Comparison of SARS-CoV-2 Detection in Nasopharyngeal Swab and Saliva Samples from Patients Infected with Omicron Variant

To compare the detection of the SARS-CoV-2 Omicron variant in nasopharyngeal-swab (NPS) and oral saliva samples. 255 samples were obtained from 85 Omicron-infected patients. SARS-CoV-2 load was measured in the NPS and saliva samples by using Simplexa™ COVID-19 direct and Alinity m SARS-CoV-2 AMP assays. Results obtained with the two diagnostic platforms showed very good inter-assay concordance (91.4 and 82.4% for saliva and NPS samples, respectively) and a significant correlation among cycle threshold (Ct) values. Both platforms revealed a highly significant correlation among Ct obtained in the two matrices. Although the median Ct value was lower in NPS than in saliva samples, the Ct drop was comparable in size for both types of samples after 7 days of antiviral treatment of the Omicron-infected patients. Our result demonstrates that the detection of the SARS-CoV-2 Omicron variant is not influenced by the type of sample used for PCR analysis, and that saliva can be used as an alternative specimen for detection and follow-up of Omicron-infected patients.

Comparison of SARS-CoV-2 Detection in Nasopharyngeal Swab and Saliva Samples from Patients Infected with Omicron Variant.

To compare the detection of the SARS-CoV-2 Omicron variant in nasopharyngeal-swab (NPS) and oral saliva samples. 255 samples were obtained from 85 Omicron-infected patients. SARS-CoV-2 load was measured in the NPS and saliva samples by using Simplexa™ COVID-19 direct and Alinity m SARS-CoV-2 AMP assays. Results obtained with the two diagnostic platforms showed very good inter-assay concordance (91.4 and 82.4% for saliva and NPS samples, respectively) and a significant correlation among cycle threshold (Ct) values. Both platforms revealed a highly significant correlation among Ct obtained in the two matrices. Although the median Ct value was lower in NPS than in saliva samples, the Ct drop was comparable in size for both types of samples after 7 days of antiviral treatment of the Omicron-infected patients. Our result demonstrates that the detection of the SARS-CoV-2 Omicron variant is not influenced by the type of sample used for PCR analysis, and that saliva can be used as an alternative specimen for detection and follow-up of Omicron-infected patients.

Safety and immune response kinetics of GRAd-COV2 vaccine: phase 1 clinical trial results.

Despite the successful deployment of efficacious vaccines and therapeutics, the development of novel vaccines for SARS-CoV-2 remains a major goal to increase vaccine doses availability and accessibility for lower income setting. We report here on the kinetics of Spike-specific humoral and T-cell response in young and old volunteers over 6 months follow-up after a single intramuscular administration of GRAd-COV2, a gorilla adenoviral vector-based vaccine candidate currently in phase-2 of clinical development. At all three tested vaccine dosages, Spike binding and neutralizing antibodies were induced and substantially maintained up to 3 months, to then contract at 6 months. Potent T-cell responses were readily induced and sustained throughout the study period, with only minor decline. No major differences in immune response to GRAd-COV2 vaccination were observed in the two age cohorts. In light of its favorable safety and immunogenicity, GRAd-COV2 is a valuable candidate for further clinical development and potential addition to the COVID-19 vaccine toolbox to help fighting SARS-CoV-2 pandemic.

Monitoring of SARS-CoV-2 circulation using saliva testing in school children in Rome, Italy.

