Comparison of CRISPR/Cas and Argonaute for nucleic acid tests.

Guided, programmable, and target-activated nucleases, exemplified by Cas in the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system and Argonaute (Ago), are emerging as a new generation of nucleic acid tests (NATs). A specific approach for comparison of these two nucleases side by side in terms of similarities, differences, and complementarities is instrumental for the sensible design of novel NATs.

RETROSPECTIVE ANALYSIS OF THE COVID-19 PANDEMIC IN AN URBAN SAFETY-NET HOSPITAL IN ATLANTA, GA

SESSION TITLE: COVID-19 Infections: Issues During and After Hospitalization SESSION TYPE: Original Investigations PRESENTED ON: 10/17/2022 01:30 pm - 02:30 pm PURPOSE: We present a retrospective study at one of the largest public, safety-net hospitals in the United States to highlight the importance of codifying the impact of COVID-19 disparities in marginalized populations. We used the following metrics to draw conclusions: patient demographics, vaccination status, comorbid conditions, length of stay (LOS), readmission rates, and clinical outcome. METHODS: For this retrospective study, we used Slicer Dicer software (Epic Verona, WI), an Epic self-service reporting tool, to query clinical data and identified a cohort of 9,040 patients ≥ 18 years old diagnosed with COVID-10 at Grady Memorial Hospital in Atlanta from 1/1/21 to 12/31/21. Statistical significance was defined as p<0.05. RESULTS: Of the 9,040 patients, 54.7% were female (4,942) and 45.3% were male (4,096). The cohort median age was 51 (range 18 – 100) and 80.5% were African American (7,278/9,040). Double-dose vaccination rate was only 24.5% (2,215/9,040). 38.3% of patients diagnosed with COVID-19 were admitted (3,467/9,040) and among these patients 3.0% were re-admitted (107/3,467). The most prevalent comorbidities were essential hypertension (45.2%), diabetes (21.7%), and asthma (13.2%). Patients with these comorbidities were more likely to be discharged as opposed to being admitted. Patients with the following comorbidities were more likely to be admitted: Pulmonary hypertension (70% admission rate), COPD (64.9%), heart failure (61.0%), cancer (60.8%), atrial fibrillation (57.1%). Median LOS from admission was 4 days and there was no statistical difference among different comorbidities. We found higher mortality in COVID-19 patients with cancer (12.9%), atrial fibrillation (12.6%), heart failure (11.1%), pulmonary hypertension (10.1%) and COPD (9.1%) compared to patients with diabetes (7.5%), hypertension (6.7%), HIV (4.8%), DVT/PE (4.6%), or asthma (2.7%). When examining overall mortality based on self- reported race, we found that African American patients had a statistically significant higher mortality compared to Caucasian patients (p-value= 0.00454). CONCLUSIONS: Current retrospective study, which included COVID-19 patients with different comorbidities showed that COVID-19 patients with pulmonary hypertension have worse clinical outcomes compared to other comorbid conditions. CLINICAL IMPLICATIONS: Our findings suggest the importance of investigating COVID-19 disparities in marginalized populations to better understand the impact in these communities. All individuals should be encouraged to get vaccinated against COVID-19, especially those found to be at high risk of severe illness such as pulmonary hypertension. In this retrospective study, we found higher hospital admission rate and worse outcomes in patients with cancer, atrial fibrillation, heart failure, and pulmonary hypertension, as well as higher mortality among the African American patient population. DISCLOSURES: No relevant relationships by nicolas bakinde No relevant relationships by Suvrat Chandra No relevant relationships by Michelle Lee no disclosure on file for Mario Ponce;

UP IN SMOKE: PATTERNS OF THC USE AND CANNABINOID HYPEREMESIS SYNDROME AT AN ACADEMIC MEDICAL CENTER

Background/Aim The prevalence of marijuana use has increased in the United States as many states have legalized its use. Cannabinoid hyperemesis syndrome (CHS) is an adverse effect that 17-30% of chronic users of marijuana will experience. The impact of the COVID- 19 pandemic on healthcare disruptions has been well established. The effect of the pandemic on vice-associated conditions has been described with increases in alcohol and substance related hospitalizations and mortality. Few studies have evaluated the effect of the COVID- 19 pandemic on CHS with regards to prevalence, admissions, readmissions, and healthcare burden. We sought to identify the impact of the COVID-19 pandemic on CHS using admissions and readmissions as metrics to evaluate healthcare burden. Methods Using Slicer-Dicer, an electronic medical record based self-service query tool, all cases of CHS requiring hospital admissions and those resulting in readmissions were recorded at the university's 3 hospital centers and stratified by gender, age, and location. Data was separated into pre-COVID (August 5, 2018 to April 5, 2020) and post-COVID (April 6, 2020 to October 5, 2021). Additionally, all positive cases of tetrahydrocannabinol (THC) tested were recorded and stratified by postal code. The primary outcome was identification of CHS cases requiring admission from the emergency department pre-COVID and post-COVID. The secondary outcome was identifying any differences in admission and readmission rates pre- COVID and post-COVID. Results A significant increase in total THC positive cases (p = <0.001) was seen with 2485 pre-COVID and 2936 post-COVID cases. 68 patients were diagnosed with CHS pre-COVID and 75 post-COVID. Cases requiring admission were 27.9% pre-COVID CHS and 30.7% post-COVID with a significant increase in admissions from one campus from 0% to 30.4% (p = 0.025). Pre-COVID CHS cases requiring readmission after an index admission was 31.6% and post-COVID was 26.1%. No significance was seen when stratifying the cohorts by gender and age. Discussion Our study shows a significant increase in CHS diagnoses and admissions with an associated significant increase in THC-positivity when comparing the pre-COVID and post-COVID cohorts. This is consistent with prior studies describing an increase in alcohol and substance use during the pandemic. Data from the Centers for Disease Control and Prevention also reveal a 30% increase in substance related deaths in 2020 when compared to 2019. Some suggest that factors related to the pandemic including social isolation stress, substance use in isolation, and decreased access to substance use treatment or programs are contributors. This study highlights the importance to identify this association to better understand and respond to pandemic-associated risk factors for substance use disorders to help alleviate its effect on healthcare burden.

