Impact of the SARS-CoV-2 nucleocapsid 203K/204R mutations on the inflammatory immune response in COVID-19 severity.

BACKGROUND: The excessive inflammatory responses provoked by SARS-CoV-2 infection are critical factors affecting the severity and mortality of COVID-19. Previous work found that two adjacent co-occurring mutations R203K and G204R (KR) on the nucleocapsid (N) protein correlate with increased disease severity in COVID-19 patients. However, links with the host immune response remain unclear. METHODS: Here, we grouped nasopharyngeal swab samples of COVID-19 patients into two cohorts based on the presence and absence of SARS-CoV-2 nucleocapsid KR mutations. We performed nasopharyngeal transcriptome analysis of age, gender, and ethnicity-matched COVID-19 patients infected with either SARS-CoV-2 with KR mutations in the N protein (KR patients n = 39) or with the wild-type N protein (RG patients n = 39) and compared to healthy controls (n = 34). The impact of KR mutation on immune response was further characterized experimentally by transcriptomic and proteomic profiling of virus-like-particle (VLP) incubated cells. RESULTS: We observed markedly elevated expression of proinflammatory cytokines, chemokines, and interferon-stimulated (ISGs) genes in the KR patients compared to RG patients. Using nasopharyngeal transcriptome data, we found significantly higher levels of neutrophils and neutrophil-to-lymphocyte (NLR) ratio in KR patients than in the RG patients. Furthermore, transcriptomic and proteomic profiling of VLP incubated cells confirmed a similar hyper-inflammatory response mediated by the KR variant. CONCLUSIONS: Our data demonstrate an unforeseen connection between nucleocapsid KR mutations and augmented inflammatory immune response in severe COVID-19 patients. These findings provide insights into how mutations in SARS-CoV-2 modulate host immune output and pathogenesis and may contribute to more efficient therapeutics and vaccine development.

Incidence, predictors and outcomes of redo pancreatectomy in infants with congenital hyperinsulinism: a 16-year tertiary center experience.

PURPOSE: Recurrent severe hypoglycemic attacks often persist even after performing pancreatectomy for medically unresponsive congenital hyperinsulinism (CHI). In this study, we present our experience with redo pancreatectomy for CHI. METHODS: We reviewed all children who underwent pancreatectomy for CHI between January 2005 and April 2021 in our center. A comparison was made between patients whose hypoglycemia was controlled after primary pancreatectomy and patients who required reoperation. RESULTS: A total of 58 patients underwent pancreatectomy for CHI. Refractory hypoglycemia after pancreatectomy occurred in 10 patients (17%), who subsequently underwent redo pancreatectomy. All patients who required redo pancreatectomy had positive family history of CHI (p = 0.0031). Median extent of initial pancreatectomy was lesser in the redo group with borderline level of statistical significance (95% vs. 98%, p = 0.0561). Aggressive pancreatectomy at the initial surgery significantly (p = 0.0279) decreased the risk for the need to redo pancreatectomy; OR 0.793 (95% CI 0.645-0.975). Incidence of diabetes was significantly higher in the redo group (40% vs. 9%, p = 0.033). CONCLUSION: Pancreatectomy with 98% extent of resection for diffuse CHI, especially with positive family history of CHI, is warranted to decrease the chance of reoperation for persistent severe hypoglycemia.

Severe Rheumatic Mitral Stenosis, Worse Left Atrial Mechanics is Closely Associated with Echo Criteria for Intervention.

Background: Rheumatic mitral valve (MV) stenosis is associated with progressive left atrial (LA) fibrosis and functional impairment, Pulmonary artery systolic pressure (PASP) and right ventricular (RV) dysfunction. The aims of the study were to determine in those patients with severe MV stenosis if LA mechanical function as assessed by speckle tracking echocardiography could identify those with increased PASP, atrial fibrillation (AFib), and RV dysfunction. Subjects and Methods: Patients with severe MV stenosis were identified from the institutional echo database. Echocardiograms were read off line and measurements included atrial and ventricular strain. Patients were divided into tertiles of LA reservoir strain (LASr) values and data compared between the groups. Results: Ninety-seven patients, 67 females, mean age 47.4 ± 11.9 years, had MV mean gradient of 8.3 ± 5.1 mmHg, MV area by pressure half time of 1.3 ± 0.3 cm2 and LASr of 11.18% ± 6.4%. Those patients in the lowest LASr tertile had more AFib (72%, P = 0.0001), PASP >50 mm Hg (39%, P = 0.005), and worst RV impairment. In multivariable logistic regression analysis, LASr, age, and mean MV gradient were the independent predictors of AFib and PASP >50 mm Hg. Cutoffs, determined by receiver operating characteristic curve analysis had high specificity for the composite outcome of Afib and PASP >50 mmHg (85% for LASr <7.7%). Conclusion: In severe MV stenosis LASr, age and mean MV gradient, are independent predictors of Afib and PASP >50 mmHg. LASr <7.7% has high sensitivity and specificity in identifying those who meet ESC guideline 2017 criteria for valve intervention, suggesting its potentially helpful addendum to the surveillance of patients with MV stenosis.