OBJECTIVES: To describe the trend of SARS-CoV-2 RNA in saliva samples from children attending nine schools in Rome in the local surveillance unit RM3 during the period of September 2021-March 2022, in parallel with the trend of SARS-CoV-2 RNA observed in nasopharyngeal swabs (NPSs) from the population in the same catchment area that was routinely tested at our laboratory in the same period. METHODS: Saliva samples were collected using the Copan LolliSpongeTM device and analyzed by Aptima® SARS-CoV-2 Assay on the Panther® System. NPSs were tested using either Aptima® SARS-CoV-2 Assay or Alinity m SARS-CoV-2 Assay. RESULTS: The percentage of positivity in the two populations was different; of the 2222 saliva samples from students, 0.99% had positive results, whereas the percentage was higher (33.43%) in the 8994 NPSs representing the population from local surveillance unit RM3. Interestingly, the trend of SARS-CoV-2 RNA in saliva samples from students was consistent with that observed in NPSs from the population in same catchment area, although with peaks slightly anticipated. CONCLUSION: Overall, screening of saliva in the schools represents a good system to monitor SARS-CoV-2 circulation in the population, allowing early detection and quick isolation of students who are asymptomatic with positive test results and thus prevention of virus transmission.

Rapid and qualitative identification of SARS-CoV-2 mutations associated with variants of concern using a multiplex RT-PCR assay coupled with melting analysis.

OBJECTIVES: Considering the spread of new genetic variants and their impact on public health, it is important to have assays that are able to rapidly detect SARS-CoV-2 variants. METHODS: We retrospectively examined 118 positive nasopharyngeal swabs, first characterized by the Sanger sequencing, using the Simplexa® SARS-CoV-2 Variants Direct assay, with the aim of evaluating the performance of the assay to detect N501Y, G496S, Q498R, Y505H, E484K, E484Q, E484A, and L452R mutations. RESULTS: A total of 111/118 nasopharyngeal swabs were in complete agreement with the Sanger sequencing, whereas the remaining seven samples were not amplified due to the low viral load. The evaluation of the ability of the assay to detect the E484Q mutation was performed using a viral isolate of the SARS-CoV-2 Kappa variant, showing concordance in 15/15 samples. Simplexa® SARS-CoV-2 Variant Direct assay was able to detect mutation pattern of Alpha, Beta, Gamma, Delta, and Omicron variants with 100% specificity and 94% sensitivity, whereas 100% sensitivity and specificity for the Kappa variant was observed. CONCLUSION: The assay can be useful to obtain faster results, contributing to a prompt surveillance of SARS-CoV-2 variants; however, it requires to be confirmed by the Sanger method, especially in the case of pattern of mutations that are different from those expected and also requires updates as new variants emerge.

Longitudinal dynamics of SARS-CoV-2 anti-receptor binding domain IgG antibodies in a wide population of health care workers after BNT162b2 vaccination.

OBJECTIVES: With the availability of vaccines, commercial assays detecting anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies evolved toward quantitative assays directed to the spike glycoprotein or its receptor-binding domain (RBD). The objective was to perform a large-scale, longitudinal study involving health care workers (HCWs), with the aim of establishing the kinetics of immune response throughout the 9-month period after receipt of the second dose of the BNT162b2 vaccine. METHODS: Quantitative determination of immunoglobulin (Ig) G antibodies against the RBD of the S1 subunit of the spike protein of SARS-CoV-2 on the Alinity systems. RESULTS: The highest levels of anti-RBD IgG were measured after 1 month from full vaccination (median: 1432 binding antibody units/ml [BAU/ml]); subsequently, a steep decrease (7.4-fold decrease) in IgG levels was observed at 6 months (median: 194.3 BAU/ml), with a further 2.5-fold decrease at 9 months (median: 79.3 BAU/ml). Furthermore, the same data, when analyzed for sex, showed significant differences between male and female participants at both 1 and 9 months from vaccination, but not at 6 months. CONCLUSION: Our results confirm the tendency of anti-RBD antibodies to decrease over time, also when extending the analysis up to 9 months, and highlight a better ability of the female sex to produce antibodies 1 month and 9 months after vaccination. Overall, these data, obtained in a wide population of HCWs, support the importance of having increased the vaccine doses.

Characterization of raloxifene as a potential pharmacological agent against SARS-CoV-2 and its variants.

