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Background: Amidst the persistent global health threat posed by the evolving SARS-CoV-2 virus throughout the four-year-long COVID-19 pandemic, the focus has now turned to the Omicron variant and its subvariant, JN.1, which has rapidly disseminated worldwide. This study reports on the characteristics and clinical manifestations of patients during the surge of the JN.1 variant in Saudi Arabia; it also investigates the evolution of SARS-CoV-2 variants in organ transplant patients and identifies patient risk factors., Methods: A total of 151 nasopharyngeal samples from patients with PCR-confirmed SARS-CoV-2 infection were collected between September 2023 and January 2024. Demographic and clinical data of the patients were obtained from electronic health records. All confirmed positive samples underwent sequencing using Ion GeneStudio and the Ion AmpliSeq™ SARS-CoV-2 panel., Results: During the surge of the JN.1 variant, the average age of the patients was 40 years, ranging from 3 to 93 years, and nearly 50% of the patients were male. Our investigation revealed that the J.N variant predominantly infected patients with comorbidities or organ transplant recipients (57.6%). Moreover, patients with comorbidities or organ transplants exhibited a higher number of mutations. In our organ transplant cohort, an increased total number of spike mutations was associated with a lower risk of developing severe disease (OR = 0.96, 95% CI: 0.93-0.98)., Conclusions: Although JN.1 may not prove to be particularly harmful, it is crucial to recognize the persistent emergence of concerning variants, which create new pathways for the virus to evolve. The ongoing evolution of SARS-CoV-2 is evident in the continuous divergence of these variants from the original strain that marked the onset of the pandemic nearly four years ago., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Abnormal transcriptomic profiles of coagulation genes have been linked to coagulopathies in patients with coronavirus disease 2019 (COVID-19). The objective of the present study was to explore the frequency of genotypes and potential association of polymorphisms in genes encoding coagulation factors with the disease severity in COVID-19 patients.The patients were clinically categorized into four groups of COVID-19 disease severity (asymptomatic, mild, moderate, and severe). Three variants of genes, involving the coagulation genes rs3136516 (F2 gene), rs6042 (F7 gene), and rs6123 (PROS1 gene), were studied. Polymorphisms were genotyped by Sanger DNA sequencing.Most of the subjects had moderate COVID-19 infection (n = 53, 62.4%), followed by mild (n = 16, 18.8%), and severe infections (n = 15, 17.6%). The frequency of the rs3136516 AG genotype was considerably higher in non-ICU patients compared to ICU patients (51.3% vs 34.1%, OR 3.167, 95% CI 1.094-9.170, P  = .031). Furthermore, the dominant genetic model (AA + AG vs GG) was significantly associated with a decreased probability of admission to the ICU in COVID-19 patients (OR 0.340, 95% CI 0.127 - 0.905, and P  = .028). No other variants of the coagulation genes studied were found to be associated with the severity of COVID-19 disease, admission to the ICU, and mortality ( P  > .05).The rs3136516 AG genotype could predispose COVID-19 patients to increased disease severity and therefore admission to the ICU, while the dominant genetic model (AA + AG vs GG) of rs3136516 exerts a protective role., Competing Interests: Declaration of Conflicting InterestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

RNA viruses, including SARS-CoV-2, rely on genetic mutation as a major evolutionary mechanism, leading to the emergence of variants. Organ transplant recipients (OTRs) may be particularly vulnerable to such mutations, making it crucial to monitor the spread and evolution of SARS-CoV-2 in this population. This cohort study investigated the molecular epidemiology of SARS-CoV-2 by comparing the SARS-CoV-2 whole genome, demographic characteristics, clinical conditions, and outcomes of COVID-19 illness among OTRs ( n = 19) and non-OTRs with ( n = 38) or without ( n = 30) comorbid conditions. Most patients without comorbidities were female, whereas most OTRs were male. Age varied significantly among the three groups: patients with comorbidities were the oldest, and patients without comorbidities were the youngest. Whole-genome sequencing revealed that OTRs with mild disease had higher numbers of unusual mutations than patients in the other two groups. Additionally, OTRs who died had similar spike monoclonal antibody resistance mutations and 3CLpro mutations, which may confer resistance to nirmatrelvir, ensitrelvir, and GC37 therapy. The presence of those unusual mutations may impact the severity of COVID-19 illness in OTRs by affecting the virus's ability to evade the immune system or respond to treatment. The higher mutation rate in OTRs may also increase the risk of the emergence of new virus variants. These findings highlight the importance of monitoring the genetic makeup of SARS-CoV-2 in all immunocompromised populations and patients with comorbidity.

