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.

Epidemiology and Clinical Characteristics of People with Confirmed SARS-CoV-2 Infection during the Early COVID-19 Pandemic in Saudi Arabia.

This study provides epidemiologic and clinical characteristics of 492 consecutive patients diagnosed with SARS-CoV-2 infection at King Faisal Specialist Hospital and Research Centre in Saudi Arabia between March and September 2020. Data were collected from electronic case reports. The cohort was 54% male, with 20.4% aged >60 years, 19.9% aged 31-40 years, and 17% aged 41-50 years. The median incubation period was 16 days, with upper and lower 95% quartiles of 27 and 10 days, respectively. Most patients (79.2%) were symptomatic. Variables significantly different between symptomatic and asymptomatic patients were age, blood oxygen saturation percentage, hemoglobin level, lymphocyte count, neutrophil to lymphocyte (NTL) ratio, and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) level. Asymptomatic patients were mostly younger, with lower body mass index and ALT and AST levels but higher lymphocyte counts, NTL ratio, and CD4, CD8, natural killer cell, IgG, and IgM levels. Factors associated with increased risk of mortality were age (>42 years) and comorbidities, particularly diabetes mellitus and hypertension. Patients who were not given an antiviral regimen were associated with better prognosis than patients who received an antiviral regimen (HR, 0.07; 95% CI, 0.011-0.25). These findings will help clinicians and policymakers adopt best management and treatment options for SARS-CoV-2 infection.

Epidemiological characteristics, clinical course, and laboratory investigation of pediatric COVID-19 patients in a tertiary care center in Saudi Arabia.

Background: Since the initial emergence of the novel SARS-CoV-2 coronavirus responsible for the 2019 coronavirus disease (COVID-19) pandemic, many studies have been exploring the nature and characteristics of this virus and its associated clinical manifestations. The present study aimed to describe the clinical presentation and outcomes of COVID-19 infections in pediatric patients. Methods: A retrospective review of findings associated with 143 pediatric patients (age <14 years) with a confirmed COVID-19 diagnosis who had undergone inpatient or outpatient treatment at King Faisal Specialist Hospital and Research Center in Riyadh, Saudi Arabia, between March 2020 and October 2020, was conducted. The analyzed data included patient demographic information, pre-existing medical conditions, symptoms, interventions, and outcomes. Results: The median age of this patient population was 7 years. Of these 143 patients, 67 (46.8%) had known pre-existing medical conditions including bronchial asthma (12.8%), chronic lung disease (CLD) (3%), congenital heart disease (CHD) (17%), primary immunodeficiencies (1.5%), malignancies (9.8%), and 7.5% were post-transplant patients. Thirty-seven patients (26%) were overweight or obese. Sixty-three of these patients (51%) were symptomatic, with the most common symptom being fever (55%). Ultimately, 45 patients (31%) required admission to the hospital, with a median duration of hospitalization of 9.6 days for admitted patients. There were no documented cases of infection-related mortality among this pediatric cohort, although 11 patients experienced post-infectious complications that primarily manifested as a loss of taste and smell. Conclusion: These findings suggest that pediatric COVID-19 patients tend to experience mild forms of the disease, without any significant differences in disease severity as a function of patient gender or immune status.

SARS-CoV-2 genetic diversity and variants of concern in Saudi Arabia.

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.

Seroprevalence of SARS-CoV-2 antibodies in healthcare workers at a tertiary care hospital in Riyadh, Saudi Arabia.

Background: The SARS-CoV-2 pandemic has led to a strain on medical resources. The development of countermeasures to prevent its spread is evolving. Healthcare workers (HCWs) are at high risk for contracting and transmitting the disease. Methods: Serology testing of volunteer HCWs was performed at King Faisal Specialist Hospital and Research Center in Riyadh (the Center) in order to determine the prevalence of SARS-CoV-2 antibodies, as well as the associated risk factors, in the hope of implementing adequate prevention and control measures. Results: 1076 subjects participated in this study, of whom 24.3% were seropositive. The majority were nurses (379, 35%) or physicians (245, 22.2%). 392 (36.4%) of the 1076 subjects were caregivers for COVID-19 patients, and 463 (43.0%) reported contact with infected employees. There was a statistically significant association between taking care of COVID-19 patients and being diagnosed with COVID-19 (chi-square test, p = 0.046). There was a significant association between being in contact with infected employees and having a positive IgG result (chi-square test, p < 0.001). Conclusions: A baseline analysis of SARS-CoV-2 seropositivity in HCWs at a large tertiary care hospital in Riyadh was performed as the first part of a prospective study of HCWs. The reported seropositivity was 24.3% - higher than that of other hospitals in Riyadh. IgG testing was very useful in the detection of previous SARS-CoV-2 infection, as it has high negative predictive value.

Test-based de-isolation in COVID-19 immunocompromised patients: Cycle threshold value versus SARS-CoV-2 viral culture.

BACKGROUND: Immunocompromised patients with coronavirus disease 2019 (COVID-19) have prolonged infectious viral shedding for more than 20 days. A test-based approach is suggested for de-isolation of these patients. METHODS: The strategy was evaluated by comparing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load (cycle threshold (Ct) values) and viral culture at the time of hospital discharge in a series of 13 COVID-19 patients: six immunocompetent and seven immunocompromised (five solid organ transplant patients, one lymphoma patient, and one hepatocellular carcinoma patient). RESULTS: Three of the 13 (23%) patients had positive viral cultures: one patient with lymphoma (on day 16) and two immunocompetent patients (on day 7 and day 11). Eighty percent of the patients had negative viral cultures and had a mean Ct value of 20.5. None of the solid organ transplant recipients had positive viral cultures. CONCLUSIONS: The mean Ct value for negative viral cultures was 20.5 in this case series of immunocompromised patients. Unlike those with hematological malignancies, none of the solid organ transplant patients had positive viral cultures. Adopting the test-based approach for all immunocompromised patients may lead to prolonged quarantine. Large-scale studies in disease-specific populations are needed to determine whether a test-based approach versus a symptom-based approach or a combination is applicable for the de-isolation of various immunocompromised patients.

Development and validation of an in-house, low-cost SARS-CoV-2 detection assay.

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 (R2 = 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.

Portable RT-PCR System: a Rapid and Scalable Diagnostic Tool for COVID-19 Testing.

Combating the ongoing coronavirus disease 2019 (COVID-19) pandemic demands accurate, rapid, and point-of-care testing with fast results to triage cases for isolation and treatment. The current testing relies on reverse transcriptase PCR (RT-PCR), which is routinely performed in well-equipped laboratories by trained professionals at specific locations. However, during busy periods, high numbers of samples queued for testing can delay the test results, impacting efforts to reduce the infection risk. Besides, the absence of well-established laboratories at remote sites and low-resourced environments can contribute to a silent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These reasons compel the need to accommodate point-of-care testing for COVID-19 that meets the ASSURED criteria (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable). This study assessed the agreement and accuracy of the portable Biomeme SARS-CoV-2 system against the gold standard tests. Nasopharyngeal and nasal swabs were used. Of the 192 samples tested using the Biomeme SARS-CoV-2 system, the results from 189 samples (98.4%) were in agreement with the reference standard-of-care RT-PCR testing for SARS-CoV-2. The portable system generated simultaneous results for nine samples in 80 min with high positive and negative percent agreements of 99.0% and 97.8%, respectively. We performed separate testing in a sealed glove box, offering complete biosafety containment. Thus, the Biomeme SARS-CoV-2 system can help decentralize COVID-19 testing and offer rapid test results for patients in remote and low-resourced settings.