The new coronavirus SARS-CoV-2 is the causative agent of the COVID-19 pandemic, which so far has caused over 6 million deaths in 2 years, despite new vaccines and antiviral medications. Drug repurposing, an approach for the potential application of existing pharmaceutical products to new therapeutic indications, could be an effective strategy to obtain quick answers to medical emergencies. Following a virtual screening campaign on the most relevant viral proteins, we identified the drug raloxifene, a known Selective Estrogen Receptor Modulator (SERM), as a new potential agent to treat mild-to-moderate COVID-19 patients. In this paper we report a comprehensive pharmacological characterization of raloxifene in relevant in vitro models of COVID-19, specifically in Vero E6 and Calu-3 cell lines infected with SARS-CoV-2. A large panel of the most common SARS-CoV-2 variants isolated in Europe, United Kingdom, Brazil, South Africa and India was tested to demonstrate the drug's ability in contrasting the viral cytopathic effect (CPE). Literature data support a beneficial effect by raloxifene against the viral infection due to its ability to interact with viral proteins and activate protective estrogen receptor-mediated mechanisms in the host cells. Mechanistic studies here reported confirm the significant affinity of raloxifene for the Spike protein, as predicted by in silico studies, and show that the drug treatment does not directly affect Spike/ACE2 interaction or viral internalization in infected cell lines. Interestingly, raloxifene can counteract Spike-mediated ADAM17 activation in human pulmonary cells, thus providing new insights on its mechanism of action. A clinical study in mild to moderate COVID-19 patients (NCT05172050) has been recently completed. Our contribution to evaluate raloxifene results on SARS-CoV-2 variants, and the interpretation of the mechanisms of action will be key elements to better understand the trial results, and to design new clinical studies aiming to evaluate the potential development of raloxifene in this indication.

Changes in the Circulation of Common Respiratory Pathogens among Hospitalized Patients with Influenza-like Illnesses in the Lazio Region (Italy) during Fall Season of the Past Three Years.

A descriptive analysis of common respiratory pathogens (CRPs) detected in nasopharyngeal swabs (NPSs) from hospitalized patients with influenza-like illness during the fall seasons of the past three years, 2019-2021, in the Lazio region, Italy, was conducted to assess whether or not CRP circulation changed because of COVID-19 during the fall season. The results observed in a total of 633 NPSs subjected to molecular diagnosis for CRPs by multiplex PCR assay during the autumn seasons (exactly from week 41 to week 50) were compared with each other. In 2019, in 144 NPSs, the more represented CRPs were rhinovirus/enterovirus (7.6%) and influenza A/B (4.2%). In 2020, 55 (21.6%) out of 255 NPSs resulted positive for SARS-CoV-2 and, except for one case of Legionella pneumophila, the CRPs detected were exclusively rhinovirus/enterovirus (4.7%). In 2021, among 234 NPSs, 25.6% resulted positive for SARS-CoV-2, 14.5% for respiratory syncytial virus (RSV), and 12.8% for rhinovirus/enterovirus. Compared with 2019, in 2020, CRP circulation was severely limited to a few cases; in 2021, instead, infections by RSV (detected also among adults), rhinovirus/enterovirus, and other respiratory pathogens were observed again, while influenza was practically absent. The comparison of the CRPs detected in the NPSs depicts a different circulation in the Lazio region during the last three fall seasons. CRP monitoring has a direct impact on the prevention and control strategies of respiratory infectious diseases, such as the non-pharmacological interventions implemented in response to the COVID-19 pandemic. Future studies should investigate the impact of specific interventions on the spread of respiratory infections.

Differential Dynamics of SARS-CoV-2 Binding and Functional Antibodies upon BNT162b2 Vaccine: A 6-Month Follow-Up.