SARS-CoV-2 genomes from Saudi Arabia implicate nucleocapsid mutations in host response and increased viral load.

Monitoring SARS-CoV-2 spread and evolution through genome sequencing is essential in handling the COVID-19 pandemic. Here, we sequenced 892 SARS-CoV-2 genomes collected from patients in Saudi Arabia from March to August 2020. We show that two consecutive mutations (R203K/G204R) in the nucleocapsid (N) protein are associated with higher viral loads in COVID-19 patients. Our comparative biochemical analysis reveals that the mutant N protein displays enhanced viral RNA binding and differential interaction with key host proteins. We found increased interaction of GSK3A kinase simultaneously with hyper-phosphorylation of the adjacent serine site (S206) in the mutant N protein. Furthermore, the host cell transcriptome analysis suggests that the mutant N protein produces dysregulated interferon response genes. Here, we provide crucial information in linking the R203K/G204R mutations in the N protein to modulations of host-virus interactions and underline the potential of the nucleocapsid protein as a drug target during infection.

Recurrence of left ventricular pseudoaneurysm after multiple mitral valve replacements.

Left ventricular pseudoaneurysm (LVPA) formation is a potentially lethal complication of myocardial infarction (MI) and mitral valve (MV) replacement that requires prompt diagnosis and treatment. A female patient who had been complaining of exertional dyspnea underwent a two-dimensional transthoracic echocardiogram (TTE) which revealed a functioning mechanical MV with severe paravalvular leak, severe tricuspid regurgitation (TR) and severely elevated pulmonary artery systolic pressure. Moreover, echo-lucent space at the postero-lateral portion of the left ventricle near the MV was seen, suggestive of a large LVPA. Transesophageal echocardiography (TEE) and computed tomography (CT) angiography confirmed these findings. Afterwards, the patient had a surgical repair for the LVPA along with mitral and tricuspid valve (TV) replacement. Three months later, the patient presented with symptoms of congestive heart failure. The LVPA had recurred at the same location of the previous pseudoaneurysm and given the high risk for reoperating on the patient, close monitoring and medical management were deemed as a better option.

Quick and Easy Assembly of a One-Step qRT-PCR Kit for COVID-19 Diagnostics Using In-House Enzymes.

One-step reverse-transcription quantitative polymerase chain reaction (qRT-PCR) is the most widely applied method for COVID-19 diagnostics. Notwithstanding the facts that one-step qRT-PCR is well suited for the diagnosis of COVID-19 and that there are many commercially available one-step qRT-PCR kits in the market, their high cost and unavailability due to airport closures and shipment restriction became a major bottleneck that had driven the desire to produce the key components of such kits locally. Here, we provide a simple, economical, and powerful one-step qRT-PCR kit based on patent-free, specifically tailored versions of Moloney murine leukemia virus reverse transcriptase and Thermus aquaticus DNA polymerase and termed R3T (Rapid Research Response Team) one-step qRT-PCR. We also demonstrate the robustness of our enzyme production strategies and provide the optimal reaction conditions for their efficient augmentation in a one-step approach. Our kit was routinely able to reliably detect as low as 10 copies of the synthetic RNAs of SARS-CoV-2. More importantly, our kit successfully detected COVID-19 in clinical samples of broad viral titers with similar reliability and selectivity to that of the Invitrogen SuperScript III Platinum One-step qRT-PCR and TaqPath one-step RT-qPCR kits. Overall, our kit has shown robust performance in both laboratory settings and the Saudi Ministry of Health-approved testing facility.

A Robust, Safe, and Scalable Magnetic Nanoparticle Workflow for RNA Extraction of Pathogens from Clinical and Wastewater Samples.

