Biomarkers and HPV DNA in First-Void Urine.

The potential of first-void (FV) urine as a non-invasive liquid biopsy for detection of human papillomavirus (HPV) DNA and other biomarkers has been increasingly recognized over the past decade. In this study, we investigated whether the volume of this initial urine stream has an impact on the analytical performance of biomarkers. In parallel, we evaluated different DNA extraction protocols and introduced an internal control in the urine preservative. Twenty-five women, diagnosed with high-risk HPV, provided three home-collected FV urine samples using three FV urine collection devices (Colli-Pee) with collector tubes that differ in volume (4, 10, 20 mL). Each collector tube was prefilled with Urine Conservation Medium spiked with phocine herpesvirus 1 (PhHV-1) DNA as internal control. Five different DNA extraction protocols were compared, followed by PCR for GAPDH and PhHV-1 (qPCR), HPV DNA, and HBB (HPV-Risk Assay), and ACTB (methylation-specific qPCR). Results showed limited effects of collection volume on human and HPV DNA endpoints. In contrast, significant variations in yield for human endpoints were observed for different DNA extraction methods (p < 0.05). Additionally, the potential of PhHV-1 as internal control to monitor FV urine collection, storage, and processing was demonstrated.

A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities.

One goal of microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods the authors previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, the authors compared the relative performance of two total nucleic acid extraction protocols with the authors' previously benchmarked protocol. The authors included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here the authors present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection and well-to-well contamination between these protocols.

A knowledge gap unmasked: viral transmission in surgical smoke: a systematic review.

BACKGROUND: Concerns regarding the aerosolized transmission of SARS-CoV-2 via SS have caused significant apprehension among surgeons related to the use of minimally invasive surgery (MIS) during the COVID19 pandemic. While a limited number of studies have previously demonstrated the presence of viral material in SS, no comprehensive systematic review exists on the subject of viral transmission in SS. Methods A systematic review of the literature was conducted as per PRISMA guidelines. MEDLINE, EMBASE, and CENTRAL databases were searched for publications reporting the primary outcome of the presence of viral particles in SS and secondary outcomes of indices suggesting transmission of viable virus particles in SS producing clinically important infection. All human, animal, and in vitro studies which used accepted analytic techniques for viral detection were included. A meta-analysis was not complete due to methodologic heterogeneity and inconsistent reporting of outcomes of interest. RESULTS: 23 publications addressed the presence of viral components in SS, and 19 (83%) found the presence of viral particles in SS. 21 publications additionally studied the ability of SS to induce clinically relevant infection in host cells, with 9 (43%) demonstrating potential for viral transmission. CONCLUSION: Evidence exists for viral transmission via SS. However, HPV remains the only virus with documented transmission to humans via SS. While meaningful translation into practical guidelines during the COVID pandemic remains challenging, no evidence exists to suggest increased risk in MIS.

Determining a sampling regime for PCR detection of respiratory tract viral infection at coronial post-mortem examinations.

Death due to respiratory infection is commonly encountered at autopsy. With only one opportunity to obtain samples for identification of a causative agent, it is important to ensure that sampling regimes are optimized to provide the greatest detection, without the expense and redundancy that can arise from over-sampling. This study was performed retrospectively using data from Coronial autopsies over the period 2012-2019 from which swabs from the nasopharyngeal region, trachea and lung parenchyma, in addition to samples of lung tissue, had been submitted for multiplex PCR detection of respiratory pathogens. From 97 cases with all four samples, there were 24 with at least one positive result for viral infection. Some cases had multiple positive results and a total of 27 respiratory tract viruses were identified, of which rhinovirus, influenza A virus and respiratory syncytial virus were the most common. Seventeen of the 27 viral infections (63%) were identified in all four samples. However, in nearly all cases (96%) the nasopharyngeal swab detected the infective agent when the multiplex PCR panel had detected infection in any of the four sample types. A nasopharyngeal swab is considered to be an optimal sample for detection of respiratory tract viral infection. As the samples analyzed were acquired before the appearance of the COVID-19 virus, the applicability of this finding for COVID-19 screening is not established.

Nanopore Targeted Sequencing for the Accurate and Comprehensive Detection of SARS-CoV-2 and Other Respiratory Viruses.