To investigate the dynamic association among binding and functional antibodies in health-care-workers receiving two doses of BNT162b2 mRNA COVID-19-vaccine, SARS-CoV-2 anti-RBD IgG, anti-Trimeric-S IgG, and neutralizing antibodies (Nabs) were measured in serum samples collected at 2 weeks, 3 months, and 6 months from full vaccination. Despite the high correlation, results for anti-RBD and anti-Trimeric S IgG were numerically different even after recalculation to BAU/mL following WHO standards indications. Moreover, after a peak response at 2 weeks, anti-RBD IgG levels showed a 4.5 and 13 fold decrease at 3 and 6 months, respectively, while the anti-Trimeric S IgG presented a less pronounced decay of 2.8 and 4.7 fold. Further different dynamics were observed for Nabs titers, resulting comparable at 3 and 6 months from vaccination. We also demonstrated that at NAbs titers ≥40, the area under the receiver operating characteristic curve and the optimal cutoff point decreased with time from vaccination for both anti-RBD and anti-Trimeric S IgG. The mutating relation among the anti-RBD IgG, anti-Trimeric S IgG, and neutralizing antibodies are indicative of antibody maturation upon vaccination. The lack of standardized laboratory procedures is one factor interfering with the definition of a correlate of protection from COVID-19.

Comparison of Allplex™ SARS-CoV-2 Assay, Easy SARS-CoV-2 WE and Lumipulse quantitative SARS-CoV-2 antigen test performance using automated systems for the diagnosis of COVID-19.

Diagnostic methods based on SARS-CoV-2 antigen detection are a promising alternative to SARS-CoV-2 RNA amplification. We evaluated the automated chemiluminescence-based Lumipulse® G SARS-CoV-2 Ag assay as compared to real time assays (combined results from RT-PCR Allplex™ SARS-CoV-2 assay and Easy SARS-CoV-2 WE kit) on 513 nasopharyngeal swabs (NPS). Among these, 53.6% resulted positive to RT-PCR, considered as the reference test. Compared to the reference test, overall sensitivity and specificity of Lumipulse® G SARS-CoV-2 Ag assay were 84.0%, and 89.1%, respectively, and overall agreement between the antigen and molecular assays was substantial (κ = 0.727). When stratifying samples into groups based on ranges of RT-PCR cycle threshold (Ct), the antigen test sensitivity was >95% for samples with Ct <30. Linear regression analysis showed strong and highly significant correlation between the Lumipulse Ag concentrations and the RT-PCR Ct values (RdRp gene), irrespective of whether the Ct values from molecular test were combined in a unique regression analysis or analysed separately. Overall, chemiluminescence-based antigen assay may be reliably applied to NPS samples to identify individuals with high viral loads, more likely to transmit the virus.

Predicting the protective humoral response to a SARS-CoV-2 mRNA vaccine.

OBJECTIVES: Simple and standardized methods to establish correlates to vaccine-elicited SARS-CoV-2 protection are needed. METHODS: An observational study on antibody response to a mRNA vaccine (Comirnaty) was performed on health care workers (V, n=120). Recovered COVID-19 patients (N, n=94) were used for comparison. Antibody response was evaluated by a quantitative anti-receptor binding domain IgG (anti-RBD) commercial assay and by virus microneutralization test (MNT), in order to establish a threshold of anti-RBD binding antibody units (BAU) able to predict a robust (≥1:80) MNT titer. RESULTS: Significant correlation between BAU and MNT titers was found in both V and N, being stronger in V (rs=0.91 and 0.57 respectively, p<0.001); a higher incremental trend starting from MNT titer 1:80 was observed in the V group. The 99% probability of high MNT titer (≥1:80) was reached at 1,814 and 3,564 BAU/mL, and the area under the receiver operating characteristic (ROC) curve was 0.99 (CI: 0.99-1.00) and 0.78 (CI: 0.67-0.86) in V and N, respectively. CONCLUSIONS: A threshold of 2,000 BAU/mL is highly predictive of strong MNT response in vaccinated individuals and may represent a good surrogate marker of protective response. It remains to be established whether the present results can be extended to BAU titers obtained with other assays.

Frequency and Duration of SARS-CoV-2 Shedding in Oral Fluid Samples Assessed by a Modified Commercial Rapid Molecular Assay.