The global demand for rapid identification of circulating SARS-CoV-2 variants of concern has led to a shortage of commercial kits. Therefore, this study aimed to develop and validate a rapid, cost-efficient genome sequencing protocol to identify circulating SARS-CoV-2 (variants of concern). Sets of primers flanking the SARS-CoV-2 spike gene were designed, verified and then validated using 282 nasopharyngeal positive samples for SARS-CoV-2. Protocol specificity was confirmed by comparing these results with SARS-CoV-2 whole-genome sequencing of the same samples. Out of 282 samples, 123 contained the alpha variant, 78 beta and 13 delta, which were indicted using in-house primers and next-generation sequencing; the numbers of variants found were 100% identical to the reference genome. This protocol is easily adaptable for detection of emerging variants during the pandemic.

Background: SARS-CoV-2 infects through the respiratory route and triggers inflammatory response by affecting multiple cell types including type II alveolar epithelial cells. SARS-CoV-2 triggers signals via its Spike (S) protein, which have been shown to participate in the pathogenesis of COVID19., Aim: Aim of the present study was to investigate the effect of SARS-CoV2 on type II alveolar epithelial cells, focusing on signals initiated by its S protein and their impact on the expression of inflammatory mediators., Results: For this purpose A549 alveolar type II epithelial cells were exposed to SARS CoV2 S recombinant protein and the expression of inflammatory mediators was measured. The results showed that SARS-CoV-2 S protein decreased the expression and secretion of IL8, IL6 and TNFα, 6 hours following stimulation, while it had no effect on IFNα, CXCL5 and PAI-1 expression. We further examined whether SARS-CoV-2 S protein, when combined with TLR2 signals, which are also triggered by SARS-CoV2 and its envelope protein, exerts a different effect in type II alveolar epithelial cells. Simultaneous treatment of A549 cells with SARS-CoV-2 S protein and the TLR2 ligand PAM3csk4 decreased secretion of IL8, IL6 and TNFα, while it significantly increased IFNα, CXCL5 and PAI-1 mRNA expression. To investigate the molecular pathway through which SARS-CoV-2 S protein exerted this immunomodulatory action in alveolar epithelial cells, we measured the induction of MAPK/ERK and PI3K/AKT pathways and found that SARS-CoV-2 S protein induced the activation of the serine threonine kinase AKT. Treatment with the Akt inhibitor MK-2206, abolished the inhibitory effect of SARS-CoV-2 S protein on IL8, IL6 and TNFα expression, suggesting that SARS-CoV-2 S protein mediated its action via AKT kinases., Conclusion: The findings of our study, showed that SARS-CoV-2 S protein suppressed inflammatory responses in alveolar epithelial type II cells at early stages of infection through activation of the PI3K/AKT pathway. Thus, our results suggest that at early stages SARS-CoV-2 S protein signals inhibit immune responses to the virus allowing it to propagate the infection while in combination with TLR2 signals enhances PAI-1 expression, potentially affecting the local coagulation cascade., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Al-Qahtani, Pantazi, Alhamlan, Alothaid, Matou-Nasri, Sourvinos, Vergadi and Tsatsanis.)