CRISPR-Cas, Argonaute proteins and the emerging landscape of amplification-free diagnostics.

Polymerase Chain Reaction (PCR) is the reigning gold standard for molecular diagnostics. However, the SARS-CoV-2 pandemic reveals an urgent need for new diagnostics that provide users with immediate results without complex procedures or sophisticated equipment. These new demands have stimulated a tsunami of innovations that improve turnaround times without compromising the specificity and sensitivity that has established PCR as the paragon of diagnostics. Here we briefly introduce the origins of PCR and isothermal amplification, before turning to the emergence of CRISPR-Cas and Argonaute proteins, which are being coupled to fluorimeters, spectrometers, microfluidic devices, field-effect transistors, and amperometric biosensors, for a new generation of nucleic acid-based diagnostics.

Emerging Argonaute-based nucleic acid biosensors.

Recent advances in Argonaute (Ago)-mediated biotechnology have provided new insights into the development of programmable and highly sensitive nucleic acid detection platforms. This study provides an overview of recent research on Ago-based nucleic acid detection. The potential applications of these emerging nucleic acid biosensors and the challenges associated with their use have been discussed.

Characterization of a Programmable Argonaute Nuclease from the Mesophilic Bacterium Rummeliibacillus suwonensis.

Prokaryotic Argonautes (pAgos) from mesophilic bacteria are attracting increasing attention for their genome editing potential. So far, it has been reported that KmAgo from Kurthia massiliensis can utilize DNA and RNA guide of any sequence to effectively cleave DNA and RNA targets. Here we find that three active pAgos, which have about 50% sequence identity with KmAgo, possess typical DNA-guided DNA target cleavage ability. Among them, RsuAgo from Rummeliibacillus suwonensis is mainly explored for which can cleave both DNA and RNA targets. Interestingly, RsuAgo-mediated RNA target cleavage occurs only with short guide DNAs in a narrow length range (16-20 nt), and mismatches between the guide and target sequence greatly affect the efficiency of RNA target cleavage. RsuAgo-mediated target cleavage shows a preference for a guide strand with a 5'-terminal A residue. Furthermore, we have found that RsuAgo can cleave double-stranded DNA in a low-salt buffer at 37 °C. These properties of RsuAgo provide a new tool for DNA and RNA manipulation at moderate temperatures.

Argonaute-integrated isothermal amplification for rapid, portable, multiplex detection of SARS-CoV-2 and influenza viruses.

Isothermal amplification methods are a promising trend in virus detection because of their superiority in rapidity and sensitivity. However, the generation of false positives and limited multiplexity are major bottlenecks that must be addressed. In this study, we developed a multiplex Argonaute (Ago)-based nucleic acid detection system (MULAN) that integrates rapid isothermal amplification with the multiplex inclusiveness of a single Ago for simultaneous detection of multiple targets such as SARS-CoV-2 and influenza viruses. Owing to its high specificity, MULAN can distinguish targets at a single-base resolution for mutant genotyping. Moreover, MULAN also supports portable and visible devices with a limit of detection of five copies per reaction. Validated by SARS-CoV-2 pseudoviruses and clinical samples of influenza viruses, MULAN showed 100% agreement with quantitative reverse-transcription PCR. These results demonstrated that MULAN has great potential to facilitate reliable, easy, and quick point-of-care diagnosis for promoting the control of infectious diseases.

A virus-derived microRNA targets immune response genes during SARS-CoV-2 infection.

SARS-CoV-2 infection results in impaired interferon response in patients with severe COVID-19. However, how SARS-CoV-2 interferes with host immune responses is incompletely understood. Here, we sequence small RNAs from SARS-CoV-2-infected human cells and identify a microRNA (miRNA) derived from a recently evolved region of the viral genome. We show that the virus-derived miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer, which are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3'UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 can potentially employ a virus-derived miRNA to hijack the host miRNA machinery, which could help to evade the interferon-mediated immune response.

Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARS-CoV-2 and HPV.

Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.