BACKGROUND: RT-PCR on nasopharyngeal (NPS)/oropharyngeal swabs is the gold standard for diagnosis of SARS-CoV-2 infection and viral load monitoring. Oral fluid (OF) is an alternate clinical sample, easy and safer to collect and could be useful for COVID-19 diagnosis, monitoring viral load and shedding. METHODS: Optimal assay conditions and analytical sensitivity were established for the commercial Simplexa™ COVID-19 Direct assay adapted to OF matrix. The assay was used to test 337 OF and NPS specimens collected in parallel from 164 hospitalized patients; 50 bronchoalveolar lavage (BAL) specimens from a subgroup of severe COVID-19 cases were also analysed. RESULTS: Using Simplexa™ COVID-19 Direct on OF matrix, 100% analytical detection down to 1 TCID50/mL (corresponding to 4 × 103 copies (cp)/mL) was observed. No crossreaction with other viruses transmitted through the respiratory toute was observed. Parallel testing of 337 OF and NPS samples showed highly concordant results (κ = 0.831; 95 % CI = 0.771-0.891), and high correlation of Ct values (r = 0.921; p < 0.0001). High concordance and elevated correlation was observed also between OF and BAL. Prolonged viral RNA shedding was observed up to 100 days from symptoms onset (DSO), with 32% and 29% positivity observed in OF and NPS samples, respectively, collected between 60 and 100 DSO. CONCLUSIONS: Simplexa™ COVID-19 Direct assays on OF have high sensitivity and specificity to detect SARS-CoV-2 RNA and provide an alternative to NPS for diagnosis and monitoring SARS-CoV-2 shedding.

Lumipulse G SARS-CoV-2 Ag assay evaluation using clinical samples from different testing groups.

OBJECTIVES: Compared to RT-PCR, lower performance of antigen detection assays, including the Lumipulse G SARS-CoV-2 Ag assay, may depend on specific testing scenarios. METHODS: We tested 594 nasopharyngeal swab samples from individuals with COVID-19 (RT-PCR cycle threshold [Ct] values ≤ 40) or non-COVID-19 (Ct values >40) diagnoses. RT-PCR positive samples were assigned to diagnostic, screening, or monitoring groups of testing. RESULTS: With a limit of detection of 1.2 × 104 SARS-CoV-2 RNA copies/mL, Lumipulse showed positive percent agreement (PPA) of 79.9% (155/194) and negative percent agreement of 99.3% (397/400), whereas PPAs were 100% for samples with Ct values of <18 or 18-<25 and 92.5% for samples with Ct values of 25-<30. By three groups, Lumipulse showed PPA of 87.0% (60/69), 81.1% (43/53), or 72.2% (52/72), respectively, whereas PPA was 100% for samples with Ct values of <18 or 18-<25, and was 94.4, 80.0, or 100% for samples with Ct values of 25-<30, respectively. Additional testing of RT-PCR positive samples for SARS-CoV-2 subgenomic RNA showed that, by three groups, PPA was 63.8% (44/69), 62.3% (33/53), or 33.3% (24/72), respectively. PPAs dropped to 55.6, 20.0, or 41.7% for samples with Ct values of 25-<30, respectively. All 101 samples with a subgenomic RNA positive result had a Lumipulse assay's antigen positive result, whereas only 54 (58.1%) of remaining 93 samples had a Lumipulse assay's antigen positive result. CONCLUSIONS: Lumipulse assay was highly sensitive in samples with low RT-PCR Ct values, implying repeated testing to reduce consequences of false-negative results.

Saliva Is a Valid Alternative to Nasopharyngeal Swab in Chemiluminescence-Based Assay for Detection of SARS-CoV-2 Antigen.