Objective: To describe the chronological genomic evolution of SARS-CoV-2 and its impact on public health in the Middle East and North Africa (MENA) region., Methods: This study analysed all available SARS-CoV-2 genomic sequences, metadata and rates of COVID-19 infection from the MENA region retrieved from the Global Initiative on Sharing All Influenza Data database from January 2020 to August 2021. Inferential and ‎descriptive statistics were conducted to describe the epidemiology of SARS-CoV-2., Results: Genomic surveillance of SARS-CoV-2 in the MENA region indicated that the variants in January 2020 predominately belonged to the G, GR, GH or O clades and that the most common variant of concern was Alpha. By August 2021, however, the GK clade dominated (57.4% of all sequenced genomes), followed by the G clade (18.7%) and the GR clade (11.6%). In August, the most commonly sequenced variants of concern were Delta in the Middle East region (91%); Alpha (44.3%) followed by Delta (29.7%) and Beta (25.3%) in the North Africa region; and Alpha (88.9%), followed by Delta (10%) in the fragile and conflict-affected regions of MENA. The mean proportion of the variants of concern among the total sequenced samples differed significantly by country (F=1.93, P=0.0112) but not by major MENA region (F=0.14, P=0.27) or by vaccination coverage (F=1.84, P=0.176)., Conclusion: This analysis of the genomic surveillance of SARS-CoV-2 provides an essential description the virus evolution and its impact on public health safety in the MENA region. As of August 2021, the Delta variant showed a genomic advantage in the MENA region. The MENA region includes several fragile and conflict-affected countries with extremely low levels of vaccination coverage and little genomic surveillance, which may soon exacerbate the existing health crisis within those countries and globally., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Even with the widespread availability of vaccines for the COVID-19 disease, there is no sign of decline in the rate of spread of the disease. Based on findings of different studies across the globe, the disease is characterized by poor outcomes in specific sociodemographic categories such as age, gender and presence of symptoms., Methods: In this study, we carried out a multivariable logistic regression analysis on a national database (HESN+) of confirmed COVID-19 cases in Saudi Arabia to determine predictors of hospitalization and mortality for these patients., Results: Data was extracted for 328,301 confirmed COVID- 19 patients (mean age (SD) = 37.79 (1.68)) with 34.92% females and 65.08% males. Of these, 59.87% were Saudi Arabian citizens and 40.13% were non-Saudi. 68.91% of cases were discovered in Riyadh (n = 67,384), Makkah (n = 72,590) and the Eastern Province (n = 79,666). 72.2% of all cases were diagnosed and treated by the Ministry of Health (MOH). Of all confirmed cases, 95.28% showed one or more symptoms associated with COVID-19. 5.48% of these were hospitalized and 1.11% died. Predictors of mortality and hospitalization, respectively, included age (OR; 1.088 and 1.03), being male (OR; 1.443 and 1.138), nationality (OR; 2.11 and 1.993), presence of symptoms (OR; 1.816 and 4.386), and the health care sector in which patients received treatment (MOH OR; 1.352 and 4.731)., Conclusion: We found that COVID-19-related hospitalization or mortality was higher among males, older adults, and patients showing one or more symptoms, and mortality likelihood was more than fourfold for patients treated by the MOH. Immigrants were also more likely to be hospitalized or die from COVID-19 infection compared to Saudi nationals., Competing Interests: Conflicts of interest The authors declare that there is no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Coronaviridae is a large family of enveloped, positive-strand RNA viruses that has plagued the world since it was discovered in humans in the 1960s. The recent severe acute respiratory syndrome coronavirus (SARS-CoV)-2 pandemic has already exceeded the number of combined cases and deaths witnessed during previous SARS-CoV and Middle East respiratory syndrome-CoV epidemics in the last two decades. This narrative review focuses on genomic mutations in SARS-CoV-2 and their impact on the severity and progression of COVID-19 in light of reported data in the literature. Notable SARS-CoV-2 mutations associated with open reading frames, the S glycoprotein, and nucleocapsid protein, currently circulating globally, are discussed along with emerging mutations such as those in the SARS-CoV-2 VUI 202012/01 variant in the UK and other European countries, the 484K.V2 and P.1 variants in Brazil, the B.1.617 variant in India, and South African variants 501Y.V2 and B.1.1.529 (omicron). These variants have the potential to influence the receptor binding domain, host-virus fusion, and SARS-CoV-2 replication. Correlating these mutations with disease dynamics could help us understand their pathogenicity and design appropriate therapeutics.

Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/μL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, β, and δ SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.

