Characteristics and clinical manifestations of patients, including organ transplant patients, during the surge of JN.1: Insights from Saudi Arabia.

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.

Genomic Surveillance and Mutation Analysis of SARS-CoV-2 Variants among Patients in Saudi Arabia.

The genome of severe acute respiratory coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), has undergone a rapid evolution, resulting in the emergence of multiple SARS-CoV-2 variants with amino acid changes. This study aimed to sequence the whole genome of SARS-CoV-2 and detect the variants present in specimens from Saudi Arabia. Furthermore, we sought to analyze and characterize the amino acid changes in the various proteins of the identified SARS-CoV-2 variants. A total of 1161 samples from patients diagnosed with COVID-19 in Saudi Arabia, between 1 April 2021 and 31 July 2023, were analyzed. Whole genome sequencing was employed for variant identification and mutation analysis. The statistical analysis was performed using the Statistical Analytical Software SAS, version 9.4, and GraphPad, version 9.0. This study identified twenty-three variants and subvariants of SARS-CoV-2 within the population, with the Omicron BA.1 (21K) variant (37.0%) and the Delta (21J) variant (12%) being the most frequently detected. Notably, the Omicron subvariants exhibited a higher mean mutation rate. Amino acid mutations were observed in twelve proteins. Among these, the spike (S), ORF1a, nucleocapsid (N), and ORF1b proteins showed a higher frequency of amino acid mutations compared to other the viral proteins. The S protein exhibited the highest incidence of amino acid mutations (47.6%). Conversely, the ORF3a, ORF8, ORF7a, ORF6, and ORF7b proteins appeared more conserved, demonstrating the lowest percentage and frequency of amino acid mutations. The investigation of structural protein regions revealed the N-terminal S1 subunit of the S protein to frequently harbor mutations, while the N-terminal domain of the envelope (E) protein displayed the lowest mutation frequency. This study provides insights into the variants and genetic diversity of SARS-CoV-2, underscoring the need for further research to comprehend its genome evolution and the occurrence of mutations. These findings are pertinent to the development of testing approaches, therapeutics, and vaccine strategies.

Diagnosis of Human Cytomegalovirus Drug Resistance Mutations in Solid Organ Transplant Recipients-A Review.

Human cytomegalovirus (HCMV) infection may be asymptomatic in healthy individuals but can cause severe complications in immunocompromised patients, including transplant recipients. Breakthrough and drug-resistant HCMV infections in such patients are major concerns. Clinicians are first challenged to accurately diagnose HCMV infection and then to identify the most effective antiviral drug and determine when to initiate therapy, alter drug dosage, or switch medication. This review critically examines HCMV diagnostics approaches, particularly for immunocompromised patients, and the development of genotypic techniques to rapidly diagnose drug resistance mutations. The current standard method to identify prevalent and well-known resistance mutations involves polymerase chain reaction amplification of UL97, UL54, and UL56 gene regions, followed by Sanger sequencing. This method can confirm clinical suspicion of drug resistance as well as determine the level of drug resistance and range of cross-resistance with other drugs. Despite the effectiveness of this approach, there remains an urgent need for more rapid and point-of-care HCMV diagnosis, allowing for timely lifesaving intervention.

Association of SARS-CoV-2 Nucleocapsid Protein Mutations with Patient Demographic and Clinical Characteristics during the Delta and Omicron Waves.

SARS-CoV-2 genomic mutations outside the spike protein that may increase transmissibility and disease severity have not been well characterized. This study identified mutations in the nucleocapsid protein and their possible association with patient characteristics. We analyzed 695 samples from patients with confirmed COVID-19 in Saudi Arabia between 1 April 2021, and 30 April 2022. Nucleocapsid protein mutations were identified through whole genome sequencing. 𝜒2 tests and t tests assessed associations between mutations and patient characteristics. Logistic regression estimated the risk of intensive care unit (ICU) admission or death. Of the 60 mutations identified, R203K was the most common, followed by G204R, P13L, E31del, R32del, and S33del. These mutations were associated with reduced risk of ICU admission. P13L, E31del, R32del, and S33del were also associated with reduced risk of death. By contrast, D63G, R203M, and D377Y were associated with increased risk of ICU admission. Most mutations were detected in the SR-rich region, which was associated with low risk of death. The C-tail and central linker regions were associated with increased risk of ICU admission, whereas the N-arm region was associated with reduced ICU admission risk. Consequently, mutations in the N protein must be observed, as they may exacerbate viral infection and disease severity. Additional research is needed to validate the mutations' associations with clinical outcomes.

SARS-CoV-2 spike gene Sanger sequencing methodology to identify variants of concern.

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.

Molecular Epidemiology of SARS-CoV-2 and Clinical Manifestations among Organ Transplant Recipients with COVID-19.

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.

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2.

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, ß, 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.