Diagnostic methods based on SARS-CoV-2 antigens detection are a promising alternative to SARS-CoV-2 RNA amplification. We evaluated the automated chemiluminescence-based Lumipulse® G SARS-CoV-2 Ag assay on saliva samples, using Simplexa™ COVID-19 Direct assay as a reference test. Analytical performance was established on a pool of healthy donors' saliva samples spiked with the 2019-nCoV/Italy-INMI1 isolate, whereas clinical performance was assessed on fresh saliva specimens collected from hospitalized patients with suspect or confirmed COVID-19 diagnosis. The limit of detection (LOD) was 0.65 Log TCID50/mL, corresponding to 18,197 copies/mL of SARS-CoV-2 RNA. Antigen concentrations and SARS-CoV-2 RNA were highly correlated (r = 0.99; p < 0.0001). Substantial agreement (80.3%) and significant correlation (r = -0.675; p = 0.0006) were observed between Lumipulse® G assay results and Ct values on clinical samples, with 52.4% sensitivity and specificity 94.1%. Sensitivity exceeded 90.0% when calculated on samples with Ct < 25, and specificity was 100% when excluding samples from recovered patients with previous COVID-19 diagnosis. Overall, chemiluminescence-based antigen assay may be reliably applied to saliva samples to identify individuals with high viral loads, more likely to transmit the virus. However, the low positive predictive value in a context of low SARS-CoV-2 prevalence underscores the need for confirmatory testing in SARS-CoV-2 antigen-positive cases.

Risk and predictive factors of prolonged viral RNA shedding in upper respiratory specimens in a large cohort of COVID-19 patients admitted to an Italian reference hospital.

BACKGROUND: Limited data are available about the predictors and outcomes associated with prolonged SARS-CoV-2 RNA shedding (VS). METHODS: A retrospective study including COVID-19 patients admitted to an Italian hospital between March 1 and July 1, 2020. Predictors of viral clearance (VC) and prolonged VS from the upper respiratory tract were assessed by Poisson regression and logistic regression analyses. The causal relation between VS and clinical outcomes was evaluated through an inverse probability weighted Cox model. RESULTS: The study included 536 subjects. The median duration of VS from symptoms onset was 18 days. The estimated 30-day probability of VC was 70.2%. Patients with comorbidities, lymphopenia at hospital admission, or moderate/severe respiratory disease had a lower chance of VC. The development of moderate/severe respiratory failure, delayed hospital admission after symptoms onset, baseline comorbidities, or D-dimer >1000ng/mL at admission independently predicted prolonged VS. The achievement of VC doubled the chance of clinical recovery and reduced the probability of death/mechanical ventilation. CONCLUSIONS: Respiratory disease severity, comorbidities, delayed hospital admission and inflammatory markers negatively predicted VC, which resulted to be associated with better clinical outcomes. These findings highlight the importance of prompt hospitalization of symptomatic patients, especially where signs of severity or comorbidities are present.

Virological Characterization of the First 2 COVID-19 Patients Diagnosed in Italy: Phylogenetic Analysis, Virus Shedding Profile From Different Body Sites, and Antibody Response Kinetics.

BACKGROUND: The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remains unclear. We report the detection of viral RNA from different anatomical districts and the antibody profile in the first 2 COVID-19 cases diagnosed in Italy. METHODS: We tested for SARS-CoV-2 RNA clinical samples, either respiratory and nonrespiratory (ie, saliva, serum, urine, vomit, rectal, ocular, cutaneous, and cervico-vaginal swabs), longitudinally collected from both patients throughout the hospitalization. Serological analysis was carried out on serial serum samples to evaluate IgM, IgA, IgG, and neutralizing antibody levels. RESULTS: SARS-CoV-2 RNA was detected since the early phase of illness, lasting over 2 weeks in both upper and lower respiratory tract samples. Virus isolate was obtained from acute respiratory samples, while no infectious virus was rescued from late respiratory samples with low viral RNA load, collected when serum antibodies had been developed. Several other specimens came back positive, including saliva, vomit, rectal, cutaneous, cervico-vaginal, and ocular swabs. IgM, IgA, and IgG were detected within the first week of diagnosis, with IgG appearing earlier and at higher titers. Neutralizing antibodies developed during the second week, reaching high titers 32 days after diagnosis. CONCLUSIONS: Our longitudinal analysis showed that SARS-CoV-2 RNA can be detected in different body samples, which may be associated with broad tropism and different spectra of clinical manifestations and modes of transmission. Profiling antibody response and neutralizing activity can assist in laboratory diagnosis and surveillance actions.