SARS-CoV-2 vaccination in solid organ transplant recipients is associated with suboptimal immune response and risk for breakthrough infection. It is not known whether they are at risk of severe post-vaccine breakthrough infections in the presence of SARSCoV-2 variant of concern. We describe a case series of four fully vaccinated solid organ transplant recipients who developed SARS-CoV-2 variants of concern breakthrough infections. Three patients received BNT162b2 mRNA (Pfizer-BioNTech) and one patient received ChAdOx1 (AZD12220) COVID-19 vaccines. The patients were infected with SARS-CoV-2 variants circulating in Saudi Arabia. Two patients were infected with Alpha variant and had severe pneumonia requiring intensive care admission and ventilatory support and subsequently died. The other two patients recovered; one patient was infected with Beta variant required low supplemental oxygen via nasal flow and the other patient was infected with Delta variant and required high supplemental oxygen nasal flow. Younger patients had a better outcome than older patients. Future large studies are required to confirm our observations and to compare the different vaccine efficacy among solid organ transplants in the era of SARS-CoV-2 variants of concern., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: In December 2019, a new severe acute respiratory syndrome coronavirus, SARS-CoV-2, emerged in China, causing coronavirus disease 2019. The present study investigated genetic profiles and variations of SARS-CoV-2 distributed in different regions of Saudi Arabia to begin to understand the pathogenesis and transmission of SARS-CoV-2 in this country and analyzed associations of these variations with host factors., Methodology: In total, 774 SARS-CoV-2 genomic sequences obtained and annotated by the Global Initiative on Sharing All Influenza Data (GISAID) were captured and analyzed., Results: The most common SARS-CoV-2 clades in Saudi Arabia were GH followed by O, GR, G, and S. Statistically significant associations were detected between clades and patient outcome. Age, as a host factor, was significantly associated with many variables, including virus geographical location, clade, and patient outcome. The most common variants detected were the NSP12_P323L mutation 94.9%, followed by the D614G mutation (76%) and the NS3_Q57H mutation (71.4%). The concerned variants B.1.1.7, B.1.351, and P.1 were not detected in our population. D614G was associated with higher morbidities than the wild-type virus, including higher rates of death and hospitalization. The NS3_Q57H mutation was the only variant associated with better patient outcome than the wild type. Risk of death was highest with the NSP12_P323L mutation (OR = 1.84; 95% CI = 0.37-9.30) and lowest with the NS3_Q57H mutation (OR = 0.43; 95% CI = 0.25-0.727)., Conclusions: SARS-CoV-2 has evolved uniquely and independently in Saudi Arabia. Our findings provide evidence to begin linking the evolutionary implications to host factors and their effects on the virus severity and transmission., Competing Interests: No Conflict of Interest is declared, (Copyright (c) 2021 Dalia Abdullah Obeid, Madain Saleh Alsanea, Rawan Talal Alnemari, Ahmed Ali Al-Qahtani, Sahar Isa Althawadi, Maysoon Saleh Mutabagani, Reem Saad Almaghrabi, Faten Mohammed Alhadheq, Basma Mohammed Alahideb, Fatimah Saeed Alhamlan.)

Background: One major challenge for detecting the virus that causes COVID-19 is commercial SARS-CoV-2 testing kit or reagent availability. To allow every laboratory or hospital access to an in-house assay, we developed a low-cost SARS-CoV-2 detection assay protocol using in-house primers and reagents/equipment on hand in most biology or diagnostic laboratories: a SYBR Green-based RT-PCR. RNA extraction has also become a major bottleneck due to limited supplies and the required labor. Thus, we validated an alternative RNA extraction protocol., Methods: We designed and synthesized in-house primers according to SARS-CoV-2 genome sequences retrieved from GISAID database. One hundred and ninety patient samples were collected by nasopharyngeal swab, coded, and used to develop and validate the assay protocol. RNA extraction was performed using TRI reagent-based RNA protocol to inactivate the virus; thus, testing was conducted in a conventional biosafety level 2 laboratory., Results: The sensitivity and specificity of the primers were evaluated using 190 patient samples previously tested for SARS-CoV-2. The positive amplicons were sequenced to confirm the results. The assay protocol was developed, and the specificity of each RT-PCR product was confirmed using melting curve analyses. Of 190 samples, the SYBR Green-based RT-PCR assay detected SARS-CoV-2 target genes in 88 samples, with no false-positive results. These findings indicate that the sensitivity of our assay was 97.7% and specificity of 100% with those of the diagnostic laboratory that tested the same samples using a Rotor-Gene PCR cycler with an Altona Diagnostics SARS-CoV-2 kit (R 2 = 0.89)., Conclusions: These approaches are reliable, repeatable, specific, sensitive, simple, and low-cost tools for the detection of SARS-CoV-2 in a conventional biosafety level 2 laboratory, offering alternative approaches when commercial kits are unavailable or not affordable., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Pantazi, Al-Qahtani, Alhamlan, Alothaid, Matou-Nasri, Sourvinos, Vergadi and Tsatsanis.)