The ongoing global novel coronavirus pneumonia COVID-19 outbreak has engendered numerous cases of infection and death. COVID-19 diagnosis relies upon nucleic acid detection; however, currently recommended methods exhibit high false-negative rates and are unable to identify other respiratory virus infections, thereby resulting in patient misdiagnosis and impeding epidemic containment. Combining the advantages of targeted amplification and long-read, real-time nanopore sequencing, herein, nanopore targeted sequencing (NTS) is developed to detect SARS-CoV-2 and other respiratory viruses simultaneously within 6-10 h, with a limit of detection of ten standard plasmid copies per reaction. Compared with its specificity for five common respiratory viruses, the specificity of NTS for SARS-CoV-2 reaches 100%. Parallel testing with approved real-time reverse transcription-polymerase chain reaction kits for SARS-CoV-2 and NTS using 61 nucleic acid samples from suspected COVID-19 cases show that NTS identifies more infected patients (22/61) as positive, while also effectively monitoring for mutated nucleic acid sequences, categorizing types of SARS-CoV-2, and detecting other respiratory viruses in the test sample. NTS is thus suitable for COVID-19 diagnosis; moreover, this platform can be further extended for diagnosing other viruses and pathogens.

Isolation, Sequence, Infectivity, and Replication Kinetics of Severe Acute Respiratory Syndrome Coronavirus 2.

Since its emergence in Wuhan, China, in December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected ≈6 million persons worldwide. As SARS-CoV-2 spreads across the planet, we explored the range of human cells that can be infected by this virus. We isolated SARS-CoV-2 from 2 infected patients in Toronto, Canada; determined the genomic sequences; and identified single-nucleotide changes in representative populations of our virus stocks. We also tested a wide range of human immune cells for productive infection with SARS-CoV-2. We confirm that human primary peripheral blood mononuclear cells are not permissive for SARS-CoV-2. As SARS-CoV-2 continues to spread globally, it is essential to monitor single-nucleotide polymorphisms in the virus and to continue to isolate circulating viruses to determine viral genotype and phenotype by using in vitro and in vivo infection models.

COVID-19 not detected in peritoneal fluid: a case of laparoscopic appendicectomy for acute appendicitis in a COVID-19-infected patient.

PURPOSE: COVID-19 greatly affected millions and affected the way we practice with heightened posture in the way we treat surgical patients. Surgical consensus guidelines are recommending caution in the use of laparoscopy for the theoretical possibility of viral transmission from aerosolization of tissue and peritoneal fluid during surgery. However, there has yet to be proof of COVID-19 being present in peritoneal fluid, justifying the consensus statements. We aim to assess the presence of COVID-19 in peritoneal fluid. METHODS: We performed a laparoscopic appendicectomy for a COVID-19-infected patient with acute appendicitis. Peritoneal fluid and peritoneal washings were collected and sent for COVID-19 PCR. RESULTS: The peritoneal fluid sample collected on entry and at the end of the operation was negative for COVID-19 on PCR. The patient had an uneventful recovery from surgery. CONCLUSIONS: This case revealed that COVID-19 was not detected in peritoneal fluid and peritoneal washings in a patient infected with COVID-19. This study provides novel preliminary data in the investigation of COVID-19 transmission from laparoscopy-related aerosolization.

Consistency analysis of COVID-19 nucleic acid tests and the changes of lung CT.

BACKGROUND: COVID-19, the latest outbreak of infectious disease, has caused huge medical challenges to China and the entire globe. No unified diagnostic standard has been formulated. The initial diagnosis remains based on the positive of nucleic acid tests. However, early nucleic acid tests were identified to be negative in some patients, whereas the patients exhibited characteristic CT changes of lung, and positive test results appeared after repeated nucleic acid tests, having caused the failure to diagnose these patients early. The study aimed to delve into the relationships between initial nucleic acid testing and early lung CT changes in patients with COVID-19. METHOD: In accordance with the latest COVID-19 diagnostic criteria, 69 patients diagnosed with COVID-19 treated in the infected V ward of Xiaogan Central Hospital from 2020/1/25 to 2020/2/6 were retrospectively analyzed. The consistency between the first COVID-19 nucleic acid test positive and lung CT changes was studied. In addition, the sensitivity and specificity of CT and initial nucleic acid were studied. RESULT: The Kappa coefficient of initial nucleic acid positive changes and lung CT changes was -1.52. With a positive nucleic acid test as the gold standard, the sensitivity of lung CT was 12.00 %, 95 % CI: 4.6-24.3; with the changes of CT as the gold standard, the sensitivity of nucleic acid positive was 30.16 %, 95 % CI: 19.2-43.0. CONCLUSION: The consistency between the initial positive nucleic acid test and the CT changes in the lungs is poor; low sensitivity was achieved for initial nucleic acid detection and CT changes.