Molecular diagnosis and surveillance of pathogens such as SARS-CoV-2 depend on nucleic acid isolation. Pandemics at the scale of COVID-19 can cause a global shortage of proprietary commercial reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. An open-source method, magnetic-nanoparticle-aided viral RNA isolation from contagious samples (MAVRICS), built upon readily available reagents, and easily assembled in any basically equipped laboratory, is thus developed. The performance of MAVRICS is evaluated using validated pathogen detection assays and real-world and contrived samples. Unlike conventional methods, MAVRICS works directly in samples inactivated in phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities. MAVRICS allows wastewater biomass immobilized on membranes to be directly inactivated and lysed in TRIzol followed by RNA extraction by magnetic nanoparticles, thereby greatly reducing biohazard risk and simplifying processing procedures. Using 39 COVID-19 patient samples and two wastewater samples, it is shown that MAVRICS rivals commercial kits in detection of SARS-CoV-2, influenza viruses, and respiratory syncytial virus. Therefore, MAVRICS is safe, fast, and scalable. It is field-deployable with minimal equipment requirements and could become an enabling technology for widespread testing and wastewater monitoring of diverse pathogens.

Depression and Obsessive-Compulsive Disorders Amid the COVID-19 Pandemic in Saudi Arabia.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is a global pandemic with significant morbidity and mortality. The Saudi government adopted mandatory home quarantine and curfew hours for all residents, excluding essential service workers. During the lockdown, the public's fear of infection can adversely impact mental health, causing psychological distress. The objective of this research is to assess frequency of depression and obsessive-compulsive disorder (OCD) among the general population during COVID-19 pandemic in Saudi Arabia. METHODOLOGY: This was a cross-sectional study using the Patient Health Questionnaire (PHQ-9) and Obsessive-Compulsive Inventory-Revised assessment test (OCI-R) in Arabic and English languages distributed via social media platforms. Chi-square test was used with significance determined at p<0.005. RESULTS: We received a total of 2187 responses. Our results showed that being female, single, and unemployed had a higher frequency of suffering from depression whereas higher income levels and higher education was associated with less depression frequency. Increasing age, males, married individuals, higher income groups, higher levels of education and employed individuals were more likely to have OCD during COVID-19 pandemic.  Conclusion: The COVID-19 pandemic period is associated with significant mental health risks among the Saudi population. The results can provide further scientific support to better understand the impact of quarantine on psychological distress and mental health during emergency and disaster situations.

iSCAN: An RT-LAMP-coupled CRISPR-Cas12 module for rapid, sensitive detection of SARS-CoV-2.

The COVID-19 pandemic caused by SARS-CoV-2 affects all aspects of human life. Detection platforms that are efficient, rapid, accurate, specific, sensitive, and user friendly are urgently needed to manage and control the spread of SARS-CoV-2. RT-qPCR based methods are the gold standard for SARS-CoV-2 detection. However, these methods require trained personnel, sophisticated infrastructure, and a long turnaround time, thereby limiting their usefulness. Reverse transcription-loop-mediated isothermal amplification (RT-LAMP), a one-step nucleic acid amplification method conducted at a single temperature, has been used for colorimetric virus detection. CRISPR-Cas12 and CRISPR-Cas13 systems, which possess collateral activity against ssDNA and RNA, respectively, have also been harnessed for virus detection. Here, we built an efficient, rapid, specific, sensitive, user-friendly SARS-CoV-2 detection module that combines the robust virus amplification of RT-LAMP with the specific detection ability of SARS-CoV-2 by CRISPR-Cas12. Furthermore, we combined the RT-LAMP-CRISPR-Cas12 module with lateral flow cells to enable highly efficient point-of-care SARS-CoV-2 detection. Our iSCAN SARS-CoV-2 detection module, which exhibits the critical features of a robust molecular diagnostic device, should facilitate the effective management and control of COVID-19.

Ventricular tachycardia: beginning and ending fate of a benign invasive cardiac lipoma.

Cardiac lipomas are rare benign heart tumors. Their clinical manifestations primarily depend on their dimension and location. We describe a case of a 14-year-old boy complaining of palpitations. ECG showed non-sustained ventricular tachycardia (NSVT). Echocardiography and cardiac MRI showed a large apical mass suggestive of a cardiac lipoma. The patient underwent open-heart surgery which detected a pericardial mass, partially attached to the myocardium, and crossed by the distal segment of LAD. This critical anatomical relationship prevents its complete resection. The biopsy confirmed mature fat tissue. Postoperatively, Propranolol was started for NSVT episodes. After 8 months, he presented with VT recurrence. A subcutaneous ICD (S-ICD) was implanted. Two months later, he was admitted for an electrical storm with six appropriate shocks. Since then, amiodarone was added. Lipomas are not always benign and their surgery might be very difficult and unhelpful. A multidisciplinary team is crucial for their diagnosis and management.